The improved characterisation of risk factors for rheumatoid arthritis (RA) suggests they could be combined to identify individuals at increased disease risks in whom preventive strategies may be evaluated. We aimed to develop an RA prediction model capable of generating clinically relevant predictive data and to determine if it better predicted younger onset RA (YORA). Our novel modelling approach combined odds ratios for 15 four-digit/10 two-digit HLA-DRB1 alleles, 31 single nucleotide polymorphisms (SNPs) and ever-smoking status in males to determine risk using computer simulation and confidence interval based risk categorisation. Only males were evaluated in our models incorporating smoking as ever-smoking is a significant risk factor for RA in men but not women. We developed multiple models to evaluate each risk factor's impact on prediction. Each model's ability to discriminate anti-citrullinated protein antibody (ACPA)-positive RA from controls was evaluated in two cohorts: Wellcome Trust Case Control Consortium (WTCCC: 1,516 cases; 1,647 controls); UK RA Genetics Group Consortium (UKRAGG: 2,623 cases; 1,500 controls). HLA and smoking provided strongest prediction with good discrimination evidenced by an HLA-smoking model area under the curve (AUC) value of 0.813 in both WTCCC and UKRAGG. SNPs provided minimal prediction (AUC 0.660 WTCCC/0.617 UKRAGG). Whilst high individual risks were identified, with some cases having estimated lifetime risks of 86%, only a minority overall had substantially increased odds for RA. High risks from the HLA model were associated with YORA (P<0.0001); ever-smoking associated with older onset disease. This latter finding suggests smoking's impact on RA risk manifests later in life. Our modelling demonstrates that combining risk factors provides clinically informative RA prediction; additionally HLA and smoking status can be used to predict the risk of younger and older onset RA, respectively.
Rheumatoid arthritis (RA) is a common, incurable disease with major individual and health service costs. Preventing its development is therefore an important goal. Being able to predict who will develop RA would allow researchers to look at ways to prevent it. Many factors have been found that increase someone's risk of RA. These are divided into genetic and environmental (such as smoking) factors. The risk of RA associated with each factor has previously been reported. Here, we demonstrate a method that combines these risk factors in a process called “prediction modelling” to estimate someone's lifetime risk of RA. We show that firstly, our prediction models can identify people with very high-risks of RA and secondly, they can be used to identify people at risk of developing RA at a younger age. Although these findings are an important first step towards preventing RA, as only a minority of people tested had substantially increased disease risks our models could not be used to screen the general population. Instead they need testing in people already at risk of RA such as relatives of affected patients. In this context they could identify enough numbers of high-risk people to allow preventive methods to be evaluated.
Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.
The Infectious Diseases BioBank (IDB) has consistently archived peripheral blood mononuclear cell (PBMNC) RNA for transcriptome analyses. RNA is particularly labile, and hence, these samples provide a sensitive indicator for assessing the IDB's quality-assurance measures. Independent analyses of 104 PBMNC RNA specimens from 26 volunteers revealed that the mean RNA integrity number (RIN) was high (9.02), although RIN ranged between scores of 7 and 10. This variation of RIN values was not associated with ischemic time, PBMNC quality, number of samples processed per day, self-medication after immunization, freezer location, donor characteristics, differential white blood cell counts, or daily variation in RNA extractions (all P>0.05). RIN values were related to the date of collection, with those processed during mid-summer having highest RIN scores (P=0.0001). Amongst specimens with the lowest RIN scores, no common feature could be identified. Thus, no technical explanation for the variation in RNA quality could be ascertained and these may represent normal physiological variations. These data provide strong evidence that current IDB protocols for the isolation and preservation PBMNC RNA are robust.
A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair.
Inhibitors of kappa B (IκBs) -α, -β and -ε effect selective regulation of specific nuclear factor of kappa B (NF-κB) dimers according to cell lineage, differentiation state or stimulus, in a manner that is not yet precisely defined. Lymphocyte antigen receptor ligation leads to degradation of all three IκBs but activation only of subsets of NF-κB-dependent genes, including those regulated by c-Rel, such as anti-apoptotic CD40 and BAFF-R on B cells, and interleukin-2 (IL-2) in T cells. We report that pre-culture of a mouse T cell line with tumour necrosis factor-α (TNF) inhibits IL-2 gene expression at the level of transcription through suppressive effects on NF-κB, AP-1 and NFAT transcription factor expression and function. Selective upregulation of IκBε and suppressed nuclear translocation of c-Rel were very marked in TNF-treated, compared to control cells, whether activated via T cell receptor (TCR) pathway or TNF receptor. IκBε associated with newly synthesised c-Rel in activated cells and, in contrast to IκBα and -β, showed enhanced association with p65/c-Rel in TNF-treated cells relative to controls. Studies in IκBε-deficient mice revealed that basal nuclear expression and nuclear translocation of c-Rel at early time-points of receptor ligation were higher in IκBε−/− T and B cells, compared to wild-type. IκBε−/− mice exhibited increased lymph node cellularity and enhanced basal thymidine incorporation by lymphoid cells ex vivo. IκBε−/− T cell blasts were primed for IL-2 expression, relative to wild-type. IκBε−/− splenic B cells showed enhanced survival ex vivo, compared to wild-type, and survival correlated with basal expression of CD40 and induced expression of CD40 and BAFF-R. Enhanced basal nuclear translocation of c-Rel, and upregulation of BAFF-R and CD40 occurred despite increased IκBα expression in IκBε−/− B cells. The data imply that regulation of these c-Rel-dependent lymphoid responses is a non-redundant function of IκBε.
The tumour necrosis factor (TNF) receptor‐associated factor (TRAF) family is important in activating multiple inflammatory and immune related processes induced by cytokines such as TNFα and interleukin‐1. These genes therefore represent strong candidate susceptibility factors for rheumatoid arthritis (RA). A study was undertaken to investigate the association between single nucleotide polymorphisms (SNPs) spanning six TRAF genes and RA in a British population.
Twenty‐three haplotype tagging (ht) SNPs and 26 random SNPs spanning the six TRAF genes were initially tested for association in a cohort of 351 unrelated patients with RA and 368 controls. Any SNPs demonstrating an association were genotyped in further samples. Sequenom MassARRAY technology was preferentially used for genotyping. Both single point and haplotypic analyses were performed.
Forty‐four SNPs were successfully genotyped and conformed to Hardy‐Weinberg expectation. A single SNP, rs7514863, mapping upstream of the TRAF5 gene and affecting a putative transcription factor binding site, demonstrated a significant association across the entire cohort of 1273 cases with RA compared with 2463 healthy controls (OR for minor T allele 1.2 (95% CI 1.06 to 1.36), p = 0.005). The association was stronger in the subgroup carrying at least one copy of the shared epitope alleles (OR 1.43 (95% CI 1.18 to 1.73), p = 0.0003).
These findings provide evidence for the association of an SNP upstream of a strong candidate RA susceptibility gene, TRAF5, in a large cohort of patients and controls. Further association and functional studies are required to investigate the role of this variant, or one in linkage disequilibrium with it, in RA disease causation.
Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.
Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.
We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.
Over the past decade and a half, advances in our understanding of the pathogenesis of immune-mediated diseases such as rheumatoid arthritis (RA) have translated directly into benefit for patients. Much of this benefit has arisen through the introduction of targeted biological therapies. At the same time, technological advances have made it possible to define, at the cellular and molecular levels, the key pathways that influence the initiation and persistence of chronic inflammatory autoimmune reactions. As our understanding grows, it is likely that this knowledge will be translated into a second generation of biological therapies that are tailor-made for the patient. This review summarizes current perspectives on RA disease pathogenesis, with particular emphasis on what RA T cells look like, what they are likely to see, and how they contribute to persistence of the chronic inflammatory response.
Self-reactive T cells with low signalling capacity through the T-cell receptor were recently observed in the SKG mouse model of rheumatoid arthritis (RA) and have been linked to a spontaneous mutation in the ZAP-70 signal transduction molecule. Here we hypothesize that similar mechanisms also drive RA, associated with an abnormal innate and adaptive immune response driven by nuclear factor-κB activation and tumour necrosis factor secretion. Similar to the essential role played by pathogens in SKG mice, we propose that HLA-associated immunity to chronic viral infection is a key factor in the immune dysregulation and joint inflammation that characterize RA.
The T-cell receptor ζ (TCRζ) chain is a master sensor and regulator of lymphocyte responses. Loss of TCRζ expression has been documented in infectious, inflammatory, and malignant diseases, suggesting that it may serve to limit T-cell reactivity and effector responses at sites of tissue damage. These observations prompted us to explore the relationship between TCRζ expression and effector function in T cells. We report here that TCRζdim lymphocytes are enriched for antigen-experienced cells refractory to TCR-induced proliferation. Compared to their TCRζbright counterparts, TCRζdim cells share characteristics of differentiated effector T cells but use accessory pathways for transducing signals for inflammatory cytokine gene expression and cell contact-dependent pathways to activate monocytes. TCRζdim T cells accumulate in inflamed tissues in vivo and have intrinsic migratory activity in vitro. Whilst blocking leukocyte trafficking with anti-TNF therapy in vivo is associated with the accumulation of TCRζdim T cells in peripheral blood, this T-cell subset retains the capacity to migrate in vitro. Taken together, the functional properties of TCRζdim T cells make them promising cellular targets for the treatment of chronic inflammatory disease.
Cell therapy, pioneered for the treatment of malignancies in the form of bone marrow transplantation, has subsequently been tested and successfully employed in autoimmune diseases. Autologous haemopoietic stem cell transplantation (HSCT) has become a curative option for conditions with very poor prognosis such as severe forms of scleroderma, multiple sclerosis, and lupus, in which targeted therapies have little or no effect. The refinement of the conditioning regimens has virtually eliminated transplant-related mortality, thus making HSCT a relatively safe choice. Although HSCT remains a nonspecific approach, the knowledge gained in this field has led to the identification of new avenues. In fact, it has become evident that the therapeutic efficacy of HSCT cannot merely be the consequence of a high-dose immuno-suppression, but rather the result of a resetting of the abnormal immune regulation underlying autoimmune conditions. The identification of professional and nonprofessional immunosuppressive cells and their biological properties is generating a huge interest for their clinical exploitation. Regulatory T cells, found abnormal in several autoimmune diseases, have been proposed as central to achieve long-term remissions. Mesenchymal stem cells of bone marrow origin have more recently been shown not only to be able to differentiate into multiple tissues, but also to exert a potent antiproliferative effect that results in the inhibition of immune responses and prolonged survival of haemopoietic stem cells. All of these potential resources clearly need to be investigated at the preclinical level but support a great deal of enthusiasm for cell therapy of autoimmune diseases.
Multipotent mesenchymal stromal cells isolated from bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/or management of autoimmune diseases. In vitro studies have shown that they exhibit a dose-dependent antiproliferative effect on T and B lymphocytes, dendritic cells, natural killer cells and various B cell tumour lines – an effect that is both cell contact and soluble factor dependent. Animal models of autoimmune disease treated with multipotent mesenchymal stromal cells have mostly exhibited a positive clinical response, as have a limited number of patients suffering from acute graft versus host disease. This review summarizes the findings of a 1-day meeting devoted to the subject with the aim of coordinating efforts.
Cope discusses a new study in rats suggesting that oxidative burst inducers might have a role to play in treating inflammatory arthritis.
The development of spontaneous autoimmunity in inbred strains of rodents has allowed us to investigate the molecular basis of chronic inflammatory disease in ways that would not be possible in humans. Recently, two new mouse models of autoimmune inflammatory polyarthritis have been reported that demonstrate how alterations in signalling thresholds sufficient to perturb central T-cell tolerance lead to inflammatory arthritis. These mice provide new insights into the complexities of what may turn out to be a heterogeneous group of diseases that we call rheumatoid arthritis. They will also provide unique tools for dissecting precisely how chronically activated T cells contribute to the effector phase of arthritis through mechanisms that may be less dependent on antigen receptor signalling.
autoimmune arthritis; signalling; T cells; thymic selection
The European Workshop for Rheumatology Research met this year in Leiden, The Netherlands. The Workshop provided a platform to feast on new technologies and how they have taken research programmes forward. While there will be the inevitable delay during which mechanisms are devised for analysing the huge amount of information generated by these technologies, there is a lot already to look forward to. Highlights included genomic, reverse genomic and proteomic approaches to understanding disease pathogenesis and to identifying new therapeutic targets. Opportunities for exploring whether pharmacogenomics has a place in the clinic are now a reality, and phage display technology has been applied to in vivo arthritis models to identify human synovial microvascular 'post codes'.
diagnostics; inflammation; prognostics; research; workshop
The strong association between specific alleles encoded within the MHC class II region and the development of rheumatoid arthritis (RA) has provided the best evidence to date that CD4+ T cells play a role in the pathogenesis of this chronic inflammatory disease. However, the unusual phenotype of synovial T cells, including their profound proliferative hyporesponsiveness to TCR ligation, has challenged the notion that T-cell effector responses are driven by cognate cartilage antigens in inflamed synovial joints. The hierarchy of T-cell dysfunction from peripheral blood to inflamed joint suggests that these defects are acquired through prolonged exposure to proinflammatory cytokines such as tumour necrosis factor (TNF)-α. Indeed, there are now compelling data to suggest that chronic cytokine activation may contribute substantially to the phenotype and effector function of synovial T cells. Studies reveal that chronic exposure of T cells to TNF uncouples TCR signal transduction pathways by impairing the assembly and stability of the TCR/CD3 complex at the cell surface. Despite this membrane-proximal effect, TNF selectively uncouples downstream signalling pathways, as is shown by the dramatic suppression of calcium signalling responses, while Ras/ERK activation is spared. On the basis of these data, it is proposed that T-cell survival and effector responses are driven by antigen-independent, cytokine-dependent mechanisms, and that therapeutic strategies that seek to restore T-cell homeostasis rather than further depress T-cell function should be explored in the future.
inflammation; rheumatoid arthritis; signal transduction; T cells; TNF
Genetic susceptibility to rheumatoid arthritis (RA), a common autoimmune disease, is associated with certain HLA-DR4 alleles. Treatments are rarely curative and are often tied to major side effects. We describe the development of a humanized mouse model wherein new, less toxic, vaccine-like treatments for RA might be pretested. This model includes four separate transgenes: HLA-DR*0401 and human CD4 molecules, a RA-related human autoantigenic protein (HCgp-39), and a T-cell receptor (TCRαβ) transgene specific for an important HCgp-39 epitope, eliciting strong Th1 responses in the context of HLA-DR*0401.
autoimmunity; HCgp-39; HLA-DR4 transgenic mice; rheumatoid arthritis; T-cell receptor transgenic mice
T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-ζ chains and to a lesser extent via the CD3-ε/γ heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca2+ mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.
T lymphocytes; signal transduction; T cell receptor complex; ζ chains; transmembrane adaptor proteins
Repeated injections of adult mice with recombinant murine TNF prolong the survival of NZB/W F1 mice, and suppress type I insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. To determine whether repeated TNF injections suppress T cell function in adult mice, we studied the responses of influenza hemagglutinin-specific T cells derived from T cell receptor (HNT-TCR) transgenic mice. Treatment of adult mice with murine TNF for 3 wk suppressed a broad range of T cell responses, including proliferation and cytokine production. Furthermore, T cell responses of HNT-TCR transgenic mice also expressing the human TNF-globin transgene were markedly reduced compared to HNT-TCR single transgenic littermates, indicating that sustained p55 TNF-R signaling is sufficient to suppress T cell function in vivo. Using a model of chronic TNF exposure in vitro, we demonstrate that (a) chronic TNF effects are dose and time dependent, (b) TNF suppresses the responses of both Th1 and Th2 T helper subsets, (c) the suppressive effects of endogenous TNF produced in T cell cultures could be reversed with neutralizing monoclonal antibodies to TNF, and (d) prolonged TNF exposure attenuates T cell receptor signaling. The finding that anti-TNF treatment in vivo enhances T cell proliferative responses and cytokine production provides evidence for a novel regulatory effect of TNF on T cells in healthy laboratory mice. These effects are more pronounced in chronic inflammatory disease. In addition, our data provide a mechanism through which prolonged TNF exposure suppresses disease in animal models of autoimmunity.
The Study Group for Risk Factors for Rheumatoid Arthritis was established by the EULAR Standing Committee on Investigative Rheumatology to facilitate research into the preclinical and earliest clinically apparent phases of rheumatoid arthritis (RA). This report describes the recommendation for terminology to be used to define specific subgroups during different phases of disease, and defines the priorities for research in this area. Terminology was discussed by way of a three-stage structured process: A provisional list of descriptors for each of the possible phases preceding the diagnosis of RA were circulated to members of the study group for review and feedback. Anonymised comments from the members on this list were fed back to participants before a 2-day meeting. 18 participants met to discuss these data, agree terminologies and prioritise important research questions. The study group recommended that, in prospective studies, individuals without RA are described as having: genetic risk factors for RA; environmental risk factors for RA; systemic autoimmunity associated with RA; symptoms without clinical arthritis; unclassified arthritis; which may be used in a combinatorial manner. It was recommended that the prefix ‘pre-RA with:’ could be used before any/any combination of the five points above but only to describe retrospectively a phase that an individual had progressed through once it was known that they have developed RA. An approach to dating disease onset was recommended. In addition, important areas for research were proposed, including research of other tissues in which an adaptive immune response may be initiated, and the identification of additional risk factors and biomarkers for the development of RA, its progression and the development of extra-articular features. These recommendations provide guidance on approaches to describe phases before the development of RA that will facilitate communication between researchers and comparisons between studies. A number of research questions have been defined, requiring new cohorts to be established and new techniques to be developed to image and collect material from different sites.