The Effect Of Human Antibacterial Peptide LL-37 In The Pathogenesis Of Chronic Obstructive Pulmonary Disease

BackgroundChronic obstructive pulmonary disease is one of the leading causes of morbidity and mortality worldwide, and its prevalence is increasing. Progressive airflow limitation and symptoms in COPD are associated with an abnormal inflammatory response of the lung to noxious particles or gases. Previous research has shown that both the innate and acquired immune systems are involved in the pathogenesis of COPD, but the effects of inflammatory cytokines and proteinases released by neutrophils and macrophages seem to be more important. It was presumed that the innate immune system was more important than the acquired immune system in this process. LL-37 is the only human cathelicidin identified so far. As an integral part of the innate immune system, this protein is involved in the first line of host defence. Besides antibacterial activity, its chemotactic activity, damage repairing, influencing apoptosis and its cytotoxicity are attracting people's attention. We presume that with the wide spectrum and multi-effect biological function, LL-37 may play important role in the pathogenesis of COPD. Research on the state of LL-37 may be helpful to reveal the effect of the innate immune system in the process of COPD.ObjectiveExplore the effect of LL-37 in the pathogenesis of COPD through quantifying the levels of LL-37 in serum, induced sputum and lung tissue of COPD patients and in cellular level, investigating the expression of LL-37 on human bronchial epithelial cells (BEP2D), alveolar epithelial cells (A549) exposuring to cigarette smoke extraction (CSE),lipopolysaccharides (LPS) and the influence of LL-37 synthetic peptide on inflammatory cytokines releasing, apoptosis and its cytotoxicity.Methods1. Peripheral blood and induced sputum were collected in 28 COPD patients,14 healthy smokers and 18 healthy no-smokers. Pulmonary function was measured. Health status was assessed using the St. George's Respiratory Questionnaire (SGRQ). 6MWD was used to evaluate patients'exercise tolerance. Serum and induced sputum levels of LL-37 were measured by ELISA.2.9 COPD patients and 10 controls with operation because of lung tumor, lung bullae and lung cyst were recruited and the protein expression of LL-37 was detected by immunohistochemistry.3. Bronchial epithelial cells (BEP2D), alveolar epithelial cells (A549) were cultured and stimulated by different concentration of CSE and LPS. The protein expression of LL-37 was detected by laser scanning confocal microscopy.4. BEP2D and A549 were stimulated by different concentration of LL-37 synthetic peptide. Apoptosis was determined by flow cytometry, the levels of IL-8,TNF-αin the supernatants were evaluated by ELISA and the levels of lactic acid dehydrogenase (LDH) were detected by semi-automatic biochemistry analyzer.Results1. LL-37 levels were significantly higher in induced sputum samples ofⅠ+ⅡCOPD andⅢ+ⅣCOPD patients compared with healthy non-smokers (P<0.05) and healthy smokers (P<0.05). Expression of LL-37 was also elevated inⅢ+ⅣCOPD patients compared withⅠ+ⅡCOPD patients (P<0.05).There were no statistically significant differences in serum LL-37 levels between healthy non-smokers, healthy smokers,Ⅰ+ⅡCOPD andⅢ+ⅣCOPD patients. Sputum LL-37 in COPD patients showed significant inverse correlations with FEV1%(r=-0.32, P=0.01) and 6MWD (r=-0.27, P=0.01), and a significant positive correlation with SGRQ scores (r=0.35, P=0.01).2. LL-37 in pulmonary tissue were mainly expressed on bronchial epithelial cell, alveolar epithelial cell, inflammatory cell(including neurophils,monocyte-macrophage and lymphocyte) and fibroblast. The expressing intensity of positive cell in both airway district and pulmonary alveoli disrict in COPD group significantly increase compared with control group (P<0.01).3. The exposure to different concentration of CSE and LPS enhanced protein expression of LL-37 significantly (P<0.05).4. LL-37 synthesis polypeptide can induce apoptosis of BEP2D,A549,showing dose and time-dependent effect. LL-37 synthesis polypeptide can promot the releasing of inflammatory factor IL-8 and increase the secretion of LDH.ConclusionIn COPD patients, increased induced sputum levels of LL-37 were associated with airflow limitation, health status and exercise tolerance, suggesting that LL-37 may be a valid biomarker for disease activity and progression, and could potentially be a novel therapeutic target in COPD. Through stimulation by CSE and LPS, the expression of LL-37 was increased in bronchial epithelial cell and alveolar epithelial cell. LL-37 synthesis polypeptide can induce apoptosis of bronchial epithelial cell and alveolar epithelial cell and promote the releasing of inflammatory factor, suggesting that LL-37 may play important role in the pathogenesis of COPD.