Claire L. Gorman

Polymorphisms in the CD3Z Gene Influence TCRζ Expression in Systemic Lupus Erythematosus Patients and Healthy Controls

TCRζ (CD247) functions as an amplification module in the TCR signaling cascade and is essential for assembly and surface expression of the TCR/CD3 complex. The TCRζ-chain is down-regulated in many chronic infectious and inflammatory diseases, including systemic lupus erythematosus (SLE). It is unclear whether reduced TCRζ expression is a cause or a consequence of chronic inflammatory responses. We have addressed this question by adopting a combined genetic and functional approach. We analyzed TCRζ protein expression using a FACS-based expression index and documented considerable, but longitudinally stable, variation in TCRζ expression in healthy individuals. The variation in TCRζ expression was associated with polymorphisms in the CD3Z 3′-untranslated region (UTR) in SLE patients and healthy controls. Detailed mapping of the 3′-UTR revealed that the minor alleles of two single nucleotide polymorphisms (SNPs) in strong disequilibrium (rs1052230 and rs1052231) were the causal variants associated with low TCRζ expression (p = 0.015). Using allelic imbalance analysis, the minor alleles of these 3′-UTR SNPs were associated with one-third of the level of mRNA compared with the major allele. A family-based association analysis showed that the haplotype carrying the low-expression variants predisposes to SLE (p = 0.033). This suggests that a genetically determined reduction in TCRζ expression has functional consequences manifested by systemic autoimmunity.

Nitric oxide production of T lymphocytes is increased in rheumatoid arthritis

Experimental and clinical evidence for T cell involvement in the pathology of rheumatoid arthritis (RA) is compelling, and points to a local dysregulation of T cell function in the inflamed joint. Nitric oxide (NO) has been shown to regulate T cell function under physiological conditions, but overproduction of NO may contribute to lymphocyte dysfunction characteristic of RA. Several investigations in patients with RA have documented evidence of increased NO synthesis, but these studies have focused largely on macrophage-derived NO and its impact on innate immune and inflammatory responses. In this study, we set out to explore the contribution that T cells make to NO production. We find that T cells from RA patients produce >2.5 times more NO than healthy donor T cells (p<0.001). Although NO is an important physiological mediator of mitochondrial biogenesis, mitochondrial mass is similar in RA and control T cells. In contrast, increased NO production is associated with increased cytoplasmic Ca(2+) concentrations in RA T cells (p<0.001). In vitro treatment of human peripheral blood lymphocytes, or Jurkat cells with TNF increases NO production (p=0.006 and p=0.001, respectively), whilst infliximab treatment in RA patients decreases T cell derived NO production within 6 weeks of the first infusion (p=0.005). Together, these data indicate that TNF induced NO production in T lymphocytes may contribute to perturbations of immune homeostasis in RA.

Immune-mediated pathways in chronic inflammatory arthritis

Rheumatoid arthritis (RA) is the prototype chronic inflammatory arthropathy; its precise aetiology is unknown. Over recent years, a number of crucial advances in our understanding of the disease have had a major impact on the treatment of patients with RA.

Tracking antigen-experienced effector T cells in vitro and in vivo.

The TCR complex is a multisubunit complex, comprising at least eight transmembrane units. The clonotypic TCR alpha and beta chains are responsible for antigen recognition, whilst the invariant chains of the CD3 complex (delta, epsilon and gamma) and two zeta (zeta) polypeptides couple antigen recognition to downstream signal transduction pathways. TCRzeta (CD247) functions as an amplification module in the TCR signalling cascade and is also essential for the assembly and surface expression of the TCR/CD3 complex. Loss of TCRzeta expression is common in chronic infectious and inflammatory diseases, as well as in cancer. Previous work has indicated that TCRzeta(low)-expressing cells phenotypically resemble antigen-experienced effector T cells. Here, we describe the derivation of a flow cytometry-based TCRzeta expression index for the purpose of more precisely defining TCRzeta expression, in addition to utilising a simple transmigration assay in the demonstration that TCRzeta(dim) T cells have intrinsic migratory properties that may explain their accumulation at sites of inflammation.

Nitric oxide differentially regulates T-cell function in rheumatoid arthritis and systemic lupus erythematosus.

Experimental and clinical evidence for T-cell involvement in the pathogenesis of rheumatoid arthritis (RA) is compelling, and points to a local dysregulation of T-cell function in the inflamed joint. Nitric oxide (NO) has been shown to regulate T-cell function under physiological conditions, but overproduction of NO may contribute to lymphocyte dysfunction in RA. NO has recently been recognized as a key signaling intermediate for T-cell activation and mitochondrial biogenesis. NO is synthesized from L-arginine by NO synthetases (NOS). Three distinct isoforms of NOS are known, including neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) enzymes. We previously detected the expression of eNOS and nNOS and the absence of iNOS in human PBL, while during inflammation macrophages and monocytes express iNOS. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown origin characterized by the involvement of multiple organs. Several studies carried out on patients with both RA and SLE have documented increased endogenous NO synthesis, but its contribution to T-lymphocyte mitochondrial biogenesis and T-cell dysregulation is not known. We investigated the role of NO in T-cell mitochondrial biogenesis in RA and SLE. The mitochondrial mass, NO production and cytoplasmic Ca2+ levels were measured by flow cytometry. Mitochondria were visualized using transmission electron microscope. T cells from RA patients produce >2.5 times more NO than T cells from healthy donors (P < 0.001). Unexpectedly, the mitochondrial mass was found to be similar in RA and control T cells (P = 0.65), whilst increased NO production was associated with increased cytoplasmic Ca2+ concentrations in RA T cells (P < 0.001). We observed that T-cell NO production decreased in most RA patients following anti-TNF treatment. Although lupus T cells produced comparable amounts of NO to normal T cells, lupus monocytes produced twice as much NO as normal monocytes (P = 0.015). We also observed increased mitochondrial mass (47.7 ± 2.8%; P = 0.00017) and increased cytoplasmic (38 ± 6.4%; P = 0.0023) Ca2+ content in T cells from SLE patients when compared with control donors. Electron microscopy revealed that T cells of lupus patients contained 8.76 ± 1 mitochondria, while control donors contained 3.18 ± 0.28 mitochondria per cell (P = 0.0009). In addition, lupus lymphocytes harbor several-fold enlarged mega-mitochondria. These data suggest that monocytes are the primary source of NO in SLE, while T lymphocytes are the primary source of NO in RA. Although the iNOS pathway is not as rapid as eNOS or nNOS, it is thought to be capable of generating much larger quantities of NO (nanomolar range) than the constitutive NOS isoforms (picomolar range), explaining the differences in T-cell mitochondrial biogenesis in SLE and RA. Since mitochondria can take up, store and release Ca2+, increased mitochondrial mass may account for altered Ca2+ handling in SLE. Furthermore increased NO production may contribute to T-cell dysfunction in both SLE and RA.

Rheumatoid arthritis: a disease of chronic, low-amplitude signals transduced through T cell antigen receptors?

SummaryTechnology has advanced to the stage where it is now possible to identify genes that confer low to moderate risk of developing autoimmune diseases such as rheumatoid arthritis (RA). This has been facilitated by the growing appreciation that these hard to detect genetic signals can only be defined in large cohorts of well characterized patients. In RA, the association between disease susceptibility and genes encoded within the MHC has been known for decades. Recent studies have identified several new candidate genes that provide further insights into the molecular nature of aberrant immune responses in chronic inflammatory diseases. Here, we describe some of these new genes. Based on their known functions we propose that in a subgroup of patients with RA inheritance of allelic variants at distinct loci could lead to dysregulation of adaptive immune responses characterized by chronic, low-amplitude signaling transduced by antigen T cell receptors.ZusammenfassungTechnologische Fortschritte haben es möglich gemacht, Gene, welche mit einem niedrigen bis mäßigem Risiko für die Entwicklung von Autoimmunerkrankungen wie rheumatoide Arthritis (RA) assoziiert sind, zu identifizieren. Dies wurde durch die zunehmende Einsicht ermöglicht, dass diese schwer detektierbaren genetischen Signale nur in großen Kohorten von gut charakterisierten Patienten definiert werden können. Bei der RA ist der Zusammenhang der Krankheitssuszeptibilität und MHC-Genen seit Jahrzehnten bekannt. Aktuelle Studien haben verschiedene neue Kandidatengene, welche weitere Erkenntnisse über die molekulare Natur von gestörten Immun-Antworten bei chronischen entzündlichen Erkrankungen ergeben, identifiziert. In dieser Arbeit beschreiben wir einige dieser neuen Gene. Aufgrund ihrer bekannten Funktionen nehmen wir an, dass in einer Subgruppe von RA-Patienten die Vererbung von allelischen Varianten bei bestimmten Gen-Loci zu einer Dysregulation von adaptiven Immun-Antworten führt, welche durch chronische Signale mit niedriger Amplitude über Antigen T-Zellrezeptoren übertragen werden.

What does the immunogenetic basis of rheumatoid arthritis teach us about the immunobiology of the disease

Rheumatoid arthritis is a chronic inflammatory autoimmune disease in which, although the exact etiology is unknown, the contribution from genetic factors is approximately 60%. major histocompatibility complex alleles make the largest contribution to this genetic effect. The remainder is probably made up of an, as yet undefined, number of genes (∼50–200) with low disease penetrance. Recent advances in genetic technology are now enabling us to start to identify some of these more moderate risk-conferring candidate genes. Evidence from functional studies of such genes is beginning to provide insight into the exact nature of the pathways and processes involved in disease susceptibility and expression. In this review, we will discuss how a growing number of genetic polymorphisms might underpin the immunological and molecular anomalies characteristic of rheumatoid arthritis. Specifically, we will focus on one particular pathway, T-cell activation, with an emphasis on the genetic polymorphism that influences antigen presentation and recognition in antigen-presenting cells, as well as those genes that influence the thresholds of antigen-receptor signaling in T lymphocytes.