Possible role of apoptosis in the pathogenesis of bleomycin-induced scleroderma.

To elucidate the role of apoptosis in cutaneous sclerosis, we examined the induction of apoptosis and expression of Fas, Fas ligand, as well as caspase-3 in a murine model of bleomycin-induced scleroderma. Dermal sclerosis was induced by local injections of bleomycin (1 mg per mL) in C3H/HeJ mice. Induction of apoptosis was examined by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling) assay and DNA gel electrophoresis. TUNEL positivity was prominently detected on keratinocytes and infiltrating mononuclear cells, but not endothelial cells and fibroblasts, in the lesional skin. DNA fragmentation revealed laddering at 3 to 4 wk following bleomycin treatment. Immunohistochemistry showed increased expression of Fas in infiltrating mononuclear cells at early phases following bleomycin exposure, whereas constitutive expression in fibroblasts. Fas ligand expression was increased in mononuclear cells as well as fibroblasts in the sclerotic skin. Results of reverse transcription-polymerase chain reaction analysis revealed that expression of Fas ligand mRNA was upregulated and reached a maximum at 3 wk, whereas Fas mRNA was continuously detected. mRNA expression as well as activity of caspase-3 was also enhanced at 3 wk. Administration of neutralizing anti-Fas ligand antibody together with local bleomycin treatment reduced the development of dermal sclerosis, associated with the reduction of TUNEL-positive mononuclear cells and also with the blockade of apoptosis. Caspase-3 activity in the lesional skin was also significantly reduced after anti-Fas ligand treatment. These findings suggest that excessive apoptosis, which is mediated by Fas/Fas ligand pathway and caspase-3 activation, is involved in the pathogenesis of bleomycin-induced scleroderma, possibly by playing an inflammatory role.