SPECIFIC HUMAN PANCREATIC ISLET CELL PROTEINS RECOGNIZED BY ANTIBODIES IN DIABETIC CHILDREN

Circulating antibodies that bind to the surface of viable pancreatic islet cells have been demonstrated in sera from many newly diagnosed insulin-dependent diabetic (IDD) patients. These antibodies can mediate a complement-dependent cytotoxic reaction and seem to bind mainly to the insulin-producing /3-cells. These observations indicate that a n autoimmune attack specific for p-cells may be involved in the pathogenesis of the disease. However, the target antigens for the circulating antibodies are unknown. We have studied (1 ) whether autoantibodies in sera from newly diagnosed diabetic children can immunopresipitate human islet cell proteins and (2) whether binding of the antibodies can aflect the function of /3-cells. Human islets isolated from the pancreases of five cadaver kidney donors were labeled biosynthetically with methionine and lysed in 1 % NP-40. After ultracentrifugation to remove cellular debris, the supernatant was incubated with normal human serum, followed by adsorption to formalin-fixed StnphyIococcus uurrus. Aliquots of the preabsorbed lysate were incubated with sera from ten newly diagnosed diabetic children or eight normal controls and the immune complexes were isolated by binding to Protein A Sepharose and analysed by SDS polyacrylamide gel electrophoresis, followed by autoradiography. To study the effect of the diabctic antibodics on p-cell function, !he immunoglobulin fraction was isolated from the heat-inactivated sera of six newly diagnosed diabetic children and six normal controls by precipitation with 10% polyethylene glycol. After extensive dialysis against Swimms medium, the immunoglobulin fractions, a t a final concentration of 10%) ( v / v ) , were used to perifuse dispersed rat islet cells supported in small Bio-Gel P-2 polyacrylamide columns. The dynamics of insulin release was determined by radioimmunoassay of fractions collected at various time intervals. Sera from eight out of ten diabetic children precipitated a protein of M,. 64 000 from human pancreatic islet cell lysates. An additional protcin at M,. 38 000 was precipitated by all four sera tested on islets isolated from a HLA-DR 3 positive pancreatic donor. Neither of these bands were precipitated by control sera, nor were they detected in immunoprecipitates of human lymphocyte lysates processed in parallel. While there was little effect a t 5.5 mmol I-' glucose, the immunoglobulin fractions of the sera from six diabetic children were found to inhibit insulin release from dispersed rat islet cells perifused with 30 mmol I-' glucose. The results suggest that diabetic antibodies may be directed against 64 000 or 38 000 islet cell-specific proteins and that the binding of such antibodies to /3-cells can block glucose-stimulated insulin release.