Repulsive guidance molecule-A (RGM-A) inhibits leukocyte migration and mitigates inflammation

Abstract

Directed cell migration is a prerequisite not only for the development of the central nervous system, but also for topically restricted, appropriate immune responses. This is crucial for host defense and immune surveillance. Attracting environmental cues guiding leukocyte cell traffic are likely to be complemented by repulsive cues, which actively abolish cell migration. One such a paradigm exists in the developing nervous system, where neuronal migration and axonal path finding is balanced by chemoattractive and chemorepulsive cues, such as the neuronal repulsive guidance molecule-A (RGM-A). As expressed at the inflammatory site, the role of RGM-A within the immune response remains unclear. Here we report that RGM-A (i) is expressed by epithelium and leukocytes (granulocytes, monocytes, and T/B lymphocytes); (ii) inhibits leukocyte migration by contact repulsion and chemorepulsion, depending on dosage, through its receptor neogenin; and (iii) suppresses the inflammatory response in a model of zymosan-A-induced peritonitis. Systemic application of RGM-A attenuates the humoral proinflammatory response (TNF-α, IL-6, and macrophage inflammatory protein 1α), infiltration of inflammatory cell traffic, and edema formation. In contrast, the demonstrated anti-inflammatory effect of RGM-A is absent in mice homozygous for a gene trap mutation in the neo1 locus (encoding neogenin). Thus, our results suggest that RGM-A is a unique endogenous inhibitor of leukocyte chemotaxis that limits inflammatory leukocyte traffic and creates opportunities to better understand and treat pathologies caused by exacerbated or misdirected inflammatory responses.

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 with levels of RGM-A mRNA expression present in the murine brain. Along with lymphatic tissue such as spleen, pronounced RGM-A mRNA levels were detected in organs hosting an intrinsic immune compartment, including the brain (microglia), lungs (alveolar macrophages), and intestines (Peyer's patches). (B) Western blot analysis of RGM-A protein expression in pooled murine tissues compared with levels of expression in the brain. Values correspond to relative RGM-A mRNA levels. All data are mean ± SEM; n = 5. (C) FACS analysis verified the expression of RGM-A by leukocyte subsets including CD15⁺ granulocytes (PMNs), CD14⁺ monocytes, CD3⁺ T lymphocytes, and some CD19⁺ B lymphocytes (PMNs). Isotype controls demonstrated no relevant signal.

Fig. 2.

RGM-A is expressed at the epithelial barrier. Given the repulsive bioactivity of RGM-A, we investigated its presence in cells relevant for epithelial or endothelial barriers. (A) In the lung, RGM-A expression is confined to cytokeratin-positive epithelial cells. (B) In contrast, RGM-A expression is absent on vWF-positive endothelium. (C) Isotype controls demonstrated no relevant signal. (D) The absence of RGM-A in endothelial barriers was further corroborated by the lack of RGM-A expression by the major endothelial cell lines HMEC-1 and HUVEC, which serve as cell sources to investigate features of endothelial function (8).

Fig. 3.

RGM-A inhibits active PMN migration in vitro. (A) fMLP (10 ng/mL) induced chemotaxis-dependent migration of PMNs through a CaCo epithelial cell monolayer. 1 × 10⁶ granulocytes were placed in the apical compartment and transmigration of PMNs measured after 60 min. Measurement of MPO as representative marker was used to quantify basolateral PMN transmigration. (B) PMN transmigration in the presence of distinct concentrations of RGM-A or BSA, showing suppression of PMN migration by RGM-A in a dose-dependent fashion. (C) RGM-A–specific effects on PMN migration in the presence of RGM-A antibody (AB) and heat-inactivated RGM-A (HI-RGM-A). (D) PMN transmigration in the presence of distinct concentrations of RGM-A in the apical compartment, basolateral compartment, or both compartments of a transmigration chamber. All data are mean ± SEM; n = 6 per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

RGM attenuates PMN migration through its receptor neogenin in vitro. (A) Flow cytometry (overlay histogram) of freshly isolated human PMNs stained with isotype-matched control antibody (green), anti-CD45 antibody (red), and anti-neogenin antibody (purple). (B) Immunohistochemical staining of freshly isolated human PMNs with anti-neogenin (FITC) or isotype control and anti-CD45 (Cy3) antibodies, with the DAPI label used for nuclear counterstaining. Isotype control antibodies demonstrated no detectable signal levels (Left, overlay; original magnification 200×). Demonstration of CD45⁺ PMNs to coexpress abundant neogenin (Right; original magnification 600×). (C) PMN transmigration in the presence of distinct concentrations of functionally blocking neogenin antibodies compared with isotype IgG controls. Data are mean ± SEM; n = 6 per group. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Neo1^−/− PMNs did not respond to RGM-A, revealing an intact, regular migration response (MPO) as demonstrated by WT-derived PMNs after apical BSA application. (E) To investigate a direct contact-repulsion property of RGM-A on PMNs, we analyzed PMN binding to differentially coated surfaces (± RGM-A). The contact-repulsive RGM-A effect on PMN blocked PMN surface binding and was neogenin-dependent. PMN binding was dose-dependently blocked by RGM-A coating (top row). This was completely reversible on preincubation of PMNs with the neogenin antibody (middle row), whereas anti-HLA does not interfere with RGM-A–mediated contact repulsion. One representative experiment out of three experiments conducted is shown.

Fig. 5.

RGM-A reduces leukocyte migration and dampens inflammation during ZyA-induced peritonitis. After WT mice were injected i.p. with 1 mg of ZyA (1 mg/mL), they were given recombinant murine RGM-A (i.v. 1 μg in 0.2% BSA) or vehicle. (A) Cell counts in peritoneal lavage fluid after 4 h, 8 h, and 24 h. (B) MPO activity in peritoneal lavage after 4 h, 8 h, and 24 h. (C) Cell counts of granulocytes, lymphocytes, and monocytes in peritoneal fluid after 4 h in mice with ZyA-induced peritonitis and vehicle or RGM-A injection. All data are mean ± SEM; n = 6 per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

RGM-A fails to attenuate ZyA-induced peritonitis in neo1^−/− mice. After neo1^−/− mice were injected i.p. with 1 mg of ZyA (1 mg/mL), they received i.v. recombinant murine RGM-A (1 μg in 0.2% BSA) or vehicle. (A) Cell count in peritoneal lavage obtained after 4 h. (B) MPO activity in peritoneal lavage fluid obtained after 4 h. (C) Cytokine levels of TNF-α measured by ELISA in peritoneal fluid of WT neo1^−/− mice 4 h after ZyA-induced peritonitis. (D) IL-1β, (E) IL-6, and (F) MIP-1α levels in peritoneal fluid WT compared with Neo^−/− mice.