Involvement Of Annexin Ⅰ In The Dexamethasone-mediated Upregulation Of A549 Cells Phagocytosis Of Apoptotic Eosinophils

BackgroundEosinophils are recently confirmed as one of the major effector cells in the immuno-inflammation changes in the airway in asthma. By infiltrating into the bronchial mucosa and subsequent release of histotoxic substances, eosinophils may cause epithelial damage, an event thought to be important in the development of airway hyperresponsiveness. On the other hand, the bronchial epithelial cells have the ability to engulf the apoptotic eosinophils, which may be crucial to the solution of inflammation. The process is regulated by many factors and the regulators of this process may have therapeutically potential.Annexin I is a member of annexin superfamily of 13 structurally related proteins that are able to bind to cell membrane in a calcium-dependent manner. Interestingly Annexin I may also be an endogenous ligand mediating the engulfment of apoptotic cells. Fan et al found that annexin I served as both ligand and receptor in promoting the phagocytosis of Jurkat T lymphocytes and human peripheral T lymphocytes by macrophages. In addition, annexin I is expressed on the membrane surface of A549 cell and is up-regulated by dexamethasone, indicating that annexin I may be involved in the dexamethasone's upregulation of the engulfment of apoptotic eosinophils in A549 cell.ObjectiveIn the present study, we observed the real time process of phagocytosis of apoptotic eosinophils by A549 cells and investigated whether Annexin I was involved in the dexamethasone-mediated upregulation of phagocytosis of apoptotic eosinophils by A549 cells. In the meanwhile, we investigated the effect of phagocytosis of apoptotic eosinophils on IL-6 and IL-8 release in A549 cells. Method1. Eosinophil isolation and apoptosis inductionAfter removal of red blood cells using red blood cell lysis buffer, the eosinophils were isolated by micromagnetic beads coated with anti-CD16, anti-CD15 and the magneticactivated separation column. Flow cytometric analysis assessed the apoptosis eosinophils rate after culturing for 48 hours.2. Real time process of phagocytosis of apoptotic eosionphils by A549 cells.A549 Cells were put into the heated-plate system. After this, apoptotic eosinophils were put into the dishes through the hole. The whole process of phagocytosis was automatically recorded every one minute. The curve length, area and density of A549 cells before and after phagocytosis were measured by MetaMorph software.A549 cells transfected by GFP engulf apoptotic eosinophils labled with 7-AAD, which was scanned by laser confocal scanning microscope.3. Quantitative observation of phagocytosis of apoptotic eosionphils by A549 cells.The coverslip with A549 cells were transferred to new 12-well plates coated with Parafilm membrane one by one. Washed apoptotic eosinophils were resuspended in DMEM and added to the coverslip (1×104/slide) with A549 cells for 60 minutes at 37°C under 5%CO2. After interaction, the noningested eosinophils were washed using oscillator with Ca2+ and Mg2+ free PBS solution supplemented with EDTA before fixation. The ingested eosinophils were visualized by o-dianisidine staining. At least 200 A549 cells which were conter-stained with haematoxylin in duplicate wells in randomly selected fields were counted by a blinded investigator, and the proportion of ingested eosinophils was expressed as a percentage.4. Western blotting analysis of annexin IIn order to provide a sufficient number of cells for measurement of cell surface annexin I induction, subconfluent A549 cells were seeded in DMEM medium in 100mm dishes. The supernatants washed with EDTA were collected and followed by western blot analysis.5. Cytokine productionA549 cells unstimulated or stimulated with dexamethasone (10-6 M) were incubated in DMEM in 48 wells plates alone or togather with apoptotic eosinophils. IL-6 and IL-8 were assayed in supernatants from cocultures of A549 cells and apoptotic eosinophils by RIA kit according to the manufacturer's instruction. Concentration of TGF-β1 was measured by ELISA.6. Statistical methodAll experiments were performed for five independent times. Data were expressed as the means±standard error and were analyzed for significant differences by unpaired Student's t test and one-way ANOVA with SPSS 10.0. Differences were considered statistically significant if P value <0.05.Result1. The curve length, area, and density of A549 cells were changed after phagocytosisThe whole process of phagocytosis of apoptotic eosinophils by A549 cells was automatically recorded every one minute. The curve length, area and density of A549 cells before and after phagocytosis were measured by MetaMorph software. The results showed that the curve length and area of A549 cells were reduced after phagocytosis (P<0.001), while the density of A549 cells were increased (P<0.001).Phagocytosis was further confirmed by laser confocal scanning microscope.2. Dexamethasone promotes engulfment of apoptotic eosinophils by A549A549 epithelial cells recognized and ingested apoptotic eosinophils that were visualized as dark-staining peroxidase-positive cells. Effects of dexamethasone stimulation with various duration of preincubation on the engulfment of apoptotic eosinophils by A549 cells showed that the effect was not observed at shorter incubation time (< 1 h), but was evident at longer incubation time. The capacity of engulfment reached the highest at the 4h of preincubation (55%±2.2%) and reduced to the 38%±1.4% at the 24h of preincubation which was still significantly higher than that in control (0h).3. Dexamethasone treatment increased annexin I on cell surface of A549Western blot analysis showed the effect of dexamethasone treatment on the expression of annexin I on cell surface of A549. The result showed that the intensity was maximal at 4h after dexamethasone treatment and returned to the basal levels at 24h. Correlation analysis shown that the amount of cell surface annexin I was positively correlated with the capacity of engulfment induced by dexamethasone (r = 0.779, p<0.001).4. Increased phagocytosis of apoptotic eosinophils by A549 cells stimulated by treatment with dexamethasone was inhibited by annexin I mAbTo investigate the functional contributions of A549 cell surface annexin I, we tried to use the annexin I mAb to inhibit the phagocytosis of apoptotic eosinophils by A549 cells. The capacity of phagocytosis of A549 cells treated with dexamethasone and annexin I mAb (10μg/ml; 30 min at 37°C) was compared with that of A549 cells treated with dexamethasone only, previously shown to significantly enhance phagocytosis. The results showed that pretreatment with dexamethasone and annexin I mAb downregulated the phagocytosis of apoptotic eosinophils.5. Annexin I expressed on apoptotic eosinophil surface was involved in its phagocytosis by A549Apoptotic eosinophils were washed with EDTA to remove annexin I before presented to A549 cells. The result showed removal of annexin I with EDTA and preincubation with annexin I mAb inhibited phagocytosis (p<0.001).6. Dexamethasone-promoted uptake of apoptotic eosinophils did not change the proinflammatory factor secretion in A549 cellsThere was no difference in the release of proinflammatory factors and TGF-β1 between the A549 cells stimulated with apoptotic eosinophils and non-stimulated A549 cells. Conclusion1. The curve length, area, and density of A549 cells were changed after phagocytosis. The curve length and area of A549 cells were reduced after phagocytosis, while the densities of A549 cells were increased.2. Dexamethasone promotes engulfment of apoptotic eosinophils by A549 through annexin I.3. Dexamethasone-promoted uptake of apoptotic eosinophils did not change the proinflammatory factor secretion in A549 cells.