The role of IgG subclass in the pathogenesis of bullous pemphigoid

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We examined the role of IgG subclass in the mechanism of bullous pemphigoid (BP) as follows : (1)Whether BP IgG subclass binds to the different binding site of BP antigen or not. (2)Which BP IgG subclass induces directly dermo-epidermal separation. (3)Whether BP IgG subclass bound to BP antigen passes some signal into the cell or not. (4)Whether complement activated by BP IgG1 induces cell lysis or not. (5)Whether cultured keratinocytes secrete any chemical substance (ex.protease) or not, after BP IgG subclass binds to BP antigen on the surface of cultured keratinocytes.First, we examined the binding site of BP IgG subclass on BP antigen, using blocking immunofluorescense, immunoblot and ELISA.The result suggested that BP IgG1, IgG2 and IgG4 could bind to the same epitope of BP antigen, but considerable variation occured between patients. Second, we observed vertical section of luciffer yellow loaded keratinocytes, by using confocal laser scanning microscopy. The vertical image showed that there was no difference between BP IgG and normal IgG.Third, using confocal laser scanning microscopy, we chased the time course of fluorescent intensity change of fluo-3/am, snarf-1/am and dioc-5 which indicated intracellular Ca^<2+> concentration ([Ca^<2+>]i), intracellular pH and membrane potential, respectively. The result showed BP IgG1 induced a transient increase of [Ca^<2+>]i, even though fluo-3 loaded keratinocytes were incubated in Ca removed medium. There was no significant change about membrane potential. Forth, BP IgG1 induced a transient increase of [Ca^<2+>]i, when complement was added. But at the same time, it wasn't observed the cytolysis of luciffer yellow loaded keratinocytes. Fifth, we examined the presence of urokinase receptor (UKR) in the lesional skin of various skin diseases. The result showed that UKR was observed in the intercellular space of pemphgus vulgaris, but not observed at the basement membrane zone of BP.