Blocking lymphotoxin-beta receptor activation diminishes inflammation via reduced mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression and leucocyte margination in chronic DSS-induced colitis

The lymphotoxin-beta receptor (LTbetaR) pathway is critical for maintenance of organized lymphoid structures and is involved in the development of colitis. To investigate the mechanisms by which LTbetaR activation contributes to the pathology of chronic inflammation we used a soluble LTbetaR-Ig fusion protein as a competitive inhibitor of LTbetaR activation in the mouse model of chronic colitis induced by oral administration of dextran sulphate sodium. Strong expression of LTbeta which constitutes part of the LTalpha(1)beta(2) ligand complex was detected in colonic tissue of mice with chronic colitis. Treatment with LTbetaR-Ig significantly attenuated the development and histological manifestations of the chronic inflammation and reduced the production of inflammatory cytokines such as TNF, IL-1beta, and IL-6. Moreover, LTbetaR-Ig treatment significantly down-regulated mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression, leading to reduced leucocyte rolling and sticking in postcapillary and collecting venules and reduced extravasation into the intestinal mucosa as quantified by in vivo fluorescence microscopy. Thus, LTbetaR pathway inhibition ameliorates DSS-induced experimental chronic colitis in mice by MAdCAM-1 down-regulation entailing reduced lymphocyte margination and extravasation into the inflamed mucosa. Therefore, a combined treatment with reagents blocking T cell-mediated perpetuation of chronic inflammation such as LTbetaR-Ig together with direct anti-inflammatory reagents such as TNF inhibitors could constitute a promising treatment strategy for chronic colitis.     

Combined screening of thymocytes using apoptosis-specific cDNA array and promoter analysis yields novel gene targets mediating TCDD-induced toxicity.

We have used pathway-specific cDNA arrays coupled with analysis of gene promoter regions to identify novel genes that may mediate the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the thymus. C57BL/6 mice were injected ip with 50 microg/kg TCDD, and 3, 6, or 24 h later, RNA was extracted from the thymus and subjected to microarray analysis. Several members of the TNF and TNFR family were induced following TCDD exposure, including receptor/ligand pairs Ltbeta-R/LIGHT, OX40/OX40L and TNF-alpha/TNFR1. In addition, Fas and CD30 were also upregulated. Pro-apoptotic bcl-2 gene family members Bax and Hrk, among others, were also induced, as were pro-survival bcl-2 family genes Bcl-x and Bcl-w. Cell-cycle regulator p21Cip1 was also induced. In addition, we analyzed the promoter regions of genes induced by TCDD for the presence of dioxin-responsive elements (DREs). The Fas and LIGHT gene promoters were found to contain DREs as analyzed by Matinspector Web-based search algorithm. Furthermore, binding of the aryl hydrocarbon receptor (AhR) to the DREs present on these genes was confirmed by chromatin immunoprecipitation. Given that several of the genes, including Fas, LIGHT, and CD30 are involved in negative selection of T cells in the thymus, our studies suggest that TCDD-induced upregulation of these genes may enhance negative selection leading to thymic atrophy.     

Herpesvirus entry mediator (HVEM) attenuates signals mediated by the lymphotoxin β receptor (LTβR) in human cells stimulated by the shared ligand LIGHT

Signals mediated by members of the tumor necrosis factor receptor superfamily modulate a network of diverse processes including initiation of inflammatory responses and altering cell fate between pathways favoring survival and death. Although such pathways have been well-described for the TNF-α receptor, less is known about signaling induced by the TNF superfamily member LIGHT and how it is differentially altered by expression of its two receptors LTβR and HVEM in the same cell. We used cell lines with different relative expression of HVEM and LTβR to show that LIGHT-induced signals mediated by these receptors were associated with altered TRAF2 stability and RelA nuclear translocation. Production of the inflammatory chemokine CXCL10 was primarily mediated by LTβR. Higher expression of HVEM was associated with cell survival, while unopposed LTβR signaling favored pathways leading to apoptosis. Importantly, restoring HVEM expression in cells with low endogenous expression recapitulated the phenotype of cells with higher endogenous expression. Together, our data provide evidence that relative expression of HVEM and LTβR modulates canonical NF-κB and pro-apoptotic signals stimulated by LIGHT.     

The histopathological and electron-microscopic examination of the stereotactic pulsed radiofrequency and conventional radiofrequency thermocoagulation lesions in rat brain

Abstract

Objective: Neurodestructive procedures have been used for treating intractable pain for a long time. Pulsed radiofrequency (RF) is a newly defined energy type. Pulsed RF may be used in the treatment of patients with some pain syndromes in whom the pain could not be controlled by the alternative techniques. The objective of the present study was to examine the histological and electron microscopical changes in rat brain after pulsed RF application.

Methods: Forty-five male rats were used in these experiments. Lesions were applied stereotactically to the target areas of the rat brains. Two different RF energy type were used as representative models of pulsed-RF and conventional-RF procedures. The rats were kept alive for 21 days and then killed. The effect of pulsed RF lesions on cerebral tissue ultrastructure was studied.

Results: In the pulsed RF group, intracytoplasmic edema, clarity of the mitochondrial cristas and opening in the cell membrane pores were observed on the electron microscopic examination. In the conventional RF group, these findings were more prominent. In the pulsed RF group, the ratio of the effected neurons was 5.5% on light microscopic examination. In the conventional RF group, the ratio of the effected neurons was 14.26% and central necrosis was observed additionally.

Discussion: Pulsed RF caused ultrastructural changes in the neurons. The pulsed RF may possibly cause a depression on the cell membrane potential by opening the cell membrane pores and resulting in the ion entrance into the cell cytoplasm and intracytoplasmic edema. However, it seems that all these changes were reversible.     

Investigation of osteoclastogenic signalling of the RANKL substitute LIGHT

LIGHT (TNFSF14) is a member of the TNF superfamily and is known to substitute for RANKL to induce osteoclast differentiation. LIGHT binds HVEM and LTβR, but it is not known whether these receptors play a role in osteoclast formation or whether LIGHT acts via RANKL signalling pathways. We found that both RANKL and LIGHT strongly induced phosphorylation of Akt and NFκB but not JNK in mouse osteoclast precursor cells. The addition of an Akt inhibitor showed decreased osteoclast differentiation and resorption mediated by both RANKL and LIGHT. RT-PCR and FACS analysis showed that CD14⁺ human osteoclast precursors expressed HVEM and LTβR; expression levels of HVEM increased in the course of osteoclastogenesis and a decrease in LIGHT expression was associated with an increase in HVEM suggesting that there is a feedback loop related to this receptor. Our findings show that LIGHT is not inhibited by the soluble RANKL receptor OPG and that LIGHT is a potent osteoclastogenesis factor that activates the Akt, NFκB and JNK pathways.     

LIGHT/HVEM/LTβR Interaction as a Target for the Modulation of the Allogeneic Immune Response in Transplantation

The exchange of information during interactions of T cells with dendritic cells, B cells or other T cells regulates the course of T, B and DC‐cell activation and their differentiation into effector cells. The tumor necrosis factor superfamily member LIGHT (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes) is transiently expressed upon T cell activation and modulates CD8 T cell‐mediated alloreactive responses upon herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR) engagement. LIGHT‐deficient mice, or WT mice treated with LIGHT‐targeting decoy receptors HVEM‐Ig, LTβR‐Ig or sDcR3‐Ig, exhibit prolonged graft survival compared to untreated controls, suggesting that LIGHT modulates the course and severity of graft rejection. Therefore, targeting the interaction of LIGHT with HVEM and/or LTβR using recombinant soluble decoy receptors or monoclonal antibodies represent an innovative therapeutic strategy for the prevention and treatment of allograft rejection and for the promotion of donor‐specific tolerance.     

Transgenic expression of decoy receptor 3 protects islets from spontaneous and chemical-induced autoimmune destruction in nonobese diabetic mice

Decoy receptor 3 (DCR3) halts both Fas ligand- and LIGHT-induced cell deaths, which are required for pancreatic beta cell damage in autoimmune diabetes. To directly investigate the therapeutic potential of DCR3 in preventing this disease, we generated transgenic nonobese diabetic mice, which overexpressed DCR3 in beta cells. Transgenic DCR3 protected mice from autoimmune and cyclophosphamide-induced diabetes in a dose-dependent manner and significantly reduced the severity of insulitis. Local expression of the transgene did not alter the diabetogenic properties of systemic lymphocytes or the development of T helper 1 or T regulatory cells. The transgenic islets had a higher transplantation success rate and survived for longer than wild-type islets. We have demonstrated for the first time that the immune-evasion function of DCR3 inhibits autoimmunity and that genetic manipulation of grafts may improve the success and survival of islet transplants.     

Regulation of proliferation, survival and apoptosis by members of the TNF superfamily

Tumor necrosis factor (TNF) was first identified in 1984 as a cytokine with anti-tumor effects in vitro and in vivo. Extensive research since then has shown that there are at least 18 distinct members of the TNF super family and they exhibit 15-25% amino acid sequence homology with each other. These family members bind to distinct receptors, which are homologous in their extracellular domain. These cytokines have been implicated in a wide variety of diseases including tumorigenesis, septic shock, viral replication, bone resorption, rheumatoid arthritis, diabetes, and other inflammatory diseases. TNF blockers have been approved for human use in treating some of these conditions in the United States and other countries. Various members of the TNF super family mediate either proliferation, survival, or apoptosis of cells. Although distinct receptors, all members share a common cell signaling pathway that mediates the activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (e.g. c-jun N-terminal kinase). Regulation of cell growth and activation of NF-kappaB and of c-jun N-terminal kinase by the TNF super family is mediated through sequential activation/association of a set of cell signaling proteins named TNF receptor-associated factors, Fas-associated death domain and FADD-like ICE, caspases, receptor-interacting protein, NF-kappaB-inducing kinases, and IkappaBalpha kinases. Both apoptotic and antiapoptotic signals are activated simultaneously by the same cytokine in the same cell. Together these cytokines regulate cell growth/survival/apoptosis in a complex dance of changing partners and overlapping steps.