Repulsive guidance molecule-A (RGM-A) inhibits leukocyte migration and mitigates inflammation Valbona Mirakaja,b, Sebastian Browna, Stefanie Lauchera, Carolin Steinlc, Gerd Kleinc, David Köhlera,b, Thomas Skutellad, Christian Meisele, Benedikt Brommerf, Peter Rosenbergera,b,1,2, and Jan M. Schwabf,1,2 aDepartment of Anesthesiology and Intensive Care Medicine, Tübingen University Hospital, 72076 Tübingen, Germany; bClinic of Anesthesiology, Intensive Care Medicine and Pain Therapy, Frankfurt University Hospital, Frankfurt, Germany; cSection for Transplantation Immunology and Immunohematology, Center for Medical Research, 72072 Tübingen, Germany; dInstitute for Anatomy and Cell Biology, School of Medicine, University of Heidelberg, D-69120 Heidelberg, Germany; eDepartment of Medical Immunology, Charité–Universitätsmedizin Berlin, 13353 Berlin, Germany; and fDepartment of Neurology and Experimental Neurology, Spinal Cord Injury Research, Charité–Universitätsmedizin Berlin, Humboldt University, 10117 Berlin, Germany Edited by Dennis A. Carson, University of California San Diego, La Jolla, CA, and approved February 23, 2011 (received for review October 17, 2010) Directed cell migration is a prerequisite not only for the develop- A is thought to be shed from the cell membrane by cleavage at ment of the central nervous system, but also for topically restricted, amino acid 412 or instability cleavage at the putative autopro- appropriate immune responses. This is crucial for host defense and teolytic cleavage site (amino acid 168), which is sensitive to pH immune surveillance. Attracting environmental cues guiding leu- drop-induced cleavage (14–17). During neuronal development, kocyte cell traffic are likely to be complemented by repulsive cues, RGM-A is a chemorepulsive environmental cue that instructs nav- which actively abolish cell migration. One such a paradigm exists in igating outgrowing axons (15–17) and also acts on neuronal mi- the developing nervous system, where neuronal migration and gration and patterning, as demonstrated by neural tube closure fi axonal path nding is balanced by chemoattractive and chemo- defects in RGM-A knockout mice (17). In addition, RGM-A repulsive cues, such as the neuronal repulsive guidance molecule-A fl regulates neuronal survival and differentiation by binding to its (RGM-A). As expressed at the in ammatory site, the role of RGM-A dependence receptor neogenin (18–20). After development of within the immune response remains unclear. Here we report that i the CNS is complete, RGM-A is present in the myelin sheath RGM-A ( ) is expressed by epithelium and leukocytes (granulocytes, fi ii (21, 22). RGM-A reexpression is con ned to the parenchymal monocytes, and T/B lymphocytes); ( ) inhibits leukocyte migration fi fl IMMUNOLOGY by contact repulsion and chemorepulsion, depending on dosage, lesion environment, speci cally in areas of in ammation after through its receptor neogenin; and (iii) suppresses the inflam- rodent or human CNS injury (21, 22). Because axonal outgrowth fl matory response in a model of zymosan-A–induced peritonitis. and the transmigration of leukocytes into in amed tissue share Systemic application of RGM-A attenuates the humoral proinflam- the conserved biological mechanisms of cytoskeleton remodeling matory response (TNF-α, IL-6, and macrophage inflammatory (9), we hypothesized that RGM-A also repulses leukocyte mi- protein 1α), infiltration of inflammatory cell traffic, and edema gration in response to chemotactic factors. formation. In contrast, the demonstrated anti-inflammatory effect Our results demonstrate that besides being expressed in the of RGM-A is absent in mice homozygous for a gene trap mutation in developing CNS and in lesions in the adult CNS, RGM-A is the neo1 locus (encoding neogenin). Thus, our results suggest that strongly expressed by epithelium, leukocytes [ie, polymorpho- RGM-A is a unique endogenous inhibitor of leukocyte chemotaxis nuclear leukocytes (PMNs), monocytes, and T/B lymphocytes], and that limits inflammatory leukocyte traffic and creates opportunities tissues that are sensitive to infection. Both in vitro and in vivo, to better understand and treat pathologies caused by exacerbated RGM-A inhibits PMN migration through its receptor neogenin or misdirected inflammatory responses. and modifies the inflammatory milieu by attenuating the proin- flammatory response. In addition, RGM-A is a potent inhibitor of uiding migrating cells toward their destination is of prime leukocyte recruitment to sites of inflammation, mitigating hall- Gimportance in cell biology and physiology and is a prerequisite marks of inflammation, such as edema formation and proin- for tissue homeostasis, host defense, and immune surveillance (1, 2). flammatory cytokine expression, in a model of mouse peritonitis. Attracting cues, such as chemokines to guide leukocyte cell traffic, RGM-A is essential to limiting the ability of leukocytes to trans- have been well characterized in recent years (3–5) and are likely to migrate through epithelial cell layers, thereby attenuating the in- be complemented by repulsive cues, which actively abolish cell mi- flammatory milieu (“cytokine storm”), and also to reducing the gration. One such a paradigm exists in the developing nervous sys- number of leukocytes subsequently recruited. Thus, we have iden- tem, where neuronal migration is regulated by chemoattractive and tified a previously uncharacterized function of the neuronal guid- chemorepulsive cues, such as the neuronal repulsive guidance ance cue RGM-A outside the CNS. Our findings identify RGM-A molecule A (RGM-A). The migration of leukocytes is a tightly or- as a potent immunomodulatory protein in vitro and in vivo. Its anti- chestrated and controlled multiphasic biological cascade elicited by fl fl in ammatory functions through binding to its receptor neogenin. chemotactic signals during an acute in ammatory process (3, 4). These results support a conserved molecular role for RGM-A as Excessive immune cell migration into an inflammatory site might a guidance cue for neurons and leukocyte chemotaxis. result in tissue destruction and organ dysfunction (functio laesa). Recently, neuronal guidance cues were identified that control and attenuate inappropriate migration and infiltration of leukocytes into fl – Author contributions: P.R. and J.M.S. designed research; V.M., S.B., S.L., C.S., G.K., D.K., T.S., acutely in amed areas (6 12). Given that the neuronal repellent C.M., B.B., and P.R. performed research; V.M., G.K., T.S., P.R., and J.M.S. contributed new RGM-A is expressed at inflammatory sites but shares no sequence ho- reagents/analytic tools; S.B., S.L., C.S., G.K., D.K., T.S., C.M., B.B., P.R., and J.M.S. analyzed mology with any other known guidance cues, we explored whether data; and P.R. and J.M.S. wrote the paper. it inhibits leukocyte migration in vitro and in vivo (7, 12, 13). The authors declare no conflict of interest. RGM was the first candidate for characterization as a topo- This article is a PNAS Direct Submission. graphic guidance cue and was originally described as a glycosylpho- 1P.R. and J.M.S. contributed equally to this work. sphatidylinositol-anchored proline- and cysteine-rich glycocprotein 2To whom correspondence may be addressed. E-mail: [email protected] or peter. (13). Vertebrates comprise three homologs of RGM: RGM-A, [email protected]. RGM-B (DRAGON), and RGM-C (hemojuvelin). The human This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. RGM-A isoform is located on chromosome 15q26.1 (14). RGM- 1073/pnas.1015605108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1015605108 PNAS | April 19, 2011 | vol. 108 | no. 16 | 6555–6560 Downloaded by guest on September 28, 2021 A C CD45+pan-leukocytes CD15+granulocytes (PMN) A Epithelium Overlay DAPI RGM-A Cytokeratin CD45 CD45& Isotype CD15 CD15& Isotype RGMA RGMA RGM-A mRNA (Relative Expression) B Brain Lung KidneyLiver SpleenIntestine Endothelium CD45 Isotype FITC CD15 CD45 & RGM-A Isotype PC5/PE CD15 & RGM-A Overlay DAPI RGM-A vWF B CD14+monocytes CD3+ &CD19+ T-/B- lymphocytes C RGM-A Isotype controls & DAPI -Actin CD14 CD14& Isotype CD3& CD3& Isotype RGMA CD19 CD19&RGMA IgG vWF IgG RGM-A IgG Cytokeratin Brain Lung Liver Spleen Intestine Kidney D HMEC HUVEC CD14 Isotype FITC CD3 & CD19 CD14 & RGM-A Isotype PE CD3 & CD19 & RGM-A Endothelial Cell lines Fig. 1. RGM-A is expressed outside the CNS postdevelopmentally. (A)RGM-A mRNA in murine tissues quantified by quantitative real-time RT-PCR compared Isotype controls with levels of RGM-A mRNA expression present in the murine brain. Along with RGM-A-FITC lymphatic tissue such as spleen, pronounced RGM-A mRNA levels were detected in organs hosting an intrinsic immune compartment, including the brain Fig. 2. RGM-A is expressed at the epithelial barrier. Given the repulsive (microglia), lungs (alveolar macrophages), and intestines (Peyer’s patches). (B) bioactivity of RGM-A, we investigated its presence in cells relevant for epi- fi Western blot analysis of RGM-A protein expression in pooled murine tissues thelial or endothelial barriers. (A) In the lung, RGM-A expression is con ned compared with levels of expression in the brain. Values correspond to relative to cytokeratin-positive epithelial cells. (B) In contrast, RGM-A expression is RGM-A mRNA levels. All data are mean ± SEM; n =5.(C) FACS analysis verified absent on vWF-positive endothelium. (C) Isotype controls demonstrated no the expression of RGM-A by leukocyte subsets including CD15+ granulocytes relevant signal. (D) The absence of RGM-A in endothelial barriers was further (PMNs), CD14+ monocytes, CD3+ T lymphocytes, and some CD19+ B lymphocytes corroborated by the lack of RGM-A expression by the major endothelial cell (PMNs). Isotype controls demonstrated no relevant signal. lines HMEC-1 and HUVEC, which serve as cell sources to investigate features of endothelial function (8). Results effective host defense and also to avoid tissue damage due to an RGM-A Is Expressed Outside the CNS. To perform an immunologic aberrant, exacerbated inflammatory response (3, 4).