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. 2012 Jan 10;109(2):547-52.
doi: 10.1073/pnas.1110996109. Epub 2011 Dec 27.

Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils

Rafael Sanguinetti Czepielewski  1 , Bárbara Nery Porto, Lucas Bortolotto Rizzo, Rafael Roesler, Ana Lúcia Abujamra, Larissa Garcia Pinto, Gilberto Schwartsmann, Fernando de Queiroz Cunha, Cristina Bonorino
Affiliations

Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils

Rafael Sanguinetti Czepielewski et al. Proc Natl Acad Sci U S A. .

Abstract

Neutrophil migration to inflamed sites is crucial for both the initiation of inflammation and resolution of infection, yet these cells are involved in perpetuation of different chronic inflammatory diseases. Gastrin-releasing peptide (GRP) is a neuropeptide that acts through G protein coupled receptors (GPCRs) involved in signal transmission in both central and peripheral nervous systems. Its receptor, gastrin-releasing peptide receptor (GRPR), is expressed by various cell types, and it is overexpressed in cancer cells. RC-3095 is a selective GRPR antagonist, recently found to have antiinflammatory properties in arthritis and sepsis models. Here we demonstrate that i.p. injection of GRP attracts neutrophils in 4 h, and attraction is blocked by RC-3095. Macrophage depletion or neutralization of TNF abrogates GRP-induced neutrophil recruitment to the peritoneum. In vitro, GRP-induced neutrophil migration was dependent on PLC-β2, PI3K, ERK, p38 and independent of Gαi protein, and neutrophil migration toward synovial fluid of arthritis patients was inhibited by treatment with RC-3095. We propose that GRPR is an alternative chemotactic receptor that may play a role in the pathogenesis of inflammatory disorders.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
GRP induces neutrophil recruitment to the peritoneal cavity of mice. Mice were injected i.p. with (A) different doses of GRP (0.06–6 μg/cavity) and recovered cells were counted after 4 h, **P < 0.01 compared with saline-treated group; (B) GRP (0.6 μg/cavity), RC-3095 (6 μg/cavity), or RC-3095 (6 μg/cavity) + GRP (0.6 μg/cavity) and control group, saline, ***P < 0.001 compared with GRP-injected group; and (C) GRP (0.6 μg/cavity). After 1.5, 4, 8, 16, 24, 48, and 72 h, animals were killed, cells were cytocentrifuged, stained with Diff-Quick, and counted. Filled circles, neutrophils (Nφ) in saline groups; open circles, GRP-treated groups. Filled squares, mononuclear cells (MN) in controls; open squares, mononuclear cells counted in GRP-treated groups. ***P < 0.001 compared with saline-injected group; (D) GRP (0.6 μg/cavity). After 1.5, 4, 8, 16, 24, 48, and 72 h, cells in the peritoneal fluid were analyzed by FACS. Gates 1 to 4 were determined on the basis of FSC × SSC distribution and staining with anti-CD14, CD11c, CD4, and B220. G1 = lymphocytes; G2 = larger lymphocytes and DCs; G3 = macrophages; G4 = neutrophils. Filled forms, saline groups; open forms, GRP-treated groups. Data representative of four independent experiments (n = 4 for each group of treatment) and expressed as the mean ± SE of the percentage or number of cells.
Fig. 2.
Fig. 2.
GRP-induced neutrophil recruitment in vivo depends on macrophages and TNF-α production. (A) Mice were injected i.p. with chlodronate liposomes (500 μg per cavity) (open bars) or saline (filled bars), 24 h before GRP (0.6 μg per cavity) or RC-3095 (6 μg per cavity) + GRP (0.6 μg per cavity) injection. After 4 h of i.p. GRP with/without RC-3095 injection, differential counts in the peritoneal fluid were determined. ***P < 0.001 compared with GRP-injected group. (B) Mice were pretreated with infliximab or IgG control before i.p. injection of GRP (0.6 μg per cavity). After 4 h, differential counts in the peritoneal fluid were determined. (C) Murine peritoneal macrophages or human monocytes were isolated, stimulated with GRP (1 nM) or medium, and 24 h later the supernatant was analyzed for cytokines by cytometric bead array. Open bars, control; filled bars, GRP-treated cells; ND, not detectable. (D) Peritoneal macrophages or human monocytes were stimulated with GRP (1 nM) or medium alone, and 2 h later the supernatant was harvested and analyzed for TNF or chemokine production by ELISA. Data are representative of three independent experiments, performed in triplicate for each sample, and expressed as mean ± SE.
Fig. 3.
Fig. 3.
GRP has a direct chemoattractant effect on neutrophils. (A) Human neutrophils were allowed to migrate toward GRP (0.001–10 nM) with 2% FCS for 2 h. **P < 0.01 and ***P < 0.001 compared with negative control. (B) Neutrophils were preincubated with RC-3095 (1–20 nM) for 1 h at 37 °C under 5% CO2 and stimulated to migrate toward GRP (1 nM) for 2 h at 37 °C under 5% CO2. (C) Neutrophils were placed in the upper chamber alone, or together with GRP (1 nM), and exposed in the lower chamber to medium alone or GRP (1 nM) with 2% FCS for 2 h. ***P < 0.001 compared with negative control and ###P < 0.001 compared with GRP-treated group. Data are representative of four independent experiments, performed in triplicate for each sample, and expressed as mean ± SE.
Fig. 4.
Fig. 4.
GRP induces neutrophil migration independently of Gαi protein and dependently on PLC-β, PI3K, and MAPK. Neutrophils were preincubated with: (A) Pertussis toxin 100 ng/mL for 2 h; (B) U-73122 1 μM (PLC-β inhibitor); (C) LY294002 50 μM (PI3K inhibitor), SB203580 10 μM (p38 inhibitor), or PD98059 30 μM (ERK inhibitor) for 1 h, and stimulated to migrate toward GRP (1 nM) or LTB4 (1 nM) for 2 h. (D) Neutrophils were stimulated with GRP (1 nM) for 5 min and stained for phosphorylated proteins (AKT, p38, and ERK 1/2). Filled histograms are control neutrophils and black solid lines are GRP-stimulated neutrophils. Phosphorylation of protein pathways are presented as fold increase relative to unstimulated neutrophils (Left column). Data are representative of three independent experiments, performed in triplicate for each sample, and expressed as mean ± SE. ***P < 0.001 compared with negative control and ###P < 0.001 compared with LTB4- or GRP-treated group.
Fig. 5.
Fig. 5.
Synovial fluid-induced migration of neutrophils is partially inhibited by RC-3095. (A) Human neutrophils were preincubated with RC-3095 (10 nM) or SB225002, a CXCR2 antagonist (300 nM), for 1 h and allowed to migrate toward GRP (1 nM) or synovial fluid (SF) of rheumatoid arthritis (RA) patients. (B) Samples of RA patients’ SF were assayed for the presence of GRP by ELISA (n = 6). (C) Neutrophils were preincubated with SB225002 (300 nM) for 1 h at 37 °C under 5% CO2 and stimulated to migrate toward GRP (1 nM) for 2 h. Data are representative of three independent experiments, performed in triplicate for each sample, and expressed as mean ± SE. ***P < 0.001 compared with negative control; ###P < 0.001 compared with SF-treated group.
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