Inflammatory Regulation And Sex-based Differences On Phagocytosis Of Apoptotic Cells By Mouse Macrophages

Background and objectives:The resolution of inflammation involves the apoptosis and removal of inflammatory cells, which can be regulated by inflammatory mediators. However, mechanisms underlying the removal of apoptotic immune cells remain to be clarified. The most knowledge on regulation of phagocytosis is based on analysis in vitro. Establishment of a method to evaluate phagocytosis in vivo is essential to study phagocytic clerance of apototic cells in physiological conditions. Given that defective clearance of apoptotic cells many result in autoimmune diseases with a significant predominance in female. It should be worthwhile to investigate sexual difference in the clearance of apoptotic cells.Materials and methods:Mouse neutrophils were isolated and induced spontenous apoptosis by culturing at 37℃for 24 h. Mouse thymocytes were induced apoptosis with Dexamethasone (DEX) at 37℃. Mouse peritoneal macrophages were isolated and characterized with phycoeruthrin (PE)-conjugated F4/80 antibodies. Phagocytosis assays were performed in vitro and vivo. Flow cytometry was used to identify macrophages, apoptotic cells and phagocytes engulfing apototic cells. The level of mRNA was analyzed by quantitative real-time RT-PCR (Q-PCR). Cytokine concentration was measured by ELISA.Results:Lipopolysaccharide (LPS) specifically inhibits phagocytosis of apoptotic cells by macrophages. We found that LPS induced TNF-αproduction and supressed expression of Gas6 in macrophages. The neutralizing antibodies to TNF-αand exogenous Gas6 reversed significantly the LPS-mediated the inhibition of phagocytosis. The results suggest that LPS inhibits the phagocytosis of apototic cells by macrophages through induction of TNF-αand suppression of Gas6 in macrophages.A novel method for evaluating the phagocytosis in vivo was established. Fluorescein isothiocyanate (FITC) -labeled apoptotic cells were injected into mouse peritoneal cavity. The peritoneal cells were collected at the specified duration. The fluorescence of surface-bound apoptotic cells was quenched by trypan blue (TB). F4/80+/FITC+ cells were determined as the macrophages that engulfed apoptotic cells. This is a simple and reliable method to analyze the phagocytosis in vivo.The phagocytic capacity of macrophages is different between male and female mice. This diffrence was associated with ages of mice. In adult mice (2-24 months), phagocytic capacity of macrophages of male mice was higher than that of females. However, opposite results were observed in prepubertal (< 1 month) and aged (> 24 months) mice. Furthermore, we provided evidence that the sex- and age-based diffrences in the phagocytosis of apoptotic cells by macrophages should not result from hormone levels in enviroments.Conclusions:LPS inhibits the engulfing of apoptotic cells by mouse peritoneal macrophages through regulating production of TNF-a and Gas6 in macrophages. Data provide novel insights into the regulation of phagocytic clearance of apoptotic cells by inflammatory mediators. We describe a novel method to assess phagocytic ability of peritoneal macrophages in vivo, which should be useful to investigate the regulation of phagocytosis in vivo. Phagocytic capacity of macrophages varies in different gender and ages, which may provide novel insights into the etiology of sex- and age-based differences on prevalence of autoimmune diseases.