Soluble mediators from mononuclear cells increase the synthesis of glycosaminoglycan by dermal fibroblast cultures derived from normal subjects and progressive systemic sclerosis patients

Abstract

Dermal fibroblast cultures from patients with progressive systemic sclerosis (PSS) synthesize up to 5 times more glycosaminoglycan (GAG) than normal cultures. In an in vitro model of fibroblast-lymphocyte interactions, we show that the supernatants of activated mononuclear cells (MNC) modulate GAG synthesis, as measured by the incorporation of 3H-glucosamine into GAG following incubation of the confluent fibroblast monolayers with active supernatant preparations. GAG accumulation was selectively increased up to 18 times in normal dermal fibroblast cultures. Cell viability was dot affected, and 3H-thymidine uptake and cell numbers were depressed in cultures treated with the supernatants. In contrast to normal dermal fibroblast cultures, PSS fibroblasts responded to MNC supernatants by only a 1-2-fold increase in GAG. Supernatants of concanavaPresented in part at the 47th Annual Meeting of the American Rheumatism Association,

Persistence of scleroderma‐like phenotype in normal fibroblasts after prolonged exposure to soluble mediators from mononuclear cells

“…Under defined growth conditions and with a standard inoculum of 40,000 fibroblasts in the log phase of growth, normal and PSS cultures are in confluence by day 6 (16). Using strains, we have shown that no further increases in cell numbers occur by day 4 of culture (unpublished observations).…”

“…Fresh medium and supernatants of MNC not activated with Con A were used as controls. The fibroblasts were incubated with the MNC-SN for 72 hours (16) and then assayed for GAG accumulation as described below. Active MNC-SN were pooled and stored at -70°C until further use.…”

“…1 (January 1986) repeatedly been shown to alter the behavior of fibroblasts in culture. MNC-SN may modulate the growth (11,12) and synthesis of collagen (13)(14)(15) and GAG (16,17) by fibroblasts. Normal as well as PSS fibroblasts have been shown to respond t o these modulatory influences (16).…”

Heterogeneous synthetic phenotype of cloned scleroderma fibroblasts may be due to aberrant regulation in the synthesis of connective tissues

“…These numbers were used to calculate the levels of GAG and CSP synthesis per cell. In each of the triplicate wells, GAG synthesis was estimated as the counts per minute of 'Hglucosamine incorporated into total GAG (cells + culture medium) and precipitable by cetylpyridinium chloride, as described previously (15). The CSP synthesis was determined in triplicate wells as the cpm of 3H-proline incorporated into total acid-soluble, collagenase-sensitive material, using the method of Peterkofsky and Diegelman (16), with our modifications as described elsewhere (17).…”

“…It is possible that a combination of selection and activation events could account for its appearance. A variety of mechanisms, including serum factors (6,22) and products of mononuclear cells (15,17,23), have been proposed as effectors of fibrotic changes in PSS and other diseases.…”