SCLERODERMA-DERIVED HUMAN FIBROBLASTS RETAIN ABNORMAL PHENOTYPIC AND FUNCTIONAL-CHARACTERISTICS FOLLOWING RETROVIRAL TRANSDUCTION WITH THE SV40 TST ANTIGEN

In this study an amphotropic retrovirus has been used to efficiently t ransduce normal human (NF) and scleroderma (systemic sclerosis; SSc) d ermal fibroblasts (SScF) with a sequence encoding a temperature-sensit ive mutant of the SV40 large T antigen (tsA58-U19). From the primary o utgrowths of skin explants, cultures were generated whose growth was s tringently temperature-dependent. When grown at a low, permissive temp erature (35 degrees C), both normal and SSc-transduced cells continuou sly divided with similar doubling times, whereas at a high, nonpermiss ive temperature (39.5 degrees C), division of both the NF and SScF cel ls was rapidly arrested. These cells have been passaged more than 50 t imes, have the typical morphological appearance of fibroblasts, and ha ve retained an anchorage-dependent phenotype. The transduced normal ce lls (tsT-NF) synthesized the matrix molecules collagen and fibronectin and expressed phenotypic antigens characteristic of their nontransduc ed counterparts, including MHC Class I, VLA beta 1 (CD29), Hermes 1 (C D44), VLA-4 alpha (CD49d), ICAM-1 (CD54) and LFA-3 (CD58) and the cell surface ectoenzymes neutral endopeptidase (CD10), aminopeptidase N (C D13), and dipeptidyl peptidase IV (CD26), Analysis of the transduced S Sc fibroblasts (tsT-SScF) showed that these cells exhibited certain ma jor features of the SSc pathology, notably the abnormally high synthes is of type I collagen, increased expression of ICAM-1, and depressed l evels of CD26. Moreover, these phenotypic characteristics were retaine d even after prolonged culture in vitro. The tsT-SScF cells also retai ned their responsiveness to cytokines, since interferon-gamma (IFN-gam ma) and tumor necrosis factor-alpha (TNF-alpha) both produced a marked increase in ICAM-1 expression. Our findings show that infection of SS cF with the SV40 tsT antigen extends the life span of these cells and does not ablate their abnormal phenotypic and functional characteristi cs. (C) 1995 Academic Press, Inc.