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.

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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Two soluble tumour-necrosis-factor-a(TNF)-binding proteins are derived from the extracellular domains of the pS.5 and p7.5 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 p>. 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-growthfactor 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 p.55 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 SDSPAGE 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 BIAcorem Biosensor, by measurement of their ability to inhibit TNF cytotoxicity on WEHI cells, and '251-TNF binding to U937 cells. 44, 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 Tumour-necrosis factor a (TNF), a pleiotropic cytokine produced primarily by mononuclear phagocytes, plays a pivotal role in a wide variety of immune and inflammatory responses. These include septic shock, cachexia [l], cerebral malaria [2] and rheumatoid arthritis (RA) [3, 41. TNF exerts its biological effects by interaction with high-affinity cell surface receptors, which also bind the structurally related cytokine, lymphotoxin (LT) [ 5 ] . Two distinct human TNF receptors (TNF-R) have been cloned and characterized, each of which binds TNF and LT. These are a 5.5-kDa species designated p.55 TNF-R [6-8] and a 75-kDa species designated p7.5 TNF-R [9]. The ligand-binding region resides in the ex-

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.