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Two soluble tumour-necrosis-factor-alpha(TNF)-binding proteins are derived from the extracellular domains of the p55 and p75 TNF receptors. They are considered to play a pivotal regulatory role in TNF-mediated inflammatory processes, including diseases such as rheumatoid arthritis, by competing with the cell surface receptors for TNF and lymphotoxin (LT, tumour-necrosis factor beta). The extracellular domains of the two receptors each contain four similar cysteine-rich repeats of about 40 amino acids, in common with several other cell surface proteins including the p75 nerve-growth-factor receptor and the CD40 and Fas antigens. The aim of this study was to characterize the involvement of the four cysteine-rich repeats of the human p55 TNF receptor in TNF and LT binding by both membrane-bound and soluble forms of the receptor. Individual repeats were systematically deleted by PCR mutagenesis and the variants transiently expressed in COS cells. Immunoprecipitated receptor variants exhibited the expected sizes on SDS/PAGE gels, and bound a panel of conformation-dependent anti-(TNF receptor) antibodies. Binding of TNF by the four soluble derivatives was compared with binding by the wild-type soluble receptor using a TNF-affinity column and a BIAcore Biosensor, by measurement of their ability to inhibit TNF cytotoxicity on WEHI cells, and 125I-TNF binding to U937 cells. delta 4, which lacks the fourth cysteine-rich repeat, bound TNF comparably with the full-length soluble receptor. TNF-binding affinity was unaltered by deletion of the fourth membrane-proximal cysteine-rich repeat, as determined by Scatchard analysis of the transmembrane derivatives. We conclude that the fourth cysteine-rich repeat is not required for TNF binding. In contrast, both the soluble and the transmembrane derivatives lacking any one of the first, second or third repeats failed to bind TNF. Although we cannot entirely exclude the possibility that this may be due to indirect conformational change, rather than the removal of essential epitopes, our results suggest that the first three repeats are each required for TNF binding by both the soluble and the cell-surface receptor.

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Tumor necrosis factor (TNF) mediates its pleiotropic effects via high-affinity cell surface receptors. In man, molecular cloning has identified two distinct, independent TNF receptors (TNFR) of 55 and 75 kDa. It is unclear, however, whether the multiple effects of TNF are suggested between the receptor types. In the mouse, previous studies had shown functional heterogeneity of TNFR, since the WEHI 164 fibroblast line is sensitive to the cytotoxic effects of both murine and human TNF, whereas the murine T cell line, CT6, proliferates in response to murine but not human TNF. In this study, the cloning of a cDNA encoding the murine homologue of the p55 TNFR is reported. This receptor binds murine and human TNF with equal affinity and is expressed on WEHI 164 and a number of other cell lines, but only low levels of mRNA and no protein is detectable on CT6 cells. CT6 cells, however, express a second TNFR of approximately 75 kDa, identified by cross-linking analysis, which is also found on WEHI 164 cells, and binds only murine TNF. These studies establish that there are also two TNFR in the mouse, and suggests that there may be segregation of the cytotoxic and proliferative responses between different receptors, at least in these cell lines.

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In both thyroid autoimmune diseases Graves' and Hashimoto's thyroiditis, the epithelial thyroid follicular cells (TFC) have been shown to express HLA class II molecules, and can restimulate autoreactive T cells cloned from the diseased tissue. This aberrant class II expression is important in the mechanism of perpetuation of the disease process, therefore we have compared the effect of interferon gamma (IFN gamma) and tumour necrosis factor (TNF alpha) on the HLA-DR alpha mRNA expression of thyroid follicular cells derived from Graves' disease (GD) and a non autoimmune disease, non toxic goitre (NTG). Our results indicate that TNF alpha synergises with IFN gamma in the induction of HLA class II mRNA. There was no consistent difference in DR alpha mRNA expression between the GD and NTG thyroid follicular cell preparations in response to induction by a combination of these lymphokines at various concentrations. Our data suggest that the differences in the level of expression of class II molecules observed in vivo in Graves' disease and non toxic goitre, which is much higher in the former, is probably due to local release of lymphokines by infiltrating T lymphocytes, although other factors may be involved.

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The expression of HLA class II mRNA was investigated in the joints of patients with active rheumatoid arthritis (RA) in order to evaluate patterns of synthesis. Northern hybridization analysis showed that HLA class II gene transcripts in RA joints were of the correct sizes, and subsequent analyses were performed by slot blotting. All active RA samples expressed high levels of HLA-DR, DP, and DQ mRNA with DP and DQ less than DR. Synovial fluid or membrane cells, chiefly a mixture of T cells and macrophages, were placed in culture, in the absence of any stimulation. The levels of mRNA remained at a high level in vitro. The half of HLA-DR mRNA in joint cells was very brief (approximately 30 min), indicating that prolonged synthesis was due to restimulation of the cells. The effect of lymphokines on HLA class II regulation on joint cell was assessed. Gamma interferon was capable of augmenting HLA-DR to some extent, but paradoxically interleukin 2 at concentrations optimal for stimulating T cells, diminished HLA-DR expression.

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HLA class II expression is notable in rheumatoid arthritis. We have investigated the mechanism of HLA class II regulation in the joints and found local synthesis, as judged by mRNA levels to be high. The role of antigen presentation in maintaining class II mRNA was explored, and blocking presentation by using monoclonal antibodies to HLA class II inhibited synthesis of mRNA for HLA-DR alpha chain. HLA class II expression is maintained by cytokines and so cytokine production in rheumatoid joints was investigated. It was chosen to use mRNA analysis by slot blotting as a screening assay, and the expression of many cytokines was detected. Levels of these were maintained in culture in the absence of extrinsic stimulation.

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A cDNA clone encoding the alpha chain of the human T-cell antigen receptor was isolated by screening a library from the human T-cell line Jurkat with a mouse alpha-chain cDNA clone. This human alpha-chain clone, together with a human antigen receptor beta-chain cDNA clone, was used to determine the stage of T-cell development at which antigen receptor mRNAs first appear. Blot-hybridization analysis of mRNA isolated from a panel of human thymic tumor lines clearly demonstrated that beta-chain transcripts could be detected in all T-lineage cells. However, alpha-chain transcripts were only found in the most phenotypically mature lines, which express the antigen receptor-associated molecule T3. Furthermore, beta-chain transcripts were abundant in RNA prepared from purified T3-negative thymocytes, whereas alpha-chain transcripts were virtually absent. From these results we conclude that alpha-chain expression occurs later in thymic ontogeny than that of the beta chain and propose that it controls surface expression of the antigen receptor-T3 complex.

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A number of cDNA clones homologous to the human T-cell receptor beta-chain gene have been isolated from a library constructed from the human leukaemic cell line Jurkat. The nucleotide sequences of two of these clones demonstrate that Jurkat synthesizes an RNA, probably derived from an aberrantly rearranged C beta 1 gene, which cannot encode a functional beta-chain. This transcript contains a novel joining region and similar transcripts appear to be relatively abundant in several T-cell lines.

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The cultured human B lymphoblastoid cell line Maja synthesises two forms of the gamma heavy chain of immunoglobulin G (IgG) that differ in apparent molecular weight. The lower-molecular-weight form is secreted into the culture medium as a water-soluble product in association with light chains and comigrates on dodecyl sulphate polyacrylamide gels with serum IgG gamma chains. The higher-molecular-weight form is not detected in culture supernatants. In distinction to the secreted form, the higher-molecular-weight form is labelled by a lipophilic, photoactivatable nitrene and is inserted asymmetrically in a transmembrane orientation into rough microsomes. It is concluded that Maja cells synthesise secretory (gamma s) and membrane-associated (gamma m) forms of IgG heavy chains. Both forms of the gamma heavy chain are glycosylated, and can contain one or two asparagine-linked glycan units. The gamma m and gamma s heavy chains differ by about 10 000 in apparent molecular weight. This difference resides exclusively in the polypeptide moiety. Although part of the difference comprises a transmembrane peptide and a cytoplasmic tail of apparent molecular weight about 2000 for gamma m chains, a substantial segment of unique peptide is most probably present on the non-cytoplasmic side of the bilayer. The ionophore monensin inhibits the intracellular transport of gamma s and gamma m chains at a stage when they are sensitive to the enzyme endo-beta-N-acetylglucosaminidase H. In contrast, HLA-A and HLA-B antigens reach a stage at which they are insensitive to this enzyme in the presence of monensin, although their surface expression is inhibited by the ionophore. The implications of these results for the intracellular transport of membrane-associated glycoproteins are discussed.

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The biosynthesis of the HLA-DR antigens was studied in the B lymphoblastoid cell line BRI 8. Three chains, of molecular weights 33,000 (alpha), 31,000 (p31), and 26,000 (beta) were detected intracellularly in pulse-labeled cells by immunoprecipitation with anti-(HLA-DR) sera. The alpha and beta chains were inserted asymmetrically into the rough endoplasmic reticulum as transmembrane polypeptides with the majority of the polypeptide chains oriented in the lumen. At this stage, both chains carried "high mannose" oligosaccharide units which were processed to the complex form during subsequent intracellular transport to the cell surface. The Mr = 31,000 polypeptide was also glycosylated but was structurally distinct from the alpha chain and was probably oriented differently in the lipid bilayer, with a much greater proportion of its polypeptide chain exposed in the cytoplasm. It ws not, therefore, a precursor of the alpha chain. The mature HLA-DR antigens at the plasma membrane comprised polypeptides of Mr = 34,000 and 28,000. These chains corresponded to the processed alpha and beta chains. Although the Mr = 31,000 component was only detected intracellularly, it was not ruled out that some or all of it may have been processed and exposed on the cell surface with an apparent molecular weight indistinguishable from that of the alpha chain.

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The biosynthesis of the HLA-A and -B antigens was studied in the B lymphoblastoid cell line BRI 8 and the Burkitt lymphoma line DAudi. The heavy chains of the HLA-A and -B antigens were inserted asymmetrically into the rough endoplasmic reticulum of BRI 8 cells as transmembrane polypeptides. During the first 5 min after synthesis, the majority of the heavy chains became associated with beta 2-microglobulin. At this stage, the heavy chains carried a high mannose (core) oligosaccharide. Subsequent processing of the oligosaccharide unit occurred during the next 20 to 25 min, resulting in the conversion of the oligosaccharide from the high mannose to the complex form. About 30 to 40 min after synthesis, the mature antigens were expressed at the cell surface. Glycosylation was not required for asymmetric insertion, intracellular transport, or surface expression of the HLA-A and -B antigens. However, studies with Daudi cells indicated that combination with beta 2-microglobulin was necessary for subsequent processing and intracellular transport of the heavy chains after their synthesis in the rough endoplasmic reticulum.

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Microsomal metabolites of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one (Structure I) were separated by high-pressure liquid chromatography, and their structures were established on the basis of their ultraviolet and mass spectra, together with considerations of their general chemical properties. This was assisted by comparisons with metabolites formed in the same way from the synthetic 15-hydroxy (Structure III), 16-hydroxy (Structure II), and 11-hydroxymethyl (Structure IV) derivatives, which themselves occur as metabolites of Structural I. Products derived from attack at the two benzo-ring double bonds occurred, but no K-region products were found. Only metabolites having a non-bay region 3,4-dihydrodiol system were mutagenic and bound to DNA after in vitro microsomal activation, and it was concluded that the 3,4-dihydro-3,4-diol (Metabolite e) was the main form and that the 3,4-diols of the monools (Structure II to IV) were minor proximate forms of this carcinogen. In a two-stage experiment, the synthetic 16-ol (Structure II) was shown to be almost as carcinogenic as was Structure I itself in mice; the 15-ol (Structure III) and 11-hydroxymethyl derivative (Structure IV) were much less active. The same order was also observed in the mutagenicity of these compounds in the Ames test.

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After metabolic activation the carcinogen 15,16-dihydro-11-[3H]methylcyclopenta[a]phenanthren-17-one binds to DNA in vitro, and this binding is prevented by 7,8-benzoflavone. Radioactivity cannot be removed from the DNA with organic solvents or by chromatography on Sephadex G-50, even after heat denaturation of the DNA. Enzymatic hydrolysis yields radioactive fractions, which elute from a column of Sephadex LH-20 immediately after the natural nucleosides. At least two species of reactive metabolites are involved in this bending, those with a half-life of a few hr and others with greater stability. After extraction from the aqueous incubation mixture, they could be detected in discrete polar fractions from separations of the complex metabolite mixture by high-pressure liquid chromatography. Their ability to bind to DNA decreased with time at ambient temperature, and they were rapidly deactivated by acid. 7,8-Benzolflavone acted by suppressing the formation of polar metabolites derived from enzymatic oxidation of the aromatic double bonds. The inhibitor had no effect on the enzymes hydroxylating saturated carbon; hence it is unlikely that metabolism of the methyl group is important in conversion of this carcinogen to its proximate form, although the presence of the 11-methyl group is essential for carcinogenic activity in this series.

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Fifty-four polycyclic compounds, 29 of the cyclopenta[a]phenanthrene series, 11 chrysenes, and 14 benz[a]anthracenes, have been tested for mutagenicity by Ames's method, using Salmonella typhimurium TA100. Without exception all 37 carcinogens and a known initiator were mutagens. Of the 16 noncarcinogens 7 were mutagenic, but none of these has yet been tested for initiating, as opposed to carcinogenic, activity. There appeared to be little quantitative correspondence between carcinogenic and mutagenic potency, however, and possible reasons for this are discussed. The aryl hydrocarbon hydroxylase inhibitor 7,8-benzoflavone strongly inhibited the mutagenicity of certain compounds when it was added to the incubations.

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