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 G(alphai)- 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.

Abstract-The CXC ligand (CXCL)12/CXC receptor (CXCR)4 chemokine-receptor axis controls hematopoiesis, organ development, and angiogenesis, but its role in the inflammatory pathogenesis of atherosclerosis is unknown. Here we show that interference with Cxcl12/Cxcr4 by a small-molecule antagonist, genetic Cxcr4 deficiency, or lentiviral transduction with Cxcr4 degrakine in bone marrow chimeras aggravated diet-induced atherosclerosis in apolipoprotein E-deficient (Apoe Ϫ/Ϫ ) or LDL receptor-deficient (Ldlr Ϫ/Ϫ ) mice. Chronic blockade of Cxcr4 caused leukocytosis and an expansion of neutrophils and increased neutrophil content in plaques, associated with apoptosis and a proinflammatory phenotype. Whereas circulating neutrophils were recruited to atherosclerotic lesions, depletion of neutrophils reduced plaque formation and prevented its exacerbation after blocking Cxcr4. Disrupting Cxcl12/Cxcr4 thus promotes lesion formation through deranged neutrophil homeostasis, indicating that Cxcl12/Cxcr4 controls the important contribution of neutrophils to atherogenesis in mice (Circ Res. 2008;102:209-217.)

Edited by Masayuki MiyasakaKeywords: CD74 CXCR4 MIF Chemokine receptor GPCR Invariant chain a b s t r a c t MIF is a chemokine-like inflammatory mediator that triggers leukocyte recruitment by binding to CXCR2 and CXCR4. MIF also interacts with CD74/invariant chain, a single-pass membrane-receptor. We identified complexes between CD74 and CXCR2 with a role in leukocyte recruitment. It is unknown whether CD74 also binds to CXCR4. We demonstrate that CD74/CXCR4 complexes formed when CD74 was expressed with CXCR4 in HEK293 cells. Expression of CD74-variants lacking an ERretention signal showed CD74/CXCR4 complexes at the cell surface. Importantly, endogenous CD74/ CXCR4 complexes were isolated by co-immunoprecipitation from monocytes. Finally, MIF-stimulated CD74-dependent AKT activation was blocked by anti-CXCR4 and anti-CD74 antibodies and AMD3100, whereas CXCL12-stimulated AKT activation was not reduced by anti-CD74. Thus, CD74 forms functional complexes with CXCR4 that mediate MIF-specific signaling.

The phagosomal lumen in macrophages is the site of numerous interacting chemistries that mediate microbial killing, macromolecular degradation, and antigen processing. Using a non-hypothesisbased screen to explore the interconnectivity of phagosomal functions, we found that NADPH oxidase (NOX2) negatively regulates levels of proteolysis within the maturing phagosome of macrophages. Unlike the NOX2 mechanism of proteolytic control reported in dendritic cells, this phenomenon in macrophages is independent of changes to lumenal pH and is also independent of hydrolase delivery to the phagosome. We found that NOX2 mediates the inhibition of phagosomal proteolysis in macrophages through reversible oxidative inactivation of local cysteine cathepsins. We also show that NOX2 activity significantly compromises the phagosome's ability to reduce disulfides. These findings indicate that NOX2 oxidatively inactivates cysteine cathepsins through sustained ablation of the reductive capacity of the phagosomal lumen. This constitutes a unique mechanism of spatiotemporal control of phagosomal chemistries through the modulation of the local redox environment. In addition, this work further implicates the microbicidal effector NOX2 as a global modulator of phagosomal physiologies, particularly of those pertinent to antigen processing.phagocytosis | cathepsin | disulfide reduction | antigen processing | lysosome U nlike many specialized lineages of the mononuclear phagocyte system, tissue macrophages function in a diverse array of homeostatic and immune physiologies. Critical to many of these functions is the phagosome. Over the past decade, proteomic characterization of the phagosome in conjunction with biochemical analysis of phagosomal chemistries in reconstituted systems has given great insight into the function of this organelle (1, 2). More recently, measurement of phagosomal properties in live cells has enabled the in situ dissection of the complex crosstalk between spatiotemporally intimate phagosomal chemistries (3, 4). In particular, cross-talk influencing the control of phagosomal proteolysis has recently received much attention (4). It has become increasingly apparent that a tightly controlled, limited level of proteolysis within the endolysosomal system, as found in dendritic cells (DCs), is essential for efficient antigen processing (5, 6). Macrophages possess a reported 20-to 60-fold higher level of lysosomal proteolysis than DCs, which is implicated in limiting their efficiency as antigen-presenting cells (7-9). Nonetheless, macrophages are capable of productively presenting antigen to T cells and play an important role in the secondary immune response (10). This is presumably aided by the stringent control of the macrophage's lysosomal protease activities in its antigenprocessing compartments.Control of lysosomal proteases such as cathepsins occurs at several regulatory levels, including transcription, trafficking, prodomain removal, regulatory proteins (e.g. cystatins), and vacuolar pH (11). Modification of the redox...

Alternatively activated macrophages, generated in a T-helper 2 environment, have demonstrated roles in wound repair and tissue remodeling in addition to being charged with immune tasks. Because the hydrolytic chemistries of the phagosomal lumen are central to many of these functions, we investigated their modification after alternative activation with IL-4 and IL-13. Most significantly, we found striking up-regulation of the proteolytic levels within the phagosome of IL-4-activated macrophages. Two synergistic mechanisms were determined to underlie this up-regulation. First, IL-4-activated macrophages displayed increased expression of cathepsin S and L, providing greater proteolytic machinery to the phagosome despite unchanged rates of lysosomal contribution. Secondly, decreased phagosomal NADPH oxidase (NOX2) activity, at least partially resulting from decreased expression of the NOX2 subunit gp91 phox , resulted in a more reductive lumenal microenvironment, which in turn, enhanced activities of local cysteine cathepsins.Decreased NOX2 activity additionally increased the phagosome's ability to reduce disulfides, further enhancing the efficiency of the macrophage to degrade proteins containing disulfide bonds. Together, these changes initiated by IL-4 act synergistically to rapidly and dramatically enhance the macrophage's ability to degrade phagocytosed protein, which, we reason, better equips this cell for its roles in wound repair and tissue remodeling. IntroductionThe ability of the macrophage to dramatically remodel its cell biology in response to environmental cues and immune signals permits this ubiquitous cell lineage to function in a wide range of homeostatic and immune physiologies. Classic activation of the macrophage in response to microbial products and T-helper 1 cytokines has been long known to reprogram the macrophage's biology to enhance microbial killing and its ability to present antigen. [1][2][3] Modulation of the macrophage's key organelle, the phagosome, contributes to this functional reprogramming. [4][5][6] During the past 2 decades, alternative activation of macrophages by the T-helper 2 cytokines IL-4 and IL-13 has been shown to result in a unique phenotype that enables the macrophage to perform distinct physiologic functions. In addition to their demonstrated roles in helminthic infection, alternatively activated macrophages (AAMØs) are gaining considerable attention for their roles in tissue remodeling, wound repair, and control of inflammation. 7,8 Concomitant with these roles, several features of AAMØs have been identified that are consistent with the clearance of apoptotic cells and debris, such as up-regulation of certain phagocytic receptors and modification of endolysosomal dynamics. 7,[9][10][11][12] Hitherto changes to the functional chemistries within the phagosome that facilitate the AAMØ's function have not been elucidated.The lumenal chemistries of phagosomes play a central role in many macrophage functions. In addition to microbial killing and antigen processing, the ph...

Background-The CC chemokine CCL5/Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) is upregulated in mononuclear cells or deposited by activated platelets during inflammation and has been implicated in atherosclerosis and neointimal hyperplasia. We investigated the influence of the transcriptional regulator Y-box binding protein (YB)-1 on CCL5 expression and wire-induced neointimal hyperplasia. Methods and Results-Analysis of the CCL5 promoter revealed potential binding sites for YB-1, and interaction of YB-1 with a sequence at position Ϫ204/Ϫ173 was confirmed by DNA binding assays. Both YB-1 expression and CC chemokine ligand-5 (CCL5) mRNA expression were increased in neointimal versus medial smooth muscle cells, as analyzed by real-time polymerase chain reaction. Overexpression of YB-1 in smooth muscle cells (but not macrophages) enhanced CCL5 transcriptional activity in reporter assays, mRNA and protein expression, and CCL5-mediated monocyte arrest. Carotid arteries of hyperlipidemic apolipoprotein E-deficient mice were subjected to intraluminal transfection with a lentivirus encoding YB-1 short hairpin RNA or empty vector directly after wire injury. Double immunofluorescence revealed YB-1 expression in neointimal smooth muscle cells but not macrophages and colocalization with neointimal CCL5, which was downregulated by YB-1 short hairpin RNA. Neointima formation was decreased significantly after YB-1 knockdown compared with controls and was associated with a diminished content of lesional macrophages. A reduction of lesion formation by YB-1 knockdown was not observed in apolipoprotein E-deficient mice deficient in the CCL5 receptor CCR5 or after treatment with the CCL5 antagonist Met-RANTES, which indicates that YB-1 effects were dependent on CCL5. Conclusions-The transcriptional regulator YB-1 mediates CCL5 expression in smooth muscle cells and thereby contributes to neointimal hyperplasia, thus representing a novel target with which to limit vascular remodeling.

MIF was recently redefined as an inflammatory cytokine, which functions as a critical mediator of diseases such as septic shock, rheumatoid arthritis, atherosclerosis, and cancer. MIF also regulates wound healing processes. Given that fibroblast migration is a central event in wound healing and that MIF was recently demonstrated to promote leukocyte migration through an interaction with G-protein-coupled receptors, we investigated the effect of MIF on fibroblast migration in wounded monolayers in vitro. Transient but not permanent exposure of primary mouse or human fibroblasts with MIF significantly promoted wound closure, a response that encompassed both a proliferative and a pro-migratory component. Importantly, MIF-induced fibroblast activation was accompanied by an induction of calcium signalling, whereas chronic exposure with MIF down-regulated the calcium transient, suggesting receptor desensitization as the underlying mechanism.

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