Antiinflammatory role of MUC1 mucin during infection with nontypeable Haemophilus influenzae.

MUC1 (or Muc1 in nonhuman species) is a membrane-tethered mucin expressed on the apical surface of mucosal epithelia (including those of the airways) that suppresses Toll-like receptor (TLR) signaling. We sought to determine whether the anti-inflammatory effect of MUC1 is operative during infection with nontypeable Haemophilus influenzae (NTHi), and if so, which TLR pathway was affected. Our results showed that: (1) a lysate of NTHi increased the early release of IL-8 and later production of MUC1 protein by A549 cells in dose-dependent and time-dependent manners, compared with vehicle control; (2) both effects were attenuated after transfection of the cells with a TLR2-targeting small interfering (si) RNA, compared with a control siRNA; (3) the NTHi-induced release of IL-8 was suppressed by an overexpression of MUC1, and was enhanced by the knockdown of MUC1; (4) the TNF-α released after treatment with NTHi was sufficient to up-regulate MUC1, which was completely inhibited by pretreatment with a soluble TNF-α receptor; and (5) primary murine tracheal surface epithelial (MTSE) cells from Muc1 knockout mice exhibited an increased in vitro production of NTHi-stimulated keratinocyte chemoattractant compared with MTSE cells from Muc1-expressing animals. These results suggest a hypothetical feedback loop model whereby NTHi activates TLRs (mainly TLR2) in airway epithelial cells, leading to the increased production of TNF-α and IL-8, which subsequently up-regulate the expression of MUC1, resulting in suppressed TLR signaling and decreased production of IL-8. This report is the first, to the best of our knowledge, demonstrating that the inflammatory response in airway epithelial cells during infection with NTHi is controlled by MUC1 mucin, mainly through the suppression of TLR2 signaling.

Analysis of the proteome of human airway epithelial secretions.

BACKGROUND: Airway surface liquid, often referred to as mucus, is a thin layer of fluid covering the luminal surface that plays an important defensive role against foreign particles and chemicals entering the lungs. Airway mucus contains various macromolecules, the most abundant being mucin glycoproteins, which contribute to its defensive function. Airway epithelial cells cultured in vitro secrete mucins and nonmucin proteins from their apical surface that mimics mucus production in vivo. The current study was undertaken to identify the polypeptide constituents of human airway epithelial cell secretions to gain a better understanding of the protein composition of respiratory mucus. RESULTS: Fifty-five proteins were identified in the high molecular weight fraction of apical secretions collected from in vitro cultures of well-differentiated primary human airway epithelial cells and isolated under physiological conditions. Among these were MUC1, MUC4, MUC5B, and MUC16 mucins. By proteomic analysis, the nonmucin proteins could be classified as inflammatory, anti-inflammatory, anti-oxidative, and/or anti-microbial. CONCLUSIONS: Because the majority of the nonmucin proteins possess molecular weights less than that selected for analysis, it is theoretically possible that they may associate with the high molecular weight and negatively charged mucins to form a highly ordered structural organization that is likely to be important for maintaining the proper defensive function of airway mucus.

Suppressive effect of fluticasone propionate on MMP expression in the nasal mucosa of allergic rhinitis patients in vivo.

The effects of intranasal corticosteroids on matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases (TIMPs)) in the nasal mucosa of patients with allergic rhinitis (AR) are not known. Nasal mucosa biopsy specimens were obtained from AR patients, with or without the administration of fluticasone propionate (FP) nose drops, and from healthy volunteers as controls. The specimens were analyzed by immuno-histochemistry and ELISA for MMP-2, MMP-9, TIMP-1 and TIMP-2. The MMP-9 levels in nasal mucosa extracts in the AR patients were significantly higher than in the controls. A significant suppressive effect of FP on the MMP-9 levels was shown. The control subjects showed no MMP- or TIMP-positive cells, whereas such positive cells were clearly present in the AR patients. No MMP- or TIMP-positive cells were detected after topical application of FP. These results suggest that the suppressive effect of FP on MMP expression is, in part, responsible for its clinical efficacy in AR.

Epinastine hydrochloride antagonism against interleukin-4-mediated T cell cytokine imbalance in vitro.

BACKGROUND: Interleukin (IL)-4 is well accepted to be a cytokine that plays many roles in the regulation of immune responses. Although the primary pharmacological target of antihistamines has been regarded as the histamine H1 receptor, there is little information about the influence of antihistamines on IL-4-mediated immune responses. The present study was undertaken to examine whether H1 receptor antagonists could modulate IL-4-mediated immune responses in vitro. METHODS: CD4+ T cells from normal human peripheral blood (1 x 10(6) cells/ml) were incubated with various concentrations of epinastine hydrochloride (EP) or chlorpheniramine (CH) for 30 min and then stimulated with 10.0 ng/ml IL-4. After 24 h, culture supernatants were collected and assayed for IL-5, IL-6, IL-13 and interferon-gamma by ELISA. The influence of EP on transcription factor activation and mRNA expression for cytokines was also examined. RESULTS: Addition of EP into cell cultures at more than 20.0 ng/ml significantly suppressed the production of IL-5, IL-6 and IL-13, which were increased by IL-4 stimulation. EP at more than 20.0 ng/ml also suppresses nuclear factor-kappaB activation, signal transducers and activators of transcription 6 phosphorylation and mRNA expression, which were upregulated by IL-4 stimulation. However, the ability of CD4+ T cells to produce interferon-gamma was decreased by IL-4 stimulation, which was dramatically restored by treatment with EP at more than 15.0 ng/ml. On the other hand, CH, a first-generation H1 receptor antagonist, could not inhibit cytokine production from CD4+ T cells in response to IL-4 stimulation, even when 90.0 ng/ml of the agent was added to cell cultures. CONCLUSION: The present results strongly suggest that EP, a second-generation H1 receptor antagonist, interferes with IL-4-activated signaling in CD4+ T cells and results in favorable modification of the allergic disease state or conditions.

Effect of tranilast on matrix metalloproteinase production from neutrophils in-vitro.

Tranilast is an anti-allergic agent that blocks the release of chemical mediators, such as histamine and leukotrienes from mast cells, and has been reported to suppress keloid and hypertrophic scar formation. Since matrix metalloproteinases (MMPs) play an essential role in tissue remodelling, this study was undertaken to determine whether tranilast suppresses MMP production from neutrophils after lipopolysaccharide (LPS) stimulation in-vitro. Neutrophils from five healthy donors (1 x 10(5) cells/mL) were stimulated with 1.0 microg mL(-1) LPS in the presence or absence of various concentrations of tranilast for 24 h. MMP-7, MMP-8, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 levels in the culture supernatants were assayed by ELISA. In addition, the influence of tranilast on MMP mRNA expression and transcriptional factor activation in cells cultured for 12 h and 4 h was also evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Tranilast inhibited MMP and TIMP-1 production from neutrophils when cells were treated with the agent at more than 5.0 x 10(-5) M. It also suppressed MMP mRNA expression and transcriptional factor activation induced in neutrophils by LPS stimulation. The results suggest that tranilast inhibits the formation of keloid scarring through the suppression of factors such as MMPs and TIMP, which are essential for tissue remodelling, from inflammatory cells.

Quantitative analysis of glucocorticoid receptors in nasal polyps / 中国耳鼻咽喉头颈外科

OBJECTIVE The purpose of our study was to measure the glucocorticoid receptor(GR)protein level in the nuclei of nasal polyp cells and compare the results in nasal polyposis patients with chronic rhinosinusitis alone and with chronic rhinosinusitis accompanied with asthma, before and after glucocorticoid(Glu)therapy. METHODS We used enzyme-linked immunosorbentassay(ELISA) techniques to quantitatively measure the activated- GR protein level in the nuclei of the nasal polyp cells. Nasal polyp tissues were obtained from patients with chronic rhinosinusitis alone and with chronic rhinosinusitis accompanied with bronchial asthma. In the latter, polyp tissues were obtained before and after Glu therapy seperatively. RESULTS Our data showed no significant differences between the activated- GR protein level of the nasal polyposis patients with chronic rhinosinusitis alone and with chronic rhinosinusitis accompanied with bronchial asthma before Glu therapy. However, the activated-GR protein level was significantly upregulated after Glu therapy in the patients with chronic rhinosinusitis accompanied with bronchial asthma. CONCLUSION The result of our research clearly showed that Glus upregulated the activated-GR protein level in the nuclei of nasal polyp cells and that the upregulation is essential for Glus anti-inflammatory action.

Suppression of matrix metalloproteinase production from nasal fibroblasts by fluticasone propionate in vitro.

OBJECTIVE: To examine the influence of fluticasone propionate (FP) on matrix metalloproteinase (MMP) production from nasal polyp fibroblasts in vitro. MATERIAL AND METHODS: Fibroblasts derived from five nasal polyps were stimulated with tumor necrosis factor (TNF)-alpha in the presence of various concentrations of FP. The influence of FP on MMP production was assessed by examining the levels of MMP-2 and -9 in culture supernatants using ELISA. We also examined the influence of FP on MMP mRNA expression using reverse transcriptase polymerase chain reaction. RESULTS: The addition of FP caused significant suppression of MMP-2 and -9 production from nasal polyp fibroblasts in response to TNF-alpha stimulation. MMP mRNA expression was also suppressed by the addition of FP to cell cultures. The minimum concentration of the agent required to cause suppression was 10(-5) M. CONCLUSION: These results suggest that the inhibitory action of FP on tissue remodeling may underlie the clinical efficacy of corticosteroids in nasal polyposis.

Suppressive activity of macrolide antibiotics on nitric oxide production by lipopolysaccharide stimulation in mice.

BACKGROUND: Low-dose and long-term administration of macrolide antibiotics into patients with chronic airway inflammatory diseases could favorably modify their clinical conditions. However, the therapeutic mode of action of macrolides is not well understood. Free oxygen radicals, including nitric oxide (NO), are well recognized as the important final effector molecules in the development and the maintenance of inflammatory diseases. PURPOSE: The influence of macrolide antibiotics on NO generation was examined in vivo. METHODS: Male ICR mice, 5 weeks of age, were orally administered with either roxithromycin, clarithromycin, azithromycin or josamycin once a day for 2-4 weeks. The mice were then injected intraperitoneally with 5.0 mg/kg lipopolysaccharide (LPS) and the plasma NO level was examined 6 h later. RESULTS: Although pre-treatment of mice with macrolide antibiotics for 2 weeks scarcely affected NO generation by LPS injection, the administration of macrolide antibiotics, except for josamycin, for 4 weeks significantly inhibited LPS-induced NO generation. The data in the present study also showed that pre-treatment of mice with macrolide antibiotics for 4 weeks significantly suppresses not only production of pro-inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, but also inducible nitric oxide synthase mRNA expressions, which are enhanced by LPS injection. CONCLUSION: These results strongly suggest that suppressive activity of macrolide antibiotics on NO generation in response to LPS stimulation in vivo may, in part, account for the clinical efficacy of macrolides on chronic inflammatory diseases.