Mechanism of Serum-Mediated Endothelial Injury in Scleroderma: Identification of a Granular Enzyme in Scleroderma Skin and Sera

• Published in: Clinical immunology and immunopathology Vol. 83; no. 1; pp. 32 - 40
• Main Authors: Kahaleh, M.Bashar, Fan, Pan-Sheng
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.04.1997; New York, NY Academic Press; Boston
• Subjects: Adult; Biological and medical sciences; Biopsy; Cell Division - drug effects; Cells, Cultured; Cytotoxicity, Immunologic; Dermatology; Endothelium - cytology; Endothelium - injuries; Female; Fibroblasts - cytology; Gene Expression; Granzymes; Growth Inhibitors - blood; Humans; Leukocytes, Mononuclear - enzymology; Male; Medical sciences; Scleroderma, Systemic - blood; Scleroderma, Systemic - immunology; Scleroderma, Systemic - pathology; Serine Endopeptidases - genetics; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; Skin - pathology; Skin involvement in other diseases. Miscellaneous. General aspects; Tissue Distribution; Human; Immunopathology; Connective tissue disease; Skin disease; Endothelial cell; Serine endopeptidases; Enzyme; Pathogenesis; Autoimmune disease; Peptidases; Granzyme A; Granzyme B; Systemic disease; Hydrolases; Serum; Lesion; Scleroderma
• ISSN: 0090-1229; 1090-2341
• DOI: 10.1006/clin.1996.4322

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.