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: PKC alpha and PKC epsilon. 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 INF-alpha to IL-6 and IL-8 release. PKC alpha and PKC epsilon 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 INF-alpha was measured and PKC isoform activities assessed. We found that HDE stimulates PKC alpha activity by 1 hour, and within 6 hours the activity returns to baseline. PKC alpha-specific inhibitor or PKC alpha DN 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 INF-alpha neutralizing antibody does not further inhibit cytokine release. In contrast, PKC epsilon activity was enhanced by 6 hours after HDE treatment. TNF-alpha blockade abrogated this effect. HDE-stimulated IL-6, but not IL-8 release in PKC epsilon DN cells. The concentration of INF-alpha released by HDE-stimulated HBE is sufficient to have a potent cytokine-eliciting effect. A time course of INF-alpha release suggests that TNF-alpha is produced after PKC alpha activation, but before PKC epsilon. 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.