Effects Of Autophagy On Clearance Of The Apoptotic Cells By Macrophages

Objective To investigate the morphological characteristics of the autophagic structures after macrophages phagocytize the apoptotic cells.To explore the effects of autophagy on clearance of the apoptotic cells by macrophages and the relationship between autophagy and heterophagy.Methods Apoptosis of lymphocytes and Jurkat cells was induced with cyclophosphamide.The apoptotic lymphocytes and Jurkat cells were added into dishes where there were macrophages respectively.Cell number of the cleaned apoptotic cells were counted.The morphological changes of macrophages phagocytizing the apoptotic cells were viewed with scanning electron microscope and Giesma staining.The structural features of the proautophagosome, autophagosomes,autophagolysosomes and amphisomes in macrophages phagocytized the apoptotic cells were examined with transmission electron microscope,the cross-section areas of the autophagic structures and cytoplasm were measured with image analyzer,the ratio of the autophagic structures and cytoplasm was calculated. The autophagosomes of macrophages were labeled with MDC staining,viewed and counted under confocal laser scanning microscope.Autophagy of macrophages was inhibited with 3-MA,then the changes of cleance of the apoptotic cells were examined.Results Macrophages phagocytized actively the apoptotic cells, apoptotic nuclei,apoptotic bodies and other cell debris to form heterophagosomes. Being compared with the control group,number of macrophages engulfed the apoptotic cells and the autophagosomes in these cells increases,the ratios of the cross-section areas of the autophagic structures and cytoplasm of macrophages are greater.There are the apoptotic bodies or other cell debris in many autophagosomes. These autophagosomes are large and near the cell membrane.In addition,some autophagosomes contain cytoplasm-like substance or the injured mitochondrion and endoplasmic reticulum,they were located near the mitochondrion or rough endoplasmic reticulum.The autophagosomes containing the whole apoptotic cell or apoptotic nucleus were not observed.Some phagosomes fused with the autophagomes to form amphisome.Amphisome contains the fragments of apoptotic cell and integrated autophagosome wrapped by inner layer of membrane.The clearance ratio of the apoptotic cells decreased when autophagy of macrophages were inhibited.Conclusion Autophagic abilitiy of macrophages enhances significantly when the cells phagocytize the apoptotic cells.Autophagy plays important direct and indirect roles in clearance of the apoptotic cells by macrophages.Relations between autophagy and heterophagy is establisted though formation of the amphisome.