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As TNF is one of the earliest signals that can be detected in the leukocyte-derived inflammatory cascade which drives subsequent cytokine production, we are interested in determining whether TNF is one of the initiating factors controlling liver remodeling and regeneration following chronic liver damage. One of the early responses is the expression of lymphotoxin-β by hepatic progenitor oval cells. The aim of this study was to determine whether hepatic expression of LT-β was controlled by TNF and to understand the basis of this regulation. We previously showed that LT-β expression is transcriptionally controlled via the TNF-induced, inflammatory NF-κB pathway in T lymphocytes. Here we show that TNF is able to upregulate LT-β expression in hepatic cells at the transcriptional level by the binding of NF-κB p50/p65 heterodimers and Ets1 to their respective sites in the LT-β promoter., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A library of new thalidomide C4/5 analogues containing either a phenyl or alkyne tether were synthesized using Sonogashira or Suzuki cross coupling reactions from their aryl halogenated precursors. All thalidomide analogues were tested for their ability to inhibit the expression of the proinflammatory cytokine Tumor Necrosis Factor (TNF). More explicitly the use of a novel reporter system utilizing the promoter region of the TNF gene in a human T-cell line provided a rapid and effective measure of NFkappaB transcriptional activity. Several compounds either containing either an aryl-isobutyl or aryl-isopropoxy group were the most effective in inhibiting TNF expression, and were several times more active than thalidomide itself. Five of the more active derivatives indicated an apoptotic response while one of these compounds, containing an aldehyde tether, showed possible influence of cell cycling effects., (Copyright 2009 Elsevier Ltd. All rights reserved.)

A library of new thalidomide analogues containing an olefin functionality were synthesised using a Heck cross coupling reaction from their aryl halogenated precursor. All analogues were tested for their ability to inhibit the synthesis of the proinflammatory cytokine Tumour Necrosis Factor (TNF). Compounds 22, 29, 33 and 37 were the most effective in this assay inhibiting TNF expression 50%, 69%, 52% and 50%, respectively.

Lymphotoxin-beta (LT-beta) is a transmembrane protein expressed mainly on cells of the lymphoid lineage. It associates with LT-alpha on the cell surface to form the heterotrimeric LTalpha1,beta2 complex, which binds the LT-beta receptor. Membrane lymphotoxin is a crucial signal for the appropriate development of lymph nodes and Peyer's patches, and in the formation of B and T cell compartments in the spleen. In this study we report the characterization of mechanisms governing both basal as well as PMA- and TNF-inducible regulation of the human LT-beta promoter. Using a Jurkat T cell line, induction with either PMA or TNF resulted in an increase in mRNA levels compared with uninduced values. This induction corresponded to an increase in transcriptional activity of the human LT-beta promoter. Mutational and deletion analysis demonstrated the importance of Ets and NF-kappaB motifs in the regulation of basal transcription. Furthermore, the ability of PMA to induce activity was lost in the Ets mutant constructs. Interestingly, the same mutation had little effect on the ability of TNF to induce transcription of the LT-beta promoter. TNF inducibility was localized to the NF-kappaB site positioned at -83 of the promoter sequence. Thus, it appears that the Ets site, although playing a major role in PMA induction, did not mediate TNF inducibility. Therefore, our study suggests that alternative signaling pathways may be present to induce the expression of LT-beta in response to different immunological or inflammatory stimuli.

Lymphotoxin-beta (LT- beta) is a tumor necrosis factor (TNF)-related membrane-bound cytokine that forms a heterotrimeric surface lymphotoxin (LT) complex with LT-alpha on the surface of lymphoid cells. Although knockout studies have revealed a role in lymph node biogenesis during development, the regulation and function of surface LT in mature cell types are poorly understood. The present study aims to understand the physiologic signals that regulate the components of surface LT in Jurkat T cells. We show that the previously observed upregulation of surface LT by phorbol myristate acetate (PMA) is markedly abrogated by cotreatment with ionomycin through posttranscriptional mechanisms. In addition, the observation of striking similarities between the mRNA accumulation kinetics of LT-alpha and LT-beta during these treatments indicates tight coupling of expression under certain conditions. In investigating the reported upregulation of LT-beta during inflammation, we tested the effects of various proinflammatory and anti-inflammatory cytokines on LT-beta expression. Our data demonstrate an upregulation of LT-beta mRNA by the inflammatory cytokines TNF and LT-alpha.

Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis by activated monocyte/macrophages, contributing to an anti-inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-alpha transcription. Site-directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-alpha promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-alpha 3'-untranslated region. We conclude that glucocorticoids suppress LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding sites in the -106 to -88 bp region of the TNF-alpha promoter.

The authors have previously demonstrated that the tumour necrosis factor (TNF) -308 G/A polymorphism affects the binding of transcription factors. In transient transfection assays in PMA stimulated U937 monocytes and Jurkat T cells, the A-containing TNF2 promoter has a 2-3-fold greater transcriptional activity than the TNF1 promoter in the presence of the TNF 3'UTR. In this study it was found that a difference in TNF1 and TNF2 promoter activities was only observed in U937 and Jurkat cells, and not in Raji (B cell line), HeLa (epithelial carcinoma cell line), HepG2 (hepatoma cell line) or THP-1 (monocyte), suggesting cell-type specific transcription factors or modifications may be involved in the formation of the -308 protein/DNA complex. Physiological stimulators, TNF and interferon gamma (IFN-gamma) did not cause differential promoter activity between TNF1 and TNF2, but LPS did with only the TNF2 promoter/3'UTR construct being significantly responsive to lipopolysaccharide (LPS) in U937 cells. In U937 cells, the -308 polymorphism affected transcription following differentiation by phorbol myristate acetate (PMA), retinoic acid, PMA plus LPS and PMA plus retinoic acid with an increase in nuclear factor binding to both TNF1 and TNF2 in the -323 to -285 region being observed. The greatest difference between TNF2 and TNF1 promoter activities (5-fold) was observed following PMA plus retinoic acid treatment of transfected U937 cells for 48h. During this time, U937 differentiated into cells with a macrophage-like morphology. An understanding of the cell type and stimuli specific requirements for differential expression of the -308 polymorphism may help elucidate the role the TNF -308 polymorphism plays in diseases where elevated TNF levels are thought to be important., (Copyright 2000 Academic Press.)

A biallelic G (TNF1 allele) to A (TNF2 allele) polymorphism 308 nucleotides upstream from the transcription initiation site in the tumor necrosis factor (TNF) promoter is associated with elevated TNF levels and disease susceptibilities observed in human subjects. The TNF2 allele is strongly associated with the high-TNF-producing autoimmune MHC haplotype HLA-A1, B8, DR3, with elevated serum TNF levels and a more severe outcome in infectious diseases, such as cerebral malaria. A number of groups have set out to determine whether the -308 polymorphism could affect transcription factor binding and hence influence TNF transcription and expression levels. Although some studies have failed to show any functional difference between the two allelic forms, others have shown that the -308 polymorphism effected transcription factor binding to the region encompassing -308, with the region in the TNF2 allele showing altered binding characteristics. The -308 polymorphism also has been found by some groups to be functionally significant in reporter gene assays in Raji B cells, Jurkat T cells, and U937 pre-monocytic cells. Up to fivefold differences can be measured between TNF1 and TNF2 allelic constructs when the TNF 3'UTR is present, indicating a role in the expression of the polymorphism. Although controversial, the majority of the data support a direct role for the TNF2 -308 allele in the elevated TNF levels observed in TNF2 homozygotes and HLA-A1, B8, DR3 individuals. Elevated TNF levels due to the -308 polymorphism may alter the immune response such that it confers susceptibility to certain autoimmune and infectious diseases.

Glucocorticoids suppress many functions in activated monocyte/macrophages, including the release of TNF-alpha. This is likely to contribute to the efficacy of glucocorticoids in some inflammatory diseases, such as rheumatoid arthritis, where TNF-alpha contributes to pathogenesis. Glucocorticoids suppress the activity of reporters which include TNF-alpha promoter regions and modify the activity of NF-kappa B family transcription factors in activated human monocytic cell lines, suggesting effects of glucocorticoids on TNF-alpha gene transcription. In addition, glucocorticoids have been reported to antagonise the enhanced translational efficiency of TNF-alpha mRNA which occurs at least after stimulation of murine monocytic cells. It is likely, therefore, that glucocorticoids act at several points in stimulated monocyte/ macrophages to reduce TNF-alpha secretion. Understanding glucocorticoid control of TNF-alpha secretion may explain some of the variability in response to GC in inflammatory diseases and may reveal means of inducing glucocorticoid-like anti-inflammatory effects in monocyte/macrophages without exposing other tissues to the adverse effects of glucocorticoids.

Since the tumor necrosis factor alpha (TNF-alpha) gene was found to be located in the central major histocompatibility complex (MHC) there has been much speculation concerning a genetic association between particular TNF alleles and disease susceptibility. A relationship between the MHC haplotype A1, B8, DR3, TNF-alpha expression levels and susceptibility to autoimmune disease has been suggested by several groups. The identification of the -308 polymorphism and its association with the HLA A1, B8, DR3 haplotype have led to speculation that the polymorphism may play a role in the altered expression of TNF-alpha. We have demonstrated that the region (-323 to -285) encompassing -308 in the TNF2 allele binds nuclear factors differently to the same region in the promoter of the more common TNF1 allele. The G/A -308 polymorphism affected the affinity of factor binding and resulted in a factor binding to TNF2 but not TNF1. The observed differential binding was shown to be functional, with the 38bp region from TNF2 causing a two-fold greater activity of a heterologous promoter over that due to the same region in TNF1. To further substantiate the functional consequences of the TNF-alpha -308 polymorphism, we analysed both allelic forms of the TNF-alpha promoter region (-993 to +110) in a transient transfection assay, using luciferase as a reporter gene. The results showed that when present with the 3'UTR the -308A allelic form gave a two-fold greater level of transcription than the 308G form in PMA-stimulated Jurkat and U937 cells. This suggests that the -308 G/A polymorphism may play a role in the altered TNF-alpha gene expression observed in individuals with the HLA A1, B8, DR3 haplotype.

We have identified a region of the human TNF-alpha promoter between nucleotides -323 and -285, capable of influencing transcriptional activity. This region encompasses the -308 polymorphism and contains a 10 bp sequence homologous to the consensus binding site of activator protein-2 (AP-2). Protein complexes derived from U937 and Jurkat cells were found to bind to this element. Competitive EMSA using a consensus AP-2 oligonucleotide indicated that AP-2 may be involved. Functional assays demonstrate that this region can repress activity of a heterologous promoter in the Jurkat T-cell line, but act as an inducible enhancer of transcription in U937 cells.

The location of the TNF and other genes in the central MHC and their possible relevance to disease susceptibility provided an impetus to develop useful typing markers. The 4AOHW undertook to assess the various markers available, including DNA sequence-based systems. A panel of well-characterized lymphoblastoid cell lines were typed by Nco I RLFP analysis, SSO typing, and TNF microsatellite typing. RFLP and SSO typing were relatively reproducible as judged by the blind replicates. The two techniques provided the same results with only one exception, and it would be reasonable to prefer SSO typing because of its advantages in terms of cost and time. Microsatellite typing was much more discriminating but, as expected, less robust in that some discrepancies were apparent. As a result of the workshop and subsequent testing, alleles and haplotypes were allocated to most cells within the 4AOHW panel, including 10W cells typed in previous studies. While there was evidence that microsatellites may be relatively stable, they have the potential to identify recent mutations within ancestral haplotypes.

To characterize the genomic structure and gene content between HLA-B and TNF we studied three overlapping yeast artificial chromosome (YAC) clones derived from the Boleth cell line carrying the 62.1 MHC ancestral haplotype (AH). In particular, we have evaluated the stability of haplospecific sequence motifs (haplospecific geometric elements) in YACs. By PCR and gene scanning we show that the two larger YAC clones (318G11 and 8F2) exhibit features characteristic of the 62.1 AH carried by the parental cell line as well as those of a separate cell line (BSM) which also carries 62.1. It is concluded that a highly polymorphic duplicated region is represented faithfully in spite of the fact that it contains sequences that could be interpreted as simple repeats liable to mutations. In contrast, microsatellites AFM031 and AFM158, which are also located in the MHC, showed greater instability and the structure in the YACs was not consistent with genomic structure. Likewise, different examples of the same ancestral haplotype show differences at these microsatellite loci. We conclude that polymorphic repeat sequences may exhibit a range of stabilities in vivo and that this may be reflected in their stability in YACs. Northern blotting using the largest YAC clone revealed several novel transcripts which await localization.

It remains unclear which MHC loci are involved in susceptibility to autoimmune diseases and immune deficiencies. We have chosen to evaluate whether different alleles of tumour necrosis factor-alpha (TNF-alpha) are important, as TNF has been implicated in the etiology of many immunological disorders. We have shown previously that a restriction fragment length polymorphism in the TNF region correlates with MHC ancestral haplotypes. We therefore examined the effect of ancestral haplotype on the activity of TNF-alpha in culture supernatants of lymphoblastoid cell lines. The results demonstrate that TNF-alpha activity in supernatants of 8.1 (A1, B8, DR3) cell lines was higher than that present in the supernatants from cells homozygous for eight different MHC ancestral haplotypes, and indicate that polymorphisms in TNF-alpha, or in other MHC genes that regulate TNF, may be responsible for the 8.1 phenotype.

The HLA-B locus is extremely polymorphic. We have sequenced a region, CL, telomeric of HLA-B that also shows a high degree of allelic variation which we have shown previously by RFLP analysis. The polymorphism can be accounted for by sequence variation in duplicated, reiterated sequence elements called geometric elements. Comparison of the CL1 and CL2 sequences from the 57.1, 8.1, 18.2 and 7.1 ancestral haplotypes revealed that the lengths of the elements vary, both between the duplicated loci within a haplotype and between haplotypes, apparently because certain sequences are inserted or deleted. It is possible, using the polymerase chain reaction, to amplify these elements in genomic DNA from ancestral haplotypes for which sequence data of the CL region are not available and to obtain gel patterns which are characteristic of different ancestral haplotypes. The most striking feature of the data is the fact that the majority of the CL patterns are haplospecific; i.e. have a particular pattern that is unique for a particular ancestral haplotype and can be used to type these ancestral haplotypes. At least 12 different allelic patterns have been identified within a panel of 29 cell lines representing 16 ancestral haplotypes. For these 16 ancestral haplotypes, all examples of each haplotype have the same CL pattern. The haplotypic nature of the patterns confirms that ancestral haplotypes are conserved chromosomal segments and that coding and non-coding sequences are identical by descent from a remote ancestor.

Polymorphisms were sought between HLA B and tumor necrosis factor (TNF) using three genomic probes. Extensive polymorphism was detected within a panel of 50 cell lines including 37 homozygotes representing 21 different ancestral haplotypes (AH). Following Taq I digestion of genomic DNA, we observed three allelic patterns with probe X (R17A) and four with probe V (R9A). Seven different allelic patterns were found with probe Y (M20A) after Taq I + Rsa I digestion. Family studies showed that the Y, X, and V alleles were inherited and segregated with HLA haplotypes. A striking feature of the allelic patterns detected by these probes was that cells with the same AH had identical Y, X, and V alleles (i.e., the alleles were haplotypic). Of 15 different Y-X-V haplotypes observed, 11 were found to be specific for a particular AH (i.e., were haplospecific). Four were shared by more than one AH, but in these instances there were extensive similarities in other regions within the major histocompatibility complex (MHC), for example, the Japanese 46.2 (HLA Bw46-DRw8) and the Chinese 46.1 (Bw46-DR9) share all alleles between HLA C and C4 and differ only in class II, suggesting their relatively recent divergence by recombination between C4 and DR. Surprisingly, two insulin-dependent diabetes mellitus (IDDM)-resistant but race-specific AHs 52.1 (Bw52-DRB1*1502, Japanese) and 7.1 (B7-DRB1*1501, Caucasoid) carry the same Y-X-V haplotype, suggesting the possibility of localizing gene(s) relevant to IDDM. The present study confirms that MHC AHs have been conserved en bloc, including the region between HLA B and TNF.