Effect Of LMPs On Airway Epithelial Cells And Its Mechanisms

Background and Objective:Chronic obstructive pulmonary disease(COPD) is characterized by persistent airflow limitation in chronic airway inflammation, with progressive development of airflow limitation, relating to enhanced reaction of airway and lungs to toxic particles or gases. Because of lack of further research of etiology and pathogenesis of COPD, new treatment is extremely limited. Recent studies have found that COPD is closely related to immunomodulation, occurrence and development of disease is directly related to airway epithelial injury and repair. Airway epithelium is the first line of defense against the outside environment. Increased airway epithelial cell apoptosis exposes epithelial basal layer allowing increased contact with toxic inflammatory mediators, causing the spread of inflammation and airway remodeling.Lymphocyte-derived microparticles(LMPs) are small vesicles formed via membrane budding when lymphocytes are activated, undergo injury or apoptosis. Our previous clinical research has found that LMPs are elevated in bronchoalveolar lavage fluid(BALF) of COPD patients, and the number of LMPs is closely related to the severity of the disease. Further animal studies have confirmed LMPs could induce airway inflammation in C57BL/6 mice[1]. Accordingly, we hypothesized that LMPs may play an important role in occurrence and development of COPD, acting on airway epithelial cells.Oxidative stress is closely related to apoptosis. A lot of reactive oxygen species(ROS) produced by oxidative stress modify membrane phospholipid fatty acid by lipid peroxidation, and change the membrane protein structure, membrane mobility, and intracellular signal transduction pathways etc., ultimately leading to cell apoptosis or necrosis. Arachidonic acid(AA) is one of the essential fatty acids, belongs to long-chain polyunsaturated fatty acids of Omega-6 family. It is widely found in animal cells. It is also an important component of cell membranes. The relationship between AA and oxidative stress is very complex and related to cell types. AA and oxidative stress together lead to tissue damage. LMPs stimulate oxidative stress in umbilical vein endothelial cells. We will further explore whether oxidative stress could play an important role in airway epithelial injury induced by LMPs.LMPs are a kind of active debris, containing a variety of substances such as membrane lipid, receptors, many cytokines, enzymes, nucleic acids and so on. Moreover, LMPs carry receptors and adhesion molecules. They establish special interaction with target cells. LMPs also contain a variety of cytokines, enzymes, m RNA and DNA. LMPs can transfer substances to the target cells, such as organelles, receptors and other proteins. LMPs can also mediate target cells to secrete cytokines. LMPs can promote cell-cell interaction, and deliver biological information as messengers. Therefore, the key components playing a toxic role in airway epithelial cells have been not clear.Our study will be about the role of LMPs in airway epithelial cells and its mechanisms, and finally find the key components of LMPs. Methods:1. Study of effect of LMPs on airway epithelial cells1.1 Extraction and identification of LMPsHuman CEM-T lymphocytes were purchased from Beijing Cell Library, Chinese Academy of Sciences. CEM-T cells with normal morphology were added with a final concentration of 5 μg/ml of actinomycin D and stimulated for 24 h. LMPs were extracted from supernatent by standard muticentrifugation procedure. Supernatant from last centrifugation was retained as control. Sizes and annexin V-positive rate of LMPs were determined via flow cytometry. Concentrations of LMPs were determined via Bradford assay. 10 μM of Di I pre-stained CEM-T lymphocytes for 20 min, then cytochalasin D was added to stimulate cells for 24 h, resulting in Dil-LMPs.1.2 Effect of LMPs on human airway epithelial cells1.2.1 Morphological observationMorphological changes of airway epithelial cells before and after LMPs stimuation were observed by ordinary optical microscopy and transmission electron microscopy.71.2.2 Detection of cell growth and proliferationMTT was used to detect the effect of low concentrations of LMPs(1 μg/ml and 2 μg/ml) or high concentrations of LMPs(5-40 μg/ml) on airway epithelial cell growth, and the relationship between growth rate and stimulation time(24 h, 48 h or 72 h) under 20 μg/ml of LMPs stimulation. After cells were stimulated by LMPs for 24 h, 48 h or 72 h, Using Ki67 staining determined the relationship between cell growth and stimulation time.1.2.3 Apoptosis detectionBy Annexin V-FITC/PI staining, cell apoptosis under different stimulation concentrations(10 μg/ml and 20 μg/ml LMPs) or different stimulation time(24 h and 48 h) was analyzed via flow cytometry.1.2.4 Immunofluorescence staining to study the interaction between LMPs and cellsBronchial epithelial cells and alveolar epithelial cells were used. Cells were stimulated by different concentrations of Dil-LMPs(5,10, 20 μg/ml), or for different time(30 min, 1 h, 2 h, 4 h, 8 h and 16 h). Dil-LMPs and lysosome were observed for their relationship between their localization and stimulation concentrations or stimulation time via fluorescence microscopy or confocal microscopy. To analyze the internalization of Dil-LMPs, cells were pretreated with 20 μM of cytochalasin D(phagocytic inhibitor) or 20 μg/ml of chloroquine(lysosomal inhibitor), and then LMPs were added.1.3 Effect of LMPs on airway epithelium and lung epithelium in mice1.3.1 Histopathological observationC57BL/6 mice were randomly divided into two groups(n=6): control group and LMPs treatment group. Mouse bronchial rings and lung tissues were obtained by surgey and cultured in vitro, then were treated according to different groups. Morphological changes of bronchial epithelium were observed by sanning electron microscopy. Histopathology of bronchial and lung tissues were measured by HE staining.1.3.2 TUNNEL assayLMPs induced mouse bronchial rings and lung tissues for 24 h, cell apoptosis was analyzed by TUNNEL assay.2. The mechanisms of apoptosis induced by LMPs in airway epithelial cells2.1 Effect of caspase induced by LMPs in airway epithelial cells2.1.1 Caspase detection(1) Caspase-3,-8-9 activities in the airway epithelial cells before and after LMPs stimuation were measured by activity assays. Caspase-3 and cleaved caspase-3 proteins were confirmed by Western blot.(2) After airway epithelial cells were added with LMPs following by pretreated by caspase inhibitor Z-VAD-FMK(50 μM), apoptosis was analyzed by flow cytometry.2.2 Effect of LMPs on oxidative stress in the airway epithelial cells2.2.1 Detection of MDA, superoxide, ROS and GSHIntracellular MDA, superoxide, ROS and antioxidant GSH in 16 HBE and A549 cells before and after LMPs stimuation for different time were measured.2.2.2 Apoptosis detectionAfter airway epithelial cells were added with LMPs following by pretreated by antioxidant NAC(20 μM), apoptosis was analyzed by flow cytometry.2.3 Upstream and downstream mechanisms of oxidative stress induced by LMPs in airway epithelial cells2.3.1 Western blot to detect changes of MAPK familyThree members of MAPK family were detected by Western blot, such as p38, p-p38, JNK, p-JNK, ERK, p-ERK. Caspase-3 and cleaved caspase-3 proteins also were detected.2.3.2 ELISA assay to detect the change of AA(1) AA inside LMPs or all of AA in the intracellular and culture supernatant from cells before and after LMPs stimuation were performed by ELISA.(2) After airway epithelial cells(16HBE and A549 cells) were added with LMPs for 24 h following by pretreated by 20 μM of cytochalasin D, AA in the airway epithelial cells was performed by ELISA.2.3.3 Effect of AA on cell apoptosisAirway epithelial cells before and after stimulation of 100 μM AA were analyzed DNA content via flow cytometry. Caspase-3 activity was also analyzed. These two analytical methods were used to determine effect of AA on cell apoptosis.2.3.4 Effect of p38 MAPK signaling pathway on AA and ROS(1) 1 μM of SB203580(an inhibitor of p38 MAPK signaling pathway) was used to pretreat 16 HBE and A549 cells before addition of LMPs, ELISA was used to analyze content of AA in cells;(2) 1 μM of SB203580 was used to pretreat 16 HBE and A549 cells for 2 h before addition of LMPs, flow cytometry was used to analyze changes in ROS levels.2.3.5 Effect of AA on ROS16HBE and A549 cells were pretreated with 10 μM of AA inhibitor TFP for 2 h before LMPs were added, flow cytometry was used to analyze changes in ROS levels.2.3.6 Effect of AA on cell apoptosis(1) After cells were pretreated with 10 μM of TFP and then stimulated with LMPs, Western Blot was used to detect changes in the expression of caspase-3, cleaved caspase-3 proteins.(2) After cells were pretreated by 10 μM of TFP and then stimulated with LMPs, cell apoptosis was assessed by flow cytometry. 10 μM of TFP was used for stimulation alone to observe the effect of TFP itself on cell apoptosis.3. Key components of LMPs playing pro-apoptotic effect3.1 After surface antigen PSR in airway epithelial cells was neutralized, immunofluorescence staining was used to assess the change of internalization of Dil-LMPs in airway epithelial cells.3.2 Western blot was used to assess the expression of Fas L in LMPs. After neutralizing Fas L in LMPs with Fas L antibody, MTT assay was used to assess the effect of LMPs on cell growth.3.3 After inactivation of LMPs by high temperature, MTT assay was used to assess the effect of LMPs on cell growth. Results:1. Study of effect of LMPs on airway epithelial cells1.1 Extraction and identification of LMPsWe successfully obtained LMPs from CEM-T lymphocytes induced by cytochalasin D. The diameters of LMPs were less than 1 μm by flow cytometry. Annexin V-positive rate of LMPs was 75.73%, in accordance with the characteristics of MPs.1.2 Effect of LMPs on human airway epithelial cells1.2.1 Result of morphological observationLMPs caused changes in cell morphology by optical microscopy and transmission electron microscopy, such as cellular shrinkage, medullary structure, vacuolization, stained nucli, chromosome condensation, changes in membrane structure, even the formation of apoptotic body and cell necrosis.1.2.2 Result of detection of cell growth and proliferation(1) Low concentrations of LMPs(1 μg/ml and 2 μg/ml) did not affect cell growth(p>0.05). High concentrations of LMPs(5-40 μg/ml) inhibited cell growth(both p<0.001). The inhibition rate increased with gradually increasing stimulation concentrations or extended stimulation time. So LMPs inhibited cell growth in dose- and time-dependent manners.(2) Cell proliferation rate induced by LMPs decreased from 60% in the control group to 39.1%(24 h), 12.6%(48 h) or 3.3%(72 h) by Ki-67 staining. Therefore, inhibition of cell proliferation was time-dependent.1.2.3 Apoptosis detectionAnnexin V and PI staining showed only Annexin V-positive rate, double Annexin V and PI-positive rate and total Annexin V-positive rate of cells under LMPs stimulation were significantly higher compared with control. 20 μg/ml LMPs at 24 h of stimulation had a significant effect on apoptosis in airway epithelial cells. So we selected 20 μg/ml LMPs at 24 h of stimulation to do further study.1.2.4 Interaction between LMPs and cells by immunofluorescence staining(1) With the extension of incubation time, the amount of Dil-LMPs gradually increased within the cytoplasm. Dil-LMPs almost completely entered into the cytoplasm after 16 h. The way of LMPs into the cells is time-dependent.(2) The amount of Dil-LMPs gradually increased into the cytoplasm, with increasing incubation concentrations of Dil-LMPs(5 μg/ml, 10 μg/ml and 20 μg/ml), which was dose-dependent manner.(3) The amount of Dil-LMPs into the cytoplasm largely reduced after cytochalasin D(phagocytosis inhibitor) pretreated cells. The amount of Dil-LMPs into the cytoplasm greatly increased after chloroquine(lysosome inhibitor) pretreated cells. It shows that LMPs entered into cells by phagocytosis.1.3 Effect of LMPs on airway epithelium and lung epithelium in mice1.3.1 Histopathological observation and TUNNEL assayHistopathological observation: We observed bronchial and lung tissues in vitro induced by LMPs via scanning electron microscopy, HE staining and TUNNEL staining and found LMPs induced bronchial and alveolar epithelial injury, tissue disorder and cell apoptosis(TUNNEL stained in brown). It have been further confirmed cell apoptosis induced by LMPs in airway epithelial cells was universal and not related to cell types on the aspect of tissue culture and different species of origin.2. The mechanisms of apoptosis induced by LMPs in airway epithelial cells2.1 Effect of caspase induced by LMPs in airway epithelial cells2.1.1 Caspase detection(1) Caspase-8 and-9 activities reached the peak in the airway epithelial cells after LMPs treatment for 4h(compared with control, p<0.05). Caspase-3 activity reached its peak in cells after LMPs treatment for 8h(compared with control, p<0.05). Western blot further confirmed LMPs increased the expression of caspase-3 protein(compared with control, p<0.05).(2) Z-VAD-FMK(caspase inhibitor) reversed cell apoptosis mediated by LMPs(compared with LMPs group, p<0.05). Therefore, caspase-3 may involve in the mechanisms about apoptosis induced by LMPs.2.2 Effect of LMPs on oxidative stress in the airway epithelial cells2.2.1 Changes of production of MDA, superoxide, ROS and GSHLMPs increased levels of MDA, superoxide, ROS in 16 HBE and A549 cells compared with control(p<0.05). However, LMPs reduced level of GSH, compared with control, which was a statistical significance(p<0.001).2.2.2 Change of cell apoptosis20 μM of NAC(oxidative stress inhibitor) can decreased only Annexin V-positive rate induced by LMPs from 14.8% to 3.22% in 16 HBE cells, from 17.4% to 4.60% in A549 cells(both p<0.05). So, NAC could reverse LMPs-induced apoptosis.2.3 Upstream and downstream mechanisms of oxidative stress induced by LMPs in airway epithelial cells2.3.1 Changes of MAPK family proteins by Western blot(1) After LMPs treatment for 30 min and more, LMPs increased the expression of phospho-p38 protein in 16 HBE and A549 cells compared with control by Western blot(all p<0.05), and phosphorylation of p38 was gradually increased with extended stimulation time. So LMPs activated p38 MAPK signal transduction pathway in airway epithelial cells.2.3.2 Change of AA production(1) LMPs increased production of AA from 23.01 ± 2.59 μg/ml in control group to 45.42 ± 2.32 μg/ml in 16 HBE cells by ELISA(p<0.05), from 23.01 ± 0.77 μg/ml in control group to 40.51 ± 2.25 μg/ml in A549 cells(p<0.05). 20 μg/ml LMPs contained only small amounts of AA(3.49 ± 0.54μg/ml).(2) Cytochalasin D(phagocytosis inhibitor) reversed the production of AA mediated by LMPs(p<0.05). Therefore, the production of AA mediated by LMPs was endogenous.2.3.3 Effect of AA on cell apoptosis100 μM of AA increased apoptosis rate from 0% in control to 48.04% in 16 HBE cells(50.37% in A549 cells) by flow cytometry. AA increased caspase-3 activity in 3.99-fold in 16 HBE cells and in 3.17-fold in A549 cells(p<0.001). Therefore, AA significantly increased apoptosis in airway epithelial cells.2.3.4 Effect of p38 MAPK signal pathway on AA and ROSSB203580(an inhibitor of p38 MAPK signal pathway) effectively reversed 27% of AA mediated by LMPs in 16 HBE cells(p<0.05); whereas 28% in A549 cells(p<0.05). Meanwhile, SB203580 effectively reduced the increase of LMPs-mediated ROS(p<0.05). Therefore, AA and oxidative stress were located in downstream of p38 MAPK signaling pathway.2.3.5 Effect of AA on ROSIn both epithelial cells, TFP(AA inhibitor) reversed the increase of LMPs-induced ROS(p<0.05). Therefore, AA regulated oxidative stress.2.3.6 Effect of AA on cell apoptosis(1) Western Blot showed TFP reversed an increase of cleaved caspase-3 protein induced by LMPs. It shows that TFP could reverse LMPs-mediated caspase-3 activation.(2) TFP reversed apoptosis rate induced by LMPs from 9.88% to 1.95% in 16 HBE cells; whereas from 9.08% to 2.26% in A549 cells(p<0.01). However, the same dose of TFP itself didn’t induce apoptosis. So TFP reversed apoptosis mediated by LMPs, independent of TFP itself.3. Key components of LMPs playing pro-apoptotic effect(1) After PSR in cells was neutralized, the amounts of Dil-LMPs(red) into the cytoplasm in 16 HBE and A549 cells didn’t significantly change(compared with Dil-LMPs group). So PS/PSR signaling pathway was not involved in phagocytosis process.(2) Western blot showed LMPs contained Fas L. After neutralization of Fas L in LMPs, the inhibitory effect of cell growth mediated by LMPs didn’t change(p>0.05). Thus, although LMPs included Fas L, Fas/Fas L pathway was not involved in the inhibitory effect of cell growth mediated by LMPs.(3) After inactivation of LMPs by the high temperature, MTT showed the inhibitory effect of cell growth mediated by LMPs didn’t change. Therefore, heat-insistant components or other lipid components in LMPs may play a role. Conclusion:(1) LMPs induced airway epithelial damage and apoptosis.(2) LMPs entered into airway epithelial cells by cell phagocytosis.(3) The mechanisms that LMPs played a pro-apoptotic role in airway epithelial cells were: LMPs activated p38 MAPK signaling pathway, largely stimulated production of arachidonic acid, increased oxidative stress, activated caspase-3, resulting in apoptosis of bronchial epithelial cells and alveolar epithelial cells.(4) Heat-insistant components or other lipid components in LMPs may play a role.