| BackgroundThe human airway surface is covered with a layer of mucus secreted by submucosal glands and epithelial goblet cells, which protects the underlying airway epithelium from exogenous insults such as pathogens and harmful particles. Mucins (e.g., MUC5AC) are major components of mucus, which assists in clearance of inhaled foreign materials. Chronic obstructive pulmonary disease (COPD) is a major global health problem, which is projected to become the third leading cause of death worldwide by2020. Airway mucus overproduction is the prominent feature of COPD, which contributes to airway obstruction, accelerated decline of lung function, morbidity, and mortality. Cigarette smoking, which is considered a major etiologic factor of COPD, plays an essential role in airway mucus overproduction. However, cigarette smoke is not the only dominator in the process. Other stimuli relevant to COPD, such as proinflammatory cytokines (TNF-a and IL-1β), oxidative stress, neutrophil elastase and microbes, have been shown to evoke the production of airway mucm.Recently, the antimicrobial peptides (AMPs) have been implicated in the pathophysiology of COPD. AMPs are positive charged, amphiphilic, and short peptides that expressed in many tissues and cell types including skin, sweat glands, bronchial and intestinal epithelia, as well as neutrophils, monocytes, NK cells and mast cells. AMPs of mammals are considered essential components of host innate immunity and can be categorized into two families:defensins and cathelicidins. The human cationic antimicrobial peptides of18KD (hCAP-18) is the only member of human cathelidicins family, with a37-residue helical peptide (LL-37) cleaved to display potential antimicrobial activity and various biological functions involving the regulation of proliferation, apoptosis, inflammation and immune responses. Several studies have demonstrated high levels of LL-37in respiratory tract of COPD patients. Our previous study regarding the pathogenesis of COPD also showed a significantly increased presentation of LL-37in small airway epithelium and induced sputum from COPD patients with a long smoking history, and suggested a possible role of LL-37in inducing airway inflammation and alveolar apoptosis. However, we did not investigate whether LL-37impacts airway mucus overproduction relevant to smoking related COPD, and to the best of our knowledge, no current studies have yet explored this thesis.Airway mucus overproduction is mainly mediated through epidermal growth factor receptor (EGFR) signaling pathway in response to various stimuli including cigarette smoke, bacterial exoproducts, neutrophil elastase and inflammatory mediators such as TNF-a and IL-1β. Moreover, TNF-a converting enzyme (TACE), a member of "a disintegrin and metalloprotease"(ADAM) family, was demonstrated to mediate EGFR activation induced by cigarette smoke, with TACE/TGF-a/EGFR pathway established. Interestingly, LL-37has been reported to stimulate colonic mucus synthesis, as well as to activate airway epithelial cells via activation of EGFR signaling pathway which might be mediated by TACE. These results suggested a possibility of LL-37to stimulate mucus production by airway epithelial cells. It is reasonable to hypothesize that LL-37overexpressed in COPD airways can induce mucus overproduction by airway epithelial cells, thus contributing to the development of COPD.ObjectiveTo examine the expression of Cathelicidin/LL-37and MUC5AC mucin in small airway epithelium of COPD patients, and investigate the effects and the underlying mechanism of LL-37on MUC5AC mucin production in airway epithelial cells in vitro, thus elucidating the role of Cathelicidin/LL-37in airway mucus overproduction in COPD.MethodsWe recruited three groups of subjects undergoing lung resection for a solitary peripheral carcinoma or benign tumor:15smokers with COPD (COPD group),15asymptomatic smokers with normal pulmonary function (Normal Smoking group, NS group), and15lifelong non-smoking subjects with normal pulmonary function (Normal Non-smoking group, NN group). The diagnosis of COPD was established according to published guideline GOLD (2010).For each subject, two to three randomly selected tissue blocks (sample size15-25mm) were taken from the subpleural parenchyma of the lobe obtained in surgery, at least5cm far from the margin of the tumor to avoid the areas involved by tumor. The morphology of small airways located in the specimen was examined by HE and PAS staining, and the expression of LL-37and MUC5AC mucin in small airways was determined by immunohistochemical staining. The correlations between LL-37and MUC5AC mucin, LL-37and forced expiratory volume%predicted (FEV1%), MUC5AC mucin and FEV1%were analyzed.In vitro, human airway epithelial NCI-H292cells were cultured and processed as follows:i. Cells were treated with serial concentrations of LL-37and CSE for different time periods, and the viability of cells was determined by MTT methods.ii. Cells were treated with serial concentrations of LL-37or scramble LL-37(sLL-37) for different time periods in the absence and presence of CSE (0.005cig/ml). The MUC5AC mucin produced by cells was measured by ELISA.iii. Cells were treated with serial concentrations of LL-37for different time periods in the absence and presence of CSE (0.005cig/ml). The IL-8secreted by cells was measured by ELISA.iv. Cells were treated with recombinant human IL-8, or IL-8-neutralizing antibody plus LL-37, the MUC5AC mucin produced by cells was measured by ELISA.v. Cells were treated with CSE and the expression of LL-37was detected by immunofluorescence staining and ELISA.vi. Cells were treated with serial concentrations of LL-37for different time periods, and the TGF-a released by cells were determined by ELISA, the phosphorylations of EGFR and ERK were determined by Western blotting, and the TACE activity was determined by fluorescence resonance energy transfer (FRET) assay.vii. After preincubated with TAPI-1(TACE inhibitor), AG1478(EGFR inhibitor), U0126(ERK inhibitor), TGF-a-neutralizing antibody, EGFR-neutralizing antibody, amphiregulin-neutralizing antibody and HB-EGF-neutralizing antibody, cells were treated with LL-37. The TGF-a released by cells were determined by ELISA, the phosphorylations of EGFR and ERK were determined by Western blotting and the TACE activity was determined by FRET assay.viii. Cells were treated with LL-37and intracellular ROS was determined by DCFH-DA staining. Cells were preincubated with DMTU, nPG and staurosporine, and then were treated with LL-37, the TACE activity was determined by FRET assay and the MUC5AC mucin produced by cells was measured by ELISA.Resultsi. The immunohistochemical staining results showed that the LL-37was located in multiple cell types including ciliated cells and goblet cells, whereas MUC5AC mucin was mainly located in goblet cells. COPD group had the highest expression of LL-37and MUC5AC mucin among three groups, whereas NS group had higher expression of LL-37and MUC5AC mucin compared to NN group. Correlation analysis showed that LL-37was correlated with FEV1%(r=-0.606, P<0.01), MUC5AC was correlated with FEV1%(r=-0.641, P<0.01), and that LL-37was correlated with MUC5AC mucin (r=0.776, P<0.01).ii. LL-37induced MUC5AC mucin production by airway epithelial cells in a time-and concentration-dependent manner, in the absence or presence of CSE (all P<0.05). sLL-37had no effect on MUC5AC induction (P>0.05).iii. CSE induced IL-8production time-and dose-dependently (all P<0.05), and human recombinant IL-8induced MUC5AC mucin production while IL-8-neutralizing antibody prevented LL-37-induced MUC5AC mucin production in NCI-H292cells (all P<0.05).iv. CSE upregulated the expression and release of LL-37by NCI-H292cells. The high expression of LL-37was detected in the cytoplasm by immunofluorescence staining. The release of LL-37by NCI-H292cells was enhanced by CSE (P<0.05).v. LL-37induced EGFR and ERK phosphorylation. When stimulated with LL-37for15min, the phosphorylation of ERK in cells was most pronounced. LL-37induced EGFR and ERK activation dose-dependently in cells treated with LL-37for15min. AG1478inhibited LL-37-induced EGFR phosphorylation, ERK phosphorylation and MUC5AC mucin production while EGFR-neutralizing antibody and U0126inhibited LL-37-induced ERK phosphorylation and MUC5AC mucin production (all P<0.05). HB-EGF-neutralizing antibody and amphiregulin-neutralizing antibody did not effect LL-37-induced EGFR phosphorylation and MUC5AC mucin production (P>0.05).vi. LL-37induced TACE activation and TGF-a release time-and dose-dependently. The activity of TACE peaked when stimulated with LL-37for6h (all P<0.05), and maintained for at least24h.TAPI-1inhibited LL-37-induced TGF-a release, EGFR and ERK phosphorylation, and MUC5AC mucin production (all P<0.05). TGF-a-neutralizing antibody inhibited LL-37-induced EGFR phosphorylation and MUC5AC mucin production (P<0.05). EGFR-neutralizing antibody increased LL-37-induced TGF-a production (P<0.05).vii.LL-37had no effect on ROS production (P>0.05). The DMTU, nPG and Staurosporine did not effect LL-37-induced TACE activity and MUC5AC mucin production (all P>0.05).Conclusionsi. The expression of Cathelicidin/LL-37and MUC5AC mucin was upregulated in small airway epithelial cells of COPD patients, and a correlation existed between these two parameters.ii. Cathelicidin/LL-37possesses the ability to increase airway mucin production, which can be enhanced by cigarette smoke.iii. TACE-TGF-a-EGFR-ERK cascase is responsible for LL-37-induced MUC5AC mucin production. Additionally, IL-8is involved in the process.iv. Overexpressed Cathelicidin/LL-37in small airways of COPD patients may contribute to mucus overproduction, thus participating in the development of COPD. |
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