INSULIN AS A TARGET ANTIGEN IN AUTOIMMUNE DIABETES - A NATURAL REPERTOIRE AS THE SOURCE OF ANTIBODY-RESPONSE

A solid-phase immunoenzymatic technique with B1- or B29-biotinylated i nsulin coupled to avidin coated wells was used to characterize serum a nti-insulin antibodies and to locate insulin antibody-producing B lymp hocytes in different organs of mice. Low natural serum anti-insulin Ig M and IgG antibodies were found in ten different healthy inbred strain s of mice. Prediabetic nonobese diabetic (NOD) mice had significantly higher measurements than BALB/c mice (P < 0.05). Anti-insulin IgM anti body-producing B lymphocytes were found in bone marrow and spleen of N OD mice and healthy strains of mice, but not in peripheral lymph nodes , thymus, blood or pancreas. B29-fixed insulin was more frequently rec ognized than B1-fixed insulin. There was no relationship to the MHC or to other immune markers. IgG insulin antibody-producing cells were no t detected. IgG insulin antibody-producing cells appeared in the drain ing lymph node and in the blood 10 days after immunization with insuli n. IgM insulin-recognizing cells in the spleen were reduced in number during the same period (P < 0.05-0.01 for BALB/c, DBA2, B10.D2 and NOD ), suggesting migration of these cells. This was tested by in vivo sta ining of spleens with the red-fluorescent membrane linker PKH-26 on da y 7 after immunization. Cells from immunized lymph nodes were FACS-sor ted on day 10. Insulin antibody-producing B lymphocytes with red-fluor escence were found, indicat ing a splenic origin. Examination of IgG s ubclasses showed preferential production of complement-fixing IgG2b in sera and lymph node cells of immunized NOD mice (P < 0.05 vs BALB/c). We conclude that a natural repertoire of insulin recognition exists i n bone marrow and spleen of mice. Hydrophobic epitopes around B1 (B29- fixed insulin) are more frequently recognized than hydrophilic epitope s around B29 (B1-fixed insulin), indicating a genetically fixed patter n of autorecognition. Insulin-recognizing cells from the spleen functi on as the source of insulin antibody response. Preferential occurrence of complement-fixing IgG2b in NOD mice could contribute to autoimmune -mediated beta-cell damage.