Human neutrophil elastase augments fibroblast-mediated contraction of released collagen gels.

In the present study, we tested the hypothesis that neutrophil elastase (NE) might mediate remodeling of extracellular matrix by affecting fibroblast-mediated contraction of three-dimensional collagen gels. Human lung fibroblasts were cast into type I collagen gels containing NE. After gelation, the gels were released into medium and the area was measured by image analyzer. NE augmented gel contraction (p < 0.001). This was not due to cell proliferation or to degradation to soluble collagen fragments because the amounts of DNA and hydroxyproline were not altered. α1-Protease inhibitor and the synthetic inhibitor of NE, L-680,833, when added in sufficient amount to inhibit free elastase activity, blocked the contraction induced by NE. Furthermore, neutrophil granulocytes (PMN) in coculture, as well as conditioned media from PMN, resulted in an increased contractility (p < 0.001 for both). Bronchoalveolar lavage fluid (BALF) from patients with increased PMN in their lower respiratory tract and free elastase..

Sequential Activation of Protein Kinase C Isoforms by Organic Dust Is Mediated by Tumor Necrosis Factor

Dust samples collected from Nebraska swine confinement facilities (hog dust extract [HDE]) are known to elicit proinflammatory cytokine release from human bronchial epithelial (HBE) cells in vitro. This response involves the activation of two protein kinase C (PKC) isoforms: PKCα and PKCɛ. Experiments were designed to investigate the relationship between the two isoenzymes and the degree to which each is responsible for cytokine release in HBE. Experiments also examined the contribution of TNF-α to IL-6 and IL-8 release. PKCα and PKCɛ activities were inhibited using isoform-specific pharmacologic inhibitors and genetically modified dominant-negative (DN) expressing cell lines. Release of the proinflammatory cytokines IL-6, IL-8, and TNF-α was measured and PKC isoform activities assessed. We found that HDE stimulates PKCα activity by 1 hour, and within 6 hours the activity returns to baseline. PKCα-specific inhibitor or PKCαDN cells abolish this HDE-mediated effect. Both IL-6 and IL-8 release are likewise diminished under these conditions compared with normal HBE, and treatment with TNF-α–neutralizing antibody does not further inhibit cytokine release. In contrast, PKCɛ activity was enhanced by 6 hours after HDE treatment. TNF-α blockade abrogated this effect. HDE-stimulated IL-6, but not IL-8 release in PKCɛDN cells. The concentration of TNF-α released by HDE-stimulated HBE is sufficient to have a potent cytokine-eliciting effect. A time course of TNF-α release suggests that TNF-α is produced after PKCα activation, but before PKCɛ. These results suggest a temporal ordering of events responsible for the release of cytokines, which initiate and exacerbate inflammatory events in the airways of people exposed to agricultural dust