The innate immune system in drosophila and mammals senses the invasion of microorganisms using the family of Toll receptors, stimulation of which initiates a range of host defense mechanisms. In drosophila antimicrobial responses rely on two signaling pathways: the Toll pathway and the IMD pathway. In mammals there are at least 10 members of the Toll-like receptor (TLR) family that recognize specific components conserved among microorganisms. Activation of the TLRs leads not only to the induction of inflammatory responses but also to the development of antigen-specific adaptive immunity. The TLR-induced inflammatory response is dependent on a common signaling pathway that is mediated by the adaptor molecule MyD88. However, there is evidence for additional pathways that mediate TLR ligand-specific biological responses.

Toll-like receptors.

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Purification and Characterization

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Secretory products of macrophages.

Sepsis is a condition that results from a harmful or damaging host response to infection. Many of the components of the innate immune response that are normally concerned with host defences against infection can, under some circumstances, cause cell and tissue damage and hence multiple organ failure, the clinical hallmark of sepsis. Because of the high mortality of sepsis in the face of standard treatment, many efforts have been made to improve understanding of the dysregulation of the host response in sepsis. As a result, much has been learnt of the basic principles governing bacterial-host interactions, and new opportunities for therapeutic intervention have been revealed.

The immunopathogenesis of sepsis.

Publisher Summary This chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.

Plasminogen activators, tissue degradation, and cancer.

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.1' 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. 3 The 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 antigen-s 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. Guiniea 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 TI37Ra). Each animal received a total dose of 100 ,ug of antigen distributed iTito the four footpads, 0.1 ml per footpad. Tissue culture media: The basic tissue culture media used throughouit were mirnimal essential media, Eagle 12-1.25 (Microbiological Associates, Bethesda, Md.) containing 15% normal guinea pig serum and 85 units of penicillin and 85 ,ug 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 beeni anesthetized with ether arid exsangulinated by cardiac puncture. The nodes were diced into tissue culture medium, teased gently with mouse-toothed forceps, and the resulting cell sIIspensioni was pipetted into centrifuge tubes. The suspension was allowed to stand for 4 min so that the tissue fragments settled by gravity. The superiiatanit was then removed to a fresh tube. After three such settlings the cell suspensions were essentially free of tissue fragments. The majority of cells were viable as assessed by trypan-blue exclusion, and the preparations containied 90-95% lymphocytes by Wright's stain and phase microscopy. Suspensions were adjusted to a final concentration of 1.8 X 107 cells per ml. Aliquots of these suspensions were made to contain ovalbumin, or OCBC-BGG 100 ,ug/ml. Suspensions not containing antigen were also prepared. In each experiment, suspensions were incuibated in specific antigen and an unrelated antigen.

Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction.

Macrophages are pivotal effector cells of the innate immune system, which is vital for recognizing and eliminating invasive microbial pathogens1,2. When microbial products bind to pathogen-recognition receptors, macrophages become activated and release a broad array of cytokines3 that orchestrate the host innate and adaptive immune responses. Initially identified as a T-cell cytokine4,5, macrophage migration inhibitory factor (MIF) is also a macrophage cytokine and an important mediator of inflammation and sepsis6,7,8,9,10,11,12. 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-κB and the production of tumour-necrosis factor-α. 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.

MIF regulates innate immune responses through modulation of Toll-like receptor 4

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.

https://www.pnas.org/content/pnas/99/1/345.full.pdf

Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: Regulatory role in the innate immune response

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 alpha (TNF-alpha) than did wild-type mice, but normal levels of interleukin (IL)-6 and IL-10. When stimulated with LPS and interferon gamma, macrophages from MIF-/- mice showed diminished production of TNF-alpha, 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.

https://rupress.org/jem/article-pdf/189/2/341/1120634/98-1683.pdf

Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis.

Summary 1.The migration of peritoneal exudate cells from guinea pigs with delayed hypersensitivity to tuberculin purified protein derivative, ovalbumin and diphtheria toxoid is markedly inhibited by the respective antigen, and such inhibition is specific. 2.Cells from guinea pigs producing precipitating antibody are not inhibited by antigen. 3.Sera from experimental animals exhibiting delayed hypersensitivity or sera containing precipitating antibody did not sensitize normal cells to antigen. 4.The inhibition still occurs when heat-in-activated sera are used. 5.The findings are consistent with the view of the primary importance of cells in manifestations of delayed hypersensitivity reactions, and the in vitro system gives promise of further elucidation of the mechanism involved.

John R. David

Papers

Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction.

MIF regulates innate immune responses through modulation of Toll-like receptor 4

Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: Regulatory role in the innate immune response

Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis.

Delayed hypersensitivity in vitro. i. the specificity of inhibition of cell migration by antigens.