The Studies Of Phagocytosis Of Apoptotic Neutrophils Induced Cell Death In Macrophage And The Role Of CpG-DNA In The Process Of Phagocytosis

BackgroundsMacrophages and neutrophils are now recognized as the major effector cells in the airway inflammatory diseases.By infiltrating into the bronchial mucosa,the neutrophils can effectively bind and kill microorganisms that may cause damages to tissue,and then they timely undergo a vigilantly controlled programmed cell death known as apoptosis.Clearance of these apoptotic neutrophils by macrophages is important for the successful resolution of acute inflammation and tissue homeostasis. However,the process of phagocytosis of apoptotic neutrophils by macrophages and the fates of macrophages after their ingestion have not been unveiled.There are growing evidences that defective handling of apoptotic cells by macrophages plays a pivotal role in the development of chronic airway inflammatory diseases,systemic lupus erythematosus(SLE) and other autoimmune diseases. Strategies to improve clearance of apoptotic neutrophils may have therapeutic significance.Bacterial genomic DNA and synthetic oligonucleotide containing unmethylated cytosine followed by guanine(CpG-ODN) have been shown to stimulate immunocompetent cells,including macrophages/monocytes,dendritic cells (DCs),and B cells.Whether they can enhance the ability of macrophages to engulf apoptotic neutrophils and the potential mechanisms are still unclear.ObjectivesTo measure the dynamic process of phagocytosis of apoptotic neutrophils by macrophages in real time;to identify the fates of macrophages after their ingestion of apoptotic neutrophils;to investigate the role of CpG-ODN and bacterial DNA in the process of ingestion of apoptotic neutrophils by macrophages and the potential mechanisms.Methods1.Neutrophil isolation and apoptosis inductionAfter removal of red blood cells using red blood cell lysis buffer,the neutrophils were isolated by the Percoll discontinuous density gradient centrifugation.Apoptosis was assessed using flow cytometry after culturing for 20 hours2.Live cell imaging of Raw264.7 cells engulfing apoptotic neutrophils and phagocytosis induced cell death in macrophageRAW264.7 Cells grown in 35 mm~2 dishes were put into a heated-plate system on the stage of an inverted microscope equipped with a CCD camera controlled by SPOT RT software.Then apoptotic neutrophils were put into the dishes and allowed them to interact with each other.The dynamic process of phagocytosis of human apoptotic neutrophils by RAW264.7 cells in real time was recorded automatically every 30 seconds for a period of 5—12h.3.Form of macrophage cell death analyzed by transmission electric microscope After interacting with apoptotic neutrophils for 60 minutes and washing away the non-ingested ones,the macrophages were fixed,scraped and pelleted by centrifugation.Ultrathin sections were stained with uranyl acetate and lead citrate and were observed under a transmission electron microscope4.Form of macrophage cell death analyzed by confocal microscopeAfter interacting with apoptotic neutrophils for 60 minutes and washing away the non-ingested ones,the macrophages were stained with MDC/AO,AO,AO/EB respectively for 15min.Then the cells were scanned using confocal microscopy within 1 hour. 5.Quantification of IL-6 and TNF-a productionAfter interacting with apoptotic neutrophils for 60 minutes and washing away the non-ingested ones,the macrophages were cultured for additional 24h.The supernatants were collected at the indicated times points and IL-6 and TNF-a were assayed by radio-immuno assay(RIA).6.Quantification of uptake of apoptotic neutrophils by RAW264.7 cells treated with CpG-DNARAW264.7 cells were grown in 24-well plates and treated with CpG-ODN in the presence or absences of chloroquine for 24h.The cover-slips with RAW264.7 cells were transferred to 12-well plates coated with parafilm membrane.Apoptotic neutrophills were added to the coverslip with RAW264.7 cells for 60 minutes.After washing away the noningested neutrophils,the ingested neutrophils were visualized by o-dianisidine staining.RAW264.7 cells were counter-stained with haematoxylin. The cells were assayed and photographed.At least 200 RAW264.7 cells were counted in randomly selected fields.The proportion that had ingested one or more neutrophils was expressed as a percentage.7.Cell viability assay by MTTRAW264.7 cells were plated in 96 wells and incubated overnight.After 24 hours stimulation of CpG-ODN and B-DNA in the presence or absences of chloroquine for 2h ahead,Cells were then washed twice in PBS,and 180μl of fresh medium and 20μl of MTT(5 mg/ml) were added to each well,followed by incubation for an additional 4 h.The supematants were removed,and 150μl of DMSO was added to each well.MTT crystals were completely solubilized with libration for 10 min and optical density(OD) was measured at wavelengths A570 nm.The percentage of cell viability was calculated.8.Western blot analysis of total TLR9Macrophages(2×10~6) were treated with CpG-ODN and B-DNA for 24h in the presence or absence of chloroquine for 2h ahead.Then the cells were harvested and lysed with lysis buffer.Cell lysates were then subjected to Western blotting for TLR9 9.Flow cytometry analysis of surface TLR9For measuring surface expression of TLR9,cells were analyzed with a flow cytometry after 24 hours treatment with CpG-ODN and B-DNA in the presence or absence of 2μg/ml chloroquine for 2h ahead.The cells were incubated with the appropriately diluted anti-TLR9 mAb,followed by incubation with fluorescein isothiocyanate-conjugated secondary mAb.Then macrophages were analyzed for TLR9 on the flow cytometer10.Statistical analysisAll experiments were performed for three or 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.Results1.We measured for the first time the dynamic process of phagocytosis of apoptotic human neutrophils by RAW264.7 cells in vitro.We staged four steps of phagocytosis as the recognition and tethering,internalization,ingestion and exocytosis steps. Furthermore,we found that after phagocytosis,some macrophages might undergo a characteristic sequence of morphological changes,beginning with cell shrinkage, followed by membrane blebbing,and ultimately concluding in cell membrane rupture.2.The dead macrophages consisted of autophagy,apoptosis and oncosis as revealed by transmission electron microscopy and confocal microscopy combined with specific dyes.The percentages of autophagic,apoptotic,oncotic macrophages were 8.00±2.00%,12.33±2.08%,and 3.66±1.50%respectively.3.The concentration of IL-6 and TNF-a in the supernatants of cultured macrophages was increased after interacting with apoptotic neutrophils.4.We found that the ability of macrophages to ingest apoptotic neutrophils was up-regulated by CpG-ODN and B-DNA treatment but the effect was abolished by chloroquine pre-treatment. 5.Increased uptake of apoptotic neutrophils was consistent with up-regulation of total and surface Toll-like receptor-9 expression in macrophages.6.Chloroquine couldn't down-regulate the total and surface TLR9 expression in macrophages up-regulated by B-DNA and CpG-ODN treatment.Conclusions1.The phagocytosis of apoptotic neutrophils can also be divided into the recognition and tethering,internalization,digestion and exocytosis stages and the phagocytosis of apoptotic neutrophils can induce cell death in macrophages.2.After ingestion of apoptotic neutrophils,macrophages may undergo autophagy, apoptosis,oncosis that may play an important role in the pathogenesis and progression of inflammatory diseases.Autophagy of macrophage after ingestion of apoptotic cell may be one of the new mechanisms in immune response and inflammatory diseases.3.CpG-ODN and B-DNA can act through TLR9 to enhance the macrophages uptake of apoptotic neutrophils;enhancement of uptake of apoptotic neutrophils also requires endosomal maturation/acidification.