Contraction of three-dimensional collagen gels is a model of the contraction that characterizes normal healing and remodeling after injury. In the current study, we evaluated the hypothesis that a number of inflammatory factors, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and interferon (IFN)-gamma, modulate this process by induction of prostaglandin (PG) E(2) and nitric oxide (NO) production and that these secondary mediators function in an autocrine or paracrine manner to modulate contraction. Human fetal lung fibroblasts (HFL) were cultured in type I collagen gels and floated in medium containing TNF-alpha, IL-1 beta, or IFN-gamma alone or in combination (cytomix). All cytokines inhibited the contraction significantly. The potency order was IL-1 beta, TNF-alpha, IFN-gamma. The cytomix was no more potent than was IL-1 beta alone. PGE(2) production was increased by TNF-alpha (5.0 versus 0.16 ng/ml, P < 0.01), IL-1 beta (5.3 versus 0.16 ng/ml, P < 0.01), and cytomix (5.9 versus 0.16 ng/ml, P < 0.01), and was completely inhibited by indomethacin. Indomethacin (P < 0.05) and L-NG-monomethyl arginine citrate (L-NMMA) (P < 0.05) alone both partially attenuated the inhibition of contraction caused by cytokines alone or by cytomix. Indomethacin and L-NMMA together attenuated inhibition more than either alone (P < 0.05). Exogenous PGE(2) and exogenous NO donors (DETA nononate and 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride) inhibited the contraction significantly. The protein kinase A inhibitor KT5270 and the protein kinase G inhibitor Rp-pCPT-cGMPS attenuated the inhibition induced by PGE(2) and NO, respectively. In summary, PGE(2) and NO appear to function in parallel as autocrine/paracrine mediators of cytokine-driven fibroblast inhibition of the contraction of collagen gels and may contribute to remodeling during repair and inflammation in lung disorders.

Proteolytic degradation of extracellular matrix is thought to play an important role in many lung disorders. In the current study, human lung fibroblasts were cast into type I collagen gels and floated in medium containing elastase, cytomix (combination of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma), or both. After 5 days, gel collagen content was determined by measuring hydroxyproline. Elastase alone did not result in collagen degradation, but in the presence of fibroblasts, elastase reduced hydroxyproline content to 75.2% (P < 0.01), whereas cytomix alone resulted in reduction of hydroxyproline content to 93% (P < 0.05). The combination of elastase and cytomix reduced hydroxyproline content to 5.2% (P < 0.01). alpha(1)-Proteinase inhibitor blocked this synergy. Gelatin zymography and Western blot revealed that matrix metalloproteinase (MMP)-1, -3, and -9 were induced by cytomix and activated in the presence of elastase. Tissue inhibitor of metalloproteinase (TIMP)-1 and -2 were also induced by cytomix but were cleaved by elastase. We conclude that a synergistic interaction between cytomix and elastase, mediated through cytokine induction of MMP production and elastase-induced activation of latent MMPs and degradation of TIMPs, can result in a dramatic augmentation of collagen degradation. These findings support the notion that interaction among inflammatory mediators secreted by mononuclear cells and neutrophils can induce tissue cells to degrade extracellular matrix. Such a mechanism may contribute to the protease-anti-protease imbalance in emphysema.

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Following lung injury, red blood cells (RBC) may interact with extracellular matrix (ECM). Fibroblasts, the resident cell in the ECM, have the capacity to produce and secrete a variety of mediators including interleukin-8 (IL-8). In the present study we hypothesized that RBC, or soluble factors released from them, may stimulate IL-8 production by fibroblasts. Fibroblasts were cultured in a three-dimensional collagen gel culture system in the presence or absence of RBC or conditioned medium from RBC (RBC-CM). IL-8 release from fibroblasts was significantly increased when cultured with RBC or RBC-CM and both tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) further stimulated this IL-8 secretion. The enhanced production of IL-8 within fibroblasts was accompanied by increased IL-8 mRNA expression. To evaluate whether RBC-fibroblast interaction may lead to recruitment of neutrophils, a functional migration assay was performed. RBC and RBC-CM, in the presence of IL-1beta and TNF-alpha, increased the transmigration of neutrophils. Our results indicate that RBC, when interacting with ECM, may participate in the recruitment of inflammatory cells by stimulating fibroblasts to secrete IL-8. This might be an important mechanism regulating tissue repair after injury.