Mast cell chymase and tryptase during tissue turnover: analysis on in vitro mitogenesis of fibroblasts and keratinocytes and alterations in cutaneousscars

In order to shed further light on the potential role of mast cells during t issue turnover, we have investigated the number of mast cells containing on ly tryptase and those storing both tryptase and chymase by enzyme histochem istry in normal versus healing skin. Furthermore, we have studied the in vi tro effect of these enzymes on the mitogenesis of subconfluent quiescent fi broblast and HaCaT keratinocyte cultures, using flowcytometric DNA analysis . Chymase-containing mast cell numbers were markedly decreased in scars (P< 0.001), whereas the overall number of tryptase-containing mast cells was no t decreased, although these cells were smaller and stained more faintly in scars. Chymase (5 to 300 mU/ml) induced a marked, dose-dependent in vitro m itogenic response in 3T3 fibroblasts, whereas the effects of tryptase, at u p to 60 nM, were only moderate, compared to the known fibroblast mitogens E GF, TGF-alpha, alpha-thrombin and trypsin at optimal concentrations. Coincu bation of either protease with EGF or a-thrombin had additive effects. In c ontrast to fibroblasts, keratinocytes showed only minor mitogenic responses to tryptase and chymase, also in comparison to other known mitogenic stimu li, and responses to EGF and alpha-thrombin were inhibited on costimulation of cells with the proteases. These findings document for the first time a potential role of mast cell chymase in connective tissue repair, with trypt ase being less active on fibroblasts, and with inhibitory effects of both m ast cell proteases on keratinocytes.