Mechanism of serum-mediated endothelial injury in scleroderma: identification of a granular enzyme in scleroderma skin and sera.

Circulating endothelial cell growth-inhibitory factor with a molecular weight of 40-60 kDa was described in scleroderma (SSc) sera and shown to have a proteolytic action. In view of the recent demonstration of cellular immune activation in SSc, and because of the description of novel serine proteases in the granules of activated cytolytic T cells (granzymes), we hypothesized that granzymes represent the endothelial inhibitory principal in SSc sera. Granular enzymes were isolated from IL-2-activated nonadherent normal lymphocytes, and a 60-kDa granzyme was isolated using benzamidine-affinity column and molecular sieve column. A polyclonal antiserum was generated by immunizing rabbits with the isolated granzyme. Anti-granzyme antibody abolished SSc serum-mediated EC growth inhibition. Furthermore, a circulating protein similar to isolated granzyme was identified as a 60-kDa band on Western blots of benzamidine column-purified SSc sera. Immunofluorescence studies of SSc skin biopsies using anti-granzyme antibody demonstrated the presence of granzyme reactivity, while healthy control tissues were negative. Moreover, granzyme A gene expression was identified in SSc skin biopsies by a PCR method. The data suggest cytolytic mechanism involvement in the pathogenesis of scleroderma.