Structural determinants of MIF functions in CXCR2-mediated inflammatory and atherogenic leukocyte recruitment Christian Weber*†‡, Sandra Kraemer*§, Maik Drechsler†, Hongqi Lue§, Rory R. Koenen†, Aphrodite Kapurniotu¶, Alma Zernecke†, and Ju¨ rgen Bernhagen‡§ †Institute for Molecular Cardiovascular Research (IMCAR); and §Department of Biochemistry and Molecular Cell Biology, RWTH Aachen University, 52056 Aachen, Germany; and ¶Laboratory of Peptide Biochemistry, Center of Integrated Protein Science Munchen,¨ Technische Universität, D-85350 Munich, Germany Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and accepted by the Editorial Board August 15, 2008 (received for review April 25, 2008) We have recently identified the archaic cytokine macrophage ing to CCR5 (i.e., HisRS) and CCR3 (i.e., AsnRS) (9, 10), and migration inhibitory factor (MIF) as a non-canonical ligand of the fragments of TyrRS mediate pro-angiogenic activity by direct CXC chemokine receptors CXCR2 and CXCR4 in inflammatory and binding to CXCR1 through a CXCL8 (also known as interleu- atherogenic cell recruitment. Because its affinity for CXCR2 was kin-8; IL-8)-like N-terminal motif consisting of residues Glu, particularly high, we hypothesized that MIF may feature structural Leu, and Arg (ELR) (11). motives shared by canonical CXCR2 ligands, namely the conserved Macrophage migration inhibitory factor (MIF) is a long- N-terminal Glu-Leu-Arg (ELR) motif. Sequence alignment and struc- known T cell cytokine discovered more than four decades ago tural modeling indeed revealed a pseudo-(E)LR motif (Asp-44-X- that more recently has been recognized to be a key mediator of Arg-11) constituted by non-adjacent residues in neighboring loops innate immunity and pleiotropic inflammatory cytokine. MIF but with identical parallel spacing as in the authentic ELR motif. plays a pivotal role in the pathogenesis of acute and chronic Structure–function analysis demonstrated that mutation of resi- inflammatory diseases such as septic shock, rheumatoid arthritis, dues R11, D44, or both preserve proper folding and the intrinsic inflammatory lung disease, and atherosclerosis by promoting catalytic property of MIF but severely compromises its binding to and amplifying involved inflammatory reactions such as mono- CXCR2 and abrogates MIF/CXCR2-mediated functions in chemo- cyte/macrophage survival, MAPK signaling, or inflammatory taxis and arrest of monocytes on endothelium under flow condi- cytokine release (12–14). We have recently demonstrated that tions. R11A-MIF and the R11A/D44A-MIF double-mutant exhibited MIF, contrary to its historic and eponymous name, is a non- a pronounced defect in triggering leukocyte recruitment to early cognate ligand of the CXC chemokine receptors CXCR2 and atherosclerotic endothelium in carotid arteries perfused ex vivo CXCR4. Importantly, through interaction with these receptors, and upon application in a peritonitis model. The function of MIF is instrumental in inflammatory leukocyte recruitment in D44A-MIF in peritoneal leukocyte recruitment was preserved as a atherosclerosis, targeting monocytes and neutrophils through result of compensatory use of CXCR4. In conjunction, our data CXCR2 and T cells through CXCR4 (15, 16). MIF is strongly identify a pseudo-(E)LR motif as the structural determinant for over-expressed in the arterial wall of human atherosclerotic MIF’s activity as a non-canonical CXCR2 ligand, epitomizing the tissue, and blockade or genetic deletion of MIF in animal models structural resemblance of chemokine-like ligands with chemokines of both native and injury-induced atherogenesis leads to a and enabling selective targeting of pro-inflammatory MIF/CXCR2 marked reduction in arterial inflammation and lesion size, interactions. including regression of established plaques (14, 17). MIF binds to CXCR2 with low nanomolar affinity and induces CXCR2- ϩ atherosclerosis ͉ CXC chemokine ͉ cytokine ͉ ELR chemokine mediated leukocyte arrest and chemotaxis (15). CXCR2 signaling induced by the known cognate ligands, such hemokines govern leukocyte trafficking and deployment as CXCL8, requires an ELR motif (18). For example, mutagen- Cduring immune responses and inflammatory reactions by esis experiments or alanine scanning indicated that substitution ϩ signaling through corresponding Gi protein-coupled chemokine of the ELR residues in ELR CXC chemokines such as CXCL8 receptors of the CXC chemokine receptor (CXCR) and CC or CXCL7 resulted in a dramatic loss of CXCR2 binding affinity chemokine receptor (CCR) family (1–3). Molecular mimicry of or elastase release from neutrophils, whereas its introduction the chemokine system is exploited by viruses, e.g., HIV-1, which into related chemokines, namely CXCL4, but not CXCL10 or invades host cells following interaction of its capsid protein gp120 with host CXCR4, or to evade the host defense, e.g., by herpes viruses expressing macrophage inflammatory proteins, Author contributions: C.W., S.K., R.R.K., A.K., and J.B. designed research; S.K., M.D., H.L., which operate as CCR agonists/antagonists (4–6). It is increas- and A.Z. performed research; C.W., S.K., M.D., H.L., A.K., A.Z., and J.B. analyzed data; and C.W. and J.B. wrote the paper. ingly appreciated that various host proteins, which cannot be Conflict of interest statement: J.B., C.W., and A.Z. are inventors on a patent application on classified according to the consensus nomenclature of chemo- anti-MIF strategies in atherosclerosis. J.B. is a coinventor on a patent on applications of kines, also rely on direct interactions with chemokine receptors anti-MIF antibodies in inflammatory diseases. J.B. and C.W. are stockholders of a biotech- to regulate inflammatory and immune processes, thus acting as nology company that is developing anti-MIF strategies. ‘‘non-cognate’’ ligands for CXCRs and CCRs. For instance, This article is a PNAS Direct Submission. antimicrobial human ␤-defensins display chemotactic activity for Freely available online through the PNAS open access option. T and dendritic cell subsets by binding to CCR6. In turn, the *C.W. and S.K. contributed equally to this study cognate ligand of CCR6, CCL20, shares anti-microbial proper- ‡To whom correspondence may be addressed. E-mail: [email protected] or ties with ␤-defensins as a result of a similar surface topology of [email protected]. positively charged residues (7, 8). Autoantigenic aminoacyl- This article contains supporting information online at www.pnas.org/cgi/content/full/ tRNA synthetases (AaaRS) and their fragments released from 0804017105/DCSupplemental. damaged cells induce chemotactic cell migration through bind- © 2008 by The National Academy of Sciences of the USA 16278–16283 ͉ PNAS ͉ October 21, 2008 ͉ vol. 105 ͉ no. 42 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804017105 Downloaded by guest on September 28, 2021 ) θ θ θ θ A A LPCFTMQW E1 E2 B 15000 WT M r 10000 R11A R6 52 D44A 5000 31 R11A-D44A E4 L5 0 19 17 -5000 11 -10000 6 200 210 220 230 240 250 Mean residue ellipticity ( ellipticity residue Mean Wavelength (nm) B C 0.6 D 100 0.5 WT 75 R11A D44 0.4 50 D44A 0.3 WT R11 R11A-D44A 25 R11A 0.2 buffer A (475 nm) D44A ∆ ∆ ∆ ∆ 0 0.1 R11A-D44A 0.0 -25 C 01234 Unfolded protein (%) 012345678 IL-8 Time (min) E4 L5 R6 GdnHCl (M) MIF Fig. 2. Characterization of the pseudo-(E)LR MIF mutants. The pseudo-(E)LR R11 D44 mutants were prepared by an almost identical procedure as that established for WT MIF. (A) Purification of the pseudo-(E)LR mutants. Representative, D silver-stained SDS gel of the purification of R11A-MIF. Cell lysates were pre- pared by high-spin centrifugation (L and P). Upon concentration (C, FT), mutants were purified by anion exchange chromatography (MQ) and by C8 reverse-phase chromatography (washing with 20% acetonitrile [W], elution with 60% acetonitrile [E1, E2]). The molecular weight (Mr) is indicated on the Left.(B) Overall structural integrity of the pseudo-(E)LR mutants as evidenced by CD spectroscopy. Renatured mutants were compared with WT MIF. Mean residue ellipticities per residue (␪) are plotted against the wavelength. Spectra are representative of three independent recordings. (C) Pseudo-(E)LR mutants show an identical tautomerase activity as WT MIF. Monitoring of tautomerase activity by measuring the decrease in absorbance at 475 nm over a 4-min time period. Data represent means Ϯ SEM of three independent experiments. (D) Fig. 1. Structural homology between CXCL8 and MIF and their ELR and Distinct conformational stabilities of the mutants. GdnHCl-induced unfolding pseudo-(E)LR motives, respectively. The 3D structures of the CXCL8 dimer (A) of WT MIF and pseudo-(E)LR mutants as followed by CD spectroscopy. Un- and the MIF monomer (B) share an architectural homology. (A) The three folding curves are presented as the percentage of unfolded relative to native N-terminal amino acids Glu-3 (E3), Leu-4 (L4), and Arg-5 (R5) of each CXCL8 protein calculated from the change in ellipticity at 222 nm over the concen- monomer form an ELR motif known to be essential for signaling through tration of GdnHCl. Data represent means Ϯ SEM of three independent CXCR2. (B) Conformational pseudo-(E)LR motif of MIF formed by the two experiments. non-adjacent residues Arg-11 (R11) and Asp-44 (D44), which reside in an ELR-like spacing in exposed neighboring loops. Note that the N-terminal methionine of MIF is processed; thus, numbering of residues starts with Pro-1. contains neither N-terminal cysteine residues nor an ELR motif (C) Schematic illustration indicating the positions of the ELR and pseudo-(E)LR in the N-terminal sequence (Fig. 1C). However, inspection of the residues in CXCL8 and MIF, respectively. (D) Surface model of the trimeric structure of the MIF monomer (23) revealed that residues structure of MIF. Following the color code of the scheme in C, the location of Asp-44 and Arg-11 are located in neighboring loops in a parallel R11 in each subunit is depicted in red; D44 is highlighted in green.