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Genetic predisposition to coronavirus disease 2019 (COVID-19) may contribute to its morbidity and mortality. Because cytokines play an important role in multiple phases of infection, we examined whether commonly occurring, functional polymorphisms in macrophage migration inhibitory factor (MIF) are associated with COVID-19 infection or disease severity.

Macrophage migration inhibitory factor (MIF) activates CD74, which leads to severe disorders including inflammation, autoimmune diseases and cancer under pathological conditions. Molecular dynamics (MD) simulations up to one microsecond revealed dynamical correlation between a residue located at the opening of one end of the MIF solvent channel, previously thought to be a consequence of homotrimerization, and residues in a distal region responsible for CD74 activation. Experiments verified the allosteric regulatory site and identified a pathway to this site via the MIF β-strands. The reported findings provide fundamental insights on a dynamic mechanism that controls the MIF-induced activation of CD74.

Contains fulltext : 170277.pdf (publisher's version ) (Open Access)

Contains fulltext : 170277.pdf (publisher's version ) (Open Access)

Contains fulltext : 170277.pdf (publisher's version ) (Open Access)

Objective Glucocorticoids remain a mainstay in the treatment of rheumatoid arthritis (RA). Dose-dependent adverse effects highlight the need for therapies that regulate glucocorticoid sensitivity to enable dosage reduction. Macrophage migration inhibitory factor (MIF) is a proinflammatory protein that has been implicated in the pathogenesis of RA; it impairs glucocorticoid sensitivity via MAPK phosphatase 1 (MKP-1) inhibition. The intracellular protein glucocorticoid-induced leucine zipper (GILZ) mimics the effects of glucocorticoids in models of RA, but whether it represents a target for the modulation of glucocorticoid sensitivity remains unknown. We undertook this study to investigate whether GILZ is involved in the regulation of glucocorticoid sensitivity by MIF. Methods GILZ expression was studied in the presence and absence of MIF, and the role of GILZ in the MIF-dependent regulation of the glucocorticoid sensitivity mediator MKP-1 was studied at the level of expression and function. Results GILZ expression was significantly inhibited by endogenous MIF, both basally and during responses to glucocorticoid treatment. The effects of MIF on GILZ were dependent on the expression and Akt-induced nuclear translocation of the transcription factor FoxO3A. GILZ was shown to regulate the expression of MKP-1 and consequent MAPK phosphorylation and cytokine release. Conclusion MIF exerts its effects on MKP-1 expression and MAPK activity through inhibitory effects on GILZ. These findings suggest a previously unsuspected interaction between MIF and GILZ and identify GILZ as a potential target for the therapeutic regulation of glucocorticoid sensitivity. Copyright © 2014 by the American College of Rheumatology.

Objective Glucocorticoids remain a mainstay in the treatment of rheumatoid arthritis (RA). Dose-dependent adverse effects highlight the need for therapies that regulate glucocorticoid sensitivity to enable dosage reduction. Macrophage migration inhibitory factor (MIF) is a proinflammatory protein that has been implicated in the pathogenesis of RA; it impairs glucocorticoid sensitivity via MAPK phosphatase 1 (MKP-1) inhibition. The intracellular protein glucocorticoid-induced leucine zipper (GILZ) mimics the effects of glucocorticoids in models of RA, but whether it represents a target for the modulation of glucocorticoid sensitivity remains unknown. We undertook this study to investigate whether GILZ is involved in the regulation of glucocorticoid sensitivity by MIF. Methods GILZ expression was studied in the presence and absence of MIF, and the role of GILZ in the MIF-dependent regulation of the glucocorticoid sensitivity mediator MKP-1 was studied at the level of expression and function. Results GILZ expression was significantly inhibited by endogenous MIF, both basally and during responses to glucocorticoid treatment. The effects of MIF on GILZ were dependent on the expression and Akt-induced nuclear translocation of the transcription factor FoxO3A. GILZ was shown to regulate the expression of MKP-1 and consequent MAPK phosphorylation and cytokine release. Conclusion MIF exerts its effects on MKP-1 expression and MAPK activity through inhibitory effects on GILZ. These findings suggest a previously unsuspected interaction between MIF and GILZ and identify GILZ as a potential target for the therapeutic regulation of glucocorticoid sensitivity. Copyright © 2014 by the American College of Rheumatology.

Objective. Recent studies have established an essential role for macrophage migration inhibitory factor (MIF) in T cell and macrophage activation, both of which are characteristics of rat adjuvant arthritis. This study investigated the role of MIF in early adjuvant arthritis. Methods. MIF was detected in rat synovium by immunohistochemistry and enzyme-linked immunosorbent assay using specific monoclonal antibodies (MAb). Anti-MIF MAb treatment was administered, and the effects on clinical aspects of adjuvant arthritis were assessed. Results. MIF was absent from normal rat synovium prior to adjuvant injection, but was detectable on day 4 after injection (6 days before the onset of clinical disease) and was colocalized with ED-1+ macrophages throughout the evolution of the disease. Levels of MIF were increased in established adjuvant arthritis sera, and adjuvant arthritis synovial macrophages released MIF at a mean +/- SEM concentration of 607.9 +/- 201.5 pg/ml. Anti-MIF treatment led to profound, dose-dependent inhibition of the adjuvant arthritis clinical score, paw swelling, and synovial lavage leukocyte numbers (P < 0.001), and also resulted in reduced synovial macrophage and T cell accumulation. Conclusion. These findings demonstrate an important role for MIF in the evolution of rat adjuvant arthritis.

Objective. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose involvement in tumor necrosis factor alpha (TNFalpha) synthesis and T cell activation suggests a role in the pathogenesis of rheumatoid arthritis (RA). Antagonism of MIF is associated with marked inhibition of animal models of RA. Uniquely, MIF is inducible by low concentrations of glucocorticoids. We sought to investigate the expression of MIF in RA synovial tissue. Methods. MIF was demonstrated in human RA synovium by immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). Regulation of MIF expression was investigated by treatment of cultured fibroblast-like synoviocytes (FLS) with interleukin-1beta (IL-1beta), TNFalpha, or interferon-gamma (IFNgamma), and dexamethasone (DEX). Mononuclear cell TNFalpha release after exposure to FLS-conditioned medium was measured by ELISA. Results. MIF was present in RA synovial lining CD14+ macrophages and FLS. Constitutive MIF messenger RNA (mRNA) expression was demonstrated by RT-PCR of RNA from unstimulated cultured RA FLS, which also released abundant MIF. Serum, synovial fluid, and FLS intracellular MIF were significantly higher in RA patients than in controls. Synoviocyte MIF was not increased by IL-1beta, TNFalpha, or IFNgamma. In contrast, DEX 10-7M significantly reduced synoviocyte MIF, while DEX 10-10-10-12M induced a significant increase in MIF and MIF mRNA. Peripheral blood mononuclear cell TNFalpha release was induced by culture in RA FLS-conditioned medium, and this induction was significantly abrogated by monoclonal anti-MIF antibody, suggesting that MIF is an upstream regulator of TNFalpha release. Conclusion. These data represent the first demonstration of the cytokine MIF in human autoimmune disease and suggest MIF as a potential therapeutic target in RA.

Background and Aim: The progression of renal injury, initiated by either an immune or non-immune insult, is closely associated with the accumulation of leucocytes and fibroblasts in the damaged kidney. Macrophage migration inhibitory factor (MIF) regulates leucocyte activation and fibroblast proliferation in vitro. Studies have identified a pathological role for MIF in immune-initiated renal injury in the rat. In this study, we examined the role of MIF in obstructive nephropathy, where renal injury is initiated by a non-immune insult. Methods and Results: Unilateral ureteric ligation was performed on MIF wildtype (+/+) and MIF deficient (-/-) mice. Groups of five mice were killed at days 0, 1, 5 or 10 after obstruction, and kidneys were examined via immunohistochemistry and northern blotting. In MIF +/+ mice, expression of the MIF protein increased in obstructed kidneys compared to normal control kidneys. Interstitial macrophage and T cell accumulation was significantly increased in obstructed kidneys at day 5 and 10, but was unaffected by MIF deficiency. Osteopontin and macrophage colony stimulating factor (M-CSF) mRNA expression in obstructed kidneys were equally increased in both genotypes, indicating that expression of these chemokines is not influenced by MIF. No difference was detected in the development of renal fibrosis in obstructed MIF +/+ and MIF -/- kidneys, as assessed by myofibroblast accumulation and proliferation and expression of profibrotic molecules (transforming growth factor-beta 1(TGF-beta1) and collagen). Conclusion(s): These results demonstrate that MIF expression is increased in obstructive nephropathy without affecting kidney leucocyte accumulation or the development of renal fibrosis. This suggests that the progression of renal injury in obstructive nephropathy is independent of MIF.

Objective. Cytokines play an important role in the pathology of rheumatoid arthritis (RA). Macrophage migration inhibitory factor (MIF) is a cytokine with a broad spectrum of actions, including induction of monocyte tumour necrosis factor alpha (TNF-alpha). Evidence of the expression and proinflammatory activity of MIF has recently been demonstrated in RA synovium and in animal models of RA. We wished to assess the relationship between MIF expression in synovium and clinical disease. Methods. Computer-assisted analysis of the cytokine content of arthroscopically obtained biopsies of RA synovium, using paired samples from eight patients with active and inactive/treated disease, was compared with documented clinical parameters. Results. Synovial MIF immunostaining correlated strongly with disease activity as measured by CRP concentration. Reductions in clinical disease parameters, including CRP, tender and swollen joint counts, were accompanied by significant reductions in synovial MIF. Synovial TNF-alpha, transforming growth factor beta (TGF-beta) and interleukin (IL) 10 also showed a significant reduction in association with reduced disease activity, while IL-1beta and IL-1 receptor agonist did not. Conclusion. The correlation of synovial MIF with disease activity corroborates existing evidence of the role of this cytokine in RA. The demonstration that only MIF and TNF-alpha show significant variation in synovial cytokine content with clinical remission suggests that MIF is an important member of the cytokine hierarchy in RA.

Background. Angiotensin II (Ang II) plays an important role in the development of renal injury through its vasoactive and proinflammatory activities. We investigated whether some of the effects of Ang II could be mediated through the production of macrophage migration inhibitory factor (MIF). Methods. Groups of rats underwent sham surgery (sham), subtotal nephrectomy (STNx), or STNx plus treatment with irbesartan. Renal tissue was examined 12 weeks postsurgery for MIF mRNA expression and leukocyte accumulation. To determine whether Ang II had a direct effect on MIF production, mRNA synthesis and protein secretion were examined in proximal tubular epithelial (NRK52E and MCT) cell lines. Results. MIF mRNA was strongly expressed in 5.4% +/- 1.1% (mean +/- SD) of cortical tubules of sham-operated rats. This was significantly up-regulated in STNx rats (44.9% +/- 22.6%) and was abrogated by administration of irbesartan (2.8% +/- 2.4%). STNx resulted in significant glomerular and interstitial accumulation of macrophages and T cells, which correlated with glomerular and tubular MIF mRNA expression, respectively. In vitro studies of tubular epithelial cells revealed that Ang II caused a twofold increase in MIF mRNA expression in NRK52E and MCT cells, which was abrogated by irbesartan. In addition, Ang II induced a rapid release of 50% of MIF protein from NRK52E cells within 20 minutes. Conclusion. This study has demonstrated that Ang II up-regulates MIF mRNA production and MIF protein secretion by tubular epithelial cells. Ang II may promote accumulation and activation of interstitial leukocytes via induction of MIF synthesis and secretion in renal tubular epithelial cells. This may be an important mechanism by which Ang II mediates renal injury.

Objective: Macrophage migration inhibitory factor (MIF) facilitates multiple aspects of inflammatory arthritis, the pathogenesis of which has been significantly linked to the activity of neutrophils. The effects of MIF on neutrophil recruitment are unknown. This study was undertaken to investigate the contribution of MIF to the regulation of neutrophil chemotactic responses. Method(s): K/BxN serum-transfer arthritis was induced in wild-type (WT), MIF -/-, and monocyte chemotactic protein 1 (MCP-1; CCL2)-deficient mice as well as in WT mice treated with monoclonal antibodies to cytokine-induced neutrophil chemoattractant (anti-KC). Leukocyte trafficking in vivo was examined using intravital microscopy, and neutrophil function in vitro was examined using migration chambers and assessment of MAP kinase activation. Result(s): K/BxN serum-transfer arthritis was markedly attenuated in MIF-/- mice, with reductions in the clinical and histologic severity of arthritis and the synovial expression of KC and interleukin-1. Arthritis was also reduced by anti-KC antibody treatment, but not in MCP-1-deficient mice. In vivo, neutrophil recruitment responses to KC were reduced in MIF-/- mice. Similarly, MIF-/- mouse neutrophils exhibited reduced chemotactic responses to KC in vitro, despite displaying unaltered chemokine receptor expression. Reduced chemotactic responses of MIF-/- mouse neutrophils were associated with reduced phosphorylation of p38 and ERK MAP kinases. Conclusion(s): These findings suggest that MIF promotes neutrophil trafficking in inflammatory arthritis via facilitation of chemokine-induced migratory responses and MAP kinase activation. Therapeutic MIF inhibition could limit synovial neutrophil recruitment. © 2011, American College of Rheumatology.

Macrophage migration inhibitory factor (MIF) plays a pivotal role in the inflamatory response in endotoxemia and m the delayed-type hypersensitivity response, but its potential as a regulator of immunologically induced disease is unknown. We have addressed this issue by administering a neutralizing anti-MIF antibody in a rat model of immunologically induced crescentic anti-glomerular basement membrane (GBM) glomerulonephritis. Six individual experiments using paired inbred littermates were performed. Rats were primed with rabbit immunoglobulin on day -5 and then injection with rabbit anti-rat GBM serum on day 0. Pairs of animals were treated with anti-MIF or a control monoclonal antibody from the time of anti-GBM serum administration until being killed 14 d later. Control antibody-treated animals developed severe proteinuria and renal function impairment with severe histological damage due to marked leukocytic infiltration and activation within the kidney. In contrast, anti-MIF treatment substantially reduced proteinuria, prevented the loss of renal function, significantly reduced histological damage including glomerular crescent formation, and substantially inhibited renal leukocytic infiltration and activation (all P <0.001 compared with control treatment). Inhibition of renal disease by anti-MIF treatment was attributed to preventing the marked upregulation of interleukin-1beta, leukocyte adhesion molecules including intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and inducible nitric oxide synthase expression seen in the control antibody-treated animals. This inhibition of progressive renal injury was mirrored by the complete suppression of the skin delayed-type hypersensitivity response to the challenge antigen (rabbit IgG). Interestingly, anti-MIF treatment did not effect the secondary antibody response or immune deposition within the kidney, indicating that MIF participates in cellular-based immunity in this primed macrophage-dependen

Although macrophages play an important role in lipid-induced glomerular injury, we know little of the mechanisms by which hyperlipidaemia induces monocyte recruitment. This study investigated the role of M-CSF and macrophage MIF in monocyte recruitment during the development of lipid- induced glomerular injury in the susceptible ExHC rat strain. Groups of five ExHC rats were fed a high cholesterol diet (HCD) containing 3% cholesterol, 0-6% sodium cholate and 15% olive oil, and killed after 3 days, 1, 2 or 6 weeks. Control animals were killed on day 0 or after 6 weeks on a normal diet. Animals were hypercholesterolaemic 3 days after the induction of the HCD, but showed no change in plasma triglycerides over the 6-week period. Glomerular macrophage accumulation was first evident at 1-2 weeks and increased up to week 6, when macrophage-derived foam cells were seen in almost all glomeruli, and segmental lesions and mild proteinuria were also evident. Combined in situ hybridization and immunohistochemistry staining demonstrated that, coincident with the induction of hypercholesterolaemia on day 3, there was marked up-regulation of M-CSF and MIF mRNA expression by intrinsic glomerular cells (mostly mesangial cells and podocytes) which preceded monocyte recruitment. There was a highly significant correlation between the number of M-CSF and MIF-positive cells and glomerular macrophage accumulation over the 6-week period. Although some glomerular macrophages and foam cells exhibited M-CSF and MIF expression, the major source of these molecules was intrinsic glomerular cells. No local macrophage proliferation was observed during the development of glomerular lesions. In conclusion, hypercholesterolaemia caused marked up-regulation of M-CSF and MIF expression by intrinsic glomerular cells, which correlated with monocyte recruitment and the development of lipid-induced glomerular injury. This is the first study to implicate local synthesis of MIF in the pathogenesis of lipid-in

In the adult rat testis, macrophages belong to one of two subsets differentiated by expression or lack of expression of the resident macrophage surface antigen recognized by monoclonal antibody ED2. Local regulation of the testicular macrophage subsets was investigated in normal and 4-wk experimentally cryptorchid adult rats with and without s.c. testosterone implants (T-implants). Macrophage subsets ED2+ (resident-type) and ED2- (monocyte-like) were identified immunohistochemically and counted in perfusion-fixed frozen testis sections. Depletion of the spermatogenic cells by cryptorchidism had no effect on testicular macrophage numbers. Inhibition of Leydig cell and seminiferous tubule function by low-dose (3 cm) T-implants caused a 40% reduction in ED2+ resident macrophages in both scrotal and abdominal testes. High-dose (24 cm) T-implants, which inhibit Leydig cell function while maintaining normal seminiferous tubule function, also reduced the number of resident macrophages by approximately 40%, although this reduction was at least partially prevented in the abdominal testes. In the scrotal testis only, the ED2- monocyte/macrophage subset was significantly reduced in number by low-dose, but not highdose, T-implants. The concentration of the Leydig cell-secreted cytokine macrophage-migration inhibitory factor (MIF) in testicular fluid was reduced by cryptorchidism, but not by the T-implants. When data from all experimental groups were combined, ED2+ resident macrophage numbers showed a significant positive correlation with parameters of Leydig cell function (serum LH and testicular testosterone levels) but a negative correlation with MIF levels. This study indicates that Leydig cells regulate testicular macrophage numbers directly, rather than via an effect upon the seminiferous epithelium, in the adult rat testis. The data also suggest that testosterone and MIF play only a minor role, if any, in this regulation.

Macrophage migration inhibitory factor (MIF) is a potent pro- inflammatory cytokine that also counter-regulates glucocorticoid action. We investigated whether immunoneutralization of MIF could reverse established experimental crescentic glomerulonephritis and if this treatment could modulate endogenous glucocorticoid levels. Accelerated anti-GBM glomerulonephritis was induced in six littermate pairs of rats. Once crescentic disease was established on day 7, one animal in each pair was given a daily injection of neutralizing anti-MIF antibody (Ab) or irrelevant isotype control Ab for 14 days and then killed on day 21. In addition, a group of 6 animals was killed on day 7 of disease without any treatment. Animals receiving the control Ab exhibited a rapidly progressive glomerulonephritis with severe renal injury (proteinuria), loss of renal function (creatinine clearance), anemia, and marked histologic damage (including glomerular crescent formation), compared with animals killed on day 7 without treatment. In contrast, anti-MIF Ab treatment partially reversed the disease by restoring normal renal function and reducing histological damage compared with untreated animals killed on day 7 (p < 0.05). Interestingly, anti-MIF Ab treatment also prevented severe anemia (p < 0.05). Reversal of disease was associated with a significant reduction in leukocyte infiltration and activation and renal interleukin-1 (IL-1) production. Importantly, anti-MIF Ab treatment caused a significant increase in endogenous serum corticosterone levels, which correlated with the reversal of disease parameters. In conclusion, this study has demonstrated that blocking MIF activity can partially reverse established crescentic glomerulonephritis and suggests that MIF operates by both enhancing the cellular immune response and suppressing the endogenous anti-inflammatory glucocorticoid response.

Recent studies have identified a key role for macrophage migration inhibitory factor (MIF) in a number of immune cell-mediated diseases. The current study investigated the potential role of MIF in acute allograft rejection. Lewis rats underwent bilateral nephrectomy and then received an orthotopic DA renal allograft or an orthotopic Lewis renal isograft. Groups of six animals were killed at day 1 or 5 after transplantation. No immunosuppression was used. Animals receiving a renal allograft exhibited severe rejection on day 5, as shown by high levels of serum creatinine, very low rates of creatinine clearance, and severe tubulitis with a dense macrophage and T cell infiltrate. In contrast, isografts had normal renal function on day 5 with no histological evidence of rejection. Northern blotting showed that renal MIF mRNA expression was unchanged at day 1, but was increased 3.5-fold on day 5. In situ hybridization showed a marked increase in MIF mRNA expression by tubular cells and MIF mRNA expression by many infiltrating mononuclear cells in day 5 allografts. Immunostaining confirmed an increase in tubular MIF protein expression, particularly in areas of severe tubular damage with prominent leucocytic infiltration. Double staining showed that many infiltrating macrophages and T cells expressed the MIF protein in day 5 allografts. There was only a minor increase in MIF expression in day 5 isografts, demonstrating that neither surgical injury nor stress cause significant up-regulation of MIF expression in allograft rejection. In conclusion, this study has demonstrated that local MIF production is specifically increased in acute renal allograft rejection. These results suggest that MIF may play an important role in the cellular immune response mediating acute allograft rejection.

Inflammation plays an important role in mediating and exacerbating myocardial ischemia-reperfusion (I/R) injury. Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, facilitates inflammation and modulates metabolism. However, the role of MIF in mediating local inflammation subsequent to ischemic myocardial injury has not been established. We hypothesized that genetic deletion of MIF protects the heart against severe I/R injury by suppressing inflammation and/or modulating energy metabolism. We showed in the mouse I/R model that duration of both ischemia and reperfusion is a determinant for the degree of regional inflammation and tissue damage. Following a prolonged cardiac I/R (60 min/24 h) MIF KO mice had a significant reduction in both infarct size (26 +/- 3% vs 45 +/- 4%, P< 0.05) and cardiomyocyte apoptosis (1.4 +/- 0.2% vs 5.4 +/- 0.4%, P< 0.05) and preserved contractile function compared with WT. MIF KO mice with I/R had reduced expression of various inflammatory cytokines and mediators (P< 0.05), suppressed infiltration of neutrophils (- 40%) and macrophages (- 33%, both P< 0.05), and increased macrophage apoptosis (14.4 +/- 1.4% vs 5.2 +/- 0.6%, P< 0.05). Expression of toll-like receptor-4 (TLR-4), phosphorylation of c-Jun N-terminal kinase (JNK), and nuclear fraction of NF-kappaB p65 were also significantly lower in MIF KO hearts with I/R. Further, MIF KO mice exhibited a lower glucose uptake but higher fatty acid oxidation rate than that in WT (both P< 0.05). In conclusion, MIF deficiency protected the heart from prolonged/severe I/R injury by suppressing inflammatory responses. We have identified a critical role of MIF in mediating severe I/R injury. Thus, MIF inhibitory therapy may be a novel strategy to protect the heart against severe I/R injury. © 2011 Elsevier Ltd.

(MIF) is a broad-spectrum proinflammatory cytokine implicated in human rheumatoid arthritis. The synthesis of MIF by synovial cells is stimulated by glucocorticoids, and previous studies suggest that MIF antagonizes the anti-inflammatory effects of glucocorticoids. This has not been established in a model of arthritis. We wished to test the hypothesis that MIF can act to reverse the anti-inflammatory effects of glucocorticoids in murine antigen-induced arthritis (AIA). Cutaneous DTH reactions and AIA were induced by intradermal injection and intra-articular injection, respectively, of methylated bovine serum albumin in presensitized mice. Animals were treated with anti-MIF MoAbs, recombinant MIF, and/or dexamethasone (DEX). Skin thickness of DTH reactions was measured with callipers and arthritis severity was measured by blinded quantitative histological assessment of synovial cellularity. Cutaneous DTH to the disease-initiating antigen was significantly inhibited by anti-MIF MoAb treatment (P < 0-001). AIA was also significantly inhibited by anti-MIF MoAb (P < 0.02). DEX treatment induced a dose-dependent inhibition of AIA, which was significant at 0-2 mg/kg (P < 0.05). MIF treatment reversed the effect of therapeutic DEX on AIA (P < 0.001). DEX also significantly inhibited DTH reactions (P < 0.05) but rMIF had no effect on this effect of DEX. DTH and AIA are MIF-dependent models of inflammation and arthritis. The reversal of glucocorticoid suppression of AIA by MIF supports the concept that MIF is a counter-regulator of glucocorticoid control of synovial inflammation. Although DTH was observed to be MIF-dependent and glucocorticoid-sensitive, rMIF had no reversing effect on the suppression of DTH by ghicocorticoids. This suggests that inflammatory, processes in specific tissues may respond differently to MIF in the presence of ghicocorticoids.

Objective. To study the capacity of macrophage migration inhibitory factor (MIF) to regulate proliferation, apoptosis, and p53 in an animal model of rheumatoid arthritis (RA) and in fibroblast-like synoviocytes (FLS) from humans with RA. Methods. Antigen-induced arthritis (AIA) was induced in MIF-/- mice and littermate controls. FLS were obtained from patients with RA. Western blotting and immunohistochemistry were used to measure p53 in cells and tissues. Apoptosis was detected in cells by flow cytometry using TUNEL and annexin V/propidium iodide labeling. Apoptosis in tissue was detected using TUNEL. Proliferation was assessed in cultured cells and tissue by 3H-thymidine incorporation and Ki-67 immunostaining, respectively. Results. MIF inhibited p53 expression in human RA FLS. Levels of p53 were correspondingly increased in MIF-/- mouse tissues and cells. Spontaneous and sodium nitroprusside-induced apoptosis were significantly increased in MIF-/- cells. In vitro exposure of FLS to MIF reduced apoptosis and significantly induced FLS proliferation. Synoviocyte proliferation in MIF-/-mice was correspondingly reduced. A decrease in the severity of AIA in MIF-/- mice was associated with an increase in p53 and apoptosis in synovium. Evidence of in situ proliferation was scant in this model, and no difference in in situ proliferation was detectable in MIF-/- mice compared with wild-type mice. Conclusion. These results indicate a role for MIF in the regulation of p53 expression and p53-mediated events in the inflamed synovium and support the hypothesis that MIF is of critical importance in the pathogenesis of RA.

Macrophage migration inhibitory factor (MIF) promotes macrophage accumulation and leucocyte activation during inflammation. Macrophage migration inhibitory factor is upregulated in intrinsic renal cells in many types of kidney diseases, and has a pathogenic role in rat crescentic nephritis. However, little is known about the factors that regulate the production and secretion of MIF in kidney cells. In this study, we evaluated whether interferon-gamma (IFN-gamma), a cytokine implicated in the development of kidney disease and a potent inducer of MIF production in macrophages, could promote MIF synthesis and secretion from renal tubular epithelial cells. Northern blot analysis detected constitutive expression of MIF mRNA in rat tubular epithelial cells (NRK52E), which increased twofold after a 6-h stimulation with IFN-gamma. Macrophage migration inhibitory factor protein was found only in the cytoplasm of NRK52E cells. Following IFN-gamma stimulation, intracellular MIF in NRK52E cells was rapidly secreted with a maximal reduction of 50% after 20 min, which returned to normal levels after 2-4 h. Rapid secretion of MIF in response to IFN-gamma was also seen in rat mesangial cells. These findings indicate that IFN-gamma induces rapid secretion of MIF by tubular epithelial cells, and suggest that this may be an important mechanism leading to inflammatory cell accumulation and activation during kidney disease.

Macrophage migration inhibitory factor (MIF) is an important constituent of the host response to stress and infection and is the first mediator that has been identified to be released from immune cells upon stimulation with glucocorticoids. MIF also has been shown to be secreted from the anterior pituitary gland, monocytes/macrophages, and T cells activated by various proinflammatory stimuli. Once released, MIF acts to counter-regulate the inhibitory effect of glucocorticoids on inflammatory cytokine production. To characterize more precisely the role of MIF in the host response to infection, we undertook a systematic analysis of MIF expression in various organs of the rat after endotoxin (lipopolysaccharide) administration. MIF protein and mRNA were analyzed by immunohistochemistry and in situ hybridization, respectively. MIF was found to be expressed constitutively in organs such as the lung, liver, kidney, spleen, adrenal gland, and skin. Significant quantities of MIF protein were detected preformed in various cell types and appeared to be released as a consequence of endotoxemia. In virtually all tissues examined, the loss of MIF protein 6 hours after lipopolysaccharide administration was accompanied by the induction of MIF mRNA and, at 24 hours, by the restoration of immunoreactive, intracellular MIF. The constitutive production of MIF by several cell and tissue types together with its rapid release from intracellular pools distinguishes MIF from other cytokines or hormonal mediators and significantly expands the physiological role of this unique counter-regulator of glucocorticoid action.

Macrophage migration inhibitory factor (MIF), a key mediator of the delayed-type hypersensitivity response, was originally thought to be produced by activated T cells. However, recent studies have found that MIF is produced in many cell types including monocytes/macrophages and anterior pituitary cells. The current study has examined MIF expression in normal and diseased kidney using in situ hybridization, immunohistochemistry, and Northern blotting. MIF mRNA and protein are constitutively expressed in normal kidney, being largely restricted to tubular epithelial cells and some glomerular visceral and parietal epithelial cells. During the development of rat anti- glomerular basement membrane glomerulonephritis, a model of macrophage- mediated renal injury, there was marked de novo expression of MIF by intrinsic kidney cells including endothelium and glomerular and tubular epithelial cells. Up-regulation of MIF expression correlated with macrophage accumulation within the glomerulus (P < 0.001) and tubulointerstitium (P < 0.001). Of significance, the accumulation of macrophages was exclusively localized to areas of strong MIF expression, contributing to focal glomerular and tubulointerstitial lesion formation. In addition, up-regulation of MIF expression by parietal epithelial cells was associated with macrophage accumulation within Bowman's space and crescent formation. Combined in situ hybridization and immunostaining also demonstrated MIF expression by macrophages, T cells, and fibroblast-like cells within renal lesions. In conclusion, these data provide the first demonstration that renal epithelial cells are a major source of MIF in both normal and diseased kidney. Furthermore, the up-regulation of MIF expression may play an important role in macrophage accumulation and progressive renal injury in rat crescentic glomerulonephritis.

Mesangial cells are thought to promote glomerular macrophage accumulation in glomerulonephritis. This may occur through the production of macrophage migration inhibitory factor (MIF), a molecule known to regulate macrophage accumulation at sites of inflammation. To study this, glomerular MIF expression and macrophage accumulation were examined in rat anti-Thy-1 disease, a model of mesangioproliferative nephritis. In situ hybridization and immunohistochemistry showed that MIF is expressed by some podocytes in normal rat glomeruli. De novo MIF expression by glomerular endothelium was seen on day 1 of anti-Thy-1 disease. On day 6, glomerular MIF mRNA and protein expression were prominent in segmental proliferative lesions, which was also the location of most infiltrating macrophages. Double-staining identified de novo MIF mRNA and protein expression by proliferating mesangial cells within these lesions. Cytokine regulation of mesangial cell MIF expression was examined in vitro. Northern blotting showed that cultured rat mesangial cells express a single 0.6-kb species of MIF mRNA, and Western blotting detected a single protein band of 12.5 kD. Six-hour stimulation of mesangial cells with interferon-gamma or platelet-derived growth factor significantly increased MIF mRNA levels. However, the addition of recombinant MIF to mesangial cells did not affect mesangial cell proliferation or constitutive transforming growth factor-beta mRNA expression, nor did MIF induce monocyte chemoattractant protein-1 mRNA expression. In conclusion, this is the first study to demonstrate that mesangial cells can produce MIF in vivo and in vitro. It is postulated that mesangial cell MIF production in response to injury acts to promote macrophage accumulation within segmental proliferative lesions in rat anti-Thy-1 nephritis.

Advanced glycosylation endproducts (AGEs) which result from the non- enzymatic interaction of proteins and glucose are implicated in the vasculopathy of diabetes and aging. Since aminoguanidine (A) inhibits the accumulation of AGEs, we explored its effects on the development of atherosclerosis. Male New Zealand white cross rabbits fed a high cholesterol (1%) diet were randomized to control (C) or increasing doses of A treatment (25, 50 and 100 mg/kg A body weight). The animals were sacrificed after 12 weeks. Sudan IV was used to stain the lipid containing plaques of the aortic arch, thoracic and abdominal aorta and the surface area occupied by atheroma was assessed. Increasing doses of A treatment were associated with reduction in plaque formation in the aorta. At a dose of 100 mg/kg A, there was a 30, 49 and 48% reduction in plaque formation in the aortic arch, thoracic and abdominal aorta, respectively. There was a correlation between AGE levels and the degree of atheroma in these cholesterol fed rabbits (control, r = 0.75, P < 0.01; 100 mg/kg A, r = 0.59, P = 0.02). These data suggest that advanced glycation may participate in atherogenesis and raise the possibility that inhibitors of advanced glycation may retard this process.

Objective. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose involvement in tumor necrosis factor alpha (TNFalpha) synthesis and T cell activation suggests a role in the pathogenesis of rheumatoid arthritis (RA). Antagonism of MIF is associated with marked inhibition of animal models of RA. Uniquely, MIF is inducible by low concentrations of glucocorticoids. We sought to investigate the expression of MIF in RA synovial tissue. Methods. MIF was demonstrated in human RA synovium by immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). Regulation of MIF expression was investigated by treatment of cultured fibroblast-like synoviocytes (FLS) with interleukin-1beta (IL-1beta), TNFalpha, or interferon-gamma (IFNgamma), and dexamethasone (DEX). Mononuclear cell TNFalpha release after exposure to FLS-conditioned medium was measured by ELISA. Results. MIF was present in RA synovial lining CD14+ macrophages and FLS. Constitutive MIF messenger RNA (mRNA) expression was demonstrated by RT-PCR of RNA from unstimulated cultured RA FLS, which also released abundant MIF. Serum, synovial fluid, and FLS intracellular MIF were significantly higher in RA patients than in controls. Synoviocyte MIF was not increased by IL-1beta, TNFalpha, or IFNgamma. In contrast, DEX 10-7M significantly reduced synoviocyte MIF, while DEX 10-10-10-12M induced a significant increase in MIF and MIF mRNA. Peripheral blood mononuclear cell TNFalpha release was induced by culture in RA FLS-conditioned medium, and this induction was significantly abrogated by monoclonal anti-MIF antibody, suggesting that MIF is an upstream regulator of TNFalpha release. Conclusion. These data represent the first demonstration of the cytokine MIF in human autoimmune disease and suggest MIF as a potential therapeutic target in RA.

Objective. Recent studies have established an essential role for macrophage migration inhibitory factor (MIF) in T cell and macrophage activation, both of which are characteristics of rat adjuvant arthritis. This study investigated the role of MIF in early adjuvant arthritis. Methods. MIF was detected in rat synovium by immunohistochemistry and enzyme-linked immunosorbent assay using specific monoclonal antibodies (MAb). Anti-MIF MAb treatment was administered, and the effects on clinical aspects of adjuvant arthritis were assessed. Results. MIF was absent from normal rat synovium prior to adjuvant injection, but was detectable on day 4 after injection (6 days before the onset of clinical disease) and was colocalized with ED-1+ macrophages throughout the evolution of the disease. Levels of MIF were increased in established adjuvant arthritis sera, and adjuvant arthritis synovial macrophages released MIF at a mean +/- SEM concentration of 607.9 +/- 201.5 pg/ml. Anti-MIF treatment led to profound, dose-dependent inhibition of the adjuvant arthritis clinical score, paw swelling, and synovial lavage leukocyte numbers (P < 0.001), and also resulted in reduced synovial macrophage and T cell accumulation. Conclusion. These findings demonstrate an important role for MIF in the evolution of rat adjuvant arthritis.

Background. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is a potent activator of macrophages and T cells. Previous studies have shown that local MIF production is increased in acute renal allograft rejection, suggesting that it may play an important role in the rejection process. Aims. To determine if urine and serum MIF concentrations: (1) are increased in acute rejection, and (2) can be used as noninvasive tools to discriminate between acute rejection (AR) and cyclosporine nephrotoxicity (CyA toxicity). Methods. In a prospective study of nine renal allograft patients (five acute rejection and four stable), serial urine MIF concentrations were measured by ELISA in the first 14 days after transplantation. In a retrospective study, MIF concentrations in urine and serum were measured in 24 patients who were biopsied for acute renal transplant dysfunction (11 AR, 13 CyA toxicity). Urine and serum MIF were also measured in 23 stable renal transplant patients and 10 normals. Results. MIF was readily detected in the urine of normal healthy controls (106+/-61 pg/mumol creatinine). In the prospective study, the urinary MIF concentration was increased substantially on day 1 posttransplantation and subsequently fell in parallel with the serum creatinine. However, urine MIF increased before episodes of biopsy proven acute rejection. The retrospective study showed that urine MIF concentrations in patients with AR were increased 5-fold compared to normal controls (439+/-313 pg/mumol Cr; P<0.01). In contrast, urine MIF concentrations in CyA toxicity were not significantly different to normal controls (145+/-119 pg/mumol Cr; P=NS). A marked increase in MIF immunostaining was seen in biopsies of AR, but not in CyA toxicity. No significant differences were evident in serum MIF levels between normals and any transplant patient group. Conclusions. These results suggest that measurement of urine MIF concentration may be useful in monitoring renal tr

Background and Aim: The progression of renal injury, initiated by either an immune or non-immune insult, is closely associated with the accumulation of leucocytes and fibroblasts in the damaged kidney. Macrophage migration inhibitory factor (MIF) regulates leucocyte activation and fibroblast proliferation in vitro. Studies have identified a pathological role for MIF in immune-initiated renal injury in the rat. In this study, we examined the role of MIF in obstructive nephropathy, where renal injury is initiated by a non-immune insult. Methods and Results: Unilateral ureteric ligation was performed on MIF wildtype (+/+) and MIF deficient (-/-) mice. Groups of five mice were killed at days 0, 1, 5 or 10 after obstruction, and kidneys were examined via immunohistochemistry and northern blotting. In MIF +/+ mice, expression of the MIF protein increased in obstructed kidneys compared to normal control kidneys. Interstitial macrophage and T cell accumulation was significantly increased in obstructed kidneys at day 5 and 10, but was unaffected by MIF deficiency. Osteopontin and macrophage colony stimulating factor (M-CSF) mRNA expression in obstructed kidneys were equally increased in both genotypes, indicating that expression of these chemokines is not influenced by MIF. No difference was detected in the development of renal fibrosis in obstructed MIF +/+ and MIF -/- kidneys, as assessed by myofibroblast accumulation and proliferation and expression of profibrotic molecules (transforming growth factor-beta 1(TGF-beta1) and collagen). Conclusion(s): These results demonstrate that MIF expression is increased in obstructive nephropathy without affecting kidney leucocyte accumulation or the development of renal fibrosis. This suggests that the progression of renal injury in obstructive nephropathy is independent of MIF.

Objective. Cytokines play an important role in the pathology of rheumatoid arthritis (RA). Macrophage migration inhibitory factor (MIF) is a cytokine with a broad spectrum of actions, including induction of monocyte tumour necrosis factor alpha (TNF-alpha). Evidence of the expression and proinflammatory activity of MIF has recently been demonstrated in RA synovium and in animal models of RA. We wished to assess the relationship between MIF expression in synovium and clinical disease. Methods. Computer-assisted analysis of the cytokine content of arthroscopically obtained biopsies of RA synovium, using paired samples from eight patients with active and inactive/treated disease, was compared with documented clinical parameters. Results. Synovial MIF immunostaining correlated strongly with disease activity as measured by CRP concentration. Reductions in clinical disease parameters, including CRP, tender and swollen joint counts, were accompanied by significant reductions in synovial MIF. Synovial TNF-alpha, transforming growth factor beta (TGF-beta) and interleukin (IL) 10 also showed a significant reduction in association with reduced disease activity, while IL-1beta and IL-1 receptor agonist did not. Conclusion. The correlation of synovial MIF with disease activity corroborates existing evidence of the role of this cytokine in RA. The demonstration that only MIF and TNF-alpha show significant variation in synovial cytokine content with clinical remission suggests that MIF is an important member of the cytokine hierarchy in RA.

Background. Angiotensin II (Ang II) plays an important role in the development of renal injury through its vasoactive and proinflammatory activities. We investigated whether some of the effects of Ang II could be mediated through the production of macrophage migration inhibitory factor (MIF). Methods. Groups of rats underwent sham surgery (sham), subtotal nephrectomy (STNx), or STNx plus treatment with irbesartan. Renal tissue was examined 12 weeks postsurgery for MIF mRNA expression and leukocyte accumulation. To determine whether Ang II had a direct effect on MIF production, mRNA synthesis and protein secretion were examined in proximal tubular epithelial (NRK52E and MCT) cell lines. Results. MIF mRNA was strongly expressed in 5.4% +/- 1.1% (mean +/- SD) of cortical tubules of sham-operated rats. This was significantly up-regulated in STNx rats (44.9% +/- 22.6%) and was abrogated by administration of irbesartan (2.8% +/- 2.4%). STNx resulted in significant glomerular and interstitial accumulation of macrophages and T cells, which correlated with glomerular and tubular MIF mRNA expression, respectively. In vitro studies of tubular epithelial cells revealed that Ang II caused a twofold increase in MIF mRNA expression in NRK52E and MCT cells, which was abrogated by irbesartan. In addition, Ang II induced a rapid release of 50% of MIF protein from NRK52E cells within 20 minutes. Conclusion. This study has demonstrated that Ang II up-regulates MIF mRNA production and MIF protein secretion by tubular epithelial cells. Ang II may promote accumulation and activation of interstitial leukocytes via induction of MIF synthesis and secretion in renal tubular epithelial cells. This may be an important mechanism by which Ang II mediates renal injury.

Objective: Macrophage migration inhibitory factor (MIF) facilitates multiple aspects of inflammatory arthritis, the pathogenesis of which has been significantly linked to the activity of neutrophils. The effects of MIF on neutrophil recruitment are unknown. This study was undertaken to investigate the contribution of MIF to the regulation of neutrophil chemotactic responses. Method(s): K/BxN serum-transfer arthritis was induced in wild-type (WT), MIF -/-, and monocyte chemotactic protein 1 (MCP-1; CCL2)-deficient mice as well as in WT mice treated with monoclonal antibodies to cytokine-induced neutrophil chemoattractant (anti-KC). Leukocyte trafficking in vivo was examined using intravital microscopy, and neutrophil function in vitro was examined using migration chambers and assessment of MAP kinase activation. Result(s): K/BxN serum-transfer arthritis was markedly attenuated in MIF-/- mice, with reductions in the clinical and histologic severity of arthritis and the synovial expression of KC and interleukin-1. Arthritis was also reduced by anti-KC antibody treatment, but not in MCP-1-deficient mice. In vivo, neutrophil recruitment responses to KC were reduced in MIF-/- mice. Similarly, MIF-/- mouse neutrophils exhibited reduced chemotactic responses to KC in vitro, despite displaying unaltered chemokine receptor expression. Reduced chemotactic responses of MIF-/- mouse neutrophils were associated with reduced phosphorylation of p38 and ERK MAP kinases. Conclusion(s): These findings suggest that MIF promotes neutrophil trafficking in inflammatory arthritis via facilitation of chemokine-induced migratory responses and MAP kinase activation. Therapeutic MIF inhibition could limit synovial neutrophil recruitment. © 2011, American College of Rheumatology.

Although macrophages play an important role in lipid-induced glomerular injury, we know little of the mechanisms by which hyperlipidaemia induces monocyte recruitment. This study investigated the role of M-CSF and macrophage MIF in monocyte recruitment during the development of lipid- induced glomerular injury in the susceptible ExHC rat strain. Groups of five ExHC rats were fed a high cholesterol diet (HCD) containing 3% cholesterol, 0-6% sodium cholate and 15% olive oil, and killed after 3 days, 1, 2 or 6 weeks. Control animals were killed on day 0 or after 6 weeks on a normal diet. Animals were hypercholesterolaemic 3 days after the induction of the HCD, but showed no change in plasma triglycerides over the 6-week period. Glomerular macrophage accumulation was first evident at 1-2 weeks and increased up to week 6, when macrophage-derived foam cells were seen in almost all glomeruli, and segmental lesions and mild proteinuria were also evident. Combined in situ hybridization and immunohistochemistry staining demonstrated that, coincident with the induction of hypercholesterolaemia on day 3, there was marked up-regulation of M-CSF and MIF mRNA expression by intrinsic glomerular cells (mostly mesangial cells and podocytes) which preceded monocyte recruitment. There was a highly significant correlation between the number of M-CSF and MIF-positive cells and glomerular macrophage accumulation over the 6-week period. Although some glomerular macrophages and foam cells exhibited M-CSF and MIF expression, the major source of these molecules was intrinsic glomerular cells. No local macrophage proliferation was observed during the development of glomerular lesions. In conclusion, hypercholesterolaemia caused marked up-regulation of M-CSF and MIF expression by intrinsic glomerular cells, which correlated with monocyte recruitment and the development of lipid-induced glomerular injury. This is the first study to implicate local synthesis of MIF in the pathogenesis of lipid-in