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.

Hypoxic upregulation of TNF receptor type 2 expression involves NF-IL-6 and is independent of HIF-1 or HIF-2.

Tumor necrosis factor (TNF) exerts its biologic activity via two distinct membrane receptors, TNF receptor type 1 (p55TNFR) and TNF receptor type 2 (p75TNFR). Whereas the p55TNFR gene is rather constitutively expressed, transcription of p75TNFR is strongly modulated by a number of stimulatory agents. Experimental evidence suggested the involvement of p75TNFR in endothelial cell activation. Therefore, we have tested the transcriptional activity of p75TNFR under conditions of hypoxia and reoxygenation. Northern blot analysis revealed that p75TNFR mRNA is upregulated in NIH3T3 cells under hypoxia and reoxygenation. This observation directly originates from transcriptional activation of the p75TNFR gene, as shown by reporter gene analysis. Cotransfection experiments clearly showed that the transcriptional induction of the p75TNFR gene is independent of the hypoxia-induced factors, HIF-1alpha and HIF-2alpha. Using deletion mutants of the 5'-flanking region of the p75TNFR gene, we were able to identify a putative DNA binding site for the transcription factor nuclear factor-interleukin-6 (NF-IL-6) to be responsible for the transcriptional upregulation of the p75TNFR gene under conditions of hypoxia and reoxygenation.

Antimetastatic effect of CpG DNA mediated by type I IFN.

The mechanisms involved in the antimetastatic effect of CpG-containing DNA were investigated in a mouse model of experimental metastasis. Tumor cell colony formation in lungs or livers of mice after i.v. inoculation with syngeneic fibrosarcoma or thymoma cells was determined. The i.v. injection of plasmid DNA or synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs before tumor cell application strongly inhibited metastasis. Because synthetic CpG-ODN was not directly tumor cytotoxic, the target cells for this CpG-ODN effect were determined. The cytotoxic activity on standard natural killer (NK) targets as well as on fibrosarcoma cells of splenic NK cells and NKT cell-containing liver mononuclear cells derived from CpG-ODN-treated mice was strongly enhanced. Participation of NK/NKT cells in the CpG-induced antimetastatic effect was demonstrated by reduction of the antimetastatic effect in mice depleted of NK/NKT cells and beta2-microglobulin-deficient mice. Neutralization of interleukin 12, interleukin 18, or IFN-gamma did not interfere with the CpG-induced antimetastatic effect. However, in sera of CpG-ODN-treated mice, high levels of IFN-alpha were detected, and in IFN-alpha/beta receptor-deficient mice, the CpG-ODN-induced antimetastatic effect was strongly reduced. These data indicate that CpG-ODNs activate NK/NKT cells for antimetastatic activity indirectly via IFN-alpha/beta receptor activation. The exploitation of the stimulatory activity of CpG-ODN for the innate immune system might be a useful strategy for antimetastatic therapy.

Recombinant, soluble LIGHT (HVEM ligand) induces increased IL-8 secretion and growth arrest in A375 melanoma cells.

The heterotrimeric lymphotoxin alpha(1)beta(2) (LTalpha(1)beta(2)) complex and LIGHT, a new member of the tumor necrosis factor (TNF) superfamily, have been identified as membrane-anchored ligands for the LTbeta receptor (LTbetaR), a member of the TNF receptor (TNFR) superfamily. Although some of the biologic activities of this receptor have been described using either soluble LTalpha(1)beta(2) as a ligand or agonistic monoclonal antibodies (mAb), very little is known about the signaling of LIGHT via the LTbetaR. To gain more insight into the biologic functions of LIGHT, we generated a recombinant soluble form of human LIGHT (rsHuLIGHT). We demonstrate here that this rsHuLIGHT is capable of binding to the LTbetaR. Interestingly, receptor-mediated ligand precipitation analysis revealed that rsHuLIGHT bound only to human LTbetaR but not to mouse LTbetaR, indicating a species-specific receptor ligand interaction. Activation of A375 human melanoma cells by rsHuLIGHT induced an increased secretion of interleukin-8 (IL-8). Furthermore, rsHuLIGHT caused growth arrest of A375 cells even in the absence of interferon-gamma (IFN-gamma).

Functional characterization of the mouse lymphotoxin-beta receptor promoter.

The lymphotoxin beta-receptor (LT beta R), a member of the tumor necrosis factor (TNF) receptor family, plays a crucial role in lymphoid organogenesis by signaling through its functional ligand LT alpha(1)beta(2). While the receptor is expressed on a wide range of cell types e.g. fibroblasts and monocytes, the ligand is expressed only on activated T, B and NK cells. Remarkably, no cell type has been identified so far that expresses both the receptor and the ligand. In order to characterize the mouse LT beta R gene expression on a molecular level, we isolated about 1 kb of the 5' flanking region of the LT beta R gene. Primer extension analysis revealed one transcriptional start site located at - 60 upstream of the ATG-containing first exon. Northern blot analysis showed that the LT beta R is abundantly expressed in the mouse fibroblast cell line NIH 3T3, and to a lesser extent, in the mouse macrophage-like cell line RAW 264.7. To determine whether the 5' flanking region exerts functional promoter activity, we generated deletion mutants fused to the luciferase reporter gene. Transfection experiments using these reporter gene constructs showed that the isolated 5' flanking region is transcriptionally active in NIH 3T3 and RAW 264.7 cells, and determined a minimum length required for the transcriptional activity of the LT beta R promoter in these cells. Further sequence analysis of the isolated 5' flanking region identified a number of putative DNA-binding sites for transcription factors. Interestingly, incubation of NIH 3T3 cells with dexamethasone resulted in an elevated mRNA level of the LT beta R gene. This effect was abolished by using the specific glucocorticoid receptor inhibitor RU486, indicating an increased transcriptional activity of the LT beta R promoter after glucocorticoid stimulation.

Development of allergic contact dermatitis requires activation of both tumor necrosis factor-receptors.

We investigated the role of the TNF receptors, type I (p55TNFR) and type II (p75TNFR), in a mouse model of contact hypersensitivity, i.e., a model of a delayed type hypersensitivity (DTH) allergic reaction. Mice deficient for p55TNFR or p75TNFR were used to investigate the functions of these receptors in development of the DTH reaction. We show that both TNF receptors have a strong influence on the overall outcome of the DTH reaction, with the two TNF receptors exerting distinct functions. Dendritic cells of mice lacking p55TNFR had a defect in allergen uptake but showed normal migration into regional lymph nodes. In contrast, dendritic cells of p75TNFR-deficient mice showed diminished migration into regional lymph nodes after allergen contact, whereas the allergen uptake was independent of the p75TNFR. Thus, both TNF receptors are required for the development of a complete DTH reaction.

A novel p75TNF receptor isoform mediating NFkappa B activation.

We report the identification of a novel p75TNF receptor isoform termed icp75TNFR, which is generated by the use of an alternative transcriptional start site within the p75TNFR gene and characterized by regulated intracellular expression. The icp75TNFR protein has an apparent molecular mass of approximately 50 kDa and is recognized by antibodies generated against the transmembrane form of p75TNFR. The icp75TNFR binds the tumor necrosis factor(TNF) and mediates intracellular signaling. Overexpression of the icp75TNFR cDNA results in TNF-induced activation of NFkappaB in a TNF receptor-associated factor 2 (TRAF2)-dependent manner. Thus, our results provide an example for intracellular cytokine receptor activation.

Tumor necrosis factor induces tumor necrosis via tumor necrosis factor receptor type 1-expressing endothelial cells of the tumor vasculature.

Activation of endothelial cells, fibrin deposition, and coagulation within the tumor vasculature has been shown in vivo to correlate with the occurrence of tumor necrosis factor (TNF)-induced tumor necrosis in mice. In the present study we investigated which target cells mediate the TNF-induced necrosis in fibrosarcomas grown in wild type (wt), TNF receptor type 1-deficient (TNFRp55-/-), and TNF receptor type 2-deficient (TNFRp75-/-) mice. TNF administration resulted in tumor necrosis exclusively in wt and TNFRp75-/-, but not in TNFRp55-/- mice, indicating a dependence of TNF-mediated tumor necrosis on the expression of TNF receptor type 1. However, using wt and TNFRp55-/- fibrosarcomas in wt mice, we found that TNF-mediated tumor necrosis was completely independent of TNF receptor type 1 expression in tumor cells. Thus we could exclude any direct tumoricidal effect of TNF in this model. Soluble TNF induced leukostasis in wt and TNFRp75-/- mice but not in TNFRp55-/- mice. TNF-induced leukostasis in TNFRp55-/- mice was restored by adoptive bone marrow transplantation of wt hematopoietic cells, but TNF failed to induce tumor necrosis in these chimeric mice. Because TNF administration resulted in both activation and focal damage of tumor endothelium, TNF receptor type 1-expressing cells of the tumor vasculature, likely to be endothelial cells, appear to be target cells for mediating TNF-induced tumor necrosis.

Isolation and functional characterization of the mouse p75 TNF receptor promoter.

Tumor necrosis factor (TNF) is a pleiotropic cytokine that plays an important role in immunological and inflammatory responses. It exerts its biological effects via two distinct membrane receptors of apparent molecular weight of 55 (p55TNFR) and 75 kDa (p75TNFR), respectively. Most cell lines and primary tissues express both receptor types. While the p55TNFR gene is constitutively expressed at rather low levels, the transcription of p75TNFR is strongly modulated by a number of stimulatory agents. To characterize the mouse p75TNFR gene expression on a molecular level, we screened a mouse genomic library using the 5' end of the p75TNFR cDNA as a probe. A 6.3kb genomic clone containing about 6 kb of 5' flanking region and 300 bp of 3' sequence including the translational start site and the first exon was isolated and subcloned. Primer extension analysis revealed three transcriptional start sites located at -35, -39, and -564 bp upstream of the ATG-containing first exon. To determine whether the 5' flanking region exerts functional promoter activity, we generated deletion mutants fused to the luciferase reporter gene. Transfection of mouse fibroblasts (NIH3T3) with these constructs showed functional promoter activity of the isolated 5' region. By further sequence analysis of the 5' flanking region a number of putative DNA-binding sites for transcription factors, e.g., Sp1, CREB, Yi, YY1, and IFN gamma-responsive element, were identified.

Design, synthesis, and characterization of HIV-1 enhancer-binding polypeptides derived from bacteriophage 434 repressor.

We have designed and synthesized HIV-1 enhancer-binding polypeptides that were derived from bacteriophage 434 repressor. These peptides were 39-54 residues long and contained either the recognition helix or the entire helix-turn-helix motif of the DNA-binding domain of 434 repressor. The dissociation constant of the complex formed between the standard peptide (R42) and a synthetic 70-bp HIV enhancer DNA was ca. 10(-8) M. The specificity of the interaction of R42 with the two HIV enhancers was demonstrated by competitive band shift assays, stepwise displacement of the p50 subunit of transcription factor NF-kappa B from its two HIV enhancer binding sites, and DNase I footprinting; R42 seemed to protect best the two TTTCC sequences of the HIV enhancers against digestion by DNase I. R42 analogues with mutated recognition helix had lower DNA binding specificity. It remains to be investigated whether our artificial HIV enhancer-binding polypeptides are active in vivo.

Activation of transcription by binding of NF-E1 (YY1) to a newly identified element in the first exon of the human DR alpha gene.

A previously unrecognized element, located downstream of the start site of transcription in the first exon of the DR alpha gene, has been defined that enhances promoter activity up to eightfold in a position-dependent manner. Mutations in this DNA-binding site abolished binding of a nuclear factor in human B cell nuclear extract and decreased the activity of the DR alpha promoter to a basal level. Significant sequence homology of this element was found in the DNA of the DR beta, DP alpha and -beta, and DQ alpha genes, always located downstream of the transcriptional start site. The nuclear factor binds to the DR alpha and DP alpha element but not to the element in the DQ alpha gene. It was identified as NF-E1 (YY1). This protein, previously identified by its binding to the Ig kappa 3' enhancer and the Ig heavy chain mu E1 site, thus also appears to be quite important in the regulation of MHC class II gene expression.

The DNA-binding properties of an artificial 42-residue polypeptide derived from a natural repressor.

Bacteriophage 434 repressor recognizes the operator sequences ACAAG and ACAAT. As the same or similar sequences occur in the enhancer region of HIV-1, 434 repressor was a potential HIV enhancer-binding protein. We found that the interaction of the DNA-binding domain of 434 repressor with a 57-bp HIV enhancer DNA was very weak whereas a 42-residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly. The results of footprint and cell-free in vitro transcription studies showed that the 42-residue peptide bound preferably to the enhancer region of HIV-1 and acted as an artificial repressor. Replacement of an essential glutamine of the recognition helix by glutamic acid resulted in a partial shift of the sequence specificity of the 42-residue peptide.

Rapid partial degradation of DDT by a cytochrome P-450 model system.

Heme compounds, in combination with a reducing agent and oxygen, can express various activities of cytochrome P-450 enzymes. In the present study it was found that a mixture of hemin and excess cysteine was able to degrade the insecticide DDT partially. The major products were three water-soluble, non-toxic conjugates of DDT metabolites with cysteine which had lost two or three of the five chlorine atoms of DDT per molecule and whose structures were elucidated by gas chromatography/mass spectrometry. In 0.05 M NH4HCO3, pH 7.7/ethanol (5:6, by vol.), the degradation reaction catalyzed by the hemin-cysteine model system was at least 8 x 10(4) times faster than the uncatalyzed reaction. In the presence of a designed 24-residues polypeptide or beta-casein, two DDT-binding proteins, an additional fourfold increase in the rate of DDT degradation was observed. Although the concentrations of DDT and cysteine occurring in an organism would be expected to be lower than those in the experiments described, the formation of water-soluble conjugates of DDT metabolites with cysteine (and other amino acids) could also play a role in metabolism and excretion of DDT in vivo.

Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli.

This paper reports the expression of an artificial functional polypeptide in bacteria. The gene of a designed 24-residue DDT-binding polypeptide (DBP) was inserted between the BamHI and PstI cleavage sites of plasmid pUR291. The hybrid plasmid, pUR291-DBP, was cloned in Escherichia coli JM109. After induction by isopropyl-beta-D-thiogalactopyranoside a fusion protein was expressed in which DBP was linked to the COOH-terminus of beta-galactosidase. DBP, which is stable to trypsin, was obtained by tryptic digestion of the fusion protein and subsequent fractionation of the tryptic peptides by reversed-phase h.p.l.c. Recombinant and chemically synthesized DBP showed identical chromatographic properties, amino acid composition, and chymotryptic digestion patterns. Both the beta-galactosidase-DBP fusion and isolated recombinant DBP bound DDT. The fusion protein was 25 times as potent as the designed 24-residue DBP in activating a cytochrome P-450 model system using equimolar catalytic amounts of the two proteins.