A functional macrophage migration inhibitory factor promoter polymorphism is associated with reduced diffusing capacity.

Cigarette smoke exposure is the leading modifiable risk factor for chronic obstructive pulmonary disease (COPD); however, the clinical and pathologic consequences of chronic cigarette smoke exposure are variable among smokers. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine implicated in the pathogenesis of COPD. Within the promoter of the MIF gene is a functional polymorphism that regulates MIF expression (-794 CATT5-8 microsatellite repeat) ( rs5844572 ). The role of this polymorphim in mediating disease susceptibility to COPD-related traits remains unknown. We performed a cross-sectional analysis of DNA samples from 641 subjects to analyze MIF-794 CATT5-8 ( rs5844572 ) polymorphism by standard methods. We generated multivariable logistic regression models to determine the risk of low expressing MIF alleles for airflow obstruction [defined by forced expiratory volume in 1 s (FEV1)/forced vital capacity ratio <0.70] and an abnormal diffusion capacity [defined by a diffusion capacity for carbon monoxide (DLCO) percent predicted <80%]. We then used generalized linear models to determine the association of MIF genotypes with FEV1 percent predicted and DLCO percent predicted. The MIF-794 CATT5 allele was associated with an abnormal diffusion capacity in two cohorts [odds ratio (OR): 9.31, 95% confidence interval (CI): 1.97-4.06; and OR: 2.21, 95% CI: 1.03-4.75]. Similarly, the MIF-794 CATT5 allele was associated with a reduced DLCO percentage predicted in these two cohorts: 63.5 vs. 70.0 ( P = 0.0023) and 60.1 vs. 65.4 ( P = 0.059). This study suggests an association between a common genetic polymorphism of an endogenous innate immune gene, MIF, with reduced DLCO, an important measurement of COPD severity.

Macrophage migration inhibitory factor (MIF): genetic evidence for participation in early onset and early stage rheumatoid arthritis.

Macrophage migration inhibitory factor (MIF) is an upstream pro-inflammatory cytokine that is associated with the pathogenesis of autoimmune inflammatory diseases including rheumatoid arthritis (RA). Two polymorphisms in the upstream region exist in the MIF gene and are associated with RA susceptibility or severity in different populations. In this case-control study, we investigated whether MIF polymorphisms are associated with RA susceptibility or activity in a western Mexican population .The relationship of MIF levels with clinical features of disease also was assessed. Genotyping of the -794 CATT5-8 (rs5844572) and the -173 G>C (rs755622) polymorphisms was performed by PCR and PCR-RFLP respectively on 226 RA patients and 210 healthy subjects. Serum MIF levels were determined by ELISA. We found a significant association between the -794 CATT5-8 6,7 MIF genotype with RA. Moreover, we detected an association between the -794 CATT7 allele with early onset RA. The -794 CATT7 and -173(*)C alleles, which are in linkage disequilibrium, were associated with high disease activity on RA patients. A positive correlation between circulating MIF levels and C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, anti-citrullinated protein/peptides antibodies and TNFα was detected. MIF levels appear to be associated with disease progression rather than disease activity, which is distinct from the established relationship between disease activity and TNFα levels. In conclusion, the MIF gene and protein are associated with RA in a western Mexican population, with a main contribution onto early onset and early stages of disease.

MIF is a common genetic determinant of COVID-19 symptomatic infection and severity.

BACKGROUND: Genetic predisposition to coronavirus disease 2019 (COVID-19) may contribute to its morbidity and mortality. Because cytokines play an important role in multiple phases of infection, we examined whether commonly occurring, functional polymorphisms in macrophage migration inhibitory factor (MIF) are associated with COVID-19 infection or disease severity. AIM: To determine associations of common functional polymorphisms in MIF with symptomatic COVID-19 or its severity. METHODS: This retrospective case-control study utilized 1171 patients with COVID-19 from three tertiary medical centers in the USA, Hungary and Spain, together with a group of 637 pre-pandemic, healthy control subjects. Functional MIF promoter alleles (-794 CATT5-8,rs5844572), serum MIF and soluble MIF receptor levels, and available clinical characteristics were measured and correlated with COVID-19 diagnosis and hospitalization. Experimental mice genetically engineered to express human high- or low-expression MIF alleles were studied for response to coronavirus infection. RESULTS: In patients with COVID-19, there was a lower frequency of the high-expression MIF CATT7 allele when compared to healthy controls [11% vs. 19%, odds ratio (OR) 0.54 [0.41-0.72], P < 0.0001]. Among inpatients with COVID-19 (n = 805), there was a higher frequency of the MIF CATT7 allele compared to outpatients (n = 187) (12% vs. 5%, OR 2.87 [1.42-5.78], P = 0.002). Inpatients presented with higher serum MIF levels when compared to outpatients or uninfected healthy controls (87 ng/ml vs. 35 ng/ml vs. 29 ng/ml, P < 0.001, respectively). Among inpatients, circulating MIF concentrations correlated with admission ferritin (r = 0.19, P = 0.01) and maximum CRP (r = 0.16, P = 0.03) levels. Mice with a human high-expression MIF allele showed more severe disease than those with a low-expression MIF allele. CONCLUSIONS: In this multinational retrospective study of 1171 subjects with COVID-19, the commonly occurring -794 CATT7MIF allele is associated with reduced susceptibility to symptomatic SARS-CoV-2 infection but increased disease progression as assessed by hospitalization. These findings affirm the importance of the high-expression CATT7MIF allele, which occurs in 19% of the population, in different stages of COVID-19 infection.

Elevated levels of macrophage migration inhibitory factor in women with metabolic syndrome.

Metabolic syndrome is a complex clinical disorder characterized by obesity, a disturbance of glucose metabolism, dyslipidemia, and hypertension, leading to increased cardiovascular risk. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced both by innate immune cells and by adipocytes, and it plays an important role in inflammatory and cardiovascular diseases. The goal of this study was to evaluate the expression of circulating MIF in patients with metabolic syndrome. A study was conducted involving 172 persons who attended the Jeju National University Hospital Health Promotion Center. Among the 172 subjects, 88 patients with metabolic syndrome and 84 healthy control subjects were included. Serum MIF levels were considerably higher in patients with metabolic syndrome than in healthy subjects (mean±SEM, 1413.0-pg/ml±102.6 vs. 1077.0-pg/ml±-91.3, p=0.016). Among the metabolic syndrome patients, MIF levels were significantly increased in women (1403.0-pg/ml±114.2 vs. 921.3 pg/ml±117.3, p=0.005), but not in men. Even after further linear regression adjustment for age and body mass index, the expression of MIF for women with metabolic syndrome was still clearly elevated when compared to healthy subjects (p=0.011). Circulating MIF concentrations showed a gender disparity between healthy and metabolic syndrome subjects. An elevation of systemic MIF in women with metabolic syndrome may contribute to pathogenesis of metabolic syndrome or to the development of metabolic syndrome-related diseases, such as atherosclerosis and type 2 diabetes mellitus.

Regulatory role for macrophage migration inhibitory factor in acute respiratory distress syndrome.

Migration inhibitory factor (MIF) is known to exert significant pro-inflammatory effects and has the potential to override the anti-inflammatory action of glucocorticoids. In this study we have identified significant quantities of MIF in the alveolar airspaces of patients with acute respiratory distress syndrome (ARDS). We show in alveolar cells from patients with ARDS that MIF augments pro-inflammatory cytokine secretion (TNF alpha and IL-8), anti-MIF significantly attenuates TNF alpha and IL-8 secretion and MIF overrides, in a concentration-related fashion, the anti-inflammatory effects of glucocorticoids. These findings suggest that MIF may act as a mediator sustaining the pulmonary inflammatory response in ARDS and that an anti-MIF strategy may represent a novel therapeutic approach in inflammatory diseases such as ARDS.

Protection from septic shock by neutralization of macrophage migration inhibitory factor.

Identification of new therapeutic targets for the management of septic shock remains imperative as all investigational therapies, including anti-tumor necrosis factor (TNF) and anti-interleukin (IL)-1 agents, have uniformly failed to lower the mortality of critically ill patients with severe sepsis. We report here that macrophage migration inhibitory factor (MIF) is a critical mediator of septic shock. High concentrations of MIF were detected in the peritoneal exudate fluid and in the systemic circulation of mice with bacterial peritonitis. Experiments performed in TNFalpha knockout mice allowed a direct evaluation of the part played by MIF in sepsis in the absence of this pivotal cytokine of inflammation. Anti-MIF antibody protected TNFalpha knockout from lethal peritonitis induced by cecal ligation and puncture (CLP), providing evidence of an intrinsic contribution of MIF to the pathogenesis of sepsis. Anti-MIF antibody also protected normal mice from lethal peritonitis induced by both CLP and Escherichia coli, even when treatment was started up to 8 hours after CLP. Conversely, co-injection of recombinant MIF and E. coli markedly increased the lethality of peritonitis. Finally, high concentrations of MIF were detected in the plasma of patients with severe sepsis or septic shock. These studies define a critical part for MIF in the pathogenesis of septic shock and identify a new target for therapeutic intervention.

Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts.

Conditioned media obtained from fibroblasts cultured from rheumatoid and certain other inflammatory synovia were observed to stimulate [3H]thymidine incorporation in an indicator murine fibroblast line. Synovial fibroblasts derived from the joints of patients with osteoarthritis did not display this property. This effect persisted in culture for many weeks and occurred in the absence of co-stimulatory immune cells. Antibody neutralization studies implicated a role for basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 1 beta (IL-1 beta) in the increased proliferative activity of synovial fibroblast-conditioned media. Synovial cell synthesis of bFGF, TGF beta 1, GM-CSF, IL-1 beta, and IL-6 was confirmed by 35S-methionine labeling and immunoprecipitation. The constitutive production of inflammatory and mitogenic cytokines by synovial fibroblasts may represent the result of long-term, phenotypic changes that occurred in vivo. Persistent cytokine production by synovial fibroblasts may play an important role in the continued recruitment and activation of inflammatory cells in chronic arthritis and in the formation of rheumatoid pannus.

The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor.

For over 25 years, the cytokine known as macrophage migration inhibitory factor (MIF) has been considered to be a product of activated T lymphocytes. We recently identified the murine homolog of human MIF as a protein secreted by the pituitary in response to endotoxin administration. In the course of these studies, we also detected MIF in acute sera obtained from endotoxin-treated, T cell-deficient (nude), and hypophysectomized mice, suggesting that still more cell types produce MIF. Here, we report that cells of the monocyte/macrophage lineage are an important source of MIF in vitro and in vivo. We observed high levels of both preformed MIF protein and MIF mRNA in resting, nonstimulated cells. In the murine macrophage cell line RAW 264.7, MIF secretion was induced by as little as 10 pg/ml of lipopolysaccharide (LPS), peaked at 1 ng/ml, and was undetectable at LPS concentrations > 1 microgram/ml. A similar stimulation profile was observed in LPS-treated peritoneal macrophages; however, higher LPS concentrations were necessary to induce peak MIF production unless cells had been preincubated with interferon gamma (IFN-gamma). In RAW 264.7 macrophages, MIF secretion also was induced by tumor necrosis factor alpha (TNF-alpha) and IFN-gamma, but not by interleukins 1 beta or 6. Of note, MIF-stimulated macrophages were observed to secrete bioactive TNF-alpha. Although previously overlooked, the macrophage is both an important source and an important target of MIF in vivo. The activation of both central (pituitary) and peripheral (macrophage) sources of MIF production by inflammatory stimuli provides further evidence for the critical role of this cytokine in the systemic response to tissue invasion.

An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction.

30 years ago, investigations into the molecular basis of the delayed-type hypersensitivity reaction (DTH) provided evidence for the first lymphokine activity: a lymphocyte-derived mediator called macrophage migration inhibitory factor (MIF), which inhibited the random migration of peritoneal macrophages. Despite the long-standing association of MIF with the DTH reaction and the cloning of a human protein with macrophage migration inhibitory activity, the precise role of MIF in this classic cell-mediated immune response has remained undefined. This situation has been further complicated by the fact that two other cytokines, interferon gamma and IL-4, similarly inhibit macrophage migration and by the identification of mitogenic contaminants in some preparations of cloned human MIF. Using recently developed molecular probes for mouse MIF, we have examined the role of this protein in a classical model of DTH, the tuberculin reaction in mice. Both MIF messenger RNA and protein were expressed prominently in DTH lesions, as assessed by reverse transcription polymerase chain reaction, in situ hybridization, and immunostaining with anti-MIF antibody. The predominant cellular origin of MIF appeared to be the monocyte/macrophage, a cell type identified recently to be a major source of MIF release in vivo. The administration of neutralizing anti-MIF antibodies to mice inhibited significantly the development of DTH, thus affirming the central role of MIF in this classic immunological response.

Application of DNA amplification to pneumocystosis: presence of serum Pneumocystis carinii DNA during human and experimentally induced Pneumocystis carinii pneumonia.

Pneumocystis carinii pneumonia is a leading cause of morbidity and mortality in patients with the acquired immunodeficiency syndrome (AIDS). Much remains unknown about the basic biology of P. carinii and studies of this infection have been hampered by the lack of cultivation methods. We developed a sensitive and specific assay for P. carinii by utilizing DNA amplification of the P. carinii dihydrofolate reductase (DHFR) gene. By this method, P. carinii DNA was detected in the lungs of rats with experimentally induced P. carinii pneumonia 2 wk before the onset of histopathological changes. DNA amplification analysis of serum demonstrated that by 10 wk of corticosteroid treatment, 12 of 12 (100%) infected rats had circulating DHFR DNA. P. carinii DHFR DNA also was detected in the serum of patients with AIDS and active P. carinii pneumonia (12 of 14 sera collected prospectively). Patients with advanced AIDS but without a history of P. carinii pneumonia were negative by this assay (0 of 6 sera examined). Serum polymerase chain reaction may facilitate investigations into the natural history and epidemiology of P. carinii infection, provide insight into the pathogenesis of parasite dissemination, and offer a useful, noninvasive diagnostic test for the detection of human pneumocystosis.

The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat.

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

Cytotoxicity of a perfluorocarbon blood substitute to macrophages in vitro.

Murine macrophage and macrophage-depleted splenocyte cultures were incubated under ambient oxygen with a commercially available perfluorocarbon blood substitute. The perfluorocarbon preparation was found to be selectively cytotoxic to macrophages. This finding may be significant in view of the preliminary therapeutic usage of these preparations. In addition, perfluorocarbons may be useful as a means of selectively removing macrophages from tissue and organ cultures.

Hemoglobin-AGE: a circulating marker of advanced glycosylation.

Advanced glycosylation end products (AGEs) form spontaneously from glucose-derived Amadori products and accumulate on long-lived tissue proteins. AGEs have been implicated in the pathogenesis of several of the complications of aging and diabetes, including atherosclerosis and renal disease. With the use of recently developed AGE-specific antibodies, an AGE-modified form of human hemoglobin has been identified. Termed hemoglobin-AGE (Hb-AGE), this modified species accounts for 0.42 percent of circulating hemoglobin in normal individuals but increases to 0.75 percent in patients with diabetes-induced hyperglycemia. In a group of diabetic patients treated with the advanced glycosylation inhibitor aminoguanidine, Hb-AGE levels decreased significantly over a 1-month period. Hemoglobin-AGE measurements may provide an index of long-term tissue modification by AGEs and prove useful in assessing the contribution of advanced glycosylation to a variety of diabetic and age-related complications.

Macrophage migration inhibitory factor (MIF) promotes cell survival by activation of the Akt pathway and role for CSN5/JAB1 in the control of autocrine MIF activity.

The phosphoinositide-3-kinase (PI3K)/Akt signaling pathway plays an important role in cell survival and the development of cancer. Macrophage migration inhibitory factor (MIF) is a critical inflammatory cytokine that was recently associated with tumorigenesis and that potently inhibits apoptosis. This may involve inhibition of p53-dependent genes, but the initiating molecular mechanism of how MIF controls survival/apoptosis is unknown. Here, we show that MIF prevents apoptosis and promotes tumor cell survival by directly activating the Akt pathway. MIF enhanced Akt activity in primary and immortalized fibroblasts (MEF and NIH/3T3), HeLa cervix carcinoma cells and various breast cancer cell lines. Activation was abolished by kinase inhibitors Ly294002 and PP2 and in Src/Yes/Fyn(SYF)(-/-) and CD74(-/-)(MEFs), while being enhanced in CD74-overexpressing MEFs, demonstrating that the MIF-induced Akt pathway encompasses signaling through the MIF receptor CD74 and the upstream kinases Src and PI3K. Akt was activated by exogenous rMIF and autocrine MIF action, as revealed by experiments in MIF(-/-)MEFs and antibody blockade. siRNA knockdown of CSN5/JAB1, a tumor marker and MIF-binding protein, showed that JAB1 controls autocrine MIF-mediated Akt signaling by inhibition of MIF secretion. Akt activation by MIF led to phosphorylation of the proapoptotic proteins BAD and Foxo3a. Apoptosis inhibition by MIF was functionally associated with Akt activation as it was abolished by overexpression of the Akt pathway inhibitor PTEN and occurred independently of p53. This was shown by studying DNA damage-induced apoptosis in fibroblasts, the Fas death pathway in HeLa cells that do not express functional p53, and etoposide-induced apoptosis in breast carcinoma cells expressing mutant p53. Importantly, dependence of breast cancer cell survival on MIF correlated with Akt activation and the PTEN status of these cells. Thus, MIF can directly promote cell survival through activation of the PI3K/Akt pathway and this effect is critical for tumor cell survival.

Ras transformation requires metabolic control by 6-phosphofructo-2-kinase.

Neoplastic cells transport large amounts of glucose in order to produce anabolic precursors and energy within the inhospitable environment of a tumor. The ras signaling pathway is activated in several cancers and has been found to stimulate glycolytic flux to lactate. Glycolysis is regulated by ras via the activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFK2/FBPase), which modulate the intracellular concentration of the allosteric glycolytic activator, fructose-2,6-bisphosphate (F2,6BP). We report herein that sequential immortalization and ras-transformation of mouse fibroblasts or human bronchial epithelial cells paradoxically decreases the intracellular concentration of F2,6BP. This marked reduction in the intracellular concentration of F2,6BP sensitizes transformed cells to the antimetabolic effects of PFK2/FBPase inhibition. Moreover, despite co-expression of all four mRNA species (PFKFB1-4), heterozygotic genomic deletion of the inducible PFKFB3 gene in ras-transformed mouse lung fibroblasts suppresses F2,6BP production, glycolytic flux to lactate, and growth as soft agar colonies or tumors in athymic mice. These data indicate that the PFKFB3 protein product may serve as an essential downstream metabolic mediator of oncogenic ras, and we propose that pharmacologic inhibition of this enzyme should selectively suppress the high rate of glycolysis and growth by cancer cells.

Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes.

Nitric oxide (an endothelium-derived relaxing factor) induces smooth muscle relaxation and is an important mediator in the regulation of vascular tone. Advanced glycosylation end products, the glucose-derived moieties that form nonenzymatically and accumulate on long-lived tissue proteins, have been implicated in many of the complications of diabetes and normal aging. We demonstrate that advanced glycosylation products quench nitric oxide activity in vitro and in vivo. Acceleration of the advanced glycosylation process in vivo results in a time-dependent impairment in endothelium-dependent relaxation. Inhibition of advanced glycosylation with aminoguanidine prevents nitric oxide quenching, and ameliorates the vasodilatory impairment. These results implicate advanced glycosylation products as important modulators of nitric oxide activity and endothelium-dependent relaxation.

Advanced glycosylation endproducts block the antiproliferative effect of nitric oxide. Role in the vascular and renal complications of diabetes mellitus.

Advanced glycosylation endproducts (AGEs) accumulate on long-lived tissue proteins such as basement membrane collagen and have been implicated in many of the long-term complications of diabetes mellitus. These products originate from glucose-derived Schiff base and Amadori products but undergo a series of complex rearrangement reactions to form ultimately protein-bound, fluorescent heterocycles. AGEs can react with and chemically inactivate nitric oxide (NO), a potent endothelial cell-derived vasodilator and antiproliferative factor. Since mesenchymal cell proliferation is an early and characteristic lesion of diabetic vasculopathy and glomerulopathy, we investigated the possibility that collagen-bound AGEs functionally inactivate the antiproliferative effect of NO. In model cell culture systems, AGEs were found to block the cytostatic effect of NO on aortic smooth muscle and renal mesangial cells. The inactivation of endothelial cell-derived NO by basement membrane AGEs may represent a common pathway in the development of the accelerated vascular and renal disease that accompany long-term diabetes mellitus.

Nonenzymatic addition of glucocorticoids to lens proteins in steroid-induced cataracts.

A frequent manifestation of long-term glucocorticoid administration is the occurrence of posterior subcapsular cataracts. The molecular basis for this effect has not yet been elucidated. The addition of prednisolone to the rat lens in culture results in a time- and concentration-dependent lens opacification that correlates with the formation of covalent prednisolone-lens protein adducts. Prednisolone adduct formation was analyzed by [3H]prednisolone incorporation and by immunoprecipitation with antiserum specific for proteins modified by the nonenzymatic addition of prednisolone. In the rat lens, these adducts were localized in both the water-soluble and urea-soluble lens protein fractions. Gel electrophoresis and fluorography revealed that the most extensively modified proteins were two crystallins subunits. Lens proteins from 33 normal and cataractous human lenses were fractionated and analyzed for the presence of prednisolone-protein adducts by competitive radioimmunoassay. Adducts were detected only in those samples derived from glucocorticoid-induced cataractous lenses. We conclude that elevated glucocorticoid levels lead to the formation of glucocorticoid-lens protein adducts both in vitro and in vivo. Lens protein modification by glucocorticoids may lead to sufficient biochemical or structural alterations so as to result in cataract formation. The ability of glucocorticoids to form adducts with proteins in vivo also may play a role in some of the other toxic manifestations of long-term glucocorticoid therapy.

Advanced glycation end products increase retinal vascular endothelial growth factor expression.

Advanced glycation end products (AGEs) are linked with the development of diabetic retinopathy; however, the pathogenic mechanisms are poorly defined. Vascular endothelial growth factor (VEGF) levels are increased in ischemic and nonischemic diabetic retina, and VEGF is required for the development of retinal and iris neovascularization. Moreover, VEGF alone can induce much of the concomitant pathology of diabetic retinopathy. In this study, we found that AGEs increased VEGF mRNA levels in the ganglion, inner nuclear, and retinal pigment epithelial (RPE) cell layers of the rat retina. In vitro, AGEs increased VEGF mRNA and secreted protein in human RPE and bovine vascular smooth muscle cells. The AGE-induced increases in VEGF expression were dose- and time-dependent, inhibited by antioxidants, and additive with hypoxia. Use of an anti-VEGF antibody blocked the capillary endothelial cell proliferation induced by the conditioned media of AGE-treated cells. AGEs may participate in the pathogenesis of diabetic retinopathy through their ability to increase retinal VEGF gene expression.

The proinflammatory mediator macrophage migration inhibitory factor induces glucose catabolism in muscle.

Severe infection or tissue invasion can provoke a catabolic response, leading to severe metabolic derangement, cachexia, and even death. Macrophage migration inhibitory factor (MIF) is an important regulator of the host response to infection. Released by various immune cells and by the anterior pituitary gland, MIF plays a critical role in the systemic inflammatory response by counterregulating the inhibitory effect of glucocorticoids on immune-cell activation and proinflammatory cytokine production. We describe herein an unexpected role for MIF in the regulation of glycolysis. The addition of MIF to differentiated L6 rat myotubes increased synthesis of fructose 2,6-bisphosphate (F2,6BP), a positive allosteric regulator of glycolysis. Increased expression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) enhanced F2,6BP production and, consequently, cellular lactate production. The catabolic effect of TNF-alpha on myotubes was mediated by MIF, which served as an autocrine stimulus for F2, 6BP production. TNF-alpha administered to mice decreased serum glucose levels and increased muscle F2,6BP levels; pretreatment with a neutralizing anti-MIF mAb completely inhibited these effects. Anti-MIF also prevented hypoglycemia and increased muscle F2,6BP levels in TNF-alpha-knockout mice that were administered LPS, supporting the intrinsic contribution of MIF to these inflammation-induced metabolic changes. Taken together with the recent finding that MIF is a positive, autocrine stimulator of insulin release, these data suggest an important role for MIF in the control of host glucose disposal and carbohydrate metabolism.