Changes Of Microparticles In The Bronchoalveolar Lavage Fluid Of Patients With Chronic Obstructive Pulmonary Disease

Background and Objective:Microparticles (MPs) are small membranous vesicles released by budding of the cellmembranes, which can occur either by apoptosis or in response to various stimuli. They aretiny membrane fragments which contain phospholipids and their diameters are ranged from50nm to2μm. MPs are enriched with cell surface receptors and mRNAs, with a variety ofbiological functions[1][2][3]. Types of cells that can produce MPs known as “Origin cells”.According to the sources, MPs can be divided into Macrophage-derived microparticles(MMPs), T Lymphocyte-derived microparticles (TLMPs), B Lymphocyte-derivedmicroparticles (BLMPs), Platelet microparticles (PMPs), Endothelial microparticles(EMPs) and so on. MPs are assumed to reflect a dynamic equilibrium status about the cellproliferation, apoptosis or stimulation[4]. When this balance is disturbed due to variousreasons, the resultant MPs will be produced in a large amount and appear in the bloodcirculation. MPs can deliver bioactive molecules to the target organs or even other MPs, andstimulate the productions of cytokines, growth factors, and tissue factors to disturb theinternal microenvironment of the host[5][6][7], which can affect the occurrence andprogression of the diseases to a large extent. Emerging evidence has being shown that MPsare pro-inflammatory and procoagulant and are able to regulate immune functions. Theyplay important roles in the tumor, inflammation, thrombosis, sepsis, malaria, autoimmune diseases, blood diseases and others[1].MPs could have enormous impacts on the lung because of the presence of two sets ofblood supplies. Our previous study found that LMPs could inhibit the growth of humanbronchial epithelial cells, and arrested their growth cycle in G1phase, which isaccompanied with the upregulation of P21and P27via regulation of AKT/FOXO1signaling pathway[2][3]. Our results suggested that micorparticles could play certain roles inthe course of respiratory diseases. Thus, it is necessary to conduct an in-depth study. COPDis a common chronic inflammatory airway disease. Its high incidence, case fatality rate andseriousness affect the patients’ ability to work and quality of life. It has been establishedthat macrophages, lymphocytes and endothelial cells as well as platelets play importantroles in the process of COPD disease. It remains unknown about the contents and dynamicchanges of microparticles generated from these cells in COPD patients so far. The researchof the microparticles in COPD will greatly our understandings of the field. This studyexplored the contenst and dynamic changes of various kinds of MPs in the BALF,including the MMPs, TLMPs, BLMPs, PMPs and EMPs in the COPD patients so as toprovide a novel insight for the evaluation of indicators and the clinical prognosis of COPDprogress.MethodIn this study, we collected BALF from the patients and the control group and detectedthe absolute counts of MMPs, TLMPs, BLMPs, EMPs, PMPs within100μl of lavage fluidby flow cytometry.1. Collection and process of BALFs1.1BALF collection: BALFs were collected from COPD patients at different stagesincluding inpatients and outpatients, and control subjects. All the research participants wereintramuscularly injected with atropine (0.5mg) and were implanted with local anesthesia bythe use of2%lidocaine solution on airway before performance of fiberoptic bronchoscopy. The bronchial lavages were then obtained in the third or fourth level of subsegmental lavageof20ml sterile saline (37℃, PH:7.3). Lavaged fluid of maximum absorption wasrecovered for about50%[1][2][3][4][5]. Of note, the broncho-alveolar lavage should be operatedgently for fear of increasing the suffers of participants. The BALFs that contained carbonfoam, blood or sputum specimens deemed to be unqualified. The standard lavage fluidswere quickly kept in cooler for further inspection[6].1.2BALF pretreatment: The collected lavage fluids were centrifuged at2000x g,4℃for10min. The supernatant was collected and the relatively turbid liquid at the bottom wasdiscarded. The procedure was repeated for three times. The processed supernatant wasmoved into an EP tube for100μl/tube.2. Quantitative measurement of various groups of MMPs, TLMPs, BLMPs, PMPs,EMPs by flow cytometryBALFs of the AECOPD group, the stable COPD group and the control group weredivided into six tubes (100μl/tube) per group under dark in avoidance of light. The first tubewas added with5μl of anti-human CD14antibody in order to mark MMPs; the second tubewas added with5μl of anti-human CD4in order to label CD4+TLMPs; the third tube wasadded with5μl of anti-human CD8antibody to mark CD8+TLMPs; the forth tube wasadded with20μl of anti-human CD20antibody to mark BLMPs; the fifth tube was addedwith5μl of E plus5μl of anti-human CD142antibody to label PMPs; and the six tube wasadded with5μl of anti-human CD41plus5μl of anti-human CD142antibody to markEMPs. After thorough mix and incubation, the samples were ready for flow cytometry[7][8][9]. Results1. BALF collectionThe cases that were fulfillment with the selection criteria for the study of the dynamicchanges of TLMPs and MMPs included35AECOPD,12stable COPD, and31controlsubjects. The cases that were qualified for the selection criteria for the study of BLMPsincluded33AECOPD,12stable COPD, and31control subjects. The cases that weresatisfactory with the selection criteria for the study of the dynamic changes of PMPs andEMPs included35AECOPD,12stable COPD, and20control subjects.2. The changes of the counts of MMPs2.1Changes of MMPs: As compared to the control group [45(1-2296)], bothAECOPD group[906（25-15537）] and stable COPD group[737（33-10149）] had substantialhigher number of MMPs (P <0.05), but no statistical significance in the difference of thenumber of MMPs was found between AECOPD group and the stable COPD group (P>0.05). Statistical significant difference in the number of MMPs was reveal after comparisonmade among those three groups (P <0.05).2.2Changes of CD8+TLMPs: The number of CD8+TLMPs in AECOPD group[3683(100-35159)] was substantially higher than that in the stable COPD group [1020.5(46-3297)] and the control group [29(3-1012)](P<0.05). The stable COPD group hadsubstantial higher numbers of CD8+TLMPs as compared to the control group (P<0.05).Statistical significance was found after comparison made among those three groups(P<0.05).2.3Changes of CD4+TLMPs: There was no statistical significance in thedifference of the number of CD4+TLMPs after comparison made among the AECOPDgroup [494(5-15585)], the stable COPD group [953(4-6247) and the control group [435(3-9863)](P>0.05).2.4Changes of BLMPs: The numbers of BLMPs in the AECOPD group [46(6-1012)]were less than those in the control group [237(1-4685)] and the stable COPD group [114.5(37-876)](P<0.05), but no significance was found after comparison made between thestable COPD group and the control group (P>0.05). Statistical significance was foundamong those three groups (P<0.05). 2.5Changes of PMPs: The number of PMPs in AECOPD group [81(29-370)] wassignificantly lower than that in stable COPD [378.5(56-1260)] and the control group [623.5(74-1531)](P<0.05). However, no statistical significance in the difference of the number ofPMPS was found between the stable COPD group and the control group (P>0.05).Statistical significance was found among those three groups (P<0.05).2.6Changes of the EMPs: The numbers of EMPs in AECOPD group [202(12-1873)]were significantly less than those in the stable COPD [493(124-1892)] and the controlgroup [693.5(43-1847)](P<0.05). The difference of the numbers of EMPS between thestable COPD group and the control group was not statistically significant (P>0.05).Statistical significance was found among those three groups (P<0.05).3. Analysis of the correlations of MPs among COPD patientsThere were correlations of MPS among three groups of COPD patients. In AECOPDgroup, both BLMPs and CD4+TLMPs had low negative correlation (rs=-0.344, P<0.05),and no clear-cut association was found among the remaining MPs (P>0.05). In stable COPDgroup, the number of PMPs was significantly correlated with that of BLMPs (rs=0.755,P<0.05), and no correlation was found among the rest of MPs (P>0.05). In the control group,no correlation was found among all of MPs (P>0.05).4. The relationship between the numbers of MPs in the BALF of COPD patients andthe other factors such as age, sex, smoking status,body surface area was analyzed. We foundno correlation between the changes of those six MPs and the factors including the age, sex,body surface area and smoking in both AECOPD and stable COPD patients.Conclusions1. The numbers of BLMPs, EMPs and PMPs were significantly reduced in COPDpatients, but the numbers of MMPs and CD8+TLMPs were increased, which was muchmore obvious in the acute aggravating period of the disease. The results suggest that thechanges may be related to disease stage.2. There was no statistical significance in the difference between the number ofCD4+TLMPs in the BALF of COPD patients and that of the control group.3. In AECOPD patients, the reduction in the number of BLMPs had negativecorrelation with the number of CD4+TLMPs; while in stable COPD patients, the reduction in the number of PMPs was significantly correlated with the number of BLMPs.4. The patient’s age, sex, body surface area and smoking or not were not correlatedwith the changes of MPs.