Targeting The First Extracellular Loop Of CCR5 Inhibits The Development Of Type 1 Diabetes

Type 1 diabetes is an autoimmune disease characterized by the destruction of pancreatic islet β-cells by invading leukocytes and the consequent deterioration of the insulin-dependent glucose homeostasis. Although the pathway of the disease has not yet clarified, many studies show that chemokines and chemokine receptors are critically involved in this autoimmune disease by activating migration monocytes and recruitment of T cells into islets. Depending on the juxtaposition of the first two cysteine residues in amino acid sequence, chemokines are divided into four classes: the CXC, CC, C and CX3C families. Chemokines act through specific receptors that belong to the 7-transmenbrane spanning, G-protein-coupled receptor family. Recent observations suggested CC chemokines might play an important role in autoimmune diseases such as experimental allergic encephalomyelitis, active multiple sclerosis and type 1 diabetes.Objective: We used a specific polyclonal antibody targeting the first extracellular loop of CCR5, in the well-established NOD mouse transfer model, which has higher incidence of diabetes than the spontaneous one, to explore: 1) whether the development of diabetes could be affected by blocking part of CCR5; 2) whether they can serve as the potential therapeutic targets? Methods: Two diabetic female NOD mice, with blood glucose of 27.2mmol/L and 29.4mmol/L respectively, were killed and suspensions of splenocytes were washed twice in RPMI1640 medium containing 10% fetal calf serum. Each NOD.Scid mouse was injected i.p. with 1×107 splenocytes. 20 NOD.Scid mice were randomly divided into three groups. One group was injected i.p. with 200 μl PBS. The other two were injected with 10μg of anti-CCR5 Ab (diluted in 200 μl PBS) per mouse each time. PBS, anti-CCR5Ab were injected i.p. into NOD.Scid mice respectively 1 h before cell transfer and then the injection continued for 4 wks three times a week after transfer (a total of 12 injections of 120 μg of Ab per mouse). Blood glucose levels were measured to observe the anti-diabetogenic effect of the antibody. Histological examination, immunohistochemical analysis and ELISA analysis were performed to find the underlying mechanism. Results:(1) Early administration of anti-CCR5Ab prevents diabetes. Within 70 days, all of the control mice developed diabetes while 4 of the 6 mice treated with anti-CCR5 Ab were diabetes-free. (2) Late administration of anti-CCR5Ab does not affect the development of diabetes and insulitis. (3) Early administration of anti-CCR5Ab inhibits insulitis. The results of H&E staining and histological examination showed that the mice treated with PBS (7 wks after transfer) had extensive insulitis, whereas anti-CCR5-treated mice revealed no insulitis). The difference of inflammation scores between two groups was significent. (mean±SD: PBS: 2.5±0.2 n=4 mice/group; anti-CCR5: 0.3±0.1 n=5 mice/group; p<0.001 anti-CCR5 vs PBS, Student's-t test)(4) Immunohistochemical analysis of pancreatic sections prepared from diabetic recipient mice showed that the islet cells were intensively stained after incubation with anti-CCR5 Ab. The pancreatic islet of normal NOD.Scid mice failed to be stained with the same concentration of anti-CCR5 Ab.(5) Effects of Anti-CCR5 on the secretion of islet Ag-reactive Th1 cells. The supernatant of cultured T cells derived from anti-CCR5-treated mice contained little amounts of IFN-γ (0.24ng/ml) and IL-2 (25.2pg/ml) whereas that from control mice contained much more amounts of IFN-γ (18.03ng/ml) and IL-2 (57.7pg/ml). There was no significant difference in IL-10 (anti-CCR5 vs control Ab 27.3pg/ml vs 31.6pg/ml) produced by islet Ag-specific T cells.Conclusion:Early administration of polyclonal antibody, targeting the first extracellular loop of CCR5, could induce the resistance to type 1 diabetes in the adoptive transfer model. The mechanisms by which anti-CCR5 Ab prevents the diabetes may be included the remission in insulitis by direct inhibition of migration of monocytes int...