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

GRP Induces Neutrophil Migration in Vivo.

It has been previously shown that GRPR antagonist RC-3095 has antiinflammatory activity in animal models of inflammation (9, 10, 16). We hypothesized that GRP could have proinflammatory potential, so we tested whether GRP would have a dose-dependent effect on neutrophil recruitment in vivo. We performed a kinetic analysis, looking at different time points after GRP injection. I.p. injection of human GRP induced neutrophil recruitment after 4 h in a dose-dependent fashion, the highest numbers being obtained with 0.6 μg per cavity (Fig. 1A). Pretreatment of mice with RC-3095 immediately before injection of GRP abolished neutrophil accumulation in the peritoneum (Fig. 1B), indicating that GRP-induced neutrophil migration occurs specifically through activation of GRPR. The chemoattractant effect of GRP in vivo was restricted to neutrophils, because no recruitment of macrophages or B or T cells was observed in any of the time points, from 1.5 to 72 h (Fig. 1 C and D), as assessed by both Diff-Quick staining and flow cytometry. In our hands, a peritoneal lavage of an unmanipulated mouse yielded between 5 and 10 million cells, and injection of GRP did not significantly alter total numbers of cells. The absolute numbers of neutrophils migrating to the peritoneum after GRP i.p. were thus comparable between both methods. Collectively, these results indicate that GRP has a selective effect over neutrophils in vivo, inducing their migration through activation of GRPR.

GRP-Induced Neutrophil Recruitment in Vivo Depends on Macrophages and TNF-α Production.

Neutrophil migration to sites of inflammation in vivo is mediated by the release of cytokines and chemokines by resident cells. We decided to investigate the role of macrophages on neutrophil migration induced by GRP in vivo. We performed macrophage depletion by i.p. injection of chlodronate liposomes in mice, later injecting GRP or saline i.p. Depletion of macrophages almost completely inhibited GRP-induced neutrophil migration (Fig. 2A), suggesting that macrophages play a role in neutrophil recruitment to the peritoneum in response to GRP injection.

TNF-α is one of the major cytokines released by macrophages in response to inflammatory stimuli, enhancing neutrophil migration by increasing the expression of adhesion molecules on endothelial cells and neutrophils, facilitating cell arrival to inflamed sites (17). To investigate the role of TNF-α in our system, we pretreated mice with a therapeutic TNF-α–specific neutralizing antibody (infliximab), a chimeric (mouse/human) antibody that cross-reacts with murine TNF-α (18). Mice were treated with infliximab or human IgG, immediately before GRP injection, and blocking of TNF-α significantly inhibited GRP-induced neutrophil recruitment to the peritoneum (Fig. 2B). To determine whether GRP could induce the production of proinflammatory cytokines, we performed cytokine analysis (IL-4, IL-6, IL-10, IL-17A, INF-γ, and TNF-α) in response to GRP stimulation of murine peritoneal macrophages and human monocytes. Twenty-four hours after stimulation with 1 nM GRP (19, 20), none of these mediators were induced (Fig. 2C). An alternative explanation for our observations was that GRP induced chemokine production by macrophages, which in turn could up-regulate TNF-α production in vivo, enhancing GRP-induced neutrophil recruitment. Chemokines peak earlier than some cytokines (17) and it was possible that a dose other than 1 nM was necessary to induce these mediators in macrophages/monocytes, so we used a range of concentrations of GRP for stimulation (10–0.001 nM), for 2 h. Supernatants were analyzed for TNF-α, KC/IL-8, MIP-2, and MCP-1 production. Results shown in Fig. 2D reveal that in 2 h, GRP induces TNF-α in murine macrophages, at 0.1 nM, and MCP1 in human monocytes, at 10 nM. Together, these results suggest that in vivo neutrophil recruitment through GRPR depends on macrophage presence and TNF-α production, and that TNF/chemokine production by macrophages/monocytes can be triggered by GRP.

GRP Has a Direct Chemoattractant Effect on Neutrophils.

It has recently been demonstrated that neutrophils express GRPR (12). Chemokines (17) and leukotrienes (21) and molecules released by damaged tissues (22, 23) act as chemoattractants, acting directly on neutrophils to induce migration. We investigated whether GRP, a neuropeptide, would induce neutrophils to migrate up a gradient of GRP in vitro, in a Transwell system. Nanomolar amounts of GRP induce neutrophil migration in a dose-dependent manner (Fig. 3A) in a bell-shaped curve. These results reflect previous studies on macrophages (19), lymphocytes (20), and mast cells (24), showing that GRP modulates immune cell function in a nanomolar range. To investigate the effects exerted by RC-3095 over migration, we performed a dose–response curve of this inhibitor using the optimal dose of GRP determined previously. We observed that RC-3095 does not inhibit GRP-induced migration while in equivalent nanomolar amounts (Fig. 3B). However, a 10-fold increase of the inhibitor blocks the effect of GRP on neutrophil migration, and a 20-fold increase completely abolishes it.

To exclude the possibility that GRP was simply inducing chemokinesis, and not chemotaxis, we performed a checkerboard analysis. For this, the optimal concentration of GRP (1 nM) was placed in the upper wells, lower wells, or both upper and lower wells of the Transwell chamber (25). GRP was only able to induce influx of neutrophils when it was added to the lower wells, confirming chemotaxis rather than chemokinesis (Fig. 3C).

Neutrophil Migration Induced by GRP Is Independent of Gαi Proteins, but Dependent on PLC-β, PI3K, and MAPK.

The classical pathway of neutrophil migration involves triggering GPCR receptors, which release Gβγ subunits from Gαi proteins, activating specific signaling pathways (26). However, GRPR preferentially couples to Gαq proteins (27), which have not been identified with neutrophil migration. Given the complexity of GPCR biology, we hypothesized that GRPR could associate with Gαi protein to induce migration. To investigate this, we pretreated neutrophils with pertussis toxin (PTx), which irreversibly inhibits Gαi proteins (28) and allowed cells to migrate toward GRP or leukotriene B₄ (LTB₄). Pretreating neutrophils with PTx abrogated their migration to LTB₄ but did not affect migration toward GRP (Fig. 4A), suggesting that Gαi proteins are not involved in neutrophil chemotaxis induced by GRP.

Because GRPR is coupled to Gαq protein, and triggering Gαq-coupled receptors leads to activation of phospholipase C-β (PLC-β) (29), we investigated its involvement on GRP-induced migration. The pretreatment of cells with a selective inhibitor of PLC-β, U-73122, abolished neutrophil migration toward GRP (Fig. 4B), indicating that GRP activates PLC-β to induce chemotaxis.

Neutrophil migration induced by GPCR ligation is known to be dependent on PI3K and MAPK signaling routes (30). To determine downstream signaling pathways activated in GRP-induced neutrophil migration, we pretreated the cells with selective inhibitors of PI3K (LY29004), as well as of ERK 1/2 (PD98059) and p38 MAPK (SB203580). Blocking these pathways completely inhibited recruitment toward GRP (Fig. 4C), indicating that GRP activates PI3K and MAPK, similarly to other chemoattractant molecules (31). We also investigated whether treatment of neutrophils with GRP would activate these signaling pathways, analyzing phosphorylation of MAPKs and AKT (as a reading of PI3K activation) (32). Results showed in Fig. 4D support the findings that GRP rapidly signals through GRPR, activating these pathways, which promote neutrophil chemotaxis.

Neutrophil Migration Toward Synovial Fluid (SF) of Rheumatoid Arthritis (RA) Patients Can Be Blocked by RC-3095.

Previous studies revealed an antiinflammatory effect of RC-3095 in murine arthritis (9) and sepsis models (10). This effect was attributed to the inhibition of IL-1β and TNF-α production. Other studies reported an increase of GRP concentrations in SF of arthritis patients, correlating with severity of disease (33, 34).

We hypothesized that at least part of the effect of RC-3095 in arthritis could be explained by an inhibition of neutrophil recruitment to the arthritic joint. We analyzed neutrophil migration toward SF from RA patients. The concentrations of GRP in samples of SF from patients varied between 0.8 and 1.8 ng/mL in our samples (n = 6) (Fig. 5B). Analysis in the Transwell system indicated that SF induced neutrophil migration, comparably to GRP alone (Fig. 5A). This migration was abrogated by RC-3095, indicating that GRP in the SF can activate GRPR and trigger neutrophil chemotaxis. Interestingly, the inhibition of neutrophil migration by RC-3095 was comparable to that observed with pretreatment of neutrophils with a CXCR2 antagonist (SB225002). The concentration of IL-8 in SF samples of RA was greater than 300 pg/mL (311.12 ± 3.45 pg/mL). To exclude the possibility that GRP in SF could be binding directly to CXCR2, we allowed neutrophils pretreated with SB225002 to migrate toward GRP. This pretreatment did not inhibit GRP-induced neutrophil migration (Fig. 5C), suggesting the effect of GRP was restricted to GRPR binding.

Mice and Patients.

C57BL/6 females, 6–8 wk old, were purchased from FEPPS and housed in accordance with National Institutes of Health Guidelines. Synovial fluid from RA patients were provided by the Rheumatology Service of São Lucas Hospital (PUCRS). All patients signed an informed consent term approved under Ethics protocol no. 858/05-CEP. This study was approved by the Ethics committee at PUCRS under protocol no. CEUA 10/00186.

Human Neutrophil Isolation and Chemotaxis Assay.

Neutrophils were isolated from heparin-treated venous blood of healthy human volunteers using Histopaque-1077 (Sigma-Aldrich). Neutrophil chemotaxis was assayed in a Transwell system (Corning). Stimuli was added to the bottom wells, and neutrophils were added to top wells and incubated for 2 h at 37 °C. For checkerboard analysis, neutrophils were placed in the upper chamber alone, or with GRP (1 nM), and exposed in the lower chamber with medium alone or GRP (1 nM). Involvement of PLC-β, PI3K, MAPK (p38 and ERK), GαI, and CXCR2 was tested by pretreating neutrophils with selective inhibitors at 37 °C for 1 h.

In Vivo Neutrophil Migration Assay.

For neutrophil migration, GRP (0.06–6 μg per cavity) and/or RC-3095 (6 μg per cavity) or endotoxin-free saline were injected i.p., in 200 μL. After 4 h, peritoneal cavities were rinsed with 3 mL PBS. Differential counting of leukocytes was carried out on Diff-Quick-stained slides.

Murine Macrophage and Human Monocyte Purification.

Peritoneal macrophages were obtained by peritoneal and adherent cells stimulated with GRP (0.001–10 nM) for 2 or 24 h. Human monocytes were isolated from peripheral blood mononuclear cells obtained by density gradient centrifugation with Histopaque-1077, and adherent cells were stimulated with GRP for 2 or 24 h. Supernatants were collected and frozen until cytokine/chemokine analysis.

Flow Cytometry.

Peritoneal cells were stained with anti-CD14 PE, CD11c PE-Cy7, CD4 FITC, and B220 PE-Cy5.5. Murine macrophages and human monocytes were stimulated with GRP for 24 h and culture supernatants analyzed for cytokines by Th1/Th2/Th17 Cytometric Bead Array (BD Biosciences), according to the manufacturer's instructions.

p-Akt, p-p38, and p-ERK 1/2 in human neutrophils were measured by flow cytometry using BD Phosflow protocol for human whole blood sample. Data were obtained using FACSCantoII (Beckton Dickinson) and BD FACSDiva software, and analyzed using Flowjo v 7.5.

ELISA.

GRP (Phoenix Pharmaceuticals), TNF, and chemokines were determined according to the supplier's instructions.

Statistical Analysis.

Data are presented as mean ± SE. Results were analyzed using GraphPad Prism 5. Statistical differences among the experimental groups were evaluated by ANOVA with Tukey or Bonferroni correction or with Student's t test. The level of significance was set at P < 0.05.