It has long been thought that delayed hypersensitivity is mediated by cells or cell-associated substances. In an attempt to investigate the mechanism of delayed hypersensitivity, in vitro studies using the inhibition of cell migration by specific antigen as an assay have been carried out. In this in vitro system, it has been shown that peritoneal exudate cells taken from guinea pigs exhibiting delayed hypersensitivity and placed in capillary tubes are inhibited from migration by specific antigen." 2 Moreover, when mixed populations of normal and sensitive cells were prepared, it was observed that if as few as 2.5 per cent of the cells in a population were from a sensitive animal, the whole population (97.5% normal cells) would be inhibited by antigen.3The results of recent experiments suggest that the lymphocyte is the specifically sensitive cell in this system. It was found that lymph node cells (approximately 95% lymphocytes) obtained from sensitive guinea pigs will, when mixed with a population of normal peritoneal exudate cells, cause the whole population to be inhibited by specific antigen.4 It is of note that such sensitive lymphoid cells, when assayed alone in culture, are not themselves inhibited from migrating by antigen.Experiments were initiated in an attempt to determine the manner by which these sensitive lymphoid cells achieved their effect. The results, here reported, demonstrate that, following incubation of sensitive lymphoid cells with specific antigen for 24 hr, a nondialyzable substance is detected in the cell-free supernatants which inhibits the migration of normal peritoneal cells.Materials and Methods.-Sensitization: The antigens used were ovalbumin (Worthington) and o-chlorobenzoyl chloride conjugated to bovine gamma globulin (OCBC-BGG) by the method of Benacerraf and Levine,5 kindly supplied by Dr. Y. Borel. Guinea pigs of the Hartley strain weighing 500-800 gm were sensitized with the appropriate antigen diluted in saline and emulsified in an equal volume of complete adjuvant (Difco H37Ra). Each animal received a total dose of 100 ,ug of antigen distributed into the four footpads, 0.1 ml per footpad.Tissue culture media: The basic tissue culture media used throughout were minimal essential media, Eagle 12-125 (Microbiological Associates, Bethesda, Md.) containing 15% normal guinea pig serum and 85 units of penicillin and 85 jAg of streptomycin/ml.Preparation of lymph node cell suspensions: Twelve to twenty one days after sensitization axillary, inguinal and popliteal lymph nodes were obtained aseptically from guinea pigs which had been anesthetized with ether and exsanguinated by cardiac puncture. The nodes were diced into tissue culture medium, teased gently with mouse-toothed forceps, and the resulting cell suspension was pipetted into centrifuge tubes. The suspension was allowed to stand for 4 min so that the tissue fragments settled by gravity. The supernatant was then removed to a fresh tube. After three such settlings the cell suspensions were essentially free of tissue fragments. ...

The importance of the macrophage in innate immunity is underscored by its secretion of an array of powerful immunoregulatory and effector molecules. We report herein that macrophage migration inhibitory factor (MIF), a product of activated macrophages, sustains macrophage survival and function by suppressing activation-induced, p53-dependent apoptosis. Endotoxin administration to MIF ؊/؊ mice results in decreased macrophage viability, decreased proinflammatory function, and increased apoptosis when compared with wild-type controls. Moreover, inhibition of p53 in endotoxin-treated, MIF-deficient macrophages suppresses enhanced apoptosis and restores proinflammatory function. MIF inhibits p53 activity in macrophages via an autocrine regulatory pathway, resulting in a decrease in cellular p53 accumulation and subsequent function. Inhibition of p53 by MIF coincides with the induction of arachidonic acid metabolism and cyclooxygenase-2 (Cox-2) expression, which is required for MIF regulation of p53. MIF's effect on macrophage viability and survival provides a previously unrecognized mechanism to explain its critical proinflammatory action in conditions such as sepsis, and suggests new approaches for the modulation of innate immune responses.apoptosis ͉ arachidonic acid ͉ Cox-2 ͉ sepsis

To study the biologic role of migration inhibitory factor (MIF), a pleiotropic cytokine, we generated a mouse strain lacking MIF by gene targeting in embryonic stem cells. Analysis of the role of MIF during sepsis showed that MIF−/− mice were resistant to the lethal effects of high dose bacterial lipopolysaccharide (LPS), or Staphylococcus aureus enterotoxin B (SEB) with d-galactosamine and had lower plasma levels of tumor necrosis factor α (TNF-α) than did wild-type mice, but normal levels of interleukin (IL)-6 and IL-10. When stimulated with LPS and interferon γ, macrophages from MIF−/− mice showed diminished production of TNF-α, normal IL-6 and IL-12, and increased production of nitric oxide. MIF−/− animals cleared gram-negative bacteria Pseudomonas aeruginosa instilled into the trachea better than did wild-type mice and had diminished neutrophil accumulation in their bronchoalveolar fluid compared to the wild-type mice. Thioglycollate elicited peritoneal exudates in uninfected MIF−/− mice, but showed normal neutrophil accumulation. Finally, the findings of enhanced resistance to P. aeruginosa and resistance to endotoxin-induced lethal shock suggest that the counteraction or neutralization of MIF may serve as an adjunct therapy in sepsis.

Macrophages are pivotal effector cells of the innate immune system, which is vital for recognizing and eliminating invasive microbial pathogens. When microbial products bind to pathogen-recognition receptors, macrophages become activated and release a broad array of cytokines that orchestrate the host innate and adaptive immune responses. Initially identified as a T-cell cytokine, macrophage migration inhibitory factor (MIF) is also a macrophage cytokine and an important mediator of inflammation and sepsis. Here we report that MIF is an essential regulator of macrophage responses to endotoxin (lipopolysaccharide) and Gram-negative bacteria. Compared with wild-type cells, MIF-deficient macrophages are hyporesponsive to lipopolysaccharide and Gram-negative bacteria, as shown by a profound reduction in the activity of NF-kappaB and the production of tumour-necrosis factor-alpha. This reduction is due to a downregulation of Toll-like receptor 4 (TLR4), the signal-transducing molecule of the lipopolysaccharide receptor complex, and is associated with decreased activity of transcription factor PU.1, which is required for optimal expression of the Tlr4 gene in myeloid cells. These findings identify an important role for MIF in innate immunity and provide a molecular basis for the resistance of MIF-deficient mice to endotoxic shock.