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, differentiatio n, turnover of extracellular matrix components, oncogene expression, a nd other aspects of cellular phenotype. Unlike lung fibroblasts of cer tain species, unstimulated human lung fibroblast lines produce little or no TGF-beta in culture. However, TGF-beta has been reported to auto regulate its own production in certain human tumor cells and in rodent cell lines. To test whether this phenomenon is operative in fibroblas ts from normal human lung tissue, confluent cultures of IMR90 normal f etal lung fibroblasts were exposed to TGF-beta. Cultures were exposed briefly to purified TGF-beta1 under serum-free conditions and secretio n of newly synthesized TGF-beta over the ensuing 72 h was determined b y immunoblotting and bioassays made specific with the use of neutraliz ing antibodies. Steady-state levels of mRNA for TGF-beta1 were detecte d by Northern and slot blot hybridization analysis of total cellular R NA. 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. Incre ased levels of TGF-beta were detected in conditioned medium 9 h after the start of the exposure. Thereafter, TGF-beta continued to accumulat e 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 TG F-beta1 auto-induced TGF-beta secretion. Increasing concentrations of exogenous TGF-beta (1 to 10 ng/ml) raised the auto-induction of secret ed TGF-beta in a concentration dependent manner. All of the TGF-beta r eleased by stimulated IMR90 fibroblasts was in latent form, confirming that it is newly synthesized cytokine. Furthermore, incubation of con ditioned medium with anti-TGF-beta neutralizing antibodies inhibited t he 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 fibroblas ts. 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 tha t cytokine signal amplification occurs in the pulmonary interstitium.