Macrophage migration inhibitory factor deficiency attenuates macrophage recruitment, glomerulonephritis, and lethality in MRL/lpr mice

Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disease of unknown etiology. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is operative in innate and adaptive immunity and important in immune-mediated diseases such as rheumatoid arthritis and atherosclerosis. The functional relevance of MIF in systemic autoimmune diseases such as SLE is unknown. Using the lupus-prone MRL/lpr mice, we aim to examine the expression and function of MIF in this murine model of systemic autoimmune disease. These experiments revealed that renal MIF expression was significantly higher in MRL/lpr mice compared with nondiseased control mice (MRL/MpJ), and MIF was also markedly up-regulated in skin lesions of MRL/lpr mice. To examine the effect of MIF on development of systemic autoimmune disease, we generated MRL/lpr mice with a targeted disruption of the MIF gene (MIF(-/-)MRL/lpr), and compared their disease manifestations to MIF(+/+)MRL/lpr littermates. MIF(-/-)MRL/lpr mice exhibited significantly prolonged survival, and reduced renal and skin manifestations of SLE. These effects occurred in the absence of major changes in T and B cell markers or alterations in autoantibody production. In contrast, renal macrophage recruitment and glomerular injury were significantly reduced in MIF(-/-)MRL/lpr mice, and this was associated with reduction in the monocyte chemokine MCP-1. Taken together, these data suggest MIF as a critical effector of organ injury in SLE.     

Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is overexpressed in prostate cancer, but the mechanism by which MIF exerts effects on tumor cells remains undetermined. MIF interacts with its identified membrane receptor, CD74, in association with CD44, resulting in ERK 1/2 activation. Therefore, we hypothesized that increased expression or surface localization of CD74 and MIF overexpression by prostate cancer cells regulated tumor cell viability. Prostate cancer cell lines (LNCaP and DU-145) had increased MIF gene expression and protein levels compared with normal human prostate or benign prostate epithelial cells (p < 0.01). Although MIF, CD74, and CD44 variant 9 expression were increased in both androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells, cell surface of CD74 was only detected in androgen-independent (DU-145) prostate cancer cells. Therefore, treatments aimed at blocking CD74 and/or MIF (e.g., inhibition of MIF or CD74 expression by RNA interference or treatment with anti-MIF- or anti-CD74- neutralizing Abs or MIF-specific inhibitor, ISO-1) were only effective in androgen-independent prostate cancer cells (DU-145), resulting in decreased cell proliferation, MIF protein secretion, and invasion. In DU-145 xenografts, ISO-1 significantly decreased tumor volume and tumor angiogenesis. Our results showed greater cell surface CD74 in DU-145 prostate cancer cells that bind to MIF and, thus, mediate MIF-activated signal transduction. DU-145 prostate cancer cell growth and invasion required MIF activated signal transduction pathways that were not necessary for growth or viability of androgen-dependent prostate cells. Thus, blocking MIF either at the ligand (MIF) or receptor (CD74) may provide new, targeted specific therapies for androgen-independent prostate cancer.     

ISO-1 binding to the tautomerase active site of MIF inhibits its pro-inflammatory activity and increases survival in severe sepsis

MIF is a proinflammatory cytokine that has been implicated in the pathogenesis of sepsis, arthritis, and other inflammatory diseases. Antibodies against MIF are effective in experimental models of inflammation, and there is interest in strategies to inhibit its deleterious cytokine activities. Here we identify a mechanism of inhibiting MIF pro-inflammatory activities by targeting MIF tautomerase activity. We designed small molecules to inhibit this tautomerase activity; a lead molecule, "ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)," significantly inhibits the cytokine activity in vitro. Moreover, ISO-1 inhibits tumor necrosis factor release from macrophages isolated from LPStreated wild type mice but has no effect on cytokine release from MIFdeficient macrophages. The therapeutic importance of the MIF inhibition by ISO-1 is demonstrated by the significant protection from sepsis, induced by cecal ligation and puncture in a clinically relevant time frame. These results identify ISO-1 as the first small molecule inhibitor of MIF proinflammatory activities with therapeutic implications and indicate the potential of the MIF active site as a novel target for therapeutic interventions in human sepsis.     

Activation of the JNK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on CXCR4 and CD74

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family and controls essential processes such as inflammation, cell differentiation, and apoptosis. JNK signalling is triggered by extracellular signals such as cytokines and environmental stresses. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine with chemokine-like functions in leukocyte recruitment and atherosclerosis. MIF promotes MAPK signalling through ERK1/2, while it can either activate or inhibit JNK phosphorylation, depending on the cell type and underlying stimulation context. MIF activities are mediated by non-cognate interactions with the CXC chemokine receptors CXCR2 and CXCR4 or by ligation of CD74, which is the cell surface expressed form of the class II invariant chain. ERK1/2 signalling stimulated by MIF is dependent on CD74, but the receptor pathway involved in MIF activation of the JNK pathway is unknown. Here we comprehensively characterize the stimulatory effect of MIF on the canonical JNK/c-Jun/AP-1 pathway in fibroblasts and T cell lines and identify the upstream signalling components. Physiological concentrations of recombinant MIF triggered the phosphorylation of JNK and c-Jun and rapidly activated AP-1. In T cells, MIF-mediated activation of the JNK pathway led to upregulated gene expression of the inflammatory chemokine CXCL8. Activation of JNK signalling by MIF involved the upstream kinases PI3K and SRC and was found to be dependent on CXCR4 and CD74. Together, these data show that the CXCR4/CD74/SRC/PI3K axis mediates a rapid and transient activation of the JNK pathway as triggered by the inflammatory cytokine MIF in T cells and fibroblasts.     

Critical modifications of the ISO-1 scaffold improve its potent inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity

Based on the scaffold of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of the proinflammatory cytokine MIF, two critical modifications and chiral resolution have significantly improved the potency of the inhibition. Compound (R)-17 is 20-fold more potent than ISO-1 and inhibits MIF tautomerase activity with an IC(50) of 550 nM.     

MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment

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.     

Role for nephritogenic T cells in lupus glomerulonephritis: progression to renal failure is accompanied by T cell activation and expansion in regional lymph nodes

Autoreactive T cells are critical in the initiation and maintenance of autoantibody responses that are a hallmark of systemic lupus erythematosus. However, the direct contribution of T cells in end-organ disease like lupus glomerulonephritis (GN) is poorly understood. In this study, we investigated the role of T cells in progression of lupus GN in NZM2328 mice, a murine model of spontaneous systemic lupus erythematosus. At 26 wk of age, NZM2328 female mice showed glomerular immune complex deposits and acute proliferative GN. This was associated with up-regulation of MHC class II and the detection of T cells and CD11c(+) dendritic cells in the glomeruli. The regional lymph nodes (LN) showed preferential activation of T cells and an oligoclonal T cell response with skewed expansion of certain Vbeta families. This suggests an Ag-driven response occurring in the regional LN of nephritic mice during acute GN. In contrast, male NZM2328 mice developed glomerular immune complexes and acute GN, but rarely progressed to fatal chronic GN. Significantly, male kidneys at 40 wk of age did not have detectable dendritic cells and T cells in the glomeruli. Thus, glomerular immune complex deposition initiates an immune response against renal Ags in the regional LN, leading to T cell recruitment into the kidney during acute proliferative GN. This T cell activation and infiltration are influenced by gender-dependent end-organ factors and may determine the progression of acute GN to chronic GN and renal failure.     

Generation of human regulatory gammadelta T cells by TCRgammadelta stimulation in the presence of TGF-beta and their involvement in the pathogenesis of systemic lupus erythematosus

As a component of the innate immune cell population, γδ T cells are involved in tumor immunosurveillance and host defense against viral invasion. In this study, we demonstrated a novel function of human γδ T cells as regulatory cells by detecting their suppressive effect on the proliferation of autologous naive CD4(+) T cells. These regulatory γδ T cells (γδ Tregs) could be generated in vitro by stimulating with anti-TCRγδ in the presence of TGF-β and IL-2. Similar to CD4(+)Foxp3(+) Tregs, γδ Tregs also expressed Foxp3. Additionally, they primarily belonged to the Vδ1 subset with a CD27(+)CD25(high) phenotype. Furthermore, these γδ Tregs showed an immunoregulatory activity mainly through cell-to-cell contact. Importantly, this γδ regulatory population decreased in the peripheral blood of systemic lupus erythematosus patients, suggesting a potential mechanism in understanding the pathogenesis of systemic lupus erythematosus.     

LPS-mediated cell surface expression of CD74 promotes the proliferation of B cells in response to MIF

Macrophage migration inhibitory factor (MIF) is a chemokine-like inflammatory cytokine, which plays a pivotal role in the pathogenesis of inflammatory and cardiovascular diseases as well as cancer. We previously identified MIF as a novel B cell chemokine that promotes B cell migration through non-cognate interaction with the CXC chemokine receptor CXCR4 and CD74, the surface form of MHC class II invariant chain. In this study, we have analyzed the regulation of the MIF receptors under inflammatory conditions by investigating the impact of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) on CD74 and CXCR4 expression in B lymphocytes. We found that both LPS and TNF-α stimulation of primary B cells and the human B myeloma cell line RPMI-8226 enhanced protein expression as well as mRNA levels of CD74 in a time- and dose-dependent manner. By contrast, no effect on CXCR4 expression was observed. Selective inhibition of IκBα phosphorylation significantly attenuated LPS-induced expression of CD74, suggesting the contribution of NF-κB signaling pathways to the regulation of CD74 expression. Importantly, individual or simultaneous blockade of MIF or CD74 using specific neutralizing antibodies markedly affected B cell proliferation after LPS exposure. Taken together, our findings unveil a connection between the pro-proliferative activity of MIF/CD74 signaling in B cells and inflammation, offering novel target mechanisms in inflammatory cardiovascular or autoimmune pathogenesis.     

Macrophage migration inhibitory factor and CD74 regulate macrophage chemotactic responses via MAPK and Rho GTPase

Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment to sites of inflammation. However, whether this stems from a direct effect on leukocyte migration is unknown. Furthermore, the role of the MIF-binding protein CD74 in this response has not been investigated. Therefore, the aim of this study was to examine the contributions of MIF and CD74 to chemokine-induced macrophage recruitment. Intravital microscopy studies demonstrated that CCL2-induced leukocyte adhesion and transmigration were reduced in MIF(-/-) and CD74(-/-) mice. MIF(-/-) and CD74(-/-) macrophages also exhibited reduced chemotaxis in vitro, although CD74(-/-) macrophages showed increased chemokinesis. Reduced CCL2-induced migration was associated with attenuated MAPK phosphorylation, RhoA GTPase activity, and actin polymerization in MIF(-/-) and CD74(-/-) macrophages. Furthermore, in MIF(-/-) macrophages, MAPK phosphatase-1 was expressed at elevated levels, providing a potential mechanism for the reduction in MAPK phosphorylation in MIF-deficient cells. No increase in MAPK phosphatase-1 expression was observed in CD74(-/-) macrophages. In in vivo experiments assessing the link between MIF and CD74, combined administration of MIF and CCL2 increased leukocyte adhesion in both MIF(-/-) and CD74(-/-) mice, showing that CD74 was not required for this MIF-induced response. Additionally, although leukocyte recruitment induced by administration of MIF alone was reduced in CD74(-/-) mice, consistent with a role for CD74 in leukocyte recruitment induced by MIF, MIF-treated CD74(-/-) mice displayed residual leukocyte recruitment. These data demonstrate that MIF and CD74 play previously unappreciated roles in CCL2-induced macrophage adhesion and migration, and they indicate that MIF and CD74 mediate this effect via both common and independent mechanisms.     

Cutting edge: fasting-induced hypoleptinemia expands functional regulatory T cells in systemic lupus erythematosus

Fasting is beneficial in the prevention and amelioration of the clinical manifestations of autoimmune diseases including systemic lupus erythematosus. The mechanisms responsible for these effects are not well understood. During fasting, there is a dramatic reduction of the levels of circulating leptin, an adipokine with proinflammatory effects. Leptin also inhibits CD4(+)CD25(+)Foxp3(+) regulatory T cells, which are known to contribute significantly to the mechanisms of peripheral immune tolerance. In this study, we show that fasting-induced hypoleptinemia in (NZB × NZW)F(1) lupus-prone mice induced an expansion of functional regulatory T cells that was reversed by leptin replacement. The specificity of the findings was indicated by the lack of these effects in leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice. These observations help to explain the beneficial effects of fasting in autoimmunity and could be exploited for leptin-based immune intervention in systemic lupus erythematosus.     

Nasal anti-CD3 antibody ameliorates lupus by inducing an IL-10-secreting CD4+ CD25- LAP+ regulatory T cell and is associated with down-regulation of IL-17+ CD4+ ICOS+ CXCR5+ follicular helper T cells

Lupus is an Ab-mediated autoimmune disease. One of the potential contributors to the development of systemic lupus erythematosus is a defect in naturally occurring CD4(+)CD25(+) regulatory T cells. Thus, the generation of inducible regulatory T cells that can control autoantibody responses is a potential avenue for the treatment of systemic lupus erythematosus. We have found that nasal administration of anti-CD3 mAb attenuated lupus development as well as arrested ongoing lupus in two strains of lupus-prone mice. Nasal anti-CD3 induced a CD4(+)CD25(-)latency-associated peptide (LAP)(+) regulatory T cell that secreted high levels of IL-10 and suppressed disease in vivo via IL-10- and TFG-beta-dependent mechanisms. Disease suppression also occurred following adoptive transfer of CD4(+)CD25(-)LAP(+) regulatory T cells from nasal anti-CD3-treated animals to lupus-prone mice. Animals treated with nasal anti-CD3 had less glomerulonephritis and diminished levels of autoantibodies as measured by both ELISA and autoantigen microarrays. Nasal anti-CD3 affected the function of CD4(+)ICOS(+)CXCR5(+) follicular helper T cells that are required for autoantibody production. CD4(+)ICOS(+)CXCR5(+) follicular helper T cells express high levels of IL-17 and IL-21 and these cytokines were down-regulated by nasal anti-CD3. Our results demonstrate that nasal anti-CD3 induces CD4(+)CD25(-)LAP(+) regulatory T cells that suppress lupus in mice and that it is associated with down-regulation of T cell help for autoantibody production.     

Macrophage migration inhibitory factor induces macrophage recruitment via CC chemokine ligand 2

Macrophage migration inhibitory factor (MIF) was originally identified for its ability to inhibit the random migration of macrophages in vitro. MIF is now recognized as an important mediator in a range of inflammatory disorders. We recently observed that the absence of MIF is associated with a reduction in leukocyte-endothelial cell interactions induced by a range of inflammatory mediators, suggesting that one mechanism whereby MIF acts during inflammatory responses is by promoting leukocyte recruitment. However, it is unknown whether MIF is capable of inducing leukocyte recruitment independently of additional inflammatory stimuli. In this study, we report that MIF is capable of inducing leukocyte adhesion and transmigration in postcapillary venules in vivo. Moreover, leukocytes recruited in response to MIF were predominantly CD68(+) cells of the monocyte/macrophage lineage. Abs against the monocyte-selective chemokine CCL2 (JE/MCP-1) and its receptor CCR2, but not CCL3 and CXCL2, significantly inhibited MIF-induced monocyte adhesion and transmigration. CCL2(-/-) mice displayed a similar reduction in MIF-induced recruitment indicating a critical role of CCL2 in the MIF-induced response. This hypothesis was supported by findings that MIF induced CCL2 release from primary microvascular endothelial cells. These data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues. This function may be critical to the ability of MIF to promote diseases such as atherosclerosis and rheumatoid arthritis, in which macrophages are key participants.     

CD4+CD25+ regulatory T cells control innate immune reactivity after injury

Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1(-/-)) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-alpha, IL-1beta, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1(-/-), CD4(-/-), or CD8(-/-) mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8(-/-) mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4(-/-) mice produced cytokines at significantly higher levels, equivalent to those in Rag1(-/-) mice. Moreover, reconstitution of Rag1(-/-) or CD4(-/-) mice with WT CD4(+) T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4(+) T cells into CD4(+)CD25(+) and CD4(+)CD25(-) subpopulations before their adoptive transfer into Rag1(-/-) mice showed that CD4(+)CD25(+) T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4(+)CD25(-) T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4(+)CD25(+) T regulatory cells in controlling host inflammatory responses after injury.     

Helicobacter pylori CagA-dependent macrophage migration inhibitory factor produced by gastric epithelial cells binds to CD74 and stimulates procarcinogenic events

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has recently been implicated in carcinogenesis. Helicobacter pylori, which is closely linked to gastric cancer, induces the gastric epithelium to produce proinflammatory cytokines, including MIF. MIF can bind to CD74, which we have previously shown to be highly expressed on the surface of gastric epithelial cells (GEC) during H. pylori infection. In this study, we sought to investigate the role of the H. pylori-induced MIF on epithelial proliferation and procarcinogenic events. Upon establishing a role for the H. pylori CagA virulence factor in MIF production, MIF binding to CD74 on GEC was confirmed. rMIF and H. pylori were shown to increase GEC proliferation, which was decreased when cagA- strains were used and when CD74 was blocked by mAbs. Apoptosis was also decreased by MIF, but increased by cagA- strains that induced much lower amounts of MIF than the wild-type bacteria. Furthermore, MIF binding to CD74 was also shown to decrease p53 phosphorylation and up-regulate Bcl-2 expression. This data describes a novel system in which an H. pylori virulence factor contributes to the production of a host factor that in turn up-regulates procarcinogenic events by the gastric epithelium.     

FcalphaRI (CD89) alleles determine the proinflammatory potential of serum IgA

The human IgA FcR (FcalphaRI; CD89) mediates a variety of immune system functions including degranulation, endocytosis, phagocytosis, cytokine synthesis, and cytokine release. We have identified a common, nonsynonymous, single nucleotide polymorphism (SNP) in the coding region of CD89 (844A-->G) (rs16986050), which changes codon 248 from AGC (Ser(248)) to GGC (Gly(248)) in the cytoplasmic domain of the receptor. The two different alleles demonstrate significantly different FcalphaRI-mediated intracellular calcium mobilization and degranulation in rat basophilic leukemia cells and cytokine production (IL-6 and TNF-alpha) in murine macrophage P388D1 cells. In the absence of FcR gamma-chain association in P388D1 cells, the Ser(248)-FcalphaRI allele does not mediate cytokine production, but the Gly(248)-FcalphaRI allele retains the capacity to mediate a robust production of proinflammatory cytokine. This allele-dependent difference is also seen with FcalphaRI-mediated IL-6 cytokine release by human neutrophils ex vivo. These findings and the enrichment of the proinflammatory Gly(248)-FcalphaRI allele in systemic lupus erythematosus populations in two ethnic groups compared with their respective non-systemic lupus erythematosus controls suggest that FcalphaRI (CD89) alpha-chain alleles may affect receptor-mediated signaling and play an important role in the modulation of immune responses in inflammatory diseases.     

IL-10 regulates murine lupus

MRL/MpJ-Tnfrsf6(lpr) (MRL/MpJ-Fas(lpr); MRL-Fas(lpr)) mice develop a spontaneous lupus syndrome closely resembling human systemic lupus erythematosus. To define the role of IL-10 in the regulation of murine lupus, IL-10 gene-deficient (IL-10(-/-)) MRL-Fas(lpr) (MRL-Fas(lpr) IL-10(-/-)) mice were generated and their disease phenotype was compared with littermates with one or two copies of an intact IL-10 locus (MRL-Fas(lpr) IL-10(+/-) and MRL-Fas(lpr) IL-10(+/+) mice, respectively). MRL-Fas(lpr) IL-10(-/-) mice developed severe lupus, with earlier appearance of skin lesions, increased lymphadenopathy, more severe glomerulonephritis, and higher mortality than their IL-10-intact littermate controls. The increased severity of lupus in MRL-Fas(lpr) IL-10(-/-) mice was closely associated with enhanced IFN-gamma production by both CD4(+) and CD8(+) cells and increased serum concentration of IgG2a anti-dsDNA autoantibodies. The protective effect of IL-10 in this lupus model was further supported by the observation that administration of rIL-10 reduced IgG2a anti-dsDNA autoantibody production in wild-type MRL-Fas(lpr) animals. In summary, our results provide evidence that IL-10 can down-modulate murine lupus through inhibition of pathogenic Th1 cytokine responses. Modulation of the level of IL-10 may be of potential therapeutic benefit for human lupus.     

Developments in the clinical understanding of lupus

Advances in genetics and new understanding of the molecular pathways that mediate innate and adaptive immune system activation, along with renewed focus on the role of the complement system as a mediator of inflammation, have stimulated elaboration of a scheme that might explain key mechanisms in the pathogenesis of systemic lupus erythematosus. Clinical observations identifying important comorbidities in patients with lupus have been a recent focus of research linking immune mechanisms with clinical manifestations of disease. While these advances have identified rational and promising targets for therapy, so far the therapeutic trials of new biologic agents have not met their potential. Nonetheless, progress in understanding the underlying immunopathogenesis of lupus and its impact on clinical disease has accelerated the pace of clinical research to improve the outcomes of patients with systemic lupus erythematosus.     

Macrophage CD74 contributes to MIF-induced pulmonary inflammation

Background

MIF is a critical mediator of the host defense, and is involved in both acute and chronic responses in the lung. Neutralization of MIF reduces neutrophil accumulation into the lung in animal models. We hypothesized that MIF, in the alveolar space, promotes neutrophil accumulation via activation of the CD74 receptor on macrophages.

Methods

To determine whether macrophage CD74 surface expression contributes MIF-induced neutrophil accumulation, we instilled recombinant MIF (r-MIF) into the trachea of mice in the presence or absence of anti-CD74 antibody or the MIF specific inhibitor, ISO-1. Using macrophage culture, we examined the downstream pathways of MIF-induced activation that lead to neutrophil accumulation.

Results

Intratracheal instillation of r-MIF increased the number of neutrophils as well as the concentration of macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived chemokine (KC) in BAL fluids. CD74 was found to be expressed on the surface of alveolar macrophages, and MIF-induced MIP-2 accumulation was dependent on p44/p42 MAPK in macrophages. Anti-CD74 antibody inhibited MIF-induced p44/p42 MAPK phosphorylation and MIP-2 release by macrophages. Furthermore, we show that anti-CD74 antibody inhibits MIF-induced alveolar accumulation of MIP-2 (control IgG vs. CD74 Ab; 477.1 ± 136.7 vs. 242.2 ± 102.2 pg/ml, p < 0.05), KC (1796.2 ± 436.1 vs. 1138.2 ± 310.2 pg/ml, p < 0.05) and neutrophils (total number of neutrophils, 3.33 ± 0.93 × 10⁴ vs. 1.90 ± 0.61 × 10⁴, p < 0.05) in our mouse model.

Conclusion

MIF-induced neutrophil accumulation in the alveolar space results from interaction with CD74 expressed on the surface of alveolar macrophage cells. This interaction induces p44/p42 MAPK activation and chemokine release. The data suggest that MIF and its receptor, CD74, may be useful targets to reduce neutrophilic lung inflammation, and acute lung injury.