Human bronchial epithelial cells modulate collagen gel contraction by fibroblasts.

Connective tissue contraction is an important aspect of both normal wound healing and fibrosis. This process may contribute to small airway narrowing associated with certain airway diseases. Fibroblast-mediated contraction of a three-dimensional collagen gel has been considered a model of tissue contraction. In this study, the ability of primary cultured human bronchial epithelial cells (HBEC) obtained by bronchial brushings to modulate fibroblast gel contraction was evaluated. Human lung fibroblasts (HFL1) were cast into type I collagen gels. The gels were floated both in dishes containing a monolayer of HBEC or in dishes without HBEC. Contraction assessed by measuring the area of gels was increased at all time points from 24 h up to 96 h of coculture. At 48 h, coculture of HBEC with fibroblasts resulted in significantly more contraction than fibroblasts alone (36.6 +/- 1.2 vs. 20.4 +/- 1.7%, P < 0.05). Lipopolysaccharide (LPS, 10 micrograms/ml) stimulation of the HBEC augmented the contraction (44.9 +/- 1.0%, P < 0.05 vs. HBEC). In the presence of indomethacin, the augmentation by LPS was increased further (52.2 +/- 4.3%, P < 0.05 vs. HBEC with LPS), suggesting that prostaglandins (PGs) are present and may inhibit contraction. Consistent with this, PGE was present in HBEC-conditioned medium. Bronchial epithelial cell conditioned medium had an effect similar to coculturing. SG-150 column chromatography revealed augmentive activity between 20 and 30 kDa and inhibitory activity between 10 and 20 kDa. Measurement by enzyme-linked immunosorbent assay confirmed the presence of the active form of transforming growth factor (TGF)-beta 2. The stimulatory activity of conditioned medium was blocked by adding anti-TGF-beta antibody. These data demonstrate that, through the release of factors including TGF-beta 2 which can augment and PGE which can inhibit, HBEC can modulate fibroblast-mediated collagen gel contraction. In this manner, HBEC may modulate fibroblast activities that determine the architecture of bronchial tissue.

Lipopolysaccharide increases fibronectin production and release from cultured lung fibroblasts partially through proteolytic activity.

Fibronectin is a major product of fibroblasts and can mediate diverse functions including wound healing. Chronic bacterial infections are generally associated with a marked decreased in the ability to repair. We therefore hypothesized that bacterial endotoxin, lipopolysaccharide (LPS), might alter fibroblast fibronectin production. LPS augmented fibronectin production by fibroblasts and also stimulated the release of fibronectin from cell layers. An increase in new protein synthesis appeared to account for part of the increased fibronectin, because the inhibitor of protein synthesis, cycloheximide, inhibited the increase in total production of fibronectin. Cycloheximide did not attenuate the increased release of fibronectin into the culture medium. This increased release appeared to be caused, at least in part, by fragmentation of fibronectin by proteases contained in LPS preparations. In this regard all preparations of LPS tested were found to cleave fibronectin. Finally, zymograms indicated that LPS could also cleave gelatin with at least two bands of proteolytic activity but that it did not cleave bovine serum albumin or ovalbumin. These results indicate that the ability of bacterial products to alter fibronectin production and to degrade this macromolecule may account for altered wound repair that occurs with chronic bacterial infection.

Transforming growth factor-beta stimulates the expression of desmosomal proteins in bronchial epithelial cells.

Transforming growth factor-beta 1 (TGF-beta 1) has been shown to induce squamous differentiation of cultured airway epithelial cells. It has also been shown to increase expression of matrix proteins and integrin receptors in cell culture of these and other cells. However, it is unknown if TGF-beta 1 affects expression of genes encoding intercellular junctional proteins. Therefore, we have investigated the effect of TGF-beta 1 on the expression of proteins and mRNAs for desmoplakins (DPs) I and II, desmosomal plaque proteins. Fibronectin, known to be induced by TGF-beta 1 was used as a positive control and tubulin as a negative control. Twenty-four hours after TGF-beta 1 stimulation, DP I and II mRNA levels assessed by Northern blotting analysis had increased significantly (DP I mRNA, 1.8-fold, P less than 0.05; DP II mRNA, 2.4-fold, P less than 0.04), thereby indicating pretranslational regulation of DP expression. By comparison, mRNA for fibronectin increased 8.1-fold whereas mRNA for tubulin was unchanged. Immunofluorescence using the monoclonal anti-DP I and II antibodies revealed dramatic increased expression of punctate DP structures after exposure to TGF-beta 1. Immunoblot analyses with polyclonal anti-DP I antibodies showed increased levels of both DP I (250 kD) and DP II (215 kD), with the DP I increase being more pronounced (DP I, 2.5-fold; DP II, 1.4-fold at 48 h relative to controls), suggesting translational regulation by TGF-beta 1. This study therefore demonstrates the ability of TGF-beta 1 to alter cellular phenotype by altering expression of proteins involved in intercellular junctions.(ABSTRACT TRUNCATED AT 250 WORDS)