title: Mechanism of serum-mediated endothelial injury in scleroderma: identification of a granular enzyme in scleroderma skin and sera.
abstract: 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.
authors.affiliation: Department of Medicine, Medical College of Ohio, Toledo 43699, USA.
authors.forename: M B
authors.initials: MB
authors.lastname: Kahaleh
authors.forename: P S
authors.initials: PS
authors.lastname: Fan
mesh_terms: D000328:Adult; D001706:Biopsy; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D004727:Endothelium / Q000166:cytology / Q000293:injuries*; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression; D053804:Granzymes; D006131:Growth Inhibitors / Q000097:blood; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D008297:Male; D012595:Scleroderma, Systemic / Q000097:blood* / Q000276:immunology / Q000473:pathology; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012867:Skin / Q000473:pathology; D014018:Tissue Distribution
chemical_list: D006131:Growth Inhibitors; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human