Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils Rafael Sanguinetti Czepielewskia,b,1, Bárbara Nery Portoa,1, Lucas Bortolotto Rizzoa, Rafael Roeslerc,d,e, Ana Lúcia Abujamrac,d,e, Larissa Garcia Pintof, Gilberto Schwartsmannd,e,g, Fernando de Queiroz Cunhaf, and Cristina Bonorinoa,b,2 aLaboratory of Cellular and Molecular Immunology, Biomedical Research Institute (IPB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), 90610-900, Porto Alegre, RS, Brazil; bDepartment of Cellular and Molecular Biology (FABIO), PUCRS, 90619-900, Porto Alegre, RS, Brazil; cLaboratory of Neuropharmacology and Neural Tumor Biology, Department of Pharmacology, Institute for Basic Health Sciences, Federal University of Rio Grande do Sul, 90050-170, Porto Alegre, RS, Brazil; dCancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003, Porto Alegre, RS, Brazil; eNational Institute for Translational Medicine, Porto Alegre, RS, Brazil; gDepartment of Internal Medicine, School of Medicine, Federal University of Rio Grande do Sul, 90035-003, Porto Alegre, Brazil; and fDepartment of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, São Paulo, Brazil Edited* by Philippa Marrack, Howard Hughes Medical Institute, National Jewish Health, Denver, CO, and approved November 30, 2011 (received for review July 13, 2011) Neutrophil migration to inflamed sites is crucial for both the we report that GRP can be an endogenous inflammatory media- initiation of inflammation and resolution of infection, yet these tor, acting as a chemoattractant through GRPR. In addition, it cells are involved in perpetuation of different chronic inflammatory activates specific signaling pathways that promote neutrophil mi- diseases. Gastrin-releasing peptide (GRP) is a neuropeptide that acts gration. We propose that GRP triggers neutrophil recruitment through G protein coupled receptors (GPCRs) involved in signal both indirectly, through macrophages, as well as directly, binding transmission in both central and peripheral nervous systems. Its to GRPR in these cells. receptor, gastrin-releasing peptide receptor (GRPR), is expressed by various cell types, and it is overexpressed in cancer cells. RC-3095 is Results a selective GRPR antagonist, recently found to have antiinflamma- GRP Induces Neutrophil Migration in Vivo. It has been previously tory properties in arthritis and sepsis models. Here we demonstrate shown that GRPR antagonist RC-3095 has antiinflammatory ac- that i.p. injection of GRP attracts neutrophils in 4 h, and attraction is tivity in animal models of inflammation (9, 10, 16). We hypothe- fl blocked by RC-3095. Macrophage depletion or neutralization of TNF sized that GRP could have proin ammatory potential, so we abrogates GRP-induced neutrophil recruitment to the peritoneum. tested whether GRP would have a dose-dependent effect on In vitro, GRP-induced neutrophil migration was dependent on PLC- neutrophil recruitment in vivo. We performed a kinetic analysis, β2, PI3K, ERK, p38 and independent of Gαi protein, and neutrophil looking at different time points after GRP injection. I.p. injection fl of human GRP induced neutrophil recruitment after 4 h in a dose- migration toward synovial uid of arthritis patients was inhibited μ by treatment with RC-3095. We propose that GRPR is an alternative dependent fashion, the highest numbers being obtained with 0.6 g A IMMUNOLOGY chemotactic receptor that may play a role in the pathogenesis of per cavity (Fig. 1 ). Pretreatment of mice with RC-3095 imme- inflammatory disorders. diately before injection of GRP abolished neutrophil accumula- tion in the peritoneum (Fig. 1B), indicating that GRP-induced neutrophil migration occurs specifically through activation of europeptides are used by neurons as signaling molecules to GRPR. The chemoattractant effect of GRP in vivo was restricted Nregulate synaptic transmission and plasticity (1). Nonetheless, to neutrophils, because no recruitment of macrophages or B or T these molecules can be versatile, also acting as chemical messengers cells was observed in any of the time points, from 1.5 to 72 h (Fig. 1 outside the nervous system. Recent reports showed that neuro- C and D), as assessed by both Diff-Quick staining and flow peptides are produced as a result of immune pathologies (2), whereas cytometry. In our hands, a peritoneal lavage of an unmanipulated others appear to induce cytokine production by immune cells (3). mouse yielded between 5 and 10 million cells, and injection of Gastrin-releasing peptide (GRP) is a neuropeptide that induces GRP did not significantly alter total numbers of cells. The absolute gastrin secretion in the gastric tract (4). It acts by binding to the numbers of neutrophils migrating to the peritoneum after GRP i.p. gastrin-releasing peptide receptor (GRPR or BB2), a member of were thus comparable between both methods. Collectively, these the G protein coupled receptor (GPCR) superfamily expressed in results indicate that GRP has a selective effect over neutrophils the gastric, respiratory, and nervous systems, as well as endocrine glands and muscle (5). GRPR mediates gastrointestinal motility in vivo, inducing their migration through activation of GRPR. and hormone and neurotransmitter release in the gut, intestine, GRP-Induced Neutrophil Recruitment in Vivo Depends on Macrophages colon, and other organs (6). It has roles in the nervous system, α fl controlling the circadian cycle, anxiety, fear, stress, and modulation and TNF- Production. Neutrophil migration to sites of in amma- of memory (7). It is overexpressed in cancer cells, and the pro- tion in vivo is mediated by the release of cytokines and chemo- duction of GRP together with GRPR overexpression results in kines by resident cells. We decided to investigate the role of autocrine growth stimulation (6). Selective GRPR antagonists were macrophages on neutrophil migration induced by GRP in vivo. produced as candidate anticancer drugs, including RC-3095 (8). We performed macrophage depletion by i.p. injection of More recently, RC-3095 has been demonstrated to have anti- inflammatory effects in arthritis (9) and sepsis (10, 11) models, down-regulating the production of proinflammatory cytokines IL- Author contributions: R.S.C., B.N.P., A.L.A., and C.B. designed research; R.S.C., B.N.P., L.B.R., 1β, IL-6, and TNF-α. Interestingly, GRPR has been found to be and L.G.P. performed research; B.N.P., R.R., A.L.A., G.S., and F.d.Q.C. contributed new re- expressed in immune cells (12). Inflammation is a protective im- agents/analytic tools; R.S.C., B.N.P., R.R., L.G.P., G.S., F.d.Q.C., and C.B. analyzed data; and R.S.C., B.N.P., and C.B. wrote the paper. mune response initiated by exposure of innate immune cells to fl molecular patterns that signal infection or injury (13), and the The authors declare no con ict of interest. migration of neutrophils to sites of inflammation can promote *This Direct Submission article had a prearranged editor. tissue damage (14), although it is also critical for healing of the 1R.S.C. and B.N.P. contributed equally to this work. affected areas (15). 2To whom correspondence should be addressed. E-mail: [email protected]. The mechanisms underlying the actions of GRPR-binding drugs This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in inflammatory scenarios have not been elucidated. In this study, 1073/pnas.1110996109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1110996109 PNAS | January 10, 2012 | vol. 109 | no. 2 | 547–552 Downloaded by guest on September 27, 2021 Fig. 1. GRP induces neutrophil re- cruitment 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(0.6μg/cavity), RC- 3095 (6 μg/cavity), or RC-3095 (6 μg/cav- ity) + 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 cytocentri- fuged, stained with Diff-Quick, and coun- ted. 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(0.6μg/cavity). After 1.5, 4, 8, 16, 24, 48, and 72 h, cells in the perito- neal 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 = lym- phocytes; 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 per- centage or number of cells. chlodronate liposomes in mice, later injecting GRP or saline i.p. GRP Has a Direct Chemoattractant Effect on Neutrophils. It has re- Depletion of macrophages almost completely inhibited GRP-in- cently been demonstrated that neutrophils express GRPR (12). duced neutrophil migration (Fig. 2A), suggesting that macro- Chemokines (17) and leukotrienes (21) and molecules released by phages play a role in neutrophil recruitment to the peritoneum in damaged tissues (22, 23) act as chemoattractants, acting directly response to GRP injection. on neutrophils to induce migration. We investigated whether TNF-α is one of the major cytokines released by macrophages in GRP, a neuropeptide, would induce neutrophils to migrate up response to inflammatory stimuli, enhancing neutrophil migration a gradient of GRP in vitro, in a Transwell system. Nanomolar by increasing the expression of adhesion molecules on endothelial amounts of GRP induce neutrophil migration in a dose-depen- cells and neutrophils, facilitating cell arrival to inflamed sites (17).