Immunomodulators in Respiratory Disease Treatment
Supplemental Readings Barnes PI: Clinical outcome of adding long-acting beta-agonlsts to inhaled corticosteroids. Respir Med 2001; 95(Suppl B):S12-S16. Bjermer L: History and future perspectives of treating asthma as a systemic and small airways disease. Respir Med 2001; 95:703719. Duvivier OH, Votion 0, Vandenput S et al: Aerosol therapy in the equine species. Vet] 1997; 154:189-202.
Hoffman AM: Inhaled medications and bronchodilator usage in the horse. Vet CUn North Am Equine Pract 1997; 13:519-530. Rush BR, Raub ES,Rhoads WS et al: Pulmonary function in horses with recurrent airway obstruction after aerosol and parenteral administration of beclomethasone dipropionate and dexamethasone, respectively. Am] Vet Res 1998; 59:1039-1043.
Immunomodulators in Respiratory Disease Treatment M. JULIA B.F. FLAMINIO
Ithaca, New York he respiratory tract has an efficient mechanical and immunologic defense apparatus for the removal of pathogens and particles that reach the airways from the exterior environment. This mechanism of protection includes adhesive and enzymatic properties of the mucus covering the airways, the mucociliary escalator, the normal bacterial flora that competes with pathogenic agents, and the alveolar and mucosal immune systems. Nevertheless, the function of many of these elements may be impaired under stress, strenuous exercise, long-distance transportation, and infection. Therefore the use of immunomodulators is a rational approach to activate the immune defense for the prevention, attenuation, and early treatment of respiratory disease, before intense cellular damage occurs. Immunomodulator or biologic response modifier is a substance that enhances or suppresses immune responses. Immunostimulant is an agent that activates immune cells and promotes the release of endogenous immune mediators (cytokines) to assist in the treatment of immunodeficiency disorders, chronic infections, or cancer. In general, activation of the immune response involves the amplification of phagocytosis and intracellular killing of organisms by neutrophils and macrophages, antigen presentation, cytotoxic and antiviral activity of T cells, cytokine release and antibody production, creating resistance to infections or neoplastic conditions. Although immunomodulators generate a nonspecific response to antigen, they exert an effect on the components of both innate and acquired arms of the immune system. Immunomodulators (Table 8.11-1) function predominantly via activation of macrophages in the liver, spleen, bone marrow, and lungs (pulmonary intravascular macrophages). After the immunotherapeutic agent is phagocy-
tosed, intracellular signaling pathways are activated for gene expression, and the duration of the active status follows the persistence of the product within the macrophages. Therefore multiple doses should give pulses of immune stimulation. The effectivenessof many immunostimulants depends on the animal's own ability to respond with the production of endogenous cytokines, such as interleukin-I (IL-I), IL-6, tumor necrosis factor (TNF), and interferon (lPN). Systemic reactions after cytokine release vary from mild fever and transient depression to toxic symptoms that include alteration of vascular permeability, hypotension, pulmonary edema, diarrhea, infiltrative/granulomatous cell reaction, and collapse. Paradoxically, these are the same mediators that promote the desirable responses of enhanced immune function. For this reason, immunostimulants must mediate short-term responses. The selection of a specific immunomodulator should be based on the available information on mechanisms of action and effectiveness investigated by immunologic assays (Box 8.11-1) and clinical trials, in addition to an acceptable degree of safety. The stimulation of the immune response without harmful inflammation and tissue damage is imperative. Effectiveness means regression of the clinical process, prevention of recurrence, and enhancement of overall survival time. In chronic or advanced cases, severe inflammation may overcome the desirable effects of immunomodulators, and best clinical results are obtained when these products are used in the initial phase of disease and in prophylaxis. Nevertheless, the selection of immunomodulators is still challenged by insufficient information on mechanisms of action in vitro, lack of clinical trials, deleterious and unknown side effects, lack of response by some individuals, and extra-label use with extreme expectations of efficacy.
Table 8.11-1 Immunomodulators that Have Been Used in Horses Source
Propionibacterium acnes (Eq'Stirrr') Mycobacterium spp. (Equimmune IVb)
Virus products Serum products Cytokines Synthetic products
Parapoxvirus ovis (Baypamun HKC) Caprine Serum (Caprine Serum Fractlonv) lnterferon-e-Za (Roferon-N) levamisole (Levasole,' Ripercol 19)
1 ml/114 kg 1.5 rnl/horse 2 ml/horse 2 ml/horse 0.1 U/kg 2 mg/kg
q48-72h ql-3w q48h q7-10d q24h, 5 days q48h
IV IV 1M 1M PO PO
Every 48 to 72 hours; ql-Sw, every 1 to 3 weeks; q7-lOd, every 7 to 10 days; Iv, intravenous; 1M, intramuscular; PO, by mouth. *See text for details. -Immunovet. Neogen Co., Lansing, Mich. bVetrepharm Research Inc., Athens, Ga. (Bayer AG, Leverkusen, Germany.
BOX 8.11-1 In Vitro Immunologic Testing Used to Characterize Immunodeficiencies and Immunomodulatory Differences Total and differential white cell counts in peripheral blood Total and differential white cell counts in BAlF Serum immunoglobulin isotypes, concentrations, and electrophoresis Mucosal immunoglobulin isotypes and concentrations lymphocyte subpopulation phenotyping Mitogen Iymphoproliferation Cytokine production Phagocytosis and oxidative burst activity CTl and LAK cell responses NK cell activation BALF, Bronchoalveolar lavage fluid; CTL, cytotoxic lymphocyte; LAK, lymphocyte-activated killer; NK,
PROPIONIBACTERIUM ACNES Inactivated Propionibacterium acnes, formerly known as Corynebacterium parvum, has demonstrated immunostimulatory activity in in vitro and in vivo studies in the last 40 years, including macrophage activation, enhanced natural killer (NK) cell activity, increased CD8+ T lymphocyte expression with interferon release, inhibition of tumor growth, and nonspecific resistance to pathogenic challenge in mice, human patients, and domestic animals. EqStim (Immunovet, Neogen Co., Lansing, Mich.) is licensed by the United States Department of Agriculture (USDA) as a biologic response modifier for adjunct therapy in the treatment of primary and secondary viral and bacterial infections of the respiratory tract of the horse in
association with other conventional therapy. It contains 0.4 mg/ml of nonviable P. acnes in a 12.5% ethanol-insaline solution. The recommended dose is 1 ml per 114 kg of body weight, intravenously, two to three doses every 48 or 72 hours, before stress is induced or as an adjunct to conventional therapy. Mild fever after administration of the product is expected and indicates immune response. In healthy young horses, a series of three intravenous injections of P. acnes results in immunomodulatory responses. In the peripheral blood, total and proportional CD4+ T lymphocyte population are increased, as are nonopsonized phagocytosis and lymphocyte-activated killer (LAK) cell activity. In the bronchoalveolar lavage fluid (BALF), a decrease occurs in total leukocyte counts, in particular the proportion and absolute number of lymphocytes, although the proportion of CD4+ T lymphocytes increases in comparison with CD8+ cells. The absolute number of macrophages in the BALF decreases, whereas the proportion and activation of macrophages increases. The LAK cell activity also increases in BALE An open, randomized clinical trial demonstrated that administration of two doses of P. acnes before shipping reduced more than 60% of the incidence of transport-stress induced respiratory disease compared with placebotreated group. In two blinded, randomized clinical studies of horses with naturally occurring respiratory disease (characterized by fever associated with respiratory difficulty, and/or nasal discharge, cough, lymphadenopathy) treated with conventional therapy, 79% to 96% of the horses that received P. acnes recovered within 14 days of treatment, compared with 47% and 35%, respectively, of the horses from the placebo group.
MYCOBACTERIUM CELL WALL EXTRACT The bacillus Mycobacterium bovis strain attenuated by Calmette and Guerin has been used all over the world as a vaccine against tuberculosis. In addition, Mycobac-
Immunomodulators in Respiratory Disease Treatment
terium extracts are one of the most potent stimulants of macrophage function, resulting in subsequent release of cytokines (1L-1, TNF, colony-stimulating factors) and in lymphocyte activation. To this date, limited in vitro data exists that describes the effects of this immunomodulator in horse cells. The immunotherapeutic agent Equimmune IV (Vetrepharm Research Inc., Athens, Ga.) is an USDA licensed oil-in-water emulsion of purified Mycobacterium spp. cell wall extract for the treatment of equine respiratory disease complex (ERDC) resulting from virus and/or bacteria. The recommended dose is 1.S ml per animal intravenously. It has been approved for use in pregnant mares. Side effects after the immunotherapy include reaction at the injection site, fever, lethargy, and decreased appetite, likely related to the induced endogenous cytokine release. Four horses with a history of coughing were reported to develop severe inflammatory reaction in the respiratory tract (increased bronchial sounds, crackles, and wheezes in the lung fields, increased cell counts in BAL) after the immunomodulator administration, characterized by interstitial pneumonia, multi focal pulmonary granulomas and bronchiolitis, and subsequent development of lung fibrosis. In humans, although intravesical attenuated Bacillus Calmette-Guerin (BCG) immunotherapy has been used successfully in the treatment of bladder carcinoma, about 1% of patients develop systemic BCG infection (pneumonitis, hepatitis, renal insufficiency) or hypersensitivity reactions (disseminated pulmonary and hepatic granulomas characterized by noncaseating epithelioid granuloma with Langhans-type giant cells and lymphocytes) a few weeks after immunotherapy. A randomized, double-blind clinical study of Mycobacterium spp. cell wall extract was conducted in horses with naturally occurring respiratory disease (fever, cough, nasal discharge, increased respiratory sounds, ocular discharge, decreased appetite, or performance). Results suggested that 83% of the horses receiving one intravenous dose of purified extract recovered from respiratory clinical signs in a shorter period of time (7 days) than the placebo group. In addition, only 40% of the horses receiving placebo recovered by 10 days of treatment.
PARAPOXVIRUS OVIS The use of poxvirus as an immunostimulant originated from observations after the smallpox eradication program, in which vaccinated human patients presented improvement of viral diseases and tumors. Since then, the mechanism of action of parapoxviruses in the immune system has been studied, and the viral envelope is thought to contain proteins that promote the activation of NK cells, enhance phagocytic activity, and increase the release of interferon-a and IL-2. Baypamun HK (Bayer AG, Leverkusen, Germany) is the form of purified and chemically inactivated parapoxvirus ovis strain D 1701 that is commercially available in Europe for use in horses and other domestic species. It is indicated for the prophylaxis of stress-induced respiratory diseases caused by transportation, hospitalization, and weaning; for the metaphylaxis and therapy of infectious diseases; and for enhancement of the immunization re-
sponse. The generally recommended dose is 2 ml per animal intramuscularly two or three doses 48 hours apart, before the stress is induced, immediately after birth or with conventional treatment for respiratory disease. Limited swelling at the injection site may occur. In the horse, a blinded field study suggested that prophylactic administration of Parapoxvirus ovis to foals 6 and 4 days before weaning, plus at S days thereafter, assisted in preventing and reducing the incidence of respiratory disease from 24% to 7.9%. In a controlled field trial, Thoroughbred foals from the same farm received three doses of the immunotherapeutic drug or placebo immediately after birth, and 24 or 48 hours thereafter. These foals were monitored for 4 weeks, and 20% to 30% of the foals from the placebo group developed respiratory infections, whereas the foals in the groups receiving the imrnunomodulator did not. In addition, parapoxvirus ovis was suggested to minimize but not to prevent respiratory clinical signs (based on nasal exudate scores) in horses naturally challenged by contact with virulent equine herpes virus-lor -4 (EHV-l or EHV-4). A significantly greater proportion of young horses under stress induced by weaning, transportation, and commingling was more resistant to EHV-1 and EHV-4 infection, in addition to development of respiratory clinical signs when receiving parapoxvirus ovis, compared with the ones receiving placebo. To this date, limited in vitro data exist that describe the effects of this immunomodulator in horse cells.
CAPRINE SERUM FRACTION Caprine Serum Fraction Immunomodulator (CSFI; Centaur, Inc., Overland Park, Kan.) is a USDA conditionally licensed immunomodulator for adjunct treatment of lower respiratory tract disease in horses. It is a sterile, filtered, purified, and standardized fraction of goat serum preserved in phenol and thimerosal. The label recommendation is 2 ml intramuscular injections, two applications, 7 to 10 days apart. Potential side effects are swelling and heat at the site of injection for 48 to 72 hours. A clinical efficacy trial in horses with unspecified lower respiratory tract disease (characterized by tracheal exudation) suggested improvement in the airway inflammation evaluated by an endoscopic examination score after two doses of the immunomodulator. Phenotypical analysis of leukocyte subpopulations, phagocytosis and oxidative burst activity, LAK cell activity, and IL-2 receptor expression were evaluated in peripheral blood and BALF leukocytes after administration of two intramuscular injections of placebo or caprine serum fraction to six healthy yearling fillies. The results suggested immunomodulatory activity by the reduction of monocyte and CD8+ T lymphocyte counts in peripheral blood. In addition, the cellularity of bronchoalveolar lavage fluid was reduced after administration of the immunomoduIator, particularly B lymphocyte counts and macrophages. Immune function tests including LAK cell activity, phagocytosis and oxidative burst activity, and IL-2 receptor expression did not detect changes after the administration of caprine serum fraction. Localand systemic reactions to the series of intramuscular injections of the product were characterized by mild swelling reaction at the intramuscular
injection site in one filly for 48 hours, and limb edema 24 hours after administration of the product in another filly. Elevation in body temperature in response to the immunotherapy was not detected by the scheduled daily physical examination. A recent study examined the clinical application of this immunomodulator in horses diagnosed with "suppurative lower respiratory disease" based on endoscopic finding of bronchial discharge and at least one clinical evidence of respiratory disease (nasal discharge, abnormal lung sounds, cough, decreased performance). All horses were treated with antibiotics concomitantly and occasionally with dimethyl sulfoxide (DMSO). Two randomized dose response studies suggested that two intramuscular injections 1 week apart (day 0 and day 7) resulted in significant clinical improvement (clinical score based on exudate production, dyspnea, lung sounds, cough) by day 14 in the 60 mg or 120 mg CSFI-treated group compared with the placebo and lower dose-treated horses. No difference exists between 60 mg and 120 mg CSFI-treated groups. In a field study, 75% of horses treated with various antibiotics and two doses of CSFI recovered from their respiratory disease by day 21 compared with 35% of the animals receiving antibiotics and placebo. Hence some evidence exists that clinical recovery from lower respiratory tract infection may benefit from this immunomodulator. Further studies are warranted to characterize the efficacy of this product in vivo.
INTERFERON-ALPHA The presence of viral products induces mononuclear phagocytes to produce endogenous interferon-a (lFN-a) in the early stages of infection. This type I interferon binds to a common receptor expressed on most of the cells and triggers intracellular signaling pathways that result in potent antiviral, immunomodulatory, and antiproliferative activities. Its antiviral effect is characterized by inhibition of viral protein synthesis, viral RNA degradation, activation of cytolytic activity of NK and LAK cells, increase in major histocompatibility (MHC) class I expression of virus-infected cells, enhanced IFN-'Y expression by lymphocytes, macrophage activation, and dendritic cell maturation. Interestingly, once the antiviral and antiproliferative activities are triggered by IFN-a, they can be transferred cell-to-cell by direct contact in the absence of the product. Oral IFN-a acts directly on oropharyngeal-associated lymphoid tissues by activation of the antiviral state of lymphocytes. The altered lymphocytes act as amplifiers of this mechanism by transferring this enhanced biologic effects to naive lymphocytes homed in distant tissues, such as the respiratory tract. Oral administration of low doses of IFN-a has therapeutic benefit for acute and chronic viral infections in human patients and animals. In a double-blind, randomized block design study of horses with inflammatory airway disease (characterized by poor performance and exudate in the upper and lower airway) in active training, 50 U of natural, human IFN-a given orally for 5 consecutive days reduced airway inflammation, pharyngeal lymphoid hyperplasia, nasal discharge, and cough compared with horses receiving placebo. Horses that received oral IFN-a
recovered their BALF to a noninflammatory cytologic profile, and the proportion of lymphocyte subpopulations caused by the immunomodulator was unchanged. The use of commercially available recombinant human IFN-a-2a (Roferon-A, Roche Laboratories Inc., Nutley, Nj.), which contains only one subtype of IFN-a in contrast to the natural form, failed to reduce virus shedding and respiratory disease (fever, nasal and ocular discharge) in experimental herpes-virus-I infection in horses. Whether the less protective effect of the immunotherapy regime used was due to inappropriate dosage or to differences in the response to the recombinant form is unknown. High doses of IFN-a are more likely to cause side effects because of induced self-destructive inflammatory responses and immunosuppression.
OTHER IMMUNOMODULATORS Some immunomodulatory agents have been used for years in clinical patients, although their mechanism of action and effectiveness have not been established. Levamisole phosphate is a synthetic antihelrnintic, which immunomodulatory properties have been characterized poorly in vitro and in vivo. However,cell mediated response and phagocytic activity may improve in immunocompromised individuals after immunotherapy. ExtralabeI use of levamisoIe (Levasole, Ripercol L), 2 mg/kg, orally, every 48 hours in horses as an adjunct for treatment of respiratory disease is based on clinical reports of prevention and treatment of chronic respiratory infections in children and neonate animals. Other immunostimulants largely used in human medicine for the prophylaxis and treatment of respiratory disease include combined lyophilized fractions of several common respiratory tract bacterial pathogens and bacterial ribosomal fractions. In addition, the expansion in the use of pure cytokines such as Ilo2, IL-12, IL-18, TGF-~, TNF and IFN-"I as immunomodulatory agents became possible because of advances in recombinant DNAtechnology. As the field of veterinary immunology evolves and more clinical trials are available, the understanding of the complex interactions among the immune mediators in developing a balanced immune response allows a more effective use of immunomodulators in the prevention and treatment of respiratory diseases without adverse effects, either to enhance protection against pathogens or to decrease airway hyperreactivity.
Supplemental Readings Biron CA:Interferons a and ~ as immune regulators-a new look. Immunity 2001; 14:661-664. Cormack S, Alkemade S, Rogan 0: Clinical study evaluating a purified mycobacterial cell wall extract for the treatment of respiratory disease. Equine Pract 1991; 13:18. Evans DR, Rollins lB, Huff GK et al: Inactivated Propionibacterium acnes (Immunoregulin) as adjunct to conventional therapy in the treatment of equine respiratory diseases. Equine Pract 1988; 10:17. Flaminio M, Rush B,Shuman W: Immunologic function in horses after nonspecific immunostimulant administration. Vet Immunol Immunopathol 1998; 63:303-315.
Immunomodulators in Respiratory Disease Treatment
Lindner A, von Wittke P, Thein P et al: Effect of paramunity inducer on the incidence of diseases and the plasma cortisol content in Thoroughbred foals before and after weaning. Tierarztl Prax 1993; 21:47-50. Moore BR: Clinical application of interferons in large animal medicine.] Am Vet Med Assoc 1996; 208:1711-1715. Moore BR, Krakowka S, Cummins 1M et al: Changes in the airway inflammatory cell populations in Standardbred racehorses after interferon-alpha administration. Vet Immunol1996, 49:347-358. Nestved A: Evaluation of an immunostimulant in preventing shipping related respiratory disease.] Equine Vet Sci 1996; 16:78.
Rush BR, Flaminio M]BF: Immunomodulation in horses. Vet Clin North Am Equine Praet 2000; 16:183-197. Vail CD, Nestved A], Rollins ]B et al: Adjunct treatment of equine respiratory disease complex (ERDC) with Propionibacterium awes, immunostimulant, EqStim. ] Equine Vet Sci 1990; 10:399. Ziebell KL, Steinmann H, Kretzdorn D et al: The use of Baypamun N in crowding associated infectious respiratory disease: efficacy of Baypamun N (freeze dried product) in 4-10 month old horses.] Vet Med [B] 1997; 44:529-536.