Hypotonic Environment Affect The Ability Of Mouse Bone Marrow-derived Macrophages And Its Mechanism

Objective:Macrophages were reported to be involved in various immune and inflammatory responses. However, there is little evidence that hypotonic environment is associated with the function of macrophages. The purpose of our experiment is to investigate the role of hypotonic environment in the function of mouse bone marrow-derived macrophages (BMDMs), and the underlying mechanism of its involvement.Here, we report that, the expression of C1C-3in mouse bone marrow-derived macrophages (BMDMs) was confirmed at both mRNA and protein levels. And the endocytosis activity of BMDMs was up-regulated under hypotonic environment whereas the expression of CD80, CD86, and MHC class Ⅱ and the secretion of TNF-α or IL-10by BMDMs were not affected. Furthermore, the increased endocytosis activity of BMDMs could be significantly inhibited by C1C-3siKNA. Collectively, our data identify hypotonic environment as a microenvironment associated with the endocytosis of BMDMs and reveal a mechanism thatMethods:1. To investigate the effect of hypotonic environment on cell function of BMDMs, pinocytosis of FITC-dextran and phygocytosis of IgG-coated latex were detected.2. To examine the effect mediated by hypotonic environment on APC function of macrophages, CD80, CD86, and MHC class Ⅱ expression in BMDMs were detected by flow cytometry.3. To examine the effect mediated by hypotonic environment on secretion of cytokines by BMDMs, IL-10and TNF-a were detected by ELISA assay.4. To determine whether C1C-3and C1C-2were expressed in BMDMs, the mRNA and protein expression in BMDMs were test by RT-PCR and Western blot respectively. The immunofluorescence was used to determine the localization of C1C-3in BMDMs. After BMDMs were stimulated in hypotonic solution, the change in the expression level and location of C1C-3were tested.5. To investigate whether C1C-3participate into the increased endocytosis activity of BMDMs under hypotonic condition, small interfering RNA (siRNA) was used to suppress the expression of C1C-3.Results:1. Hypotonic environment promotes endocytosis by BMDMsIt was found that both FITC-dextran pinocytosis and IgG-coated latex phygocytosis were increased by stimulating with hypotonicity2. Hypotonic environment has no effect on the expression of CD80, CD86, MHC class Ⅱ on BMDMsThere were no obvious differences between hypotonic group and control group detected by flow cytometry.3. Hypotonic environment has no effect on secretion of IL-10, TNF-a by BMDMsBMDMs were stimulated by LPS, IL-10and TNF-α were observed raised obviously, whereas no significant changes was observed on the secreting of both cytokines stimulatied by hypotonicity.4. ClC-3predominantly expressed in cytoplasm of BMDMIt was found that ClC-3mRNAs and ClC-3proteins could be expressed in BMDMs and predominantly expressed in cytoplasm of BMDMs. When BMDMs were stimulated in hypotonic solution, the change in the expression level of ClC-3was not significant whereas they were observed to gather around the cytomembrane and lumped5. Hypotonic environment increases the endocytosis ability of BMDMs via ClC-3It was found that both FITC-dextran and IgG-coated latex uptake were significantly decreased in ClC-3siRNA group compared with normal control group and control siRNA transfection group.Conclusion:1. Hypotonic environment promotes endocytosis by BMDMs2. ClC-3is necessary for up-regulation the endocytosis activity of BMDMs in vitro.