5. The Interaction of MIF and Glucocorticoids
MIF is one of cytokine known to be upregulated by glucocorticoids (GCs), which suggests MIF plays a key role in regulating host global responses to infections, as GCs are released from the hypothalamus-pituitary-adrenal axis [17, 35]. GCs elicit MIF secretion both in vitro and in vivo, but the manner in which MIF is regulated by GC appears to be complex. Although very low concentrations of dexamethasone (1014 M) stimulate MIF secretion [19, 36], this induction is not accompanied by changes in MIF gene expression, and posttranslational mechanisms have been proposed [37]. On the other hand, Leech et al. showed that mRNA expression by RA broblast-like synoviocytes (FLS) was induced by the stimulation of lower concentrations of dexamethasone (M) [16]. Upon release, MIF exerts an inhibitory eect on GC activity. For example, recombinant MIF counteracts GC-induced suppression of cytokine production in macrophages and T lymphocytes [9, 19]. In vivo, MIF overcomes the protective eect of GCs in murine models of endotoxic shock [19]. And in an antigen-induced arthritis model, exogenous MIF reverses the inhibitory eect of GC on arthritis inammation, but does not aect GC-induced inhibition of delayed-type hypersensitivity, which suggests there are differences in the sensitivity of inammatory processes to MIF [38].

4. A MIF Receptor

The signal transduction pathways utilized by MIF during its activation of cells and cellular processes are incompletely dened, but one MIF receptor is known to be CD74, the cell surface form of the class II invariant chain [29]. The interaction of MIF with CD74 has been conrmed in pulldown experiments, and confocal microscopic examination showed the two proteins to be colocalized within cells [29]. MIF-induced cellular activation appears to be mediated via MAPK and a transcription factor, activator protein 1 (AP-1); that is, MIF appears to signal via classical receptor-dependent activation of MAPK upon binding to CD74 [29]. In addition, recent studies have identied recruitment of transmembrane CD44 as a potential accessory protein required for MIFCD74 signal transduction [30, 31]. These data show that the serine phosphorylation of the CD74 intracytoplasmic domain by MIF stimulation is dependent upon CD44. Of interest, more recently, crucial roles of chemokine receptors in MIF-CD74 pathway were elucidated. Bernhagen et al. have shown that the chemokine receptors CXCR2 and CXCR4 are functional receptors for MIF [32]. MIF triggered Gi and integrin-dependent arrest and chemotaxis of monocytes and T cells, rapid integrin activation, and calcium inux through CXCR2 or CXCR4. Also, CXCR2 and CD74 formed a receptor complex, and monocyte arrest elicited by MIF in inamed or atherosclerotic arteries involved both CXCR2 and CD74 [32]. Recent advances in understanding of MIF signaling pathway may have an important contribution for new therapeutic strategies for inammatory/immune diseases. It has been demonstrated that an orally bioavailable MIF antagonist, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5isoxazole acetic acid methyl ester (ISO-1) [33], inhibited the interaction between MIF and CD74 and reduced functional and histological indices of glomerulonephritis, CD74(+) and CXCR4(+) leukocyte recruitment, and proinammatory

6. Biological Activities of MIF
6.1. Chemotactic Activities. Although MIF was rst identied as an inhibitor of macrophage migration [39], induction of leukocyte rolling, adhesion, and transmigration by LPS and other inammatory mediators is diminished in MIF/ mice [40]. Similarly, blockade or depletion of MIF reduces leukocyte accumulation in models of infection/endotoxemia, arthritis and atherogenesis [4143]. Nonetheless, MIF clearly induces adhesion and migration of monocyte-lineage cells in postcapillary venules, and that function is mediated by the chemokine CCL2 (MCP-1), which is induced in ECs by MIF, itself [44]. In addition, expression and secretion of MIF by vascular smooth muscle cells (VSMCs) is increased when the cells are stimulated with oxidized lowdensity lipoprotein, and recombinant MIF enhances the migration of VSMCs [45]. This suggests that MIF acts in an autocrine and paracrine fashion to modulate the migration of VSMCs, and may be associated with the development of advanced lesions during the course of atherogenesis. MIF also induces chemotaxis in broblasts [46] and ECs [47], and the eects of neutralizing MIF in experimental autoimmune myocarditis indicates that it also stimulates migration T cells [48]. In addition, lipopolysaccharideinduced leukocyte-EC interaction was promoted by endogenous MIF, via endogenous GC-independent mechanisms
Arthritis [49]. More recently, Cheng et al. have shown that endogenous MIF promotes leukocyte recruitment via eects on endothelial expression of several adhesion molecules, including E-selectin, intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM)-1, and chemokines, including IL-8 (CXCL8) and CCL2 whereas exogenous MIF facilitates leukocyte recruitment induced by TNF by promoting endothelial P-selectin expression, which contributed to leukocyte rolling [50]. Thus MIF appears to have broad eects on the recruitment of leukocytes, VSMCs, broblasts, and leukocyte-EC interactions mediated by several inammatory molecules in inammatory/noninammatory disorders. 6.2. Induction of Inammatory Cytokines by MIF. MIF stimulates release of the proinammatory cytokines TNF-, IL1, IL-6, IL-8, and IL-12 from macrophages and up-regulates matrix metalloproteinase (MMP) -1, MMP-3, MMP-9 and MMP-13 in RA FLS [16, 5157]. In addition, MIF upregulates the adhesion molecules VCAM-1 and ICAM-1 on ECs and monocytes [58, 59]. In vivo, MIF deciency or neutralization has protective eects against lethal bacterial sepsis and septic shock induced by Gram-negative endotoxin [17] or Gram-positive exotoxin [60]. In chronic inammatory diseases, MIF reduction is associated with lower levels of circulating or local TNF and IL-1 production [61, 62], suggesting MIF is a crucial regulator of inammatory cytokine expression. In vitro, MIFdecient cells exhibit impaired TNF production in response to LPS, an eect mediated via MIF regulation of TLR4dependent cellular responses. In dierent experimental models of sepsis, blocking MIF activity, either through MIF gene disruption or with anti-MIF antibodies, reduces cytokine production by downregulating TLR4 expression [63]. MIF up-regulates macrophage TLR4 expression via the transcription factor PU.1 [63]. In contrast to other proinammatory cytokines, MIF does not induce nuclear translocation of nuclear factor(NF-)B p50 or p60 proteins at concentrations that activate ERK, and inhibitors of the NF-B pathway do not inhibit MIF-induced biological eects on FLS [64]. In addition, a recent study using an experimental diabetes model showed that MIF/ mice are less susceptible to disease induction, and the reduced susceptibility was associated with lower levels of lymphocyte proliferation and adhesion and decreased splenic production of IL-23 [65]. Because IL-23 can enhance IL-17 production [66, 67], it may be that MIF regulates IL-17 indirectly via its eects on IL-23, which is likely involved in RA and other inammatory disorders. 6.3. Other Activities of MIF. It was recently conrmed that MIF potently stimulates nitric oxide production, which can directly mediate cell injury [68, 69] and enhance macrophage activities such as intracellular killing, phagocytosis, and H2 O2 production [70]. MIF also acts as a potent angiogenic factor in autoimmune diseases [71], exerting its angiogenic eects through induction of such angiogenic mediators as vascular endothelial growth factor (VEGF) [72, 73].

7. MIF Expression and Function under Immune/Inammatory Conditions
7.1. MIF in RA. MIF has been implicated in a number of inammatory and immune-mediated diseases, including RA [16] and other inammatory arthritis. MIF levels are increased in synovial uid and synovial tissue from RA patients and patients with juvenile idiopathic arthritis [74 76]. Of interest, synovial MIF immunostaining correlated strongly with disease activity, and reductions in clinical disease parameters were accompanied by signicant reductions in synovial MIF [74]. Also, Kim et al. have shown that serum inammatory markers such as ESR and CRP were correlated with SF levels of MIF, and the SF levels of MIF were found to be higher in patients with bony erosion than in those without. In addition, MIF levels correlated with VEGF levels in both sera and synovial uids of patients with RA [72]. MIF activates RA FLSs to produce IL-8, cyclooxygenase 2 (COX-2), MMP-1, and MMP-3 via tyrosine kinase-, protein kinase C-, and AP-1-dependent pathways, which contribute to inammation and tissue destruction [52, 57]. Also, in another function of MIF in synovial inammation, the eects of MIF on FLS activation and proliferation are dependent on extracellular signal-regulated kinase (ERK) and mitogen-activated protein (MAP) kinase [64, 74]. Moreover, in animal models for inammatory arthritis such as adjuvant-induced arthritis, collagen-induced arthritis, and antigen-induced arthritis (AIA), it was clearly shown that MIF was involved in the pathogenesis of inammatory arthritis, and the development and severity of the arthritis and the inltration of inammatory cells into joint tissues were signicantly suppressed by administration of an antiMIF polyclonal antibody [20, 38, 57, 77], suggesting that MIF may be functionally active during the development of arthritis. Furthermore, exogenous MIF inhibited p53 expression in RA FLS and also increased p53 protein was detected in cells and synovial tissue derived from MIF/ mice with AIA, suggesting that MIF exerts an antiapoptotic eect in association with its inhibition of p53 in arthritic joints [78]. Finally, recent nding also indicates that MIF may be one of crucial target against anti-TNF therapy in patients with RA [79]. Taken together, these observations indicate that MIF may play important roles in the evolution of the synovitis and joint destruction in RA via modulation of inammation, angiogenesis and chemotaxis of inammatory cells. Although FLS and synovial macrophages are an important cellular source of MIF secretion in synovial tissues of RA [16], MIF was also secreted by dendritic cells (DC) in patients with RA [27]. In monocyte-derived dendritic cells (DC) from RA patients, TLRs signicantly enhance production of proinammatory mediators, including MIF, thereby amplifying the proinammatory loop seen in arthritis. Moreover, stimulation of DC with TLR4 ligands elicited higher levels of MIF production than stimulation of immature DC from healthy controls or RA patients [27]. Also TLR4 stimulation is known to induce the MIF secretion [10]. Intriguingly, TLR2 and TLR4 are both highly expressed in the synovial tissue of RA patients, and TLR4 ligands are abundant in

4 the serum and synovial uid of RA patients, suggesting TLR signaling likely occurs in the synovial compartment of these patients [26]. The MIF gene is located on chromosome 22q11.2 [80]. The identication of a single-nucleotide polymorphism at position -173 (MIF-173C allele) and a CATT58 tetranucleotide repeat element in the promoter region of the MIF gene has sparked research into the role of these variants in inammatory conditions. It has also been demonstrated that the presence of specic alleles of the MIF CATT tetranucleotide repeat correlates with the severity of RA [81]. In addition, Barton et al. found that the MIF-173C allele and the MIF CATT repeat are associated with susceptibility to inammatory arthritis, but they were unable to nd a correlation with disease severity [82]. Martinez showed that the -173C allele in the MIF promoter region is associated with an increased predisposition toward RA, mainly in earlyonset patients [83]. The fact that a substantial amount of evidence points toward a role for MIF in the pathogenesis of RA prompted investigation of the potential association between the two MIF genetic variants and the susceptibility to and severity of RA, using a large cohort of welldocumented, prospectively followed up patients with RA. Radstake et al. showed that the MIF polymorphisms (-173C and CATT alleles) are associated with the rate of radiologic joint damage, but not with RA susceptibility [84]. Increased MIF levels were shown to correlate strongly with radiologic joint damage, and carriership of the MIF-173C allele or MIF CATT allele was associated with markedly higher levels of circulating plasma MIF, suggesting that MIF expression is genetically determined and can be used as a novel prognostic tool in RA [84]. Consistent with that idea, circulating MIF levels were increased in individuals with juvenile idiopathic arthritis carrying the MIF-173C functional variant, and increased susceptibility to the disease was associated with carriership of either the MIF-173C or CATT allele [75, 76]. It was therefore suggested that for juvenile idiopathic arthritis, the MIF-173C allele is a predictor of poor outcome [75]. 7.2. MIF in SLE. SLE is an autoimmune disease characterized by multiorgan damage with inltration of inammatory cells/immune cells, and the production of autoantibodies. Although the pathogenesis of SLE has not been fully elucidated, recent progress has provided evidence that several cytokines/chemokines are detectable in the sera and also damaged organ of SLE patients during active disease. Besides RA, expression and function of MIF were clearly demonstrated in SLE and related conditions. Foote et al. have shown that serum MIF concentrations were positively associated with SLE disease damage. Also, serum MIF was positively associated with current corticosteroid dose and negatively associated with serum creatinine concentration [85]. A marked increase in both glomerular and tubular MIF expression was seen in lupus nephritis, and also in focal segmental glomerulosclerosis (FGS) and mesangiocapillary proliferative GN [86]. In immunohistochemical study, the prominent macrophage and T-cell inltrate showed were largely restricted to areas with marked upregulation of

Cytokines LPS TLRs

Arthritis

Stimuli

Inammatory/immune cells Pannus Kidney Blood vessels Gut Brain
IL-1, 6, 8, 12 TNF-, CCL2, VEGF inammatory mediators

Glucocorticoids

Amplication of the proinammatory loop by MIF
Figure 1: The orchestration of complicated cytokine networks by MIF in inammatory/immune responses. MIF is a multipotent cytokine involved in the regulation of immune and inammatory responses via other various mediators, and plays a key role in regulating a number of inammatory and immune-mediated diseases seen in pannus, blood vessels, kidney, gut, and brain.
MIF expression, contributing to glomerular hypercellularity, glomerular focal segmental lesions, crescent formation, tubulitis, and granulomatous lesions. In addition, the positive association of functional polymorphisms of MIF (-173C and CATT alleles), and the prevalence of SLE was shown by S nchez et al. [87]. a In the lupus-prone mice, it has been demonstrated that renal MIF expression was signicantly higher in MRL/lpr mice compared with nondiseased control mice. Also, MRL/lpr mice with MIF/ exhibited signicantly prolonged survival, and reduced renal and skin manifestations. In addition, renal macrophage recruitment and glomerular injury were signicantly reduced in MRL/lpr mice MIF/ , in association with reduction in CCL2 (macrophage chemoattractant protein-1) [88]. In addition, antiapoptotic eect was seen in the active lesion of inammatory arthritis as described above. This function of MIF may be crucial in the development of SLE, because apoptosis and clearance of apoptotic cells/material are considered key processes in the etiology of SLE. Matsumoto et al. reported that urinary excretion of MIF is increased in patients with focal glomerular sclerosis and that urinary MIF levels, are higher in patients with active glomerular lesions [89]. Immunocytochemical and in situ hybridization studies have shown that MIF is produced by local resident glomerular cells [22], and that administration of a neutralizing anti-MIF antibody dramatically suppresses an immunologically induced disease model of rapidly progressive crescentic glomerulonephritis (GN) [61]. In addition, both MIF mRNA and protein were detected in intrinsic renal cells and glomerular ECs and were markedly up-regulated in more severe forms of GN (e.g., crescentic GN) [22, 86, 90]. The urine MIF concentration was increased

Table 1: Biological activities of MIF. Chemotactic activities Monocytes stimulation/inhibition: dependent on its concentration
5 disease activity, including Birmingham vasculitis activity scores, CRP levels and ESR. Furthermore, MIF levels were signicantly diminished in MPA patients exhibiting clinical improvement after treatment. Similarly, serum MIF levels were elevated in patients with antineutrophil cytoplasmic antibody- (ANCA-) related vasculitis [95], as well as in Wegeners granulomatous and Kawasaki disease [96]. These ndings indicate that MIF expression is not specic to RA, but may also function as an important regulator of systemic vasculitis. Serum levels of endothelium-related molecules such as adhesion molecules and EC-derived cytokines are reportedly increased in patients with vasculitis [102, 103]. Indeed, vasculitis aecting small vessels may be associated with dysregulated EC function [104]. In patients with MPA, for example, the origin of the elevated serum MIF appears to be ECs and/or inammatory cells such as monocytes and PMNs [10, 25]. Once secreted, MIF likely participates in regulating EC proliferation [47]. There is also a positive correlation between serum MIF levels and MPO-ANCA titers in MPA patients [94]. Although there are no data on the capacity of MPO-ANCA to stimulate secretion of any cytokine, including MIF, we would expect it to be related to disease activity and MIF levels, because there appears to be positive relation between MPO-ANCA titers and vasculitis disease activity [105]. As mentioned earlier, MIF up-regulates ICAM-1 on ECs [58], as well as the expression and secretion of other inammatory cytokines, including TNF- and IL-8 [16, 57]. This in turn would be expected to enhance recruitment of leukocytes to sites of inammation, which involves adhesion molecule-dependent interactions with ECs. We also recently observed that serum MIF levels are signicantly higher in RA patients with vasculitis (rheumatoid vasculitis; RV) than in those without it (manuscript in preparation). We found that in RV patients, there are significant positive correlations between MIF levels and vasculitis disease activity scores and serum levels of immune complex and a signicant negative correlation between MIF levels and serum complement levels. Notably, MIF levels in RV patients also correlated signicantly with levels of thrombomodulin, expression of which is associated with endothelial damage and/or vascular inammation. Collectively, these ndings suggest dysregulated orchestration of the activities of MIF, adhesion molecules, and cytokines expressed by ECs and/or leukocytes plays a crucial role in the development of systemic vasculitis (e.g., MPA and RV). 7.4. MIF in Other Rheumatic Diseases and Related Conditions. Serum MIF levels and dermal broblast-derived MIF synthesis are both up-regulated in scleroderma, suggesting that MIF participates in the amplifying proinammatory loop that leads to sclerodermal tissue remodeling [97]. Serum MIF levels are also signicantly elevated in patients with primary Sj grens syndrome, especially in those with increased o globulins [98]. It has been shown that MIF signaling stimulates B cell proliferation [106], and that neutralization of MIF signicantly inhibits antibody production in vivo [9]. Increased production of MIF might therefore contribute to hypergammaglobulinemia and possibly reect the disease

T lymphocytes ECs Vascular smooth muscle cells Fibroblasts Angiogenic activities Antiapoptotic activities Stimulation of cell proliferation Induction of mediators Cytokines TNF- IL-1, 6, 8, 12 CCL2 VEGF ICAM-1 VCAM-1 E-selectin P-selectin MMP-1, 3, 9, 13
Growth factor Adhesion molecules
Proteinases Nitric oxide Superoxide
Chemotactic activities of MIF against monocytes/macrophages may be dependent on its concentration. VEGF; vascular endothelial growth factor, ICAM-1; intercellular cell adhesion molecule-1, VCAM-1; vascular cell adhesion molecule-1, MMP; matrix metalloproteinase.
3.4-fold in proliferative nephropathies and especially in crescentic GN (4.5-fold), but not nonproliferative nephropathies [91].There was a signicant correlation between the urine MIF concentration and renal MIF expression, but not with serum MIF, indicating a renal origin for the excreted urine MIF. The urine MIF concentration also correlated with the degree of renal dysfunction, histologic damage, and leukocytic inltration. Mesangial and tubular epithelial cells, as well as glomerular capillary ECs, are the major sources of MIF in GN [22, 90]. The secreted MIF then promotes macrophage activation and secretion of macrophage-derived cytokines, including IL-1 and broblast growth factor, which may induce mesangial cell proliferation [10, 92]. It thus appears that when combined with other factors, MIF expressed in the inamed kidney contributes to the development of the renal damage in GN. 7.3. MIF in Systemic Vasculitis. We recently showed that serum MIF levels are signicantly increased in some patients with systemic vasculitis [94]. Notably, signicantly elevated levels of serum MIF were seen in patients with microscopic polyangiitis (MPA), which is a small vessel vasculitis, but not in patients with medium vessel vasculitis, such as polyarteritis nodosa, or large vessel vasculitis, such as giant cell arteritis and Takayasu arteritis. The elevated MIF levels seen in MPA patients correlated positively with indexes of
Table 2: Involvement of MIF in various pathological conditions Diseases References RA, including rheumatoid vasculitis [16, 27, 52, 57, 72, 74, 79] Crohns disease [54, 93] Juvenile idiopathic arthritis [75, 76] Systemic lupus erythematosus [8587] Crescentic glomerulonephritis [86, 91] Focal glomerular sclerosis [89] Microscopic polyangiitis [94, 95] Wegeners granulomas [95] Kawasaki disease [96] Systemic sclerosis [97] Sj grens syndrome o [98] Ankylosing spondylitis [99] Adult-onset Stills disease [100] Relapsing polychondritis [101]

activity of Sj grens syndrome. Ankylosing spondylitis (AS) o is a chronic inammatory disease mainly aecting the spine and sacroiliac joints. MIF levels were signicantly higher in the AS patients than in normal individuals, which correlated with the Bath Ankylosing Spondylitis Metrology Index a composite clinical index for AS [99]. Furthermore, important pathogenic contributions of MIF have been suggested by studies in adult-onset Stills disease [100], ocular inammation [107], relapsing polychondritis [101], experimental autoimmune encephalomyelitis, a model of multiple sclerosis [71], inammatory bowel disease, Crohns disease, and experimental colitis [54, 93].

8. Conclusion

The biological activities of MIF and its relevance in various diseases are summarized in Tables 1 and 2. The central involvement of this multifunctional cytokine highlights its importance in the pathogenesis of inammatory diseases (Figure 1). Moreover, it suggests that blocking MIF may be a useful therapeutic strategy for treating these diseases.

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