PLATELET-DERIVED GROWTH-FACTOR (PDGF)-AA, (PDGF)-AB, AND (PDGF)-BB INDUCE DIFFERENTIAL CHEMOTAXIS OF EARLY-PASSAGE RAT LUNG FIBROBLASTS IN-VITRO

Platelet-derived growth factor (PDGF) isoforms are chemoattractants an d mitogens for cells of mesenchymal origin that could be important med iators of pulmonary fibrogenesis. We have previously reported that par ticle-activated alveolar macrophages secrete homologues of PDGF that a re composed of all three PDGF isoforms (PDGF-AA, -AB, and -BB). This m ixture of macrophage-derived PDGF, once dissociated from the PDGF-alph a-macroglobulin complex, induces chemotaxis of rat lung fibroblasts (R LF) in the nanomolar range, In addition, we have reported that PDGF is oforms induce differential proliferation of RLF (PDGF-BB > PDGF-AB > P DGF-AA), In the present study, we sought to determine the relative che motactic potency of the three PDGF isoforms and correlate these respon ses to the relative abundance of the two types of PDGF cell-surface re ceptors: PDGF-alpha receptor (PDGF-R alpha) and PDGF-beta receptor (PD GF-R beta), We also investigated the chemotactic activity of combinati ons of two PDGF isoforms simultaneously. Isolates of early-passage RLF were assayed for chemotaxis in 48-microwell chambers. Swiss mouse 3T3 cells were assayed in parallel as a positive control cell line for PD GF-R alpha and PDGF-R beta expression, RLF responded differentially to the PDGF isoforms: PDGF-AB and PDGF-BB were potent chemoattractants a nd stimulated maximal chemotactic responses between 4 and 8 ng/ml PDGF whereas PDGF-AA elicited a weak chemotactic response that was maximal ly 15% of that obtained with either B-chain isoform, PDGF-AB End PDGF- BB were also the most potent chemoattractants for Swiss 3T3 cells, and their response to these B-chain isoforms was similar to 40% greater t han that obtained for RLF Swiss 3T3 cells showed a 3-fold greater resp onse to PDGF-AA when compared with RLF Northern analysis demonstrated that RLF strongly expressed a 5.2 kb PDGF-R beta transcript and only a weak expression for a 6.5 kb PDGF-alpha transcript, In contrast, Swis s 3T3 cells strongly expressed both PDGF-R beta and PDGF-R alpha. Radi oligand binding assays for [I-125]PDGF-BB showed that RLF possessed ma inly PDGF-R beta at the cell surface (B-max = similar to 70 fmol/10(6) cells; K-d = 0.1 nM), and preincubation with 50 ng/ml PDGF-AA prior t o binding decreased receptor number < 10%, indicating a low level of c ell-surface PDGF-R alpha. Swiss 3T3 cells also bound [I-125]PDGF-BB wi th a K-d of 0.1 nM, but possessed a B-max of similar to 175 fmol/10(6) cells, and preincubation with 50 ng/ml PDGF-AA reduced the B-max to s imilar to 80 fmol/10(6) cells while increasing receptor affinity 3-fol d. Further analysis of the radioligand binding data showed that Swiss 3T3 cells possessed approximately equal numbers of cell-surface PDGF-R alpha and PDGF-R beta subunits. Finally, we observed that combination s of two different PDGF isoforms mixed at equal concentrations produce d additive chemotactic effects, These data indicate that all three PDG F isoforms are chemoattractants for fibroblasts, and the chemotactic r esponse to PDGF-AA, as well as PDGF-AB and PDGF-BB, depends on the abu ndance of cell-surface PDGF-R alpha relative to PDGF-R beta.