Nicholas Manolios

Pairwise, cooperative and inhibitory interactions describe the assembly and probable structure of the T-cell antigen receptor.

The T‐cell antigen receptor (TCR) is a multi‐subunit complex consisting of clonotypic heterodimers (TCR‐alpha beta or TCR‐gamma delta) that are non‐covalently linked to at least four invariant chains (CD3‐delta, ‐epsilon, ‐gamma; and zeta or eta). The ordered process of assembly and the final number of individual chains that comprise the TCR is unclear. In this study, we examined the molecular basis of subunit interactions and the component requirements leading to the formation of a complete TCR. Analysis of transient cotransfections in monkey kidney fibroblasts (COS cells) showed assembly between selective chain pairs. Multiple chain cotransfections demonstrated the formation of stable higher order partial complexes. Assembly of such subcomplexes was facilitated by cooperative interactions between clonotypic and invariant CD3 chains. When zeta was cotransfected with any TCR component, no pairwise interaction was detected. Only when there was coexpression of all of the other TCR chains (TCR‐alpha, ‐beta, CD3‐ epsilon, ‐gamma, ‐delta) did zeta assemble with the TCR complex. Not all chain pairs formed stable heterodimers. For one such pair, lack of assembly is due to the inhibitory effects of negatively charged residues within their transmembrane domains. The combined effects of these interactions probably determine the assembly and the quaternary structure of the TCR complex.

Transmembrane helical interactions: zeta chain dimerization and functional association with the T cell antigen receptor.

Members of the zeta family of receptor subunits (zeta, eta and gamma) are structurally related proteins found as components of the T cell antigen receptor (TCR) and certain Fc receptors. These proteins share the ability to form disulfide‐linked dimers with themselves and with other members of the family. Comparison of the amino acid sequences of zeta and gamma reveals a significant degree of homology, which is highest within their membrane‐spanning domains. Analysis of their transmembrane sequences on a helical wheel projection suggests that all of the identical amino acids are clustered on one face of a potential alpha‐helix. This face contains the only cysteine residue within zeta, suggesting that this conserved region may function to mediate dimerization. Indeed, replacing the transmembrane domain of the Tac antigen (alpha chain of the interleukin‐2 receptor) by that of the zeta chain resulted in the formation of disulfide‐linked dimers of Tac. The conserved aspartic acid residue found in the zeta and gamma transmembrane sequences was found to play a role in disulfide linkage. Replacing the aspartic acid with a lysine but not with an alanine or valine residue allowed formation of disulfide‐linked dimers. The ability of the aspartic acid residue to support dimerization was dependent upon its position within the helix. Thus, these observations indicate that residues within the zeta transmembrane domain play a critical role in the formation of disulfide‐linked dimers. Expression of zeta mutants in zeta‐deficient T cells revealed that the zeta transmembrane domain is also responsible for reconstituting transport of functional TCR complexes to the cell surface and differentiated the requirements for disulfide‐linked dimerization per se from assembly of the TCR complex.

X-Chromosome Inactivation in Monozygotic Twins with Systemic Lupus Erythematosus

The hypothesis that a low concordance rate in monozygotic (MZ) twins with systemic lupus erythematosus (SLE) may be accounted for by differences in X-chromosome inactivation was examined. Five MZ twin pairs, four discordant and one concordant, were recruited, zygosity confirmed by DNA fingerprinting, and their pattern of X-chromosome inactivation in DNA samples prepared from peripheral blood and buccal cells were examined. X-chromosome inactivation was assessed by the methylation status of the CpG region near trinucleotide repeats in exon 1 of the androgen receptor gene on X-chromosome after digestion with the methylation-sensitive enzyme HpaII or HhaI and PCR amplification. X-chromosome inactivation patterns were found to be the same between affected and non-affected twins in all four discordant twin pairs, with random patterns in two pairs and skewed patterns in the others. The concordant twins demonstrated the same random patterns. X-chromosome inactivation was also examined from buccal smear DNA and shown to have the same pattern as that noted from peripheral blood DNA in one informative twin pair. Differences in X-chromosome inactivation patterns were not observed in these five MZ twin pairs. The results could not support the hypothesis that differences in X-chromosome inactivation is the mechanism accounting for the low concordance rate noted in MZ twins with SLE.

High endothelial venule morphology and function are inducible in germ-free mice: A possible role for interferon-γ

Cytokines may facilitate lymphocyte traffic by modulating HEV structure and lymphocyte binding function in accordance with local tissue requirements. This study investigated whether the morphology of HEVs and their lymphocyte binding ligand are altered following antigenic challenge and evaluated the role of IFN-gamma in the induction of such changes. The morphology and lymphocyte binding function of mesenteric LN HEVs of GFM exposed to environmental pathogens were compared to those from GFM and conventional mice. Lymph nodes from all mice had microscopically identifiable HEVs. The morphology of HEVs from GFM was not uniform; many HEVs contained flat endothelial cells with sparse cytoplasm and prominent interendothelial gaps. The number of lymphocytes within the lumen and the HEV wall was low. In contrast, HEVs from GFME and conventional mice were characterized by cuboidal endothelial cells with plentiful cytoplasm and large numbers of lymphocytes in the vessel wall and lumen. There was no delineation of interendothelial cell borders. Lymphocyte binding to HEVs of lymph node sections from GFM was reduced (mean +/- SEM: 1.08 +/- 0.15) compared to that of conventional mice (1.91 +/- 0.20), P less than 0.003. GFME had augmented lymphocyte binding (2.23 +/- 0.26) to levels comparable with those of conventional mice. GFM injected intraperitoneally with IFN-gamma, IFN-alpha beta, or diluent resulted in minor changes in HEV morphology. By contrast, lymphocyte binding to HEV of GFM was more than doubled by the injection of IFN-gamma (1.95 +/- 0.25), P less than 0.01, but not IFN-alpha beta (0.54 +/- 0.07) or the relevant diluent controls (0.89 +/- 0.11, 0.56 +/- 0.06, respectively). It appears that the HEV binding ligand is inducible, and its expression is regulated by at least one immunomodulator, IFN-gamma. Although short-term exposure of HEVs to IFN-gamma influenced HEV function it caused only minor changes in morphology.

Renal disease and rheumatic manifestations.

Renal disease has long been recognized as a cause of rheumatic complaints. With the advent of renal replacement therapy, whether dialysis or renal transplantation, the older problems have been supplanted by a new generation of disorders, affecting bone, joints, and soft tissues. This review includes a discussion of recent surveys identifying a high prevalence both of ligamentous laxity in transplant patients and dialysis-associated arthropathy in long-term dialysis patients. Also discussed are studies investigating the natural history of early posttransplant avascular necrosis.

Aberrant Lymphocyte Migration Patterns in Systemic Lupus Erythematosus (MRL/l, MRL/n) Mice are Independent of the Micro-Environment

Mice with systemic lupus erythematosus (SLE) have unusual patterns of lymphocyte traffic characterised by diminished uptake of intravenously injected autoimmune cells into lymph nodes. This study examines the influence of the lymphocyte micro-environment on this aberrant migratory behaviour. To evaluate lymph node lymphocyte-endothelial interactions which can affect lymphocyte distribution without the in vivo influence of liver and spleen, the in vitro high endothelial venule (HEV) binding assay was used. Lymph node HEV binding of autoimmune MRL-lpr/lpr (MRL/l) and MRL(-)+/+ (MRL/n) lymphocytes was increased when compared with CBA/T6 lymphocytes and contrasted with diminished lymph node uptake noted in vivo. This was independent of the lymph node source (MRL/l, MRL/n, CBA/T6) onto which the lymphocytes were overlaid. To examine the influence of the microenvironment on in vivo traffic, 21Cr-labelled lymph node cells from normal CBA/T6 mice were injected into sex-matched MRL/l, MRL/n and CBA/T6 recipients. The distribution of cells was the same in each recipient strain suggesting that the micro-environment had little influence on the lymphocyte trafficking profiles of autoimmune mice. This study supports the view that aberrant lymphocyte migration in autoimmune mice results from defects intrinsic to the lymphocyte population and not the micro-environment.

Arachidonic acid metabolites in normal and autoimmune mice do not influence lymphocyte-high endothelial venule interactions

In peripheral lymphoid organs the number of lymphocytes and the proportion of functional lymphocyte subsets are regulated by multiple factors including the control of lymphocyte migration by selective lymphocyte‐high endothelial venule (HEV) interactions. In this study, prostaglandin E2 (PGE2) levels from normal and autoimmune mouse lymph node cells were measured. The contribution of eicosanoids to lymphocyte‐HEV interactions in normal (CBA/T6) and autoimmune (MRL/n) mice was examined. There was no association between PGE2 production in normal or autoimmune mice and the age of onset of disease activity in the latter strains. Arachidonic acid metabolites, in particular PGE2 and leukotriene B4 (LTB4), did not have any effects on lymphocyte‐HEV binding. Likewise, lymphocytes treated in vivo and/or in vitro with arachidonic acid metabolite inhibitors (acetyl salicylic acid, indomethacin, BW755C) did not alter lymphocyte‐HEV binding interactions in both normal and autoimmune mice. No clinical significance could be attributed to lymph node PGE2 production and the age of onset of autoimmune disease. In summary, these findings cast doubt on the role of arachidonic acid metabolites in lymphocyte‐HEV binding interactions.