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Inflammation provokes significant abnormalities in host metabolism that result from the systemic release of cytokines. An early response of the host is hyperglycemia and resistance to the action of insulin, which progresses over time to increased glucose uptake in peripheral tissue. Although the cytokine TNF-α has been shown to exert certain catabolic effects, recent studies suggest that the metabolic actions of TNF-α occur by the downstream regulation of additional mediators, such as macrophage migration inhibitory factor (MIF). We investigated the glycemic responses of endotoxemic mice genetically deficient in MIF (MIF−/−). In contrast to wild-type mice, MIF−/− mice exhibit normal blood glucose and lactate responses following the administration of endotoxin, or TNF-α. MIF−/− mice also show markedly increased glucose uptake into white adipose tissue in vivo in the endotoxemic state. Treatment of adipocytes with MIF, or anti-MIF mAb, modulates insulin-mediated glucose transport and insulin receptor signal transduction; these effects include the phosphorylation of insulin receptor substrate-1, its association with the p85 regulatory subunit of PI3K, and the downstream phosphorylation of Akt. Genetic MIF deficiency also promotes adipogenesis, which is in accord with a downstream role for MIF in the action of TNF-α. These studies support an important role for MIF in host glucose metabolism during sepsis.

Immune system impairment and increased susceptibility to infection among alcohol abusers is a significant but not well-understood problem. We hypothesized that acute ethanol administration would inhibit leukocyte recruitment and endothelial cell activation during inflammation and infection. Using LPS and carrageenan air pouch models in mice, we found that physiological concentrations of ethanol (1–5 g/kg) significantly blocked leukocyte recruitment (50–90%). Because endothelial cell activation and immune cell-endothelial cell interactions are critical regulators of leukocyte recruitment, we analyzed the effect of acute ethanol exposure on endothelial cell activation in vivo using the localized Shwartzman reaction model. In this model, ethanol markedly suppressed leukocyte accumulation and endothelial cell adhesion molecule expression in a dose-dependent manner. Finally, we examined the direct effects of ethanol on endothelial cell activation and leukocyte-endothelial cell interactions in vitro. Ethanol, at concentrations within the range found in human blood after acute exposure and below the levels that induce cytotoxicity (0.1–0.5%), did not induce endothelial cell activation, but significantly inhibited TNF-mediated endothelial cell activation, as measured by adhesion molecule (E-selectin, ICAM-1, VCAM-1) expression and chemokine (IL-8, MCP-1, RANTES) production and leukocyte adhesion in vitro. Studies exploring the potential mechanism by which ethanol suppresses endothelial cell activation revealed that ethanol blocked NF-κB nuclear entry in an IκBα-dependent manner. These findings support the hypothesis that acute ethanol overexposure may increase the risk of infection and inhibit the host inflammatory response, in part, by blocking endothelial cell activation and subsequent immune cell-endothelial cell interactions required for efficient immune cell recruitment.

Fibrocytes are a distinct population of blood-borne cells that display a unique cell surface phenotype (collagen I+/CD11b+/CD13+/CD34+/CD45RO+/MHC class II+/CD86+) and exhibit potent immunostimulatory activities. Circulating fibrocytes rapidly enter sites of tissue injury, suggesting an important role for these cells in wound repair. However, the regulatory processes that govern the differentiation of blood-borne fibrocytes and the mechanisms that underlie the migration of these cells to wound sites are currently not known. We report herein that ex vivo cultured fibrocytes can differentiate from a CD14+-enriched mononuclear cell population and that this process requires contact with T cells. Furthermore, we demonstrate that TGF-β1 (1–10 ng/ml), an important fibrogenic and growth-regulating cytokine involved in wound healing, increases the differentiation and functional activity of cultured fibrocytes. Because fibrocytes home to sites of tissue injury, we examined the role of chemokine/chemokine receptor interactions in fibrocyte trafficking. We show that secondary lymphoid chemokine, a ligand of the CCR7 chemokine receptor, acts as a potent stimulus for fibrocyte chemotaxis in vitro and for the homing of injected fibrocytes to sites of cutaneous tissue injury in vivo. Finally, we demonstrate that differentiated, cultured fibrocytes express α smooth muscle actin and contract collagen gels in vitro, two characteristic features of wound-healing myofibroblasts. These data provide important insight into the control of fibrocyte differentiation and trafficking during tissue repair and significantly expand their potential role during wound healing.

Macrophage migration inhibitory factor (MIF) has been shown to be a pivotal cytokine that mediates host inflammatory and immune responses. Recently, immunoneutralization of MIF has been found to inhibit tumor growth in mice; however, the contributing mechanisms underlying this effect have not been well defined. We investigated whether MIF plays a regulatory role in the expression of CTL activity. In a mouse model of the CTL response using the OVA-transfected tumor cell line EL4 (EG.7), we found that cultures of splenocytes obtained from EG.7-primed mice secrete high levels of MIF following Ag stimulation in vitro. Notably, parallel splenocyte cultures treated with neutralizing anti-MIF mAb showed a significant increase in the CTL response directed against EG.7 cells compared with control mAb-treated cultures. This effect was accompanied by elevated expression of IFN-γ. Histological examination of the EG.7 tumors from anti-MIF-treated animals showed a prominent increase in both CD4+ and CD8+ T cells as well as apoptotic tumor cells, consistent with the observed augmentation of CTL activity in vivo by anti-MIF. This increased CTL activity was associated with enhanced expression of the common γc-chain of the IL-2R that mediates CD8+ T cell survival. Finally, CD8+ T lymphocytes obtained from the spleens of anti-MIF-treated EG.7 tumor-bearing mice, when transferred into recipient tumor-bearing mice, showed increased accumulation in the tumor tissue. These data provide the first evidence of an important role for MIF in the regulation and trafficking of anti-tumor T lymphocytes in vivo.

Macrophage migration inhibitory factor (MIF) is a product of activated T cells, anterior pituitary cells, and macrophages. MIF plays an important role in LPS-induced shock and delayed-type hypersensitivity. Furthermore, MIF exhibits a proinflammatory spectrum of action, promoting TNF-α production by macrophages, and counter-regulates glucocorticoid suppression of cytokine production. Here, we report that purified recombinant MIF activates murine macrophages to kill Leishmania major, with maximal effects at concentrations above 1 μg/ml. This MIF-mediated activation is specific, since it can be blocked completely by anti-MIF mAb. The MIF-mediated activation is dependent on TNF-α produced endogenously by macrophages, because the administration of anti-TNF-α antiserum markedly reduced the MIF effect. No MIF-mediated activation was observed in macrophages derived from TNF receptor p55 knockout mice, thus demonstrating the requirement of the smaller TNF receptor molecule for autocrine TNF-α signaling. A highly specific inhibitor of the inducible nitric oxide synthase (iNOS), l-N6-(1-iminoethyl)lysine, dihydrochloride, also inhibited the action of MIF, suggesting an important role for iNOS in the antiparasitic properties of MIF. In line with this, no MIF-mediated activation was detected analyzing macrophages derived from iNOS-deficient mice. The effect of MIF was blocked completely by the macrophage-deactivating cytokines IL-10, IL-13, and TGF-β. Finally, the expression of MIF mRNA and protein was up-regulated in lymph nodes of mice during the first week after infection with L. major. MIF therefore represents a cytokine involved not only in the recruitment of proinflammatory cells during infection but also in the complex regulation of the antimicrobial activity of these cells.

Numerous studies demonstrate the anti-inflammatory effects of cholinergic agonists, including nicotine. Investigations by our laboratory reveal that cholinergic stimulation inhibits endothelial cell activation, in part, through the NF-κB pathway. Monocyte chemoattractant protein-1 (MCP-1) is produced by endothelial cells and is a target of the JAK/STAT3 pathway. Human umbilical vein endothelial cells (HuVECs) were isolated and used to investigate the potential role of the JAK/STAT3 pathway in regulating MCP-1 production by activated endothelial cells following cholinergic stimulation. LPS induced MCP-1 expression in a dose-dependent manner. Soluble IL-6 receptor (IL-6sR), but not IL-6 itself induced significant MCP-1 expression. MCP-1 production following LPS or IL-6sR stimulation was mediated, in part, through the JAK/STAT3 pathway. Cholinergic agonists, nicotine and GTS-21, significantly reduced both LPS and IL-6sR-induced MCP-1 production. Cholinergic agonists also attenuated STAT3 phosphorylation following LPS and IL-6 stimulation. Finally, IL-6 blockade partially inhibits LPS-stimulated MCP-1 production and STAT3 phosphorylation. Together, our observations show that both LPS and IL-6sR induce MCP-1 production in endothelial cells, in part, through the JAK/STAT3 pathway that is inhibited by cholinergic mediators. This work was supported by NIH NIGMS R01GM070727-02.