Amblyomma americanum (L): Tick macrophage migration inhibitory factor peptide immunization lengthens lone star tick feeding intervals in vivo

Immunizations of New Zealand White rabbits with specific macrophage migration inhibitory factor (MIF) tick peptide (PEP) produced circulating anti-tick PEP antibodies in the hosts. Antibody titers of greater than 1:5000 to tick MIF peptide were observed for crude sera from PEP-immunized rabbits. PEP- and BSA-vaccinated rabbits were infested with Amblyomma americanum adults. Feeding intervals, female weights, egg masses and percent egg hatch were measured for ticks feeding on control and immunized hosts. Feeding intervals were significantly lengthened to 13.3 days for PEP-vaccinated hosts compared to BSA-vaccinated controls at 12.4 days, while female engorgement weights and egg masses were unchanged. By immunizing hosts using specific tick PEP, we were able to alter the length of time the ticks fed on their hosts.     

Identification of Macrophage Migration Inhibitory Factor Isoforms in Bovine Brain

In the course of the study of the primary structures and molecular mechanisms of action of immunologically active compounds of the nervous system we have isolated from the soluble fraction of total bovine brain two heat-stable proteins. The purification procedure was mainly based on DEAE-Servacel ion-exchange chromatography and reversed-phase HPLC. The proteins were identified by the N-terminal Edman microsequence analysis and database searching as macrophage migration inhibitory factor (MIF). The N-terminal sequences for MIF1 and MIF2 were found to be identical. According to mass spectral analysis, the molecular masses for MIF1 and MIF2 were determined respectively as 12,369.21 and 12,299.7 Da. In addition, we have also isolated a third peptide having the same N-terminal sequence and Mr 9,496.2 that seems to be a proteolytic fragment of MIF. Using p-hydroxyphenylpyruvate as a substrate, we have not revealed tautomerase activity of either MIF1 or MIF2. As both the immunologic and enzymatic activities were reported to be expressed by the oligomeric structure of MIF, we suggest that the present study may give additional information on MIF in terms of structural properties of this protein. A comparatively simple purification procedure is presented that may be widely used for simultaneous isolation in one run of MIF isoforms.     

Macrophage migration inhibitory factor expression and protein localization in <Emphasis Type="Italic">Amblyomma americanum</Emphasis> (Ixodidae)

Amblyomma americanum (L.) ticks continue to emerge as disease vectors in many areas of the United States. Tick macrophage migration inhibitory factor (MIF) was first identified in A. americanum females and has been demonstrated to inhibit macrophage movement to the same extent as human MIF. This study was conducted to further characterize and elucidate the physiological role for MIF in tick feeding. A relative quantitative PCR assay was developed to determine the level of MIF gene expression during tick feeding. In addition, RNAi techniques were used to silence MIF prior to blood feeding. Physiological parameters of tick engorgement weight, length of feeding interval, and egg masses were observed to check for phenotypic manifestations of RNA silencing. Specific tick MIF antibody was used to localize MIF protein in frozen tick tissue sections. Tissue specific gene expression indicated that the midgut tissues were the most highly enriched for the MIF. Levels of gene expression did not parallel MIF protein pools seen in tissue sections. Of particular importance was the finding that unfed tick salivary glands appear to contain vesicles that are specific for MIF protein. This is the first demonstration of a pool of MIF that could be secreted during the first hours of tick feeding. While MIF silencing was demonstrated at the molecular level, no physiological phenotype was apparent. The MIF protein pools already available in the tissues may be sufficient to accomplish female tick feeding. Our studies show that the most prominent source of MIF during tick feeding is the midgut tissue. Future studies will address the role of MIF in blood feeding and nutrient digestion in the immature life stages of the tick.     

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.     

MIF proteins are not glutathione transferase homologs.

Although macrophage migration inhibitory factor (MIF) proteins conjugate glutathione, sequence analysis does not support their homology to other glutathione transferases. Glutathione transferases are not detected with MIF proteins in searches of protein sequence databases, and MIF proteins do not share significant sequence similarity with glutathione transferases. Homology cannot be demonstrated by multiple sequence alignment or evolutionary tree construction; such methods assume that the proteins being analyzed are homologous.     

Macrophage migration inhibitory factor in the regulation of myoblast proliferation and differentiation

Obesity is documented to be a state of chronic mild inflammation associated with increased macrophage infiltration into adipose tissue and liver and skeletal muscle. As a pleiotropic inflammatory mediator, macrophage migration inhibitory factor (MIF) is associated with metabolic disease, so MIF may signal molecular links between adipocytes and myocytes. MIF expression was modified during myoblast differentiation, but the role of MIF during this process is unclear. C2C12 cells were transfected with MIF to investigate their role during differentiation. MIF expression attenuated C2C12 differentiation. It did not change proliferation, but downregulated cyclin D1 and CDK4, causing cell accumulation in the G1 phase. p21 protein was increased significantly and MyoD, MyoG, and p21 mRNA also increased significantly in the C2C12 cells treated with ISO-1, suggesting that inhibition of MIF promotes differentiation. MIF inhibits the myoblast differentiation by affecting the cell cycle progression, but does not affect proliferation.     

The expression of macrophage migration inhibitory factor in the non-small cell lung cancer

ObjectiveThe objective was to investigate the expression of macrophage migration inhibitory factor (MIF) in non-small cell lung cancer (NSCLC), as well as the effects of macrophage MIF on tumor cells.MethodsThe human NSCLC cell strains H358 and H524 were selected as research objects. The Real-Time Polymerase Chain Reaction (RT-PCR) and Western Blot were utilized to detect the expression levels of MIF in human NSCLC cell strains. The lentiviral plasmid was utilized for MIF-mRNA interference. The expression levels of MIF before and after transfection were compared. The cell strains were cultured and proliferated for cell count and comparison.ResultsH358 showed MIF high expression while H524 showed MIF low expression. Once the H358 cells were constructed as silent MIF expression, compared with the original H358 cells, the difference was statistically significant. Once the H524 cells were constructed as high MIF expression, compared with original H524 cells, the difference was statistically significant. Being cultured for respective 3, 5, and 7 days, the transfected H358 cells showed a significant decrease in proliferative activity compared with original H358 cells, while the transfected H524 cells showed a significant increase in proliferative activity compared with original H524 cells.ConclusionMIF has high expression in H358 cells while low expression in H524 cells. The expression of MIF could enhance the proliferative activity of NSCLC tumor cells.     

Macrophage migration inhibitory factor (MIF) promotes rat airway muscle cell proliferation and migration mediated by ERK1/2 and FAK signaling

Macrophage migration inhibitory factor (MIF) is an inflammatory mediator that contributes to asthmatic airway remodeling; however, little is known regarding the effects of MIF on airway smooth muscle cells (ASMCs). In the present study, we found that an enhanced expression of MIF promoted ASMC proliferation, increased the population of cells in the S/G2 phase, downregulated P21 expression, and upregulated cyclin D1, cyclin D3, and Cdk6 expression. In addition, the apoptosis of ASMCs was significantly decreased in response to MIF overexpression, compared with the negative control. Moreover, MIF facilitated the migration of ASMCs by upregulating the expression of matrix metalloproteinase (MMP)‐2. Finally, we showed that MIF increased the phosphorylation of extracellular regulated protein kinases (ERK) 1/2 and focal adhesion kinase (FAK), which are associated with proliferation and migration. In conclusion, this study demonstrated that MIF overexpression promotes the proliferation and migration of ASMCs by upregulating the activity of the ERK1/2 and FAK signaling pathways in these cells, further indicating that inhibition of MIF may prove to be an effective strategy for treating asthma patients with airway remodeling.     

Macrophage migration inhibitory factor in zinc-allergic systemic contact dermatitis

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose expression has been found to be critical to the generation of the antigen-specific immune response. Recent studies suggested that MIF plays a role in the initiation and maintenance of allergic disease. The aim of this study was to investigate whether MIF is involved in the pathogenesis of zinc-allergic systemic contact dermatitis. A 49-year-old Japanese woman developed facial edema, blepharedema and pruritic edematous erythema with papules over the entire body. Based of the results of a metal patch test, drug lymphocyte stimulating test and drug challenge test, diagnosis of zinc-allergic systemic contact dermatitis was made. Serum MIF and TNF-alpha levels of the patient, 20 healthy controls and other 6 patients who showed positive reaction to metal patch test were measured by an ELISA. Moreover we examined MIF production of peripheral blood mononuclear cells (PBMCs) from our patient, 3 healthy controls and other 2 patients who showed positive reaction to metal patch test at various metal concentrations. The patient's serum showed high MIF and TNF-alpha levels compared to healthy controls and other metal allergy patients. Furthermore, zinc stimulation of patient's PBMC showed higher MIF and TNF-alpha secretion compared with healthy subjects. The MIF content of 2 patients with other metal allergy was not significantly increased after metal stimulation. Our data suggest that zinc in the peripheral blood of zinc-allergic patients induce PBMCs to produce increased MIF levels, which could lead to systemic contact dermatitis.     

Dermacentor variabilis: characterization and modeling of macrophage migration inhibitory factor with phylogenetic comparisons to other ticks, insects and parasitic nematodes

We have identified and characterized the full length cDNA sequence of macrophage migration inhibitory factor (MIF) from the American dog tick, Dermacentor variabilis. The nucleotide and putative amino acid sequences from this study shared a high level of sequence conservation with other tick MIFs. The bioinformatics analysis showed across species conservation of the MIF amino acid sequence in ticks, insects and nematodes. The multiple sequence alignment identified Pro 1, 3, 55; Thr 7, 112; Asn 8, 72; Ile 64, 96; Gly 65, 110, Ser 63 and Leu 87 amino acids to be highly conserved among the sequences selected for this study. Tick MIF does not have the oxidoreductase domain as found in MIFs from other animals suggesting that tick MIF is not capable of performing as an oxidoreductase. The phylogenetic analysis revealed that tick MIFs share a closer evolutionary proximity to parasitic nematode MIFs than to insect MIFs.     

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.     

巨噬细胞移动抑制因子在IgA肾病中的表达及意义

目的研究巨噬细胞移动抑制因子(MIF)在不同病变程度IgA肾病中的表达变化,探讨其对IgA肾病进展的影响。方法应用免疫组织化学双标记技术检测正常对照组及不同病变程度IgA肾病患者肾组织内MIF和人巨噬细胞标记抗原CD68的表达。肾组织病理改变采用常规病理学方法观察。详细收集每例患者肾活检时的24小时尿蛋白定量(TUPr)及内生肌酐清除率(CCr),并与免疫病理结果进行相关分析。结果对照组和IgA肾病轻度组仅有少量MIF和CD68表达。中、重度病变组较对照组及轻度病变组MIF和CD68的表达显著增加(P<0.05);MIF和CD68的表达之间具有显著相关性(P<0.05);肾组织内MIF、CD68及MIF /CD68 表达与TUPr及CCr具有显著相关性(P<0.05)。结论肾组织内MIF表达上调所导致的巨噬细胞浸润增加是IgA肾病进展的重要机制之一。     

Toxoplasma gondii: Impaired maturation and pro-inflammatory response of dendritic cells in MIF-deficient mice favors susceptibility to infection

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Toxoplasma gondii, we investigated host resistance in MIF−/− mice (BALB/c background) during natural oral infection. We focused on the potential involvement of MIF in Dendritic Cell (DC) maturation and IL-12 production. Following oral T. gondii infection, wild type mice developed a strong IL-12 response with an adequate maturation of their draining mesenteric lymph node DC (MLNDC) population and were resistant to challenge with either 40 or 100 cysts (ME49 strain). In contrast, similarly infected MIF−/− mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to both type of challenges. Lack of maturation and IL-12 production of DCs in response to T. gondii antigens was confirmed by in vitro studies, and these effects were reversed following treatment with recombinant MIF. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. gondii infection.     

Accelerated protein aggregation induced by macrophage migration inhibitory factor under heat stress conditions

Kinetics of thermal aggregation of model protein substrates (glycogen phosphorylase b from rabbit skeletal muscle and yeast alcohol dehydrogenase) were investigated under heat stress conditions (41-48 degrees C) in the presence of macrophage migration inhibitory factor (MIF), a heat-stable hydrophobic protein (12.5 kD). Anti-chaperone MIF activity found by turbidimetry manifests itself in significantly accelerated protein aggregation and increased limiting value of apparent optical absorption at 360 nm and t --> infinity in the sub-stoichiometric range of MIF concentrations. The aggregation kinetics is shown to have cooperative character. Possible reversibility of aggregation after removal of denaturing conditions was demonstrated using alcohol dehydrogenase aggregation at a temperature close to the physiological level (41.5 degrees C). This reversibility is caused by solubility of aggregates and stabilization of oligomeric structure of the substrate as a result of MIF binding to the partially denatured protein. The data suggest that in spite of distinct anti-chaperone effect, the chaperone-like activity of MIF can be observed in the case of heat stress removal and restoration of the system to normal conditions.     

Macrophage migration inhibitory factor-deficient mice are resistant to ovariectomy-induced bone loss

A link between macrophage migration inhibitory factor (MIF) and estrogen has recently emerged. We examined the involvement of MIF in osteoporotic changes in bone after ovariectomy (OVX), and revealed that MIF-deficient mice (MIF-KO) were completely protected from this phenomenon. The increase in osteoclast number per bone surface and serum IL-1β levels, which were observed in wild-type mice after OVX, did not occur in MIF KO. Our data suggest that MIF plays an important role in the pathogenesis of postmenopausal osteoporosis, and could be a novel target for the treatment of this disease.