Suppression of lymphokine production: I. Macrophage-mediated inhibition of MIF production

Macrophages have been found to suppress the in vitro production by stimulated T lymphocytes of a lymphokine, migration inhibitory factor. When macrophages isolated from primary MSV-induced tumors were added to antigen-stimulated MSV-immune spleen cells, a complete suppression of MIF production was observed. This suppression was nonspecific, since MIF production by antigen-stimulated alloimmune spleen cells and by PHA-stimulated normal spleen cells was also inhibited. Suppressor macrophages could also be induced by inoculation with Corynebacterium parvum, whereas light mineral oil-induced peritoneal macrophages had no detectable effect on MIF production. The failure to detect MIF in the supernatants of stimulated cultures containing activated macrophages appeared to be due to inhibition of lymphokine production rather than to absorption or inactivation of MIF or to interference with the assay for detection of MIF. Macrophages were able to suppress MIF production only when added during the first 4–5 hr of culture and they had no effect when added later. These data show that activated macrophages can nonspecifically suppress lymphokine production and that this appears to be due to inhibition of an early step in lymphocyte stimulation.     

Effect of stem cell inhibition factor and macrophage inhibition factor on mouse spleen cell exocolonization and migration]

The migration activity of the spleen cells from intact mice is inhibited by the stem cell inhibitory factor (SCIF) released by lymphocytes treated with antilymphocytic globulin. The degree of the migration inhibition is proportional to the activity of SCIF in the colony-formation inhibition. The macrophage-migration inhibitory factor (MIF), obtained in the H-2 system exhibited a stimulating effect on the colony formation in mice when used in vitro for the treatment of bone marrow transplants. This activity of MIF corresponds to its migration-inhibitory effect on the spleen cells. Incubation of the bone marrow cells with MIF for 30 minutes is more effective than the 2-hour treatment. The observed effects are interpreted as an indication of non-identity of SCIF and MIF.     

Autophagy inhibition in breast cancer cells induces ROS-mediated MIF expression and M1 macrophage polarization

Autophagy can function as a survival mechanism for cancer cells and therefore, its inhibition is currently being explored as a therapy for different cancer types. For breast cancer, triple negative breast cancer (TNBC) is the subtype most sensitive to the inhibition of autophagy; but its inhibition has also been shown to promote ROS-dependent secretion of macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine. In this work, we explore the role of MIF in breast cancer, the mechanism by which autophagy inhibition promotes MIF secretion and its effects on neighboring cancer cell signaling and macrophage polarization. We analyzed MIF mRNA expression levels in tumors from breast cancer patients from different subtypes and found that Luminal B, HER2 and Basal subtypes, which are associated to high proliferation, displayed high MIF levels. However, MIF expression had no prognostic relevance in any breast cancer subtype. In addition, we found that autophagy inhibition in 66cl4 TNBC cells increased intracellular Reactive Oxygen Species (ROS) levels, which increased MIF expression and secretion. MIF secreted from 66cl4 TNBC cells induced the activation of MIF-regulated pathways in syngeneic cell lines, increasing Akt phosphorylation in 4T1 cells and ERK phosphorylation in 67NR cells. Regarding MIF/ chemokine receptors, higher levels of CD74 and CXCR2 were found in TNBC tumor cell lines when compared to non-tumorigenic cells and CXCR7 was elevated in the highly metastatic 4T1 cell line. Finally, secreted MIF from autophagy deficient 66cl4 cells induced macrophage polarization towards the M1 subtype. Together, our results indicate an important role for the inhibition of autophagy in the regulation of ROS-mediated MIF gene expression and secretion, with paracrine effects on cancer cell signaling and pro-inflammatory repercussions in macrophage M1 polarization. This data should be considered when considering the inhibition of autophagy as a therapy for different types of cancer.     

Macrophage migration inhibitory factor: a downregulator of early T cell-dependent IFN-gamma responses in Plasmodium chabaudi adami (556 KA)-infected mice

Neutralization of macrophage migration inhibitory factor (MIF) increases anti-tumor cytotoxic T cell responses in vivo and IFN-γ responses in vitro, suggesting a plausible regulatory role for MIF in T cell activation. Considering that IFN-γ production by CD4(+) T cells is pivotal to resolve murine malaria and that secretion of MIF is induced by Plasmodium chabaudi adami parasites, we investigated the effect of MIF deficiency on the infection with this pathogen. Infections with P. c. adami 556 KA parasites were more efficiently controlled in MIF-neutralized and MIF-deficient (knockout [KO]) BALB/c mice. The reduction in parasitemia was associated with reduced production of IL-4 by non-T/non-B cells throughout patent infection. At day 4 postinfection, higher numbers of activated CD4(+) cells were measured in MIF KO mice, which secreted more IFN-γ, less IL-4, and less IL-10 than did CD4(+) T cells from wild-type mice. Enhanced IFN-γ and decreased IL-4 responses also were measured in MIF KO CD4(+) T cells stimulated with or without IL-12 and anti-IL-4 blocking Ab to induce Th1 polarization. However, MIF KO CD4(+) T cells efficiently acquired a Th2 phenotype when stimulated in the presence of IL-4 and anti-IL-12 Ab, indicating normal responsiveness to IL-4/STAT6 signaling. These results suggest that by promoting IL-4 responses in cells other than T/B cells during early P. c. adami infection, MIF decreases IFN-γ secretion in CD4(+) T cells and, additionally, has the intrinsic ability to render CD4(+) T cells less capable of acquiring a robust Th1 phenotype when stimulated in the presence of IL-12.     

Regulating effect of bone marrow cells on human T-lymphocyte function

A study was made of the regulatory effect of human bone marrow cells in two experimental systems: lymphocyte proliferation in response to PHA, and spontaneous and PHA-induced production of macrophage migration inhibition factor (MIF) by peripheral blood lymphocytes. It was shown that bone marrow cells inhibit the proliferative activity of stimulated peripheral blood lymphocytes and induced MIF production. The effect of bone marrow cells on spontaneous MIF production was found to be inconclusive.     

The cellular basis for differential lymphokine responses to mitogen stimulation

Human mononuclear cells from some individuals produce macrophage migration inhibition factor (MIF) when stimulated with Con A while those of others produce migration stimulation factor (MStF). T cells were responsible for these different responses but T4 cells produced MIF and T8 cells produced MStF regardless of the global response which was not explained by the individual T4:T8 ratios. Admixing the T-cell subpopulations in vitro revealed that MIF responses switched to MStF responses between T4:T8 ratios of 75:25 and 50:50 with MStF responders switching at higher ratios than MIF responders. Pulse exposure to supernatants from Con A-stimulated T4-enriched cells significantly reduced migration indices resulting from stimulation of fresh cells, promoting MIF responses regardless of the responder status of the supernatant donor. In contrast, supernatants from T8-enriched cells, when obtained from MStF responders, significantly increased migration indices while there was no effect when the supernatants were obtained from MIF responders. These results suggest that soluble factors from T8 cells are primarily responsible for determining whether an individual mounts a MIF or MStF response to Con A stimulation.     

Antisense macrophage migration inhibitory factor (MIF) prevents anti-IgM mediated growth arrest and apoptosis of a murine B cell line by regulating cell cycle progression

Macrophage migration inhibitory factor (MIF) is involved in the generation of cell-mediated immune responses. Recently it has been reported that MIF also plays a role in cell proliferation and differentiation. In the present study, using a B-cell line, WEHI-231, and its stable MIF-antisense transfectant, WaM2, as a representative transfectant, we investigated the mechanism underlying regulation of the cell growth by MIF. WaM2 cells produced less MIF than vector control or parental WEHI-231 cells. Reduced and increased proportions were seen in G1 and S-phase cells, respectively, in WaM2 as compared with WEHI-231. Growth arrest and apoptosis after stimulation via surface Ig (sIg) were less prominent in WaM2 cells than those in WEHI-231. However, the addition of recombinant rat MIF did not reverse the inhibition of the growth arrest and apoptosis induced in WaM2 by cross-linking sIg. Almost the same amount of p27kip1 expression was detected in WaM2 cells as those in WEHI-231 and vector control cells. Cross-linking of sIg elevated the p27kip1 level equally in these cells irrespective of the MIF-antisense expression. Taken together, it seems that MIF plays a role in inducing apoptosis in B cells upon IgM cross-linking by regulating the cell cycle via a novel intracellular pathway.     

The mediator of cellular immunity: XI. Origin and development of MIF producing lymphocytes

Thoracic duct lymphocytes obtained from rats infected with Listeria monocytogenes were characterized with respect to size, turnover and their capacity to release macrophage migration inhibitory factor (MIF). Cells responsive to Listerial antigens (LMA) in the MIF assay were identified in lymph during the first week of an immunizing infection. These were immunoblasts or large lymphocytes, as evidenced by their sedimentation with S phase lymphocytes at unit gravity. When labeled cells from the lymph of Listeria-infected donors were infused into similarly infected recipients, donor S phase lymphocytes localized rapidly, and in substantial numbers, in peritoneal exudates induced by the unrelated organism, F. tularensis. Within this immigrant population were cells which conferred immunity against L. monocytogenes and released MIF in cultures containing LMA. Exudates harvested 36 hr or 61 hr after stimulation contained labeled lymphocytes that were smaller than the S phase cells recovered during the early post-induction period. The observed shift of radioactivity from large to smaller lymphocytes was parallelled by a shift MIF production to exudate fractions containing smaller cells. The MIF producing cells in exudates of advancing age also exhibited increasing resistance to inhibition by vinblastine. These findings suggest that MIF is released by a family of lymphocytes—large, medium and small. LMA-responsive lymphocytes are delivered to the thoracic duct soon after their formation, at a stage in development when they can be stimulated to release only low levels of MIF. These mediator producing cells circulate briefly in the blood and differentiate fully only after they extravasate into inflammatory foci.     

Human V gamma 2V delta 2 T cells augment migration-inhibitory factor secretion and counteract the inhibitory effect of glucocorticoids on IL-1 beta and TNF-alpha production

In immune cells, proinflammatory cytokine gene expression is regulated by glucocorticoids, whereas migration-inhibitory factor (MIF), a pleiotropic cytokine, has the unique property of counteracting the inhibitory effect of glucocorticoids on TNF-alpha and IL-1beta secretion. A few lines of evidence suggest that gammadelta T cells play an important role in immunoregulation. However, it is unknown whether human gammadelta T cells participate in regulating MIF secretion, and how gammadelta T cells, glucocorticoids, and cytokines converge to give a unified physiological response. In this study, we demonstrate that human Vgamma2Vdelta2 T cells augment MIF secretion. Remarkably, these Vgamma2Vdelta2 T cells, functioning similarly to MIF in part, counteracted inhibition of dexamethasone on production of IL-1beta and TNF-alpha. SCID mice reconstituted with human PBMC that were mock depleted of Vdelta2 T cells and repeatedly infected with lethal dose of Escherichia coli had shorter survival time than those reconstituted with PBMC that were depleted of Vdelta2 T cells. Thus, human Vgamma2Vdelta2 T cells are likely to play broad-spectrum roles in immunoregulation and immunopathology by influencing MIF secretion and the immunomodulatory function of glucocorticoids.     

Macrophages in resistance to rickettsial infection: susceptibility to lethal effects of Rickettsia akari infection in mouse strains with defective macrophage function

We have confirmed the requirement of macrophages in the antigen-induced T-lymphocyte proliferative response and in the generation of migration inhibition factor (MIF) by immune lymphocytes. Extending these observations, we have found that autologous and non-syngeneic, oil-induced peritoneal exudate macrophages were equally effective in restoring the proliferative response and MIF production by column-purified lymph node T cells. MIF activity was optimally restored when T cells were reconstituted with 1 to 40% exudate-derived macrophages whereas 10 to 30% macrophages were needed to optimally restore the T-cell proliferative response. Normal resident macrophages from the peritoneal cavity were also capable of restoring T-cell reactivity as were normal or BCG-activated pulmonary alveolar macrophages. It was also found that the addition of as few as 1.0% glycogen-elicited peritoneal exudate cells restored the production of MIF by T cells. Quantitative considerations demonstrated that the responsible cells in these preparations were polymorphonuclear cells rather than macrophages. In contrast, neither MIF production nor the proliferative response by T cells were restored by the addition of red blood cells. In these studies we were able to demonstrate that freeze-thawed macrophages could restore antigen-induced MIF production, but not antigen-induced cellular proliferation. The ability of freeze-thawed macrophages to stimulate T cells to produce MIF was apparently associated with the macrophage membranes and not with a soluble factor in the macrophage extracts. These results demonstrate that multiple sources of phagocytic cells may interact cooperatively with lymphocytes in reactions of cell-mediated immunity. Further, at least in the case of MIF production, this interaction involves a membrane-bound determinant that is effective even in the absence of viable macrophages.     

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.     

Modulation of granulomatous inflammation in murine schistosomiasis mansoni by enteric exposure to schistosome ova: in vitro characterization of a regulatory mechanism within the granuloma

Enteric immunization with schistosome ova results in a diminished granulomatous response. This study explored a mechanism by which enteric immunization may decrease granuloma size. Granulomas from livers of acutely infected mice were dissociated and the dispersed cells were depleted of macrophages. As defined by a direct in vitro migration inhibition factor (MIF) assay, the macrophage-depleted cells, composed of lymphocytes and eosinophils, inhibited the migration of normal peritoneal exudate cells when exposed to soluble egg antigens. Anti-Thy 1.2 or -Lyt 1.1, but not -Lyt 2.1, treatment of these cells abrogated MIF activity. Next, mice were exposed enterically to eggs 4 weeks prior to sacrifice. Cells from granulomas isolated from these animals demonstrated no MIF activity unless treated with anti-Lyt 2.1. When granuloma cells from enterically immunized mice were mixed with those from unimmunized animals, MIF activity by the latter was abrogated. Treatment of cells from immunized mice with anti-Lyt 2.1 or -Thy 1.2, but not -Lyt 1.1 prior to mixing once again permitted MIF activity. These results suggest that the diminished granulomatous response induced by enteric immunization could be mediated by Lyt 2+ suppressor T cells. These suppressor cells may regulate the MIF activity of Lyt 1+ T lymphocytes residing within these lesions.     

Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation

Autophagy is an evolutionarily conserved catabolic process that maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1β and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actual role of MIF in autophagy remains unclear. Here, we have demonstrated that incubation of human hepatoma cell line HuH-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation, autophagic flux, and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of HuH-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of HuH-7 cells. Taken together, these data suggest that cell autophagy was induced by MIF under stress conditions such as inflammation and starvation through ROS generation.     

Refractoriness to migration inhibitory factor of macrophages of LPS nonresponder mouse strains.

The responsiveness to macrophage migration inhibitory factor (MIF) of peritoneal exudate cells (PEC) from the LPS unresponsive C3H/HeJ and C57BL/10ScCR mice was assessed by the indirect agarose microdroplet macrophage migration inhibition assay. No migration inhibition with PEC from C3H/HeJ nor C57BL/10ScCR mice was detected, whereas PEC from both C3H/HeN and C57BL/10Sn mice were significantly inhibited by even a 1/32 dilution of MIF-containing supernatants. Responsiveness to MIF of C3H/HeJ PEC could, however, be induced. In vivo inoculations of Mycobacterium bovis, strain BCG, 7 days before in vitro assay rendered C3H/HeJ PEC responsive to MIF. The lack of responsiveness to MIF by C3H/HeJ PEC appeared related to some form of suppression, since a mixture of PEC from C3H/HeN mice with 10 to 15% PEC from C3H/HeJ mice resulted in undetectable migration inhibition at any MIF dilution. In contrast to the usual lack of responsiveness of their macrophage to MIF, C3H/HeJ mice were able to produce MIK in response to PPD as well as their counterpart C3H/HeN mice after BCG sensitization. These results demonstrate that macrophages from C3H/HeJ and C57BL/10ScCR mice are unable to be inhibited in their in vitro migration of MIF (possibly being directly or indirectly influenced by a suppressor cell), whereas lymphoid cells from at least one of these strains, the C3H/HeJ mice, can produce MIF in response to antigenic stimulation.     

Cell-mediated immunity to murine tumor allografts. Increase in the activities of activated thymus-derived cells following in vitro incubation

The immunity of CS7B1 spleen cells to the allogeneic tumor P815 has been studied using a direct cytotoxic assay and an assay of macrophage migration inhibition. Both assays principally measure activities of thymus-derived cells. The activity of spleen cells in either assay is markedly enhanced by overnight incubation in the absence of deliberately added antigen, and, for the MIF cell assay at least, this enhancement apparently does not depend upon cell division or change in cell size during that incubation. The effect of overnight incubation is to some degree mimicked by short-term exposure to trypsin; furthermore, this effect can be blocked by incubation in the presence of serum from the spleen cell donors. These results suggest that blocking factors exist on the surface of some small T lymphocytes taken from thes animals, and that these factors can suppress T cell activity in both of the assay systems used.     

用绿色荧光蛋白（GFP）作为报告分子筛选有效的siRNA

 建立一种利用绿色荧光蛋白（GFP）作为报告分子筛选能有效抑制目的基因表达的siRNA的方法.以巨噬细胞移动抑制因子（MIF）基因为研究对象,筛选能有效沉默MIF表达的质粒载体介导的siRNA.构建拥有同一Kozak共有翻译启始序列、翻译启始密码子ATG的MIF-GFP融合表达载体pEGFP-MIF.分别将3个靶向MIF的siRNA表达质粒与pEGFP-MIF共转化HEK293细胞,在荧光显微镜下观察HEK293细胞中GFP的表达,并用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.同时,将MIF siRNA表达质粒分别与MIF表达载体共转化HEK293细胞,用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.定量PCR结果显示,GFP表达低的细胞中,MIF mRNA的表达也明显降低;利用pEGFP-MIF和MIF表达载体筛选到的有效MIF siRNA的结果一致.因此,建立了目的基因与GFP融合表达,以GFP作为报告分子来筛选抑制目的基因表达siRNA的方法,并为进行多个基因的有效siRNA的筛选提供解决方案.     

A simple and visualized method to screen for effective siRNAs by using green fluorescence protein (GFP) as a reporter

To screen for effective small interference RNA (siRNA), a simple and visualized method was developed using the green fluorescence protein (GFP) as a reporter. Candidate siRNAs targeting macrophage migration inhibition factor genes (MIF) were identified. By using the pEGFP-N3 vector, the MIF-GFP expression plasmid, pEGFP-MIF, was constructed with the same Kozak con-sensus translation initiation site and start code ATG for the MIF-EGFP coding sequence. Based on the siRNA expression vector pSilencer-4,1,3 candidate MIF siRNA expression plasmids were constructed and co-transfected with the pEGFP-MIF into the H EK293 cells, respectively. The GFP expression in HEK293 cells could be viewed by fluorescence microscopy and the MIF mRNA expressions were determined by real-time quantitative PCR. The 3 candidate MIF siRNA expression plasmids were also co-transfected with the MIF expression plasmid into the HEK293 cells, respectively, and the MIF mRNA expres-sions were determined by real-time quantitative PCR. The results show that the down-regulated expression of the MIF mRNA was consistent with the GFP expression and the same effective MIF siRNAs were screened by using the pEGFP-MIF or MIF expression plasmid with the candidate MIF siRNAs expression plasmids. Therefore, by using the GFP as a reporter, a useful method was provided to screen for effective siRNAs tar-geting specific genes co-expressed with the GFP. This may be a good strategy for screening for effective siRNAs tar-geting different genes.     

Human uveal melanoma cells produce macrophage migration-inhibitory factor to prevent lysis by NK cells

Human uveal melanoma arises in an immune privileged ocular environment in which both adaptive and innate immune effector mechanisms are suppressed. Uveal melanoma is the most common intraocular tumor in adults and is derived from tissues in the eye that produce macrophage migration-inhibitory factor (MIF), a cytokine that has recently been demonstrated to produce immediate inhibition of NK cell-mediated lytic activity. Although NK cell-mediated lysis of uveal melanomas is inhibited in the eye, melanoma cells that disseminate from the eye are at risk for surveillance by NK cells. Moreover, uveal melanoma cells demonstrate a propensity to metastasize to the liver, an organ with one of the highest levels of NK activity in the body. Therefore, we speculated that uveal melanomas produced MIF as a means of escaping NK cell-mediated lysis. Accordingly, seven primary uveal melanoma cell lines and two cell lines derived from uveal melanoma metastases were examined for their production of MIF. MIF was detected in melanoma culture supernatants by both ELISA and the classical bioassay of macrophage migration inhibition. Melanoma-derived MIF inhibited NK cell-mediated lysis of YAC-1 and uveal melanoma cells. Cell lines derived from uveal melanoma metastases produced approximately twice as much biologically active MIF as cultures from primary uveal melanomas. Inhibition of NK cell-mediated killing by uveal melanoma-derived MIF was specifically inhibited in a dose-dependent manner by anti-MIF Ab. The results suggest that human uveal melanoma cells maintain a microenvironment of immune privilege by secreting active MIF that protects against NK cell-mediated killing.     

Macrophage migration inhibitory factor mediates hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is a devastating disease leading to progressive hypoxemia, right ventricular failure, and death. Hypoxia can play a pivotal role in PH etiology, inducing pulmonary vessel constriction and remodeling. These events lead to increased pulmonary vessel wall thickness, elevated vascular resistance and right ventricular hypertrophy. The current study examined the association of the inflammatory cytokine macrophage migration inhibitory factor (MIF) with chronic lung disease and its role in the development of hypoxia-induced PH. We found that plasma MIF in patients with primary PH or PH secondary to interstitial lung disease (ILD) was significantly higher than in the control group (P = 0.004 and 0.007, respectively). MIF involvement with hypoxia-induced fibroblast proliferation was examined in both a human cell-line and primary mouse cells from wild-type (mif ^+/+) and MIF-knockout (mif ^−/−) mice. In vitro, hypoxia-increased MIF mRNA, extracellular MIF protein accumulation and cell proliferation. Inhibition of MIF inflammatory activity reduced hypoxia-induced cell proliferation. However, hypoxia only increased proliferation of mif ^−/− cells when they were supplemented with media from mif ^+/+ cells. This growth increase was suppressed by MIF inhibition. In vivo, chronic exposure of mice to a normobaric atmosphere of 10% oxygen increased lung tissue expression of mRNA encoding MIF and accumulation of MIF in plasma. Inhibition of the MIF inflammatory active site, during hypoxic exposure, significantly reduced pulmonary vascular remodeling, cardiac hypertrophy and right ventricular systolic pressure. The data suggest that MIF plays a critical role in hypoxia-induced PH, and its inhibition may be beneficial in preventing the development and progression of the disease.