Abstract
The cytokine macrophage migration inhibitory factor (MIF) plays a critical role in inflammatory diseases and atherogenesis. We identify the chemokine receptors CXCR2 and CXCR4 as functional receptors for MIF. MIF triggered Gαi- and integrin-dependent arrest and chemotaxis of monocytes and T cells, rapid integrin activation and calcium influx through CXCR2 or CXCR4. MIF competed with cognate ligands for CXCR4 and CXCR2 binding, and directly bound to CXCR2. CXCR2 and CD74 formed a receptor complex, and monocyte arrest elicited by MIF in inflamed or atherosclerotic arteries involved both CXCR2 and CD74. In vivo, Mif deficiency impaired monocyte adhesion to the arterial wall in atherosclerosis-prone mice, and MIF-induced leukocyte recruitment required Il8rb (which encodes Cxcr2). Blockade of Mif but not of canonical ligands of Cxcr2 or Cxcr4 in mice with advanced atherosclerosis led to plaque regression and reduced monocyte and T-cell content in plaques. By activating both CXCR2 and CXCR4, MIF displays chemokine-like functions and acts as a major regulator of inflammatory cell recruitment and atherogenesis. Targeting MIF in individuals with manifest atherosclerosis can potentially be used to treat this condition.
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Acknowledgements
We thank E. Liehn, S. Knarren and L. Verschuren for assistance with the atherosclerotic mouse models; S. Kraemer for help with internalization assays; A. Ben-Baruch (Department of Cell Research and Immunology, Tel Aviv University) for HEK293-CXCR2 cells; H.W.L. Ziegler-Heitbrock (University of Leicester) for MonoMac6 cells; M. Locati (Istituto Clinico Humanitas) for L1.2 cells; H. Hengel (University of Düsseldorf) for SVECs; A. Ludwig and E. Brandt (Department of Immunology and Cell Biology, Forschungszentrum Borstel) for CXCR2 antibody RII115; D. Staunton (Department of Biomedical Engineering, Genome and Biomedical Sciences Facility, University of California at Davis) for the antibody to 327C; Anormed Inc. (Genzyme Corporate Offices) for AMD3465; and A. Ludwig, E. Morand, M. Thelen and A. Kapurniotu for helpful discussions. Supported by the Deutsche Forschungsgemeinschaft grants BE 1977/2-1, BE 1977/4-1 (J.B. and C.W.), WE 1913/7-1 (C.W.), SFB542-A7 (J.B.) and SFB542-C12 (C.W.); US National Institutes of Health grants AI43210 and AR49610 (R.B.); Australian National Health and Medical Research Council (NHMRC) program grant 334067 (M.J.H. and S.R.M.); US NIH grant AR51807-01 (M.J.H. and S.R.M.); and the Netherlands Organization for Scientific Research grant VENI 016.036.061 (R.K.).
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J.B., R.B., and L.L. are co-inventors on patents describing the use of MIF inhibition for the treatment of inflammatory disease.
Supplementary information
Supplementary Fig. 1
Structural homology between the CXCL8 dimer and the MIF monomer. (PDF 72 kb)
Supplementary Fig. 2
Adhesion and chemotaxis assays with various cellular and antibody controls. (PDF 52 kb)
Supplementary Fig. 3
Ex vivo perfusion of murine carotid arteries. (PDF 122 kb)
Supplementary Fig. 4
The monoclonal Mif antibody NIHIII.D.9 specifically recognizes Mif, but not Cxcl1/Kc or CXCL8. (PDF 109 kb)
Supplementary Video 1
This movie shows an ex vivo perfusion of the carotid artery of an Apoe−/− mouse fed an atherogenic diet for 6 weeks with calcein-AM-labeled MonoMac6 cells. Applying stroboscopic epifluorescence illumination, monocytic cells firmly adherent to the vessel wall, rolling along the artery wall and flowing at the rate of perfusion (4 μL/min) can be visualized in different segments of the perfused artery. The movie is a representative of a control artery corresponding to the data in Fig. 5a-f and Supplementary Fig. 3. (MPG 2854 kb)
Supplementary Video 2
MIF-mediated accumulation of leukocytes in carotid arteries of chimeric mice in vivo relies on CXCR2 as revealed by intravital microscopy. The first sequence in this video shows several leukocytes labeled by intravenous injection of rhodamine, which have become adherent to the carotid artery wall 4 h after intraperitoneal injection of Tnf-α in a wild-type Mif+/+ mouse reconstituted with wild-type bone marrow (Mif+/+/WT bone marrow). The following sequences show very few adherent leukocytes in the carotid arteries of wild-type mice reconstituted with Cxcr2−/−/ BM (Mif+/+/Cxcr2−/− bone marrow) or Mif−/− mice reconstituted with wild-type BM (Mif−/−/WT bone marrow) and no further reduction was seen in Mif−/− mice reconstituted with Cxcr2−/− BM (Mif−/−/Cxcr2−/− bone marrow). The movie corresponds to the data in Fig. 5g,h. (MOV 997 kb)
Supplementary Video 3
MIF induces CXCR2-dependent leukocyte recruitment in vivo in the microvasculature of the cremaster muscle visualized by intravital microscopy. The first sequence in this video shows a cremasteric postcapillary venule after a control (saline) injection (saline control). Some leukocytes are visible rolling within the venule, but no leukocytes are adherent within the venule and very few leukocytes are apparent in the extravascular tissue. The second sequence in this video shows a postcapillary venule after local injection of MIF (1μ g) and IgG isotype control antibody (MIF + control IgG). While leukocyte rolling and blood flow are not markedly altered compared with saline-injected mice, many leukocytes are visible adherent to the endothelial surface. Several leukocytes have been recruited to the tissue and are visible adjacent to the postcapillary venule. The last sequence in this video shows the effects of an antibody to Cxcr2 on MIF (1 μg)-induced leukocyte adhesion and emigration (MIF + anti-Cxcr2). The numbers of adherent leukocytes within the venule are dramatically reduced compared with mice treated with MIF alone and very few leukocytes have been recruited to the extravascular tissue. The movie corresponds to the data in Fig. 6b,c. (MOV 1468 kb)
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Bernhagen, J., Krohn, R., Lue, H. et al. MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat Med 13, 587–596 (2007). https://doi.org/10.1038/nm1567
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DOI: https://doi.org/10.1038/nm1567
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