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.

Proteases in agricultural dust induce lung inflammation through PAR-1 and PAR-2 activation

“…In the present study, we tested whether the proteases in HDE influence the stimulation of PKC activity, by depleting protease activity in HDE before stimulating BEAS-2B cells in vitro. Both PKC␣ (1 h) and PKCε (6 h) activities in BEAS-2B lysates were significantly increased after exposure to complete HDE, as previously reported (43), whereas protease-depleted HDE treatment did not activate either kinase (Fig. 7).…”

“…We tested whether and to what degree the protease activity in HDE contributed to cytokine release. As in our laboratory's previous studies, treatment of subconfluent monolayers BECs (BEAS-2B and primary NHBEs) with 5% HDE resulted in a rapid and sustained increase of IL-6 and IL-8/CXCL8 release (43) were decreased from 2,456 Ϯ 279 to 785 Ϯ 16 pg/ml after AEBSF treatment; AEBSF lowered IL-8 levels from 6,595 Ϯ 348 to 3,313 Ϯ 149 pg/ml (Fig. 2, A and B); for NHBE, AEBSF reduced IL-6 from 992 Ϯ 44 to 593 Ϯ 48 pg/ml, and IL-8 from 7,704 Ϯ 853 to 3,554 Ϯ 493 pg/ml (Fig.…”

“…Previously, our laboratory demonstrated the importance of the protein kinase isoforms PKC␣ and PKCε in the release of HDE-induced proinflammatory cytokines IL-6, IL-8, and TNF-␣ in BECs (33,43). In the present study, we tested whether the proteases in HDE influence the stimulation of PKC activity, by depleting protease activity in HDE before stimulating BEAS-2B cells in vitro.…”

“…In an effort to understand the mechanistic basis of airway inflammation associated with agricultural dusts, previous work from our group has focused on characterizing inflammatory responses induced by aqueous organic dust extracts derived from hog CAFOs [hog dust extract (HDE)] using in vitro cell culture models and in vivo murine models of dust exposure (26,29,33,35,41,43). Those studies showed that HDE treatment of bronchial epithelial cells (BECs) results in the sequential activation of protein kinase C (PKC) ␣ and ε that, in turn, leads to inflammatory cytokine/chemokine production (33,43).…”

“…Those studies showed that HDE treatment of bronchial epithelial cells (BECs) results in the sequential activation of protein kinase C (PKC) ␣ and ε that, in turn, leads to inflammatory cytokine/chemokine production (33,43). In murine studies, a single exposure to HDE resulted in marked neutrophil influx and enhanced inflammatory mediator levels in bronchoalveolar lavage fluid (BALF), and repetitive HDE exposure, while having less dramatic changes in BALF cell/cytokine levels, resulted in dramatic increases in lung histopathology characterized by mononuclear leukocyte cell aggregates and peribronchiolar and perivascular inflammation (29).…”