John R. David

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

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-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.

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

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. MIFs 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.

Development of chronic colitis is dependent on the cytokine MIF

The cytokine macrophage-migration inhibitory factor (MIF) is secreted by a number of cell types upon induction by lipopolysaccharide (LPS). Because colitis is dependent on interplay between the mucosal immune system and intestinal bacteria, we investigated the role of MIF in experimental colitis. MIF-deficient mice failed to develop disease, but reconstitution of MIF-deficient mice with wild-type innate immune cells restored colitis. In addition, established colitis could be treated with anti-MIF immunoglobulins. Thus, murine colitis is dependent on continuous MIF production by the innate immune system. Because we found increased plasma MIF concentrations in patients with Crohns disease, these data suggested that MIF is a new target for intervention in Crohns disease.

Humoral and Cellular Sensitivity to Collagen in Type II Collagen-Induced Arthritis in Rats

We have recently described a new animal model of arthritis induced by intradermal injection of a distinct type of collagen found in cartilage (type II collagen). Since immunologic sensitivity to collagen could play a role in the pathogenesis of this type II collagen-induced arthritis in rats, the ability of purified types of native collagens to induce cellular and humoral responses was quantified by antigeninduced tritiated thymidine incorporation into lymphocytes by collagen and passive hemagglutination, respectively. Rats injected intradermally with native heterologous or homologous type II collagens in adjuvant developed type-specific cellular as well as humoral reactivity. Types I and III collagens were less immunogenic than was type II. The latter collagen induced brisk cellular and humoral responses that were equivalent whether complete Freunds adjuvant or incomplete Freunds adjuvant were employed. Both responses could be induced by native type II collagens modified by limited pepsin digestion, indicating that they are not attributable to determinants in the telopeptide regions of the molecule. Thus, these studies demonstrate the unique immunogenic as well as arthritogenic properties of the type II collagen molecule and indicate that both result from a helical conformation of its structurally distinct alpha-chains. Further, they suggest that type II collagen may, by humoral or cellular mechanisms, provoke or perpetuate inflammation in other arthritic diseases.

Sandfly Maxadilan Exacerbates Infection with Leishmania major and Vaccinating Against It Protects Against L. major Infection

Bloodfeeding arthropods transmit many of the world’s most serious infectious diseases. Leishmania are transmitted to their mammalian hosts when an infected sandfly probes in the skin for a bloodmeal and injects the parasite mixed with its saliva. Arthropod saliva contains molecules that affect blood flow and modulate the immune response of the host. Indeed, sandfly saliva markedly enhances the infectivity of L. major for its host. If the salivary molecule(s) responsible for this phenomenon was identified, it might be possible to vaccinate the host against this molecule and thereby protect the host against infection with Leishmania. Such an approach represents a novel means of controlling arthropod-borne disease transmission. Here, we report that a single molecule, maxadilan, in sandfly saliva can exacerbate infection with L. major to the same degree as whole saliva, and that vaccinating against maxadilan protects mice against infection with L. major.

Cellular Sensitivity to Collagen in Rheumatoid Arthritis

We examined patients with rheumatoid arthritis for cellular sansitivity to native human Types I, II and III collagens. Mononuclear cells from 50 patients with rheumatoid arthritis, 21 with other inflammatory arthritides, 20 with osteoarthritis and 20 normal subjects were evaluated for the in vitro production of leukocyte inhibitory factor in response to collagen and a control antigen, streptokinase-streptodornase. By this assay, cells from 37 (74 per cent) and 39 (78 per cent) of the patients with rheumatoid arthritis responded to Types II and III collagens, respectively. In contrast, cells from the 41 patients with other kinds of arthritis and the normal group did not produce this lymphokine to collagens. There was no response to Type I collagen or to denatured alpha chains of these collagens. All four groups responded equivalently to streptokinase-streptodornase. These data demonstrate that most patients with rheumatoid arthritis exhibit cellular sensitivity to Types II and III collagens.

Passive Transfer by Cells of Type II Collagen-Induced Arthritis in Rats

To investigate the role of immunologic hypersensitivity to collagen in the causation of type II collagen-induced arthritis in rats, passive transfer experiments were performed. Wistar/Lewis rats used in these experiments were demonstrated to be histocompatible by prolonged skin graft survival and mixed lymphocyte cultures. Popliteal lymph node weight assays excluded a potential for graft-vs.-host reactivity in this strain. 9 of 32 naive rats developed arthritis after intravenous receipt of pooled spleen and lymph node cells from donors that had been injected intradermally with type II collagen emulsified in incomplete Freunds adjuvant. This passively transferred synovitis was evident clinically as well as histologically. In control cell transfer experiments involving a total of 97 recipients, transfer of arthritis was shown to require viable cells sensitized to type II collagen. These controls included 17 rats receiving cells from unimmunized donors, 20 recipients of cells from donors injected with incomplete Freunds adjuvant alone, and 24 recipients of cells from rats injected with type I collagen in adjuvant. Deliberate addition of solubilized type II collagen to unsensitized cells at the time of transfer or injection of heat-killed sensitized cells also did not cause arthritis in a total of 36 recipients. These latter two control groups indicate that disease transfer was not the result of antigen carry-over. Intravenous injection of sera from arthritic donors was incapable of passively transferring clinical or histologic synovitis in 30 recipients. Thus, these studies directly implicate immunologic sensitivity to the cartilage type of collagen in the etiology of this autoimmune disease.

CSF1R Signaling Blockade Stanches Tumor-Infiltrating Myeloid Cells and Improves the Efficacy of Radiotherapy in Prostate Cancer

Radiotherapy is a major frontline treatment for prostate cancer patients, yet, a large portion of these patients suffer from local tumor recurrence. Tumor-infiltrating myeloid cells (TIMs), including CD11b+F4/80+ tumor-associated macrophages (TAMs) and CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs), play critical roles in promoting tumor angiogenesis, tissue remodeling and immunosuppression. Here, we show enhanced recruitment of TAMs and MDSCs after local irradiation. Although treatment is directed to the tumor sites, the impact of irradiation is systemic as dramatic increases of MDSCs were observed in the spleen, lung, lymph nodes and peripheral blood. Of the cytokines examined, we found that macrophage colony-stimulating factor 1 (CSF1) increased by 2 fold in irradiated tumors. Enhanced macrophage migration induced by conditioned media from irradiated tumor cells was completely blocked by the selective CSF1R inhibitor, GW2580. Importantly, increased CSF1 levels were also observed in the serum of prostate cancer patients after radiotherapy. ABL1 (c-Abl), a non-receptor tyrosine kinase, known to mediate apoptosis and signal transduction under stress, is activated by irradiation. Activated ABL1 translocates to the nucleus, binds to the CSF1 promoter region and enhances CSF1 transcription. Combination therapy using a CSF1R inhibitor currently in clinical trials, PLX3397, with radiation suppressed tumor growth more effectively than radiation alone. This study highlights the importance of CSF1/CSF1R signaling in the recruitment of TIMs in response to radiotherapy and suggests their significant role in promoting tumor recurrence. Furthermore, our data supports co-targeting TIMs in conjunction with radiotherapy to achieve a more effective and durable treatment strategy for prostate cancer patients.Radiotherapy is used to treat many types of cancer, but many treated patients relapse with local tumor recurrence. Tumor-infiltrating myeloid cells (TIM), including CD11b (ITGAM)(+)F4/80 (EMR1)+ tumor-associated macrophages (TAM), and CD11b(+)Gr-1 (LY6G)+ myeloid-derived suppressor cells (MDSC), respond to cancer-related stresses and play critical roles in promoting tumor angiogenesis, tissue remodeling, and immunosuppression. In this report, we used a prostate cancer model to investigate the effects of irradiation on TAMs and MDSCs in tumor-bearing animals. Unexpectedly, when primary tumor sites were irradiated, we observed a systemic increase of MDSCs in spleen, lung, lymph nodes, and peripheral blood. Cytokine analysis showed that the macrophage colony-stimulating factor CSF1 increased by two-fold in irradiated tumors. Enhanced macrophage migration induced by conditioned media from irradiated tumor cells was completely blocked by a selective inhibitor of CSF1R. These findings were confirmed in patients with prostate cancer, where serum levels of CSF1 increased after radiotherapy. Mechanistic investigations revealed the recruitment of the DNA damage-induced kinase ABL1 into cell nuclei where it bound the CSF1 gene promoter and enhanced CSF1 gene transcription. When added to radiotherapy, a selective inhibitor of CSF1R suppressed tumor growth more effectively than irradiation alone. Our results highlight the importance of CSF1/CSF1R signaling in the recruitment of TIMs that can limit the efficacy of radiotherapy. Furthermore, they suggest that CSF1 inhibitors should be evaluated in clinical trials in combination with radiotherapy as a strategy to improve outcomes.

Leukotactic factor produced by sensitized lymphocytes.

Lymph node lymphocytes obtained from guinea pigs exhibiting delayed hypersensitivity are stimulated in vitro by specific antigen to produce a soluble factor that is chemotactic in vitro for mononuclear macrophages. The material is nondialyzable, relatively heat stable, and elutes from Sephadex G-100 in the fraction containing molecules smaller than immunoglobulins.

Macrophage Migration Inhibitory Factor Deficiency Impairs Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice

Background— Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed widely by vascular cells. However, scant in vivo evidence supports direct participation of MIF in atherogenesis. Therefore, we investigated whether deficiency of MIF modulates atherosclerotic lesion formation and composition in low-density lipoprotein receptor–deficient (LDLr−/−) mice. Methods and Results— MIF−/−LDLr−/− and LDLr−/− mice were generated and consumed an atherogenic diet for 12 or 26 weeks. MIF−/−LDLr−/− mice had significantly reduced abdominal aorta lipid deposition and intimal thickening from aortic arch throughout the abdominal aorta compared with LDLr−/− mice. Marked retardation of atherosclerosis over time in MIF-deficient mice accompanied decreased lesion cell proliferation. At 26 weeks, 20% of MIF-deficient mice developed only early, fatty streak–like lesions, whereas >80% of LDLr−/− mice developed advanced lesions containing calcification and lipid cores. Analysis of smooth muscle cells from mouse aortae demonstrated that MIF deficiency reduced smooth muscle cell proliferation, cysteine protease expression, and elastinolytic and collagenolytic activities. Conclusions— Deficiency of MIF reduces atherogenesis in LDLr−/− mice. These results provide novel insight into inflammatory pathways operating in atheromata and identify a new potential target for modulating atherogenesis.