Brain, Behavior, and Immunity 14, 77–140 (2000) doi:10.1006/brbi.2000.0588, available online at http://www.idealibrary.com on
PNIRS ABSTRACTS Stressful Life Events and Disease Activity in Multiple Sclerosis. Kurt D. Ackerman,* Bruce S. Rabin,† Rock Heyman,† and Andrew Baum,* *Department of Psychiatry, †Department of Immunopathology, and †Department of Neurology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213. Multiple sclerosis is one of the most common, disabling neurologic disorders in the United States. Its etiology is thought to involve autoimmune destruction of myelin within the CNS, most often in a relapsing and remitting course. This makes MS an excellent model for studying bi-directional neural– immune interactions. Since its original description, MS disease activity has been thought to be strongly linked to stress. Despite the large number of studies examining this association, the nature of this relationship has remained elusive. We are currently conducting a longitudinal study of MS, which is designed to clarify the relationship between life stress and disease activity and identify possible mediating factors. Twenty-five relapsing–remitting MS female subjects have completed the study to date. Subjects were followed for one year using weekly questionnaires and monthly monitoring of physiologic parameters. Stressors were identified using the Life Events and Difficulties Schedule and life event checklists. Exacerbations were recorded weekly and confirmed by a clinical neurologist. Of the 59 confirmed exacerbations, 85% were preceded by a stressful life event within 6 weeks. This finding was independent of stressor severity, with approximately 50% of stressors leading to exacerbation. A paired T test was used to compare the time from the closest stressor to a randomly selected exacerbation or control period. Exacerbations occurred 14 days following life events, versus 35 days for the control period (p ⬍ .0001). Analyses are currently underway to determine whether this effect is related autonomic, neuroendocrine, and immune changes. (This work was supported by NIH Grants 1K01MH01468 and M01RR00056.) Role of the Opioid System in Lipopolysaccharide-Induced Fever in Rats. Martin W. Adler, Khalid Benamar, Jose C. Cabassa, Ellen B. Geller, and Li Xin, Center for Substance Abuse Research and Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania. Although lipopolysaccharide (LPS) has long been known to induce fever, the mechanisms involved are not fully understood. Work in our laboratory has demonstrated that the endogenous opioid system is a prime component of thermoregulation, with the mu-opioid receptor mediating hyperthermic responses and the kappa-opioid receptor mediating hypothermic responses. We have also shown that opioids are involved in the production of IL-1-mediated fever in the rat. To explore the role of the opioid system in LPS-induced fever, we gave adult male S–D rats (Zivic–Miller) LPS (50 µg/kg, i.p.), and measured body temperature (Tb) rectally over a period of several hours. Pretreatment with morphine (M) (10 mg/kg, s.c.) blocked LPS-induced fever, although M itself produced hyperthermia. Similarly, chronic M (2 slow-release pellets, each containing 75 mg M, s.c.) also blocked LPS-induced fever 3 days after pellet implantation. CTAP, a selective mu-opioid receptor antagonist microinjected into the POAH (1 µg) 30 min prior to LPS, significantly reduced LPS-induced fever. However, intra-POAH injection of CTAP failed to affect LPS-induced fever if given 3 hr after LPS. We are exploring the possibility that the similarity in effect of both the mu agonist (M) and the antagonist (CTAP) in blocking LPS-induced fever results from different routes of administration of the 2 drugs or release of an endogenous chemical by M given parenterally. The data indicate that the mu-opioid system is involved in the induction of LPS-induced fever. (Supported by Grant DA 00376 from the National Institute on Drug Abuse.) Disruptions of Social Relationships Accentuate the Association between Distress and Dysmenorrhea in Young Women. Carmen Alonso* and Christopher Coe,* *Department of Psychology and †Harlow Center for Biological Psychology, University of Wisconsin, Madison, Wisconsin. This study examined the effects of social support on dysmenorrhea and whether social support moderates the relationship between negative emotions and painful symptoms. One hundred eighty-four female undergraduates completed self-report questionnaires on dysmenorrhea, depression, anxiety, and their 77 0889-1591/00 $35.00
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social networks. Social support did not have a direct effect on dysmenorrhea, in contrast to negative emotions, which were significantly correlated with symptoms (distressed women evinced more dysmenorrhea). Moreover, differences in the level of social support did not directly account for this relationship between anxiety, depression, and dysmenorrhea. However, disruptions in social support during the previous year did affect the reporting of dysmenorrhea. Women who had lost more than three supportive persons manifested significantly more dysmenorrhea than women with stable social relations. In addition, this type of disruption in their social networks moderated the relationship between distress and dysmenorrhea symptoms. These results indicate the importance of considering specific aspects of social support when studying its effect on health. Ongoing studies are evaluating the link between these psychosocial variables, menstrual functioning, and vulnerability to viral infection in women. Social Factors Associated with the Development of Corticosterone Resistance in Stressed Mice. Ronit Avitsur,* Jennifer L. Stark,* and John F. Sheridan,*,† *Department of Oral Biology and †Institute for Behavioral Medicine Research, Ohio State University, Columbus, Ohio 43210. Male mice establish social hierarchies, typically based on dominance of a single mouse over a number of subordinates. This social order may change if the dominant male is challenged and defeated. We have previously shown that repeated disruption of the social hierarchy (Social Disruption, SDR) reduced the responsiveness of peripheral immune cells to corticosterone. In the present study, we explored the behavioral changes that contributed to the development of corticosterone resistance in splenocytes. In these studies, an aggressive intruder was introduced into cages of C57BL/6 male mice for 2–14 h. A detailed analysis of the interactions between the intruder and the residents indicated that multiple factors were associated with the induction of corticosterone resistance. First, resident mice experiencing more attacks and exhibiting more submissive responses showed reduced sensitivity to corticosterone. Moreover, the pattern of the stress played a key role in the physiological response; corticosterone resistance was correlated with higher intensity or longer duration of fighting during the first of 6 nightly cycles of SDR. Corticosterone resistance did not develop when the intensity of the stressor or its duration was gradually increased throughout the week. Additionally, corticosterone insensitivity was more likely to develop in a cage with more established social hierarchy (i.e., with clear dominant and submissive mice). Together, these findings suggest that the physiological outcomes of stress may be modified by social factors such as the intensity of aggressive interactions at critical times and the stability of the social hierarchy. (Supported by the John D. and Catherine T. MacArthur Foundation Mind Body Network and NIH Grant MH46801-08.) Endometriosis Significantly Alters the Intestinal Microflora of Aged Rhesus Monkeys. Michael T. Bailey and Christopher L. Coe, Department of Psychology, University of Wisconsin, Madison, Wisconsin 53715. Intestinal microflora, particularly members of the genus Lactobacillus, provide a strong defense against enteric infection. Under certain circumstances, however, the complex makeup of the microflora can become disturbed, resulting in susceptibility to infection. A nonhuman primate model was used to investigate whether changes in microflora patterns occur across the life span, with a particular focus on older healthy animals as well as aged animals manifesting the inflammatory condition of endometriosis. The intestinal microflora of 31 female rhesus monkeys (Macaca mulatta) between the ages of 2 and 31 years of age were investigated by enumerating total and gram-negative aerobic and facultatively anaerobic bacterial species, as well as Lactobacilli, from coprocultures. In addition, the intestinal microflora of monkeys with endometriosis were compared to age-matched healthy controls. Although no significant differences in microflora were evident across the life span in healthy animals, there was a marked alteration in aged monkeys with endometriosis. Monkeys over 20 years shed significantly fewer Lactobacilli, but only if endometriosis was also present. Conversely, the number of shed gram-negative bacteria increased in monkeys with endometriosis at every age as compared to their healthy controls. Aged monkeys with endometriosis also tended to shed fewer total aerobic and facultatively anaerobic bacteria than younger and healthier monkeys, although the difference did not reach statistical significance. These results demonstrate that age-associated inflammatory diseases profoundly disrupt the intestinal microflora. Since we know that both neurohormones and cytokines can influence bacteria, these observations provide a new approach to evaluating inflammatory diseases within the context of PNI. (Supported by MH41659 to C.C. and NRSA Predoctoral Fellowship MH12458 to M.B.)
Neonatal Glucocorticoid Treatment Has Long-Term Differential Effects on Cytokine Production by Macrophages and T Cells in Adult Rats. Joost M. Bakker,*,† Annemieke Kavelaars,* Patrick J.G.H. Kamphuis,‡ Frank van Bel,† and Cobi J. Heijnen,* Departments of *Pediatric Immunology and †Neonatology, Wilhelmina Children’s Hospital; and ‡Medical Pharmacology, Rudolf Magnus Institute, Utrecht, The Netherlands. Major concern has arisen about the possible long-term adverse effects of glucocorticoid (GC) treatment used for the prevention of chronic lung disease in preterm infants. Previously, we have shown that neonatal GC treatment of rats increases the severity and incidence of the inflammatory autoimmune disease experimental autoimmune encephalomyelitis (EAE) in adult life. In search of mechanisms which possibly explain increased susceptibility to EAE after neonatal GC treatment, we demonstrated that such treatment reduces the corticosterone response to an immune challenge in adult rats. In addition to the hypothalamo–pituitary–adrenal axis, also macrophages and T cells play an important role in EAE. In this study we investigated the effects of neonatal GC treatment on macrophage and T cell cytokine production after in vitro stimulation. Interestingly, stimulated macrophages of GC-treated rats produce less TNFalpha and IL-1beta ( p ⬍ .05) in adult life. On the other hand, after stimulation of splenocytes obtained from adult rats increased mRNA expression was seen of the proinflammatory cytokines IFNgamma ( p ⬍ .01) and TNFbeta ( p ⬍ .05), after neonatal GC treatment. Apparently, neonatal GC treatment has permanent but differential programming effects on immune functioning in adult life. In view of the frequent clinical application of GCs to premature infants, our data suggest that long-term effects are expected of such treatments on immune functioning. Proinflammatory Cytokines Are Neuroimmune Mediators of the Neurobehavioral and Neuroendocrine Disturbances Induced by HIV-1 gp120. Ohr Barak,* Joseph Weidenfeld,† Tamir Ben-Hur,† Inbal Goshen,* Anna N. Taylor,‡ and Raz Yirmiya,* *Department of Psychology, The Hebrew University of Jerusalem, Israel; †Department of Neurology, Hadassah University Hospital, Jerusalem, Israel; and ‡Department of Neurobiology, UCLA School of Medicine, Los Angeles, California. Exposure to the HIV surface envelope protein gp120 produces marked neuropathological effects. We, as well as others, demonstrated that central administration of gp120 can also induce neurobehavioral disturbances, including fever, anorexia, body weight loss, sleep disturbances, and anhedonia. Neuroendocrine disturbances, particularly an impaired regulation of the HPA axis, were also observed following icv gp120. We have also shown the involvement of prostaglandins as mediators of some of the effects of icv gp120 administration. In the present study we evaluated the role of the proinflammatory cytokines TNF alpha and IL-1 beta in mediating the central effects of gp120 on sickness behavior and on the HPA axis. Three hours postinjection, gp120 induced the expression of TNF alpha and IL-1 beta mRNA in the hypothalamus, and caused a significant increase in the levels of serum ACTH and corticosterone. In addition, icv gp120 injection (2 mg/rat) induced a significant elevation in body temperature, and a reduction in food and saccharin intake, body weight, social and exploratory behaviors. Either pentoxifylline—an inhibitor of TNF alpha synthesis—or IL-1 receptor antagonist (IL-1ra), did not attenuate the behavioral responses to gp120. However, coadministrtion of both pentoxifylline and IL-1ra abolished some of the behavioral changes induced by gp120 injection. These findings suggest that TNF alpha and IL-1 beta play a synergistic and complementary role in the mediation of gp120-induced neurobehavioral and neuroendocrine alterations. (Supported by Grant 97-204 from the U.S.–Israel Binational Science Foundation.) Mood, Cognition, and Immunity: Related Phenomena in Children? Jacqueline A. Bartlett, S. E. Keller, and J. Hill, UMDNJ New Jersey Medical School, Department of Psychiatry, Newark, New Jersey. Purpose. Depressed mood has been related to diminished cognitive ability and to altered immunity. We investigated the possibility that the altered immunity associated with depression in children is also associated with neuropsychological function. Methods. Eighty-two healthy children recruited from the 3rd and 4th grades were evaluated with a complete medical history and physical examination, self reports of anxious and depressed mood, a structured psychiatric interview, a battery of neuropsychological tests and cellular immune measures including phenotype numbers and per cents, response to mitogen stimulation (Con A, PHA, and PWM), natural killer cell activity against K562 tumor cells, and phagocytosis and killing of staph aureus by granulocytes. Results. The children were ages 8 to 12 with a mean of 8.9 years (⫹1.4). Eighty-six percent were African–American and the rest Hispanic. Depressed mood was associated with altered response to mitogens (Con A & PHA). Inattention was related to mitogen
response (Con A, PHA, PWM) and abstracting ability was related to response to Con A. Conclusions. Both mood and cognition (as measured herein) appear to relate to immunity in children, further strengthening the purported relationships between the brain and the immune system. Implications for interplay between mood, health and learning in children will be discussed. SNS Modulation of Primary Antibody Response to KLH Is Impaired in Old Mice. Denise L. Bellinger* and Kelley S. Madden,† *Center for Neuroimmunology, Loma Linda University School of Medicine, Loma Linda University, California 92352; and †Department of Psychiatry, University of Rochester School of Medicine, Rochester, New York 14642. In aging, cell-mediated immunity declines, while humoral immunity remains relatively robust. The purpose of this study was to investigate the influence of sympathetic signaling in aging T-helper (Th)1 and Th2 predominant mouse strains on T-dependent primary antibody response to KLH. The effect of chemical sympathectomy by administration of 6-hydroxydopamine in young and old BALB/cJ (Th2) and C57Bl/6J (Th1) mice immunized with KLH was examined. Beta-adrenergic receptor expression, and agonist-induced cAMP generation in splenocytes also was examined. Chemical sympathectomy depleted splenic norepinephrine concentration (62 to 79%) in young and old mice. The effect of sympathectomy on immune response was age- and strain-dependent. In young BALB/cJ mice, sympathectomy reduced antibody production, possibly due to an increase in IL-2 and IFN-gamma production. These sympathectomy effects were severely blunted in old BALB/cJ mice. In C57Bl/6J mice, sympathectomy had no effect on primary antibody response in young mice, despite an increase in IL-4 production. Sympathectomy-induced enhancement of IL-4 production was diminished in old, C57Bl/6J mice. With aging, there was no apparent difference in the density of beta-adrenergic receptors on CD4⫹ and CD8⫹ naive and memory T cells. Unfractionated splenocytes from young adult BALB/cJ mice incubated with 10-4 M isoproterenol increased the production of intracellular cAMP, a response that was significantly reduced with age. Collectively, these studies indicate that SNS modulation of primary antibody response to KLH is impaired in old mice, an effect that may be explained by an inability of beta-adrenergic receptor activation to signal an increase in the second messenger, cAMP.e that was not observed in the other two rat strains. Influence of the Sympathetic Nervous System on Primary Antibody Response in Two Strains of Aging Mice. Cheri Lubahn,* Dianne Lorton,* and Denise L. Bellinger,† *Hoover Arthritis Research Center, Sun Health Research Institute, Sun City, Arizona; and †Center for Neuroimmunology, Loma Linda University School of Medicine, Loma Linda, California 92352. Previous findings in our laboratory revealed an age-related decline in noradrenergic sympathetic innervation of the spleen in male Fischer 344 rats. The purpose of this study was to determine whether other strains of rats also progressively lose noradrenergic sympathetic innervation of the spleen with advancing age. We examined sympathetic innervation of spleens from male Brown–Norway, Brown– Norway–F344 cross (F1), and Lewis rats using high-performance liquid chromatography with electrochemical detection for measuring norepinephrine, and fluorescence histochemistry for localization of catecholamines. Neurochemistry for measuring norepinephrine revealed a significant age-related decline in norepinephrine concentration in spleens from Lewis rats, similar in magnitude as that previously reported in F344 rats. In contrast, there was no effect of age on splenic norepinephrine concentration in Brown–Norway or Brown–Norway ⫻ F344 rats. Consistent with neurochemical analysis, an agerelated significant decline in the density of noradrenergic nerve fibers was found in spleens from Lewis rats, an effect of age that was not observed in the other two rat strains. These findings indicate that the influence of age on sympathetic innervation of the spleen is strain dependent in rats. Whether these agerelated changes in sympathetic innervation of the spleen are dependent on age-related changes in the microenvironment of the spleen remains to be determined. Timing within the Estrous Cycle Modulates Adrenergic Suppression of NK Activity and Resistance to Metastasis: Possible Clinical Implications. Shamgar Ben-Eliyahu, Guy Shakhar, Keren David, and Rivka Melamed, Department of Psychology, Tel Aviv University, Tel Aviv 69978, Israel. Clinical observations suggest that metastatic development and long-term mortality rate following surgery in breast cancer patients depend on the menstrual phase during which surgery is conducted. The
menstrual cycle modulates various physiological responses and medical conditions that involve adrenergic mechanisms (e.g., asthma). Natural killer activity (NKA), an immune function controlling metastasis, is suppressed following surgery, and in vitro by adrenaline. We therefore hypothesized that the clinical observation may be partly attributable to surgery-induced adrenergic suppression of NK-dependent resistance to metastasis, a suppression that depends on menstrual phase during surgery. To test this hypothesis in rats, F344 females at different phases of their estrous cycle were injected with a beta-adrenergic agonist, metaproterenol (MP) (0.4 or 0.8 mg/kg, s.c.), or with vehicle, before i.v. inoculation with MADB106 mammary adenocarcinoma cells. In a second experiment, the dose-dependent effects of MP on NKA were studied in vitro in blood drawn at different phases of the estrous cycle. Finally, the effects of swim-stress on the number and activity of NK cells were assessed along the estrous cycle. The findings indicate that the suppressive effects of MP on resistance to metastasis and on NKA are significantly greater during estrous phases characterized by high estradiol levels (D3/proestrus/estrus). Similarly, NKA per cell was suppressed by swim-stress only during this phase. In untreated animals (not exposed to MP or to swim-stress), no effects of the estrous cycle on NKA or metastasis were evident. These findings indicate that the estrous cycle modulates adrenergic suppression of NKA and resistance to metastasis. (Supported by NIH Grant CA73056.) LPS-Induced Sickness Behavior in Rats: Modulation by Opiate Receptor Antagonists.Yehuda Bendkowsky, Gal Chaikin, Raz Yirmiya, Edna Cohen, Gilli Wolf, and Yehuda Shavit, Department of Psychology, The Hebrew University, Mont Scopus, Jerusalem 91905, Israel. Lipopolysaccharide (LPS) offers a useful model of immune activation. It induces secretion of proinflammatory cytokines accompanied by physiological and behavioral responses, which are collectively referred to as sickness behavior. Exogenous and endogenous opiates modulate cytokine production, as well as sickness behavior. We previously studied LPS-induced sickness behavior in several strains of inbred mice, genetically different in their endogenous opiate systems. We reported that a mu-opiate receptor-deficient strain exhibited a higher fever response, while an opiate receptor-rich strain exhibited a lower fever response, compared with control mice. In the present study, we examined the effects of three opiate receptor antagonists: A kappa-specific (nor-BNI), a delta-specific (NTI), and a less specific mu-receptor antagonist (naltrexone), on LPS-induced sickness behavior. Rats were injected with either nor-BNI (3 mg/kg), NTI (3 mg/kg), naltrexone (2 mg/kg), or saline, followed by LPS (50 µg/kg) or saline, and were monitored for body temperature and locomotion using a biotelemetric system. Body weights, as consumption of food and water were also measured. LPS induced fever in all animals. The fever response was augmented by the three opiate antagonists; particularly by naltrexone. The antagonists also augmented the LPS-induced decrease in locomotion, in body weight gain, and in food consumption. These results support the hypothesis that opiates play a modulatory role in the physiological and behavioral responses to immune challenge. [Supported by a grant from the Israel Foundations Trustees (1996– 1998).] The Effects of a Back Massage on Stress, Blood Pressure, and Natural Killer Cell Activity—Pilot Study. Mary P. Bennett, Suzy H. Fletcher, Deborah A. Barnhart, Joyce H. Hudgins, and Randy R. Sims, Indiana State University School of Nursing, Indiana. According to Psychoneuroimmunology theory, complementary interventions such as massage may decrease stress, and modify the effects of stress on immune functioning. This study was conducted to determine the effect of the nursing back massage on stress and immune function in a group of healthy adult, nonpregnant, female volunteers. Natural killer (NK) cell function was evaluated using the standard chromium release assay. While this study consisted of a morning group (n ⫽ 11) and an afternoon group (n ⫽ 12), due to funding and methodological considerations, NK activity was only measured in subjects in the morning group (5 control, 6 massage). Results from these subjects only are reported here. Morning participants in the massage group had significantly decreased post stress levels compared to those in the control group. A significant decrease was found in systolic blood pressure for members in the morning massage group. Natural killer cell activity decreased somewhat for all morning participants from pre to post time points, possibly due to natural diurnal variation in NK activity. A trend for individuals in the massage group to have less decrease in NK activity was observed. The results of this study suggest that a nursing back massage can decrease stress levels and systolic blood pressure. Further investigation is needed to determine the effect of back massage on natural killer cell activity.
Major Histocompatibility Complex (MHC) Involvement in Stress Pheromone-Induced Immunomodulation. Gary W. Boehm,* Kelley S. Madden,* Sandra C. Lee,* Jonathan D. Karp,† Jan A. Moynihan,* Lee J. Grota,* Robert Ader,* and Nicholas Cohen,† *Center for Psychoneuroimmunology Research, Department of Psychiatry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; †Department of Biology, Rider University, Lawrenceville, New Jersey 08648; and Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. We have published that BALB/cJ mice exposed to odors from footshock-stressed conspecifics have suppressed cell-mediated immunity, enhanced humoral immunity, and increased opioid-mediated analgesia to thermal stimulation. These effects were not observed for C57BL/6 or BALB/cJ animals exposed to odors from stressed C57BL/6 mice. Suspecting a potential role of MHC haplotype in these strain effects, we formulated comparisons for this study using BALB/cJ (H-2d) mice and the H-2 congenic strains, BALB.B (H-2b) and BALB.K (H-2k), as donors and recipients of putative stress odors. Stressed and nonstressed odor donors from each strain were paired with recipient strains of each haplotype for all 9 possible combinations of donor–recipient haplotypes (18 recipient groups, n ⫽ 8 animals/recipient group). Mice were injected (i.p.) with 100 micrograms of keyhole limpet hemocyanin (KLH) 24 h before onset of odor exposure, and they were bled 6 days later. Antibodies were assayed by an ELISA. Recipient BALB.B mice (like the C57BL/6 animals, which share the H-2b haplotype) showed no stress pheromoneinduced increases in anti-KLH antibody response, regardless of the MHC haplotype of the strain providing the stress odor. However, BALB/cJ (H-2d) and the BALB.K congenics (H-2k) both showed significant increases in KLH-specific IgG (but not IgM) production when the stress pheromones came from stressed mice of the same H-2 haplotype. Thus, genes within the MHC complex of both odor-donor and odor-recipients are important in stress pheromone-associated immunomodulation. Mechanisms Underlying the Effect of Chemical Sympathectomy on the Cytotoxic T Lymphocyte (CTL) Response to Herpes Simplex Virus (HSV) Infection. Robert H. Bonneau*,† and Nicole A. Leo,† *Department of Microbiology and Immunology, †Program in Neuroscience, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033. Lymphoid tissues are extensively innervated by noradrenergic fibers of the sympathetic nervous system. 6-hydroxydopamine (6-OHDA)-induced chemical sympathectomy has been commonly used to assess the impact of this innervation on immune function. However, previous studies from our laboratory have demonstrated that acute chemical sympathectomy results in a transient but substantial increase in the levels of circulating corticosterone and suppresses the generation of primary and the activation of memory herpes simplex virus type 1 (HSV-1)-specific cytotoxic T lymphocytes (CTL). Using the Type I (mineralocorticoid) receptor antagonist spironolactone, the Type II (glucocorticoid) receptor antagonist RU486, and the beta-adrenergic receptor antagonist nadolol, it was demonstrated that each of these receptors plays a role in sympathectomy-induced inhibition of HSV-1 immunity. Thus, both corticosterone and norepinephrine likely mediate this immunosuppression associated with acute chemical sympathectomy. Further studies demonstrated that three treatments with 6-OHDA at 4-day intervals does not result in elevated levels of corticosterone following the third treatment. Using this paradigm, we delineated the direct impact of sympathetic innervation on HSV-specific CTL activity. Whereas the extent of HSV-specific CTLm activation and function was unaffected in these chronically-sympathectomized mice, the generation of primary HSV-specific CTL was significantly suppressed. These findings suggest that some component of sympathetic innervation contributes to the generation of HSV-specific CTL during an HSV infection. Overall, these findings not only further support a role for neuroendocrineinduced modulation of immune function, but also a need to exercise caution in attributing the effects of chemical sympathectomy to solely the absence of sympathetic innervation of lymphoid tissues. (Supported by Public Health Service Research Grant MH49616.) Beta-Adrenergic Stimulation Inhibits NF-Kappa B Binding to the iNOS Promoter. Chad S. Boomershine,*,† William P. Lafuse,* and Bruce S. Zwilling,‡ *Department of Molecular Virology, Immunology and Medical Genetics, †College of Medicine, ‡Department of Microbiology, Ohio State University, Columbus, Ohio 43210. Mycobacterium avium infection is a significant cause of morbidity and mortality in AIDS patients. The ability of activated macrophages to restrict the growth of intracellular mycobacteria is a major factor
in determining the outcome of infection. Previously we reported that catecholamine treatment inhibited the antimycobacterial activity of IFN-gamma-primed murine peritoneal macrophages. The catecholamine-mediated inhibition of mycobacterial killing was due to inhibition of nitric oxide production through beta2-adrenergic receptor stimulation. We have extended these observations by showing that addition of salbutamol, a beta2-adrenoceptor agonist, to IFN-gamma-primed M. avium-infected macrophages inhibited binding to the downstream NF-kappa-B site (NF-kappa-Bd) of the iNOS promoter as assessed by electrophoretic mobility shift assay of nuclear extracts. Supershift assays showed binding to the NFkappa-Bd site was by the RelA (p65) and p50 subunits of the NF-kappa-B family. The inhibition by salbutamol was dose dependent with an IC 50 of 0.01 µM. Western blot analysis of cytoplasmic extracts using I-kappa-B alpha and phospho-I-kappa-B alpha antibodies showed the decrease in NF-kappa-B binding was not the result of a decrease in I-kappa-B alpha phosphorylation and degradation. These observations show that catecholamine hormones can affect the outcome of macrophage-pathogen interaction by inhibiting binding of NF-kappa-B to the iNOS promoter through a mechanism independent of I-kappa-B alpha degradation. This suggests one result of sympathetic nervous system activation is the suppression of the capacity of macrophages to produce anti-microbial effector molecules by affecting NF-kappa-B binding activity. (Supported by NIH Grants AI42901, HL59795, and MH54966.) Vagus Nerve Stimulation Attenuates the Systemic Inflammatory Response to Endotoxin. Lyudmila V. Borovikova,* Svetlana Ivanova,* Minghuang Zhang,* Huan Yang,* Galina I. Botchkina,* Linda R. Watkins,† Haichao Wang,‡ Naji Abumrad,§ John Eaton,¶ and Kevin J. Tracey,*,§ *Picower Institute for Medical Research, Manhasset, NY 11030; †Department of Psychology, University of Colorado, Boulder, Colorado 80309; ‡Department of Emergency Medicine, †Department of Surgery, North Shore University Hospital, Manhasset, New York 11030; ¶Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030. Vertebrates achieve internal homeostasis during infection or injury by balancing the activities of agonist (proinflammatory) and antagonist (anti-inflammatory) pathways. Endotoxin (lipopolysaccharide, LPS) produced by all Gram-negative bacteria activates monocytes to release proinflammatory mediators (e.g., TNF and IL-1beta) that are potentially lethal (endotoxemia). Anti-inflammatory signals (e.g., corticosteroids and IL-10) feedback inhibit cytokine release, which can restore homeostasis. Humoral mechanisms by which the central nervous system (CNS) regulates systemic inflammatory responses to endotoxin (neuroimmuno-modulation) have been characterized in some detail. For instance, during endotoxemia, proinflammatory cytokines and LPS in peripheral tissues activate afferent vagus nerve fibers which stimulate the hypothalamic–pituitary–adrenal axis to release adrenocorticotropic hormone; the subsequent release of adrenal glucocorticoids inhibits monocyte cytokine synthesis. Comparatively little is known about the role of the efferent vagus nerve in the regulation of systemic inflammation. Here acetylcholine, the principle neurotransmitter of the vagus nerve, was found to significantly attenuate LPS-stimulated macrophage release of proinflammatory cytokines (TNF, IL-1beta, IL-6, and IL-18). Acetylcholine failed to inhibit release of the anti-inflammatory cytokine IL-10. Electrical stimulation of vagus nerve in vivo during lethal endotoxemia in rats inhibited TNF synthesis in liver, attenuated peak serum TNF levels and conferred protection against the development of shock. This reveals a previously unrecognized anti-inflammatory role of the vagus nerve in modulating the systemic inflammatory response to endotoxin. Sickness Behavior in Breast Cancer Survivors: Relations among Fatigue, Other Sickness Behaviors, and Markers of Immune Activation. Julienne E. Bower,*,† Patricia A. Ganz,†,‡,§ and John L. Fahey,*, ¶, ¶ *Norman Cousins Center in Psychoneuroimmunology, †Jonsson Comprehensive Cancer Center, ‡School of Medicine and §School of Public Health, and ¶Department of Medicine and ¶Department of Microbiology and Immunology, University of California, Los Angeles, California 90095. Fatigue is one of the most common side effects of breast cancer treatment and can persist for months or years after completion of treatment in some patients. However, the mechanisms underlying the onset and persistence of fatigue among women with breast cancer have not been determined. In this study, we examined the hypothesis that fatigue would be associated with other behavioral manifestations of sickness (sickness behaviors) as well as markers of immune activation. Forty breast cancer survivors (20 fatigued, 20 nonfatigued) completed psychological questionnaires and provided blood samples at visits scheduled to control for diurnal variations in immune and hormonal parameters. Participants were
5 years postdiagnosis on average, had completed cancer treatment, and were currently healthy with no evidence of recurrence. Results showed that fatigued women had significantly higher serum levels of soluble tumor necrosis factor receptor II (sTNF-RII) and interleukin-1 receptor antagonist (IL-1ra), and significantly lower levels of cortisol than nonfatigued women, controlling for health behaviors. No differences in IL-1beta were detected between fatigued and nonfatigued women. Fatigued women reported a variety of other sickness behaviors, including lethargy, decreased activity, less interest in social interaction, forgetfulness, and distractibility, as well as greater sleep disturbance. Fatigued women also endorsed higher levels of depression and hopelessness, more negative expectancies about their future health, and lower levels of perceived control over their health than non-fatigued women (all p values ⬍.05). These data suggest that fatigue may be related to ongoing activation of certain components of the immune system in breast cancer survivors. (Supported in part by the Norman Cousins Center in Psychoneuroimmunology at UCLA.) A Vulnerability to the Depressive Effects of Cytokine Therapy in Cancer Patients. Lucile Capuron,* Alain Ravaud,† and Robert Dantzer,* *INSERM U394, Neurobiologie Integrative, Institut Francois Magendie, 33077 Bordeaux cedex, France; and †Institut Bergoni, Anticancer Center from Bordeaux, Department of Medicine, 33076 Bordeaux cedex, France. Cytokine therapy is currently used for the treatment of cancers, AIDS, chronic hepatitis, and multiple sclerosis. This treatment frequently induces neuropsychiatric complications, including depressive disorders in about 30% patients. The objective of the present study was to identify possible risk factors for the development of cytokine-induced depression. Thirty six cancer patients were treated by interleukin2 and/or interferon-alpha in the Cancer Center from Bordeaux (France). Fifteen patients with renal cell carcinoma received interleukin-2 subcutaneously, 15 patients with melanoma received interferon-alpha intravenously, and 6 patients with renal cell carcinoma received both interleukin-2 and interferon-alpha. Patients’ mood was evaluated before the initiation of treatment and 3–4 weeks later using the Montgomery and Asberg Depression Rating Scale (MADRS) and COVI scale for anxiety. None of the patients was clinically depressed before treatment or had a personal history of mood disorder. There was no change in COVI scores over the course of treatment whereas MADRS scores significantly increased to the point to reflect a depressive episode in 13/36 (36%) patients. Interestingly, MADRS scores before treatment significantly predicted the magnitude of MADRS scores 3–4 weeks later (R ⫽ 0.75, y ⫽ 1.53 ⫻ 4.24; p ⬍ .0001). Those patients who had an initial MADRS score higher than 7 because of lassitude, sleep disorders and pessimistic thoughts had a 5.4 higher risk to develop depressive disorders than patients with a pre-therapeutic MADRS score inferior to 7. These results show for the first time an individual vulnerability to cytokine-induced depression and provide an useful tool to adopt selective prevention measures, such as administration of antidepressant drugs. (Supported by Ligue Nationale Contre le Cancer and Association pour la Recherche sur le Cancer.) Morphine-6-Glucuronide (M6G) Induces Potent Antinociception and Immunomodulation. Kelly A. Carrigan and Donald T. Lysle, Biological Psychology Program, University of North Carolina, Chapel Hill, North Carolina 27599-3270. There exists an extensive literature documenting morphine’s immunomodulatory effects. However, little is known about the immune alterations induced by morphine’s major metabolites such as morphine6-glucuronide (M6G). The present study evaluated the antinociceptive and immunomodulatory properties of M6G in the Lewis rat following systemic and central administration. Animals were surgically implanted with cannulas directed at the lateral ventricle for central assessment of M6G. Following recovery, animals received M6G subcutaneously (0, 1.0, 3.16, or 10 mg/kg) or intracerebroventricularly (0, .1, .316, or 1.0 µg/rat) and were tested for antinociception using a tail withdrawal procedure 30, 60, and 120 min following administration. The results show that s.c. and i.c.v. administration of M6G produces a dose-dependent and time-dependent increase in antinociception that is maximal at the 60 min time point. Furthermore, the antinociceptive effect of M6G is naltrexone reversible. Immune status as measured by splenic lymphocyte proliferation, interferon-gamma production, and natural killer cell activity was assessed 60 min following M6G administration. The results show that administration of M6G produces a significant suppression of splenic lymphocyte proliferation in response to the mitogens concanavalin A and lipopolysaccharide at the 10 mg/kg (s.c.) and 1.0 µg/rat (i.c.v.) dose. Splenic natural killer cell activity is also significantly suppressed at the 3.16 and 10.0 mg/kg (s.c.) and .316 and 1.0 µg
(i.c.v.) doses of M6G. Moreover, both routes of administration of M6G induce a dose-dependent decrease in IFN-gamma production. These results show that systemic and central administration of M6G produces pronounced immune alterations at comparable doses and time points that induce antinociception. Spinal Cord Glial Activation Mediates Exaggerated Pain from Sciatic Inflammatory Neuritis (SIN). Marucia Chacur,* Erin. D. Milligan,† Robert R. Myers,‡ Larry Gazda,† David Martin,§ Kevin J. Tracey,¶ Steven F. Maier,† and Linda R Watkins,† *Department of Pharmacology, University of Sao Paulo, Sao Paulo, SP, 05508, Brazil; †Department of Psychology, University of Colorado, Boulder, Colorado 80309; ‡Department of Anesthesiology and Neuropathology, UCSD VA, San Diego, California; §Department of Pharmacology, Amgen, Thousand Oaks, California 91320; and ¶Laboratory of Biomedical Science, North Shore University Hospital, Manhassett, New York 11030. Peripheral nerve damage can induce exaggerated pain states. We tested whether (a) immune activation around rat sciatic nerve causes exaggerated pain (hyperalgesia and allodynia) and (b) this effect is spinally mediated by activated astrocytes and microglia (glia). Behavior and histology of sciatic nerve were assessed. Responses to mechanical (allodynia; von Frey test) and thermal stimuli (hyperalgesia; Hargreaves test) were recorded prior to and after injection of zymosan (0, 4, 40,160 µg) into gelfoam enwrapping one sciatic nerve. Intrathecal (IT) injections of drugs that disrupt glial function or block local cytokine release (fluorocitrate and CNI-1493, respectively) or of the proinflammatory IL1 receptor antagonist (IL1ra; 100 µg) were used to examine potential mediation by spinal glia. After behavioral testing, the gelfoam enwrapped sciatic nerve was examined by brightfield microscopy. Sciatic neuritis, but not control procedures, produced robust, dose-dependent changes in pain responses. While thermal hyperalgesia was only transiently induced, mechanical allodynias (unilateral after 4 µg; bilateral after 160 µg) lasted ⬎ 1 week and were blocked by all IT drugs tested. To date, sciatic analyses reveal that the 160µg dose of perisciatic zymosan is correlated with increased numbers of TNF-positive macrophages, edema, and axonal degeneration. Thus, immune activation near peripheral nerves creates exaggerated pain states mediated by spinal cord glia. (Supported by NIH Grants MH01558, MH00314, and NS38020 and by Amgen.) Opioids and Immunity: Involvement of the Nucleus Caudatus in Dago-Induced Stimulatory Effect in Rats. M. Cheido, E. Alperina, and L. Devoino, Institute of Physiology of Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk 630117, Russia. At present there is evidence of the important role of opioids and neurotransmitters in the immune response formation. Our recent findings demonstrated that the alterations of the immune status in CBA mice, achieved by the activation of central mu-, delta-, and kappa-opioid receptors, are provided by their interaction with different neuromediator systems. In particular, the immunostimulation caused by administration of DAGO, a highly selective mu-agonist, are realized under involvement of the dopaminergic mechanisms (experiments with haloperidol). The present results indicate that DAGO significantly increases plaque- and rosette-forming cell numbers in male Wistar rats after immunization with SRBC. In addition, animals were subjected to the bilateral electrolytic lesions of the nucleus caudatus, the brain area rich in nigrostriatal nerve terminals. Sham lesioned groups constituted an appropriate control. A significant decrease of the immune reaction level was found in lesioned groups as compared to the control. It is noted that immunostimulating influence of DAGO was not observed in treated by DAGO operated animals. Thus, these data indicate the participation of a certain dopaminergic nigrostriatal structure, in particular the nucleus caudatus, in the process of neuroimmunomodulation through the mureceptors activation by DAGO. [Supported by RFBR (99-04-50017).] Psychosocial and Autonomic Predictors of Immunologic Recovery following HAART Therapy for HIV1 Infection: The Role of Openness/Inhibition. Steve W. Cole,* Margaret E. Kemeny,† John L. Fahey,‡ and Bruce D. Naliboff,* Departments of *Medicine, †Psychology, and ‡Microbiology and Molecular Genetics, UCLA School of Medicine, Los Angeles, California 90095-1678. Highly active antitretroviral therapy (HAART) for HIV-1 infection rapidly suppresses viral replication, but immune system recovery occurs more slowly and is highly variable across individuals. To determine the role of the nervous system in variable immunologic recovery, we examined the relationship between
psychosocial factors linked in previous samples to HIV-1 disease progression (e.g., social inhibition), sympathetic nervous system (SNS) activity, and CD4⫹ T cell recovery following HAART. Data come from a study of 55 HIV⫹ gay men who were AIDS-free at study entry. Among the 21 who began HAART during the 12–16 month follow-up, CD4⫹ T cell levels increased from an average of 22.4 to 26.8% of circulating lymphocytes ( p ⫽ .02). CD4 recovery varied substantially across individuals (range ⫽ 2.0 to ⫹30.0%, SD ⫽ 7.3%). In regression analyses controlling for CD4 level at study entry and duration of HAART treatment, social inhibition and hostility were associated with poorer CD4 recovery ( p ⫽ .041 and .009), whereas the opposing characteristics of openness and agreeableness were associated with greater recovery (p ⫽ .002 and .016). Socially inhibited individuals showed elevated SNS activity (skin conductance, blood pressure, heart rate, and inverse finger pulse amplitude and transmission times, measured at rest and in response to a model stressor) ( p ⫽ .027), and elevated SNS activity was associated with poorer CD4 recovery ( p ⫽ .005). Thus individual differences in SNS function may mediate relationships between social inhibition and impaired immunologic recovery following HAART therapy for HIV-1 infection. (Supported by UCLA AIDS Institute Clinical Science Research Grants 542476-18010, NIMH MH00820, and MH15750, and the Norman Cousins Center for Psychoneuroimmunology.) Neuroeffector Molecules Regulate Dendritic Cell Phenotypes Fostering Cellular vs Humoral Immunity: Differential Activation of NF-kB and Sp1 Transcription Factors. Steve W. Cole,* Bojana A. Rnjak,* Hoss M. Sadeghi,† and Jerome A. Zack,*,† Departments of *Medicine and †Microbiology and Molecular Genetics, UCLA School of Medicine, Los Angeles, California 90095-1678. In previous research, we found that neurotransmitter activation of the cAMP/PKA signaling pathway induced immature human dendritic cells (DC) to differentiate into mature antigenpresenting cells expressing high levels of the DC maturity marker CD83 and high levels of the lymphocyte stimulatory molecule B7-2/CD86 accompanied by low levels of B7-1/CD80, CD40, and IL-12 (CD86hi CD80low CD40low IL-12low). In contrast, DC maturation induced by proinflammatory cytokines (e.g., TNFalpha, IL-1beta) elicited a CD86low CD80hi CD40hi IL-12hi phenotype. To determine the functional significance of these distinct DC phenotypes, we conducted mixed leukocyte reactions with allogeneic naive T lymphocytes. Both DC phenotypes efficiently induced naive T lymphocytes to proliferate, but DCs induced to maturity by PKA signaling elicited lower levels of IFN-gamma and higher levels of IL-4 than did DCs induced to maturity by proinflammatory cytokines (a profile associated with heightened humoral immunity at the expense of cellular immunity). These distinct DC phenotypes were accompanied by altered expression of mRNA for CD40, CD80, CD83, CD86, and the IL-12 p35 and p40 chains, and distinct patterns of transcription factor activity. Proinflammatory cytokines induced NF-kappaB activity, and both NF-kappaB activation and DC maturation could be blocked by glucocorticoids. In contrast, PKAinduced DC maturation was associated with increased Sp1 activity but no induction of NF-kappaB, and glucocorticoids did not block PKA-induced maturation. Thus neuroeffector molecules can regulate the cellular vs humoral nature of primary immune responses by activating distinct NF-kappaB- and Sp1associated gene expression programs in maturing DCs. An Assessment of the Effects of the Serotonin Releasers Methylenedioxymethamphetamine, Methylenedioxyamphetamine, and Fenfluramine on Immunity in Rats. Thomas J. Connor, John P. Kelly, and Brian E. Leonard, Department of Pharmacology, National University of Ireland, Galway, Ireland. The purpose of the present study was to examine the effect of the serotonin releasing amphetamine derivatives methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), and fenfluramine (FEN) on immunity in rats. Similar to MDA and MDMA, FEN reduced the number of circulating lymphocytes, provoked a suppression of Con A-stimulated lymphocyte proliferation and total IFNgamma and IL-10 production in diluted whole blood cultures. Thus the nonpsychostimulant amphetamine derivative FEN, shares the ability of the psychostimulant methylenedioxy-substituted amphetamines to alter these indices of immune function in the rat. However, when Con A-stimulated cytokine production was normalized for the number of lymphocytes in culture in order to examine cytokine production at a cellular level, the effect of the amphetamine derivatives began to diverge. FEN shares with MDMA and MDA the ability to suppress production of the Th2 type cytokine IL-10. However, the effect of these drugs on Th1 type cytokine secretion was more complex. While the methylendioxy-substituted amphetamines increased the secretion of the Th1 type cytokine IL-2 without altering the related Th1 type cytokine IFN-gamma, FEN did not alter IL-2 secretion, but suppressed IFN-gamma secretion. In addition to these effects on T-cell responses, all three drugs inhibited LPS-induced TNF-alpha secretion
from diluted whole blood cultures suggesting that macrophage activity is impaired following treatment. In all, these data extend our previous findings concerning the effects of MDMA on the immune system and demonstrate that the related serotonin releasers MDA and FEN also provoke immunological changes in rats. (Supported by Enterprise Ireland and the Higher Education Authority of Ireland.) Fenfluramine Impairs Responsiveness to an in Vivo LPS Challenge in Rats: Does Serotonin Release Play a Role in the Immunosuppressive Effects? Thomas J. Connor, Andrew Harkin, John P. Kelly, and Brian E. Leonard, Department of Pharmacology, National University of Ireland, Galway, Ireland. In the present study we examined the effects of acute administration of the serotonin releaser fenfluramine (FEN) on responsiveness to an in vivo challenge with bacterial lipopolysaccharide (LPS; 100 µg/ kg; i.p.). LPS provoked a characteristic increase in circulating interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha concentrations in vehicle treated rats. FEN (10 mg/kg; i.p.) significantly impaired LPS-induced IL-1beta, IL-6 and TNF-alpha responses which was initially observed 1 h following administration, and in some cases the suppression persisted for up to 24 h. In a second study we examined the effect of administration of FEN (2.5, 5, and 10 mg/kg; i.p.) on LPS-induced proinflammatory cytokine secretion 1 h following administration and demonstrated that all three doses of FEN potently suppressed LPS-stimulated TNF-alpha secretion, but only FEN (5 and 10 mg/kg) suppressed LPS-induced IL-1beta and IL-6 secretion. In order to evaluate the role of serotonin release in FEN-induced immunosuppression we examined the effect of pretreatment with the selective serotonin reuptake inhibitor paroxetine (7.5 mg/kg; s.c.). Paroxetine pretreatment prevents FEN from entering serotonergic neurons thus preventing FEN-induced serotonin release. Although paroxetine pretreatment blocked FENinduced serotonin depletion in the frontal cortex and hypothalamus it failed to alter the suppressive effects of FEN on LPS-induced proinflammatory secretion. In conclusion, these data demonstrate that acute FEN administration impairs the secretion of the proinflammatory cytokines IL-1beta, IL-6 and TNF-alpha following an in vivo LPS challenge. However, the suppressive effects of FEN on LPS-induced proinflammatory cytokine secretion could not be attributed to its effects on the serotonergic system. (Supported by Enterprise Ireland and the Higher Education Authority of Ireland.) Interleukin 18 (IL-18), A Possible Role as Neuroimmunomodulator. Bruno Conti, Laboratory of Molecular Neurobiology, Weill Medical College of Cornell University, 785 Mamaroneck Avenue, White Plains, New York 10605. IL-18 is a proinflammatory cytokine and a stimulator of cell mediated immune response with inflammatory associated tissue damage, antitumor, and antimicrobial activity. We propose here that IL-18 may have a role in neuroimmunomodulation. Rat IL-18 was cloned from the adrenal cortex where its levels were elevated by resepine, acute cold stress, or ACTH. In contrast, ACTH did not upregulate IL-18 in spleen or duodenum where this cytokine is primarily produced by immune cells. Molecular analysis demonstrated tissue specific expression of IL-18 gene and differential usage of the promoter in adrenal gland and immune cells. Thus, the adrenal cortex is a unique source of IL-18 that might be secreted during stress providing immunostimulatory signals to the immune system, an hypothesis partially supported by finding that restraint stress elevated serum IL-18 levels in mice. Interestingly, IL-18 and GC are both produced in adrenal cortex during stress, yet have opposite action on immune function. Chronic corticosterone treatment did not affect mRNA levels and reduced that of the peptide, but did not prevent the induction by ACTH. The possibility that IL-18 and GC might contribute together to the homeostasis of immune system during stress will be discussed. We also demonstrated IL-18 mRNA in the CNS. IL-18 was localized in vivo in the rat neurohypophysis and habenula, in vitro in primary cultures of mouse astrocytes and LPS treated microglia. While adrenal IL-18 may be a neuroimmunomodulator regulated by the CNS, brain IL-18 might contribute to the local immune reactions or have other, yet unidentified, functions. The Impact of Low Levels of Corticosterone on the Generation of Herpes Simplex Virus (HSV)-Specific Primary Cytotoxic T Lymphocytes (CTL) in Vivo. Crystal S. Cotter* and Robert H. Bonneau,*,† *Program in Neuroscience and †Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033. A large body of evidence obtained from both human and animal studies has indicated that psychological stress is generally immunosuppressive. However, recent studies by others have suggested that acute
stress and/or low doses of corticosterone may actually enhance immune responsiveness. Our laboratory has established a murine model to characterize the impact of long-term restraint stress and its associated increase in corticosterone on the immune response to herpes simplex virus (HSV) infection. Recently, we have initiated studies to determine the impact of low dose corticosterone on the generation of primary, HSV-specific CTL. Mice were provided varying doses of corticosterone in the drinking water (50, 25, 10, or 0 µg/ml) beginning 3 days prior to footpad infection with HSV (1 ⫻ 104 PFU/footpad) and continuing until day 5 postinfection, when the popliteal lymph nodes were removed and CTL generation assessed. Importantly, this dose of virus does not result in a maximum CTL response, thus allowing us to observe any potential increase in immune responsiveness associated with low doses of corticosterone. As expected, higher doses of corticosterone suppressed both the lymphadenopathy associated with HSV infection and the generation of HSV-specific lytic activity in a dose-dependent manner. However, at lower doses of corticosterone, immune responsiveness was not enhanced. To extend these studies, we will determine if low dose corticosterone affects the kinetics of the CTL response and further quantitate the extent of HSV-specific CTL generation using MHC class I-gB498-505 tetrameric complexes, which allow for the precise quantification of HSV gB498-505 epitope-specific CTL. (Supported by Public Health Service Research Grant MH49616.) Effects of L-Arginine Supplementation on Human Circulating Cytokine Levels and Stress Reactivity. Mary E. Coussons-Read* and Robert C. Tyler,† *Department of Psychology and †Department of Biology, University of Colorado at Denver, Denver, Colorado 80217. There is considerable evidence that arginine-derived nitric oxide (NO) is a potent immunomodulator, and recently oral arginine supplementation has been proposed as an immunoenhancing treatment for surgical patients, AIDS patients, and others. In addition, there is evidence that NO alters HPA axis activity and stress responsiveness, which may also impact immune status. In the present study, stress reactivity and levels of circulating cytokines were assessed in healthy individuals following arginine supplementation to begin to explore the impact of this treatment on stress reactivity and immune status. Nursing students received either daily oral arginine supplementation or placebo. Blood was collected and serum cortisol, TNF-alpha, IL-1beta, and IL-6 were assessed prior to drug treatment (baseline), one week into drug treatment (drug baseline) and 3 weeks into drug treatment following a stressful academic examination (drug ⫹ stress). Initial results indicate that oral arginine treatment significantly alters serum cortisol in a gender-dependent fashion. In addition, arginine treatment modestly increased levels of TNFalpha and IL-1beta at drug baseline and significantly increased levels of IL-6 overall. In sum, these initial results suggest that arginine treatment is associated with alterations in circulating cytokine levels and has a moderate gender-dependent effect on serum cortisol. Additional studies will clarify these effects, and provide further information about the impact of oral arginine supplementation on immune status and stress reactivity. (Supported by a UCD Faculty Development Grant to M.C.R.) Cytokine-Induced Sickness Behavior: Mechanisms and Implications. Robert Dantzer, Integrative Neurobiology, Bordeaux, France. It is now well accepted that the nonspecific psychological and behavioral symptoms of infection are due to the brain effects of proinflammatory cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor-alpha, that are released by activated macrophages and monocytes. These cytokines activate nerve endings at the site of infection. The resulting neural message converges with the humoral message to lead to the production of proinflammatory cytokines by microglial cells in the brain parenchyma. In contrast to the fever response, sickness behavior is more dependent on neural than on humoral pathways for transmission of the immune message to the brain. Centrally produced cytokines act on various brain areas to induce the different facets of sickness behavior. The molecular mechanisms that mediate the action of cytokines on their brain targets have been mainly studied in the case of interleukin1. Both the type I IL-1 receptor and the IL-1 receptor accessory protein are necessary for transmission of the signal. Postreceptor signaling mechanisms likely involve MAP kinases and JNK. The central production and action of proinflammatory cytokines are countered by anti-inflammatory cytokines, glucocorticoids and neuropeptides such as vasopressin. At the functional level, cytokine-induced sickness behavior represents the expression of a central motivational state that is pivotal in organizing the host response to microbial pathogens. Whether this motivational state can be activated by nonimmune stimuli including psychological stressors is now intensively studied, specially in view of the possible relation-
ships between cytokines and certain forms of depression. (Supported by INRA, INSERM, NIMH, and the European Commission.) Timing within the Menstrual Cycle, Sex, and the Use of Oral Contraceptives Determine Adrenergic Suppression of NK Cell Activity. Keren David, Guy Shakhar, Ella Rosenne, and Shamgar Ben-Eliyahu. Department of Psychology, Tel Aviv University, Tel Aviv 69978, Israel. Physiological responses involving adrenergic mechanisms, e.g., asthma and cardiovascular response to stress, fluctuate along the menstrual cycle. Postoperative metastatic development has also been reported to depend on the menstrual phase during which the primary breast tumor is removed. Natural killer (NK) cells are involved in controlling metastases and are suppressed by adrenergic stimulation and by surgery. Since catecholamines are released during surgery, we hypothesized that the sensitivity of NK activity (NKA) to adrenergic suppression fluctuates along the menstrual cycle. Indeed, our studies in rats have shown that adrenergic suppression of NKA and of resistance to metastasis is more profound during estrous phases characterized by high levels of estradiol. In the current study, we used human blood to examine the in vitro suppression of NKA by a beta-adrenergic agonist, metaproterenol (MP), comparing women at different phases of their menstrual cycle, women using oral contraceptives, and men. NKA in each blood sample was assessed in the presence of various concentrations of MP. The results indicated marked group differences in the sensitivity of NK cells to inhibition by MP: IC 50 was 2.6-fold lower in the luteal phase compared to the follicular phase and 1.8-fold lower in oral contraceptives users compared to men, whose NKA was least affected. NK sensitivity to adrenergic stimulation along the menstrual cycle paralleled the levels of plasma estradiol we measured. No group or menstrual differences in MPfree levels of NKA were evident. These findings provide the first empirical evidence for menstrual regulation of adrenergic impact on cellular immune competence. Acute Social Stress Equally Suppresses Production of Th1- and Th2-Type Cytokines but Does Not Impair Memory Formation. Johanna de Groot,*,† Florine J. van Milligen,* Bernie M. Moonen-Leusen,* Jaap M. Koolhaas,† and Wim J. Boersma,* *Department of Immunology, Institute for Animal Science and Health, P.O. Box 65, 8200 AB, Lelystad; and †Department of Animal Physiology, Rijksuniversiteit Groningen, P.O. Box 14, 9750 AA, Haren, The Netherlands. Male Balb/c mice were exposed to a single social defeat (loss of a fight) at 3 days after vaccination with an attenuated herpesvirus (pseudorabies virus). Previous experiments have shown that the antiviral immune response is suppressed at 24 h after defeat. Now we investigated short-term effects of defeat on the production of Th1- and Th2-type cytokines, and long-term effects on the Th1/Th2-phenotype of memory lymphocytes. Groups (n ⫽ 10) of defeated and control mice were sacrificed at 1 and 3 days, and at 5 weeks after defeat. Cytokine production was measured in cultures of spleen and draining lymph node suspensions. Interestingly at 1 day after defeat, which is 4 days after vaccination, cytokines were well detectable when cultures were not restimulated, reflecting ongoing in-vivo production. Social defeat dramatically suppressed the production of IL-2, 4, 6, 10 and IFN-gamma in such nonstimulated cultures of both draining lymph nodes and spleen. However, viral restimulation partially restored cytokine production (except for IL-6), indicating that memory lymphocytes were functional and produced both Th1and Th2-type cytokines. At 3 days and at 5 weeks after defeat the cytokine production in cultures that were restimulated with virus did not differ between defeated and control mice. We conclude that a single social defeat acutely suppresses production of both Th1- and Th2-type cytokines and does not affect the Th1/Th2-phenotype of memory lymphocytes. (The Ministry of Agriculture and Fisheries of The Netherlands in part supported this study.) Social Stress Impairs Induction of Immunological Memory in Young Pigs. Johanna De Groot,* Marco Ruis,* Jaap M. Koolhaas,† Jan Willem Scholte,* and Wim J. A. Boersma,* *Department of Immunology, ID-Lelystad, Institute for Animal Science and Health, P.O. Box 65, 8200 AB Lelystad; and †Department of Animal Physiology, Haren, The Netherlands. The effect of social stress (mixing) was evaluated on efficacy of vaccination in female (n ⫽ 12) and castrated male (n ⫽ 12) pigs with a vaccination-challenge model using a herpesvirus-model (pseudo rabies virus, PRV). Pairs of two related animals of the same sex were housed in separate pens from weaning. Vaccination at 6 weeks of age was performed with attenuated live PRV (strain 783 TK⫺).
Day 3 postvaccination half of the pairs were mixed such that pairs of unrelated pigs of the same sex were created. The control pairs were left undisturbed. After mixing large variations in intensity of aggressive behavior and in saliva cortisol responses were observed. Post vaccination, mixed females gilts showed no suppressive effects in in vitro proliferation assays and in serological responses (IgM, IgG) as compared to controls. In contrast, (though large variations in individual responses were noticed), mixed males as compared to controls showed relatively weak responses. After challenge at 6 weeks post vaccination, a similar pattern developed. Mixed and control females did differ in the responses to challenge. Mixed males showed more severe clinical effects (fever, bodyweight) as compared to controls. Proliferative-, serological-, and interferon-gamma responses tend to support the notion that in these animals immunological memory formation was impaired. (This study was in part supported by the Ministry of Agriculture and Fisheries of The Netherlands.) Reliability of Delayed Cutaneous Hypersensitivity Tests. Renate Deinzer, Bernd Waschul, and Sandra Schuermann, Institute for Medical Psychology, University of Duesseldorf, 40001 Duesseldorf, Germany. Psychoimmuno research often seeks for methods to assess alterations of immune function rather than single cellular or humoral immune parameters. One method to assess cell mediated immunity is the multitest system where several preloaded antigens are applied simultaneously intradermally and delayed cutaneous hypersensitivity is examined 48 h later. This method is very convenient as it is easily applicable, nonhurting, and easy to evaluate. However, this method is not very common in psychoimmuno research. One reason may be that only few data are available on reliability of multitest systems. In the present study we therefore analyze the interrater, intrarater, and parallel test reliability of a seven antigen multitest system. Twenty-one human subjects gave their informed written consent for participation in the study. A multitest (Multitest immignost, biosyn, Fellbach, Germany) with seven antigens (tetanus, diphteria, streptococcal, tuberculin, candida, tricophyton, proteus) and one glycerine control is applied to the volar surface of each forearm. After 48 h the perpendicular diameters of the induration induced by each of the antigens and the control are assessed by two independent examiners twice. The study is designed in a way that examiners are blind to the results of each other, to the results of the other forearm of one subject, and to the results of their first assessment. As the study is still in progress reliability scores will be reported at the conference. In a pilot study with 12 subjects interrater and intrarater reliability scores for assessment of delayed cutaneous hypersensitivity response to tetanus antigen were above ρ ⫽ 0.70. Stress-Induced Changes in T Cell Distribution Are Mediated by the Selectin Family of Adhesion Molecules. Firdaus S. Dhabhar,* Stephen Robinson,† Paul S. Frenette,‡ Denisa D. Wagner,‡ and Richard O. Hynes,† *Department of Oral Biology, Ohio State University, Columbus, Ohio 43210; †Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; and ‡Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115. The continuous recirculation of lymphocytes between blood and extravascular tissue is essential for effective immune function. The past decade has seen significant advances in the knowledge of how cell adhesion molecules mediate blood leukocyte traffic into extravascular tissue. Although, a rapid redistribution of blood leukocytes is one of the oldest-known and evolutionarily conserved effects of stress, surprisingly little is known about the molecular mechanisms mediating this effect. We have shown that the skin and lymph nodes are targets to which leukocytes traffic during stress, and that this trafficking is accompanied by a significant enhancement of skin cell-mediated immunity. Since E and L selectin are adhesion molecules that mediate T cell trafficking to skin and lymph nodes respectively, we hypothesized that these molecules may mediate the stress-induced redistribution of blood T cells. We examined stressinduced changes in blood helper (Th) and cytolytic (CTL) T cell numbers using wild type (WT) and selectin-deficient homozygous gene knockout mice: E ⫺/⫺, L ⫺/⫺, and EL ⫺/⫺. Animals were restrained and tail blood samples collected at baseline and following 2 h stress. WT mice showed a robust stress-induced redistribution of Th, CTL, and B cells. In contrast, redistribution of Th cells was significantly reduced in E ⫺/⫺ mice and virtually eliminated in L ⫺/⫺ and EL ⫺/⫺ mice. Redistribution of CTLs was significantly reduced in L ⫺/⫺ and EL ⫺/⫺ mice. B cell redistribution was relatively unaffected in selectin-deficient animals. These results suggest that stress-induced changes in T cell distribution are largely mediated by E and L selectin. (Supported by The John D. and Catherine T. MacArthur Foundation and by The College of Dentistry, Ohio State University.)
Open Field Behavior and Susceptibility to Adjuvant Arthritis in the Rat. Mirjana Dimitrijevic,* Olgica Laban,* Stanislava Stanojevic,* Tatjana Miletic,* Vesna Kovacevic-Jovanovic,* Vesna Vujic-Redzic,† and Jelena Radulovic,‡ *Institute of Immunology and Virology ‘‘Torlak,’’ Immunology Research Center ‘‘Branislav Jankovic,’’ Belgrade, Yugoslavia; †Institute of Chemistry, School of Medicine, Belgrade, Yugoslavia; and ‡Max Planck Institute for Experimental Medicine, Department of Molecular Neuroendocrinology, Goettingen, Germany. Rat strains were shown to express distinct behavioral traits and different sensitivity to experimental autoimmune diseases. We have reported recently that among four rat strains tested, the high susceptibility to adjuvant arthritis (AA) was associated with the low open field (OF ) activity, while the pronounced grooming behavior was related to the complete resistance to AA induction. The present study investigated the relationship between behavioral performances in the OF test and susceptibility to AA within each strain. For that purpose Lewis and Wistar female rats were subjected to three consecutive OF tests. According to the horizontal locomotor activity (number of lines crossed) rats of both strains were divided into the high and low active animals. Ten days later rats were immunized with single intradermal injection of complete Freund’s adjuvant (0.6 mg BCG/rat) in the basis of the tail. Clinical severity (edema and arthritic nodules) of the disease was evaluated daily for each paw (0–4, maximal score ⫽ 16). The results showed that there were no differences in the incidence and severity of AA, day of onset and duration of the disease between high and low active Lewis rats. The incidence of AA in Wistar rats was 5/14 and 6/11 in the high and low active animals, respectively, but the severity of AA was similar in both groups. It was concluded that OF activity can not predict the individual susceptibility to AA and the severity of the disease within particular rat strain. (Supported by the Ministry for Science and Technology, Republic of Serbia.) Social Support Mediates the Relationship between Loneliness and Human Herpesvirus-6 (Hhv-6) Antibody Titers in Hiv⫹ Gay Men Following Hurricane Andrew. Denise A. Dixon,* Kristin Kilbourn,* Michael Antoni,* Stacy Cruess,* Nancy Klimas,† Mary Ann Fletcher,† Gail Ironson,* Andrew Baum,‡ and Neil Schneiderman,* *Behavioral Medicine Research Center, c/o VA Medical Center, University of Miami, Miami, Florida 33125; †Infectious Diseases and Clinical Immunology, Department of Medicine, University of Miami School of Medicine, Miami, Florida 33136; and ‡Behavioral Medicine and Oncology, University of Pittsburgh Cancer Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213. Prior research has determined that many stressors associated with HIV infection are related to increased loneliness and declining social support, which other work has related to immune system decrements in healthy populations. The current study investigated the impact of a severe environmental stressor, Hurricane Andrew, and the role that declining social integration after the storm played in mediating its effect on loneliness and immune status (Human Herpesvirus Type 6 [HHV-6] antibody titers) in 42 HIV⫹ gay men. Loneliness was assessed with the Revised UCLA Loneliness scale and social support with the Social Provisions Scale (SPS) and the Interpersonal Support Evaluation List (ISEL). Increased loneliness (r ⫽ .51, p ⬍ .01) and decreased social support (r ⫽ -.67, p ⬍ .001) in the months following the storm were significantly associated with increased HHV-6 antibody titers, reflecting poorer control over the virus. Hierarchical regression analyses determined that poorer social integration mediated the relationship between loneliness and HHV-6 antibody titers, even after controlling for nonspecific polyclonal B cell activation, disease status (CD3⫹/CD4⫹ cell counts), living arrangements, acute social losses (bereavement), and potential disruptions in social support resources due to the hurricane (F Change [2,30] ⫽ 4.18, p ⬍ .05). These findings suggested that specific elements of social support may explain the oftnoted negative effects of loneliness on the immune system, and generalized these findings to a medically vulnerable population during the recovery period after the hurricane. (Research support by NIMH Grant MH18917.) The Effect of a Psychosocial Intervention on Cortisol, Secretory Immunoglobulin A (sIgA), and Incidence of Upper Respiratory Infection. Christyn L. Dolbier, Robert R. Cocke, and Mary A. Steinhardt, Department of Kinesiology and Health Education, University of Texas at Austin, Austin, Texas 78712. Certain protective factors may lessen the experience of stress and thus diminish the suppressive effect of stress on immune system functioning that leads to increased susceptibility to illness. Objective. The
purpose of this study was twofold: (1) to test the effectiveness of an intervention to enhance protective factors and (2) to examine cortisol release, immune activity, and subsequent health in regard to these factors. Methods. Subjects for the study were recruited from freshman seminar classes. The experimental group (N ⫽ 32) underwent a psychosocial intervention (i.e., a freshman seminar on building self-esteem, hardiness, and problem-focused coping skills), while the control group (N ⫽ 32) did not undergo a psychosocial intervention (i.e., nonpersonal growth freshman seminars). Data collection included survey and saliva samples pre- and postintervention, and prestressor, as well as a one-month follow-up measure of upper respiratory infection. The saliva samples were examined for levels of cortisol, which is known to suppress immune function, as well as sIgA, which is associated with resistance to upper respiratory infection. Subjects in the experimental group were predicted to experience less psychological stress due to a natural stressor (a difficult final exam), have lower cortisol levels and higher sIgA levels, and have lower levels of upper respiratory infection than subjects in the control group. Results. Data analyses largely supported the hypotheses. Conclusion. Implications for stress-reducing interventions and the use of cortisol and sIgA in psychoneuroimmunology research will be discussed. The salary for teaching a freshman seminar course supported this research. Operant Feeding Responses to Interleukin-1 and LPS. Artur H. Swiergiel and Adrian J. Dunn, Department of Pharmacology and Therapeutics, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130. Operant procedures were used to identify factors that influence the hypophagia induced by peripherally administered lipopolysaccharide (LPS) and mouse interleukin-1beta (mIL-1beta). In daily 30-min sessions, mice were trained to poke their noses into a hole to receive a reinforcement of 20 mg of sweetened milk under fixed ratio (FR) 1, 4, and 32, or progressive-ratio, PR5. Five training sessions sufficed to establish a stable rate of responding under FR1 that was reliably increased by overnight food deprivation and decreased by prior restraint. Mice on an FR32 schedule were more sensitive to d-fenfluramine than food-deprived mice on FR1. LPS (0.5–5 µg/mouse) caused a dose-dependent reduction in responding. On an FR1 schedule, mIL-1beta (100 ng) depressed the number of responses by 27% in nondeprived mice, but had no significant effect in food-deprived animals. On FR4, IL-1 caused a small decrease in responding, while on an FR32 schedule, IL-1 had no effect on the number of operant responses. On a PR schedule, d-fenfluramine and overnight fasting affected responding, but IL-1 did not. These results indicate that the effects of IL-1 on operant feeding depend on the metabolic state and motivational factors. Changes in operant performance do not appear to be a significant factor in IL-1-induced hypophagia. These results suggest that different mechanisms mediate anorexia induced by fenfluramine and cytokines. [Supported by grants from NIMH (MH46261).] Effects of in Vivo Administration of Mu, Kappa, and Delta Opioid Receptor Agonists on Immune Responses and on Oral Salmonella Typhimurium Infection. Toby K. Eisenstein,*,† Xiaohui Peng,* Rahil T. Rahim,* Joseph J. Meissler, Jr.,*,† Thomas J. Rogers,*,† Alan Cowan,†,‡ Ellen B. Geller,†,‡ and Martin W. Adler,†,‡ *Department of Microbiology and Immunology, †Center for Substance Abuse Research, and ‡Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140. Previous work established that morphine given by s.c. slow-release pellet resulted in suppression of splenic plaque-forming cell (PFC) responses and in sensitization to oral infection with S. typhimurium. In this study, we used Alzet minipumps to administer other opioids to assess their immunomodulatory effects. Mice were administered various doses of the kappa agonist, U50,488H, a delta2 agonist, deltorphin-II, or a delta1 agonist, DPDPE via minipumps. Controls received saline or agonist and antagonist. Splenocytes were harvested 48 later and antibody responses tested by PFC assay. The results show that U50,488H (1 mg/kg/day) or deltorphin II (0.5 mg/kg/day) each significantly suppressed PFC responses, and specific receptor antagonists blocked the effects. In opioid-treated versus control mice which received Salmonella, potentiation of infection was determined by colony counts of Peyer’s Patches (PP), mesenteric lymph nodes (MLN), and spleens 48 h after challenge. Morphine treatment markedly potentiated Salmonella growth in all three tissues, with ⬎ 10 6 organisms cultured from PP. Other opioids were used in immunosuppressive dose ranges. After U50,488H, or DPDPE treatment, no Salmonella were detected in any of these tissues. In mice receiving deltorphin II, 1/5 mice had a low level (10 3) of organisms in the PP and MLN, but 4/5 animals had 102 organisms in the spleen. These results suggest that mu agonists
have a greater effect than delta1 or kappa agonists in potentiating Salmonella infection. Delta2 agonists have a smaller but measurable effect. (This work was supported by NIDA Grants DA06650 and DA11134.) Regular Exercise Increases Total Circulating IgG in Sprague–Dawley Rats. Gwendolyn F. Elphick, Albert Moraska, and Monika Fleshner, Department of Kinesiology and Applied Physiology, University of Colorado at Boulder, Boulder, Colorado 80309. Regular, moderate physical activity can prevent the suppressive effect of stress on the generation of antibodies against keyhole limpet hemocycanin immunoglobulin (anti-KLH Ig). Exercise in the absence of stress has little impact on anti-KLH Ig production. What remains unknown is what effect exercise and stress would have on total Ig. We examined, therefore, the effect of exercise and stress on total serum IgM and IgG. Male Sprague–Dawley rats (4 months, 10/group) were housed singly in cages with either a functional or immobile running wheel. Running animals had 4 weeks of activity prior to inescapable tail shock (IS, 100, 5 s, 1.6 mA, 60 s ITI). KLH injections were then given intraperitoneally (200 µg) immediately before shock. Control animals were immunized at the same time. Blood samples from all animals were drawn from the tail vein on days 7, 14, 21, and 28 after KLH immunization. KLHspecific and total IgG and IgM ELISAs were preformed. As previously reported, stress reduced antiKLH IgM and IgG and had no effect on total IgM and IgG in sedentary rats. Exercise prevented the stress-induced suppression in anti-KLH. Exercise alone had no effect on anti-KLH and total IgM, but did increase total IgG. Thus, although exercise alone, in the absence of stress, has no effect on an antigen specific Ig response, it does elevate total circulating IgG. This may be reflective of a global stimulatory effect of physical activity on B cell IgG output in the exercising animals. (AI45576, MH60301.) Tolerance to LPS-Induced Hypoactivity Is Affected by Reproductive Status in Female, but Not Male Meadow Voles. Christopher G. Engeland,*,† Martin Kavaliers,*,†,‡ and Klaus-Peter Ossenkopp,*,†,‡ *Neuroscience Program, †Department of Psychology, ‡Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario N6A 5C2, Canada. Lipopolysaccharide (LPS), the minimal immunogenic component of gram-negative bacteria, is released during infection and causes a variety of sickness behaviors which typically include decreased locomotor activity and weight loss. This study considered how sex and reproductive status influences the effects of LPS in meadow voles. Male and female voles were housed under either reproductively stimulatory or inhibitory photoperiods (14 or 8 h). On Days 1 and 8, voles were injected with LPS (200 µg/kg ip) or saline vehicle and locomotor activity was assessed 2 h later in an automated open field for 1 h. Although none of the animals displayed significant body weight loss 24 h after injections, the first exposure to LPS caused significant decrements in locomotor activity in both sexes, regardless of reproductive state. However, nonreproductive males and females rapidly formed a behavioral tolerance to LPS after the second injection, as shown by reduced decrements in activity relative to controls. Reproductive males also quickly developed a behavioral tolerance to the locomotor reducing effects of LPS. In contrast, reproductive females given LPS for the second time still exhibited significant hypoactivity on all locomotor measures compared to controls. These results suggest that female voles in the reproductive state become tolerant to gram-negative bacteria more slowly than in the nonreproductive state, possibly due to increased circulating estradiol and/or corticosterone levels which may modulate the immune response. (Funded by NSERC.) Systemic Propranolol Does Not Disrupt, but Alters Conditioned Enhancement of Antibody Production. Enrique Espinosa, Moises King, Carolina Hill, and Federico Bermudez-Rattoni, Department of Neuroscience, Institute of Cellular Physiology, National University of Mexico, Ciudad Universitaria, 04510 Mexico, D.F., Mexico. Antibody levels can be significantly modulated by the Central Nervous System through behaviorally conditioned enhancement of antibody production, in which both afferent (immune to brain) and an efferent (brain to immune) communications participate. When laboratory animals are exposed to a novel taste stimulus during antigen challenge, a memory of this contingency is established (acquisition), so that further exposure to the gustatory stimulus alone yields a rise in specific antibody levels in blood (evocation). In this study we administrated the beta-adrenergic antagonist propranolol during acquisition or
evocation of conditioned enhancement of antibody production. We found that beta blockade with propranolol is not able to disrupt this conditioning, even though the conditioned response thus obtained is altered. Propranolol treatment during acquisition diminished substantially the subsequent conditioned enhancement of IgG2a antibodies during evocation, but yielded a slight increase in the conditioned response when measured as changes in specific IgG1 levels. Propranolol during evocation does not abolish the CNS-promoted increase in antibody levels as a response to a taste stimulus, but greatly reduces the IgG2a component of this response. Theses results have two possible interpretations: either propranolol causes a long lasting effect in cells or pathways involved in the response of the immune system to signals from the brain, or a different conditioning took place in the presence of the beta blocker. This latter raises the possibility of a differential detection by the brain of different states of the immune system. (This work was supported by Grant IN-212996 from DGAPA, UNAM.) Mechanisms and Biological Relevance of Behaviorally Conditioned Immunosuppression. Michael S. Exton,* Bettina Meier,* Cladia Gierse,* Juergen Westermann,† and Manfred Schedlowski,* *Institute of Medical Psychology, University of Essen, 45122 Essen, Germany; and †Department of Functional and Applied Anatomy, Hannover Medical School, 30625 Hannover, Germany. The mechanisms and biological relevance of behaviorally conditioned immunomodulation remain unknown. We have previously demonstrated that the conditioned reduction of splenocyte proliferation and cytokine (IL-2, IFN-gamma) production using cyclosporin A as UCS is effected via neural innervation of the spleen. Additionally, these alterations were of sufficient magnitude to prolong heart allograft survival. Therefore, we currently examined whether the conditioned suppression of proliferation could be blocked by beta-adrenergic receptor blockade. Further, we examined whether the relevance of conditioned immunosuppression was specific to a spleen-dependent response by examining the conditioned modulation of a dermal contact sensitivity reaction. To investigate mechanisms, animals were conditioned as previously described, with the addition of implantation of osmotic mini-pumps containing either propranolol or NaCl prior to CS reexposure. Lymphocyte proliferation in the spleen and cytokine production was suppressed in saline treated conditioned animals. However, administration of propranolol prior to CS re-exposure blocked the conditioned effect. To investigate biological relevance, conditioned animals were sensitized on the abdominal skin with the contact allergen 2,4-dinitrochlorobenzene (DNCB; 5%) at the conclusion of CS–UCS pairings. Following the final CS reexposure, animals were challenged with 1% DNCB on the ear pinnae, with ear thickness and cellular infiltration monitored 24 h later. Conditioning reduced ear swelling and cellular infiltration of the ear. These data indicate behavioral conditioning alters splenic immune functions in a beta-adrenergic-dependent mechanism. However, conditioning alters the course of disease which is relatively spleen-independent, via as yet unknown mechanisms. Acute Alcohol Withdrawal in Long-Term Alcohol Abusers Affects HPA Axis Function and Peripheral Blood Mononuclear Leukocyte Distribution. Michael S. Exton,* Sven Kutscher,† Dirk J. Heise,* Marcus Banger,† and Manfred Schedlowski,* *Institute of Medical Psychology and †Department of Psychiatry and Psychotherapy, University of Essen, 45122 Essen, Germany. Although alcohol withdrawal has been demonstrated to produce a number of significant endocrine alterations, no data exists on the effects of these changes on immune functions. Therefore, the current study investigated the effect of acute alcohol withdrawal on endocrine and immune parameters in alcoholics. Nine male alcoholics admitted to the university clinic for alcohol dependence participated in the study. Blood was drawn from alcoholics at 09:00 on the first morning of acute alcohol withdrawal (day 0), and at the same time on day 1 and day 7. Blood was drawn from age and gender matched healthy control subjects (n ⫽ 9) at corresponding time points. Plasma was analyzed for cortisol, prolactin, noradrenaline, and adrenaline concentrations, and blood leukocyte subset analysis completed by flow cytometry. Additionally, levels of withdrawal, mood, and cardiovascular parameters were recorded. Alcohol dependent patients displayed significantly elevated plasma cortisol on day 0 and 1 of withdrawal, which fell to control levels by day 7. Furthermore, alcoholics showed reduced numbers of CD4⫹ and CD8⫹ T cells, activated T cells and natural killer cells compared to healthy controls. These effects were observed across the period of recording. In contrast, although monocyte numbers were lower in alcohol dependent patients on day 0, acute alcohol withdrawal significantly increased the absolute number of monocytes in patients over control numbers. These data demonstrate that alcohol dependency produces a general
suppression of leukocyte subset numbers in blood. Further, acute alcohol withdrawal returns plasma cortisol to control levels, and concomitantly increases peripheral blood monocyte numbers. Stress-Induced Neutrophil Mobilization Is Increased in Physically Active Rats. Monika Fleshner, Taro P. Smith, M. C. Olson, Ben Greenwood, Ted Leem, and Gwen F. Elphick, Department of Kinesiology and Applied Physiology, University of Colorado at Boulder, Boulder, Colorado 80309. Exposure to acute stress can decrease the size and time required to resolve an inflammatory response. Voluntary freewheel running prior to stressor exposure facilitates this effect. Rats that run for 4 weeks prior to exposure to tail shock and subcutaneous E. coli, have in increased rate of inflammation resolution. Exercised and stressed rats resolve their inflammation 2–3 times faster than stress alone. Exercise alone has no effect on inflammation. One potential explanation for this effect is that exercise and stressed rats have a greater number of neutrophils at the site of inflammation. This experiment investigated whether the increase in neutrophil density was due to local chemotactic factors or changes neutrophil mobilization. Male, Fisher 344 rats (10/group) were allowed access to either mobile or immobile (sedentary) freewheels. After 6 weeks of running, rats were exposed to inescapable tail shocks (100, 5 s, 1.6 mA). Tail vein blood samples were taken at 0, 1, 2, and 4 h after stress. White blood cell differentials, red blood cell count, hemoglobin, hematocrit, and platelet count was measured. As expected, stress increased the total number of circulating white blood cells (WBC). This was due to an increase in circulating neutrophils and monocytes. Total lymphocyte number was decreased by stress. Physically active and stressed rats had a greater increase in both WBC and neutrophil number compared to their stressed, sedentary counterparts. Stress-induced lymphocyte decrease was equal in both sedentary and physically rats. Thus, the effect of stress on neutrophil mobilization is facilitated in physically active rats. (AI45575, MH60301.) The Effects of the Serotonergic Antidepressant Fluoxetine on Natural Killer Cell Activity in Rats. Julie L. Wieseler Frank,* Matthew G. Frank,† Shelton E. Hendricks,*,‡ and Donald R. Johnson,‡ *Department of Psychology, University of Nebraska at Omaha, Omaha, Nebraska 68182; †Department of Pathology and Microbiology; and ‡Department of Psychiatry, University of Nebraska Medical Center, Omaha, Nebraska 68198. Research conducted in our lab indicates that fluoxetine (FLX) treatment increases natural killer cell activity (NKCA) in patients being treated for depression. However, in laboratory rats, reports suggest that FLX suppresses NKCA. To further understand the effects of a selective serotonin reuptake inhibitor on NKCA, the effects of FLX were evaluated in rats. Blood and splenic NKCA was evaluated in vitro (incubation with 0–10 µM FLX) or in vivo after acute or chronic (14 daily injections) of FLX (0, 1, or 10 mg/kg). Blood and splenic cells were harvested from untreated animals for in vitro experiments. Blood and spleens were harvested 2 h after one injection for acute effects and after 14 daily injections for evaluating the effects of chronic FLX. Mononuclear cells were isolated and assayed for NKCA in a standard 51Cr-release assay. Blood and splenic NKCA was enhanced when incubated with FLX in vitro. Acute administration of 10 mg/kg enhanced splenic NKCA, whereas other doses failed to enhance NKCA. Blood NKCA was unaffected by FLX. Chronic FLX did not alter blood or splenic NKCA. Our results contrast previous reports that acute and chronic FLX suppress NKCA in rats. In vitro and acute administration results are consistent with our previous suggestion that FLX and other serotonergic drugs enhance NKCA through a direct action on mononuclear cells. However, the effects of chronic injection raise questions regarding differences between the rat and the depressed human patient with respect to effects of FLX on NKCA. Effects of Endogenous Stress-Related Substances on Natural Killer Activity (NKA) in Vitro. M. Garland,* Derek N. Doherty,† Noel M. Walsh,* and Cliona R. O’Farrelly,† *Department of Psychiatry, †Education and Research Centre, St. Vincent’s University Hospital, Elm Park, Dublin 4, Ireland. Aims. Natural Killer (NK) cells bear receptors for a large number of CNS-derived peptides, hormones and neurotransmitters. Many of these have been described, generally individually, as having direct or indirect effects on NKA. This study sought to examine to relative effects of physiological concentrations of a battery of these substances on NKA. Method. Blood was drawn from 4 healthy male volunteers. Freshly prepared physiological concentrations of seven stress related products—beta-endorphin (Beta-
EP), corticotrophin releasing factor (CRF), cortisol, adrenocorticotrophin (ACTH), serotonin (5-HT), neuropeptide Y (NP-Y), and adrenaline were selected. Incubation with peripheral blood mononuclear cells (PBMCs) for 2, 12, and 24 h, prior to using the PBMCs as effectors in 4 h 51Chromium-release NK assay, using the K562 erythroleukemia cell-line as targets. Three effector:target ratios were used: 100 : 1, 50 : 1, and 25 : 1. Results. Optimal effects were seen after 24 h preincubation and at the 50:1 E: T ratio. ACTH, Adrenaline, Beta-EP, CRF, and NP-Y all produced marked stimulatory effects. Cortisol and 5-HT were inhibitory. Conclusion. The results confirm that NK cells are sensitive to a large number of stress related substances. This indicates that NK cells are sensitive to multiple psychoneuroendocrine influences in vivo. Salivary Cortisol and Natural Killer Cell Numbers in Patients with Early Breast Cancer. M. Garland,* Lucy M. Golden-Mason,† Noel M. Walsh,* and Cliona R. OFarrelly,† *Department of Psychiatry, †Education and Research Centre, St. Vincent’s University Hospital, Elm Park, Dublin 4, Ireland. Aims. Cortisol, a major stress-related hormone, strongly inhibits the activity of NK cells in vitro. Prolonged stress is associated with diminutions of the NK cell population and elevations of plasma cortisol. This study was carried out to determine if salivary cortisol (which measures the physiologically active cortisol) is associated with low NK cell numbers in patients with early breast cancer. NK cells may have an anti-metastatic function in this population. Method. 20 patients (aged 35–65) provided 3 early morning salivary samples for cortisol (fluorescence immunoassay) and simultaneously, blood for NK phenotyping (CD56⫹ CD3⫺) by flow cytometry. Samples were obtained preoperatively. Results. A weak trend toward a negative correlation was found [r(Pearson’s) ⫽ ⫺0.32, p ⫽ .18] between mean cortisol and NK number. Conclusions. Despite sampling a highly stressed population which had higher basal levels of cortisol (compared to normative data), no significant relationship was found between the latter and circulating NK cell numbers. This may suggest that other stress-related mechanisms, such as autonomic nervous system activation, may be more involved in NK cell immunomodulation. The data will be reexamined when stored peripheral blood mononuclear cells are examined for their NK activity profiles. Stress Suppresses the Adaptive Immune Response. L. S. Gazda,* M. Fleshner,† T. P. Smith,† L. R. Watkins,* and S. F. Maier,* Departments of *Psychology and †Kinesiology and Applied Physiology, University of Colorado, Boulder, Colorado 80309. Prior work has shown that exposure to inescapable tail shock (IS) after immunization with KLH reduces several aspects of T-cell function several days later. However, whether these changes are antigenspecific has been unknown. Male Fischer rats (6/group) were immunized subcutaneously at the base of the tail with 200 µg keyhole limpet hemocyanin (KLH) emulsified in incomplete Freunds adjuvant and either subjected to 100, 5-s, 1.6 mA tail shocks or remained in their home cages (HCC). The proliferative response to KLH of splenic, cervical lymph node, and draining lymph node cells was determined at days 4, 7, and 10 after IS or HCC. No differences were observed in the response to KLH from spleen or cervical lymph node cells. However, suppression of the proliferative response to KLH from draining lymph node cells was observed at all time points in IS treated animals as compared to HCC. The proliferative response was specific for KLH as there were no differences in proliferation to Con A. A separate group of Fischer rats (10/group) were similarly immunized and blood samples taken at days 7, 14,21, 28, and 35 days after IS to determine IgG and IgM production to KLH. While no differences in the levels of IgM were observed, a reliable suppression of anti- IgG was found throughout the observation period. These data support the hypothesis that IS does indeed produce antigen-specific changes in Tcell function. (Supported by NIH Grants MH 55283, MH 45045, MH 01558, and MH 00314.) The Impact of Psychological Stress on the Antibody Response to a Pneumococcal Bacterial. Ronald Glaser,* John Sheridan,† William Malarkey,‡ and Janice Kiecolt-Glaser,§ *Department of Molecular Virology, Immunology and Medical Genetics, †Department of Oral Biology, ‡Department of Medicine, §Department of Psychiatry, Ohio State University Medical Center, Columbus, Ohio 43210. Influenza and pneumonia account for significant morbidity and mortality, particularly in older individuals. These respiratory illnesses are the fourth leading cause of death in people over 75 years of age. We had previously shown that psychological stress could reduce the antibody and virus specific T-cell responses to an influenza virus vaccine. In this study, we measured antibody titers following vaccination
of caregivers of dementia patients and well-matched control subjects with a pneumococcal bacterial vaccine. The vaccine consisted of noninfectious bacterial polysaccharide capsular antigens. Pneumococcal specific IgG titers were determined by ELISA. Caregivers showed deficits relative to controls in their antibody responses to the pneumococcal pneumonia vaccine. Although the groups did not differ in either antibody titer prior to vaccination or the rise in antibody two weeks or one month following vaccination, caregivers had lower antibody titers three and six months after vaccination relative to controls. Consistent with the previous studies on viral vaccines and stress, we found a significant inhibition of the IgG antibody response to this bacterial vaccine over time in the caregivers. These data continue to support the hypothesis that immune down-regulation/dysregulation induced by various psychological stressors are large enough to affect the immune response to both viral and bacterial vaccines and add to the growing evidence supporting the hypothesis that there may be significant health implications linked to behavior mediating changes in the immune response resulting in increased risk for infectious disease. Psychological Stress, Exercise, and Isoproteronol Infusion Alter TNF-Alpha and IL-6 Production in Humans. Marion U. Goebel,*,† Paul J. Mills,* Michael G. Ziegler,‡ and Michael R. Irwin,*,§ *Department of Psychiatry University of California, San Diego, California 92103; †Department of Medical Psychology, University of Essen, 45122, Germany; ‡Department of Medicine, University of California, San Diego, California 92103; and §VA Medical Center, La Jolla, California. Proinflammatory cytokines, such as TNF-alpha and IL-6 have a pivotal role in coordinating the body’s response to inflammation. Repeatedly, studies demonstrated that acute sympathetic nervous system (SNS) activation alters cytokine responses; however, there is a noticeable heterogeneity in methods and materials. The aim of this study was to investigate the effects of three different methods of SNS activation on LPS-induced TNF-alpha and IL-6 production within the same proband. Thirty-two healthy volunteers performed in a fixed order a speech task and an exhaustive exercise task, and underwent a 30-min infusion of isoproteronol (20 and 40 ng/kg/min for 15 min each). Blood was drawn prior to and immediately after each task. Leukocyte and lymphocyte subsets were determined by flow cytometry, ex vivo lipopolysaccharide-induced TNF-alpha and IL-6 production by enzyme-linked immunosorbent assay (ELISA). All three methods induced a leukocytosis. IL-6 production was increased by both the speaking task and exercise, whereas TNF-alpha production was elevated upon exercise, however remained unaffected following the speaking task. In contrast, the isoproteronol infusion decreased TNF-alpha production, yet did not alter IL-6. IL-6 and TNF-alpha production showed different profiles following the three tasks. Purely beta-agonist stimulation by isoproteronol down regulated TNF-alpha production, indicating an anti-inflammatory effect. The enhanced production of both cytokines by exercise, and IL-6 following the speech task, suggests the induction of an inflammation-like response. Adolescent Smoking: A PNI Confound of Consequence to NK Function. Arielle R. Goldklang, Jacqueline A. Bartlett, Steven J. Schleifer, and Steven E. Keller, Department of Psychiatry, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103. Objective. We investigated immunological effects of moderate smoking as part of a longitudinal study of immunity and behavior among inner-city adolescents in order to examine if smoking per se, a common confound/concomitant of PNI studies, is associated with immune alterations. Methods. Three hundred seventy-five students between 13 and 18 years were randomly selected from high schools in Newark, New Jersey and examined utilizing a battery of psychological and immune measures. The sample included in these analyses was composed of 226 African Americans and 49 Latinos. Forty-six subjects smoked for longer than one year at the time of evaluation, including 21 females 18 African Americans, 3 Latinos and 39 African Americans (20 males, 18 females). The number of years and number of packs smoked daily were also assessed to determine whether degree or duration affected immune function. Results. As expected, older subjects smoked more and for longer than younger subjects. Neither race nor gender was observed to related to smoking. Smokers showed lower numbers of NK cells and percent NK cells than nonsmokers, independent of NKCA, and the difference remained significant after partialling out demographic variance. Neither degree nor duration was significantly related to immune differences after accounting for demographics. Conclusion. Adolescent smokers are immunologically different, even with moderate use. The NK system appears to be the most sensitive to smoking. It remains to be seen whether this immune/smoking relationship in adolescents is related to subsequent health and PNI alterations in immune function. (Source of funding: NIMH Grant R01 MH44142.)
Activation of Macrophage and Lymphocyte Functions Following in Vitro Stimulation and Intravenous Administration of the Nonpeptidic Opioid Agonist SNC 80. Ricardo Gomez-Flores,* Mary E. Hicks,* Silvia N. Calderon,† Kenner C. Rice,† and Richard J. Weber,* *Department of Biomedical and Therapeutic Sciences, Section of Medical Sciences, University of Illinois College of Medicine, Peoria IL; and †Laboratory of Medicinal Chemistry, NIDDK, NIH, Bethesda, Maryland. Opioids alter immune function by binding to opioid receptors on cells of the immune system, or indirectly by acting on receptors within the CNS. Mu-selective opioid agonists cause immunosuppression, whereas delta-opioid receptor-selective agonists have been associated with immunopotentiation. We have previously shown that intracerebroventricular administration of the nonpeptide delta-opioid receptor agonist (⫹)-4-((alpha R)-alpha-((2S, 5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl)-N, N-diethyl-benzamide (SNC 80) did not alter certain parameters of immunocompetence. In the present study, we studied the in vitro and ex vivo effects of SNC 80 on rat macrophage and lymphocyte functions. We showed that SNC 80 at concentrations of 10 ⫺7 M and 10 ⫺6 M, significantly ( p ⬍ .01) stimulated the in vitro production of tumor necrosis factor-alpha (TNF-alpha) (60–100% increase) and nitric oxide (34–67% increase) by resident and LPS-stimulated peritoneal macrophages. Similarly, intravenous administration of SNC 80 (6.8 mg/kg) significantly ( p ⬍ .01) increased the production of TNFalpha and nitric oxide (2- and 1.5-fold increases, respectively, compared with saline-injected control) by LPS-stimulated splenic macrophages. SNC 80 (10 ⫺11 to 10 ⫺7 M) was also shown to potentiate (1.5to 2-fold increase) the in vitro Con A-induced proliferation of thymic lymphocytes in a dose- and timedependent fashion. Nonpeptidic delta-selective opioids that are proteolytically stable and potentiate immune functions could be useful in the treatment of cancer, infectious diseases including AIDS, and as adjuvants for poorly immunogenic vaccines. (Supported by NIH Grants DA/AI08988, DA12095, and F32-DA05865.) c-Fos Expression Is Not Habituated in Stress-Reactive, Autonomic Neurocircuits Following Freewheel Running. B. N. Greenwood, J. L. Hinde, M. Nickerson, and M. Fleshner, Department of Kinesiology and Applied Physiology, University of Colorado, Boulder, Colorado 80309. Stress reactive, autonomic brain areas of freewheel-run rats have less of an increase in expression of the functional activation marker, c-Fos, following stress compared to sedentary rats. This is implicative of either a blunting of neural activation during stress or an ‘‘habituation’’ of c-Fos expression induced by chronic activation of stress circuits caused by running. Therefore, we examined if freewheel running alone causes habituation of c-Fos expression in stress-responsive, autonomic brain nuclei. Adult Fischer rats (F 344, 5/group) were housed singly in cages with either a functional or immobilized running wheel. Rats were divided into 3 groups: (1) 6 weeks running, (2) sedentary for 6 weeks then 36 h of running, (3) sedentary. Brains were removed after 90 min of running and processed for c-Fos protein immunoreactivity (IR). Brain areas examined were the motor cortex (FR1), cingulate gyrus (1 and 3), infralimbic cortex, amygdala (BLA), septum, hypothalamus (PVN) & habenula (LHbM). Both running groups ran equal distances prior to sacrifice. C-Fos IR was higher in the motor cortex of the 2-day run group than the 6-week run and sedentary groups. The decrease after 6-weeks of running is due to reduction in reliance on cortical activation with repeated running. There were no increases in c-Fos IR in any of the stress-reactive areas examined. Results suggest that freewheel running, per se, is not stressful, and support the hypothesis that the reduction in elevated c-Fos IR in stress-responsive, autonomic nuclei in physically active rats is due to decreased neural activation, not c-Fos habituation. (Supported by AI45576, MH60301.) Does Breastfeeding Confer Stress, Immune, and Health Benefits to the Mother? Maureen Groer, Alva Jordan, Jean Hemphill, Kristina Plaas, Mitzi Davis, and Pat Droppleman, College of Nursing, University of Tennessee Knoxville, Knoxville, Tennessee 37992-4180. Immunological, stress, and immune responses of postpartum mothers to naturalistic stress were measured in breastfeeding and bottlefeeding mothers at week 4 postpartum. Mothers completed a demographic form, the Inventory of Small Life Events (ISLE) and an Infection Symptom Checklist. Ten milliliters of blood and 10 ml of milk were also collected. The ISLE scores, the perceived stress scores, the number and type of symptoms of infection, and lactational status were independent variables. The dependent variables were serum and milk cortisol, prolactin and IgA, and lymphocyte proliferation responses to concanavalin A. Breastfeeders (n ⫽ 10) had a negative event score of 16.1, compared to
27.6 in bottlefeeders (n ⫽ 8); perceived stress scores of 4.6, compared to 5.7; nonrespiratory symptoms of 1.1 compared to 2.6; respiratory symptoms of 2.8, compared to 4.7; a lymphocyte proliferation stimulation ratio of 53.3%, compared to 41.4%; a serum prolactin of 159 ng/ml, compared to 13.6; a serum IgA of 266 mg/dl compared to 191 mg/dl; and serum cortisol of 136 ng/ml, compared to 113 ng/nl. Infection scores were correlated with negative life events (r ⫽ .923, p ⫽ .000), and milk cortisol and sIgA were inversely related (⫺.605, p ⫽ 0.1). The mean number of negative events was 48.6 for the lowest income category ($0–$10,000), and 17.8 in the highest category (⬎$40,000). The data support differences in the psychophysiology of breastfeeding compared to bottle-feeding mother. Breastfeeders may be protected from stress and its deleterious effects on immune function and health status. Paroxetine Pretreatment Significantly Decreases the Initial IL-6 Response to Interferon-Alpha Administration in Patients with Malignant Melanoma. Jane F. Gumnick,* Bradley D. Pearce,* Carmine M. Pariante,† Y. Su,* Rebecca S. Goodkin,* Andrea J. Reemsnyder,* David H. Lawson,‡ Dominique L. Musselman,* and Andrew H. Miller,* *Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia 30322; †Section of Clinical Neuropharmacology, Institute of Psychiatry (C.M.P.), London, United Kingdom SE5 8AF; and ‡Department of Internal Medicine, Emory University School of Medicine, Atlanta, Georgia 30322. Interferon-alpha (IFN-alpha) is used for the treatment of cancer and viral infections. In addition to antiproliferative and antiviral effects, IFN-alpha is associated with a high rate of central nervous system complications such as anhedonia, depressed mood, anorexia, and fatigue. Pretreatment of laboratory animals with antidepressants has been shown to reduce the depression-like behavioral syndrome associated with the administration of cytokines, including IFN-alpha. Our group has found similar effects in humans. Objective. In order to investigate the mechanism(s) by which antidepressants reduce the development of depressive symptoms during IFN-alpha therapy, we examined plasma cytokine and hormonal responses to the first injection of IFN-alpha in 12 patients initiating IFN-alpha treatment for malignant melanoma. Methods. Six patients received paroxetine for two weeks prior to the initial injection of IFN-alpha, and 6 patients received placebo. Il-6 was measured at baseline and 1, 2, and 3 h post IFN-alpha administration. Results. There was a significant main effect of treatment condition with IL6 levels being reduced in paroxetine pretreated patients compared to controls (F[1,47] ⫽ 6.49, p ⫽ .025). For example, peak Il-6 levels were 37.0 pg/ml (SE 7.55) in paroxetine-pretreated patients and 59.66 pg/ml (SE 3.690) in placebo-treated controls (t ⫽ 2.70, df ⫽ 10, p ⫽ .023). In vitro studies on a fibroblast cell line demonstrated that paroxetine enhanced dexamethasone-stimulated glucocorticoid receptor function by 345%. Glucocorticoids are known to negatively regulate IL-6 release through their receptors. Conclusions. Taken together, this data indicate that antidepressants may reduce IFN-alpha mediated behavioral toxicity by reducing IL-6 responses, possibly through enhancement of glucocorticoid-mediated negative feedback. (Supported by Shering Plough, SmithKline Beecham, NIMH Grant MH00680 and NRSA 1T32MH20018-02.) Androstenediol Efficacy Against Ocular Herpes Simplex Virus Type 1 Infection Is Not Mediated by Peripheral Natural Killer Cells. Peter Haerle and Daniel J. J. Carr, Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104. A previous study found that androstenediol (AED) antagonizes herpes simplex virus type 1 (HSV1) replication and enhances survival of mice per os at 50 but not 100 µg/ml. We hypothesized that the efficacy of AED was mediated by natural killer (NK) cells, a known effector cell that significantly contributes to controlling HSV-1 infection. Therefore, we measured splenic NK activity and cell number (defined as NK1.1 ⫹ CD3⫺) in mice (n ⫽ 6–9/group) treated with or without AED (50–100 µg/ml) and infected with or without HSV-1 (210 pfu/eye) at days 1, 3, 5, and 7 days postinfection (p.i.). The results show no differences in splenic NK activity or cell number at any of the time points measured comparing the AED-infected to the vehicle-infected group as determined by ANOVA and Tukey’s t test. However, both infected groups of mice showed a time-dependent increase in splenic NK activity in comparison to uninfected mice. These results are contrary with a previous observation showing AED administered subcutaneously augmented splenic NK activity in a dose-dependent fashion measured 6 days p.i. in comparison to vehicle-treated animals. Taken together, the site of exposure to AED appears to selectively modify splenic NK activity during HSV-1 infection. Currently, we are assessing the local NK activity within the ocular tissue and sensory ganglia (trigeminal ganglion) to determine if NK activity within the microenvironment of infection is modified by AED. (This work was supported by EY12409.)
Characterization of HMG-1 Induced Fever in Rats. Michael K. Hansen,* C. Rachal Pugh,* John D. Johnson,* Haichao Wang,† Kevin J. Tracey,† Steven F. Maier,* and Linda R. Watkins,* *Department of Psychology, University of Colorado, Boulder, CO 80309; and †Laboratory of Biomedical Science, North Shore University Hospital, Manhasset, New York 11030. High mobility group-1 (HMG-1) protein, which is a member of the nonhistone chromosomal protein family, was recently identified as a late mediator of endotoxin lethality. Bacterial lipopolysaccharide and proinflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor, stimulate the release of HMG-1. Because proinflammatory cytokines produce sickness responses, such as fever, following intracerebroventricular (i.c.v.) administration and are thought to exert their actions directly at brain sites, it was of interest to examine whether HMG-1 had any endogenous pyrogen activity of its own. Thus, male Sprague–Dawley rats received i.c.v. injections of recombinant HMG-1 (10, 100, 1000 ng/rat) or equivolume vehicle and body temperature was measured by telemetry for 10 h. At this time, rats were sacrificed and blood and hypothalamus samples were collected. The i.c.v. injection of HMG1 significantly elevated body temperature in a dose-dependent manner. The increases in core body temperature began approximately 90 min after the injection and persisted for most of the recording period. In addition, HMG-1 significantly increased IL-1 protein levels in the hypothalamus. Thus, as with many proinflammatory cytokines, HMG-1 release is enhanced by other cytokines, and HMG-1 stimulates the release of at least IL-1 levels in the hypothalamus. These data support the notion that HMG-1 is an extracellular mediator of infectious and inflammatory processes and that HMG-1 may be an additional mediator of the proinflammatory cascade that can participate in the signaling between the immune and neuroendocrine systems. (Supported by NIH Grants MH55283, MH01558, MH00314, NS38020.) Long-Term Effects of Stress and Immune Challenge on Susceptibility to Adjuvant-Induced Arthritis. Michael S. Harbuz,* Antonio J. Chover-Gonzalez,† Juan Gibert-Rahola,† Hiroshi Kinoshita,* and David S. Jessop,* *URCN, University of Bristol, Bristol, United Kingdom; and †Department of Neurosciences, University of Cadiz, Spain. Single exposure to an acute stress can induce long-lasting changes in hypothalamo–pituitary–adrenal (HPA) axis parameters in the rat. We have investigated whether exposure to acute stress (footshock) or acute immune stimulation (lipopolysaccharide; LPS) 3 weeks prior to adjuvant injection can alter the severity of inflammation in response to adjuvant-induced arthritis (AA). We have assessed the effects of these challenges on hind-paw inflammation as an index of severity and determined changes in HPA axis activity, i.e., corticotrophin-releasing factor (CRF) and arginine vasopressin (AVP) mRNAs in the parvocellular cells of the paraventricular nucleus, proopiomelanocortin (POMC) mRNA in the anterior pituitary, and plasma concentrations of corticosterone. Exposure to footshock three weeks prior to adjuvant had no effect on either the time of onset or the severity of inflammation. In response to increased inflammation, there was an activation at all levels of the HPA axis with the exception of CRF mRNA, as reported previously. In contrast to acute stress, prior exposure to LPS prevented the development of AA. Despite the absence of hind-paw inflammation in these rats there was an increase in AVP mRNA, POMC mRNA, and plasma corticosterone suggesting that the alteration in inflammation occurred independently of changes in the HPA axis. These data suggest that prior exposure to acute immune challenge, but not acute stress, can modify susceptibility to AA. We are grateful to the Oliver Bird Fund of The Nuffield Foundation for financial support. Immunohistochemical Evidence for a Time-Dependent Sensitization of Neuroendocrine Regulatory Peptides in Response to Tumor Necrosis Factor-Alpha. S. Hayley,* Z. Merali,†,‡ W. A. Staines,† and H. Anisman,* *Institute of Neuroscience, Carleton University; †Department of Cellular and Molecular Medicine and ‡School of Psychology, University of Ottawa, Ottawa, Ontario, Canada. We have previously demonstrated that the cytokine, TNF-alpha provokes a time-dependent sensitization of behavioral and neurochemical processes. It was of interest to determine if a single TNF-alpha injection has protracted effects on immunoreactivity for the hypothalamo–pituitary–adrenal (HPA) axis modulating neuropeptides, AVP and CRH, as well as nitric oxide synthase (NOS). Thus, CD-1 mice received an initial 4.0 µg injection of recombinant human TNF-alpha and were subsequently reexposed to either 1.0 µg of the cytokine or a sterile nonpyrogenic saline solution 1, 7, 14, or 28 days later. Immunoreactivity for AVP and CRH, within cell terminals of the external zone of the median eminence,
was found to vary with the passage of time after the initial TNF-alpha treatment. Moreover, TNF-alpha provoked a time-dependent change in the colocalization of these peptides within the median eminence. In the central amygdala, CRH-ir increased upon reexposure to TNF-alpha, but such effects were not modified by varied times of reexposure. No visible alterations of either AVP or CRH immunoreactivity were evident within PVN cell bodies. However, the immediate early gene, c-fos, was upregulated in the PVN 3 h following the second TNF-alpha administration. Within the dentate gyrus, the number of cells displaying NOS-ir was elevated after a single acute treatment with TNF-alpha. These data suggest that some of the sensitization effects previously demonstrated for TNF-alpha may stem from variations in the HPA regulating neurochemicals AVP, CRH, and NOS, which may subserve specific time-dependent actions of TNF-alpha. (Supported by NSERC and MRC of Canada.) Neonatal Stress Potentiates Hypothalamic-Pituitary-Adrenal (HPA) Responsivity and Impairs Wound Healing and Tumor Resistance in the Adult Rat. Deborah M. Hodgson, Sally Rosengren, and Frederick R. Walker, Laboratory of Neuroimmunology, School of Behavioural Sciences, University of Newcastle, New South Wales, Australia. Exposure to neonatal stress can permanently alter the HPA-axis, resulting in potentiation of the HPA response to stress in adulthood. Given the interactions between glucocorticoids and the immune system we investigated the long-term impact of two neonatal interventions: maternal separation (MS) and endotoxin exposure (EE) on growth, stress responsivity, wound healing and resistance to tumor colonization in adulthood in F344 rats. In Exp. 1, neonates were exposed to MS (2 h/day), or left undisturbed, for 21 days postnatally. At 60 days animals were inoculated with MADB106 tumor cells (0.5 ⫻ 10 5). In Exp. 2. neonates were injected intraperitoneally with bacterial endotoxin (100 µg LPS–Escherichia coli) or vehicle on days 3, 5, and 7 postpartum. At 60 days of age animals were anesthetized and given 5 mm subscapular biopsy wounds. Three days prior to and after biopsy or tumor administration animals underwent a 10-h period of restraint. Blood was obtained after restraint for assessment of plasma corticosterone. Wound healing was assessed for 12 days following biopsy. Tumor colonization was assessed 4 weeks after inoculation. Data analysis indicates that MS and EE produced significant (p ⬍ .05) delays in weight gain. Both EE and MS were associated with significantly ( p ⬍ .05) higher stress-induced corticosterone levels compared to controls. EE was associated with a significant ( p ⬍ .05) delay in wound healing compared to controls. Finally, MS was associated with a significant ( p ⬍ .001) potentiation of tumor colonization compared to controls. In summary, neonatal stressors impair growth, wound healing, and tumor immunity, and they are associated with alterations in the HPA response to stress in adulthood. Adenosine Mediates Interleukin-1Beta Induced Behavioral Depression in Rats. Qingjun Huang and Thomas R. Minor, Department of Psychology, University of California at Los Angeles, Los Angeles, California. Intracerbroventricular injection of a physiological dose of interleukin1-beta (IL-1beta 2 ng/rat) significantly increased the floating time in the Porsolt swim test for behavioral depression. Pretreatment with the IL-1beta receptor antagonist (IL-1Ra, 6 µg/rat, icv) blocked the increase in floating time induced by IL-1beta. To determine whether the effects of IL-1beta in this paradigm are mediated via activation of the brain adenosine receptor, we injected IL1-beta treated rats with caffeine (7 mg/kg, ip), a high affinity antagonist of the adenosine receptor with no direct effect on the IL1-beta receptor. Caffeine treatment blocked the effect of IL-1beta in the swim test. These data indicate that a physiological level of IL-1beta can induce depressed behavior in rats, and that adenosine mediates this effect of IL-1beta. (The research supported by Noman Cousins Center of PNI at UCLA.) Adenosine Mediates Reserpine-Induced Behavioral Depression in Rats. Qingjun Huang and Thomas R. Minor, Department of Psychology, University of California, Los Angeles, Los Angeles, California 90095-1563. Intraperitonial injection of reserpine (4, 6, or 8 mg/kg) increased floating time in the Porsolt swim test in a dose- and time-dependent manner in rats. Although such behavioral depression usually is attributed to drug-induced depletion of brain monoamines, the outcome might be more directly related to brain adenosine signaling associated neuronal overactivation or brain cytokine induction following excitotoxic tissue damage. We addressed these possibilities by pretreating rats with caffeine (7 mg/kg), a high affinity
adenosine receptor antagonist, prior to reserpine treatment (6 mg/kg). Caffeine partially reversed the ensuing behavioral depression as measured in the Porsolt test conducted 1 h after reserpine treatment. However, intracerebroventricular injection of the interleukin 1-beta receptor antagonist (IL1-betaRa, 6 mg icv) failed to reverse the reserpine-induced depression. These data provide additional evidence that adenosine plays a crucial role in mediating behavioral depression in a number of animal models and question whether changes in brain monoamines per se are an essential ingredient. The experiments provide no evidence for a role of IL1-beta at this early stage of reserpine’s effect, although other test time points might lead to a different conclusion. Rate of Wound Healing in Rats Exposed to Acute Restraint Stress or Uncontrollable Shock. Aimee M. Hunter and Thomas R. Minor, Department of Psychology, University of California, Los Angeles, Los Angeles, California 90095-1563. The relationship between acute stress and an in vivo measure of immune function—rate of wound healing—was examined in the learned helplessness paradigm in rats. Rats received a single session of 100 inescapable shocks during restraint, simple restraint, or no stress (home-cage control). Rats were wounded using a 3-mm diameter biopsy punch to create a uniform epidermal dorsal wound either 24 h before or 24 h after the stress session. Hydrogen peroxide was applied daily and healing rate was determined by the number of days until complete reepithelialization (absence of foaming response to hydrogen peroxide application). Overall stress retarded healing, and a single stress session 24 h prior to biopsy was more effective at retarding healing than was a single stress session occurring 24 h after biopsy. Restraint stress was more effective than was uncontrollable shock at retarding healing. Given that uncontrollable shock is delivered in a restraint tube, one possible interpretation of these data is that shock mitigates the deleterious impact of restraint stress on wound healing. Restraint Stress (RST) Alters the Cytokine Response to Coxsackievirus Infection. J. Hunzeker,* E. A. Reynolds,* J. F. Sheridan,*,† and D. A. Padgett,*,† *Department of Molecular Virology, Immunology, and Medical Genetics, and †Institute for Behavioral Medicine Research, Ohio State University Health Sciences Center, Columbus, Ohio 43210. Coxsackievirus (CV) infection has been associated with the development of myocarditis in humans. Infection with the CVB3 strain also induces myocarditis in mice with a comparable pathophysiology to that seen in humans. As such, the murine model provides a useful system in which to study stress effects on antiviral immunity and myocarditis. The purpose of this study was to examine stress effects on disease progression and immunity during CVB3 (Nancy) infection (INF) of C3H/HeJ mice. Mice were restrained in tubes for 15 h for seven cycles beginning one day before infection. CVB3 (2.44 ⫻ 10 3 CID50/mouse) was injected i.p. after completion of the first stress cycle. Fifteen days after infection, all 12 nonstressed animals were alive, whereas only 13% (2/15) of RST/INF animals survived. To examine stress effects on antiviral immunity, RT-PCR was used to study cytokine gene expression in heart tissue on days 1, 3, 5, and 7 postinfection. The data showed that RST delayed expression of T-cell-derived IL-2 and INF-g. These cytokines are important for induction of cell-mediated immunity. Subsequent analysis of chemokine expression showed that RST decreased expression of MCP-1 in hearts of infected mice. Chemokines play an important role in the recruitment of lymphocytes to inflammatory sites. Reduced chemokine expression may contribute to decreased T cell responses that are important for controlling viral pathogenesis. These results suggest that stress affects T cell cytokine responses and cell trafficking during the course of CVB3 infection leading to enhanced viral-induced pathophysiology and increased mortality. (Supported by NIH Grants R29-MH56899 and R01-MH46801.) Differential Location and Secretion of Endomorphin-2 and Beta-Endorphin in the Immune System of the Rat. David S. Jessop, Kathryn A. Elsegood, Michael S. Harbuz, Matthew D. Crabb, Toni L. Coventry, and Colin M. Dayan, Department of Clinical Medicine, University of Bristol, Bristol BS2 8HW, United Kingdom. Opioid peptides are widely expressed in the immune system but low levels of expression can present difficulties in localizing opioids to specific cell groups. In order to accurately map endomorphin (EM)2 and beta-endorphin immunoreactivity, we applied a magnetic separation protocol (MACS, Miltenyi Biotech, UK) to enrich T cells, B cells, and macrophages from rat splenocyte and thymocyte populations.
Enriched cell populations were cultured for 48 h, acid-extracted, and beta-endorphin and EM-2 measured by radioimmunoassays (RIA). Concentrations of beta-endorphin in unactivated splenic macrophages, T or B cells were 19.6, 7.5, and 8.2 pg/million cells, respectively, and 26.2 and 19.6 pg/million cells in thymic macrophages and B cells. Beta-endorphin was undetectable in thymic T cells. Culture medium concentrations of beta-endorphin secreted from unactivated splenic macrophages T and B cells were 17.0, 6.8 and 9.1 pg/million cells, respectively, and 30.3 and 17.7 pg/million cells from thymic macrophages and B cells. EM-2 was not detectable in any cell extracts but was present in culture medium from splenic and thymic macrophages (8.8 and 11.5 pg), splenic B cells (3.9 pg) and thymic B cells (7.4 pg/million cells). Splenic T and B cells secreted negligible amounts of EM-2. Utilization of the MACS technique, together with sensitive RIAs, amplifies the accuracy and power of neuropeptide mapping within immune cell groups. Higher concentrations of EM-2 in culture medium compared to cell extracts is evidence that EM-2 may be constitutively expressed in the rat immune system. (This project was supported by The Wellcome Trust.) Prior Stressor Exposure Primes Immune Response to Lipopolysaccharide. John D. Johnson, Kevin A. O’Connor, Terrence Deak, Linda R. Watkins, and Steven F. Maier, Department of Psychology, University of Colorado, Boulder, Colorado 80309-0345. Bacterial endotoxin causes proinflammatory cytokine release, and these cytokines are potent activators of the hypothalamic–pituitary–adrenal (HPA) axis. Elevation of glucocorticoids (CORT) in turn regulate the immune response. The HPA axis often adopts after exposure to stressful conditions. We tested whether prior exposure to inescapable tail shock (IS) would alter the CORT response to a subsequent injection of LPS. Rats were exposed to IS or remained as home cage controls (HCC). 1, 4, 10, or 21 days later animals were injected i.p. with 10 µg/kg LPS or sterile saline. Blood samples were taken 0, 60, 120, and 300 min later and assayed for CORT. Animals exposed to IS 1 or 4 days prior to LPS had a more rapid CORT response than did HCC animals, but did not differ in peak CORT values. Additional animals were exposed to IS or were HCC and decapitated 60 min after an i.p. injection of LPS or sterile saline. Blood was collected for measurement of ACTH and cytokines and brains were dissected for measurement of IL-1beta. IS animals had significantly higher levels of plasma ACTH, IL-1beta, TNF-alpha, and brain IL1beta 60 min after LPS compared to HCCs. Plasma IL-6 levels did not differ between groups. These results suggest that exposure to IS primes the immune response to LPS for approximately 4 days. During this time exposure to LPS results in a more rapid increase in cytokine release and consequently a more rapid HPA response. (Supported by NIH Grants MH55283, MH45045, MH01558, and MH00314.) Psychoimmune Profiles to an Academic Stressor in African American Adolescents with or without Childhood Asthma. Duck-Hee Kang* and William C. Bailey,† *School of Nursing, †Department of Pulmonary and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294. Stress-associated psychoimmune profiles in African American Adolescents (AAA) are poorly understood, while these adolescents often experience greater morbidity and mortality rates of chronic illness. We examined AAAs’ responses to examinations for their perceived stress, mood states, plasma cortisol levels, natural killer cell (NK) activity, lymphocyte proliferation (LP), neutrophil oxidative metabolite release, and cytokine production across the school year, comparing responses between students with (n ⫽ 24) and without (n ⫽ 36) childhood asthma. Students completed questionnaires and gave a blood sample four times approximately every 2 weeks: T1 (baseline), T2 (exams), T3, and T4. Standard assay protocols and ELISA were used for immune and endocrine measurements. Results indicate that asthmatic students were similar to healthy students in psychological profiles. However, they differed in that asthmatic students showed significantly lower NK activity ( p ⫽ .01), LP ( p ⫽ .001–.02), and oxidative metabolite response (except at T1) ( p ⫽ .002–.05) and significantly higher interleukin-5 and -8 ( p ⬍ .001-.01) production. During the examination period, cortisol levels were significantly increased ( p ⫽ .02) in all students. Further, NK, LP, oxidative metabolite, interleukin-5, and -10 responses indicated significant changes over four time points ( p ⬍ .001-.01), although the pattern of change was different between the two groups for certain measures. In addition, significant and persistent correlations between interleukin8 and interleukin-6 production and also between interleukin-8 and oxidative metabolite response suggest close interactions in some effector cell functions. In summary, findings of this study indicate differential immune responses between students with and without childhood asthma and significant changes in response to academic stressor on selective endocrine and immune measures in AAAs.
Peripheral Administration of gp120 Results in Acute Physiological Alterations. Joseph W. Karaszewski and Christopher L. Coe, Department of Psychology and the Harlow Center for Biological Psychology, University of Wisconsin–Madison, Madison, Wisconsin 53715. The HIV-1 envelope protein gp120 has been implicated in causing neurological damage via both peripheral and central mechanisms. The primary purpose of this study was to examine the safety and immunological responses to peripheral administration of gp120 in juvenile rhesus monkeys, prior to assessment of its neurotoxicity in newborn monkeys. Juvenile monkeys were administered 400 ng/kg gp120MN or an equivalent volume of saline daily, for either 14 or 28 days. Peripheral blood measures of toxicity and immunogenicity were examined at two-week intervals. No changes were observed in the Chem-17 panel or in any of the behavioral measures assessed, indicating a lack of toxicity at this dose. However, there was significant antibody production to both gp120MN and CD4. Decreases in the absolute numbers of white blood cells, most notably neutrophils, were also observed. Prior reports of neutropenia during acute HIV-1 infection have been noted, possibly indicating that this may serve as a good measure of primary, acute HIV-1 infection. The production of neurotoxins by macrophages in response to gp120 exposure has been demonstrated in vitro. Our study will be testing the hypothesis that neurotoxin production will also occur in vivo. Additional studies in the neonate are warranted because the virally exposed infant may develop in the context of sustained exposure to viral protein. In addition, a more immature blood brain barrier may allow the passage of gp120, or neurotoxic products of the immune system, to penetrate to susceptible brain cells resulting in long-lasting neuroimmune alterations. (An Emotion Training Grant to J.W.K. and NIMH Grant MH41659 to C.L.C. supported this research.) Cyclophosphamide Pretreatment Makes C57Bl/6J Mice Sensitive to Restraint. Jonathan D. Karp and Jason Smith, Department of Biology, Rider University, Lawrenceville, New Jersey 08648. In a recent report (Shanks & Kusnecov, 1998), antibody responses to keyhole limpet hemocyanin (KLH) were elevated by restraint in BALB/cByJ but not in C57Bl/6J mice. We sought to determine if C57Bl/6J mice could be made responsive to restraint by pharmacological immunomodulation of the mice prior to stressor exposure. Cyclophosphamide (CY) or vehicle was administered to male BALB/ cByJ or C57Bl/6J mice 72 h before immunization with KLH (100 µg, ip). Eight hours after immunization, animals were restrained or food-and-water-deprived overnight (15 h). Serum samples were collected over the next three weeks and assayed for anti-KLH antibodies by ELISA. ANOVA revealed that restraint elevated serum anti-KLH IgM and IgG titers in vehicle-treated BALB/cByJ but not C57Bl/6J mice. Pretreatment of BALB/cByJ mice with CY (15 mg/kg) exaggerated the differences between restrained and unrestrained mice. C57Bl/6J mice treated with CY (50 mg/kg, but not 15 mg/kg) prior to immunization and restraint exhibited elevated KLH IgM (but not IgG1 or IgG2a) titers compared to unrestrained CY-treated C57Bl/6 mice. These results confirm that the effects of restraint on anti-KLH antibody production are strain dependent. In addition, we show that CY pretreatment magnified the effects of restraint on serum antibody titers in BALB/cByJ mice and made previously insensitive C57Bl/6J mice at least partially sensitive to this psychosocial manipulation. These results support the hypothesis that moderate perturbation of the immune system prior to stressor exposure may increase the sensitivity of the immune system to modulation by psychosocial factors. (Supported by 1-R15-MH58457-01.) Changes in GRK-s during Autoimmunity: Consequences for Beta-2-Adrenergic Signaling. Annemieke Kavelaars,* Maria Stella Lombardi,* Anne Vroon,* Manfred Schedlowski,† and Cobi J. Heijnen,* *Laboratory of Psychoneuroimmunology, Wilhelmina Children Hospital of the University Medical Center Utrecht, Utrecht, The Netherlands; and †Department of Medical Psychology, University of Essen, Essen, Germany. G protein coupled receptor kinases (GRK) play a key role in regulating desensitization of G protein coupled receptors such as beta-2-adrenergic, substance P, opioid, and chemokine receptors. We recently reported changes in GRK levels in lymphoid cells of patients with rheumatoid arthritis. The reduced GRK level was associated with increased reactivity of these cells to beta-adrenergic agonists. The aim of this study was to investigate whether changes in GRK levels in the immune system and in nonimmune organs occur during the course of inflammatory autoimmune diseases. In a rat model of adjuvant-induced arthritis (AA), we investigated GRK activity and expression lymphoid organs as well as in nonlymphoid
organs. At the peak of the disease, we observed a profound down regulation of GRK-2, GRK-6, and GRK3 in splenocytes and lymph nodes cells from AA rats. In contrast, no changes in GRK were observed in thymus, heart, and pituitary. During the remission phase of AA, GRK levels in spleen and mesenteric lymph nodes return to baseline levels. To answer the question whether reduced GRK levels in immune organs also occur during other inflammatory diseases, we analyzed immune cells from animals with experimental autoimmune encephalomyelitis, which is the animal model for multiple sclerosis. We propose that the reduced GRK levels in immune cells during autoimmunity may contribute to the chronicity of the disease by enhancing the response to beta-2-adrenergic agonists and to other mediators that signal through receptors regulated by GRKs, for example substance P, chemokines and opioids.
Immune Function in Healthy Children. Steven E. Keller, Jacqueline A. Bartlett, Arielle R. Goldklang, and Nicole A. Andrisano, Department of Psychiatry, University of Medicine and Dentistry of New Jersey—New Jersey Medical School, Newark, New Jersey 07103. Objective. The first step in understanding pediatric sychoneuroimmunological is understanding how the immune system looks in healthy children. We present research on the immune function of healthy inner-city children, an understudied population in relation to immune-related diseases, focusing on age, gender, and racial effects on the immune. Methods. 207 children ranging from 7.41 to 13 years were recruited from an elementary school in Newark, New Jersey as part of a longitudinal study of behavior, mood, and immunity in minority children. 152 healthy African American and Latino children (83 females)were given medical histories and complete physical examinations and a battery of immunological assessments, both functional and ennumerative. Subjects who had existing medical conditions or were taking medication with known or possible immunological effects were not included in these analyses. Results. We found differences in immune measures in relation to race, gender, and developmental stage. Boys had significantly lower measures of helper, percent helper t and percent t cells, and higher percent NK cells than girls. African American children had lower measures of WBC, percent granulocytes, and killing, and higher percent lymphocytes and log means of Con A and PWM than Latinos. Developmental stage for boys (as measured by the Tanner stage of development) was related to b cells and percent t cells. No such relationships were found in the girls. Conclusion. The differences observed among immune measures expand the existing knowledge of normative data in child immunology in relation to children of minority populations. (Source of funding: NIMH Grant R29 MH48645.)
Chronic, Uncontrollable Stress Is Associated with Elevated Mucosal but Not Plasma HIV RNA Viral Load in HIV Positive Men. Margaret E. Kemeny,*,† Tara L. Gruenewald,* Michael Poles,†,‡ Julie Eliott,‡ Marie Fuerst,‡ and Peter Anton,†,‡ *Department of Psychology, †AIDS Institute, and ‡Center for HIV and Digestive Disease, UCLA, Los Angeles, California 90095. Background. Plasma HIV RNA viral load is the strongest predictor of HIV progression. The gut is the largest immune organ and is populated by activated memory T cells, and in monkeys it is a primary site for replication and spread of HIV, suggesting that mucosal HIV RNA may have an important relationship to disease progression. This study is a preliminary investigation of the association between chronic stress and plasma and mucosal viral load. Methods. HIV⫹ men on HAART medication with detectable, stable plasma viral load are being recruited into a clinical trial for adjunctive therapy. All individuals participate in a three-week screening including weekly assessments of plasma viral load, a biopsy from a flexible sigmoidoscopy for mucosal viral load, a chronic stress interview (using the contextual threat approach), and a packet of psychosocial questionnaires. Results. Plasma viral load during screening was uncorrelated with the 6 chronic stress domains. However, higher mucosal viral load was strongly correlated with higher chronic family-related stress (ρ ⫽ .67, p ⫽ .008; n ⫽ 14). There were no associations between health behaviors and viral load. Most of the family-related stress was of a long-standing (10⫹ years)and uncontrollable nature, often precipitated by disclosure of homosexuality and/or HIV status. Conclusions. Chronic, uncontrollable family stress was significantly associated with HIV RNA viral load in the gut tissue. To the extent that mucosal viral load is a reflection of total body viral burden, these observations suggest that chronic stress may be associated with HIV replication and disease progression. (This project was supported by Procter and Gamble Pharmaceuticals, CFAR Mucosal Immunology Core (AI 28697), and NIMH RSDA MH00820.)
Resting Cellular and Physiological Effects of Freewheel Running. S. L. Kennedy, T. P. Smith, M. C. Olson, S. E. Smith, B. Greenwood, T. Leem, G. F. Elphick, and M. Fleshner, Department of Kinesiology and Applied Physiology, University of Colorado, Boulder, Colorado 80309. Voluntary freewheel running can prevent the immunologically deleterious consequences of stress. To better understand the benefits of exercise, it is important to characterize the potential impact of regular, moderate physical activity on resting or baseline cellular immunity and physiology. Male, Fisher 344 rats (10/group) were allowed access to either mobile or immobile (sedentary) freewheels. After 6 weeks of running, rats were sacrificed and blood and muscle (long and medial heads of the triceps muscle and the soleus) were collected. From blood, white blood cell differentials, red blood cell count, hemoglobin, hematocrit, platelet count, and lipid profiles (low-density lipoproteins (LDL) and high-density lipoproteins (HDL)) were measured. Muscle citrate synthase (CS) activity was measured. There were no differences in the total number of circulating white blood cells (WBC), red blood cells (RBC), and eosiniphils. Freewheel running decreased the number of circulating neutrophils, monocytes, basophils, and increased the number of lymphocytes, compared to sedentary controls. Importantly, mean corpuscular content of hemoglobin was elevated in the freewheel group. There were no significant differences in the lipid profiles. However, there was a trend for lower triglygerides and LDL and elevated HDL cholesterol in the freewheel animals. Muscle CS activity in both the long and medial heads of the triceps were not significantly different between the two groups. Soleus CS levels are currently being measured. Both changes in RBC hemoglobin and lipid proteins found with freewheel running are positive adaptations indicative of training. The impact of changes in resting WBC differentials is unknown (AI45575, MH60301.) Selective Expression of Beta-2-Adrenergic Receptor mRNA in CD4⫹ Th1 Cells, but Not Th2 Cells. Adam P. Kohm,* Nicholas Morley,* Michelle A. Swanson,† and Virginia M. Sanders,*,† *Department of Cell Biology, Neurobiology, and Anatomy, †Department of Microbiology and Immunology, Loyola University Medical Center, Stritch School of Medicine, Maywood, Illinois 60153. The beta-2 adrenergic receptor protein is selectively expressed by Th1 cell clones, but not on Th2 cell clones, the functional relevance of which has been demonstrated previously. We report here that beta-2 adrenergic receptor mRNA is also differentially expressed Th effector cell clones, and more importantly, by newly generated Th1 effector cells. Likewise, these effector cells differentially expressed mRNA for the Th1-selective interleukin-18 receptor and the Th2-selective ST2L. Both anti-CD3 stimulation of Th cell clones and antigen restimulation of newly generated Th cells failed to modulate the differential expression of the beta-2 adrenergic receptor by these cell populations. These data show that the beta-2 adrenergic receptor is stably expressed by clones and newly generated Th1 cells, but not on Th2 cells. Thus, given the ability of this receptor to modulate immune cell function, not only may this receptor serve to distinguish Th1 and Th2 cells, but it may also serve as a target for future therapeutic strategies to specifically modulate Th1 cell function. [This work was supported in part by research funds from the National Institutes of Health (AI37326) and the American Cancer Society (IM-798).] Antigen-Induced Activation of CD4⫹ T Cells and B Cells Increases Sympathetic Nerve Activity in the Spleen and Bone Marrow. Adam P. Kohm,* Yeuming Tang,† Stephen B. Jones,† and Virginia M. Sanders,* *Department of Cell Biology, Neurobiology, and Anatomy, †Department of Physiology, Loyola University Medical Center, Stritch School of Medicine, Maywood, Illinois 60153. We have investigated the mechanisms by which activation of the adaptive immune system by a soluble protein antigen increases efferent sympathetic nerve activity in lymphoid organs. Severe combined immunodeficient (scid) mice were reconstituted with TNP-specific B cells enriched from spleens of unimmunized mice and a clone of KLH-specific Th2 cells. Two weeks after reconstitution, scid mice were immunized (i.p.) with either TNP-KLH in adjuvant, an irrelevant antigen (FLU-OVA), or adjuvant only and sympathetic nerve activity was measured for the next 25 h in the bone marrow, spleen, and heart as determined by norepinephrine (NE) turnover. Sympathetic nerve activity was not altered 1–8 h following TNP-KLH administration. However, sympathetic nerve activity was significantly increased in the bone marrow, spleen, and heart 18–25 h following immunization with TNP-KLH. In addition, sympathetic nerve activity was not altered at any time following immunization with either FLU-OVA or adju-
vant only. Interestingly, the antigen-induced increase in sympathetic nerve activity was prevented by pretreatment of animals with the ganglionic-blocker chlorosondamine, suggesting that the antigen-induced increase in sympathetic nerve activity is related to an increase in signals originating from the central nervous system. Thus, we conclude that a soluble protein antigen challenge enhances sympathetic nerve activity and NE release in lymphoid organs. Such evidence for the antigen-induced NE release in these tissues during the early stages of T and B cell responses support the physiologic relevance of in vitro findings demonstrating effects of NE on immune cell function. [Support: NIH AI37326 (V.M.S.) and MH53562 (S.B.J.).] Exercise and Immune Response to Influenza Vaccination in the Elderly. Marian L. Kohut,* Michael S. Nickolaus,* Megan M. Cooper,* Alison E. Magel,* and Joan E. Cunnick,† *Department of Health Human Performance, †Department of Microbiology, 235 Forker Bldg., Iowa State University, Ames, Iowa 50011. Moderate exercise may modulate immune response in the elderly. We investigated whether exercise was associated with an alteration of the immune response to influenza immunization. Older (age ⬎ 60) men (n ⫽ 22, mean age ⫽ 71), and women (n ⫽ 35, mean age ⫽ 71) were classified as sedentary, active (participate in light to moderate exercise, ⬎1 times per week), or very active (participate in vigorous exercise, ⬎2 times per week). All subjects completed a dietary questionnaire (Block Dietary Data Systems 98), the perceived stress scale (PSS), the life orientation test (LOT), and answered several questions regarding social activities. Fourteen days after receiving the 1999–2000 influenza virus vaccine, a blood sample was taken. Peripheral blood mononuclear cells (PBMC) were used to assess mitogen-induced proliferation (concanavalin A, phytohemagglutinin), influenza-specific lymphocyte proliferation, influenza-specific IL-2, IFN-gamma, IL-4, and IL-10 production. PBMCs were cultured with influenza to induce antigen-specific proliferation and cytokine production. Cytokines were measured using ELISA and proliferation was assessed with MTT. After adjusting for initial baseline differences in diet or scores on the psychosocial measures, our results showed that exercise was associated with enhanced antigen-specific lymphocyte proliferation. The proliferation was greater in both active and very active subjects compared to sedentary ( p ⬍ .05), but there was no difference between active and very active subjects. No effect of activity level was found with respect to mitogen-induced proliferation, IL-2, IFN-gamma, IL-4, or IL-10. These findings suggest that moderate or vigorous exercise may be associated with an enhancement of antigen-specific lymphocyte proliferation. Adreneric Stress Yields Rapid Translocation of Memory T-Lymphocytes from Peripheral Lymphoid Tissues to the Mucosal Surface of the Lung. Richard Kradin,*,† Carol Leary,* and Hideo Sakamoto,* *Immunopathology Unit, Massachusetts General Hospital, Boston, MA 02114; and †Mind–Body Institute, Beth Israel–Deaconess Medical Center, Boston, Massachusetts 02115. Stress yields a series of rapid adaptations to perceived environmental threat. Adrenergic stimulation leads to a release of T-lymphocytes from spleen into the circulating blood. As the lung is a portal to the environment, we hypothesized that adrenergic stress would mobilize immune lymphocytes to airway surface as part of a systemic response to danger. C57BL/6 mice were injected with epinephrine (0.1 mg/kg) or saline, at time 0. Mice were sacrificed at 10 min; blood, spleen, and bronchoalveolar lymphocytes were harvested and their immune phenotype was analyzed. Epinephrine-treated mice showed decreased numbers of splenic CD4⫹ CD45RBdim and NK1.1⫹ splenic lymphocytes; correspondingly, increased numbers of these lymphocyte subsets were observed in blood and lung (p ⬍ .01 compared to control). Compartmental lymphocyte shifts were abrogated by pre-treatment with propanolol. Normal mice were injected at the tail base with the fluorescent dye PKH-2. This yielded PKH26⫹ lymphocytes in the regional draining lymph nodes, at 24 h. At 72 h, mice were injected with epinephrine or saline i.p.; lungs were excised at 10 min, snap frozen, and examined by epifluorescence microscopy. PKH26⫹ lymphocytes were detected in the lung in epinephrinetreated mice but not in controls. We conclude that epinephrine induces rapid mobilization of memory CD4⫹ lymphocytes and NK cells from spleen and peripheral lymph node to the lung. Adrenergic mediation of lymphocyte translocation is judged to represent an immune aspect of the fight or flight response. (Supported by grants from NIH AI39054 and by the Mind–Body Institute.)
Effects of Two Inhibitory Cytokines, Interleukin-13 (IL-13) and Transforming Growth Factor-Beta 1 (TGF-Beta 1), on Spontaneous Sleep in Rabbits. Takeshi Kubota, Jidong Fang, and James M. Krueger, Department of VCAPP, Washington State University, Pullman, Washington 99164-6520. Proinflammatory cytokines, including IL-1 beta and TNF-alpha are involved in physiological sleep regulation. IL-13 and TGF-beta 1 are anti-inflammatory cytokines which inhibit proinflammatory cytokines. Therefore, we hypothesized that IL-13 and TGF-beta 1 could attenuate sleep. Three doses of IL-13 (8, 40, and 200 ng) and TGF-beta 1 (40, 100, and 200 ng) in 25 µl of pyrogen free saline were injected intracerebroventricularly during the light period. One dose of IL-13 (200 ng) or TGFbeta 1 (200 ng) was injected at dark onset. The two higher doses of IL-13 and the highest dose of TGF-beta 1 significantly inhibited nonrapid eye movement sleep (NREMS) when they were given in the light period. (IL-13 40 ng: ⫺38.2 min vs control, p ⬍ .01; IL-13 200 ng: ⫺47.2 min vs control, p ⬍ .05; TGF-beta 1 200 ng: ⫺43.4 min vs control, p ⬍ .01). IL-13 also inhibited NREMS after dark onset administration; however, this inhibitory effect was less potent than that after light period administration (⫺24.7 min vs control, p ⬍ .01). TGF-beta 1 administered at dark onset had no effect on sleep. Both IL-13 and TGF-beta 1 did not affect total time spent on REMS, EEG slow wave activity, or brain temperature. These results provide additional evidence for the hypothesis that a brain cytokine network is involved in the regulation of physiological sleep. (Supported by NIH NS 25378 and NS 31453.)
Melatonin Stimulates Human Peripheral Blood Mononuclear Cells. Eva C. Kuehlwein and Michael R. Irwin, Department of Psychiatry, University of California–San Diego, 3350 La Jolla Village Drive, San Diego, California 92122. Melatonin is hypothesized to play a role in neuroimmunomodulation. Specific binding sites for melatonin are found on lymphoid cells. In addition, pharmacologic doses of melatonin enhance humoral and cellular immunity and increase cytokine production by murine splenocytes. However, less is know about the action of melatonin on human immunocytes; two studies suggest that in vitro doses of melatonin enhances IL-2, IFN-gamma, IL-1, and IL-6 production by human lymphocytes and monocytes. This study investigates the effect of physiological doses of melatonin (10 ⫺12 through 10 ⫺6 M) on human peripheral blood mononuclear cell (PBMC) proliferation and production of IL-6, IFN-gamma and IL10 with and without stimulation with Staphylococcal enterotoxin B (SEB; 1 µg/ml). In three replicate experiments, melatonin stimulated PBMC proliferation in the absence of SEB (F ⫽ 4.01, p ⬍ .01) with all concentrations of melatonin inducing higher levels of proliferation as compared to cultures without melatonin ( p ⬍ .01). When PBMCs were highly activated with SEB, melatonin did not increase the proliferation above this ceiling. The effects of melatonin on PBMC production of IL-6, IFN-gamma, and IL-10 as measured by intracellular cytokine accumulation and stimulated supernatants will be discussed. This data supports the notion that physiologic doses of melatonin have effects on human PBMCs similar to the immuno-enhancing action reported using murine splenocytes.
The Immunoadjuvant Effects of Human Interleukin-1 Beta Can Be Augmented by Adrenalectomy in BALB/C Mice. Alba Rossi-George, Department of Psychology, Rutgers, State University of New Jersey, 152 Frelinghuysen Road, Piscataway, New Jersey 08855. Peripheral signaling of the central nervous system by the immune system represents an effort to induce behavioral and neuroendocrine adjustments that support organismic adaptation. In the present study, we investigated whether the ability of interleukin-1 (IL-1) to stimulate the hypothalamic-pituitary-adrenal (HPA) axis interfered with its ability to serve as an immunological adjuvant. In initial experiments male BALB/c mice immunized with 100 µg ovalbumin (OVA) simultaneously with human IL-1 beta treatment showed significantly enhanced IgM and IgG responses at 6 and 12 days after immunization. The dose of IL-1 beta (60 units) was also confirmed to significantly activate the HPA axis. In the next experiments, mice were adrenalectomized (ADX) or sham operated and subsequently treated with IL-1 (or saline) followed by 100 µg OVA. No mortality was observed in ADX animals treated with IL-1, since bottled water was treated with 0.1 µg/ml corticosterone. The results revealed that relative to saline-treated ADX or SHAM mice, IL-1 treatment in both surgery groups significantly enhanced the antibody response to OVA. However, it was further observed that ADX animals treated with IL-1 showed a much greater
enhancement of the antibody response than IL-1 treated SHAM animals. These relative standings extended to ex vivo antibody production by spleen cells restimulated with OVA, as well as antigen-specific proliferation (but not mitogen-stimulated proliferation). These studies demonstrate the importance of adequate immune-mediated neuroendocrine activation to limit exaggerated antigen-specific immune responses. Furthermore, they suggest ways in which immunotherapeutic use of cytokines may capitalize on their ability to stimulate the CNS.
Neurokinin-1 Receptor Antagonists Inhibit Human Immunodeficiency Virus 1 Infection of Human Mononuclear Phagocytes in Vitro. Jian-Ping Lai, Wen-Zhe Ho, Guan-Xia Zhan, Joann R. Cutilli, and Steven D. Douglas, Section of Immunology, Division of Immunologic and Infectious Diseases, Joseph Stokes Jr. Research Institute at the Children’s Hospital of Philadelphia, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104. Objective. To study the role of substance P and its receptor in HIV infection of human mononuclear phagocytes. Methods. Human monocyte derived macrophages (MDM) were treated with nonpeptide SP receptor antagonists, CP96,345 or RP67,580 (10 ⫺5 to 10 ⫺11 M) for 2 h and then infected with HIV-1 Bal strain at 37°C overnight. After extensively washing, the cells were treated with fresh media with or without the SP antagonists every 4 days and the total cellular RNA was extracted from the cells at the termination of the experiments. The supernatants were analyzed by HIV reverse transcriptase (RT) assay, and the RNA was amplified using RT-PCR for HIV-1 gag gene. Results. SP upregulated HIV-1 replication in MDM in a concentration dependent relationship, while the SP antagonists (CP96,345 or RP67,580), in noncytotoxic doses (as determined by trypan blue staining), significantly inhibited HIV-1 infection in a concentration-dependent relationship as determined by HIV RT assay. Inhibitor Conc. 10-5M 10-6M 10-7M 10-8M CP96,345 83% 54% 43% 1% RP67,580 70% 70% 54%. HIV-1(Bal) infected MDM cultures (without the SP antagonist treatment) demonstrated giant syncytium formation, whereas the SP antagonist treated MDM failed to develop the typical giant syncytia induced by HIV-1 infection. Conclusions. The SP antagonists inhibit HIV-1 infection in MDM in vitro, indicating that SP-NK-1R pathway is involved in HIV-1 infection of human MDM. Thus, SP antagonists may have the potential to limit HIV-1 infection in MDM in vivo, and may offer approaches to the design of new anti-HIV therapeutics. (Supported by NIH-MH 49981 to S.D.D. and DA 12815 to W.Z.H.)
Human Microglia Express Substance P and Its Receptor. Jian-Ping Lai, Guan-Xia Zhan, Steven D. Douglas, and Wen-Zhe Ho, Section of Immunology, Division of Immunologic and Infectious Diseases, Joseph Stokes Jr. Research Institute at the Children’s Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104. Objective. To determine whether human microglia express substance P (SP) mRNA isoforms and SP protein and mRNA for neurokinin-1 receptor (NK-1R). Methods. Human fetal brain-derived microglia were isolated and cultured as previously reported in our laboratory (N. F. Hassan et al., Neuroscience 41, 149–158, 1991). The purity and SP immune reactivity of the isolated microglia were evaluated by flow cytometry and immunohistochemical staining with anti-CD68 and anti-SP antibodies. Microglia culture supernatants were collected for SP-EIA and cellular RNA was extracted for RT-PCR amplification of SP and NK-1R. The specificities of the RT-PCR were confirmed by Southern blotting and direct DNA sequence analysis of the RT-PCR products from microglia. Results. The microglia composed of 99% of the cells in the cultures as evaluated by flow cytometry and immunohistochemical staining with anti-CD68. All four mRNA isoforms of SP gene transcripts (alpha, beta, gamma, and delta) as well as, SP protein were detected in the microglia. Supernatants of microglia cultures contained SP (640–850 pg/10 6 cells) as determined by an enzyme immunoassay. By immunohistochemical staining using anti-SP, SP reactivity was observed on the cell membrane. In addition, RT-PCR and Southern blotting also demonstrated the presence of mRNA for NK-1R. Conclusion. Human fetal brain-derived microglial cell cultures express SP and NK-1R, and SP immune reactivity was detected on the cell membrane, indicating that functional SP receptors are present on these cells. Thus, SP and its receptor, NK-1R are biologically involved in regulating the functions of microglia, and most likely have a major role in host defense of central nervous system. (Supported by NIH-MH 49981 to S.D.D., and NIH DA 12815 to W.Z.H.)
Immune Changes in Adolescent Free-Ranging Macaques Are Influenced by Maternal Rank. Mark L. Laudenslager,* Kathlyn L. Rasmussen,† Carol M. Berman,‡ Sandra T. de Blois,* and Stephen J. Suomi,† *Department of Psychiatry, University of Colorado Health Sciences Center, Denver, CO 80220; †Laboratory of Comparative Ethology, National Institute of Child Health and Human Development, Poolesville, Maryland 20837; ‡Department of Anthropology, State University of New York, Buffalo, New York 14214. Dominance status in macaques is determined along matrilines. Offspring take on the dominance status of the matriline in which they are born. Dominance status of adult macaques affects immune parameters in laboratory settings. Under free-ranging conditions does dominance status of the mother affect her offspring prior to adulthood? Free-ranging rhesus monkeys (15 males and 11 females) living on the island of Cayo Santiago, Puerto Rico, were followed from birth. Monkeys were divided into two groups based on matrilineal dominance status (15 high and 11 low ranking). Beginning at 2 years of age, blood samples were collected once a year for three years. Animals were captured in field cages and blood was collected 30 min after capture. Monkeys were held for 24 h and released the following day after blood was collected a second time. Natural cytotoxicity toward K562 (lysed by CD8 and CD16) and Raji (lysed by CD16) targets, phenotypes(CD4, CD8, and CD16), and hormones (Cortisol, DHEA-SO4, growth hormone, and prolactin) were measured. Tumor lysis decreased from day 1 to day 2. Offspring of high ranking mothers showed less change in lysis of both tumors ( p ⬍ .05). The change in lysis was related to a change in CD16 but not CD8⫹ phenotypes ( p ⬍ .05). Offspring of low ranking mothers had higher percentages of CD16⫹ cells and reduced lysis per CD16⫹ cell. Cortisol was lower in male offspring of high ranking mothers compared to male offspring of low ranking mothers but did not differ for females based on rank ( p ⫽ .06). High maternal rank confers selective advantages on offspring. (Supported by NIMH Grant MH37373 and intramural funding from the National Institutes of Child Health and Human Development.)
Acute Stress Promotes the Resolution of a Live E. Coli Challenge. Ted Leem,* Jay Campisi,* Terrence Deak,† Jocelyn Seelye,* and Monika Fleshner,* *Department of Kinesiology and Applied Physiology, †Department of Psychology, University of Colorado at Boulder, Boulder, Colorado 80309. Stress modulates the immune response. While acute stress tends to compromise specific immunity, it can also enhance innate immunity. The latter produces a marked decrease in the size and time required to resolve the inflammatory response to a subcutaneous E. coli challenge. However, these findings are limited because a reduction in inflammation size and resolution time may indicate a failure to generate an inflammatory response to nonreplicating E. coli, making it difficult to determine whether this is reflective of stress-induced immunosuppression or immunopotentiation. Replicating E. coli, therefore, was used in these experiments. Experiment 1—E. coli dose response. Adult, male Fisher (F 344) rats (4–6/group) were injected subcutaneously with either 2.5 ⫻ 10 7 CFU or 2.5 ⫻ 10 9 CFU of live E. coli. Inflammation diameter (caliper) and grade (scale: 1–4) were measured daily. Experiment 2—Effect of shock on inflammation. Rats (6/group) were either exposed to an inescapable tail shock (IS) stress or designated as controls. Immediately after IS, all animals were injected with 2.5 ⫻ 10 9 CFU of live E. coli. Inflammation was measured as in experiment 1. 2.5 ⫻ 10 9 CFU of live E. coli produced an optimal inflammatory response (grade ⫽ 2, maximum diameter ⫽ 34 mm). The effect of IS is similar to that reported with nonreplicating E. coli. The initial size of the inflammation was reduced 25% compared to nonstressed controls. Stressed rats resolved their inflammatory responses 5–7 days faster than nonstressed controls. Thus, IS promotes resolution of a live E. coli challenge. The potential role of nitric oxide is currently under investigation. (AI45576, MH60301.)
Relations between Regional Cerebral Blood Flow and Immune Activity in Chronic Pain. Mats Lekander,* Mats Fredrikson,†,‡ and Gustav Wik,* *Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden; †Department of Psychology, Uppsala University, Uppsala, Sweden; and ‡Uppsala University PET-Centre, Uppsala, Sweden. However unknown, the etiology of fibromyalgia is hypothesized as being a matter of neuroimmunoendocrine dysregulation. To study brain–immune relationships, we correlated regional cerebral blood flow measured with positron emission tomography (PET) to immune function in 5 patients with fibromyalgia.
In the right hemisphere, NK activity correlated negatively to activity in the secondary somatosensory cortex, the secondary motor cortex, and the thalamus. Moreover, negative correlations were observed between NK activity and blood flow bilaterally in the posterior cingulate cortex. This pattern is partly congruent with our previous data in healthy volunteers. The posterior cingulate cortex is involved in aspects of attention, motivation, and emotion. After hypnotic analgesia, we have previously observed lowered activity in the posterior cingulate cortex in fibromyalgia patients. Since cronic stress suppresses NK activity, we speculate that the later processing stages of emotional pain perception, acting as powerful stressors, are related to lower NK activity. The relation between blood flow in the secondary somatosensory cortex and immune activity observed in both fibromyalgia patients and healthy controls is intriguing. In concert with inner somatic organs without a specific somatotopic representation in the primary somatosensory cortex, the secondary somatosensory cortex is proposed as a putative representation area of immuno-cortical signaling. Based on a small population, future research need to address generalizability to confirm the stability of the observations. In sum, strong correlations were observed between NK activity and neural activity in sensorimotor areas probably related to pain perception, emotion, and attention. (Supported by the Swedish Council for Research in Humanities and Social Sciences.)
The Effects of Stress, Social Support, Coping, and Cortisol on Progression to AIDS. Jane Leserman,* Robert N. Golden,* John M. Petitto,† Bradley N.Gaynes,* Hongbin Gu,* James D. Folds,‡ and Dwight L. Evans,§ *Department of Psychiatry, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7160; †Department of Psychiatry, University of Florida College of Medicine, Gainesville, Florida 32610; ‡Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7160; and §Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Previously we reported that stress and social support were related to HIV disease progression in men followed 5.5 years (1999, Psychosomatic Med.). The current study examines the effects of coping and serum cortisol, in addition to stress and social support, on progression to AIDS for up to 7.5 years. We collected data on 82 HIV-infected gay men in North Carolina, part of the Coping in Health and Illness Project. All men were without symptoms or AIDS at entry and were studied every six months. Disease progression was defined using criteria for AIDS (CD4⫹ lymphocytes ⬍200/ml and/or an AIDS-indicator condition). Stressful life events was based on interview ratings (range 0–28), coping was measured using the COPE (subscales range 1–4), and support was based on the Sarason Brief Social Support Questionnaire (range 1–6). We used a Cox Regression model to predict time to AIDS, with cumulative timedependent covariates (6-month lag), adjusting for race ( p ⫽ .02), baseline CD4⫹ count ( p ⫽ .002), baseline viral load ( p ⫽ .01), and antiretroviral medication number ( p ⫽ .16). Faster progression to AIDS was related to more cumulative stressful life events ( p ⫽ .004), denial coping ( p ⫽ .02), and cortisol ( p ⫽ .01), and less cumulative social support ( p ⫽ .05). For each severe stress (4 points), one point increase in denial, 1.5 point decrease in social support, or 5 mg/dl increase in serum cortisol, the hazard of AIDS was about doubled. To conclude, there may be potential health benefits of stress management interventions, and medications to treat hypercortisolemia in some HIV infected patients. (This research was supported by NIMH and NIH Grants MH-44618, MH-33127, RR-00046, and P30HD37260.)
Substance P Enhances HIV-1 Replication in Human Fetal Microglia. Yuan Li, Steven D. Douglas, and Wen-Zhe Ho, Division of Immunologic and Infectious Diseases, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104. Objective. Substance P (SP) is a potent modulator of neuroimmunoregulator. We investigate the effect of SP on HIV-1 infection of human fetal microglia. Methods. Human fetal brain-isolated microglia were treated with SP at different concentration (10-10 to 10-6 M) for 1 h and then infected with supernatants containing recombinant luciferase-encoding HIV virions pseudotyped with M-tropic ADA Env in the presence of polybrene at 37°C. The cells were lysed for luciferase activity analysis 72 h after infection. HIV chronically infected promonocytic cells (U1) were treated with or without SP in the presence of tumor necrosis factor (TNF) alpha plus phorbol myristate acetate (PMA). Supernatants were collected for HIV RT assay 48 h posttreatment. Results. SP significantly (50–60%) increased HIV-1 replication in human fetal microglia cells infected in vitro as determined by luciferase assay, and its effect could
be blocked by pretreatment of the microglia cells with SP receptor antagonists (CP-96,345, RP-67,580, or spantide), indicating specific SP receptor-mediated regulation. In addition, SP significantly (60–75%) enhanced TNF alpha plus PMA-induced HIV replication in U1. Furthermore, SP stimulated HIV LTRdirected CAT activity in U38 cells (a promonocytic cell line that contains stable integrated, silent copies of HIV LTR linked to CAT gene). Conclusion. SP may play a potentially important role as a positive regulation of HIV infection and replication in infected microglia, chronical infected monocytes/macrophages, and may have significant in vivo implication in the immunopathogenisis of the chronical and neurologic complications of HIV-1 infection and AIDS. (Supported by NIH-MH 49981 to S.D.D. and DA 12815 to W.Z.H.)
Loss of Noradrenergic Nerves in Secondary Lymphoid Tissue from Rats with Adjuvant Arthritis Parallels Loss of Nerve Growth Factor Containing Target Lymphoid Cells. Dianne Lorton, C. Lubahn, and D. L. Bellinger, Hoover Arthritis Research Center, Sun Health Research Institute, Sun City, Arizona 85351; and Loma Linda Center for Neuroimmunology, Loma Linda University School of Medicine, Loma Linda, California 92350. Previously, we reported a decrease in norepinephrine concentration, but not total norepinephrine content, in spleens and lymph nodes from Lewis rats with adjuvant-induced arthritis (AA), suggesting that altered norepinephrine concentration results from changes in organ weight as the disease progresses. The purpose of this study was to further examine noradrenergic sympathetic innervation of spleens from Lewis rats with AA using immunocytochemistry for tyrosine hydroxylase (TH), specific immune cell markers, and nerve growth factor (NGF). Thirty days after challenge with Freund’s complete adjuvant (CFA), immunocytochemistry revealed a progressive loss of TH⫹ nerves in all white pulp compartments with increasing distance from the hilus (the site of nerve entry into the spleen) with disease progression. Conversely, the density of TH⫹ nerves was more robust at the hilus of spleens from rats with AA compared with vehicle-treated rats. With double-label immunocytochemistry, the loss of TH⫹ nerves in distally located white pulps occurred concomitantly with a reduction in the density of IgM⫹ B lymphocytes in the follicles and marginal zones, and ED3⫹ macrophages in the marginal zone of white pulps distal from the hilus. Immunocytochemistry for NGF also revealed robust immunoreactivity for NGF in macrophages-like cells in the marginal zones of spleens from vehicle-treated animals that was strikingly absent in spleens from rats with AA. Collectively, these findings suggest plasticity of splenic nerves with AA progression that may result from changes in the make-up of lymphoid compartments, and/or a decline in neurotrophic support by specific cells of the immune system.
Effects of Glucocorticoid Blockade on the Exercise-Induced Suppression of Macrophage Antigen Presentation. Thomas W. Lowder, Marc D. Zack, Michael A. Ceddia, and Jeffrey A. Woods, Department of Kinesiology, University of Illinois, Urbana, Illinois 61801. We have previously shown that exhaustive exercise significantly decreased peritoneal macrophage antigen presentation (MAP) in mice. The mechanism behind this phenomenon is unknown. Stress hormones augmented by exercise (i.e., cortisol) have been shown to affect immune cells. We postulated that cortisol was a mediating factor in the exercise-induced suppression of MAP. In this study, we attempted to block these effects during exhaustive exercise. In two preliminary experiments, male Balb/ c mice (n ⫽ 8/experiment) were gavaged with either a mifepristone (RU486; 10 mg/kg) or a vehicle while under methoxyflurane-induced inhalation anesthesia. On 4 consecutive days (2 h following gavage) mice were either exercised for 3 h on a treadmill (18–40 m/min) or remained sedentary. A 1-ml thioglycolate injection was given to all mice immediately following the first exercise bout to elicit peritoneal macrophages. Mice were sacrificed immediately following the last bout of exercise on Day 4. Peritoneal macrophages were harvested then cultured without or with suboptimal (2.5 mg/ml) concentrations of chicken ovalbumin (C-OVA) for 18 h. These cells were then washed and a T cell hybridoma specific for C-OVA was added. An ELISA was performed on cell culture supernatant to determine the presence of T cell IL-2 production, an indication of MAP. Like our previous experiments, under the vehicle condition exercise resulted in significantly suppressed (⬃25%) MAP. Moreover, RU486 was shown to be effective in ameliorating the exercise-induced suppression of MAP. These preliminary data suggest that stress-induced hormones (i.e., cortisol) may be responsible for exercise-induced suppression in MAP. (Supported by AG-13928 to J. A. Woods.)
Neuroendocrine Response to Stress Reactivity in Interstitial Cystitis. Susan K. Lutgendorf,* Karl Kreder,† Nan Rothrock,* Timothy Ratliff,† Sophie Ligier,‡ and Esther M. Sternberg,‡ *Department of Psychology, †Department of Urology, University of Iowa, Iowa City, Iowa 52242; and ‡National Institute of Mental Health, NIH, Bethesda, Maryland 20892. Interstitial cystitis (IC) is a chronic inflammatory bladder condition characterized by urinary urgency, frequency, and pain. As abnormal hypothalamic–pituitary–adrenal (HPA) function has been reported in diseases having high comorbidity with IC, we hypothesized that a dysregulated HPA response to stress might contribute to IC pathophysiology, and that HPA response might differ in IC patients with and without comorbid disease. All patients had moderate or severe disease. Eight patients with IC (IC), 5 IC patients with comorbid rheumatoid arthritis or fibromyalgia (IC-FM), and 13 age-matched controls participated in an evening reactivity session including a 60-min post-IV baseline, 25 min of mental stressors, and a rest period until 100 min poststressor onset. Cortisol and ACTH were measured at repeated intervals. Chronic symptoms were assessed with an IC symptom survey. ACTH/cortisol ratios were calculated between concurrent values at baseline, and with 15-min time-lagged cortisol values at other time-points. At baseline no difference in ACTH/cortisol ratios was observed between groups (p ⫽ .87). During recovery (⫹70 to ⫹100 min) IC patients and controls showed no change in ACTH/ cortisol from baseline. Among IC-FM patients, cortisol levels dropped during recovery, resulting in ACTH⫹70/cortisol⫹85 and ACTH⫹85/cortisol⫹100 ratios almost double those of IC patients or controls ( p ⬍ .05). Among both groups of patients, lower cortisol at baseline, recovery, and greater increases in ACTH/cortisol from baseline to recovery were related to more severe symptomatology ( p ⬍ .05). These findings suggest that subgroups of IC patients may exhibit different HPA function and that cortisol levels may be related to symptoms in this condition. Opioid-Induced Alterations of Inducible Nitric Oxide Synthase. Donald T. Lysle, Tam How, and Kelly A. Carrigan, Biological Psychology Program, Department of Psychology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3270. The use of opioids is associated with a high incidence of infectious disease. The immunologic mechanisms responsible for opioid-induced alterations in resistance to infection have not been identified. Recent data has revealed that nitric oxide plays a critical role in the immune response. The present study evaluated the effect of opioid administration on nitric oxide production. The study used rats to investigate the effect of two opioid agonists, diacetylmorphine (heroin), and morphine-6-glucuronide, on the in vivo expression of inducible nitric oxide synthase (iNOS), the enzyme responsible for nitric oxide production. Surprisingly, little is known about the immunomodulatory effects of diacetylmorphine, an opioid agonist known for its high abuse potential. Furthermore, there are few investigations of the immunomodulatory effects of the major metabolites of diacetylmorphine and morphine, such as morphine-6-glucuronide. In the current study, rats received an injection of lipopolysaccharide (LPS) to induce iNOS expression followed immediately by opioid administration. The expression of iNOS was determined in spleen, lung, and liver tissue 8 h following injection of LPS using RT-PCR. The results show that both diacetylmorphine (0,.01,.1, 1.0 mg/kg) and morphine-6-glucuronide (0, 1.0, 3.163, 10.0 mg/kg) induce a pronounced dose-dependent reduction in LPS-induced expression of iNOS in spleen, lung, and liver tissue. Both agonists also induce a reduction in plasma levels of nitrate/nitrite, the stable end products of nitric oxide metabolism. These studies suggest that the detrimental health consequences of opioids may relate to a widespread decrease in the expression of iNOS in response to infectious challenge. (Supported by DA10167, DA07481, and DA00334.) One-Trial Conditioning of the Primary Antibody Response to Hen Egg Lysozyme in Rats. Kelley S. Madden,*,† Gary W. Boehm,*,† Sandra C. Lee,*,† Lee J. Grota,*,† Nicholas Cohen,*,†,‡ and Robert Ader,*,† *Center for Psychoneuroimmunology Research, and †Department of Psychiatry, and ‡Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. The immune response to protein antigens can be modulated by behavioral conditioning. This experiment attempted to replicate studies of Alvarez-Borda et al. (1995) on the conditioned enhancement of antibody production following a single pairing of hen egg lysozyme (HEL), the unconditioned stimulus (UCS), with a conditioned stimulus (CS), saccharin. All manipulations were conducted during the dark
phase of a reversed light/dark cycle. For 4 days before conditioning or testing, male Wistar rats were adapted to 2 daily 10-min drinking periods. During the adaptation period before conditioning, a CS preexposure (CSp) group received saccharin during the morning watering. Animals were conditioned by exposure to saccharin followed immediately by 0.5 mg HEL (ip). When anti-HEL titers approached baseline, conditioned animals were reexposed to saccharin (CS and CSp groups) or water (CSo group). A noncontingent (NC) group received water and HEL on the conditioning trial and was exposed to saccharin 24 h later and on the test trial. A UCS group was immunized with HEL at conditioning and at the test trial. Pairing HEL with saccharin did not induce a taste aversion to saccharin. Nevertheless, anti-HEL IgG was increased in the CS group relative to the CSo, CSp, and NC groups 8 and 11 (but not 16) days after the test trial. There were no group differences in IgM levels. These results confirm the conditioned enhancement of antibody production following a single trial in which antigen is paired with a novel conditioned stimulus. (This work was supported by a grant from the Fetzer Institute.) Nonprogression in HIV Disease Is Related to the CD38–CD8⫹ T Cell Subset. Kevin J. Maher,* Mary Ann Fletcher,*,† George F. Solomon, §Elizabeth G. Balbin,† and Gail Ironson,†,‡ *Department of Medicine, †Department of Psychology, ‡Department of Psychiatry, University of Miami School of Medicine, Miami, Florida 33136; and §Department of Psychiatry, University of California, Los Angeles, California 90095. Objectives. The overall aim of this longitudinal study is to define the psychological and immunological factors associated with long-term survival in HIV-infected persons. Subjects. The Non-Progressor Group (NPG) consisted of 19 untreated persons who had been seropositive for ⬎8 years with CD4 T cells ⬎500 and no AIDS-related symptoms. The Comparison Group (CG) consisted of 180 seropositive individuals with CD4 cell counts ⬎150 and no category C related symptoms. A subsample of CG matched to the NPG for current CD4 cells (⬎500 at time of study) was also examined (MCG). Methods. On each subject’s blood sample, HIV-1 viral load was measured by RT/PCR and lymphocyte phenotype was determined by three-color flow cytometry. Independent T tests were used to compare immune values for the NPG, CG and MCG. Results. As anticipated, NPG were found to have significantly lower ( p ⫽ .0001) HIV viral load (mean copies/ml ⫾ SD: 8,627 ⫾ 16,807) than the CG (39,870 ⫾ 113,780). NPG were also found to have significantly elevated levels of a subset of CD8 cells, that was CD38⫺ and HLA/DR⫹ (mean cells/µl ⫾ SD: 174 ⫾ 161) when compared to the CG (69 ⫾ 106, p ⫽ .001) and MCG (59 ⫾ 77, p ⫽ .001). Interestingly, similar significant differences were also found for the CD8⫹CD38⫺ HLA/DR⫺ subset. Elevation in the CD8⫹CD38-HLA/DR⫹ subset was correlated with elevated CD4 count ( p ⫽ .01) and decreased viral load ( p ⫽ .05). Conclusions. Elevation of this CD8⫹CD38⫺ subset may confer an immunologic advantage in suppressing viral replication in some HIV infected individuals. (Supported by NIH Grant RO1 MH53791, G. Ironson, Principal Investigator, and a grant from Action for AIDS.) Aberrant Nuclear Expression of AP-1 and NFkB in Lymphocytes of Women Stressed by the Experience of Breast Biopsy. Herbert L. Mathews,* Moolky Nagabhushan,*,† and Linda Witek-Janusek,† *Department of Microbiology and Immunology, Stritch School of Medicine and †Department of Maternal Child Health, Niehoff School of Nursing, Loyola University of Chicago Medical Center, Maywood, Illinois 60153. We have investigated the expression of AP-1 and NFkB in peripheral blood lymphocytes of women scheduled for breast biopsy. Samples were collected when women were informed of the need for biopsy (prebiopsy—T1, 5–7 days prior to the actual biopsy) and 7–10 days after they learned the result of their biopsy (postbiopsy—T2). At the time of blood collection, psychological stress was evaluated using Speilberger’s State Trait Anxiety Inventory (STAI) and the Profile of Mood States (POMS). Women scheduled to undergo breast biopsy reported significant increases in anxiety (STAI) and mood disturbance (POMS). Gel shift mobility assays showed that mitogen stimulated peripheral blood lymphocytes of these women were less capable of the nuclear expression of AP-1 or NFkB at T1. Similar assessments, 7–10 days after the women learned of the results of their breast biopsy, showed these same women to have a marked reduction in anxiety and mood disturbance and an increased nuclear translocation of AP-1 and NFkB. These results show a significant decrease in nuclear AP-1 and NFkB expression during the period of emotional distress prior to biopsy with a return of nuclear transcrip-
tion activity to normal levels when distress was relieved. Several studies have correlated increased psychological stress with decreased immune function. The results of this study suggest that psychological stress may mediate immunosuppression by altering the expression of the transcription factors, AP-1 and NFkB. PNI-Based Stress Management Interventions in HIV Disease. Nancy L. McCain,* Barbara A. Munjas,* Cindy L. Munro,* R. K. Elswick,*,† Pamela M. Kimball,† Andrea Ferreira-Gonzalez,† Lisa G. Kaplowitz,† Evelyn J. Fisher,† Carleton T. Garrett,† Kevin E. Brigle,* Jo W. Robins,*,† Linda C. Kendall,* Valentina Lucas,* and Katherine L. Cochran,* Schools of *Nursing and †Medicine, Virginia Commonwealth University, Richmond, Virginia 23298. A pretest–posttest design was used in a randomized clinical trial to compare the effects of 8-week cognitive–behavioral relaxation training (CBSM) and social support groups (SSG) to a WAIT-listed control group on (a) illness-related uncertainty, perceived HIV-related stress, coping strategies, perceived social support, illness-related psychological distress, and quality of life; (b) salivary cortisol and DHEA; (c) CD4⫹ and CD8⫹/CD57⫹ lymphocyte levels, NK cell cytotoxicity, selected type 1 and type 2 cytokine production levels, and peripheral viral load; and (d) HIV disease progression and symptomatology. The CBSM group was found to have higher postintervention emotional well-being and total quality of life than did the SSG or WAIT groups, but this effect dissipated by the 6-month follow-up. At 6 months CBSM participants had lower illness-related uncertainty. SSG participants had lower social/family well-being immediately postintervention and lower social support after 6 months. Interactions with health status indicated that SSG participants with more symptomatology had lower postintervention social/family functioning than the CBSM and WAIT groups, lower social integration than the CBSM group, and lower NK cell cytotoxicity than others. Perhaps group interventions that involve confronting one’s illness are not helpful in managing psychological distress in the current era of HAART, particularly among those with more advanced illness. Analyses of cytokine production revealed a more complex pattern than was anticipated and may, in part, reflect the impact of the new era of protease inhibitors. Participants clearly demonstrated a type 0 cytokine production pattern, indicative of a greater degree of immunocompetence than was expected. [Funded by the National Institute of Nursing Research, No. R01 NR04395 (McCain) and conducted with the assistance of the General Clinical Research Center, Medical College of Virginia Hospitals/Virginia Commonwealth University (No. M01 R00065, Clore).] Synergistic Effects of Depression and Chronic Stress on Immune Function. Lynanne M. McGuire,* Janice K. Kiecolt-Glaser,*,† and Ronald Glaser,†,‡ *Department of Psychiatry, †Institute for Behavioral Medicine Research OSU Health Sciences Center, ‡Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210. In a longitudinal study, we examined the individual and joint consequences of depression and chronic stress for cellular immune function in 62 spousal caregivers of dementia patients and 59 controls. Subjects’ Beck Depression Inventory scores at two time periods, baseline and 18 months, were used to create depressed and nondepressed groups (based on a standard cut-score) within caregiver and control cohorts. The combination of depression and chronic stress (depressed caregivers) was associated with greater impairment in cellular immune function than either depression (depressed controls) or chronic stress (nondepressed caregivers) alone. Antibody titers to latent Epstein–Barr virus (EBV) virus capsid antigen (VCA) IgG were measured as an indication of the ability of the cellular immune response to control the expression of latent EBV. Antibody titers were most affected by chronic stress; caregivers’ (depressed and nondepressed) EBV antibody titers increased and controls’ (depressed and nondepressed) EBV antibody titers decreased over time. T-cell blastogenic responses to the mitogens Con A and PHA were most influenced by depression. Depressed subjects (both caregivers and controls) showed the least vigorous blastogenic responses at follow-up, whereas the nondepressed controls showed a more vigorous blastogenic response than nondepressed caregivers over time. Nondepressed caregivers had a poorer blastogenic response at follow-up relative to baseline, but their response was greater than the depressed caregivers. These results support the hypothesis that there is a synergistic effect of depression and chronic stress (depressed spousal caregivers) on certain aspects of immune response relative to the effects of depression or chronic stress alone. (Supported by MH 42096 and by NIMH Postdoctoral PNI Fellowship Training Grant T32 MH18831.)
Quantification of IL-1 Alpha, IL-1 Beta, and KGF Gene Expression in Early Wounds of Restrained Mice. Ana M. Mercado,* David A. Padgett,*,† John F. Sheridan,*,† and Phillip T. Marucha,*,†,‡ *Department of Oral Biology, †Institute for Behavioral Medicine Research, ‡Department of Periodontology, Ohio State University, Columbus, Ohio 43210. Inflammatory processes that occur early after wounding are critical for eventual closure of the wound. For example, expression of proinflammatory cytokine IL-1 promotes reepithelialization of the wound surface via induction of keratinocyte growth factor (KGF). In a murine model of cutaneous wound healing, wounds of restraint-stressed mice (RST) healed significantly slower and had reduced number of inflammatory cells than wounds of controls. To elucidate the molecular mechanisms between stress and wound healing, we studied kinetics of epidermal gene expression of IL-1 alpha, IL-1 beta, and KGF. Female, hairless SKH-1 mice 6–8 weeks old were subjected to RST 3 days before and for 5 days following dorsal application of a 3.5-mm punch wound. Wounds were excised after days 0.5, 1, 3, and 5. RNA was extracted and subjected to competitive RT-PCR. At day 1, RST mice had significantly lower IL-1 beta gene expression as compared to controls. At day 3, RST mice had significantly lower KGF gene expression as compared to controls. This down-regulation of KGF mRNA in RST animals may be related to depressed expression of IL-1 beta, an inducer of KGF in dermal fibroblasts. At day 5, RST mice had significantly elevated levels of both IL-1 alpha and IL-1 beta mRNA as compared to controls, which represents a prolonged inflammatory response in wounds of RST mice. Our results suggest that alteration in the kinetics of proinflammatory cytokine responses by stress may have an impact on the expression of growth factors required for wound reepithelialization. (Supported by NIH Grant K08-00409-02.) IL-2 Produces Short-Term Effects on Metabolism and Long-Term Effects on Thresholds for Rewarding Brain Stimulation. Maia A. Miguelez and Catherine H. Bielajew, School of Psychology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada. In order to assess the effects of a single systemic injection of interleukin-2 (IL-2) on reward thresholds, rats were trained to self-administer rewarding ventral tegmental area (VTA) stimulation. Once frequency thresholds were stable, an intraperitoneal injection of either 0.5 or 1.0 µg of rrIL-2 was administered, following which thresholds were collected over a period of 6 h on day 1 (acute phase) and subsequently every second day for one month (chronic phase). The 0.5 µg dose of IL-2 produced an immediate and significant increase in thresholds, which was maintained throughout the chronic phase (21 ⫽ 15.00; p ⬍ .001), whereas the higher dose did not elevate thresholds as markedly (21 ⫽ 4.2; p ⬍ .05). Additionally, the animals’ weights and metabolic profiles were monitored on each test day. A pronounced reduction in weight was observed 6 h postinjection, in both the experimental (stimulating) and the control (nonstimulating) groups under the lower dose condition. This loss was recuperated quickly, rendering an increased food efficiency utilization profile in the 48 h following the acute testing day. These data suggest that IL-2 has dose-dependent short-term effects on metabolic parameters and long-term effects on rewarding values, at least at the level of the VTA. Psychological Interventions and the Immune System: A Meta-analytic Review and Critique. Gregory E. Miller*,† and Sheldon Cohen,* *Department of Psychology, Carnegie Mellon University, and †Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213. Interest in the hypothesis that psychological interventions can modulate the human immune response has grown dramatically in recent years. More than 85 intervention trials have appeared in the peerreviewed scientific literature, and the publication rate has been climbing since the middle of the 1980s. While several well-designed intervention trials have yielded extremely provocative findings, a number have also failed to document significant alterations in immune system parameters. To clarify the state of scientific affairs in this literature, we performed a comprehensive meta-analytic review of published studies and developed a series of theoretical models depicting the behavioral and biological pathways through which interventions might influence the immune system. The meta-analysis found only modest support for the hypothesis that psychological interventions could alter immune system parameters. The strongest evidence emerged from hypnosis and conditioning trials. Disclosure and stress-management showed scattered, inconsistent evidence of success. Relaxation demonstrated little capacity to elicit reliable immune change. Overall, the effects were considerably more narrow in scope than might have been anticipated on the basis of previous descriptions of this literature. The presentation will identify five reasons why this might be the case. Specifically, it will be argued that existing intervention trials have
failed to enroll the theoretically appropriate subject populations, choose the appropriate measures of immune system function, employ methodologically rigorous designs, seriously consider the issue of statistical power, and test hypothesized mediational pathways. Hyperalgesia and Allodynia Produced by the HIV-1 Glycoprotein, GP120: Involvement of Glial Activation in Spinal Cord. Erin D. Milligan,* Kevin A. O’Connor,* Mike K. Hansen,* Kien T. Nguyen,* Dave Martin,† Kevin J. Tracey,‡ Steven F. Maier,* and Linda R. Watkins, *Department of Psychology, University of Colorado, Boulder, Colorado 80309; †Department of Pharmacology, Amgen, Thousand Oaks, California 91320; and ‡Laboratory of Biomedical Science, North Shore University Hospital, Manhasset, New York 11030. Glial activation by the HIV-1 envelope protein gp120 releases many substances known to induce exaggerated pain states, including interleukin-1 [IL1] and tumor necrosis factor [TNF]. Indeed, intrathecal (IT; into cerebrospinal fluid surrounding lumbosacral spinal cord) gp120 produces two distinct forms of exaggerated pain: thermal hyperalgesia and mechanical allodynia (lowered threshold for response to heat and mechanical stimuli, respectively). We determined whether IT gp120 effects could be blocked by drugs that either disrupt glial function (fluorocitrate), block local cytokine release (CNI-1493), or block the effect of glially released IL1 (IL1 receptor antagonist; IL1ra) or TNF (TNF binding protein; TNFbp). We measured behavior as well as IL1 and TNF protein in lumbosacral dorsal spinal cord and cerebrospinal fluid (CSF); mRNA for these cytokines are currently being analyzed. After baseline measures of responses to thermal (Hargreaves test) and mechanical (von Frey test) stimuli, rats received either IT fluorocitrate, CNI-1493, IL1ra, TNFbp, or equivolume vehicle followed by IT gp120 (3 µg) or vehicle. Gp120-induced thermal hyperalgesia and mechanical allodynia were blocked by all drugs tested. IT gp120 increased IL1 and TNF protein in lumbosacral spinal cord and/or CSF. Fluorocitrate and TNFbp both blocked IL1 induction and release. Preliminary RT-PCR data suggests gp120 induces increases in at least IL-1b mRNA. Hence, HIV-1 gp120 in the spinal cord likely produces thermal hyperalgesia and mechanical allodynia via the spinal release of proinflammatory cytokines by activated glia. (Supported by NIH Grants MH55283, MH01558, MH00314, and NS38020 and by Amgen.) A Priming Effect of Stress on the IL-1b Response to Bacterial Lipopolysaccharide in Sprague–Dawley Rats. Albert Moraska,* Steven Maier,† Linda Watkins,† and Monika Fleshner,* *Department of Kinesiology and Applied Physiology, and †Department of Psychology, University of Colorado at Boulder, Boulder, Colorado 80309. Acute stressor exposure enhances resolution of the inflammatory response to bacteria. The purpose of these studies was to determine if stress primes the immune system to respond more robustly to in vivo bacterial lipopolysaccharide (LPS). Methods. Exp. 1, male Sprague–Dawley (4 months) rats were given saline or LPS (300 µg/kg) intraperitoneally at 1.5, 3, 5, or 24 h prior to sacrifice. Exp. 2, rats were given saline, 20, 200, or 400 µg/kg of LPS (ip) and sacrificed 1.5 h later. Exp. 3, rats were exposed to inescapable shock (IS, 100 1.6 mA, 5-s tail shocks, 60 s ITI) or remained in home cages. Four days after IS, LPS (ip, 20 µg/kg) was given and rats sacrificed 1.5 h later. Splenocytes, mesenteric lymphocytes (MLN), and peritoneal cells were collected, dissociated, and cultured for 48 h without stimulation. IL1beta (ELISA) and nitric oxide (nitrite, Greiss reaction) were measured in culture supernatant. Results. Exp. 1, peak IL-1beta and nitrite occurred at 1.5 h post-LPS, values decreased to baseline by 24 h in all tissue compartments. Exp. 2, the LPS dose of 20 µg/kg resulted in elevated, but not maximal, production of IL-1beta in all tissue compartments; nitric oxide was elevated in spleen and peritoneal exudate. Exp. 3, IS prior to LPS increased IL-1b above LPS alone in both spleen and peritoneal cells, but not MLN. Nitric oxide levels were not differentially affected. Conclusion. IS primes immune cells to respond to in vivo LPS more robustly, suggesting positive adaptations in immunity occur with acute stressor exposure (AI45576, MH45045.). The Role of B Cell Receptor and Beta-2-Adrenergic Receptor Signaling Kinases in the Modulation of CD86 (B7-2) Expression. Afsaneh Mozaffarian,* Deborah J. Kasprowicz,† and Virginia M. Sanders,*,† *Department of Cell Biology, Neurobiology, and Anatomy, and †Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, Illinois 60153. Our laboratory has recently shown that stimulation of resting antigen-specific B lymphocytes enriched from the spleens of unimmunized mice via the B cell receptor (BCR) and/or the beta-2-adrenergic recep-
tor (beta-2-AR) upregulates CD86 protein expression on the cell surface. As compared to the stimulation of each receptor alone, stimulation of both receptors further enhances the level of CD86 expression. Additional data have shown that the increase in CD86 expression enhances IL-4/Th2 cell-dependent IgG1 and IgE antibody production, suggesting a significant role for CD86 signaling. Studies using beta2-AR knockout mice have shown that the increase in CD86 expression occurs only by stimulation through the BCR and is not affected by a specific beta-2-AR agonist, further supporting that the increase in CD86 expression occurs by signaling through both receptors. The purpose of this study is to determine the signals that are generated by stimulation of these two receptors to upregulate CD86 expression that mediate a functional change in the B lymphocyte. In order to understand this intracellular signaling mechanism in vitro, freshly isolated resting B lymphocytes isolated from the spleens of Balb/c mice were stimulated through either the BCR and/or the beta-2-AR in the presence or absence of various kinase inhibitors. Preliminary data suggest that protein kinases A and C are important in enhancing the level of CD86 expression following stimulation of both receptors. However, it appears that protein tyrosine kinases upregulate CD86 via BCR stimulation only. (This work is supported by NIH Grant AI37326 and ACS Grant RPG-96-067-04-CIM.) Central Opioid Receptors Modulate the Morphine-Induced Enhancement of the Contact Hypersensitivity Response. Christina J. Nelson, Kelly A. Carrigan, and Donald T. Lysle, Biological Psychology Program, Department of Psychology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. Investigations have shown that morphine produces substantial alterations in immune function and subsequent health outcomes. To enhance our understanding of the impact of morphine on clinically significant in vivo immune responses, the present study determined the effect of morphine on the in vivo immune response of contact hypersensitivity (CHS). The CHS response, characterized by a cutaneous inflammatory reaction, was induced via the application of 2,4-dinitro-fluorobenzene to the pinnae of rats and assessed via measurements of pinnae thickness. Initial studies revealed that subcutaneous morphine administration 1 h prior to initial antigen exposure (sensitization) produced no effect on the CHS response. In contrast, morphine administration 1 h prior to antigen reexposure (challenge) produced a significant and robust enhancement of the CHS response. Further investigations showed that both systemic naltrexone and central N-methylnaltrexone (a quaternary compound which does not readily cross the blood–brain barrier) administration prior to morphine treatment blocked the enhancement of the CHS response. These results indicate that central opioid receptors play a critical and necessary role in the effects of morphine on the CHS response. Additional studies are being completed to determine the concomitant immune changes which play a role in morphine’s effects on the CHS response. The completion of these studies will provide substantial contributions in our comprehension of the complex interaction between opioids, immune status, and, ultimately, health outcomes. Early Depressive Symptoms Correlate with an Increased Cytokine Production in Cancer Patients Treated with Interleukin-2. Pierre J. Neveu,* Lucile Capuron,* Alain Ravaud,†,‡ Norbert Gualde,†,‡ Eugene Bosmans,§ Robert Dantzer,* and Michael Maes,¶ *INSERM U394, Neurobiologie Integrative, Institut Francois Magendie, 33077 Bordeaux cedex, France; †Institut Bergoni, Comprehensive Cancer Center, 33076 Bordeaux cedex, France; ‡CNRS UMR 5540, Bordeaux, France; §Eurogenetics, Tessenderlo, Belgium; and ¶Department of Psychiatry and Neuropsychology, University Hospital of Maastricht, The Netherlands. Cancer immunotherapy with interleukin-2 [IL-2] frequently induces depressive symptoms. In addition, there is some evidence for immune activation in depressed patients, and it was suggested that cytokines could be involved in the pathogenesis of depression. We therefore investigated the possibility of relationships between immune activation and depressive symptoms in response to IL-2 therapy in cancer patients. The study was carried out on 23 patients with renal cell carcinoma that were treated with subcutaneous IL-2, alone (N ⫽ 13) or in association with interferon-alpha (N ⫽ 5), or with interferon-alpha alone (N ⫽ 5). The intensity of depressive symptomatology was evaluated during a clinical interview using the Montgomery and Asberg Depression Rating Scale (MADRS) before the start of immunotherapy and 5 days later. On the same days, blood samples were collected for each patient to measure serum concentrations of cytokines (IL-6, IL-10, IL-8, IL-1ra) and cytokine-receptors (sIL-2R, LIF-R). IL-2 treatments were associated with increased serum levels of all cytokines or cytokine receptors tested. In the same way, these treatments were accompanied by increased MADRS scores on day 5. These increases in
MADRS scores were positively correlated with the variations of serum concentrations of IL-10 and sIL2R. In addition, the intensity of depressive symptoms on day 5 was correlated with the increases of IL-10 concentrations. Interestingly, patients treated with interferon-alpha alone exhibited no depressive symptoms 5 days after the beginning of treatment and no increase of plasma levels of cytokines was observed. These findings support the hypothesis of close relationships between immune alterations and psychopathology. (Supported by Ligue Nationale contre le Cancer and Association pour la Recherche sur le Cancer.) Elevated Plus Maze, Startle, and Shock Sensitivity Behavior in Predisease and Early Stage Autoimmune Lupus Disease MRL/lpr Mice. Darci M. Nielsen and Linda S. Crnic, Department of Pediatrics and Department of Psychiatry, University of Colorado School of Medicine, Denver, Colorado 80262. Behaviors indicative of anxiety have been shown to emerge with the onset of autoimmune disease in MRL/MpJ-Faslpr (MRL/lpr) mice and NZBxNZW hybrid mice. This study sought to replicate these findings and to extend behavioral characterization of MRL/lpr and the congenic MRL/MpJ⫹/⫹ (MRL/ ⫹) control mice using the auditory startle response and shock sensitivity paradigms. Severe autoimmune disease develops at approximately three months of age in MRL/lpr mice, whereas a milder form of autoimmunity develops by twelve months in MRL/⫹ mice. Mice were tested in the elevated plus maze (EPM), acoustic startle habituation, acoustic startle sensitivity, and foot-shock sensitivity paradigms. Predisease (5 week old, N ⫽ 10) MRL/lpr mice spent more time in the EPM open arms than agematched control MRL/⫹ mice (N ⫽ 10). However, MRL/lpr mice in the early stage of autoimmunity (11 weeks old, N ⫽ 10) did not differ from either age-matched MRL/⫹ mice (N ⫽ 12) or from predisease MRL/lpr mice in the EPM. MRL/lpr mice exhibited a lower startle response compared to MRL/⫹ mice. Similarly, predisease MRL/lpr mice were less reactive to footshock than MRL/⫹ mice. These differences are not likely due to peripheral neuropathy because startle latencies were not elevated in MRL/lpr mice. Both MRL/lpr and MRL/⫹ mice and each age group exhibited startle habituation deficits, implicating impairment in the central processing of sensory information in the background strain. In contrast to expectations, MRL/lpr mice in the early stage of autoimmunity did not exhibit behaviors indicative of anxiety. (Supported by MH 49043 and HD 07197.) Inescapable Shock Increases Central and Peripheral Interleukin-1Beta. Kevin A. O’Connor, John D. Johnson, Matthew Stark, Linda R. Watkins, and Steven F. Maier, Department of Psychology, University of Colorado, Boulder, Colorado 80309. Interleukin-1beta (IL-1beta) is a pleiotropic cytokine produced by a number of cell types. Increases in peripheral IL-1beta activate immune cells, signal the brain, and activate the acute phase response. IL-1beta has been shown to act centrally to produce fever and increase plasma ACTH and corticosterone (CORT). Exposure to inescapable tail shock (IS) also induces the acute phase response. IS decreases negative acute phase proteins and increases positive acute phase proteins, core body temperature, and basal levels of CORT for several days. To determine whether IS exposure also induces proinflammatory cytokines, Sprague–Dawley rats were exposed to 5, 50, or 100 IS (0.6 mA, 5 s). Animals were sacrificed immediately, 1, or 3 h after 100 IS. Trunk blood was collected and assayed for IL-1beta and CORT. Various brain regions were dissected and assayed for IL-1beta. Plasma CORT was significantly elevated immediately after 5, 50, and 100 IS, but not 1 or 3 h after 100 IS. Plasma IL-1beta was significantly elevated after 5, 50, and 100 shocks. Plasma IL-1beta remained elevated 1 h after 100 IS and returned to normal by 3 h. IL-1beta levels in the hypothalamus were elevated immediately after 100 IS but at no other time point. Inescapable shock did not alter IL-1beta levels in the pituitary or hippocampus. These data suggest that IS may induce the acute phase response by inducing peripheral and central IL1beta. (Supported by NIH Grants MH45045, MH00314, and MH401558.) AED-Induced Restoration of Responsiveness to Influenza Vaccination in Mice. David A. Padgett,* Robert C. MacCallum,* Roger M. Loria,† and John F. Sheridan,* *Institute for Behavioral Medicine Research, Ohio State University, Columbus, Ohio 43210; and †Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia. Advancing age is accompanied by a decline in immune function and is characterized by an inability to mount a protective vaccine response. Although significant research has been targeted toward the
development of vaccines, immunosenescence leaves elderly individuals relatively unprotected against preventable, but lethal infections such as influenza. Androstenediol (AED), a metabolite of dehydroepiandrosterone (DHEA), regulates innate and adaptive immune responses. Furthermore, physiological levels of AED decline with advancing age. Therefore, we hypothesized that oral supplementation with AED would restore immune competence to aging animals. To examine whether AED could effectively reverse the age-associated decline of antiviral immunity, 3-, 10-, and 22-month old mice were treated with AEDsulfate (AED-S) for 45 days beginning 10 days prior to vaccination. Subsequently, mice were primed and boosted with suboptimal doses of a commercially available trivalent influenza vaccine. Treatment of 10-month old animals with AED-S increased the titer of circulating antiviral IgG to levels comparable with those in 3-month old healthy mice. Furthermore, AED-S treatment protected 10-month old animals from intranasal challenge with a lethal dose of influenza virus 21 days after secondary vaccination. Although AED-S treatment of 22-month old mice did not enhance vaccine responses and failed to protect against lethal challenge, the data from the 10-month old animals suggest that treatment with AED-S will prevent and/or reverse the early manifestations of immunosenescence. (This work was supported by research Grants PO1-AG11585 and R29-MH56899 from the National Institutes of Health.) N-methyl-D-aspartate Receptor Antagonism Facilitates Morphine Analgesia but Not Host Resistance Against Surgery’s Tumor-Enhancing Effects. Gayle G. Page, Normalynn Garrett, and Wendy P. Blakely, School of Nursing, Johns Hopkins University, Baltimore, MD 21205. The barrage of nociceptive afferent activity in the central nervous system and accompanying pain associated with local injection of tissue-damaging agents has been shown to be dependent upon activation of the N-methyl-D-aspartate (NMDA) receptor in the spinal cord. Given that cutting and tearing of tissues also occurs during surgery, this study was intended to assess the possible benefit of NMDA receptor blockade alone or in combination with a dose of morphine that was previously shown to attenuate the metastatic-enhancing effects of surgery. To study this issue, we used the natural killer (NK)-sensitive mammary adenocarcinoma cell line, MADB106, syngeneic to the male and female F344 rats used. All surgery animals received either the competitive NMDA antagonist LY235959 (0.4 mg/kg), morphine (10 mg/kg) or both, or the saline vehicle i.p. 30 min before surgery. Anesthesia animals received saline injections. Surgery resulted in a significant increase in the lung retention of radiolabeled MADB106 cells which was attenuated by morphine administration in both males and females. NMDA antagonism provided no benefit alone or in combination with morphine. On the other hand, when the hot plate test was used to assess the time course of acute antinociception, LY235959 in combination with morphine provided improved acute analgesia compared to LY235959 or morphine alone, as has been shown by others. Taken together, these findings indicate that, in addition to its facilitation of pain relief, NMDA antagonism may compromise NK cell function, perhaps via a dopaminergic or sympathetic pathway. (Supported by NIH Grant NR03915.) Involvement of IL-12 in the Regulation of TH1/TH2 Responses Induced by Opioids. Alberto E. Panerai, Leda Gaspani, Elena Limiroli, and Paola Sacerdote, Department of Pharmacology, University of Milan, 20129 Milan, Italy. We previously showed that in KLH immunized mice, in vivo opioids could skew the Th1/Th2 cytokines toward a Th2 dominant pattern. In order to understand whether the effect of the opioids was exerted on T cells directly or was mediated by an interaction with other cell types, we tested in vitro the effects of the mu opioid agonist Damgo and of the kappa agonist U50,488H on Th-1/Th2 cytokine production and on IL-12 production by macrophages. Mice were immunized with 100 µg KLH, and one week later splenocytes were restimulated in vitro with KLH in the presence of Damgo and U50,488H at concentrations ranging from 10-6 to 10-12 M. IL-2, IFN-gamma and IL-4 were assayed in the culture medium by ELISA. For IL-12 evaluation, thioglycollate elicited peritoneal macrophages were stimulated in vitro with 1 µg/ml LPS and 50 µ/ml IFN- gamma, with and without opioid agonists, and IL-12 (p70) measured by ELISA in the 24 h supernatant. Neither Damgo nor U50,488H were able to affect Th1/Th2 cytokine production at any dose tested. On the contrary, both Damgo and U50,488H significantly and concentration dependently decreased IL-12 production. The specificity of the effects was checked by the use of the specific kappa receptor antagonist norBNI and of the mu receptor antagonist naloxone. Our data suggest that the effects exerted in vivo by opioids on Th1/Th2 cytokines are mediated by the modulation of macrophage IL-12 production.
Treatment of Multiple Sclerosis Patients with Interferon Beta-1a (AVONEX): Improvement of Life Quality and Neuropsychological Performance. Volker Pithan,* Jan Baase,* Michael S. Exton,* Hans Wilhelm,† Volker Limmroth,† and Manfred Schedlowski,* *Institute of Medical Psychology and †Department of Neurology, University of Essen, 45122 Essen, Germany. Interferon beta has shown efficacy in reducing the number and severity of relapses in patients with relapsing–remitting multiple sclerosis (RRMS). We examined the effect of this therapy on the quality of life and neuropsychological skills in a longitudinal study of 21 patients for over 1 year. Twelve patients with RRMS and nine patients with secondary progressive (SP) MS were selected consecutively. Clinical status was defined according to the Expanded Disability Status Scale. All patients were treated with 6 MIU interferon beta-1a (IFN beta-1a; AVONEX) once a week. Neuropsychological and life quality parameters were assessed at baseline and at two-month intervals over the course of one year by a battery of cognitive and psychological tests. We found significant improvement in depression, anxiety, and an increase in activities of daily life during the first year of treatment. Patients showed significant improvements in information processing speed, short- and long-term memory and sensorimotor function. Furthermore, attack frequency was reduced in RRMS patients, and disability accumulation in SPMS patients was lessened by IFN beta-1a. Our findings indicate that IFN beta-1a not only favorably influences both RR- and SPMS disease course, but also reduces depression and contributes to improving life quality of MS patients. Effect of Insulin on Microbial Growth. Balbina J. Plotkin,* Robert J. Roose,*,† and Susan M. Viselli,† *Department of Microbiology and †Department of Biochemistry, Midwestern University, Downers Grove, Illinois 60515. Hyperinsulinemia is a common occurrence in the human host. In addition to noninsulin dependent diabetes mellitus, hyperinsulinemia occurs during the acute phase stress response associated with the early stages of the infectious process. It is not known what effect hyperinsulinemia has on bacterial growth and subsequent virulence. Enhanced growth in response to the hyperinsulinemic–hyperglycemic acute phase environment could result in a shift in the host–parasite interaction. Thus, the ability of insulin to affect the growth kinetics of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa was measured. For all organisms, insulin, in the absence of a metabolizable carbohydrate had no effect on generation time as compared to homologous control. Response to insulin, in the form of increased or decreased generation times, for both Gram-positive and Gram-negative bacteria was dependent on the concentration of insulin, the concentration of glucose present and the initial concentration of bacteria exposed to the glucose and insulin. Insulin alone had no impact on the minimum inhibitory concentration of 40 antimicrobials we tested. These results indicate that not only are organisms capable of responding to insulin, in the presence of glucose, but that the response is regulated by the concentration of organisms present when the bacteria are first exposed to the insulin and glucose. Therefore, there may be a basal level of glucose transporters on bacteria whose expression can be up regulated by insulin present in the host. Further a quorum signal may be involved in the ability and direction of the bacterial response to insulin. The Role of Proinflammatory Mediators in EAE-Associated Behavioral Syndrome. Yehuda Pollak,* Haim Ovadia,† Inbal Goshen,* and Raz Yirmiya,* *Department of Psychology, Hebrew University of Jerusalem, Mount Scopus, Jerusalem, Israel; and †Department of Neurology, Hadassah-Hebrew University Hospital, Jerusalem, Israel. Experimental autoimmune encephalomyelitis (EAE) is a demyelinating autoimmune disease that serves as a model for the human disease multiple sclerosis. Previously, we documented a transient sickness behavior syndrome that precedes and accompanies the neurological symptoms of the disease. Due to the well-known role of proinflammatory mediators in sickness behavior, we tested the effect of dexamethasone, a potent anti-inflammatory steroid, on EAE-associated reduction in body weight, food and sucrose intake, and social exploration. Dexamethasone, injected daily from the onset of neurological symptoms, attenuated the behavioral symptoms of EAE within 1–2 days of treatment, independently of its effect on the neurological symptoms, suggesting a role for inflammatory processes in mediating the behavioral alterations. To further explore the role of specific cytokines in the behavioral syndrome, we conducted RT-PCR study and measured the expression of TNF-alpha mRNA in different critical time
points along the behavioral syndrome (i.e., onset, peak, initial recovery, and complete recovery of EAEassociated sickness behavior). Compared to control healthy mice, TNF-alpha mRNA expression was high during all time points, regardless of the behavioral dynamics, suggesting a possible role in the initiation, but not the termination of the behavioral symptoms. In agreement with this conclusion, treatment of EAE mice with pentoxifylline, a TNF-alpha synthesis inhibitor, had no effect on the behavioral syndrome. In a preliminary study, indomethacin, a prostaglandins synthesis inhibitor, fully abrogated EAE-induced loss of body weight, without affecting the neurological symptoms or the mortality, suggesting a role of prostaglandins in some of the components of the EAE-associated sickness behavior. [This research was supported by Grant 94-204 from the United States–Israel Binational Science Foundation (BSF).]
gp120 Impairs Contextual Fear Conditioning: A Potential Role in AIDS Related Dementia Complex. C. Rachal Pugh,* John D. Johnson,* David Martin,† Steven F. Maier,* Jerry W. Rudy,* and Linda R. Watkins,* *Department of Psychology, University of Colorado, Boulder, Colorado 80309; and †Amgen, Thousand Oaks, California. Human Immunodeficiency Virus-1 (HIV-1) and its coat protein gp120 enter the brain early during the disease process. Here they remain relatively unaffected by current AIDS treatments that do not readily cross the blood brain barrier. In tissue culture studies, gp120 has been shown to activate both microglia and astrocytes. In vivo activation of glia in other contexts (e.g., by bacterial cell walls) leads to the release of the proinflammatory cytokines interleukin-1 beta (IL-1beta), and tumor necrosis factor alpha (TNFalpha). We have previously shown a role for proinflammatory cytokines in learning and memory. Specifically, central IL-1beta administration, as well as agents that induce IL-1 release, impair contextual but not auditory-cue fear. The present experiments explored the hypothesis that gp120 may produce learning impairments by inducing central cytokine release. First, we found that like central IL-1beta administration, intracerebroventricular (ICV) gp120 administration (4 µg) impaired contextual but not auditory-cue fear. ICV gp120 also caused significant site-specific increases in IL-1beta protein in the hippocampus and frontal cortex, but not in the hypothalamus. This is interesting because contextual fear depends on the integrity of the hippocampal formation. In addition, ICV alpha melanocyte stimulating hormone (alphaMSH; an endogenous IL-1 antagonist), IL-1 receptor antagonist (IL-1ra), and TNF binding protein (TNFbp) were all able to prevent the gp120-induced impairment in contextual fear. Taken together, these results suggest that gp120 may produce some of the learning impairments associated with AIDS related dementia complex by inducing central cytokine release. (Supported by NIH Grants MH55283, MH 00314, MH01558, and NIH F31 MH12148-01.)
Social Stress Increases Susceptibility to Endotoxic Shock in Mice. Ning Quan,* Ronit Avistur,* David A. Padgett,*,† Phillip T. Maucha,*,† and John F. Sheridan,*,† *Department of Oral Biology, †Institute of Behavioral Medicine Research, Ohio State University Health Science Center, Columbus, Ohio 43210. The influence of social stress on the susceptibility to endotoxic shock was investigated. Social disruption (SDR) stress was induced by introducing to established stable cohorts of mice an aggressive intruder mouse for 2 h/per day. After a week of SDR stress, animals were injected intraperitoneally (i.p.) with 1 mg/mouse of the endotoxin lipopolysaccharide. Within 24 h after the LPS injection, there was 30% mortality in SDR animals but no mortality in control animals. By 48 h, there was 70% mortality in the SDR and 30% mortality in the control animals. Postmortem examination of SDR mice revealed widespread disseminated intravascular coagulation in the brain and lung, extensive meningitis in the brain, severe hemorrhage in the lung, necrosis in the liver, and lymphoid hyperplasia in the spleen. To investigate the mechanisms for the increased susceptibility of SDR animals to endotoxic shock, LPS-stimulated proliferation of splenocytes was analyzed for their responsiveness to glucocorticoids. The LPS-induced proliferation in SDR splenocytes was resistant to the effect of corticosterone which inhibited proliferation of splenocytes from control animals. The expression of proinflammatory cytokines after the injection of LPS was determined by Realtime PCR and ELISA. Compared with the control animals, both the mRNA and the protein expression of IL-1 and TNF-alpha were increased in SDR mice. Taken together, these results show that SDR stress increases the susceptibility to endotoxic shock in mice and suggest that the development of glucocorticoid resistance and increased production of proinflammatory cytokines are the mechanisms for this behavior-induced susceptibility to endotoxic shock.
Alcoholism, Sleep, and Nocturnal Cytokine Production. Laura S. Redwine,* Martica Hall,† and Michael Irwin,* *Department of Psychiatry, University of California San Diego, San Diego, California 92161; and †Department of Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania. Disrupted sleep elicits immune changes and may impact health. Alcoholics show considerable disturbances of sleep and are at risk for immune alterations including decreased proinflammatory cytokine, IL-6, and increased inhibitory Th2 cytokine, IL-10 production. The purpose of this study was to examine the relationships between sleep and nocturnal production of proinflammatory (IL-6), Th1 (interferon, IFN), and Th2 (IL-10) cytokines. Fourteen alcoholic and 10 control African–American males underwent sleep EEG recording and serial blood sampling at 23:00, 03:00, and 07:00 h. Levels of secreted IL-6, IFN, and IL-10 were measured from isolated peripheral blood mononuclear cells stimulated with ConA. For IL-6, a mixed design repeated measures ANOVA showed a significant group effect (F ⫽ 5.0, p ⫽ .04) and a group ⫻ time interaction (F ⫽ 4.3, p ⫽ .02) in which alcoholics showed lower levels of IL-6 compared to controls. For IFN, a trend toward a group ⫻ time interaction (F ⫽ 2.8, p ⫽ .07) suggested a nocturnal rise of IFN in control subjects, which was not present in alcoholics. The Th1/ Th2 (IFN/IL-10) cytokine ratio revealed a time effect (F ⫽ 3.1, p ⫽ .05) and a trend toward a group ⫻ time interaction (F ⫽ 2.9, p ⫽ .06), suggesting a shift toward a Th2 cytokine response in alcoholics versus controls across the nocturnal period. These data further implicate sleep in the regulation of immune function. Alcoholic subjects who have prominent sleep disturbances show lower nocturnal levels of IL6, an attenuated rise of IFN across the nocturnal period, and increased dominance of Th2 responses during sleep as compared to controls. (This work was supported in part by NIAAA Grant AA10215, NIMH Grant 5T32-18399, NIMH Grant 2P30-MH30914, and NIH Grant M01 RR00827.) Alcoholism, Sleep, and Nocturnal Changes in Adhesion Molecule Density. Laura S. Redwine,* Alan Maisel,† and Michael Irwin,* *Department of Psychiatry and †Division of Cardiology and Department of Medicine, University of California San Diego, San Diego, California 92161. Cellular adhesion molecules modulate attachment and migration of leukocytes across the endothelium and to inflammatory sites and are thought to play a role in pathogenesis of atherosclerosis and possibly infectious disease. L-selectin mediates tethering and Mac-1 adhering immune cells for migration. This study examined the relationship between sleep and nocturnal changes in L-selectin and Mac-1 in alcoholics versus controls. Disordered sleep has recently been implicated in alterations in adhesion molecule density. Alcoholics, who are at risk for atherosclerotic and infectious diseases have considerable sleep disturbances. Seventeen males stratified into alcoholic and control groups underwent blood sampling at 03:00 and 06:30 h on baseline and sleep deprivation nights. L-selectin and MAC-1 densities were measured in FMLP (f-met-leu-phe) stimulated peripheral whole blood by flow cytometry. For monocyte MAC-1 density, a mixed design repeated measures ANOVA showed a significant group ⫻ night interaction (F ⫽ 15.2, p ⫽ .003), where alcoholics failed to have a sleep deprivation induced reduction in MAC-1 density compared to controls. For L-selectin there was a significant night effect (F ⫽ 18.9, p ⫽ .001) and a trend toward a group effect (F ⫽ 3.2, p ⫽ .10), demonstrating an increase in lymphocyte L-selectin density in both groups on sleep deprivation night, although alcoholics had overall lower Lselectin density than controls. These data further suggest that sleep is involved in the regulation of adhesion molecule density, with an increase in L-selectin during sleep deprivation, albeit lower density in alcoholics. Also, alcoholic subjects who have prominent sleep disturbances showed an attenuated reduction of MAC-1 on sleep deprivation night. (This work was supported in part by NIAAA Grant AA10215, NIMH Grant 5T32-18399, NIMH Grant 2P30-MH30914, and NIH Grant M01 RR00827.) Sleep Deprivation Induces Peptidoglycan Recognition Protein (PGRP) in Brain. Abdur Rehman, Ping Taishi, Jidong Fang, Jeannine H. Majde-Cottrell, and James M. Krueger, Department of VCAPP, Washington State University, Pullman, Washington 99164-6520. PGRP was recently described as a peptidoglycan (PG) binding protein (1); its production is induced by bacteria. Further, PGRP is found in a variety of tissues including brain and is likely identical to another recently cloned protein, tag-7 (2), that was characterized as a new cytokine. PG and muramyl peptides (MPs), the monomeric building blocks of PG, are thought to be one of the bacterial-derived signals that initiates the excess sleep responses that form part of the acute phase response. Prolonged sleep deprivation is associated with bacteremia. We thus, thought it possible that sleep deprivation might
upregulate PGRP in brain. Adult Sprague–Dawley rats (N ⫽ 9 control; N ⫽ 9 experimental) were used. Experimental animals were deprived of sleep for 8 h beginning 1 h after light onset. At the end of the deprivation period, rats were sacrificed, their brains were removed and dissected. RNA was extracted from the brain stem, hypothalamus, and hippocampus. RT-PCR was performed to determine PGRP mRNA levels semiquantitatively. Porphobilinogen deaminase (PBD) was used as the housekeeping gene and ratios of PGRP/PBD were determined in triplicate for each sample. PGRP was constitutively expressed in all brain regions. After sleep deprivation there was about a 17% increase in hypothalamic PCRP/PBD ratios ( p ⬍ .03) and about a 40% increase in brainstem PGRP/PBD ratios ( p ⬍ .003). No differences were observed in the hippocampus. Results are consistent with the notion that PG and MPs are involved with sleep regulation. (1) Proc. Natl. Acad. Sci. USA 95,10 078, 1198; (2) J. Biol. Chem. 273,18,633, 1998. (Supported by NIH-NS 25378, NS 31453, and HD 36520.) Endotoxin Induces Psychological Disturbances in Healthy Volunteers: Involvement of Cytokines, Cortisol, and Personality Characteristics. Abraham Reichenberg,* Thomas Pollmacher,† Monika Haack,† Thomas Kraus,† Andreas Schould,† and Raz Yirmiya,* *Department of Psychology, Hebrew University, Mount Scopus, Jerusalem 91905, Israel; and †Max Planck Institute of Psychiatry, Munich, Germany. The present study investigated the associations between endotoxin-induced cytokine and cortisol secretion and psychological variables. Twenty healthy male volunteers completed a battery of psychological tests 1, 3, and 9 h following injection of a low dose of endotoxin (0.8 ng/kg) or saline in 2 experimental sessions. Blood samples were collected at hourly intervals, and food and water consumption were continuously monitored. Personality was assessed at baseline by the NEO-FFI. Endotoxin had no significant effects on subjective ratings of physical sickness symptoms, but it caused a mild increase in rectal temperature, and a transient decrease in spontaneous food intake. Endotoxin increased the circulating levels of TNF-a, sTNF receptors, IL-6, IL-1ra, and cortisol. Following endotoxin administration, the subjects showed a transient significant increase in the levels of anxiety and depressed mood. In addition, verbal and nonverbal memory functions were significantly impaired, whereas attention and executive functions were not affected. Significant positive correlations were found between the secretion of cytokines and endotoxin-induced anxiety, depressed mood, and severity of memory impairments. In contrast, cortisol secretion was positively correlated with depressed mood only. Personality characteristics (neuroticism, extraversion, and agreeableness) and immune activation predicted endotoxin-induced increase in state depression only indirectly, via state anxiety. These findings suggest that cytokines might be pivotally involved in the cognitive and emotional disturbances that characterize many physical diseases and they might represent a novel target for neuropsychopharmacological research. (Supported by The German– Israeli Foundation for Scientific Research and Development.) Effect of Hydration Status and Fluid Supplementation on Thermoregulatory Responses during EndotoxinInduced Fever in Rabbits. Charlotte A. Richmond, School of Nursing, University of Texas Health Science Center at San Antonio, San Antonio, Texas. Substantial evidence now exists that vasopressin (AVP) acts as a neurotransmitter in the brain, exerting an antipyretic action during fever. During fever, increased metabolism, evaporative heat loss and decreased oral intake increase risks for dehydration. Historically, fever treatment includes encouragement of fluids. In view of the antipyretic activity of AVP, it is possible that dehydration, a potent stimulator of AVP, has a beneficial role during fever and that fluid supplementation may attenuate that response by inhibiting AVP release. Objective. To determine in rabbits injected with lipopolysaccharide (LPS) (1) the effects of dehydration on core temperature patterns during the febrile response and (2) the effects of perenteral fluid administration on core temperature response in euhydrated and dehydrated conditions during the febrile response. Results. Dehydrated rabbits display larger fevers that are biphasic in nature. The larger fevers may be due to a hyperthermic response, rather than a febrile response, since ear vasoconstriction was lacking during the second phase of fever. Compared to euhydrated rabbits, there was no difference in the core temperature response when euhydrated rabbits were supplemented with fluids. However, there was a large reduction of the core temperature response when dehydrated rabbits received fluids. In both euhydrated and dehydrated rabbits, there was a large reduction in heart rate during fluid supplementation experiments. Conclusion. Dehydrated rabbits may utilize different strategies for antipyresis than do euhydrated rabbits and fluid supplementation may influence endotoxin-induced fever by altering cardiovascular mechanisms. Fluid supplementation has a beneficial role during fever, regardless of hydration status.
Psychological and Immunological Response to a Mindfulness-Based Stress Reduction Program in HIVInfected Individuals. F. Patrick Robinson,*,† Herbert L. Mathews,*,‡ and Linda Witek-Janusek,*,† *Center for Biobehavioral Immunology, †Niehoff School of Nursing, and ‡Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 60153. Individuals with HIV disease are challenged with numerous psychosocial stressors. Stress-induced immunosuppression may hasten HIV disease progression and increase susceptibility to opportunistic infections. It was hypothesized that stress reduction would decrease stress and improve immune function and health in individuals infected with HIV. Therefore, the purpose of this study was to determine the effects of a structured, 8-week mindfulness-based stress reduction (MBSR) program on perceived stress, mood, anxiety, natural killer cell activity (NKCA), and functional well-being of HIV infected individuals. Psychological status and well being were measured using the Profile of Mood States (POMS), Perceived Stressor Scale, State-Trait Anxiety Inventory, FAHI functional assessment scale, and the Symptom Checklist. Individuals with HIV infection were recruited from the Chicago area community and were assigned to either the MBSR intervention or control group. Immunological and psychological data were collected at 1–2 weeks prior to the intervention, after 4 weeks of MBSR, after 8 weeks of MBSR (immediate postintervention), and at 3 months postintervention. Results indicate an overall improvement in total mood (POMS) and functional well being (FAHI) from pre- to postintervention. Subjects reported pre- to postintervention decreases in depression, tension, confusion, and fatigue. Immunologically, subjects in the intervention group exhibited a pre- to postintervention increase in NK cell number and an increase in NKCA. Thus, HIV-infected individuals may benefit both psychologically and immunologically from participation in MBSR. Chronic Stress and the Th1/Th2 Cytokine Response to an Influenza Virus Vaccine: Social Networks Count. Theodore F. Robles,* Janice K. Kiecolt-Glaser,† Ronald Glaser,‡ and John F. Sheridan,‡,§ *Department of Psychology, †Department of Psychiatry, Ohio State University Institute of Behavioral Medicine Research, Ohio State University College of Medicine; ‡Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University Institute of Behavioral Medicine Research, Ohio State University College of Medicine; and §Department of Oral Biology, Ohio State University Institute of Behavioral Medicine, Ohio State University College of Dentistry. The number of different social ties (social network diversity) has been associated with vulnerability to common cold infections. In this study, we found that persons experiencing chronic stress who had fewer diverse social ties exhibited greater Th2 (IL-10) type responses to an influenza virus vaccine. In addition, having fewer diverse social ties may blunt the Th1 (IFN-gamma) responses to the vaccine. Peripheral blood leukocyte IFN-gamma and IL-10 responses to stimulation with the vaccine antigen were assessed in blood samples from 50 current and former caregivers and 50 control subjects obtained prior to, one month, and three months after influenza virus vaccination. Male control subjects with high network diversity, based on a median split of Social Network Index scores, showed a greater IFN-gamma response across draws compared to low network diversity controls ( p ⫽ .007). Caregivers with low network diversity showed a greater IL-10 response across draws ( p ⫽ .003) compared with high network diversity caregivers. These findings suggest that dysregulation of the immune response to a vaccine previously associated with the chronic stressor of caregiving may be mediated by one’s social networks. Moreover, the combination of chronic stress and low social network diversity may exacerbate age-related polarization of Th-1 and Th-2 responses to vaccines and viral infection. (This research was supported by MH42096 and a National Science Foundation Graduate Fellowship.) Neuroendocrine-Immune Responses Associated with Chronic Spinal Cord Injury and The Effect of Massage Intervention on Enhancing Cellular Immune Responses and Neuroendocrine Functioning. Sandra L. Rogers,* Charles E. Levy,† Jason A. Kahan,* and Stefanie Brezny,* *College of Medicine, Division of Occupational Therapy, †College of Medicine, Division of Physical Medicine and Rehabilitation, Ohio State University, Columbus, Ohio 43210. This study investigated whether the consequences of living with a spinal cord injury (SCI) would chronically alter neuroendocrine functioning and cellular immune responses. We also examined whether therapeutic massage could influence neuroendocrine and immune responses after SCI. Twenty individuals greater than 6 months post-SCI and 15 age- and gender-matched controls were compared for differences
in neuroendocrine function, cellular immune responses (functional and quantitative measures), level of independence in the community, self-reports of mood, and general health. The SCI participants also completed an intensive, one month long massage intervention. Blood samples were assessed at 3 time points: before exposure to massage, after exposure to massage, and after one month of no-intervention. SCI participants had more common illnesses and lower levels of independence then the control group. Interestingly, they had lower anger scores and higher scores on vigor than their peers. Cellular immune responses and neuroendocrine functioning showed that the SCI participants displayed diminished proliferative responses, and higher soluble IL-2 receptor (sIL-2R) levels, plasma cortisol levels and plasma cAMP levels when compared to controls. The massage protocol was associated with enhanced proliferative responses, diminished soluble IL-2 receptor (sIL-2R) levels, plasma cortisol levels, and plasma cAMP levels. The decreases in anxiety and anger were also correlated with increases in lymphocyte proliferation. These data demonstrate that SCI chronically alters cellular immune responses while massage provides a physiological benefit in increased cellular immune and neuroendocrine function and decreases in tension and anxiety following SCI. These results could have implications for intervention and infection following SCI.
Sex Differences in Glucocorticoid Sensitivity of Proinflammatory Cytokine Production at Baseline and after Psychosocial Stress. Nicolas Rohleder,* Nicole C. Schommer,* Dirk H. Hellhammer,* Renate Engel,* and Clemens Kirschbaum,† *Center for Psychobiological and Psychosomatic Research, University of Trier, Trier, Germany; and †Institute for Physiological Psychology, Department of Psychoneuroendocrinology and Psychoneuroimmunology, University of Duesseldorf, Duesseldorf, Germany. Objective. Men and women show marked differences in susceptibility to specific immunological disorders. These gender differences seem to be mediated by interactions of the hypothalamus–pituitary– adrenal (HPA) axis and the hypothalamus–pituitary–gonadal (HPG) axis. The aim of the present study was to investigate the impact of psychosocial stress and HPA axis activation on the glucocorticoid (GC) sensitivity of proinflammatory cytokine production in men and women. Methods. A total of 45 healthy subjects were investigated in the present study. Eighteen women in the luteal phase of their menstrual cycle and 27 men were exposed to a psychosocial stress test (Trier social stress test; TSST). Free cortisol levels were measured repeatedly after exposure to the stressor, GC sensitivity was assessed by dexamethasone (DEX) inhibition of lipopolysaccharide (LPS) stimulated production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). Results. The TSST induced significant increases in salivaryfree cortisol, but no significant differences between men and women could be found. GC sensitivity in contrast, as well as LPS stimulated cytokine production showed large gender differences. GC sensitivity in men showed a marked increase 1 h after stress, while GC sensitivity in women decreased significantly. Concordantly, LPS-induced cytokine production decreased in response to stress in men, but increased in women. Conclusions. The results demonstrate that despite similar HPA axis responses of men and women to psychosocial stress, the gender of the subject may exert differential effects on the immune system by modulating the sensitivity of relevant target tissues, e.g. the sensitivity of proinflammatory cytokine production.
Restraint Stress Enhances the Susceptibility to Bacterial Infection during Cutaneous Wound Healing. Isolde G. Rojas,* David A. Padgett,*,† John F. Sheridan,*,† and Phillip T. Marucha,*,†,‡ *Department of Oral Biology, †Institute for Behavioral Medicine Research, and ‡Department of Periodontology, College of Dentistry, Ohio State University, Columbus, Ohio 43210. Opportunistic infection is a complication of any surgical procedure or injury. Psychological stress has been shown to delay wound healing and decrease immune/inflammatory responses required for bacterial clearance. To determine if stress increases the susceptibility to infection, SKH-1 mice were subjected to cycles of restraint stress (RST) beginning three days prior to the placement of a cutaneous wound. Wounds were excised and processed for bacterial quantification (12–18 mice/time point). Wound myeloperoxidase (MPO) was also quantitated as a marker of neutrophil infiltration. RST caused a 2- to 5log increase in opportunistic bacteria at the wound as compared to controls during the first 5 days of healing ( p ⬍ .05). By day 7, 85% of the wounds from RST mice had bacterial counts predictive of infection (⬎100,000 CFU/gram tissue) as compared to 27% from control mice ( p ⬍ .001). The isolated organisms included S. aureus and group G. Streptococcus. RST mice also had a sevenfold increase in
wound MPO by day 5 after wounding as compared to controls ( p ⬍ .05). To assess the role of RSTinduced glucocorticoids in bacterial clearance, mice were treated with the glucocorticoid receptor antagonist, RU486. RST mice treated with RU486 had nearly a one-log reduction in opportunistic bacteria as compared to untreated, RST mice ( p ⬍ .05). The results showed that stress impairs bacterial clearance during wound healing resulting in increased incidence of opportunistic infections. Stress also resulted in an extended neutrophilic phase that could be contributing to increased tissue damage and delayed wound healing. Stress-induced glucocorticoids played a significant role in enhancing the susceptibility to infection. (Supported by NIH Grants DE11014, 1P50AG17811, and 1P01AG16321.)
Modulation of Disease Onset and Relapse in SJL Mice Induced with Experimental Allergic Encephalomyelitis (EAE): Effects of Tricyclic Antidepressant Treatment and Stress. Alba Rossi-George, Scott Siegel, Stanley Kreydin, and Alexander W. Kusnecov, Department of Psychology, Rutgers, State University of New Jersey, 152 Frelinghuysen Road, Piscataway, New Jersey 08855. Experimental allergic encephalomyelitis (EAE) is the predominant animal model for studying autoimmune processes that may be relevant to multiple sclerosis, and in susceptible strains of mice (e.g., SJL) can result in a remitting–relapsing disease progression. Using this model, we asked whether chronic treatment with the tricyclic antidepressant Imipramine (20 mg/Kg) would modulate disease severity in female SJL mice. Mice were immunized with 150 µg of the encephalitogenic peptide segment (139– 151) of myelin proteolipid protein (PLP) in Complete Freund’s Adjuvant (including an injection of pertussis toxin [200 ng]). Drug or vehicle treatment (for 10 days before and/or after) relative to immunization was as follows: Saline–Saline; Imipramine–Saline; Saline–Imipramine; Imipramine–Imipramine; and Untreated (i.e., nonhandled). The most pertinent results were a dramatic increase in disease severity in the Saline–Saline group relative to nonhandled animals. Because the latter did not differ from the Imipramine–Imipramine group, we hypothesized that brief stressor exposure (daily handling and injection) augments EAE, and that this can be inhibited with Imipramine. Whether the effects of Imipramine are due to central or peripheral influences remains to be determined, although they are consistent with other research involving rats and guinea pigs. In any case, additional experiments confirmed that chronic daily exposure to a unique environment significantly augmented disease severity during relapse, whereas footshock had no effect. These results suggest that within the context of a chronic autoimmune disease, varying degrees of emotional arousal exert differential effects on EAE severity and relapse, effects that may depend on interactions with central monoamine neurotransmitters.
Reduced Anxiety Behavior Following Ablation of Amygdala Neurons Expressing Substance P Receptor. Janeen L. Salak-Johnson,*,† Scott D. Rogers,* Matthew Schwei,* James P. Pomonis,* and Patrick W. Manthy,* *Department of Preventive Sciences, Psychiatry, and Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455; and †Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801. The neurokinin substance P (SP), localized in brain regions that coordinate stress responses, may play a role in modulating anxiety. Effects of ablation of substance P receptor (SPR)-expressing neurons by administration of a neurotransmitter-toxin conjugate, substance P-saporin (SP-SAP), in the amygdala (a brain region known to modulate stress and anxiety responses) were examined immunohistochemically and behaviorally 30 days following treatments. Rats were bilaterally injected in basolateral amygdala nuclei with 5 µl sterile saline, 10-6M saporin (SAP), or 10-6M SP-SAP. Both SPR-immunofluorescence levels and number of SPR-IR positive neurons in amygdalar subnuclei decreased following SP-SAP treatment. SP-SAP did not induce significant gliosis or nonspecific neuronal death. Rats were tested in elevated plus maze (EPM) and open field (OF). Anxiety level and exploratory behavior displayed by SP-SAP-treated rats were altered; they had significantly more entries into and spent more time in EPM open arms than did saline- or SAP-injected rats. They also had higher locomotor activity and exploratory behavior. Increasing illumination over EPM open arms increased anxiety level of control rats they immediately seek and spent more time in closed arm than did SP-SAP-treated rats. SP-SAP rats were unaffected by increased illumination. SP-SAP-treated rats spent less time ‘‘frozen’’ in OF than did control rats, but other behaviors did not differ among treatment groups. These results suggest that SPR expressing neurons in the amygdala plays a pivotal role in generation of anxiety behaviors and that SP may play a modulatory role in stress-induced anxiety behavior.
The Diurnal Cortisol Slope as a Predictor of Immune Reactivity to Interpersonal Stress. Sandra E. Sephton,* Firdaus S. Dhabhar,† Catherine Classen,‡ and David Spiegel,‡ *Department of Psychiatry and Behavioral Sciences, University of Louisville School of Medicine, Louisville, KY 40202; †College of Dentistry, Ohio State University, Columbus, OH 43210; and ‡Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305. Dysregulation of the HPA axis has been associated with adverse health consequences including early breast cancer mortality. Here we explore relationships between HPA axis dysregulation and stress-reactivity in a group of 36 women with metastatic breast cancer. Diurnal salivary cortisol profiles were measured one day prior to an interpersonal laboratory stress task. The diurnal cortisol slope was explored as a predictor of endocrine and immune responses to emotional stress. Patients with relatively flat, or arrhythmic, cortisol profiles evidenced suppression of resting CD4 and CD8 T lymphocyte counts in peripheral blood and poorer basal NK cell function. Morning blood cortisol levels were lower among patients with flat baseline slopes, but blood cortisol responses to interpersonal stress were enhanced. An elevated cortisol stress-response was not apparent in the unbound cortisol fraction measured in saliva. Greater stress-induced increases in peripheral blood counts of NK cells and cytotoxic T lymphocytes were evident among patients with flattened cortisol slopes. Flattened diurnal cortisol profiles were associated with some elevation of immune cell count even 24 h after the task. Results indicate that responses to interpersonal stress are enhanced, but may fail to be turned off after the cessation of stress in cancer patients with HPA axis dysregulation as measured by the diurnal cortisol slope. Coculture of Brain Slice and BV-2 Microglia Cells Increases Microglial Phagocytosis and Decreases MHC Class II Expression. Lisa L. Shafer,* John A. McNulty,* and M. Rita Young,†,‡ *Department of Cell Biology, Neurobiology, and Anatomy, and †Department of Pathology, Loyola University Chicago– Medical Center, Maywood, Illinois 60153; and ‡Research Services, Hines VA Hospital, Hines, Illinois 60141. Activation of microglia has been implicated in several neurodegenerative diseases such as Multiple Sclerosis, Alzheimer’s, as well as in aging. The factors that mediate microglial activation leading to subsequent neurodegeneration are poorly understood. Both direct contact and indirect communication through soluble mediators have been suggested to result in microglial activation. The goal of this study was to investigate the influence of brain-derived soluble factors on microglial activity. This was accomplished by culturing brain slice organ cultures on a semipermeable membrane across form underlying BV2 cells, a murine microglia cell line. The BV-2 microglia cells were then analyzed for various hallmarks of microglia activation such as phagocytosis, MHC class II expression, and TNF-alpha production. These classical markers of activation have generally been thought to all increase upon stimulation. The results of these studies showed that in the presence of brain slices, the BV-2 microglia cells increase their phagocytic capability, as assessed by DiI-acLDL uptake. However, under these same coculture conditions, MHC class II and LPS stimulated TNF-alpha expression is decreased. This bidirectional effect of brain slices on BV-2 microglia cells was selective since it was not observed in the RAW264.7 macrophage cells when they were cocultured with brain slices. The present findings suggest that soluble brainderived factors can alter the function of microglia cells, but the response of microglia is uncharacteristic in that it is not uniform for classical activation markers. This study reveals a communication process that regulates microglial activity and can influence their contribution to neurodegenerative diseases. (Supported by VA Merit Review Grant.) In Utero Exposure to Immunostimulation Selectively Disrupts Cognitive Indices in Adult Offspring: A Plausible Model for Testing the Viral Hypothesis of Schizophrenia. Guy Shakhar, Lee Zuckerman, Shamgar Ben-Eliyahu, and Ina Weiner, Department of Psychology, Tel Aviv University, Tel Aviv 69978, Israel. Various findings suggest that schizophrenia is a neurodevelopmental disorder associated with abnormal embryonic developmental. Epidemiological studies have associated schizophrenia with maternal exposure to viral infection. Since cytokines, and the corticosteroid response they elicit, have been shown to disrupt neurodevelopment, it can be hypothesized that elevated cytokine levels, caused by viral infec-
tions during pregnancy, may be a factor in the etiology of schizophrenia. To test this hypothesis in rats, we studied the effects of maternal immunostimulation on latent inhibition (LI) in the adult offspring. Disrupted LI has received increasing recognition as a model of a cognitive deficit in schizophrenia, namely, the inability to ignore insignificant stimuli. LI is impaired in subtypes of schizophrenic patients and in rats treated with amphetamine. Poly-IC, which mimics the immune response to viral infection, was administered intravenously (4 mg/kg) during gestation day 15. Compared to salinetreated controls, Poly-IC caused anorexia for approximately one day, without significantly increasing miscarriage rate. At adulthood, male offspring failed to show LI, i.e., were not affected by previous learning about the insignificance of the stimulus. In addition, these rats were not affected by a reversal of stimulus-reinforcement contingencies in a reversal discrimination task. In contrast, spatial recognition in the Morris water maze was intact in these rats. These findings suggest that prenatal antiviral immune response can lead to selective long-term cognitive deficits which may be viewed as a loss of impact of past experience on present learning, and we suggest a model for testing the viral hypothesis of schizophrenia. Postoperative Pain Management: Effects on the Immune system. Yehuda Shavit,* Benzion Beilin,† Hanna Bessler,‡ Evelyn Trabekin,§ and Boris Mordashov,† *Department of Psychology, Mount Scopus, Hebrew University, Jerusalem 91905; †Department of Anesthesiology and ‡Research Institute, Golda Campus, Rabin Medical Center, Petah Tiqva, 49372; and §Department of Anesthesiology, Schneider Medical Center, Petah Tiqva, 49372, Israel. Surgery-associated painful experiences result in immune suppression and increased rate of infection. Several perioperative pain management techniques are commonly used, including opiates on regular schedule, preemptive analgesia, patient controlled analgesia (PCA-IV), and patient controlled epidural analgesia (PCEA). The present study compared the effects of these pain management techniques on immune alterations in the postoperative period. Patients hospitalized for abdominal surgery were randomly assigned to one of four postoperative pain management techniques: Group 1—On regular schedule (Pethidine ⫹ Phenergan-IM); Group 2—Morphine PCA-IV; Group 3—PCEA (Bupivacaine ⫹ Fentanyl); Group 4—Preemptive epidural ⫹ PCEA. Postoperative pain was assessed using the Visual Analogue Scale. Blood samples were collected before, immediately, 24, 48, and 72 h following surgery. The following immunological assays were performed: Production of IL-1, IL-2, IL-6, and TNF; NK cell cytotoxic activity (NKCA); and mitogenic responses. Patients in the PCA, PCEA, and preemptive ⫹ PCEA groups exhibited lower pain scores compared with the regular schedule group. Mitogenic responses and NKCA were suppressed postoperatively in all groups, except for the preemptive ⫹ PCEA group. Levels of IL-1 increased in the regular schedule and PCA groups, while remained unchanged in the PCEA and preemptive ⫹ PCEA groups. Patients of the PCEA and the preemptive ⫹ PCEA groups exhibited lower pain scores and the least deleterious effects of surgery on the immune response. (This work was supported by a grant from the Ministry of Health, Israel.) Neuroendocrine Modulation of Mononuclear Cell Trafficking: Beta-Chemokine Gene Expression Is Required for Wound Healing. John F. Sheridan,*,† Raymond Tseng,* Patricia A. Guerra,* Isolde G. Rojas,* Bucky Jones,* Ning Quan,* Phillip T. Marucha,† and David A. Padgett,*,† *Department of Oral Biology, †Institute for Behavioral Medicine Research, Ohio State University Health Sciences Center, Columbus, Ohio 43210. Stress slows wound healing in humans and experimental animal models. Delayed healing has been associated with diminished inflammatory cell trafficking to the wound. Treatment of stressed animals with a glucocorticoid (GC) receptor antagonist has been shown to restore wound cellularity and healing. MIP-1alpha (a beta chemokine) has been shown to be a major signal for macrophage trafficking, and MIP-1alpha gene expression can be suppressed by GC. The purpose of this study was to examine the role of MIP-1 alpha in the trafficking of macrophages to sites of tissue damage. A 3.5-mm punch biopsy wound was made on the dorsum of MIP-1alpha knockout (KO) mice and healing was compared in KO and control mice. Wounds on KO mice healed significantly more slowly than control mice (an average of 2 days longer). On days 1 and 2 after wounding, similar histological changes were observed in KO and control mice. The early leukocyte infiltration consisted mainly of neutrophils. On day 3, numerous macrophages infiltrated control wounds, as expected, and the number of neutrophils dimin-
ished. In contrast, the KO infiltrate remained mostly neutrophilic. Measurement of wound tissue myeloperoxidase (a surrogate measure of neutrophil infiltration) showed significantly higher amounts in KO wounds on day 5. MIP-1alpha levels in control wounds reached 6000 pg/ml on day 3, which correlated temporally with macrophage infiltration. KO wounds were negative for MIP-1alpha and few macrophages were present. These data suggest that MIP-1alpha is key factor in macrophage recruitment into wounds, and that appropriate tissue repair requires macrophage recruitment. (This research was supported by NIH Grants MH46801, DE13749, and the John D. and Catherine T. MacArthur Foundation Mind– Body Network.) Suppression of the Immune Response to HSV-1 Following Acute Administration of Morphine. Patricia A. Sheridan* and Jan A. Moynihan,† *Interdepartmental Program in Neuroscience and †Department of Psychiatry and Microbiology and Immunology, Center for Psychoneuroimmunology Research, University of Rochester, Rochester, New York 14642. Previous studies have shown that the administration of exogenous opioids such as morphine, as well as increases in endogenous opioid levels, modulate T-cell, B-cell, natural killer (NK) cell, and macrophage functions. However, few studies have examined the impact of acute administration of morphine on the immune response to a replicating viral antigen. In this study, we investigated the effect of a single acute exposure to morphine on the immune response to a herpes simplex virus-1 (HSV-1) infection. Male Balb/cByJ mice received either morphine (10 mg/kg) or saline injected subcutaneously and were infected intraperitoneally with 4.8 ⫻ 107 plaque forming units of HSV-1 Patton strain 4 h after the administration of drug or vehicle. Mice were sacrificed 24 h or 3 days post infection (p.i.) and spleens were removed and cultured with UV-inactivated HSV-1. The supernatants were collected and interferon (IFN)-gamma and interleukin (IL)-10 protein production was assayed using cytokine specific ELISAs. An NK cell assay was also performed with splenocytes from mice 3d p.i. using YAC-1 target cells. A significant suppression of IFN-gamma and IL-10 production was observed for the morphine treated mice; morphine treated mice also had significantly decreased splenic NK cell activity. The results of this study are clinically important because they indicate that even a single exposure to morphine around the time of infection can significantly alter the immune response to the pathogen. [Research supported by the Fetzer Institute and NIMH Training Grant (T32 MH18822).] Virus-Induced Corticosterone Release through CRH-Independent Pathways. Marni N. Silverman,* Bradley D. Pearce,* Melanie C. Ruzek,† Katia Karalis,‡ Maria Venihaki,‡ Christine A. Biron,† and Andrew H. Miller,* *Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA 30322; †Department of Molecular Microbiology and Medicine, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912; and ‡Division of Endocrinology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115. From studies using purified cytokines or cytokine inducers, activation of hypothalamic corticotropin releasing hormone (CRH) is considered to be a primary mechanism by which cytokines stimulate the HPA axis with the resultant release of corticosterone (CORT). Few studies have examined the neuroendocrine pathways by which cytokines induce CORT during viral infection. Our previous data have demonstrated that murine cytomegalovirus (MCMV) infected mice exhibit a peak CORT response 36 h postinfection. This CORT response is mediated by IL-6 and is essential for protection against cytokine (TNF)-mediated lethality during MCMV infection. To determine the role of CRH in mediating MCMVinduced CORT, ACTH, and CORT responses were characterized in mice genetically deficient in CRH (CRH KO). In addition, induction of CRH mRNA expression was examined in the PVN of normal infected mice. At 36 h after infection, CRH KO mice did not exhibit the increase in ACTH (65 ⫾ 14 pg/ml) seen in infected wild type mice (246 ⫾ 56 pg/ml). This suggests that MCMV may drive the HPA axis through CRH dependent release of ACTH. However, this pathway is not absolutely required for CORT release, since infected CRH KO mice exhibited a CORT response (220 ⫾ 57 ng/ml) comparable to that exhibited by infected wild type mice (217 ⫾ 74 ng/ml). Moreover, no increase in CRH mRNA expression was detected at 36 h postinfection in the PVN of normal infected (607 ⫾ 45 nCi/g) versus uninfected mice (615 ⫾ 49 nCi/g). Taken together, these data suggest that although the classic CRH to ACTH to CORT pathway may be activated during MCMV infection, CORT responses to MCMV are not strictly dependent upon CRH. (Support provided through Grants MH47674 and NIDDKR0171261.)
Interleukin 10 Is a Mediator in the Hypothalamic–Pituitary–Adrenal Axis. Eric M. Smith,*,† Patrick Cadet,‡ George B. Stefano,‡ Mark R. Opp,* and Thomas K. Hughes, Jr.,† *Department of Psychiatry and Behavioral Sciences, †Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555; and ‡Neuroscience Research Institute, College at Old Westbury, State University of New York, Old Westbury, New York 11568. Interleukin-10 (IL-10) is a broadly active cytokine that inhibits IL-1 and tumor necrosis factor activities in the immune system. We have shown that IL-10 is produced in pituitary, hypothalamic, and neural tissues in addition to lymphocytes, and we have wondered what IL-10’s role is in these tissues. RTPCR was used to show that the IL-10 receptor mRNA was expressed in AtT20 pituitary tumor cells, so the neuroendocrine effects are likely mediated by specific receptors. In experiments to determine the function of IL-10 in the HPA axis, we found that IL-10 enhances corticotropin releasing factor (CRF) and corticotropin (ACTH) production in hypothalamic and pituitary tissues, respectively. Antibody to IL-10 decreased the amount of ACTH produced constitutively by AtT20 pituitary tumor cells and that induced with corticotropin releasing factor (CRF). The inhibition may be at the level of transcription, since IL-10 inhibits AP-1 and NF kappa B transcription factors’ activity in the AtT20 cells. IL-10’s effects may also be indirect in part. Treatment of AtT20 cells with IL-10 enhanced the expression of macrophage migration inhibitory factor (MIF) mRNA. Treatment of rat median eminence (ME) fragments with IL-10 for 10 min specifically stimulated CRF release. Our other data show that IL-10 has an effect on the endogenous corticosterone response in both immune and physiologic stress situations. Thus in addition to its more widely recognized role in immunity, IL-10’s neuroendocrine activities described here point to its role as an important regulator in communication between the immune and neuroendocrine systems. Potential Benefits of a Physically Active Lifestyle on the Immune Response to KLH. T. S. Smith, S. L. Kennedy, D. T. Smith, and M. Fleshner, Department of Kinesiology and Applied Physiology, University of Colorado, Boulder, Colorado 80309. The aging immune system is characterized by a progressive decline in the responsiveness to exogenous antigens and tumors in combination with a paradoxical increase in autoimmunity. Moreover, older chronically ill populations may be the most at risk for compromises in immune function. Regular moderate exercise may offset some of the immune function abnormalities found in healthy and chronically ill older populations. We decided, therefore, to test the effects of regular exercise on the in vivo specific antibody and delayed type hypersensitivity (DTH) responses to a benign protein in young, older, and older chronically ill populations. Subject groups were divided according to age (young 18–35 and old 60–75), training status (trained and untrained), and chronic illness (healthy and stable coronary heart disease). Subjects were intramuscularly injected with 100 mg of KLH. Three weeks later, all subjects were injected intradermally with KLH (1.0 mg). The diameter of the skin DTH response was measured 24 and 48 h after challenge. Blood draws were obtained at day 0, 7, 14, 21, and 28 after KLH. AntiKLH IgG and IgM were measured using ELISA. Anti-KLH DTH response was reduced 60–80% in the older subjects compared to the younger subjects. Exercise training increased the anti-KLH DTH response in the young subjects. Data for the older subjects and antibody data are currently being collected. Thus, the use of KLH in the human population will allow us to address whether regular exercise helps offset the negative effects of aging and chronic illness on the immune system. (AI45576, MH60301.) Distressing Life Events, Hostility, CD4⫹ Cell Decline, and Viral Load Increase in HIV⫹ Persons. George F. Solomon,* Gail Ironson,†,‡ Elizabeth Balbin,† and Mary Ann Fletcher,†,§ *Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, California 90095; and †Department of Psychology and Behavioral Medicine, ‡Department of Psychiatry, §Department of Medicine, University of Miami, Miami, Florida 33124. Objective. This study sought to determine whether stressful life events, the reaction to them, and the emotional context in which they occurred had an impact on progression of HIV disease as measured by change in CD4⫹ cell count and viral load. Methods. 140 HIV⫹ subjects with initial CD4⫹ cell counts mostly between 150–400/mm3 were evaluated on a number of psychological instruments, CD4 levels and viral load and were reevaluated 6 months later for changes in those prognostic markers. Relevant psychological instruments included an Impact of Life Events Scale and the Cook Medley Hostil-
ity Scale. Results. Although the occurrence of major life events did not predict CD4⫹ cell change, life events with a distressing impact significantly did predict decline in CD4⫹ cells 6 months later (r ⫽ .19, p ⫽ .025). Similarly, distressing life events predicted an increase in viral load controlling for change in CD4 count (partial r ⫽ .22, t ⫽ 2.62, p ⫽ .01) The prevalence of distressing life events within the 6 months prior to measurement was 42%. Major life events reported in this subsample were: major financial difficulties (85%), death of loved one or close friend (46%), and difficulties in a primary relationship (39%). In the subsample with distressing life events, hostility significantly predicted decline in CD4⫹ cells (r ⫽ .33, p ⫽ .02), but did not predict change in viral load. Conclusions. Distressing life events in the context of a hostile response diathesis have a negative impact, at least short term (6 months), on markers of disease progression in HIV seropositive persons. (This research was funded by NIH R01 MH 53791-03, Gail Ironson, P.I.) Increases in the Gene Expression of CRF and Proinflammatory Cytokines in Depression Model of Rats. Cai Song,*,† B. E.Leonard,* and S. Rivest,† *Pharmacology Department, National University of Ireland, Galway, Ireland; †Laboratory of Molecular Endocrinology, CHUL Research Centre, Laval University, Quebec, Canada. An increase in the release of interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha; have been reported in the blood and CSF of depressed patients. These cytokines stimulate the HPA axis to secrete CRF and cortisol, and change neurotransmission. However, the molecular connection between immune and neuroendocine is unclear. In this study, using in situ hybridization method, the expression of TNFalpha mRNA was found in the subcortical organ, median eminence, and area of postrema after lipopolysaccharide (LPS) challenge. However, in olfactory bulbectomized (OB) rats, a rodent model of depression, this expression was significantly stronger in these brain regions. The expression of CRF mRNA in the paraventricular nucleus of the hypothalamus was triggered by the LPS administration. This gene expression was also significantly stronger in OB rats compared to sham operated animals. In the OB rat, endotoxin significantly enhanced the expression of IkB mRNA an inhibitory factor for the transcription of inflammatory cytokines in the endothelium of the brain blood vessels and parenchymal microglia. In addition, the LPS challenge significantly elevated plasma concentrations of corticosterone in both sham and OB rats. However, this increase in OB rats was significantly greater and lasted longer than in the sham operated controls. This study demonstrated that an immune challenge may activate the gene expression of CRF and proinflammatory cytokines in the brain before activating the HPA axis and changing neurotransmission. The stronger gene expressions that were observed in OB rats may indicate that the lesion or change in the limbic system synergistically increases inflammatory transcription in the brain. (This study was supported by Laxdale Ltd., Scotland Company, UK.) Comparing Effects of IL-1 and IL-2 Caused Changes in Behavior, Endocrine, and Neurotransmitter Release in the Rat. Cai Song,* Z. Merali,† T. Connor,‡ B. E. Leonard,‡ and H. Anisman,¶ *Department of Psychology, University of British Columbia, Vancouver, Canada; †Psychology School, University of Ottawa, Canada; ‡Pharmacology Department, National University of Ireland, Galway, Ireland; and §Psychology Department, Carleton University, Ottawa, Canada. It is established that immune activation may provoke neuroendocrine and neurotransmitter changes through cytokine secretion. However, cytokines from different sources may play different role. In this study the effects of macrophage produced interleukin (IL)-1 and lymphocyte produced IL-2 on behavior, endocrine, neurotransmitter release, and immune function have been investigated. Behavioral effects: In the home cage, central IL-1beta administration resulted in freezing behavior; in a novel environment, open field, IL-1 increased locomotor activity, grooming, escape, and defecation. In the elevated plus maze, IL-1 reduced the time spent in the open arms. In Morris Water Maze, IL-1 impaired animal learning. However, IL-2 had no effect on behavior alone, but normalized some similar behavior that olfactory bulbectomized (model of depression) showed in these apparatuses. Endocrine and immune aspects: IL-1 significantly elevated plasma concentration of corticosterone and suppressed lymphocyte proliferation. IL-2 did not change corticosterone concentration, but increased lymphocyte proliferation. Neurotransmitter releases: systemic IL-1 administration significantly elevated the 5-HIAA and HVA concentrations in the nucleus accumbens of the rat. Application of a mild stressor to the IL-1 treated rat synergetically enhanced 5-HIAA and HVA releases. By contrast, IL-2 significantly reduced the release of 5-HIAA, DA, and HVA. These effects were exacerbated upon application of the stressor. However,
at the dose of 1 µg, the animals treated with IL-1 and IL-2 together showed the same changes as results caused by IL-1 treated alone. This study demonstrated that IL-1 and IL-2 have many opposite effects, while IL-2 could not antagonize IL-1 effects in the nucleus accumbence. (This study was supported by Ontario Mental Health Foundation and Laxdale Ltd., Scotland, UK.) Plasma C4a Levels Are Elevated after Exercise in Patients with Chronic Fatigue Syndrome. Bristol Sorensen,* James F. Jones,† and Monika Fleshner,* *Department of Kinesiology and Applied Physiology, University of Colorado at Boulder, Boulder, CO 80309; and †National Jewish Medical and Research Center, Denver, Colorado 80306. Patients with Chronic Fatigue Syndrome (CFS) often report an increase in flu-like symptoms and malaise after exertion. We used an exercise challenge in an attempt to induce these symptoms. We measured levels of complement split products in the plasma over 24 h. We also evaluated changes in patient reported symptoms 2 weeks prior to and 1 week postexercise. Fifteen subjects with CFS and 15 age-matched, normal controls exercised for 20 min on a stationery bike at 60% of their predicted max workload in Watts. Blood was drawn immediately before, immediately postexercise, and 1, 6, and 24 h postexercise. RIAs were performed for plasma C3a, C4a, and C5a levels. Mean pre- and postexercise symptom scores were evaluated for each group. C4a levels were higher in CFS subjects at the 6 h postexercise time point. CFS patients also had an increase in flu-like symptoms after exercise compared to controls. Elevations in C4a indicate classical pathway activation. The observation that C4a is elevated 6 h postexercise suggests an altered host response. Whether there is an increased production or an inadequate clearance of C4a is unknown. The consequence of increased C4a in the periphery and/or the central nervous system is also unknown. Effect of Methionine-Enkephalin and Opioid Antagonists on Local Inflammatory Reaction in AO Rats. Stanislava Stanojevic,* Mirjana Dimitrijevic,* Tatjana Miletic,* Vesna Kovacevic-Jovanovic,* Vesna Vujic-Redzic,† and Jelena Radulovic,‡ *Institute of Immunology and Virology Torlak, Immunology Research Center Branislav Jankovic, Belgrade, Yugoslavia; †Institute of Chemistry, School of Medicine, Belgrade, Yugoslavia; and ‡Max Planck Institute for Experimental Medicine, Department of Molecular Neuroendocrinology, Goettingen, Germany. We have reported that methionine-enkephalin (met-enk) differentially influenced peroxide production in macrophages of DA and AO rats. Additional in vivo experiments showed that local administration of met-enk suppressed Concanavalin A (Con A)- induced paw edema in DA rats, while naloxone and naltrindole did not antagonize that effect. Taking into account strain-related differences in opioid sensitivity, the present experiment investigated the effect of met-enk and opioid antagonists on local inflammatory reaction in AO rats. AO rats were given intraplantar (ipl.) injection of (a) 0, 0.1, 1, 10, and 100 µg of met-enk mixed with 400 µg of Con A; (b) 0 and 100 µg of naloxone or naltrindole followed by 100 µg of met-enk and/or 400 µg of Con A; and (c) 0 and 100 µg of naloxone or naltrindole; or 100 µg of met-enk. The hind paw diameters of tarsometatarsal joints were measured by means of constant tension caliper. The results showed that (a) 100 µg of met-enk ipl. potentiated Con A induced paw inflammation, (b) naloxone antagonized the effect of met-enk and did not influence inflammatory edema when given alone, (c) naltrindole had additive effect with met-enk and increased the diameters of inflamed paws when injected alone, and (d) only naltrindole induced edema in noninflamed paws. It may be concluded that met-enk potentiated Con A induced paw inflammation, naloxone exerted pure antagonistic, and naltrindole displayed partial agonistic activity in AO rats. Met-enk and opioid antagonists showed different effects on paw inflammation in two inbred rat strains. (Supported by the Ministry for Science and Technology, Republic of Serbia.) Social Stress Induces Glucocorticoid Resistance in Murine Splenic Macrophages. J. L. Stark,* R. Avitsur,† K. A. Campbell,‡ D. A. Padgett,†,‡,§ and J. F. Sheridan,†,‡,§ *Neuroscience Graduate Studies Program, †Section of Oral Biology, ‡Department of Molecular Virology, Immunology, and Medical Genetics, and §Institute for Behavioral Medicine Research, Ohio State University, Columbus, Ohio 43210. Although stress-induced increases in systemic glucocorticoids generally have immunosuppressive effects, previous studies from this laboratory indicate that chronic social stress of male C57BL/6 mice
enhances inflammatory responses. To test the hypothesis that peripheral immune cells become less sensitive to corticosterone, mice were exposed to six 2-h evening cycles of social disruption stress (SDR). Splenocytes from SDR and control mice were cultured with LPS and multiple concentrations of corticosterone. Cell proliferation was measured using a nonradioactive commercial assay and cytokines in cell supernatants were assessed by ELISA. Following stimulation with LPS, cells from SDR mice exhibited decreased sensitivity to the antiproliferative effects of corticosterone. In addition, there was enhanced IL-6 production by these cells. Since LPS stimulates both B cells and macrophages, experiments were undertaken to determine which cell type(s) was resistant to corticosterone. B cells were depleted from splenocyte suspensions using anti-CD19 magnetic beads, while macrophages were removed with antiCD11b beads. Following LPS stimulation, the B cell-deficient population remained resistant to corticosterone, whereas the macrophage-deficient population became corticosterone sensitive. Furthermore, in the macrophage-depleted cultures, splenocytes from SDR mice produced 10-fold less IL-6. These results demonstrate that chronic social stress makes splenic macrophages less susceptible to inhibition by corticosterone. This decreased sensitivity to glucocorticoids may underlie the enhanced inflammatory responses observed during in vivo infectious challenges of SDR mice. [Supported by grants from the NIMH (F31-MH11792 to J.L.S. and RO1-MH46801-08 to J.F.S.) and the John D. and Catherine T. MacArthur Foundation Mind–Body Network.] Behavioral, Hormonal, and Immunological Consequences of Colony Housing in Long Evans Rats. Volker Stefanski and Gunther Knopf, Silke Schulz University of Bayreuth, Department of Animal Physiology, 95440 Bayreuth, Germany. The effects of colony formation and colony housing on several endocrine and immunological measures were investigated in Long Evans rats. The study aimed to elaborate the impact of a chronic stressful situation on the immune system of individuals living under a relatively natural social environment. Three rat colonies, each containing 5–6 males and 5–6 females, were observed over 10 weeks. The colonies were established by joining together one male, one female pair groups at an age of about 3 months. Nineteen pair groups served as controls. Blood samples were taken weekly from all males plus additional four precolony samples. Immunological measurements included enumeration of blood immune cells (all major leukocyte and lymphocyte subsets) as well as lymphocyte proliferation assays. Glucocorticoids, catecholamines, and CBG titers were determined every second week. Agonistic and sexual behavior elements of all individuals were recorded. Many immunological differences became evident between colony and control males. Colony males had higher granulocyte and lower CD4 T cell counts as well as a reduced lymphocyte proliferative response. Catecholamine concentrations were elevated in the colony males, while glucocorticoid titers were unaffected. Most changes occurred quickly after colony formation and persisted until the end of the study. A clear dominant position of one male emerged in all colonies. Although these dominant males displayed fundamentally different behavior and body weight development, most immunological changes were relatively similar to subdominant males. This study shows that major and long-lasting immunological changes occur under relatively natural social conditions. (Supported in part by the Volkswagen Foundation.) Gene Expression of N-Acetyltransferase (NAT) and Hydroxyindol-O-Methyltransferase (HIOMT)—The Key Enzymes in Melatonin Synthesis—in Lymphoid Organs: Comparison with Other Tissues. Jasminka Stefulj,* Michael Hoertner,* Meenakshi Ghosh,* Albert Woelfler,* Ingo Rinner,* Johann Semmler,† Konrad Schauenstein,* J. Ross Stevenson,* and Peter M. Liebmann,* *Department of General and Experimental Pathology, University of Graz, Austria; and †Department of Medical Biochemistry, University of Graz, Austria. Besides in the pineal gland, melatonin (MEL) has been shown to be produced in cells of the retina, gastrointestinal tract, reproductive organs, and the immune system, where it could act as intracellular mediator or paracrine signal in addition to its endocrine effects. In order to compare the potential for MEL synthesis of lymphoid organs with that of other extrapineal tissues we used semiquantitative RTPCR to determine the tissue distribution of mRNA encoding HIOMT, i.e., the enzyme catalyzing the last step of the MEL biosynthesis. At 35 amplification cycles positive signals were obtained with RNA from gut, muscle, brain cortex, striatum, liver, stomach fundus, and spinal cord. Although all tested tissues gave identical intensities of the RT-PCR product of a housekeeping gene at 30 cycles, RNA from spleen, thymus, platelets, and lung yielded detectable HIOMT signal only after 45 PCR cycles. These
results show HIOMT mRNA to be present in a wide range of tissues, corroborating and extending the notion of extrapineal MEL production. However, comparatively low levels of HIOMT mRNA may suggest a limited significance of MEL synthesis in lymphoid organs. [Supported by the Austrian Science Foundation (P12679-med).] Effects of Alpha-Adrenergic Stimulation on L-Selectin Expression in Different Immune Compartments in the Rat Model. J. Ross Stevenson, Peter M. Liebmann, Michael Hoertner, Ingo Rinner, Peter Felsner, Albert Woelfler, and Konrad Schauenstein, Institute for Pathophysiology, University of Graz, Graz, Austria. L-selectin is responsible for initial adhesion of leukocytes on vascular endothelium as a prerequisite for lymph node specific homing of T-lymphocytes and migration of granulocytes into sites of inflammation. As alpha-adrenergic stimulation is known to cause redistribution of leukocytes, the present experiments were undertaken to investigate whether this redistribution is correlated with changes in L-selectin expression on leukocytes. Toward this, we measured this molecule by flow cytometry on leukocytes 12 h after rats were given implants of tablets that release noradrenalin and propranolol, or of tablets without drugs. The results were as follows: (1) Mean L-selectin level on B cells, CD8⫹ T cells, and granulocytes and the percent of NK cells expressing L-selectin in the blood were lower in the hormone treated than placebo treated rats. (2) The percent of all classes of lymphocytes that expressed L-selectin was lower in the peripheral lymph nodes after the alpha-adrenergic treatment. This was especially true of the T cells. (3) The percent of all classes of lymphocytes expressing L-selectin was higher in the spleen after hormone treatment. Conclusion. The observed redistribution under alpha-adrenergic stimulation seems not to be explained by changes in the expression of this adhesion molecule. Other factors must also be involved. [Supported by the Austrian National Bank (P6730) and Austrian Science Foundation (P12679).] Prolonged Alpha-Adrenergic Treatment of Rats Leads to a Loss of Lymphocytes in Central and Peripheral Lymphoid Organs Due to Apoptosis. J. Ross Stevenson,* Peter Felsner,* Juergen Westermann,† Peter M. Liebmann,* Michael Hoertner,* Ingo Rinner,* Albert Woelfler,* and Konrad Schauenstein, *Institute for Pathophysiology, University of Graz, A-8010 Graz, Austria; and †Department of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany. Previously, we have shown that catecholamines suppress the reactivity of PBL in the rat by alphamediated mechanisms. Here we investigated the effects of alpha-adrenergic treatment on the distribution of leukocyte subsets in different immune compartments. We found that following a 12-h treatment with s.c. implanted tablets containing noradrenaline and propranolol, blood leukocyte counts increased due to increased numbers of granulocytes and NK cells, while absolute numbers of total lymphocytes were significantly diminished. This loss in lymphocytes was likewise observed in the spleen, thymus, and lymph nodes. Use of the TUNEL technique on tissue sections revealed an increased number of apoptotic cells in all lymphoid organs due to alpha-adrenergic treatment. In the spleen this enhanced apoptosis was confined to the white pulp suggesting that the effect is selective for lymphocytes. Furthermore, in spleen and lymph nodes apoptosis induction was most prominent within germinal centers and accompanied by a significant reduction of germinal center areas in these organs. We conclude that the quantitative changes in lymphocytes as observed after alpha-adrenergic in vivo stimulation are primarily due to induction of lymphocyte apoptosis, rather than redistribution events. [Supported by the Austrian National Bank (P6730)and Austrian Science Foundation (P12679).] Coping Styles, Perceived Stress Levels, and T-lymphocyte Responses to Mitogens. Jeffrey R. Stowell,* Janice K. Kiecolt-Glaser,* and Ronald Glaser,† *Department of Psychiatry and †Department of Medical Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210. Coping style is thought to moderate the impact of stress on the immune system. Data from current and former caregivers of dementia patients (n ⫽ 102) and comparison controls (n ⫽ 83) were used to determine the relationships among coping style, perceived stress, and the blastogenic responses of peripheral blood lymphocytes to the mitogens PHA and Con A. Findings suggest that coping styles have greater effects on these T-cell responses when the person is experiencing a high level of stress. Consistent with previous findings, caregivers reported greater perceived stress on the Perceived Stress Scale than controls,
and females reported greater perceived stress and use of emotion-focused coping on the COPE than males. Perceived stress was not directly related to the immune measures, but it moderated the influence of coping style on the immune measures. Thus, the active coping styles of problem solving and mental accommodation were associated with a more vigorous blastogenic response under conditions of high stress than low stress. Avoidance coping, generally considered a maladaptive coping style, also had a greater effect on the response to PHA and Con A under conditions of high stress, and was associated with a poorer T-cell response. These results suggest that coping styles may be able to influence some immune responses, with active coping style associated with a greater T-cell response to mitogens and avoidance coping style associated with a less vigorous response, particularly under conditions of high stress. (Supported by MH42096.) The Effect of Antidepressants on Behavioral and Immunological Measures in the Wistar–Kyoto Rat. Kimberly A. Szandrowski,* William P. Pare,† and Shanaz M. Tejani-Butt,* *Department of Pharmacology and Toxicology, University of the Sciences in Philadelphia, Philadelphia, PA 19104; and †Veterans Administration Medical Center, Perry Point, Maryland 21902. The Wistar–Kyoto (WKY) rat is ulcer prone and exhibits depressive behavior when exposed to various stress paradigms. We have also found that stress alters immune measures in this rat strain, similar to those reported for clinical depression. In this study, the effects of repeated antidepressant drug treatments on behavioral and immune measures were examined. Rats were treated with desipramine, a norepinephrine uptake blocker, nomifensine, a norepinephrine and dopamine uptake blocker, paroxetine, a serotonin uptake blocker, or saline for 12 days. On days 10 and 11, the Porsolt forced-swim test was conducted. On day 13, rats were exposed to a 2-h cold-water restraint before sacrifice. Trunk blood, spleen, thymus, and adrenals were collected, and stomachs were examined for ulcers. While desipramine and nomifensine decreased immobility and increased swim time, paroxetine was without effect in the Porsolt forcedswim test, indicating that depressive behavior in WKY rats may be modified by antidepressants that alter synaptic levels of norepinephrine and/or dopamine but not serotonin. Desipramine decreased ulcer scores; desipramine and paroxetine reduced thymus weight ( p ⬍ .05) with no effect on adrenal weight. Desipramine reduced the stress effect on neutrophil and lymphocyte counts ( p ⬍ .05) compared to nomifensine and paroxetine. Stress decreased splenocyte response to Con A and LPS (p ⬍ .025 and p ⬍ .01, respectively). While desipramine alone reversed the stress effects on Con A stimulation, all three drugs reversed the stress effects on LPS stimulation. Thus, the results indicate that antidepressant drugs not only reverse depressive behavior in WKY rats, but they also reverse immune alterations as a result of stress. Stressful Life Events and Size of Social Network Predict Delayed Type Hypersensitivity Response among Women with Metastatic Breast Cancer. Julie M. Turner-Cobb,*† Joshua Denison Rabinowitz,* Cheryl Koopman,* Sandra E. Sephton,*‡ and David Spiegel,* *Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305-5718; †Department of Psychology, University of Kent at Canterbury, Canterbury, Kent CT2 7LZ, United Kingdom; and ‡Department of Psychiatry and Behavioral Sciences, University of Louisville School of Medicine, Louisville, Kentucky 40292. This study examined the relationships between stressful life events, social support, and antigen-specific cell-mediated immunity using the delayed type hypersensitivity (DTH) response measure. Participants were 84 women drawn from a larger sample of 125 women with documented metastatic breast carcinoma recruited into a randomized, prospective study of the effects of supportive–expressive group therapy on cancer survival. At baseline, prior to randomization, all participants completed standardized psychosocial measures of life stress (using the weighted Life Events Questionnaire) and social support (using the Yale Social Support Index and the Single Item Measure of Social Support), and their antigen specific immune response was measured using the delayed type hypersensitivity (DTH) test to seven skin test antigens. No main effect was found for life stress or social support with number of positive antigens. However, as hypothesized, the interactive effect of life stress and social support network size with number of positive antigens to the DTH test was significant. Number of positive antigens to the DTH test was greater for those women who had experienced a high frequency of life events but who reported a larger network of support. For those women who had experienced fewer stressful life events and who reported larger social networks, the opposite relationship was observed, revealing a weaker antigen specific immune response. These results are consistent with the notion that social support may concur immune
benefits under high stress conditions. Implications of this interaction for cancer are discussed, including the influence of possible acute stress reactions to the DTH test. A New Model for Neurodegeneration: Silencing of Survival Signals (SOSS). Homer D. Venters,* Qiang Liu,* Roger VanHoy,* Robert Dantzer,† and Keith W. Kelley,* *Laboratory of Immunophysiology, Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801; and †INRA-INSERM U394, Institute Franc¸ois Magendie, Rue Camille Saint-Sae¨ns, 33077 Bordeaux Cedex, France. Increased expression of both the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), and the survival peptide, insulin-like growth factor-I (IGF-I), occurs in numerous diseases of the central nervous system (CNS), including Alzheimer’s disease, multiple sclerosis, the AIDS-dementia complex and cerebral ischemia. Traditionally, IGF-I has been characterized as neuroprotective and TNF-alpha neurotoxic. In contrast to conventional, unrelated roles for IGF-I and TNF-alpha in the CNS, we hypothesized that the signaling pathways of these two cytokines may interact during neurodegeneration. In this model, silencing of survival signals (SOSS) would constitute a new role for TNF-alpha in neurodegeneration. Here we show that concentrations of TNF-alpha as low as 10 pg/ml markedly inhibit the ability of IGF-I to promote survival of primary murine cerebellar granule neurons (CGN). TNF-alpha reduces IGF-I-induced tyrosine phosphorylation of insulin receptor substrate-2 (IRS-2) and suppresses IRS-2precipitable phosphatidylinositol 3′-kinase (PI 3-kinase). Preliminary data suggest that SOSS may be mediated by the p55 TNF receptor. These experiments indicate that TNF-alpha promotes IGF-I receptor resistance in CGN and inhibits the capacity of the IGF-I receptor to tyrosine phosphorylate the IRS-2 docking molecule and to subsequently activate the downstream survival enzyme PI 3-kinase. This crosstalk between discrete cytokine receptors indicates that during SOSS-induced neuronal degeneration, a proinflammatory cytokine inhibits receptor signaling by a survival peptide. Assessment of Gingival Crevicular Fluid Constituents a Novel Tool in Psychoimmunology. Bernd Waschul and Renate Deinzer, Department of Medical Psychology, Heinrich-Heine-University, Dusseldorf, Germany. The present poster delineates the method of gingival crevicular fluid (GCF) sampling as a novel tool in PNI research. Assessment of immune parameters in GCF is a very common method in dentistry. It may, however, be also of interest in psycho-neuro-immunological research. Most important, GCF analysis allows for noninvasive assessment of local immune responses to bacterial challenges. Crevicular fluid sampling commonly is done by a filter paper strip inserted into the gingival crevice of a selected tooth for a certain period of time (mostly less than 30 s). Samples can be taken with high specificity at sites of gingival or periodontal inflammation. GCF contains virtually all immune parameters of interest in psycho-neuro-immunology. Currently in our lab we analyze the effects of academic stress and public speech on crevicular interleukin-1-beta, interleukin-1-receptor antagonist (Il-1-RA), prostaglandin E 2 (PGE 2), and immunoglobulin A (IgA) by high sensitive ELISA and EIA kits. It seems that these parameters are of special importance in the pathogenesis of periodontitis. Recently, in a controlled study we already could show that subjects under academic stress and when challenged with continuous plaque accumulation showed a much stronger increase in crevicular Il-1beta than control subjects without academic exams. Our studies give but one idea of the potentials of GCF assessment in PNI; we suggest, if it comes to the analysis of local immune responses, GCF assessment may be a promising new tool in psychoimmunology. Alteration of Cytokine Production, Leukocyte Function, and Induction of Cachexia in Rats Physically Dependent on Heroin. Richard J. Weber,* Ricardo Gomez-Flores,* J. E. Smith,† and Thomas. J. Martin,† *Department of Biomedical and Therapeutic Sciences, Section of Medical Sciences, University of Illinois College of Medicine, Peoria, Illinois; and †Bowman Gray School of Medicine, Wake Forest University, Winston–Salem, North Carolina. Heroin addicts exhibit an increased incidence of infectious disease, which is thought to be due to decreased immune function. In rats made physically dependent by intravenous injections of heroin, we observed a significant 1.5- to 2.3-fold suppression of splenic T lymphocyte proliferative responses. In addition, increased production of nitric oxide and TNF-alpha (2.2- and 6-fold, respectively), was observed in the heroin-treated group. Plasma TNF-alpha levels in heroin-treated rats were four times higher than
the saline-treated controls. Plasma endotoxin (4.65 EU/ml) was detected in heroin-treated animals, supporting the existence of infection. Natural killer cell activity was not affected by heroin treatment, and plasma levels of interferon-alpha, and the interleukins (IL) IL-1-beta, IL-2, IL-6, and IL-10 were normal. We also observed a significant 12-fold decrease in plasma leptin, accompanied with weight loss in herointreated animals. Heroin dependence and T-cell immunodeficiency may indicate a subclinical infection and cause the increased production of nitric oxide and TNF-alpha from macrophages, and explain the increase in plasma TNF-alpha. These immune sequelae may promote cachexia and weight loss. Immunodeficiency associated with heroin abuse may be associated with infectious diseases and weight loss in addicts and serve as a cofactor in the high incidence of AIDS in this population. (Supported by NIDA/ NIH DA12095, DA 06634, DA 00114, and DA-00247.) The Effect of Restraint Stress on the Acute Phase of Theiler’s Virus Infection. C. Jane Welsh,*,† Amy Sieve,‡ Dana D. Dean,* Ralph Storts,† Thomas H. Welsh,*,§ and Mary. W. Meagher,‡ *Department of Veterinary Anatomy and Public Health, †Department of Veterinary Pathobiology, ‡Department of Psychology, and §Department of Animal Science, Texas A & M University, College Station, Texas 77843. Stress has been implicated in the pathogenesis of multiple sclerosis (MS). Previous investigations of Theiler’s virus-induced demyelination (TVID), a mouse model of MS, have demonstrated that restraintstress reduced viral clearance and increased mortality in virus- infected CBA mice. We have also shown that restraint-stress produces elevated serum corticosterone in CBA mice. In the current study, the chronic restraint stress model was used to further investigate the effect of stress on the acute phase of TVID. Male CBA mice were restraint-stressed for 12 h overnight and then infected with 5 ⫻ 10 5 pfu of Theiler’s virus. The restraint-stress was applied for two further nights and then the mice were sacrificed. Both infected/stressed and noninfected/stressed mice suffered significant weight loss, thymic atrophy, and adrenal enlargement compared to the control groups ( p ⬍ .01). Both stress ( p ⬍ .04) and infection ( p ⬍ .05) produced a significant decrease in spleen cell numbers, with the most pronounced reductions occurring in the stressed/infected mice compared to control infected mice. Interestingly, infected/stressed mice developed enhanced NK cell activity in spleen compared to stressed/noninfected and control groups ( p ⬍ .002). Analysis of circulating white blood cells indicated a decrease in lymphocytes ( p ⬍ .007) and an increase in neutrophils ( p ⬍ .002) in the stress groups compared to the control groups. In conclusion, restraint-stress is thought to increase the mortality of mice during acute infection with Theiler’s virus, by corticosterone-induced immunosuppression which interferes with viral clearance mechanisms. (This work was supported by grants from National Multiple Sclerosis Society Grant RG 3128, Multiple Sclerosis Research Pilot Grant Initiative funded by the Dorothy and Bill Stearman Research Fund, the Texas A & M Interdisciplinary Programs Initiatives.) Mechanisms of Neuroendocrine Regulation of Experimental Autoimmune Encephalomyelitis. Caroline C. Whitacre, Kennichi C. Dowdell, Richard M. Wardrop, Ingrid E. Gienapp, and Scott Stuckman, Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210. Experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), is a demyelinating autoimmune disease of the CNS mediated by CD4⫹ T lymphocytes directed against myelin proteins. We have previously shown that restraint stress (RST) is effective in suppressing the clinical signs, relapses, histopathologic changes, and myelin-specific Th1/Th2 cytokine secretion when stress is initiated prior to EAE challenge. Corticosterone (Cort) was shown to mediate disease suppression, since administration of exogenous Cort decreased EAE clinical signs while treatment with RU486 or aminoglutethimide reversed disease suppression. In this study we explored three immunologic processes through which Cort could be acting to effect suppression of EAE antigen presentation, expression of cell surface adhesion molecules and alteration of apoptosis. MHC Class II⫹ cells (B cells and macrophages) derived from restrained mice stimulated significantly less proliferation of transgenic myelin basic protein (MBP) reactive T cells than did antigen presenting cells from control nonrestrained mice. Moreover, we observed a decrease in expression of the cell surface markers CD11a and CD25 on T cells from restrained mice and fewer CD11b⫹ (macrophages) and CD4⫹ (T) cells in the CNS of RST mice. Finally, T cells from restrained mice were observed to undergo increased apoptosis as detected by TUNEL staining relative to control nonrestrained mice. Thus, these effects appear to collectively
contribute to a reduction in CNS infiltrates, decreased demyelination, and suppression of EAE clinical signs under conditions of elevated Cort. (Supported by NIH Grants AI43367, AI35960.) Cytokine Production and Natural Killer Cell Activity in Women Stressed by Breast Cancer Diagnosis. Linda Witek-Janusek*,† and Herbert L. Mathews,*,‡ *Center for Biobehavioral Immunology, †Department of Maternal Child Health, Niehoff School of Nursing, and ‡Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago Medical Center, Maywood, Illinois 60153. Undergoing breast biopsy for cancer diagnosis is an emotional experience that may impair immune function. This study determined the effect of psychological state on cytokine production and natural killer cell activity (NKCA) in women pre- and post-breast biopsy. Perceived stress, anxiety, and mood disturbance were heightened pre-biopsy in women anticipating biopsy of their breast compared to control women not undergoing biopsy. Post breast biopsy, stress, anxiety, and mood disturbance normalized. NKCA was depressed both pre- and postbiopsy compared to nonbiopsied control women. However, postbiopsy NKCA was greater than prebiopsy NKCA in the same women. IL-6 production by peripheral blood mononuclear cells of women in the biopsy group was increased both pre- and postbiopsy compared to control women. Conversely, IFN-g, IL-4, and IL-10 were depressed pre- and postbiopsy relative to control women. No change in IL-2 production was observed. Thus, impending breast biopsy produced stress, anxiety, and mood disturbance, which was relieved postbiopsy. Associated with the experience of biopsy was depressed NKCA and altered cytokine production. These changes in immune function continued to be observed despite the normalization of psychological measures and suggest that biopsy stress has a prolonged effect on immune function. In conclusion, psychological and immune dysregulation begin early in a woman’s encounter with breast cancer diagnosis and may influence cancer control. An in Vitro Model to Study the Interaction between Neurons and Antigen-Presenting Cells. Danielle A. W. Wolvers and John Bienenstock, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada. Evidence arises that nerves and antigen-presenting cells (APC) might interact. In the skin, nerve endings in the epidermis are in close proximity to Langerhans cells (LC). In the mucosa and muscle layers of the gut and stomach, we found APC in close association with nerves. Moreover, the antigen presenting capacity of APC is influenced by neuropeptides like CGRP. To study the interaction between APC and neurons we have set up an in vitro model. Superior cervical ganglia of newborn mice are used to generate sympathetic neuron cultures. APC from different sources are then cocultured with these neurons. Both LC isolated from skin and dendritic cells (DC) isolated from lymph nodes associate with neurons in culture as demonstrated by their attachment to axons and occasionally neuronal cell bodies. APC do not attach to the culture dish without the presence of neurons nor do they attach to culture dishes covered with fibroblasts. Electron microscopic analysis revealed an intimate contact between the two cell types after 24 h of coculture, with mitochondrial presence on the neuronal side, but no further signs of specialization. Using Campenot’s compartmentalized culture system with neurons growing in a center compartment and APC added to adjacent compartments, we demonstrated an active crossing of axons into APC containing compartments after a week of culture, suggesting that a neurotrophic factor is secreted by APC. Blocking studies with antibodies to candidate neurotrophics are currently being performed to determine which factor is responsible for the neuronal outgrowth into APC containing compartments. The Effect of Stress on the Primary and Memory Immune Response to Herpes Simplex Virus (HSV) Infection at Mucosal Sites. Keith M. Wonnacott and Robert H. Bonneau, Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033. Psychological stress affects many aspects of innate and adaptive immunity. Our laboratory is specifically interested in characterizing the effects of psychological stress on the generation and function of the immune response to HSV infection. The present studies used a murine model to determine the effects of restraint stress on the development of an HSV-specific primary immune response following vaginal infection and the restimulation of a memory cytotoxic T lymphocyte (CTL) response to an intranasal infection. In the first model, female mice in the diestrous phase of the estrous cycle were infected intravaginally with HSV-2. A subset of these mice was restrained, beginning one day prior to infection, for 14
h a day for 8 days. Using this paradigm we found that restraint stress increased both viral infection and lethality indicating decreased immune-based protection in the vagina. In the second model, mice were immunized intranasally with a recombinant vaccinia virus that expresses a CTL recognition epitope from glycoprotein B of HSV (gB498-505) and one month later were challenged intranasally with HSV-2. Using this model it was previously shown that memory CTL are elicited and protect against lethal HSV2 infection. In these studies, we found that mice subjected to restraint during this intranasal HSV infection exhibited an increased level of mortality and spread of virus into the central nervous system as compared to control, nonrestrained mice. These studies suggest that stress has a potentially important role in altering immune responses to viral infections at mucosal sites. (Supported by Public Health Service Research Grant MH49616.) Underlying Mechanisms Responsible for the Increased Interleukin-6 in the Brain of Aged Mice. ShiMing Ye and Rodney W. Johnson, Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801. Interleukin (IL)-6 is increased in the brain of aged mice, suggesting that it may play a role in the neuropathophysiological manifestations of old age. The purpose of this study was to investigate the underlying mechanisms responsible for the age-associated increase in brain IL-6. In an initial study, gel mobility shift analysis (GMSA) showed that binding of NF-kB to the regulatory element of the IL-6 gene was increased in brain of aged mice (24-month-old) relative to adult mice (3-month-old). Inhibitors of NF-kB decreased IL-6 gene expression in glia from aged brain. Because the anti-inflammatory cytokine IL-10 inhibits NF-kB activity, we hypothesized that a decrease in IL-10 was responsible for the increase in NF-kB activity and IL-6 expression in aged mouse brain. Consistent with this hypothesis, coronal brain sections as well as glia from aged mice produced more IL-6 and less IL-10 than brain sections or glia from adults when incubated in the absence or presence of LPS. When glia from aged mice were incubated with recombinant murine IL-10, GMSA showed that NF-kB binding to the response element of the IL-6 promoter was decreased, and constitutive levels of IL-6 mRNA and protein were reduced. These studies suggest that IL-10 constrains IL-6 expression in the adult brain, but in the aged brain it decreases and thus enables a cascade of intracellular events that increase the expression of the IL-6 gene. Interleukin-2 Influences Exploration and Increases Sensitivity to the Stimulatory Effects of a Selective Dopamine Uptake Inhibitor. Steven S. Zalcman and Hong-Lin Niu, UMD–New Jersey Medical School, Department of Psychiatry, Medical Science Building, 185 S. Orange Ave., Newark, New Jersey 071032714. Interleukin (IL)-2 is a cytokine that is linked with psychopathological outcomes, including schizophrenia and cognitive disorders. It also influences mesocorticolimbic dopamine activity in rodents and induces behavioral changes that are at least partly mediated by central dopaminergic processes. We presently report that a single IP injection of murine IL-2 enhanced novelty-induced activity (incl., locomotion, investigatory behavior). Less pronounced alterations of activity were evident in mice that were preexposed to the test cage prior to receiving IL-2. Activity was persistently elevated in mice receiving repeated intermittent injections of IL-2 (i.e., 5 daily IP injections; 0.4 µg/mouse). Indeed, throughout the treatment period, levels of eventual habituation to the test cage were significantly higher in IL-2-treated mice than in those receiving saline. IL-2 treatment also increased sensitivity to the behavior-activating effects of GBR 12909, a selective dopamine reuptake inhibitor. This effect was very long-lasting, since the dopamine agonist was administered 6-weeks after cessation of cytokine treatment. The latter finding, to our knowledge, is the first demonstration that IL-2 can produce a long-lasting increase in sensitivity to the behavior-activating effects of a stimulant, and parallels the finding of Zalcman et al. (Brain Res. 847, 276–283, 1999), i.e., that IL-6 increases sensitivity to the locomotor-stimulating effects of amphetamine. We suggest that immune-derived products can influence the development of psychopathological outcomes that are characteristic of aberrant mesocorticolimbic dopamine activity via sensitization-like processes. (Supported by a Foundation of UMDNJ grant.)