Macrophage Migration Inhibitory Factor Counter-regulates Anti-inflammatory Effects Of Glucocorticoids And Affects Insulin Resistance Of Endothelial Cells

PartⅠMacrophage Migration Inhibitory Factor Counter-regulates Anti-inflammatory Effects of GlucocorticoidsInflammation is a reflexive response to infection, the binding of antibodies to antigens within the body, mechanical irritation, or injury. Whereas restricted inflammation is beneficial, excessive or persistent inflammation incites tissue destruction and disease. Glucocorticoids (GC) are indicated for the treatment of acute inflammations including septic shock, systemic inflammatory response syndrome, acute respiratory distress syndrome and chronic ones including asthma, rheumatoid arthritis, systemic lupus erythematosus, glomerular nephritis, multiple sclerosis and psoriasis. The efficacy of GCs in alleviating inflammatory disorders results from the pleiotropic effects of the glucocorticoid receptor (GR) on multiple signaling pathways. Pleiotropy can, however, also have adverse effects: growth retardation in children, immunosuppression, hypertension, inhibition of wound repair, osteoporosis, and metabolic disturbances.Annexin 1 as an inhibitor of phospholipase A2 (PLA2) is responsible for its anti-inflammatory actions including the production of arachadonic acid. It is now apparent that a cytosolic form of PLA2 (cPLA2) as well as a secretory form (sPLA2) exist. cPLA2 exhibits partiality for the arachidonyl-containing phospholipids. cPLA2 is predominantly involved in the production of inflammatory lipid mediators, and arachadonic. Annexin has now been shown to inibit PLA2 activity directly, rather than by substrate depletion. Annexin 1 is also found to inhibit the activity of cycloocygenase 2 (COX-2). Annexin 1 expression can be up-regulated with GC treatment through the GR, which contributes to its anti-inflammatory activity.Macrophage migration inhibory factor (MIF) is a cytokine which is synthesized by macrophages, activated T cells and anterior pituitary gland corticotropic cells. MIF is released by innate immune cells such as monocytes and macrophages when exposed to microbial products and pro-inflammatory mediators and has the ability of inhibiting migration of macrophage. TNF-α, IL-1 as well as MIF compose the first line of defense of innate immune. GCs regulate a wide variety of systemic immunologic and inflammatory response. Exogenous GCs suppress the whole immune system including activation of T cells, expression of inflammatory cytokine and adhesion molecules, migration of cells and productioin of effector molecules. Endogenous GCs regulate immune system in a concentration and diphase-dependent manner. Until recently, no endogenous neuroendocrine or immune mediator had been identified that would function as a counter-regulatory partner of GCs to modulate immune response except MIF. MIF is closely related with local and systemic inflammation and plays a pathogenic role in antoimmune diseases. However, very little is known about the molecular basis of MIF-GC interactions within the immune system.Based on the backgrounds of above researches, we address the question that whether the counter-regulatory effect of MIF on GCs is related with Annexin 1, as which is induced by GCs to exert an anti-inflammatory response.Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4) production were measured by ELISA. Protein expression of Annexin 1, cytosolic phospholipase A2 alpha (cPLA2α) and phospho-cPLA2αwere evaluated by Western blotting with recombinant MIF or without endogenous MIF expression using ribonucleic acid interference (RNAi) method.The results showed that recombinant MIF counter-regulated the inhibition of dexamethasone (Dex) on PGE2 and LTB4 production in RAW 264.7 macrophages stimulated with lipopolysaccharides (LPS) in a dose-dependent manner. Stimulation of RAW 264.7 macrophages with LPS resulted in a down-regulation of Annexin 1, while Dex or Dex plus LPS led to a significant up-regulation of Annexin 1 expression. The effect of Dex on Annexin 1 was counter-regulated by the administration of recombinant MIF. RNAi-mediated knockdown of the intracellular MIF further increased Annexin 1 expression of RAW 264.7 macrophages after incubation with Dex, and accordingly resulted in a low production of PGE2 and LTB4 through inhibiting the activation of cPLA2α.Annexin 1 plays a key role in MIF counter-regulation of Dex inhibition on eicosanoids production, which indicates that exogenous Annexin 1 may partially reduce the effects of MIF on GCs, thus becoming a potentially effective steroid sparing therapy on inflammatory diseases. PartⅡMacrophage Migration Inhibitory Factor Affects Insulin Resistace of Endothelial CellsVascular endothelial cells have a key role of stabilizing the internal environment of blood vessels. They keep the balance of many functions of endothelium including contraction or dilation of vessels, stimulating or inhibiting proliferation and migration of vascular smooth muscle cells, blood coagulation or fibronolysis and so on. Vascular endothelial cell is one of the target organs of insulin. Insulin exerts its biological effect of vasodilation by binding to the cognate cell surface receptor on the endothelium and promoting the release of nitrogen monoxidum. Nitric oxide (NO), the most powerful endothelium-derived relaxing factor, has the major role of maintaining angiotasis. Insulin-resistant conditions including obesity, type 2 diabetes, and hypertension, a feature of endothelial dysfunction, is followed by endothelial insulin resistance due to reduction of NO.It has been presumed that endothelial dysfunction is the basis on induction of endothelial insulin resistance, and inflammation is the triggering factor of endothelial dysfunction. It has been demonstrated that interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), these two inflammatory cytokines, could induce endothelial insulin resistance by affecting endothelial function. They achived this effects through interacting with certain proteins which belong to insulin-mediated signaling pathway.Macrophage migration inhibitory factor (MIF) addressed in the front part of this thesis is a powerful inflammatory cytokine. It not only has the ability of counter-regulating the anti-inflammatory effects of glucocorticoids, but also has been found to have the close relationship with cardiovascular and metabolic diseases in recent years including atherosclerosis, diabetic mellitus, obesity and hypertension. Based on the close relationship of inflammation, insulin resistance and atherosclerosis, we addressed the hypothesis whether MIF would be involved in the pathogenesis of endothelial insulin resistance, and if involved, what the mechanism would be. We have not found similar research work at home and abroad, so we studied this subject in the following experiments.Firstly, we evaluated the effects of insulin on production and release of NO in human umbilical vein endothelial cells (HUVEC). Secondly, we examined the effects of both the MIF and insulin on NO in HUVEC cells in order to identify the synergistic or antagonistic action of them. Thirdly, we evaluated the effects of MIF on insulin-mediated signaling pathway by studying the phosphorylation of insulin receptor substrate-1 and endothelial nitric oxide synthase and the activation of PI 3-KINASE p85 subunit to elucidate the molecular mechanisms of interaction of MIF and insulin.The results showed that insulin increased the production and release of NO in HUVEC cells in a dose- and time-dependent fashion. However, the production and release of NO significantly reduced after stimulation with both MIF and insulin. However, antagonism of MIF almost restored the production and release of NO by insulin. So there is an antagonistic but synergistic action between MIF and insulin, that is to say exposure of HUVEC cells to MIF resulted in inhibition of insulin-stimulated production of NO. Next study showed that MIF affects insulin signaling involved in NO production in HUVEC cells. This event was associated with impaired IRS-1 phosphorylation at Tyr612, one site essential for engaging the p85 subunit of PI 3-KINASE, resulting in defective activation of PI 3-KINASE and eNOS. This inhibitory effect of MIF was reversed by the MIF antagonist ISO-1. MIF increased IRS-1 phosphorulation at Ser312 and Ser616, which could inactivate the IRS-1.Our data suggest that MIF increased IRS-1 phosphorylation at Ser312 and Ser616, thus impairing the vasodilator effects of insulin mediated by the IRS-1/PI 3-kinase/eNOS pathway. MIF induced insulin resistance in endothelial cells may play an important role in the pathophysiology of cardiovascular disease associated with hypertension and insulin resistance. The characterization of the molecular mechanism involved in MIF-induced insulin resistance in the endothelium may help to design efficacious pharmacological molecules to treat endothelial dysfunction associated with insulin resistance states.