POTENTIAL AUTOANTIGENS IN IDDM - EXPRESSION OF CARBOXYPEPTIDASE-H ANDINSULIN BUT NOT GLUTAMATE-DECARBOXYLASE ON THE BETA-CELL SURFACE

Insulin, carboxypeptidase-H (CP-H), and glutamate decarboxylase (GAD) have been identified as potential autoantigens in insulin-dependent di abetes mellitus (IDDM). Previous studies have described immunoreactive insulin as a surface molecule on the plasma membrane of rat islet cel ls and suggested that cell-surface insulin was derived during exocytos is by the fusion of insulin secretory granules with the beta-cell plas ma membrane. These findings predict that insulin and other secretory g ranule-derived proteins such as the putative autoantigen CP-H may be c olocalized with insulin at specific sites of exocytosis on the beta-ce ll surface. In studies to test this hypothesis, cell-surface staining of dispersed rat islet cells occurred in a granule-like pattern with a ntibodies for CP-H and insulin. The specificity of the CP-H antiserum was confirmed by immunoblotting and indicated that the antiserum was e ssentially monospecific for CP-H. Confocal laser microscopy confirmed that immunoreactive staining for CP-H and insulin was confined to the beta-cell surface. Colocalization of CP-H and insulin on the cell surf ace of beta-cells was demonstrated by double staining with antibodies to CP-H and insulin, and the percentage of beta-cells positive for bot h of these autoantigens increased twofold with increases in insulin se cretion. In contrast, islet cells failed to reveal cell-surface staini ng for GAD(65), another putative autoantigen in IDDM, under either bas al or insulin stimulatory conditions or following exposure of islet ce lls to the cytokines interleukin-1 beta, tumor necrosis factor-alpha a nd recombinant human interferon-gamma. These results demonstrate that the insulin secretory granule-derived proteins, insulin and CP-H, colo calize on the cell surface of beta-cells during exocytosis and in this manner could be recognized by components of the immune system under c ertain disease settings, These findings further delineate a cellular m echanism whereby the functional activity of the pancreatic beta-cell, i.e., resting versus actively secreting, may correlate with cell-surfa ce localization of antigens and raises the possibility that other unid entified granule derived antigens also may colocalize at sites of exoc ytosis on the beta-cell membrane.