Focal dermal-epidermal separation and fibronectin cleavage in basement membrane by human mast cell tryptase

Mast cell proteases are believed to participate in the basement membrane de struction in blistering diseases. Thus, normal human skill specimens were i ncubated with purified human skin tryptase or compound 48/80 (a mast cell d egranulator) for up to 24 h, Thereafter, the specimens were studied immunoh istochemically. Tryptase caused, in the presence and absence of 1,10-phenan throline, focal dermal-epidermal separation above laminin and almost comple te disappearance of the staining of the extra domain A region of cellular f ibronectin in and beneath the basement membrane, The immunopositivity of th e cell-binding region of fibronectin, laminin, and collagens TV and VII, ho wever, was unaltered. Compound 48/80 induced almost complete dermal-epiderm al separation above intact laminin and only focal reduction in the extra do main A region of cellular fibronectin staining. These alterations by compou nd 48/80 were prevented partially by N alpha-p-tosyl-L-lysine chloromethyl ketone or 1,10-phenanthroline alone but completely when both inhibitors wer e present suggesting the involvement of tryptic serine proteinases, probabl y also tryptase, and metalloproteinases. Preventive effect of N-tosyl-L-phe nylalanine chloromethyl ketone was weak suggesting minor function of chymot ryptic serine proteinases, When tryptase was incubated with heparin and pur e plasma fibronectin, an abrupt decrease in the adherence of cultured kerat inocytes on to plastic surface coated with these substances and a gradual p lasma fibronectin cleavage to 173, 161, and 28 kDa fragments in sodium dode cyl sulfate-polyacrylamide gel electrophoresis were found. In conclusion, t ryptase can cause focal dermal-epidermal separation above laminin in skin s pecimens but it is not known to what extent the decreased keratinocyte adhe rence in vitro and fibronectin cleavage are related to this dermal-epiderma l separation.