Auto-induction of Transforming Growth Factor- β in Human Lung Fibroblasts

Abstract

The type β transforming growth factors (TGF-βs) 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-β in culture. However, TGF-β 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-β. Cultures were exposed briefly to purified TGF-β1 under serum-free conditions and secretion of newly synthesized TGF-β 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-β1 were detected by Northern and slot blot hybridization analysis of total cellular RNA. The 2.5 kb TGF-β1 mRNA species rose within 1.5 h of exposure of IMR90 cells to TGF-β1 and reached maximal levels after 16 h. Increased levels of TGF-β were detected in conditioned medium 9 h after the start of the exposure. Thereafter, TGF-β continued to accumulate at an elevated rate (90 ± 7 versus ≥ 15 pg/106 cells/h in uninduced cells) for up to 72 h. As little as 1 ng/ml TGF-β1 auto-induced TGF-β secretion. Increasing concentrations of exogenous TGF-β (1 to 10 ng/ml) raised the auto-induction of secreted TGF-β in a concentration dependent manner. All of the TGF-β released by stimulated IMR90 fibroblasts was in latent form, confirming that it is newly synthesized cytokine. Furthermore, incubation of conditioned medium with anti-TGF-β neutralizing antibodies inhibited the activity of secreted TGF-β. Bioassay data were also confirmed by Western blots demonstrating a specific 24 kD type 1 TGF-β 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.