AUTOINDUCTION OF TRANSFORMING GROWTH-FACTOR-BETA IN HUMAN LUNG FIBROBLASTS

The type beta transforming growth factors (TGF-betas) are a family of potent cytokines with diverse effects on proliferation, differentiation, turnover of extracellular matrix components, oncogene expression, and other aspects of cellular phenotype. Unlike lung fibroblasts of certain species, unstimulated human lung fibroblast lines produce little or no TGF-beta in culture. However, TGF-beta has been reported to autoregulate its own production in certain human tumor cells and in rodent cell lines. To test whether this phenomenon is operative in fibroblasts from normal human lung tissue, confluent cultures of IMR90 normal fetal lung fibroblasts were exposed to TGF-beta. Cultures were exposed briefly to purified TGF-beta1 under serum-free conditions and secretion of newly synthesized TGF-beta over the ensuing 72 h was determined by immunoblotting and bioassays made specific with the use of neutralizing antibodies. Steady-state levels of mRNA for TGF-beta1 were detected by Northern and slot blot hybridization analysis of total cellular RNA. The 2.5 kb TGF-beta1 mRNA species rose within 1.5 h of exposure of IMR90 cells to TGF-beta1 and reached maximal levels after 16 h. Increased levels of TGF-beta were detected in conditioned medium 9 h after the start of the exposure. Thereafter, TGF-beta continued to accumulate at an elevated rate (90 +/- 7 versus less-than-or-equal-to 15 pg/10(6) cells/h in uninduced cells) for up to 72 h. As little as 1 ng/ml TGF-beta1 auto-induced TGF-beta secretion. Increasing concentrations of exogenous TGF-beta (1 to 10 ng/ml) raised the auto-induction of secreted TGF-beta in a concentration dependent manner. All of the TGF-beta released by stimulated IMR90 fibroblasts was in latent form, confirming that it is newly synthesized cytokine. Furthermore, incubation of conditioned medium with anti-TGF-beta neutralizing antibodies inhibited the activity of secreted TGF-beta. Bioassay data were also confirmed by Western blots demonstrating a specific 24 kD type 1 TGF-beta protein in increased amounts in conditioned medium from auto-induced fibroblasts. Parallel evaluation of adult human lung fibroblast lines indicated that auto-induction occurred in them as well. These studies establish that auto-induction occurs in normal lung fibroblasts, suggesting that cytokine signal amplification occurs in the pulmonary interstitium.