Increase in hypotonic stress-induced endocytic activity in macrophages via ClC-3

Abstract

Extracellular hypotonic stress can affect cellular function. Whether and how hypotonicity affects immune cell function remains to be elucidated. Macrophages are immune cells that play key roles in adaptive and innate in immune reactions. The purpose of this study was to investigate the role and underlying mechanism of hypotonic stress in the function of bone marrow-derived macrophages (BMDMs). Hypotonic stress increased endocytic activity in BMDMs, but there was no significant change in the expression of CD80, CD86, and MHC class II molecules, nor in the secretion of TNF-α or IL-10 by BMDMs. Furthermore, the enhanced endocytic activity of BMDMs triggered by hypotonic stress was significantly inhibited by chloride channel-3 (ClC-3) siRNA. Our findings suggest that hypotonic stress can induce endocytosis in BMDMs and that ClC-3 plays a central role in the endocytic process.

Fig. 1

Hypotonic environment promotes endocytosis in BMDMs. (A) The purity of BMDMs analyzed by flow cytometry. IgG was used as an isotype control. (B) Apoptotic fraction of cells detected by annexin V staining (x-axis)/propidium iodide staining (y-axis) after treatment with hypotonic solution. (C, D) Gray fill represents hypotonic group. (C) Pinocytotic activity of BMDMs in hypotonic environment shown by mean fluorescence intensity (MFI) of cells incubated with FITC-dextran. (E, F) Uptake of IgG-coated latex beads analyzed by light microscopy and calculated as phagocytosis index. Photos taken from a representative experiment. The data represent mean ± SD from five independent experiments (n = 5 for each group). ***P < 0.001 for group comparisons.

Fig. 2

Expression of CD80, CD86, and MHC class II molecules in BMDMs was not affected by hypotonic stress. (A) Histograms from representative experiments. Gray fill represents hypotonic group. (B) MFI values of CD80, CD86, and MHC class II analyzed by flow cytometry (n = 5 for each group).

Fig. 3

Secretion of TNF-α or IL-10 by BMDMs was not affected by hypotonic stress. (A, B) Cytokine levels quantified by ELISA. LPS group served as positive control. (n = 3 for earch group). ***P < 0.001 for LPS group compared to control and hypotonicity groups.

Fig. 4

Expression and localization of ClC-2 and ClC-3 in BMDMs. (A) ClC transcripts detected in BMDMs by RTPCR. Heart tissue was used as positive control. (B) ClC protein expression in BMDMs measured by Western blot. Heart tissue served as positive control. For specific controls, antibodies against ClCs were pre-incubated with corresponding peptide antigens. (C) Localization of ClC-3 in BMDMs shown by double-staining immunofluorescence (original magnification × 1000). Scale bar = 20 μm. Nuclei (blue) and ClC-3 (Green) were counterstained with Hoechst 33342 and ClC-3 antibody, respectively. (D, E) ClC-3 expression in BMDMs after hypo-tonic exposure examined by Western blot. Photos taken from a representative experiment. The data represent mean ± SD from three independent experiments. (F) Localization of ClC-3 in BMDMs stimulated with hypotonic solution visualized by double-staining immunofluorescence (original magnification × 1000). Scale bar = 20 μm. ClC-3 collected in clusters around the cytomembrane, shown by arrows (n = 3 for each group).

Fig. 5

ClC-3 plays a role in endocytic activity of BMDMs under hypotonic stress. (A) The efficiency of ClC-3 siRNA transfection measured by flow cytometry. IgG was used for isotype control. (B, C) Protein expression of ClC-3 after transfection detected by Western blot. (D) MFI values for normal control, ClC-3 siRNA and control siRNA groups analyzed by flow cytometry. Pinocytotic activity in BMDMs was decreased by interference with ClC-3. (E, F) Uptake of IgG-coated latex analyzed as phagocytosis index in normal control, ClC-3 siRNA and control siRNA groups. Phagocytic activity in BMDMs was down-regulated by interference with ClC-3. Photos taken from a representative experiment. The data represent mean ± SD from three independent experiments (n = 3 for each group). *P < 0.05, ***P < 0.001 for differences between treatment groups.