The Expressional Changes And Mechanisms Of MUC1in Chronic Obstructive Pulmonary Disease Development

【Background】Chronic Obstructive Pulmonary Disease (COPD) is one of the chronic diseaseswith worldwide high incidence and mortality. COPD is characterized by persistentairflow limitation which is usually progressive and associated with an enhancedchronic inflammatory response in the airway and the lung caused by smoking orinhaled noxious particles or gas. Much evidence showed that airway chronicinflammation is critical for COPD. The mechanisms for the inflammation generationare complicated with involvements of various inflammatory cells such asneutrophils and macrophages. These cells release inflammatory mediators likeTNF-α and IL-8which lead to lung parenchymal tissue destruction and consequentCOPD.MUC1(MUC1in humans and Muc1in non-human species) is a type Itransmembrane glycoprotein ubiquitously expressed on the apical surface ofepithelial cells in respiratory, gastrointestinal and reproductive tracts and on someimmune cells. MUC1contains three major domains, an extracellular (EC) domain, atransmembrane (TE) domain and a cytoplasmic tail (CT) domain. Functions ofMUC1include protection and lubrication on epithelial surfaces. The cytoplasmic taildomain of MUC1mediates the cell signal transductions and cell adhesion. Recent studies showed that MUC1was over expressed in acute inflammation after thePseudomonas aeruginosa or respiratory syncytial virus infection, which wascorrelated with TNF-α and neutrophil elastase increases. This mentions aninvolvement of MUC1/Muc1as an important endogenous molecule during infectionand inflammation.However, the expressional pattern of MUC1in COPD patients’ lung andwhether it is correlated with the changes of inflammatory factor levels is remained tobe elucidated.【Objective】In this study, the expressional changes of MUC1in different sources ofspecimens were studied, including airway mucosa and sputum specimen fromCOPD patients in various phases, lung and bronchoalveolar lavage fluid from COPDmodel mice induced by cigarette smoke exposure or acute lung inflammation modelmice induced by inhaled lipopolysaccharide (LPS), and A549cells treated withcigarette smoke extract and LPS. The levels of TNF-α and IL-8in sputum fromCOPD patients in different phases and the levels of TNF-α in bronchoalveolar lavagefluid from COPD model mice and LPS induced acute inflammation model mice weremeasured. This study indicated the correlation between Muc1change and TNF-αchange stimulated by long-term cigarette smoke exposure or acute LPS inhalation,then gives a helpful discussion on the mechanism of MUC1expression changes inCOPD patients. These results will provide new theoretical basis for COPDoccurrence and give potential target for future COPD prevention and treatment.【Methods】This study was carried out with patient specimens, animal experiments andcultured cells.(1) The MUC1levels in mucosal tissues from COPD patients takenwith fibre bronchial microscope were detected by immunohistochemistry. The levelsof MUC1, TNF-α and IL-8in sputum from COPD patients in acute and remissionphase were determined with ELISA.(2) The COPD mouse model was established by6months cigarette smoke exposure. The Muc1levels in lung and BALF weredetermined with western blot and ELISA respectively. The TNF-α levels and differential cell numbers in bronchoalveolar lavage fluid (BALF) were assayed.(3)The mice were intranasally given LPS to establish acute inflammation mouse model.The mice were sacrificed at12h,24h,48h,96h and1w after the LPS treatment totake the lung tissues and BALF. Muc1levels in BALF and lung were determined withELISA and western blot respectively and the levels of TNF-α in BALF weremeasured also.(4) The MUC1levels in cultured human lung epithelial A549cellstreated with cigarette smoke extract or LPS for24hours were assayed with Westernblot.【Results】1. MUC1levels were upregulated in mucosal tissues from COPD patients.(1) Compared with the normal control group (including non-smokers and smokers),immunohistochemistry showed that MUC1was highly positive in airway mucosaof COPD patients.(2) Sputum MUC1levels detected by ELISA with an antibody raised against CTdomain of MUC1, the levels of sputum IL-8and TNF-α were higher in acute thanin remission phase of COPD (P<0.05). Total white cells, total neutrophils andneutrophil percentage in sputum from acute phase were higher than those fromremission phase (P<0.05). The sputum IL-8levels were correlated with sputumneutrophil percentage in both acute and remission phases (acute phase, r=0.463,p=0.046; remission, r=0.547, p=0.015). The sputum TNF-α level was positivelycorrelated with neutrophil percentage in both acute and remission phases (acutephase, r=0.489，P=0.040; remission, r=0.794，P=0.000). The line multipleregression analysis showed that sputum MUC1levels were impacted by totalwhite cells in sputum, FVC and ages.2. The results on cigarette smoke exposure induced COPD model mice(1) COPD mouse model was successfully established by pure6-month cigarettesmoke exposure. The lung function tests showed that COPD model micedisplayed significant differences to the normal mice in increased airway resistanceand decreases in dynamic compliance, peak inspiratory flow and peak expiratoryflow (P<0.05). The infiltrated inflammatory cells were observed around bronchia and in lung and alveoli of lung were different in size，part of them coalesced andexpanded，which could be taken as typical emphysema pathological changes.(2) The Muc1levels in lung and BALF were higher in COPD model mice than innormal mice (P<0.05). The total cell number, total neutrophils and macrophagesand TNF-α level in BALF were significantly higher in COPD mice than innormal mice (P<0.05).3. The results from acute inflammation mouse model(1) Acute inflammation mouse model was established by intranasally giving LPS.The following phenotypes were observed in model mice lung, lung parenchymaedema and infiltrate inflammatory cells around bronchia and in lung.(2) The Muc1levels in lung were detected by western blot with an antibody raisedagainst Muc1CT domain. The lung Muc1was increased12h after LPStreatment (P<0.05, compared with normal mice) and decreased to the levelsimilar to normal mice at24h and maintained at the level non-different tonormal mice at48h,96h and1w. Muc1levels (detected with antibody raisedagainst Muc1CT domain) in BALF were low and had no changes. However, theBALF Muc1level detected with an antibody raised against Muc1EC domainwas significantly increased at12h but stated to decrease at24h and returned tothe level similar to the normal mice at1w after LPS treatment. In the BALF,the LPS treated mice displayed significant increases in total cell number,neutrophil percentage and TNF-α level at all time points after the treatment(P<0.05).4. The results from cultured A549cellsMUC1was upregulated at24h after incubations with the cigarette smokeextract or LPS in A549cells.【Conclusion】1. MUC1levels were upregulated in COPD patients’ mucosa.2. The MUC1levels in induced sputum from COPD patients were higher in acutephase than in remission phase.3. The MUC1increase might due to the stimulation of long-term cigarette smoke exposure or LPS to the lung epithelial cells.4. The MUC1increase in COPD patients might correlate with the chronicinflammation induced by long-term cigarette smoke exposure or acuteinflammation caused by bacteria infection. Meanwhile, the increases ofinflammatory mediator, such as TNF-α. and/or the neutrophil products, such asneutrophil elastase, might be involved in triggering MUC1up-regulation.