Persistence of scleroderma-like phenotype in normal fibroblasts after prolonged exposure to soluble mediators from...

Supernatants of mononuclear cells (MNC-SN) were shown to increase synthesis of glycosaminoglycan (GAG) by cultured normal dermal fibroblasts. Fibroblasts from the skin of patients with progressive systemic sclerosis (PSS, scleroderma) were hyporesponsive. We exposed fibroblasts outgrowing from explants of normal adult skin to MNC-SN for up to 30 generations in culture. MNC-SN were obtained by incubating normal MNC with concanavalin A. Four experimental, 4 normal control, and 3 PSS control lines were passaged by trypsinizing and splitting the cultures 1:2 every 7 days. At the third and fifth passages, portions of the experimental fibroblasts were removed from MNC-SN, then passaged in medium alone. Cell counts, assays for GAG, and electron microscopy were performed and increases in GAG after brief reexposure to MNC-SN were determined at the third, fifth, and eighth passages. In normal dermal fibroblasts, baseline GAG production, measured by 3H-glucosamine uptake, was low and increased as much as 15 times after reexposure to MNC-SN. In contrast, production was high in both experimental and PSS lines, and increases after reexposure to MNC-SN were consistently small. This PSS-like behavior persisted in experimental fibroblasts removed from MNC-SN at the third and fifth passages. Growth of experimental and scleroderma fibroblasts was slower than that of control fibroblasts. Ultrastructurally, both scleroderma and experimental dermal fibroblasts differed from normal fibroblasts by their oval cellular shape, indentations in nuclear membrane, numerous organelles and bundles of microfilaments, prominent Golgi, and intranuclear inclusions. These experiments indicate that normal adult dermal fibroblasts subjected to MNC-SN in vitro acquire a scleroderma-like phenotype that persists for many generations.