IgG autoantibodies from bullous pemphigoid (BP) patients bind antigenic sites on both the extracellular and the intracellular domains of the BP antigen 180

Bullous pemphigoid (BP) and gestational pemphigoid (PG) are subepidermal bl istering disorders associated with autoantibodies directed against two comp onents of hemidesmosomes: the BP antigen 180 (BP180) and the BP antigen 230 (BP230), Autoantibodies against the extracellular domain (ECD) of BP180 ar e thought to play an initiatory role in subepidermal blister formation. To characterize the targeted antigenic sites on BP180, we have assessed the re activity of sera from BP and PG patients against eukaryotic recombinant pro teins encompassing various portions of the ECD and the intracellular domain (ICD) of BP180, Twenty-two of 22 (100%) BP sera that immunoblotted BP180 i n keratinocyte extracts, bound a mutant form consisting of the entire ECD o f BP180, whereas only three of these 22 sera (14%) reacted against the ECD of BP180 lacking the NC16A membrane proximal region. Thirteen out of the 22 (59%) BP sera recognized the ICD of BP180. Circulating IgG from a represen tative BP patient that was affinity purified against the ECD of BP180 did n ot bind the ICD when reblotted, indicating that there was no antigenic cros s-reactivity between the ECD and the ICD of BP180, Reactivity against the I CD of BP180 was further ascertained by immunofluorescence microscopy studie s showing that nine of the 22 (41%) BP sera stained COS-7 cells expressing the ICD of BP180. Using deletion mutants of the ICD of BP180, the majority of the sera was found to recognize the central region of the ICD of BP180. Specifically, an immunodominant region was localized to an 87-amino acid se gment located towards the NH2-terminus of BP180, In contrast to BP sera, fi ve of six (83%) PG sera contained IgG that recognized exclusively the NC16A region, whereas none bound to the ICD of BP180. Together, the results indi cate that in BP, autoantibody reactivity to BP180 is not exclusively restri cted to the NC16A region, but that additional antigenic determinants exist on the ICD of BP180, The observed heterogeneous immune response against BP1 80 might reflect intramolecular epitope spreading. Because the ICD of BP180 harbors functionally important regions, it is possible that autoantibodies against the ICD of BP180 have pathogenic significance for the progression of the disease.