Epistatic effect of TLR-1, -6 and -10 polymorphisms on organic dust-mediated cytokine response.

Exposure to organic dust from agricultural environments is associated with inflammatory respiratory conditions. The putative causal agents in organic dust include viral, microbial and fungal components, which are recognized by the family of Toll-like receptors (TLRs) and drive host innate and adaptive responses. Our aim in this study was to determine whether responsiveness to organic dust among agricultural workers was dependent on polymorphisms in the TLR10-TLR1-TLR6 gene cluster. We stimulated whole blood from 509 agricultural workers with organic dust, triacyl lipopeptide N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)4 (Pam3CSK4) and the diacyl-lipopeptide peptidoglycan. Several of the tagging polymorphisms and haplotypes conferred hyper-responsiveness to organic dust with an increase in interleukin-6 (IL-6; P<0.005), but not tumor necrosis factor-α (TNF-α), secretion. We conclude that genetic variation in the TLR10-TLR1-TLR6 gene cluster mediates responsiveness to organic dust, but indicates different signaling pathways for IL-6 and TNF-α. These studies provide new insight into the role of the TLR10-TLR1-TLR6 gene cluster and the innate immune response to organic dust.

CXCR1/CXCR2 antagonist CXCL8(3-74)K11R/G31P blocks lung inflammation in swine barn dust-instilled mice.

Inhalation of agricultural occupational dusts from swine confinement facilities can result in lung inflammation. The innate immune response to organic barn dusts results in production of a number of pro-inflammatory factors in the lungs of barn workers such as cytokines, chemokines, and an influx of neutrophils. Many of these inflammatory factors are influenced by the chemokine CXCL8/IL-8 (KC or MIP-2 in mice). Previously, we have demonstrated that an endotoxin-independent component of swine barn dust extract (SBE) elevates lung chemokines in a protein kinase C (PKC)-dependent manner resulting in the significant formation of lung inflammatory cell infiltrates in a mouse model of SBE injury. In this study we test the ability of a CXCR1/CXCR2 antagonist, CXCL8(3-74)K11R/G31P (G31P) to block many of the features of lung-inflammation in response to challenge with SBE in an established mouse exposure system. Injection of G31P concurrent with SBE nasal instillation over a course of 3 weeks significantly reduced neutrophil accumulation in the lungs of barn dust exposed animals compared to those given SBE alone. There was a similar reduction in pro-inflammatory cytokines and chemokines IL-6, KC, and MIP-2 in SBE plus G31P-treated mice. In addition to excreted products, the receptors ICAM-1, CXCR1, and CXCR2, which all were elevated with SBE exposure, were also decreased with G31P treatment. SBE activation of PKCα and PKCε was reduced as well with G31P treatment. Thus, G31P was found to be highly effective at reducing several features of lung inflammation in mice exposed to barn dust extracts.

Protein kinase C activation is required for cigarette smoke-enhanced C5a-mediated release of interleukin-8 in human bronchial epithelial cells.

Complement-derived anaphylatoxin C5a is a glycopolypeptide important in the regulation of inflammation. Previously, we have shown that C5a receptors (C5aR) are constitutively expressed on human bronchial epithelial cells (HBECs) grown in culture. We have also shown that the expression of C5aR is increased upon exposure of HBECs to 5% cigarette smoke extract (CSE), and that this subtoxic dose of CSE significantly enhances C5a-stimulated interleukin (IL)-8 release. To determine the intracellular signaling pathway of CSE + C5a-mediated IL-8 release, we assayed protein kinase C (PKC) activity of HBECs after exposing the cells to CSE and/or C5a. No increase in PKC activity was observed when HBECs were treated with 50 nM C5a for various times. However, PKC activity was increased by 2- to 3-fold in HBECs stimulated with 5% CSE for 1 h, as compared with cells incubated with medium only. No additional increase in PKC was observed when HBECs were treated with CSE and C5a together. When HBECs were pretreated with the PKC-specific inhibitor calphostin C (1 microM), no CSE-mediated PKC activation was observed. We then correlated PKC activation with IL-8 release in the same cells. Although HBECs required stimulation by both CSE and C5a to release maximal levels of IL-8, preincubation of CSE-stimulated HBECs with calphostin C inhibited IL-8 release by CSE + C5a. These results suggest that PKC activation by CSE alone does not result in IL-8 release, but that CSE-stimulated PKC activation is required for C5a-mediated IL-8 release from HBECs.

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. alpha1-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 activity had augmentive activity for gel contraction which could be partially blocked by the inhibitors. We conclude that NE augments fibroblast-mediated contraction of collagen gels. The findings support the notion that products secreted by PMN in inflammatory disorders may lead to rearrangement of extracellular matrix and could subsequently lead to tissue dysfunction.

Expression of receptors for C5a anaphylatoxin (CD88) on human bronchial epithelial cells: enhancement of C5a-mediated release of IL-8 upon exposure to cigarette smoke.

Results are presented that demonstrate a heightened responsiveness of human bronchial epithelial cells (HBECs) toward the complement-derived anaphylatoxin C5a when these cells are exposed to cigarette smoke. This C5a response is possible because we show at both the protein and mRNA levels that HBECs constitutively express receptors for C5a (C5aR, CD88). Control (untreated) HBECs responded to C5a (50 nM) by releasing the proinflammatory cytokine IL-8 at low but significant levels. However, exposure of HBECs to 5% cigarette smoke extract (CSE) for at least 15 min resulted in an increase in the ability of an anti-human C5aR Ab to bind to the cell surface. CSE-treated HBECs responded in a dose-dependent fashion to human recombinant C5a and to a conformationally biased decapeptide agonist of C5a (YSFKPMPLaR) by releasing IL-8. The levels of IL-8 released in response to C5a were significantly greater in CSE-treated HBECs than in control HBECs. Moreover, this C5a-mediated release of IL-8 from CSE-treated HBECs was significantly reduced in the presence of the anti-human C5aR Ab. These results indicate that HBECs constitutively express C5aRs and that exposure to environmental irritants such as cigarette smoke modulates the expression and responsiveness of these C5aRs toward the C5a-mediated release of IL-8.

Aerosolized beclomethasone in chronic bronchitis. Improved pulmonary function and diminished airway inflammation.

Chronic bronchitis is associated with airways obstruction and inflammation. In order to determine whether aerosolized beclomethasone can modulate airway inflammation and diminish airway obstruction, subjects with chronic bronchitis performed spirometry and underwent bronchoalveolar lavage (BAL) before and after receiving 6 wk of therapy (five puffs four times a day) with either aerosolized beclomethasone (n = 20) or placebo (n = 10) in a double-blinded, randomized fashion. All subjects received aerosolized albuterol before each use of the study medications. Before BAL, the airways were visually assessed for the appearance of inflammation and assigned a score, the bronchitis index. BAL was performed by instilling five 20-ml aliquots of saline into each of three sites and pooling and separately analyzing the returns from the first aliquots to yield a "bronchial sample." The bronchial lavages were repeated in an additional three sites to increase the volume of fluid available for analysis. The fluid was prepared for cytologic examination by cytocentrifugation. Albumin (as a measure of epithelium permeability) and lactoferrin and lysozyme (as measures of serous cell activity) were measured in unconcentrated BAL fluid by enzyme-linked immunosorbent assay, and concentrations in epithelial lining fluid were estimated using urea as an internal marker for dilution. After treatment, the beclomethasone group, but not the placebo group, showed improvement in FVC (p = 0.02), FEV1 (p = 0.002), and 25 to 75% forced expiratory flow (p = 0.006). Associated with the improvement in spirometry, the bronchitis index fell (13.5 +/- 1.0 versus 10.75 +/- 1.1, p = 0.02) in the beclomethasone-treated group, but not the placebo-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)