Evidence for the involvement of TGF-beta and PDGF in the regulation of prolyl 4-hydroxylase and lysyloxidase in cultured rat lung fibroblasts

Lung, fibrosis is the end-point of numerous lung disorders induced by a pneumonia or by a variety of different noxes, one of which is the cytostatic drug bleomycin (BLM). Fibrosis is characterized by excessive extracellular matrix accumulation. Macrophage-fibroblast interactions are suggested to play an important role in the development of this disease. The present study was addressed to investigate one possible pathway of this interaction, the influence of soluble mediators produced by BLM-stimulated macrophages on lung fibroblast collagen synthesis and modification. Conditioned media (CM) of BLM-exposed macrophages of the cell line NR8383 submitted to rat lung fibroblast cultures increased the activity of prolyl 4-hydroxylase (P4H) in fibroblasts in a dose dependent manner. CM of stimulated macrophages increased the collagen concentration in fibroblast culture supernatant. The level of mRNAs specific for the a-subunit of P4H and that for alpha1(I) collagen were found to be increased by about twofold, that for lysyloxidase (LO) by about 2.5-fold in fibroblasts cultured in CM of stimulated macrophages. Pre-incubation of CM of BLM-exposed macrophages with neutralizing antibodies against TGF-beta or against PDGF resulted in a partial reversal of the increasing effect of the CM on P4H- and LO-activities in fibroblasts. Both growth factors, TGF-beta and PDGF, added to fibroblast cultures led to significant increases of P4H activity in the treated cells. We conclude that TGF-P and PDGF produced by stimulated macrophages are involved in the regulation of the expression of alpha1(I) collagen, of P4H-alpha-subunit and LO in lung fibroblasts. The results indicate that this is not a direct effect but involves the action of a so far unidentified mediator responsible for autocrine stimulation of collagen production.