Vol. 23, No.2 P. 88 -94
Chin Med Sci J June 2008
CHINESE MEDICAL SCIENCES JOURNAL DIABETOGENIC T CELLS INDUCE AUTOIMMUNE DIABETES IN BALB/c MICE' Xiao-lei Zou , Zeng-yu Zhao , Yun-yang Wang , Zhi-qiang Su, and Ming Xiang * Department of Pharmacology, College of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030
Key words: diabetogenic T cells ; adoptive transfer ; streptozotocin-induced diabetes mellitus Objective To investigate the role of T cell and its subsets in the induction of insulitis and type 1 diabetes mellitus (TIDM) in BALB/c mice. Methods Autoimmune diabetes mellitus was developed by intraperitoneal injection of 40 mg/kg streptozotocin (STZ) daily for 5 consecutive days in BALB/c mice as sources of donor cells. Spleen cells from diabetic mice were then cultured for 7 days in the stimulation of interleukin-2 ( IL-2 ) to harvest diabetogenic T cells, which were subsequently transferred into normal BALB/c mice recipients. MlT , ELISA , and HE staining were used to analyze the lymphocyte proliferation, cytokine ( IL-2, interferon-? , IL-4, and IL-10) levels, and pathological changes in pancreatic islets. R a u h As few as 3 x lo6 diabetogenic T cells successfully induced diabetes mellitus in recipients pretreated with STZ twice, whereas transfer of equal amount of normal splenocytes, T cell-depleted diabetogenic splenocytes, or diabetogenic CD4 + T cells alone in recipients receiving STZ twice pretreatment was proved not to induce diabetes mellitus either. A markedly increased lymphocyte proliferation, high levels of interferon-? and IL-2 in the supernatants of diabetogenic T cells were observed. In addition, a markedly enhanced lymphocyte proliferation, a high level of interferon-y secretion in semm , and numerous lymphocytes infiltration in pancreatic islets were detected in the diabetic mice induced by diabetogenic T cells transfer. (h~lusions A novel TIDM murine model is established in STZ-pretreated BALB/c mice by adoptive transfer of diabetogenic T cells. CD4 + T cells with interferon-y may promote the onset of diabetes mellitus.
YPE 1 diabetes mellitus ( TlDM) is a genetically controlled autoimmune disease caused by selective destruction of insulin-secreting p cells in pancreatic islets.' T cell has been shown to cause insulitis
Received for publication October 16, 2007. 'Corresponding author Tel: 027-62524669, Fax: 027-83657547, E-mail : [email protected]
Asupported by the National Natural Science Foundation of China ( 30200343 ) .
and ultimately be responsible for the destruction of p cells in non-obese diabetic ( NOD) mice.* Moreover, T cells infiltration in pancreatic p cells and the prevention of the disease by neonatal thymectomy or treatment with anti-T cell monoclonal antibodies (mAbs) , e. g., anti-thymocyte serum ,3 anti-Thy-1.2 mAb , anti-CD3 mAb , antLCD4 mAb ,' anti-CDS mAb , anti-TcR ap mAb, and anti-TcR Vp8 mAb , further indicated the involvement of T cells in the development of overt diabetes mellitus. Therefore, T cell-mediated immunity, which can deplete or interfere
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with the action of T cells, plays a critical role in the pathogenesis of overt diabetes mellitus. Direct evidence for both autoimmunity and T-cell involvement in insulitis and diabetes mellitus has been provided by adoptive transfer experiments using both splenocytes and highly purified T-cell populations in NOD mice.' However, there is still no report about the lymphocyte transfer of streptozotocin ( STZ ) -induced diabetes mellitus in normal homogeneity recipients especially in common line mice, although STZ has been widely used for the experimental induction of diabetes mellitus. Moreover, adoptive transfer experiment is so far mainly performed on spontaneous diabetes-prone animals, such as BroBreeding ( BB ) rats'' and NOD mice, ' so that it is not strong enough for us to explain T cell roles in the induction of diabetes mellitus with the exception for the reference of spontaneous mechanism. Therefore, in the present study we established a new TlDM murine model by transfer of diabetogenic T cells together with STZ for elucidating T cell roles in diabetes mellitus onset.
MATERIALS AND METHODS Animal and p u p Six-week-old male BALB/c mice ( 18-23 g ) were purchased from Experimental Animal
Center of Tongji Medical College, Huazhong University of Science and Technology ( Wuhan, China) and housed in standard environmental conditions with free access to food and water to acclimatize for 1 week before any experiments were started. All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. A group of 8 mice were used to develop STZ-induced diabetes mellitus and 40 mice were randomly divided into 5 groups with 8 mice per group for Experiment 1 , in the same way another 40 mice were used for Experiment 2. Another 8 mice were simultaneously fed with normal mouse chow without any treatment as normal control and 8 mice were injected with 40 mg/kg STZ for consecutive 5 days as model control. Induction of diabetes mellitw Forty mg/kg STZ ( Sigma, St Louis, MO , USA) dissolved in citrate buffer was intraperitoneally administered daily into 8 BAL,B/c mice for 5 consecutive days to develop STZ-induced autoimmune diabetes. Diabetic mice were then sacrificed as donors of diabetogenic splenocytes for the following transfer experiments 28 days after the last injection of STZ.
Cell pparations Spleens of STZ-induced diabetic mice were removed, teased apart, and pressed through mesh with PBS to produce a single-cell suspension. Lymphocytes were isolated by density gradient centrifugation as previously described" and suspended in complete RPMI1640 medium [ containing 10% fetal calf serum ( FCS) , 5 mmoVL glutamine, 0. 5 moVL p-mercaptoethanol , and 400 U/mL sodium penicillin] at the concentration of 5 x 106/mL after washed twice using PBS. Then, 5 x lo5 splenocytes ( 0. 1 mL) were placed in a total volume of 0.2 mL WMI-l640/FCS per well in a flat-bottom 96-well microculture plate with 20 U/mL interleukin-2 ( IL-2). A 7 days' incubation was carried out at 37°C and 5% CO, with fresh medium supplemented with cytokines every other day. After cultured, diabetogenic splenocytes were harvested through centrifugation and resuspended in PBS for adoptive transfer or relevant assays. Of the other experiment, T cells were partly depleted from diabetogenic splenocytes with anti-Thy-1.2 mAb plus rabbit complement. CD4 + T cells were positively selected magnetically with mAbs directly bound to MACS MicroBeads (Miltenyi Biotec , Bergisch Gladbach , Germany ) according to the manufacturer's protocol, and were counted as viable cells ( trypan blue stain negative). The purity of CLM + T cells was assessed by fluorescence-activated cell sorting (FACS) assay using FITC-labeled anti-CD3 mAb and PElabeled anti-CD4 mAb ( eBioscience, San Diego, CA, USA). FACS assay Totally, 4 x lo5 cultured splenocytes in 5% FCS/PBS were incubated with FITC-labeled anti-CD3 or PE-labeled anti-CD4 mAbs for 30 minutes at 4"C, respectively. Then, the cells were washed twice in PBS and analyzed with flow cytometry (Becton Dickinson, San Jose, CA, USA) using CellQuest software ( Becton Dickinson, San Jose, CA, USA). The result was expressed as the percentage of cells conjugated with corresponding mAb in cells assayed. Adoptive transfez Cell suspension ( 0 . 2 mL) containing 3 x lo5 to 1 x lo7 cells was intravenously injected into BALB/c recipients co-treated with low-dose of STZ (40 mg/kg) either once or twice 24 hours prior to the transfer. The precise cell number used varied dependent on the experimental design and is described in Table 1. In Experiment 2 , a group of 8 mice treated by STZ twice were in-
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jected with 3 x lo6 T cell-depleted diabetogenic splenocytes ( D-spl-T) to c o n f m the importance of T cells in diabetes transfer. Equal amount of diabetogenic CD4 + T cells ( DCD4 + T) was samely infused into another 8 mice to investigate the role of CD4' T cells in the onset of diabetes. And 3 x lo6 normal splenocytes which were isolated from normal control mice and then cultured for 7 days in the same condition as diabetogenic splenocytes ( N-spl ) were intravenously injected into STZ-twice recipients to ascertain whether donors should be overtly diabetic at the time of transfer. Additional 2 groups of recipients just received STZ twice or 3 x lo6 diabetogenic splenocytes alone.
Table 1. Group and treatment
Treatment on recipients Prior to transfer
3 x 106D-spl
1 x 107D-spl
3 x lo5D-spl
2 x 106D-spl
3 x 106D-spl
3 x 106D-spl
3 x 106N-spl
3 x lo6D-spl-T
3 x ~ o ~ D - Tc ~ +
STZ : streptozotocin; D-spl : diabetogenic splenocytes ; D-spl-T : T cell-depleted diabetogenic splenocytes; D-CW
T: diabetogenic CD4 T cells; N-spl: splenocytes from normal mice cultured same as diabetogenic splenocytes. +
hours before the end of cultivation. Then, 150 pL DMSO was added to each well and oscillated for 10 minutes. The absorbance was measured at a wavelength of 570 nm and the results were expressed as the stimulation index ( SI). SI = ODexperimentalwell/ODcontrol well' ELlsA After 2 x lo6 cells were cultured with 5 pdmL ConA for 3 days, supernatants were collected. Mice were sacrificed at 4 weeks after cell transfer, blood samples were collected, and serum was taken. The concentrations of IL-2, interferon-y ( IFN-y) , IL-4, and IL-10 in the supernatants and serum were determined respectively by using ELISA kits ( eBioscience , San Diego, CA, USA) according to the manufacturer's recommendations. Histological examination Mice were sacrificed after 4 weeks of cell transfer and pathological changes in pancreatic islets were examined with hematoxylin-eosin ( HE) staining. Eight islets from each mouse were microscopically observed and the levels of insulitis were scored according to the following criteria: 13 0 , normal islet with no sign of T cell infiltration; 1 , focal peri-islet T cell infiltration; 2 , more extensive pen-islet infiltration, but with lymphocytes less than one-third of the islet area; 3 , intra-islet T cell infiltration in one-third to one-half of the islet area; and 4 , extensive intra-islet inflammation involving more than half of the islet area. Statistical analysis Data were expressed as mean f SD and compared by student's r-test using SPSS software version 11. 5. A value of P < 0. 05 was considered statistically significant.
RESULTS Blood glucose examination The routine examination began with the last dose of STZ or cell transfer. Urinary glucose levels were monitored every other day and the mice were classified as diabetic when the Tes-Tape values consistently exceeded 1 t . Blood glucose levels ( BGLs ) from tail vein were measured twice weekly following cell transfer. Diabetes was defined when 2 consecutive BGLs showed above 16.7 mmol/L.Iz M l T Totally, 2 x lo6 cells/well were stimulated with T-cell mitogen concanavalin A ( ConA, 5 pg/mL, Sigma, USA) in 96-well plates in a total volume of 200 kL/well. Control wells were prepared similarly without ConA. After incubation for 48 hours at 37°C and 5 % CO, , MTT ( Amresco, Solon, OH, USA) was added 4
Hyperglycemia iuduced by diabetogenic splenocytes BGLs in all the groups before transfer were comparable ( P > 0.05). However, the recipients receiving 3 x lo6 diabetogenic splenocytes plus STZ twice displayed progressive hyperglycemia during the 28-day interval after transfer. In contrast, diabetogenic CD4' T cells alone failed to raise the BGLs of recipients ( Table 2 ) . Five out of eight transfer recipients developed diabetes 18.0 f 3 . 4 days after transfer of 3 x lo6 diabetogenic splenocytes plus STZ twice. Seven of eight model control mice developed diabetes 17. 9 f 4. 5 days after last injection of STZ. None of the recipients in other groups progressed to diabetes.
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Table 2. Blood glucose levels before and after transfer of diabetogenic splenocytes* &UP
One week after transfer
Normal control Model control A B C
5.84 i 0.75 7.40 i 0 . 6 3 6.98 i 0 . 9 5 7.04 i 1.21 6.71 i 0 . 8 7
7. 12 io. 85 8.75 i 1.03 6.81 i 1.20 7.23 i l . 04 5.94 i 0.63 7.38 i 0 . 6 8 7.02 i 0 . 4 7
Q M N
Two weeks after transfer
(mmoVL, n = 8 )
Three weeks after transfer
Four weeks after transfer
5.72 f 1.24 14.40 f 1.07 5.94 i o . 91 7.25 ~ 0 . 7 8 7.68 f 1.07 9.32 f 1.47 12. 18 i 0 . 9 3 7. 13 * O . 89 7.58 f 1. 16 5.81 i o . 77 6.24 i 0 . 7 1 6.98 i l . 26
5.87 f 1.05
7.02 i 1.35
8.54 i 1.08
17.34 i 0 . 9 8 * 7. 11 i 1.23 8. 17 i 1.29 6.93 iO.83 9.37 i 1.42
18.36 i 1.55 * 5.84 iO.75 9.03 i 1.04 8.75 i 1.35 12. 18 * O . 93
18.08 i 0.95 * 5.76 i 1.38 6.87i0.95 6.73 1.02 6.88 i 0 . 5 9 7.75 i O . 82
20.48 i 2.09 * 6.31 i 0 . 7 2 5.92 i 0 . 8 9 5.97 i 1.11 7.06 i 1.23 9.03 i 1.04
27.68 i l . 10'. 6.98 i 1.26 7.75 * O . 82 6.27 i 0.92 5.78 i 1.05 8.26 * O . 93
6.27 i O . 92 15.04 i 1.48 6.57 i 0 . 7 6 8.42 iO.98 5.97 i 0 . 5 2
B : Plus-minus values are means i SD. P < 0.05, * P < 0.01 compared with normal control
characteristics of diabetogenic T cells FACS analysis showed that the percentages of T cells and CD4 ' T cells in the cultured splenocytes were 85.6% and 81.7% , respectively (Fig. 1 ) . The lymphocyte proliferation of both diabetogenic T cells (3.593 f 0.215) and diabetogenic CD4 ' T cells (3.287 f 0.296 ) was significantly higher than that of normal cells (2.097 f 0.188, all P < 0.05 ) . However,
there was no significant difference between diabetogenic T cells and diabetogenic CD4 ' T cells ( P > 0.05 ) . Higher amounts of IFN-y and IL-2 were observed in the supernatants of diabetogenic T cells compared with normal splenocytes ( P < 0.05, Fig. 2 A ) , whereas the contents of IL-4 and IL-10 showed no obvious change between diabetogenic T cells and normal splenocytes ( P >O. 05, Fig. 2A).
I 81.7% I
Pigure 1. Fluorescence-activatedcell sorting (FACS) analysis of T cells (A) and CD4 + T cells ( B ) in the cultured splenocytes. FSC-H: forward-angle light scatter.
Autoimmune aud histological features of the novel TlDM model induced by diabetogenic T cells MlT assay showed that the lymphocyte proliferation of splenocytes from transferred diabetic mice (3. 164 *O. 127) was significantly higher than that of normal control mice ( 1.875 f 0 . 4 1 2 , P < 0.05 ) , and comparable to that of model control ( 3.05 1 f 0. 373 , P > 0.05 ). IFN-y secretion significantly increased in serum of transferred diabetic mice com-
pared with normal control mice ( P
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0.74 ) than that of the normal control mice ( 0 , P < 0.01 ) .
R p 2. ELISA analysis of the levels of IL-4, IL-2, IFN--y, and IL-10. A. cytokines in the supernatant of cultured cells; B. cytokine secretion in the serum of mice. IL: interleukin; IFN--y : interferon-y. * P < 0.05 compared with normal splenocytes ; * P < 0.01 compared with normal control; n = 8.
Figure 3. Histological examination of pancreatic islets. HE staining x 200 A. islet from normal control; B. islet from model control; C. islet from the transferred diabetic mice.
diabetes up to 80% in females. To confirm the role of T cells in human TlDM, our present experiments were conducted to use BALB/c mice ( an inbred strain mouse without any genetic predisposition) as recipients to develop a novel TlDM model by means of transferring splenic lymphocytes taken from STZ-induced diabetic BALB/c mice. We also investigated the mechanisms of the effector T cells involved in the cellular events leading to p cell destruction.
cytes successfully induced autoimmune diabetes in the subordinate BALB/c mice with STZ twice pretreatment. Injection of STZ twice was necessary to induce diabetes, which was consistent with the development of adoptivelytransferred diabetes in the NOD mice.” It suggested that the susceptibility of recipients to disease transfer was associated with the islet self-damage of the recipients at the time of transfer. The transfer of normal splenocytes and T-cell-depleted diabetogenic splenocytes failed to induce diabetes, indicating that diabetogenic T cells were responsible for the autoimmune diabetes. The inflammatory process of pancreatic islets induced by diabetogenic T cells and STZ was as follows: STZ made islet cells vulnerable to injury. The host immune response directly induced by transferred diabetogenic T cells could act on vulnerable islet p cells, causing the mass reduction and dysfunction of p cells. It has been established that T1DM is a T cell-mediated autoimmune disease. Ellerman et all6 demonstrated that is-
Our results showed that 3 x lo6 diabetogenic spleno-
let cell membrane antigens preferentially activated CD4 + T
DISCUSSION The pathogenesis of TlDM is very complex. Evidence derived from the NOD mouse model of T1 DM demonstrated T cells are normally required to induce the disease.14 Adoptive transfer experiments have been widely used to assess the in vivo activity of islet-specific T cells in NOD mice, Is a model for T1 DM , which automatically develop diabetes at a certain age with the cumulative incidence of
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cells and B cells rather than CD8' T cells. Calderon ef all7 indicated that CD4 + T cells were essential for development of diabetes by recognizing p-cell antigens in the context of the class I1 MHC LAg7. In contrast, Karges ef allB proposed that CD8' T cell response primed the autoimmune lesion on islet p cells and Pinkse et at" provided evidence for CD8 + T cell autoreactivity associated with recurrent autoimmunity and loss of p cell function in TlDM. In our study, diabetogenic CD4 + T cell was considered as the active factor of diabetes, but insufficient to induce hyperglycemia (data not shown). The confliction just verified the previous report that more than one subclass of T-cells of NOD mice was needed for the disease to occur in the transferred recipients." So, it was likely that in our study CD4 + T cell and CD8 + T cell invaded into host pancreas in turn to produce insulitis and hyperglycemia respectively. IFN-y has been shown to have direct cell toxic effects on rodent islet" and result in loss of tolerance to islet Ags" and autoimmune diabetes? In our experiments, the Thl/Th2 imbalance resulting from IFN-y over-expression was shown to exist in both the serum of the transferred diabetic mice and the supernatants of diabetogenic T cells (Fig. 2 ) , confirming the immune feature of the novel TlDM model and the involvement of IFN-y in the priming process of the transferred TlDM. It was likely that extrinsic IFN-y accompanied by diabetogenic T cells directly acted on islet p cells to promote the release of islet specific autoantigens, which activated autoreactive T cells to initiate host autoimmune response ; meanwhile, intrinsic IFN-y induced immune deviation in the recipients, promoting the disturbance of autoimmune tolerance. So it was reasonable to suppose that the synergistic effect of CD4 + , CD8 + T cells, and IFNy in disease transfer led to the onset of diabetes in homogeneity recipients. In conclusion, a novel TlDM murine model is established by injection of low-dose of STZ plus diabetogenic T cells. The model will provide us a tool for analysis of the role of T cells in the pathogenesis of TlDM.
the run up to autoimmune diabetes of the NOD mouse. Physiol Genomics 2005 ; 21 :201-11. 3.
Harada M, Makino S. Suppression of overt diabetes in NOD mice by anti-thymocyte serum or anti-Thy 1 , 2 antibody. Jikken Dobutsu 1986 ; 35 :501-4.
Bisikirska BC, Herold KC. Use of anti-CD3 monoclonal antibody to induce immune regulation in type 1 diabetes. Ann N Y
Acad Sci 2004 ; 1037 :1-9. Makhlouf L , Grey ST, Dong V, et al. Depleting anti-CD4 monoclonal antibody cures new-onset diabetes, prevents recurrent autoimmune diabetes, and delays allograft rejection in nono-
bese diabetic mice. Transplantation 2004 ; 77 :990-7. Udstad ST, Chilton PM, Xu H, et al. Preconditioning of NOD mice with anti-CD8 mAb and costimulatory blockade enhances chimerism and tolerance and prevents diabetes, while depletion of alpha beta-TCR' and CD4' cells negates the effect. Blood 2005 ; 105 :2577-84.
Sem# P , Btdossa P , Richard MF , et al. Anti-alphdbeta T cell receptor monoclonal antibody provides an efficient therapy for autoimmune diabetes in nonobese diabetic (NOD) mice. Eur J Immunol 1991; 21 :1163-9.
Bacelj A, Charlton B , Mandel TE. Prevention of cyclophosphamide-induced diabetes by anti-V beta 8 T-lymphocyte-receptor monoclonal antibody therapy in NOD/Wehi mice. Diabetes 1989 ; 38 :1492-5.
Fiichtenbusch M , Larger E , Thebault K, et al. Transfer of diabetes from prediabetic NOD mice to NOD-SCID/SCID mice: association with pancreatic insulin content. Horm Metab Res 2005 ; 37 :63-7.
10. Lundsgaard D, Markholst H. A co-transfer system in young prediabetic BB rats: reactivated autoreactive T cells can be partly controlled J Autoimmun 2003 ; 21 :325-38. 11. Yagi H , Suzuki S , Matsumoto M, et al. Immune deficiency of the CTS mouse. I. Deficiency of in vitro T cell-mediated immune response. Immunol Invest 1990 ; 19 :279-95. 12. Lv QJ. Methodology of new drug research in pharmacology. Beijing: Chemical Industry Press; 2007. p. 302. 13. Zhang CL, Zou XL, Peng JB , et al. Immune tolerance induced by adoptive transfer of dendritic cells in an insulin-dependentdiabetes mellitus murine model. Acta Pharmacol Sin 2007 ; 28 :98104. 14. Serreze DV, Leiter EH, Christianson GJ, et al. Major histocompatibility complex class I-deficient NOD-B2mnull mice are diabetes and insulitis resistant Diabetes 1994 ; 43 305-9. 15. Daniel D, Gill RG, Schloot N, et aL Epitope specificity, cyto-
Bach JF. Insulin-dependent diabetes mefitus as an autoimmune disease. Endocr Rev 1994; 15 :516-42.
Vukkadapu SS, Belli N,Ishii K, et al. Dynamic interaction between T cell-mediated beta-cell damage and beta-cell repair in
kine production profile and diabetogenic activity of insulin-specific T cell clone isolated from NOD mice. Eur J Immunol 1995; 25 :1056-62. 16. Ellerman KE, Like AA. Islet cell membrane antigens activate
CHINESE MEDICAL SCIENCES JOURNAL
diabetogenic CD4 + T-cells in the BB/Wor rat Diabetes 1999 ; 48:975-82.
low doses of streptozotocin is mediated by Vp8.2
T-cells. D i e ,
17. Calderon B , Suri A, Unanue ER. In CD4 + T-cell-induced diabetes, macrophages are the final effector cells that mediate islet
abetes 1995 ; 44 :354-9. 21. Hayashi T, Morimoto M, Iwata H, et al. Interferon-gamma plays a role in pancreatic islet-cell destruction of reovirus type 2.
p-cell killing: studies from an acute model. Am J Pathol2006; 169 :2137-47.
induced diabetes-like syndrome in DBMl suckling mice. Int J Exp Path01 1998; 79:313-20.
18. Karges W, Rajasalu T , Spyrantis A, et al. The diabetogenic, insulin-specific CD8 T cell response primed in the experimental autoimmune diabetes model in RIP-B7. 1 mice. Eur J Immunol 2007; 37 :2097-103. 19. Pinkse GG, Tysma OH, Bergen CA, et al. Autoreactive CD8 T cells associated with beta cell destruction in type 1 diabetes. Proc Natl Acad Sci USA 2005 ; 102 :18425-30. 20. Herold KC, Bloch TN , Vezys V , et al. Diabetes induced with
22. Cain JA, Smith JA, Ondr JK, et al. NKT cells and IFN-gamma establish the regulatory environment for the control of diabeb genic T cells in the nonobese diabetic mouse. J Immunol2006; 176: 1645-54. 23. Muller A, Schott-Ohly P, Dohle C , et aL Differential regulation of Thl -type and Th2-type cytokine profiles in pancreatic islets of C57BW6 and BALB/c mice by multiple low doses of streptom twin. Immunobiology 2002 ; 205 :35-50.