Regulator of G protein signaling 5 restricts neutrophil chemotaxis and trafficking

Abstract

Neutrophils are white blood cells that are mobilized to damaged tissues and to sites of pathogen invasion, providing the first line of host defense. Chemokines displayed on the surface of blood vessels promote migration of neutrophils to these sites, and tissue- and pathogen-derived chemoattractant signals, including N-formylmethionylleucylphenylalanine (fMLP), elicit further migration to sites of infection. Although nearly all chemoattractant receptors use heterotrimeric G proteins to transmit signals, many of the mechanisms lying downstream of chemoattractant receptors that either promote or limit neutrophil motility are incompletely defined. Here, we show that regulator of G protein signaling 5 (RGS5), a protein that modulates G protein activity, is expressed in both human and murine neutrophils. We detected significantly more neutrophils in the airways of Rgs5^-/- mice than WT counterparts following acute respiratory virus infection and in the peritoneum in response to injection of thioglycollate, a biochemical proinflammatory stimulus. RGS5-deficient neutrophils responded with increased chemotaxis elicited by the chemokines CXC motif chemokine ligand 1 (CXCL1), CXCL2, and CXCL12 but not fMLP. Moreover, adhesion of these cells was increased in the presence of both CXCL2 and fMLP. In summary, our results indicate that RGS5 deficiency increases chemotaxis and adhesion, leading to more efficient neutrophil mobilization to inflamed tissues in mice. These findings suggest that RGS5 expression and activity in neutrophils determine their migrational patterns in the complex microenvironments characteristic of inflamed tissues.

Keywords: G protein–coupled receptor; GPCR; RGS; chemotaxis; inflammation; innate immunity; leukocyte; neutrophil; regulator of G protein signaling; trafficking.

Figure 1.

Immunoreactive RGS5 is detected in mouse neutrophils. A, neutrophils were isolated from bone marrow of naïve mice using Ly6G microbeads. Flow cytometry was used to assess purity based on CD11b expression. Neutrophils were dispersed by cytospin and identified by modified Giemsa staining. The plot is from a single experiment representative of three experiments (purity of 93.2 ± 3%). B, BM-derived neutrophils were left untreated (NT) or stimulated with CXCL1 or CXCL2 (100 ng/ml) or fMLP (1 μm) for 2 h followed by cell lysis and immunoblotting. The bar graph shows the relative RGS5 expression (normalized by β-actin signal; error bars indicate mean ± S.E.) of three to five independent experiments using one to two mice/experiment. *, p = 0.04, one-way ANOVA, Dunnett's post hoc test versus control (unstimulated condition). C, CXCL2-treated neutrophils from WT or Rgs5^−/− mice were stained with anti-RGS5 (grayscale in first two images on the left and red in the color images) and counterstained with DAPI to identify nuclei (blue). The bar graph shows the percentage of cells/field containing immunoreactive RGS5 (error bars indicate mean ± S.E. of >3 fields/experiment, each containing >10 cells/field) analyzed in three independent experiments using one mouse of each genotype/experiment. ****, p < 0.0001, unpaired t test. Scale bar, 6 μm.

Figure 2.

Hematopoiesis and neutrophil localization at homeostasis in WT and Rgs5^−/− mice. A–D, serum hemoglobin (Hgb; A), platelet (B), total leukocyte (WBC; C), and differential leukocyte (D) counts were determined from peripheral blood. Error bars indicate mean ± S.E. A–C, *, p = 0.01, t test; n.s., not significant. D, **, p = 0.001; ***, p = 0.0008, two-way ANOVA, Sidak multiple comparisons. E–G, neutrophil numbers were determined in BM (tibia and femur of both lower extremities) (E), spleen (F), or lungs (G) by flow cytometry–based assessment of percentages of Ly6G^hiCD11b⁺ cells, respectively, as shown in the representative plot from bone marrow. All results are mean ± S.E.; each symbol represents an individual mouse.

Figure 3.

RGS5 deficiency results increased in airway neutrophils in acute respiratory virus infection. A, hematoxylin and eosin–stained lung sections from WT or Rgs5^−/− mice at baseline (naïve) and 5 days after inoculation with PVM. Images are from a single mouse representative of three to four mice/group. Inset, diffuse neutrophilic alveolitis characteristic of acute PVM infection (40× magnification); arrows denote neutrophils. B, virus recovery was assessed by qPCR detection of virus-specific SH gene. C, chemokines CCL3 and CXCL1 in BALF of PVM-infected WT and Rgs5^−/− mice. D–G, neutrophils in lung (D), airways (E), bone marrow (F), peripheral blood (G), or spleens (H) at day 5 of PVM infection. *, p = 0.02; **, p = 0.002; ***, p = 0.0007, Mann–Whitney; n.s., not significant. All error bars indicate mean ± S.E.; each symbol represents one mouse.

Figure 4.

Neutrophils from RGS5-deficient mice are intrinsically capable of enhanced migration. A, mice were injected i.p. with 4% sodium thioglycollate. At the indicated time points, total cells (left) and neutrophils (right) were determined by flow cytometry as in Figs. 1 and 2. Error bars indicate mean ± S.E. of six to eight mice/genotype evaluated in three to four experiments. *, p = 0.02; **, p = 0.004, two-way ANOVA, Sidak multiple comparisons; n.s., not significant. B, chemokine CXCL1 levels were evaluated in peritoneal fluid following TG injection. Results are mean ± S.E.; each symbol represents one mouse. C, surface CXCR2 in TG-elicited peritoneal neutrophils determined by flow cytometry using the antibodies as indicated (IgG2a, isotype control). Histograms are from a single mouse representative of four mice/group assessed in two to three independent experiments. D, BM-derived neutrophils were stained with violet (Rgs5^−/−) or green (WT) fluorophores. Cells were mixed at a 1:1 ratio and injected i.v. into mice previously administered TG. Numbers of cells of each genotype were determined by flow cytometry. Error bars indicate mean ± S.E. of cells from four mice/group evaluated in two independent experiments. ***, p = 0.0005, two-way ANOVA, Sidak multiple comparisons.

Figure 5.

Increased chemotaxis of RGS5-deficent neutrophils. A, chemotaxis of BM-derived neutrophils was assessed in Transwell assays as described under “Experimental procedures.” Error bars indicate mean ± S.E. of four to six experiments using cells from one to two mice of each genotype/experiment. *, p < 0.02; **, p < 0.006, two-way ANOVA, Sidak multiple comparisons. B, surface CXCR2 or CXCR4 in untreated BM-derived neutrophils was assessed by flow cytometry. Histograms represent a single mouse/genotype representative of two to three mice evaluated in separate experiments; the shaded peak represents cells stained with isotype control antibody. C, surface CXCR2 expression in BM-derived neutrophils pre- and poststimulation with CXCL2 (100 ng/ml) for the indicated times. The bar graph is CXCR2 geometric mean fluorescence intensity (GFMI) after treatment with CXCL2 for 15 min as a percentage of that measured in untreated cells (error bars indicate mean ± S.E. of four independent experiments using cells from one mouse of each genotype/experiment). n.s., not significant.

Figure 6.

RGS5 negatively regulates chemokine-induced Ca²⁺ signaling and Akt phosphorylation in neutrophils. A, Akt phosphorylation (pAkt) in BM-derived neutrophils was evaluated by immunoblotting. The blot is from a single experiment representative of seven similar experiments. Error bars indicate mean ± S.E. *, p = 0.03, Mann–Whitney. B–D, Ca²⁺ flux in response to CXCL1 (B), CXCL2 (C), or ionomycin (D). Bar graphs represent area under the curve (error bars are mean ± S.E.) of five to eight independent measurements in four separate experiments each using cells from one mouse of each genotype. *, p = 0.04, Mann–Whitney. The arrow represents time of agonist addition. n.s., not significant.

Figure 7.

RGS5-deficient neutrophils demonstrate increased chemokine-induced directional velocity and adhesion to endothelial cells. A and B, migrational velocity and directional error of BM-derived neutrophils were assessed in Dunn chambers in the presence of CXCL2 (10 μm; A) or fMLP (100 nm or 5 μm; B). Error bars indicate mean ± S.E. of three independent experiments using cells from one mouse/genotype and three to five separate chambers/condition in which 50–110 cells/chamber were individually tracked. ****, p < 0.00001, unpaired t test; **, p = 0.009, Mann–Whitney. C, neutrophil adhesion was assessed by binding to immobilized ICAM-1 as described under “Experimental procedures” in the presence of CXCL2 (10 μm) or fMLP (5 μm) in three independent experiments using cells from one mouse of each genotype. ***, p < 0.0005, one-way ANOVA, Sidak multiple comparisons. Error bars indicate mean ± S.E. D, EC adhesion was assessed in flow chambers as described under “Experimental procedures.” Error bars indicate mean ± S.E. of four independent experiments using cells from one mouse of each genotype. ****, p < 0.00001, unpaired t test. IL-8, interleukin-8.

Figure 8.

RGS5 overexpression inhibits chemotaxis of human neutrophils. A, human peripheral blood neutrophils were isolated and transduced with tat fusion proteins as described under “Experimental procedures.” Cell lysates were prepared, and intracellular expression of tat GFP fusion proteins was assessed by immunoblotting. B, chemotaxis of human neutrophils transduced with tat fusion proteins was assessed in Transwell assays. Error bars indicate mean ± S.E. of three independent experiments using neutrophils from one to two donors/experiment. **, p < 0.008, two-way ANOVA, Sidak multiple comparisons.