The pathophysiology of sepsis-induced myocardial dysfunction still remains controversial. Macrophage migration inhibitory factor (MIF) has recently been identified as a cardiac-derived myocardial depressant factor in septic shock. Putative mechanisms by which MIF affects cardiac function are unknown. In an investigation of possible mechanisms of action, a rat model of endotoxin toxicity was designed using intraperitoneal (I/P) injection of lipopolysaccharides (LPS) with or without coinfusion of neutralizing anti-MIF or isotypic-matched antibodies. Echocardiographic evaluation revealed that MIF neutralization reversed endotoxin-induced myocardial dysfunction at 24 hours after injection. RNase protection assay (RPA) and Western blot established that MIF neutralization prevented LPS-induced mRNA expression and production of heart-derived inflammatory paracrine and autocrine cytokines such as IL-1s and IL-6. Moreover, MIF immunoneutralization increased heart Bcl-2/Bax protein ratio and suppressed endotoxin-induced release of mitochondrial cytochrome-c, as demonstrated by Western blotting. Inhibition of mitochondrial loss of cytochrome-c decreased in heart caspase-3 activity at 6 and 24 hours after injection. MIF neutralization also restored the LPS-induced deficient nuclear translocation of phospho-Akt and consequently the expression of the heart survival nuclear factor GATA-4. The restoration of the translocation/expression of survival factors by MIF inhibition resulted in lowered endotoxin-induced DNA fragmentation at 24 hours, a hallmark of downstream cardiomyocyte apoptosis. Our data indicate that early inactivation of MIF significantly reverses the imbalance of proapoptotic to prosurvival pathways and reduces acute inflammation of the heart thereby improving myocardial dysfunction induced by endotoxin.

OBJECTIVE

Proinflammatory genetic profiles, resulting from the combination of single nucleotide polymorphisms in genes encoding inflammatory molecules, may contribute to the development and progression of cardiovascular diseases. We evaluated the association between history of ischemic stroke and genetic profiles determined by the synergistic effects of polymorphisms in genes encoding prototypical inflammatory proteins.

METHODS

The study included 237 individuals with history of ischemic stroke and 223 age-matched and gender-matched controls. The polymorphisms of the C-reactive protein (CRP), interleukin-6 (IL-6), macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin (E-sel), and matrix metalloproteinase-3 (MMP-3) genes were studied.

RESULTS

IL-6 GG, IL-6 GC, MCP-1 GG, ICAM-1 EE, E-sel AA, and MMP-3 5A5A genotypes were significantly and independently associated with stroke history. The odds of stroke increased with the number of high-risk genotypes: carrying 1 proinflammatory gene variant conferred a risk of 3.3 (1.6 to 6.9), whereas individuals concomitantly carrying 2 and 3 proinflammatory gene variants had adjusted odds ratios of 21.0 (7.6 to 57.5) and 50.3 (10.2 to 248.1), respectively.

CONCLUSIONS

Proinflammatory genetic profiles are significantly more common in subjects with stroke history. Synergistic effects between proinflammatory genotypes might be potential markers for cerebrovascular diseases.

The protein that has been historically called macrophage migration inhibitory factor (MIF) was one of the first cytokine activities to be discovered and was originally described to be a T lymphocyte product that inhibited the random migration of macrophages. Over the years, additional molecules with MIF "activity" have been described and the precise role of the original MIF "protein" remained enigmatic. Recent studies have led to the discovery of a pituitary mediator that appears to act as the counterregulatory hormone for glucocorticoid action within the immune system. Isolated as a product of murine anterior pituitary cells, this peptide was sequenced and found to be the mouse homolog of MIF. MIF has the unique property of being released from macrophages and T cells in response to physiological concentrations of glucocorticoids. The secretion of MIF is tightly regulated and decreases at high, anti-inflammatory steroid concentrations. Once released, MIF "overrides" or counterregulates the immunosuppressive effects of steroids on immune cell activation and cytokine production. These observations suggest that MIF fills an important gap in our understanding of how the host initiates and controls immunity. Because glucocorticoids are an integral part of the host's global response to infection or tissue invasion, the physiological role of MIF is to act at an inflammatory site or lymph node to counterbalance the profound inhibitory effects of steroids on the immune response.

The modification of proteins by SUMO (small ubiquitin-related modifier) plays important roles in regulating the activity, stability and cellular localization of target proteins. Similar to ubiquitination, SUMO modification is a dynamic process that can be reversed by SENPs [SUMO-1/sentrin/SMT3 (suppressor of mif two 3 homologue 1)-specific peptidases]. To date, six SENPs have been discovered in humans, although knowledge of their regulation, specificity and biological functions is limited. In the present study, we report that SENP7 has a restricted substrate specificity, being unable to process SUMO precursors and displaying paralogue-specific isopeptidase activity. The C-terminal catalytic domain of SENP7 efficiently depolymerized poly-SUMO-2 chains but had undetectable activity against poly-SUMO-1 chains. SENP7 also displayed isopeptidase activity against di-SUMO-2- and SUMO-2-modified RanGAP1 (Ran GTPase-activating protein 1) but had limited activity against SUMO-1-modified RanGAP1. in vivo, full-length SENP7 was localized to the nucleoplasm and preferentially reduced the accumulation of high-molecular-mass conjugates of SUMO-2 and SUMO-3 compared with SUMO-1. Small interfering RNA-mediated ablation of SENP7 expression led to the accumulation of high-molecular-mass SUMO-2 species and to the accumulation of promyelocytic leukaemia protein in subnuclear bodies. These findings suggest that SENP7 acts as a SUMO-2/3-specific protease that is likely to regulate the metabolism of poly-SUMO-2/3 rather than SUMO-1 conjugation in vivo.

Metastasis and drug resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil, methotrexate, doxorubicin, or oxaliplatin. Drug-resistant invasive cells were compared with noninvasive cells using cDNA microarray, quantitative reverse transcription-PCR, flow cytometry, immunoblots, and ELISA. Functional and cellular signaling analyses were undertaken using pharmacologic inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-Fluorouracil- and methotrexate-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behavior in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration-inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, monoclonal antibody 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 was associated with the upregulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2alpha and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from patients with colon cancer treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis could potentially counteract the emergence of the invasive metastatic behavior in clonal derivatives of drug-resistant colon cancer cells.

Macrophage migration inhibitory factor (MIF) is a leaderless protein that is secreted from cells by a specialized, nonclassical export pathway. The release of MIF nevertheless is regulated and its production in response to different inflammatory, mitogenic, and hormonal stimuli plays an important role in diverse physiologic and pathologic processes. We report herein the identification of the Golgi complex-associated protein p115 as an intracellular binding partner for MIF. MIF interacts with p115 in the cytoplasm and the stimulated secretion of MIF results in the accumulation of both proteins in supernatants, which is consistent with MIF release from cells in conjunction with p115. The depletion of p115 from monocytes/macrophages decreases the release of MIF but not other cytokines following inflammatory stimulation or intracellular bacterial infection. Notably, the small molecule MIF inhibitor 4-iodo-6-phenylpyrimidine inhibits MIF secretion by targeting the interaction between MIF and p115. These data reveal p115 to be a critical intermediary component in the regulated secretion of MIF from monocytes/macrophages.

The regulatory mechanism(s) active in the spontaneous suppression of parasite egg-induced granulomatous response was analyzed in Schistosoma mansoni-infected mice. A single injection of cyclophosphamide (CY) given to chronically infected mice with diminished granulomas and impaired ability to produce MIF restored the enhanced granulomatous response and lymphokine production by spleen cells. Drug-treated animals also showed a rise in the level of circulating anti-egg antigen antibody. After CY treatment, spleen cells were still capable of adoptively suppressing the vigorous granulomatous response and MIF production in acutely infected recipients. Adoptive suppression, however, was abrogated when spleen cells of chronically infected mice were pretreated with anti-Lyt 1.1 alloantiserum and C before transfer. Based on the present and previous observations, it is proposed that the modulated granulomatous inflammatory response in mice with chronic infection is maintained by a dynamic equilibrium between effector and regulator lymphocytes. Although the inflammation is maintained by Ly 1+ Ia- TDH cells, the intensity of the response is regulated by Ly 1+ Ia+ TH, Ly 2+ Ia+ Ts and possibly a putative precursor population of lymphocytes.

Macrophage migration inhibitory factor (MIF) is expressed and secreted in response to mitogens and integrin-dependent cell adhesion. Once released, autocrine MIF promotes the activation of RhoA GTPase leading to cell cycle progression in rodent fibroblasts. We now report that small interfering RNA-mediated knockdown of MIF and MIF small molecule antagonism results in a greater than 90% loss of both the migratory and invasive potential of human lung adenocarcinoma cells. Correlating with these phenotypes is a substantial reduction in steady state as well as serum-induced effector binding activity of the Rho GTPase family member, Rac1, in MIF-deficient cells. Conversely, MIF overexpression by adenovirus in human lung adenocarcinoma cells induces a dramatic enhancement of cell migration, and co-expression of a dominant interfering mutant of Rac1 (Rac1(N17)) completely abrogates this effect. Finally, our results indicate that MIF depletion results in defective partitioning of Rac1 to caveolin-containing membrane microdomains, raising the possibility that MIF promotes Rac1 activity and subsequent tumor cell motility through lipid raft stabilization.

BACKGROUND

Macrophage migration inhibitory factor (MIF) was recently rediscovered as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. MIF is constitutively expressed in various cells and enhances production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, and interferon gamma. Recently, it was reported that MIF mRNA was overexpressed in prostatic tumors, which suggests that MIF is a protein involved in tumor cell growth beyond inflammatory and immune responses.

METHODS

We examined the expression of MIF in the murine colon carcinoma cell line colon 26 by Western and Northern blot analyses and immunohistochemistry. Next, we investigated the effects of transforming growth factor (TGF) beta, basic fibroblast growth factor (b-FGF), and platelet-derived growth factor (PDGF) on the expression of MIF mRNA. Furthermore, we examined whether MIF is involved in tumor cell proliferation, using an MIF anti-sense plasmid transfection technique.

RESULTS

We demonstrated that MIF protein and its mRNA were highly expressed in colon 26 cells, using Western and Northern blot analyses, respectively. By immunohistochemical analysis, we found that MIF was localized largely in the cytoplasm of the tumor cells. In response to TGF-beta, b-FGF, and PDGF, MIF mRNA expression was significantly up-regulated. Following this, we transfected the cells with an anti-sense MIF plasmid, which revealed that this treatment induced significant suppression of cell proliferation.

CONCLUSIONS

Although MIF plays multifunctional roles in a broad spectrum of pathophysiological states, little has been done to investigate the role of this protein in association with tumor growth. The current results suggest the possibility that MIF induces tumor cell growth in concert with other growth factors, which encouraged us to investigate a novel approach for tumor therapy using an anti-MIF antibody and an MIF anti-sense plasmid transfection technique.

BACKGROUND

Macrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner.

RESULTS

The effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly.

CONCLUSIONS

MIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIF's potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1alpha protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF.

Macrophage migration inhibitory factor (MIF) is an important immunoregulatory molecule with a unique ability to suppress the anti-inflammatory effects of glucocorticoids. Although considered a cytokine, MIF possesses a three-dimensional structure and active site similar to those of 4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase. Moreover, a number of catalytic activities have been defined for MIF. To gain insight into the role of catalysis in the biological function of MIF, we have begun to characterize the catalytic activities in more detail. Here we report the crystal structure of MIF complexed with p-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF. The three binding sites for p-hydroxyphenylpyruvate in the MIF trimer lie at the interface between two subunits. The substrate interacts with Pro-1, Lys-32, and Ile-64 from one subunit and Tyr-95 and Asn-97 from an adjacent subunit. Pro-1 is positioned to function as a catalytic base. There is no functional group that polarizes the alpha-carbonyl of the substrate to weaken the adjacent C-H bond. Mutation of Pro-1 to glycine substantially reduces the catalytic activity. The insertion of an alanine between Pro-1 and Met-2 essentially abolishes activity. Structural studies of these mutants define a source of the reduced activity and provide insight into the mechanism of the catalytic reaction.

Atherosclerosis is a chronic inflammatory disease of the arterial wall and an increasing body of evidence suggests that the immune system actively participates in the initiation, progression and persistence of atherosclerosis. Different types of leukocytes such as T and B lymphocytes, natural killer cells (NK) and NKT cells, macrophages, dendritic cells and mast cells have been found within atherosclerosis-prone aortas. The mechanisms of monocyte recruitment have been partially characterized and involve P-selectin, E-selectin, VCAM-1, ICAM-1 and JAM-A. CXCL1, CCL5, CXCL4, CXCL7 and MIF are also implicated in monocyte trafficking into aortas. Recently it has been reported that Ly6C(high) and Ly6C(low) monocyte subsets differently use CCL2, CX3CL1 and CCL5 for their homing into atherosclerotic aortas. T and B lymphocytes constitutively migrate into the normal and atherosclerotic aortic wall in an L-selectin-dependent manner. Recent studies suggest an important role of CCL5, CXCL10, CXCL16, CXCR6 and MIF in T cell influx into the atherosclerotic wall. However, there is little information available on the mechanisms of recruitment of other types of the immune cells such as NK, NKT and mast cells. In this review we shall summarize what is known about leukocyte recruitment into the aortic wall during atherosclerosis with a focus on mouse model systems.

BACKGROUND

Identification of new therapeutic targets remains an imperative goal to improve the morbidity and mortality associated with severe sepsis and septic shock. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, has recently emerged as a critical mediator of innate immunity and experimental sepsis, and it is an attractive new target for the treatment of sepsis.

METHODS

Circulating concentrations of MIF were measured in 2 clinical trial cohorts of 145 pediatric and adult patients who had severe sepsis or septic shock caused predominantly by infection with Neisseria meningitidis or other gram-negative bacteria, to study the kinetics of MIF during sepsis, to analyze the interplay between MIF and other mediators of sepsis or stress hormones (adrenocorticotropic hormone and cortisol), and to determine whether MIF is associated with patient outcome.

RESULTS

Circulating concentrations of MIF were markedly elevated in 96% of children and adults who had severe sepsis or septic shock, and they remained elevated for several days. MIF levels were correlated with sepsis severity scores, presence of shock, disseminated intravascular coagulation, urine output, blood pH, and lactate and cytokine levels. High levels of MIF were associated with a rapidly fatal outcome. Moreover, in meningococcal sepsis, concentrations of MIF were positively correlated with adrenocorticotropic hormone levels and negatively correlated with cortisol levels and the cortisol:adrenocorticotropic hormone ratio, suggesting an inappropriate adrenal response to sepsis.

CONCLUSIONS

MIF is markedly and persistently up-regulated in children and adults with gram-negative sepsis and is associated with parameters of disease severity, with dysregulated pituitary-adrenal function in meningococcal sepsis, and with early death.

The expression levels of mRNA encoding a panel of 28 chicken cytokines and chemokines were quantified in intestinal lymphocytes following Eimeria acervulina and Eimeria tenella primary and secondary infections. Compared with uninfected controls, transcripts of the pro-inflammatory cytokines IFN-alpha, IL-1beta, IL-6, and IL-17 were increased up to 2020-fold following primary infection. By contrast, following secondary infection by either microorganism, pro-inflammatory mRNAs levels were relatively unchanged (< or = 20-fold). Transcripts encoding the Th1 and Th1 regulatory cytokines IFN-gamma, IL-2, IL-10, IL-12, IL-15, IL-16, and IL-18 were uniformly increased 14-2471-fold after E. acervulina primary infection, but either unchanged (IL-15, IL-16, IL-18), increased (IFN-gamma, IL-10, IL-12), or decreased (IL-2) following E. tenella primary infection. Following secondary infections, Th1 cytokine mRNA levels were relatively unchanged, with the exception of IL-12 which was increased 1.5 x 10(5)-fold after E. acervulina and decreased 5.1 x 10(4)-fold after E. tenella infection. Transcripts for the Th2 or Th2 regulatory cytokines IL-3 and GM-CSF were increased up to 327-fold following primary or secondary infection with both parasites, while IL-4 and IL-13 mRNAs were decreased 25- to 2 x 10(5)-fold after primary or secondary infection. The dynamics of chicken chemokine expression revealed modest changes (<100-fold) following primary or secondary infection except for lymphotactin. When lymphocyte subpopulations were similarly analyzed, IFN-gamma, IL-2, IL-3, IL-15, and MIF were most highly increased in TCR2(+) cells following E. acervulina infection, while TCR1(+) cells only expressed high levels of IL-16 following E. tenella infection. In contrast, CD4(+) cells only expressed highest levels of IL-10 after E. acervulina infection, whereas these cells produced abundant transcripts for IFN-gamma, IL-3, IL-15, and MIF after E. tenella infection. We conclude that coccidiosis induces a diverse and robust primary cytokine/chemokine response, but a more subdued secondary response.

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non-cognate ligand of the CXC-family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine-like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC-derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia-induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non-classical mechanism and originated from pre-formed MIF stores. Early hypoxia-triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia-inducible factor (HIF)-1α-induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF-1α-induced de novo synthesis of MIF. To functionally investigate the role of hypoxia-inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL(+) KDR(+) CD31(+) EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose-dependent bell-shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia-conditioned HUVECs, an effect that was completely abrogated by anti-MIF- or anti-CXCR4-antibodies. Thus, hypoxia-induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia-induced myocardial damage.

BACKGROUND

MiRNAs play important roles in diverse biological processes including tumorigenesis. However, little is known about the function and mechanism of miR-451 in nasopharyngeal carcinoma (NPC).

METHODS

Quantitative RT-PCR was used to quantify miR-451 expression in NPC cell lines and clinical tissues. Kaplan-Meier curves were used to estimate the association between miR-451 expression and survival. The MTT, colony formation, Transwell migration and invasion assays, and a xenograft model were performed. A miR-451 target was confirmed using luciferase reporter assays, quantitative RT-PCR, and Western blotting.

RESULTS

MiR-451 was significantly downregulated in NPC cell lines and clinical tissues (P < 0.01). Patients with low expression of miR-451 had poorer overall survival (HR, 1.98; 95% CI, 1.16-3.34; P = 0.01) and disease-free survival (HR, 1.68; 95% CI, 1.07-2.62; P = 0.02) than patients with high expression. MiR-451 was an independent prognostic factor in NPC in multivariate Cox regression analysis. Ectopic expression of miR-451 suppressed cell viability, colony formation, and cell migration and invasion in vitro, and inhibited xenograft tumor growth in vivo. MIF was verified as a direct target of miR-451, and MIF regulated NPC cell growth and invasion.

CONCLUSIONS

The newly identified miR-451/MIF pathway provides insight into NPC initiation and progression, and may represent a novel therapeutic target.

Cytokines play an important role in inflammation and immunity. In this study, the authors examined expression of macrophage migration inhibitory factor (MIF) in vascular endothelial cells, using human umbilical vein endothelial cells (HUVEC), by reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot, Western blot analysis, and immunohistochemistry. The RT-PCR/Southern blot showed that MIF mRNA was exceedingly upregulated by the stimulation of lipopolysaccharide (LPS) and reached the maximum 12 h after the stimulation. At the range of 10 pg/ml to 10 ng/ml of LPS, the MIF mRNA expression was induced in a dose-dependent manner, but drastically decreased at doses of more than 100 ng/ml. Western blot analysis and immunohistochemistry using an anti-human MIF antibody revealed the presence of MIF protein in cytoplasm of the unstimulated cells. The precise pathophysiological role of MIF in HUVEC has not been fully understood; however, the upregulation of MIF mRNA expression in vascular endothelial cells by LPS stimulation suggests the possibility that the cytokine plays an important role in systemic inflammatory events such as endotoxaemia.

Macrophage migration inhibitory factor (MIF) is known to exert pleiotropic functions including inhibition of macrophage migration, anchoring, and counteraction of the anti-inflammatory and immunosuppressive activity of glucocorticoids. Ninety-three primary breast cancer tissues and 64 sera of primary breast cancer patients were analyzed for the expression of MIF. The clinico-pathological significance of MIF expression was evaluated. It was found that MIF was frequently over-expressed in primary breast cancer tissues. RT-PCR and western blotting analysis confirmed that wild-type MIF is expressed, and immunohistochemical analysis showed that MIF expression was localized at tumor cells as well as stromal cells, including tumor-associated macrophages. Intratumoral MIF protein concentrations detected by enzyme-linked immunosorbent assay (ELISA) varied with a median value of 1821 ng/mg protein (range: 8 - 8126 ng/mg protein), and correlated inversely with nodal involvement (P = 0.039). No significant correlation was observed with other clinico-pathological factors including tumor size, menopausal status and hormone receptors. The circulating level of MIF protein ranged up to 105.7 ng/ml (median: 17.3 ng/ml), and it was also found to correlate inversely with the number of involved nodes (P = 0.02). A comparative study with other soluble inflammatory mediators showed that intratumoral levels of MIF were significantly associated with those of interleukin-1 beta, suggesting that interactions between tumor cells and tumor-associated macrophages play an important role in the up-regulation of MIF. The multifunctional inflammatory/immune mediator MIF was frequently expressed in primary breast cancer, and its expression level was inversely associated with nodal spread. Thus, MIF seems to play a role in tumor-stroma interactions of primary breast cancers, particularly those with a phenotype of node-negative or minimal nodal spread.

The importance of secreted cytokines and growth factors in the development and promotion of malignancies is often underestimated. Many different soluble, extracellular gene products participate in processes that collectively contribute to the growth and survival of a developing neoplasm. These secreted molecules can, directly or indirectly, play a central role in uncontrolled tumour cell division, angiogenic stimulation or suppression of tumour cell immune surveillance. One of the first cytokine activities ever described, macrophage migration inhibitory factor (MIF), is unique to these soluble mediators in that it participates in all of these pro-tumourigenic processes. Overexpressed in most tumour types examined, MIF has been shown to promote malignant cell transformation, inhibit tumour cell-specific immune cytolytic responses and strongly enhance neovascularization. Despite this broad array of activities, the elucidation of molecular and cellular mechanisms involved in MIF-dependent bioactions has remained elusive. This review will focus on recently characterized phenotypes and mechanistic effectors thought to be associated with MIF-dependent promotion of neoplastic processes and discuss their relative importance in carcinogenesis.

Dengue fever is an emerging viral disease transmitted by arthropods to humans in tropical countries. Dengue hemorrhagic fever (DHF) is escalating in frequency and mortality rates. Here we studied the involvement of macrophage migration inhibitory factor (MIF) in dengue virus (DENV) infection and its pathogenesis. Patients with DHF had elevated plasma concentrations of MIF. Both leukocytes from these patients and macrophages from healthy donors infected in vitro with DENV showed a substantial amount of MIF within lipid droplets. The secretion of MIF by macrophages and hepatocytes required a productive infection and occurred without an increase in gene transcription or cell death, thus indicating active secretion from preformed stocks. In vivo infection of wild-type and mif-deficient (Mif(-/-)) mice demonstrated a role of MIF in dengue pathogenesis. Clinical disease was less severe in Mif(-/-) mice, and they exhibited a significant delay in lethality, lower viremia, and lower viral load in the spleen than wild-type mice. This reduction in all parameters of severity on DENV infection in Mif(-/-) mice correlated with reduced proinflammatory cytokine concentrations. These results demonstrated the contribution of MIF to the pathogenesis of dengue and pointed to a possible beneficial role of neutralizing MIF as an adjunctive therapeutic approach to treat the severe forms of the disease.

Severe malarial anemia (SMA), caused by Plasmodium falciparum infections, is one of the leading causes of childhood mortality in sub-Saharan Africa. Although the molecular determinants of SMA are largely undefined, dysregulation in host-derived inflammatory mediators influences disease severity. Macrophage migration inhibitory factor (MIF) is an important regulator of innate inflammatory responses that has recently been shown to suppress erythropoiesis and promote pathogenesis of SMA in murine models. To examine the role of MIF in the development of childhood SMA, peripheral blood MIF production was examined in Kenyan children (aged <3 years, n = 357) with P. falciparum malarial anemia. All children in the study were free from bacteremia and human immunodeficiency virus type 1. Since deposition of malarial pigment (hemozoin [Hz]) contributes to suppression of erythropoiesis, the relationship between MIF concentrations and monocytic acquisition of Hz was also examined in vivo and in vitro. Circulating MIF concentrations declined with increasing severity of anemia and significantly correlated with peripheral blood leukocyte MIF transcripts. However, MIF concentrations in peripheral blood were not significantly associated with reticulocyte production. Multivariate regression analyses, controlling for age, gender, and parasitemia, further revealed that elevated levels of pigment-containing monocytes (PCM) was associated with SMA and decreased MIF production. In addition, PCM levels were a better predictor of hemoglobin and MIF concentrations than parasite density. Additional experiments in malaria-naive individuals demonstrated that hemozoin caused both increased and decreased MIF production in cultured peripheral blood mononuclear cells (PBMC) in a donor-specific manner, independent of apoptosis. However, PBMC MIF production in children with acute malaria progressively declined with increasing anemia severity. Results presented here demonstrate that acquisition of hemozoin by monocytes is associated with suppression of peripheral blood MIF production and enhanced severity of anemia in childhood malaria.

OBJECTIVE

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine derived from T cells and the pituitary gland. However, several types of solid cancers also secrete MIF, and this factor has been suggested to play an important role in carcinogenesis and the progression of malignancy. In this study, we quantified MIF mRNA expression of non-small cell lung cancer tissues and examined its relationship with clinicopathological factors.

METHODS

MIF mRNAs of both tumor and normal tissues were quantified by a real-time monitoring reverse-transcription PCR in 59 patients with non-small cell lung cancer. The relationship between the grade of MIF expression and clinicopathological factors such as smoking history, cell type, stage, and prognosis was examined to investigate the clinical significance of intratumoral expression of MIF.

RESULTS

The mean copy number of MIF mRNA per 0.08 micro g of total mRNA in tumor tissues was 144,078.00, whereas that of normal lung tissue was 25,438.46 (P < 0.0001). The amounts of MIF proteins revealed by a Western blot analysis correlated well with those of the corresponding mRNAs. Male patients and heavy smokers showed significantly higher expression of MIF. Patients with squamous cell carcinomas showed a higher expression of MIF mRNA than other subjects. In squamous cell carcinoma patients, higher expression of MIF mRNA was significantly associated with unfavorable prognosis (P = 0.0142).

CONCLUSIONS

The general intratumoral expression and close relation with smoking suggested that MIF might contribute to tumorigenesis in the lung.

Originally described as a T lymphocyte-derived factor that inhibited the random migration of macrophages, the protein known as macrophage migration inhibitory factor (MIF) was an enigmatic cytokine for almost 3 decades. In recent years, the discovery of MIF as a product of the anterior pituitary gland and the cloning and expression of bioactive, recombinant MIF protein have led to the definition of its critical biological role in vivo. MIF has the unique property of being released from macrophages and T lymphocytes that have been stimulated by glucocorticoids. Once released, MIF overcomes the inhibitory effects of glucocorticoids on TNF alpha, IL-1 beta, IL-6, and IL-8 production by LPS-stimulated monocytes in vitro and suppresses the protective effects of steroids against lethal endotoxemia in vivo. MIF also antagonizes glucocorticoid inhibition of T-cell proliferation in vitro by restoring IL-2 and IFN-gamma production. This observation has identified a pivotal role for MIF within the immune system and fills an important gap in our understanding of the control of inflammatory and immune responses. Glucocorticoids have long been considered to be an integral component of the stress response to infection or tissue invasion and serve to modulate inflammatory and immune responses. MIF is the first mediator to be identified that can counter-regulate the inhibitory effects of glucocorticoids and thus plays a critical role in the host control of inflammation and immunity.

Low oxygen tension-mediated transcription by hypoxia-inducible factors (HIF) has been reported to facilitate tumor progression, therapeutic resistance, and metastatic adaptation. One previously described target of hypoxia-mediated transcription is the cytokine/growth factor macrophage migration inhibitory factor (MIF). In studies designed to better understand hypoxia-stimulated MIF function, we have discovered that not only is MIF induced by hypoxia in pancreatic adenocarcinoma but MIF is also necessary for maximal hypoxia-induced HIF-1alpha expression. Cells lacking MIF are defective in hypoxia- and prolyl hydroxylase inhibitor-induced HIF-1alpha stabilization and subsequent transcription of glycolytic and angiogenic gene products. Moreover, COP9 signalosome subunit 5 (CSN5), a component of the COP9 signalosome previously reported to functionally interact with MIF, has recently been shown to interact with and stabilize HIF-1alpha. Our results indicate that MIF interacts with CSN5 in pancreatic cancer cells and that MIF-depleted cells display marked defects in hypoxia-induced CSN5/HIF-1alpha interactions. This functional interdependence between HIF-1alpha and MIF may represent an important and previously unrecognized pro-tumorigenic axis.