BRIEF DEFINITIVE REPORT The neuropeptide neuromedin U promotes inflammation by direct activation of mast cells Maiko Moriyama,1,2 Takahiro Sato,1 Hiromasa Inoue,3 Satoru Fukuyama,3,4 Hitoshi Teranishi,1 Kenji Kangawa,5 Tatsuhiko Kano,2 Akihiko Yoshimura,4 and Masayasu Kojima1 1Department of Molecular Genetics, Institute of Life Science, Kurume University, Fukuoka, 839-0864, Japan 2Department of Anesthesiology, Kurume University School of Medicine, Fukuoka, 830-0011, Japan 3Research Institute for Diseases of the Chest, Graduate School of Medical Sciences and 4Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan 5Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka, 565-8565, Japan Neuromedin U (NMU) is a neuropeptide that is expressed in the gastrointestinal tract and central nervous system. NMU interacts with two G protein–coupled receptors, NMU-R1 and NMU-R2. Whereas NMU-R2 localizes predominantly to nerve cells, NMU-R1 is expressed in peripheral tissues including lymphocytes and monocytes, suggesting a role of NMU in immunoregulation. However, the functions of NMU in peripheral tissues have not been clarified. In this study, using NMU-deficient mice, we first demonstrated that NMU plays an important role in mast cell-mediated inflammation. Complete Freund’s adjuvant- induced mast cell degranulation as well as edema and neutrophil infiltration, which occurred v weakly in mast cell–deficient WBB6F1-W/W mice, did not occur in NMU-deficient mice. Moreover, intraplantar injection of NMU into paws induced early inflammatory responses such as mast cell degranulation, vasodilation, and plasma extravasation in WT mice but v not in WBB6F1-W/W mice. NMU-R1 was highly expressed in primary mast cells, and NMU induced Ca2ϩ mobilization and degranulation in peritoneal mast cells. These data indicate that NMU promotes mast cell–mediated inflammation; therefore, NMU receptor antagonists could be a novel target for pharmacological inhibition of mast cell–mediated inflammatory diseases. CORRESPONDENCE Neuromedin U (NMU) is a neuropeptide of the Gq11 subfamily of G protein (5). NMU- Maiko Moriyama: originally purified from porcine spinal cord R2 is expressed in a specific region of the The Journal of Experimental Medicine [email protected] (1). The first biological activity ascribed to brain, whereas NMU-R1 is expressed abun- OR Masayasu Kojima: NMU was smooth muscle contraction of the dantly in various peripheral tissues, with rela- [email protected] uterus, but NMU now has been shown to tively high levels in the small intestine, pancreas, reduce food intake and body weight (2), and stomach. Recent studies have indicated regulate stress responses (3), and modify ion that NMU has potent pronociceptive effects transport in the gastrointestinal tract (4). De- acting on NMU-R1 and NMU-R2 expressed spite extensive study of the actions of this in the spinal cord (6–8). peptide, its receptors, NMU-R1 and NMU-R2, NMU-R1 is also expressed in spleen and have been identified only recently (2). NMU- lymphocytes (9). Recently, NMU was shown R1 and NMU-R2, which share 51% amino to promote intracellular Ca2ϩ release and the acid identity, both belong to the G protein– secretion of various cytokines in a mouse Th2 coupled receptor family. Both NMU-R1 cell clone (10). Because NMU has been and NMU-R2 mobilize intracellular Ca2ϩ shown to be expressed in APCs, including stores in response to NMU binding, suggest- dendritic cells, monocytes, and B cells (9), ing that these receptors couple to members NMU is suggested to be involved in the reg- ulation of innate and adaptive immunity. M. Moriyama and T. Sato contributed equally to this work. A number of neuropeptides, such as sub- The online version of this article contains supplemental material. stance P (SP) and neuropeptide Y, are expressed JEM © The Rockefeller University Press $8.00 217 Vol. 202, No. 2, July 18, 2005 217–224 www.jem.org/cgi/doi/10.1084/jem.20050248 in central and peripheral nerve cells and are known to dependent inflammation models such as CFA-induced va- activate mast cells directly, which trigger neurogenic in- sodilation and neutrophil infiltration in the paw. Using mast v flammation and promote further anaphylactic responses cell–deficient WBB6F1-W/W mice, we also demonstrate (11–13). Alternately, chemical mediators secreted from ac- that exogenous NMU injection triggered mast cell–depen- tivated mast cells, such as histamine and serotonin, stimu- dent early inflammatory responses. Unlike other neuroin- late neural cells. Therefore, mast cells are thought to be an flammatory peptides, NMU is expressed abundantly in epi- important interface between the immune and neuro-endo- dermis and is released by CFA injection. NMU activates crine systems. mast cells directly, inducing Ca2ϩ mobilization and degranu- In this paper, using NMU-deficient (KO) mice, we lation in vitro. We propose that NMU is an important me- demonstrate that NMU plays an important role in mast cell– diator of mast cell–mediated inflammation. Figure 1. Reduced CFA-induced inflammation in NMU-KO mice. (B) Time course of the changes in paw diameter after intraplantar injection (A) Paw swelling, neutrophil infiltration, and localization of mast cells in of CFA (n ϭ 4 for each time point). (C) Expressions of NMU and NMU-R1 ϩ ϩ the paws of CFA-treated mice. (a) Hindlegs of WT, NMU-KO, WBB6F1- / , mRNA determined by RT-PCR of BMMCs and indicated organs derived -W/Wv, and W/Wv-ϩ/ϩ mice photographed 24 h after intraplantar injection from WT mice. (D) Expressions of NMU, NMU-R1, and the indicated in- of CFA or vehicle. (b) Sections of paws removed 3 h after CFA injection were flammatory mediators in the paws of indicated mice before or after intra- stained with H&E. b-2 shows magnified photos of rectangular regions plantar CFA injection. Two different mice were examined at 3 h, from in b-1. (c) Localization of skin mast cells stained with TB. Bars, 50 ␮m. which the expression levels were determined by RT-PCR. 218 NEUROMEDIN U PROMOTES MAST CELL–DEPENDENT INFLAMMATION | Moriyama et al. BRIEF DEFINITIVE REPORT RESULTS AND DISCUSSION oped inflammation in a manner similar to WT mice (WBB6F1- Suppression of CFA-induced inflammation ϩ/ϩ and –W/Wv; Fig. 1, A, B, and D). BM-derived mast cells in NMU-deficient mice (BMMCs) expressed relatively high levels of NMU-R1 NMU-KO mice exhibit obesity and reduced nociceptive re- mRNA, compared with BM or spleen cells (Fig. 1 C). Recon- v flexes, which are reflective of the functions of NMU in the ner- stitution of skin mast cells in WBB6F1-W/W mice with BM- vous system (8,14). Although NMU-R1 is expressed in a vari- MCs from -ϩ/ϩ mice (W/Wv -ϩ/ϩ) by adoptive transfer re- ety of tissues, including the gastrointestinal tracts and immune stored the CFA-induced inflammatory responses (W/Wv -ϩ/ϩ; cells such as T cells, NK cells, and monocytes (9), the role of Fig. 1, A and B), confirming that the early phase of CFA-induced NMU in the periphery has not been clarified. Therefore, we inflammation is dependent on mast cells. first compared inflammatory responses using NMU-KO mice. The number and degranulation of skin mast cells were Intraplantar injection of CFA induced edema, neutrophil confirmed by toluidine blue (TB) staining. The total numbers infiltration, and paw swelling in WT C57BL6/J mice (WT of mast cells were not significantly different in the paws of ϩ ϩ and KO; Fig. 1, A and B). NMU mRNA expression levels WT, NMU-KO, and WBB6F1- / mice (Fig. S1 A, avail- were extremely high in the paws of WT mice, being compa- able at http://www.jem.org/cgi/content/full/jem.20050248/ rable or even higher than those in the intestine, which has DC1), suggesting that the NMU deficiency did not alter mast been thought to be the most abundant source of NMU (Fig. cell development in NMU-KO mice. However, intense de- 1 C). In contrast, paw edema and neutrophil infiltration did granulation of mast cells was observed 3 h after CFA applica- ϩ ϩ not occur in NMU-KO mice 3 h after intraplantar CFA tion in the paws of WT and WBB6F1- / mice but not in injection (WT and KO; Fig. 1, A and B). In WT mice, those of NMU-KO mice (Fig. S1 B). These data suggest a injection of CFA resulted in a rapid induction of proinflam- strong connection between NMU and the activation of mast matory cytokines including TNF-␣, IL-6, macrophage in- cells in CFA-induced peripheral inflammation. flammatory protein 2, and leukocyte adhesion molecules, We also confirmed that CFA-induced mast cell degranula- v such as intracellular cell adhesion molecule 1, on the surface tion and paw edema occurred in WBB6F1-W/W mice of endothelial cells (WT; Fig. 1 D). Induction of these acute reconstituted with either WT or NMU-KO BMMCs (Fig. inflammatory factors, however, did not occur in NMU-KO S2, available at http://www.jem.org/cgi/content/full/jem. mice (KO; Fig. 1 D), indicating that NMU plays a critical 20050248/DC1). Thus, the lack of mast cell–mediated in- role in the early inflammatory responses induced by CFA. flammation following CFA injection in NMU-KO mice re- The early inflammation induced by CFA has been shown sults from the absence of NMU release, not from an intrinsic v to be mast cell–dependent (15). Indeed, WBB6F1-W/W mice defect in mast cell effector function. These results suggest that in which mast cells are deficient did not exhibit any paw swell- NMU release from the environment after CFA treatment ac- ing, infiltration of neutrophils, or the induction of proinflam- tivates mast cells.