title: Sequential activation of protein kinase C isoforms by organic dust is mediated by tumor necrosis factor.
abstract: 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: PKCalpha and PKCepsilon. 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-alpha to IL-6 and IL-8 release. PKCalpha and PKCepsilon 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-alpha was measured and PKC isoform activities assessed. We found that HDE stimulates PKCalpha activity by 1 hour, and within 6 hours the activity returns to baseline. PKCalpha-specific inhibitor or PKCalphaDN 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-alpha-neutralizing antibody does not further inhibit cytokine release. In contrast, PKCepsilon 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 PKCepsilonDN cells. The concentration of TNF-alpha released by HDE-stimulated HBE is sufficient to have a potent cytokine-eliciting effect. A time course of TNF-alpha release suggests that TNF-alpha is produced after PKCalpha activation, but before PKCepsilon. 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.
authors.affiliation: Department of Internal Medicine, Pulmonary, Critical Care, Sleep & Allergy Section, University of Nebraska Medical Center, 985300 Nebraska Medical Center, Omaha, NE 68198-5300, USA. twyatt@unmc.edu
authors.forename: Todd A
authors.initials: TA
authors.lastname: Wyatt
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Slager
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Heires
authors.forename: Jane M
authors.initials: JM
authors.lastname: Devasure
authors.forename: Susanna G
authors.initials: SG
authors.lastname: Vonessen
authors.forename: Jill A
authors.initials: JA
authors.lastname: Poole
authors.forename: Debra J
authors.initials: DJ
authors.lastname: Romberger
mesh_terms: D000393:Air Pollutants / Q000009:adverse effects*; D000822:Animal Husbandry; D000818:Animals; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D002461:Cell Line, Transformed; D004391:Dust; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000276:immunology; D006799:Housing, Animal; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007527:Isoenzymes; D009154:Mutation; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D013552:Swine; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000393:Air Pollutants; D057134:Antibodies, Neutralizing; C508606:CXCL8 protein, human; D004391:Dust; C508600:IL6 protein, human; D018836:Inflammation Mediators; D015850:Interleukin-6; D016209:Interleukin-8; D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C496628:PRKCA protein, human; C497255:PRKCE protein, human; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon