Development Of A Fibroblast Proliferation Bioassay To Detect Mediators Of Pulmonary Fibrosis

INTRODUCTION Proliferation and enhanced synthesis of collagen by pulmonary fibroblasts have been shown to be key steps in the development of chronic silicosis (Goldstein and Fine, 1986). The regulation of lung fibroblast proliferation by cytokines released from alveolar macrophages may be an important pathogenetic mechanism in the development of the fibrotic process (Kelley, 1990). One cytokine, platelet-derived growth factor (PDGF), promotes fibroblast proliferation by inducing the movement of quiescent (Go) cells into the C1 phase of the cell cycle (Chen and Rabinovitch, 1989). Others regulate the rate of transition of fibroblasts from Gl into the S phase (Leof et al., 1982). These two classes of cytokines have been termed, respectively, competence and progression factors. One approach used to examine the release of cytokines from macrophages is the fibroblast proliferation assay in which fibroblasts are exposed to culture supernatants from macrophages exposed to various stimuli. In most of these assays, the supernatant contains fetal calf serum which provides the competence factor(s) necessary to facilitate the proliferation of fibroblasts (Bitterman et al., 1982; Bitterman et al., 1983; Elias et al., 1988). Recently, a fibroblast proliferation assay using plateletpoor plasma (lacking competence factor(s)) as a substitute for fetal calf serum has been described (Kuman et al., 1988; Bauman et al., 1990). In this assay, the release of a competence-inducing PDGF-like growth factor from rat and human macrophages can be distinguished from other cytokines that act as progression factors. In order to obtain more consistent results and with the ultimate goal to be able to discriminate between the effects of competence factors as opposed to progression factors, we have conducted experiments to determine the appropriate concentrations of plasma and PDGF required for imparting competence in the fibroblast proliferation assay. We tested lung fibroblast cells obtained from explants of rat lung tissue and also a fetal human lung fibroblast cell line obtained from American Tissue Culture Collection (ATCC153). MATERIALS AND METHODS Fibroblasts Specific pathogen-free, male Sprague-Dawley rats were use in some studies. Animals were given a lethal intraperitoneal dose of sodium pentobarbital. Fibroblasts were isolated by chopping the lung in enzymes that digest the connective tissue but liberate lung cells for further study (Rabovsky et al., 1989). After digestion, the remaining lung tissue suspension was filtered through two layers of sterile gauze and centrifuged to recover lung fibroblasts. These were resuspended in culture medium that contained 10% fetal calf serum and distributed to culture plates for growth. In other experiments, a human fetal lung fibroblast cell line, obtained from American Type Culture Collection, Rockville, MD, 20852, was used instead of rat lung fibroblasts. In these cases, a 1 ml ampule containing human fetal fibroblasts was plated into a tissue culture flask containing medium plus 10% fetal calf serum. For both types of fibroblasts, culture medium was changed 3 times per week and cultures were incubated at 37°C until confluent. Harvested rat and human lung fibroblasts were quantified using an electronic cell counter equipped with a cell sizing attachment (Coulter Electronics, Inc., Hialeah, Florida). Tritiated Thymidine Incorporation The basic procedural outline of Kumar et al. (1988) was used with modifications to evaluate tritiated thymidine incorporation into fibroblast DNA following exposure to PDGF and plasma. Both rat and human lung fibroblasts were plated at 50,000 cells/ml at a density of 250,000 cells/25cm2 culture plate. Cells were quiesced for 4 days with 2% rat plasma. As the assay was refined, fibroblasts were quiesced in plasma-free media for 48 hrs, since the mitogenic activity of 2% plasma was variable. Test medium was applied for a period of 6 hrs, followed by a 24 hr tritiated thymidine (lµCi/ml) labelling period in plasma-free media. Medium alone was used as a negative control and media with 10 or 20% fetal calf serum was used as the positive control for rat and human fibroblasts, respectively. Cell Quantification and Measurement of Mitogenesis Twenty-four hours after the addition of tritiated thymidine, the fibroblasts were washed with 5ml of fresh serum-free media, centrifuged and resuspended in phosphate-buffered saline. The cells were dissolved in 0.5m1 of O.1N NaOH and radioactivity determined in a beta counter. Incorporation of trititaed thymidine as an index of DNA synthesis was expressed as DPM/fibroblast. RESULTS In the present study, we quantified mitogenic potential by monitoring the incorporation of tritiated thymidine as