Glia maturation factor-γ mediates neutrophil chemotaxis

Abstract

Chemotaxis is fundamental to the directional migration of neutrophils toward endogenous and exogenous chemoattractants. Recent studies have demonstrated that ADF/cofilin superfamily members play important roles in reorganizing the actin cytoskeleton by disassembling actin filaments. GMFG, a novel ADF/cofilin superfamily protein that is expressed in inflammatory cells, has been implicated in regulating actin reorganization in microendothelial cells, but its function in neutrophils remains unclear. Here, we show that GMFG is an important regulator for cell migration and polarity in neutrophils. Knockdown of endogenous GMFG impaired fMLF- and IL-8 (CXCL8)-induced chemotaxis in dHL-60 cells. GMFG knockdown attenuated the formation of lamellipodia at the leading edge of cells exposed to fMLF or CXCL8, as well as the phosphorylation of p38 and PAK1/2 in response to fMLF or CXCL8. Live cell imaging revealed that GMFG was recruited to the leading edge of cells in response to fMLF, as well as CXCL8. Overexpression of GMFG enhanced phosphorylation of p38 but not of PAK1/2 in dHL-60 cells. In addition, we found that GMFG is associated with WAVE2. Taken together, our findings suggest that GMFG is a novel factor in regulating neutrophil chemotaxis by modulating actin cytoskeleton reorganization.

Figure 1.. GMFG mediates cell migration in neutrophils and dHL-60 cells.

(A) RT-PCR (upper panels) and immunoblotting analysis (lower panels) of endogenous GMFG expression in undifferentiated HL-60 and dHL-60 cells and human neutrophils. (B) RT-PCR and immunoblotting analysis of GMFG expression in dHL-60 cells 48 h after mock transfection or transfection with a mixed GMFG siRNA pool or negative-control siRNA. (C and D) Transwell migration assays in response to vehicle alone (chemokinesis) or increasing concentration of fMLF (used as fMLP throughout figures; 1–100 nM) or CXCL8 (used as IL-8 throughout figures; 1–100 ng/mL) in dHL-60 cells expressing control siRNA or GMFG siRNA. The percentage of cells that migrated into the lower well was recorded. (E and F) EZ-TAXIScan assay chemotaxis toward fMLF or CXCL8 in dHL-60 cells expressing control siRNA or GMFG siRNA. Cells were loaded onto the upper chamber, and the lower chamber was filled with 100 nM fMLF or 100 ng/mL CXCL8. Data were collected at 30-s intervals for 30 min. Representative images of migrating cells in a gradient of fMLF or CXCL8 at the indicated time-point are shown. Original scale bars, 20 μm (also see Supplemental Videos 1–8). (G and H) EZ-TAXIScan assay chemotaxis of dHL-60 cells 48 h after mock transfection or transfection with control siRNA or GMFG siRNA in response to increasing concentration of fMLF (1–100 nM) or CXCL8 (1–100 ng/mL). Mean migration speed was quantified from the captured images during the course of the EZ-TAXIScan chemotaxis assay, as described in Materials and Methods (G). The CI, defined as the cosine of the angle between the motion vector of the cells at a given time and the vector pointing to direction of the gradient, was determined from the digital time-lapse movies (H). Data are representative of at least three independent experiments and are expressed as mean ± sd. *P < 0.05 versus control.

Figure 2.. GMFG localizes to the leading edge and regulates F-actin polymerization during cell chemotaxis in neutrophils or dHL-60 cells.

(A) Localization of GFP-GMFG in neutrophils or dHL-60 cells. Cells transiently transfected with GFP-tagged GMFG were adhered to glass coverslips and stained for F-actin with phalloidin A-488. Microscopy was used to analyze GMFG and F-actin distribution. (B) Distribution of GFP-GMFG in response to chemoattranctant fMLF or CXCL8 stimulation in dHL-60 cells upon dHL-60 cells expressing GFP-GMFG was plated to glass coverslips and stimulated with 100 nM fMLF or 100 ng/mL CXCL8 for 10 min. (C and D) Distribution of F-actin and CD43 in response to fMLF (C) or CXCL8 (D) in negative-control, siRNA- or GMFG siRNA-expressing dHL-60 cells. Cells transfected with negative-control siRNA or GMFG siRNA were adhered to glass coverslips and stimulated with a uniform concentration of fMLF (100 nM) or CXCL8 (100 ng/mL) for 0, 1, or 10 min. The cells were fixed and stained with F-actin (phalloidin A-488; in green) and anti-CD43 primary antibody (in red) and confocal microscopy used to analyze distribution of staining and morphologic changes. Results shown are representative of three independent experiments. Original scale bar, 5 μm.

Figure 3.. GFP-GMFG redistributes to the cell leading edge during neutrophil and dHL-60 cell chemotaxis.

Time-lapse sequences of the migration of neutrophils and dHL-60 cells expressing control GFP vector or GFP-GMFG toward a point source of fMLF or CXCL8. Neutrophils or dHL-60 cells were transfected with GFP vector or GFP-GMFG vector for 24 h, then plated on glass-bottom dishes coated with fibrinogen, and exposed to a chemotactic gradient generated by the slow release of fMLF (100 nM) or CXCL8 (100 ng/mL) from a micropipette. Fluorescence time-lapse images were taken at 30-s intervals for 7 min. The direction of chemoattractant source is indicated by an arrow. Original scale bars, 5 μm (also see Supplemental Videos 9 and 10). Fluorescence images were captured using a Zeiss Axiovert 200 fluorescence microscope and a 40× objective, NA 1.3, with ORCA-ER C4742-95 camera-driven MetaMorph imaging software (Universal Imaging).

Figure 4.. GMFG knockdown induces temporal instability of the leading edge.

Time-lapse sequential images showing the distribution of GFP-actin in negative-control, siRNA- or GMFG siRNA-expressing neutrophils or dHL-60 cells during chemotaxis. GFP-actin-expressing neutrophils or dHL-60 cells cotransfected with control siRNA or GMFG siRNA were plated on glass-bottom dishes coated with fibrinogen and exposed to a chemotactic gradient generated by the slow release of fMLF (100 nM; A) or CXCL8 (100 ng/mL; B) from a micropipette. Fluorescence time-lapse images were recorded in a confocal microscope at 20-s intervals for 3 min. The direction of chemoattractant source is indicated by arrows. Original scale bars, 5 μm.

Figure 5.. GMFG regulates p38 and PAK phosphorylation and interacts with WAVE2 in dHL-60 cells.

(A and B) Immunoblot analysis of the phosphorylation (p) of PAK1/2, p38, and ERK1/2 at the indicated times after fMLF (100 nM) or CXCL8 (100 ng/mL) stimulation of dHL-60 cells expressing negative-control siRNA or GMFG siRNA. Equal amounts of total cellular lysates were compared using total p38 antibody (T-p38). (C) dHL-60 cells were mock-transfected or transiently transfected with His-tagged GMFG plasmid or empty vector for 24 h and then lysed. Cell lysates were subjected to immunoblot analysis using antibodies against phosphorylated p38. Equal amounts of total cellular lysates were compared using total p38 antibody. (D) Lysates prepared from dHL-60 cells transfected with Myc-tagged GMFG (+) or empty vector (–) were immunoprecipitated (IP) with anti-Myc antibody or IgG control, and then the immunoprecipitated proteins were subjected to immunoblot analysis with anti-WAVE2 antibody. The same blot was stripped and reprobed with anti-Myc antibody. All figures are representative of at least three independent analyses.