Dual regulation of macrophage migration inhibitory factor (MIF) expression in hypoxia by CREB and HIF-1

Macrophage migration inhibitory factor (MIF) is a well-described pro-inflammatory mediator that has also been implicated in the process of oncogenic transformation and tumor progression. However, despite the compelling evidence that MIF is overexpressed in, and contributes to, the pathology of inflammatory and malignant diseases the mechanisms that contribute to exaggerated expression of MIF have been poorly described. Here we show that hypoxia, and specifically HIF-1alpha, is a potent and rapid inducer of MIF expression. In addition, we demonstrate that hypoxia-induced MIF expression is dependent upon a HRE in the 5'UTR of the MIF gene but is further modulated by CREB expression. We propose a model where hypoxia-induced MIF expression is driven by HIF-1 but amplified by hypoxia-induced degradation of CREB. Given the importance of MIF in inflammatory and malignant diseases these data reveal a HIF-1-mediated pathway as a potential therapeutic target for suppression of MIF expression in hypoxic tissues.     

Blockage of macrophage migration inhibitory factor (MIF) suppressed uric acid-induced vascular inflammation,smooth muscle cell de-differentiation,and remodeling

Hyperuricemia contributes to vascular injury and dysfunction, yet the potential mechanisms are not well understood. Uric acid (UA) has been found to stimulate macrophage migration inhibitory factor (MIF) up-regulation in renal tubules from rats subjected to UA-induced nephropathy. Given that MIF is able to induce vascular smooth muscle cell (VSMC) de-differentiation (from contractile state to a secretory state), we thus hypothesized that UA-induced vascular injury is via up-regulating of MIF in VSMCs, which enhancing vascular inflammation and VSMC transition. Within a mouse model of UA injection (500?mg/kg, twice/day, 14 days), we measured circulating and vascular MIF levels under UA stimulation at 6?h, day 1, and 14. We tested the efficacy of MIF inhibitor (10?mg/kg, twice/day, 14 days) on UA-induced vascular inflammation and remodeling. High plasma level of UA induced vascular MIF release into the plasma at acute phase. In the chronic phase, the protein level of MIF is up-regulated in the vessels. MIF inhibitor suppressed vascular inflammatory responses, repressed VSMC de-differentiation, and attenuated vascular remodeling and dysfunction following UA stimulation. Knockdown of MIF in cultured VSMCs repressed UA-induced de-differentiation. Our results provided a novel mechanism for MIF-mediated vascular injury in response to UA stimulation, and suggested that anti-MIF interventions may be of therapeutic value in hyperuricemic patients.     

Characterization of Neospora caninum macrophage migration inhibitory factor

The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). BLAST-N analysis of NcMIF revealed high similarity (87%) to the Toxoplasma gondii MIF. NcMIF was cloned and expressed in Escherichia coli in 3 forms, NcMIF (mature protein), NcMIFm (mutation of proline-2 to glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). None of these recombinant NcMIFs (rNcMIF) had tautomerase, oxidoreductase, or immunologic regulatory activities. rNcMIF was unable to compete with recombinant human MIF for a MIF receptor (CD74), suggesting that NcMIF does not bind to this MIF receptor. The glycine substitution for proline-2 of NcMIF resulted in increased retention time on SEC-HPLC and decreased formation of dimers and trimers. The addition of N-terminal HIS-tag led to increased formation of trimers. Immunofluorescence staining demonstrated that NcMIF was localized to the apical end of N. caninum tachyzoites. Immunoelectron microscopy further revealed that NcMIF was present in the micronemes, rhoptries, dense granules, and nuclei. NcMIF was abundant in the tachyzoite lysate and present in excretory and secretory antigen (ESAg) preparations. Total and secretory NcMIF was more abundant in a non-pathologic clone, Ncts-8, than in the wild type isolate (NC1). Furthermore, NcMIF release by the both isolates was increased in the presence of calcium ionophore. This differential production of NcMIF by the pathologic and non-pathologic isolates of N. caninum may suggest a critical role of this molecule in the infectious pathogenesis of this parasite.     

Association of macrophage migration inhibitory factor (MIF) polymorphisms with risk of meningitis from Streptococcus pneumoniae

Macrophage migration inhibitory factor (MIF) is an upstream proinflammatory cytokine encoded by a functionally polymorphic locus. This study of 119 patients explored the potential relationship between MIF genotype and invasive Streptococcus pneumoniae infections. We observed an association between a high-expression MIF allele and occurrence of pneumococcal meningitis.     

Macrophage migration inhibitory factor (MIF) is rendered enzymatically inactive by myeloperoxidase-derived oxidants but retains its immunomodulatory function

Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF.     

Specific reduction of insulin disulfides by macrophage migration inhibitory factor (MIF) with glutathione and dihydrolipoamide: potential role in cellular redox processes

The molecular mechanism of action of MIF, a cytokine that plays a critical role in the host immune and inflammatory response, has not yet been identified. We recently demonstrated that MIF is an enzyme that exhibits oxidoreductase activity by a cysteine thiol-mediated mechanism. Here we further investigated this function by examining the reduction of insulin disulfides by wild-type human MIF (wtMIF) using various substrates, namely glutathione (GSH), dihydrolipoamide, l-cysteine, β-mercaptoethanol and dithiothreitol. The activity of wtMIF was compared to that of the relevant cysteine mutants of MIF and to two carboxy-truncated mutants. Only GSH and dihydrolipoamide were found to serve as reductants, whereas the other substrates were not utilized by MIF. Reduction of insulin disulfides by MIF was closely dependent on the presence of the Cys⁵⁷-Ala-Leu-Cys⁶⁰ (CALC) motif-forming cysteines C57 and C60, whereas C81 was not involved (activities: 51±13%, 14±5%, and 70±12% of wtMIF, respectively, and 20±3% for the double mutant C57S/C60S). Confirming the notion that the activity of MIF was dependent on the CALC motif in the central region of the MIF sequence, the C-terminal deletion mutants MIF(1–105) and MIF(1–110) were found to be fully active. The favored use of GSH and dihydrolipoamide indicated that MIF may be involved in the regulation of cellular redox processes and was supported further by the finding that MIF expression by the cell lines COS-1 and RAW 264.7 was significantly induced upon treatment with the oxidant hydrogen peroxide.     

Haemaphysalis longicornis: molecular characterization of a homologue of the macrophage migration inhibitory factor from the partially fed ticks

The macrophage migration inhibitory factor (MIF) has been identified from some vertebrates and invertebrates. MIF is related to inflammation, tumor growth, and angiogenesis in vertebrates. Here, we report the molecular characterization of a homologue of MIF from partially fed Haemaphysalis longicornis. The sequence analysis of the H. longicornis MIF (HlMIF) indicated that its deduced amino acid sequence has an identity of 77% with the MIF of the tick Amblyomma americanum. Western blot analysis using the anti-His-HlMIF antibody showed that HlMIF was up-regulated during blood feeding. Immunohistochemistry showed that the endogenous HlMIF in partially fed ticks was localized to the midgut and epidermal cells. Moreover, the functional assay revealed that the GST-HlMIF inhibited the migration of human monocytes. In conclusion, we consider that HlMIF may facilitate blood feeding by inhibiting host macrophage migration to the feeding lesion or may participate in the proliferation and differentiation of cells in the tick body.