Production of macrophage-dependent fibroblast-stimulating activity (M-FSA) by murine macrophages: Effects on BALBc 3T3 fibroblasts

Abstract

Macrophages cultured in vitro in medium containing platelet-poor plasma serum (PPPS) produce a substance (or substances) that stimulates the proliferation of fibroblasts in vitro. Two alternative assays have been used to study the production of this “macrophage-dependent fibroblast-stimulating activity” (M-FSA) by mouse peritoneal macrophages. The first determined increase in cell numbers induced in cultures of quiescent 3T3 fibroblasts (Growth Curve Assay); the second determined the incorporation of [ 3H]thymidine into the DNA of quiescent 3T3 cells induced by various culture media. Medium containing 5% blood serum (5% BS) resulted in higher final cell numbers (5-fold), and greater incorporation of [ 3H]thymidine into DNA than did medium containing 5% platelet-poor plasma serum (PPPS). Macrophages cultured in PPPS produced two different operationally defined activities. The first, M-FSA, resulted in greater final cell numbers, and was non-dialysable. The second reduced the incorporation of [ 3H]thymidine into DNA, and was found to be dialysable. Maximum levels of M-FSA were produced at a concentration of approx. 5 × 10 6 macrophages/10 ml culture medium containing 5 % PPPS. Successive 18 h cultures of macrophages in fresh changes of medium containing PPPS resulted in equivalent levels of M-FSA in each successive batch of medium. The presence of PPPS in the culture medium appeared to be an absolute requirement for the production of M-FSA. Culture in the absence of serum, in the presence of BSA, FCS, or of RBS did not result in the production of M-FSA. The dialysable factor, on the other hand, was produced non-specifically, in media containing either PPPS, FCS, RBS, BSA, or no serum. The level of production increased with increasing macrophage concentrations. At a concentration of 100 × 10 6 macrophages/10 ml medium, the stimulatory effect of M-FSA in the [ 3H]thymidine incorporation assay was completely masked. The dialysable substance did not affect the proliferation of 3T3 cells. Only the incorporation of [ 3H]thymidine into DNA was affected. It is suggested that M-FSA could be produced by macrophages, indirectly, by activation or de-inhibition of inactive (or inhibitory) substances present specifically in PPPS.