Anti-Fas antibody induces apoptosis in cultured human renal interstitial fibroblasts

Objective To detect if Fas is expressed in human renal interstitial fibrobl asts (hRIFs) and apoptosis of hRIFs can be induced by specific anti-fas ant ibody. Methods hRIFs were cultured from isolated papillae of human kidney, and ide ntified by morphologic examination, assay of antigenic components and cultu re in D-valine selective medium. Fas expression in normal hRIFs was detecte d by RT-PCR and immunocytochemistry staining. After hRIFs were incubated wi th interferon gamma (gamma -IFN 500 U/ml, 1000 U/ml, 1500 U/ml and 2000 U/m l, respectively) for 48 hours, Fas expression was determined by Northern bl ot, Western blot and flow cytometry. hRIFs pre-stimulated with gamma -IFN ( 500 U/ml, 48 hours) were incubated with anti-fas antibody (IgM, 0.5 mug/ml) for 12 hours. And apoptosis was identified by morphologic examination, DNA ladder assay and flow cytometry. Results The cultured hRIFs showed a shuttle-like shape and were positively stained by labeled antivimentin antibody but negatively stained by anti-epi thelial membrane antibody. They could not grow in the D-valine selective me dium and died partly in a week. Fas mRNA and protein were expressed in norm al hRIFs and markedly upregulated by stimulation with gamma -IFN. Apoptosis in gamma -IFN pre-stimulated hRIFs was induced by anti-fas antibody, showi ng cell nuclear shrinkage and condensation in morphologic feature, internuc leosomal DNA fragmentation in DNA ladder assay and a pick of hypo-diploid n uclei by flow cytometry. Conclusion Fas is normally expressed in hRIFs and can be markedly upregulat ed by gamma -IFN. Anti-Fas antibodies can induce apoptosis of hRIFs pre-sti mulated with gamma -IFN.