The role and clinical implications of G6PI in experimental models of rheumatoid arthritis

The antigens that trigger the pathogenic immune response in rheumatoid arthritis (RA) remain unknown. Until recently it was assumed that either viral or microbial antigens, or joint-specific antigens were the target of arthritogenic T and B lymphocytes in RA. Consequently, murine models of arthritis are induced by immunization with either joint-specific antigens such as type II collagen or microbial products such as streptococcal cell wall. In the K/BxN T-cell receptor transgenic mouse model arthritis is caused by a systemic autoimmune response to the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI). The autoreactive transgenic T cells recognize G6PI and provide help for the production of arthritogenic IgG antibodies against G6PI. More recently it was shown that G6PI immunization induces severe symmetrical peripheral polyarthritis in genetically unaltered DBA/I mice. In that model CD4+ T cells are necessary not only for the induction but also for the effector phase of arthritis. Here we review the pathomechanisms that lead from systemic autoreactivity to arthritis in these models, consider the relevance of anti-G6PI immune reactivity for RA, and discuss the insights into the pathogenesis of RA and possibly other autoimmune conditions that can be gained from these models.     

Immunization with glucose-6-phosphate isomerase induces T cell-dependent peripheral polyarthritis in genetically unaltered mice

Rheumatoid arthritis is a chronic inflammatory disease primarily affecting the joints. The search for arthritogenic autoantigens that trigger autoimmune responses in rheumatoid arthritis has largely focused on cartilage- or joint-specific Ags. In this study, we show that immunization with the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI) induces severe peripheral symmetric polyarthritis in normal mice. In genetically unaltered mice, T cells are indispensable for both the induction and the effector phase of G6PI-induced arthritis. Arthritis is cured by depletion of CD4(+) cells. In contrast, Abs and FcgammaR(+) effector cells are necessary but not sufficient for G6PI-induced arthritis in genetically unaltered mice. Thus, the complex pathogenesis of G6PI-induced arthritis in normal mice differs strongly from the spontaneously occurring arthritis in the transgenic K/B x N model where Abs against G6PI alone suffice to induce the disease. G6PI-induced arthritis demonstrates for the first time the induction of organ-specific disease by systemic autoimmunity in genetically unaltered mice. Both the induction and effector phase of arthritis induced by a systemic autoimmune response can be dissected and preventive and therapeutic strategies evaluated in this model.     

Patients with inflammatory arthritic diseases harbor elevated serum and synovial fluid levels of free and immune-complexed glucose-6-phosphate isomerase (G6PI)

In K/BxN mice, anti-glucose-6-phosphate isomerase (G6PI) IgG antibodies (Abs) cause joint-specific inflammation and destruction. Anti-G6PI Abs are also present in humans with inflammatory arthritis, especially among patients with rheumatoid arthritis (RA). A contributing factor to the induction of such autoantibodies may be upregulated expression of the corresponding antigen G6PI in affected tissues and/or increased levels of G6PI in the circulation. To determine G6PI levels and the presence of free G6PI and/or G6PI-containing immune complexes in sera and synovial fluids (SF) of patients with different arthritides, serum and SF obtained concomitantly from 91 clinically well-defined arthritis patients were assessed in a blinded manner for G6PI enzymatic assay and for G6PI protein concentration by ELISA. Sera and SF from patients with immune-based inflammatory arthritis contained significantly higher levels of G6PI enzymatic activity compared to sera or SF from patients with non-immune-based inflammatory arthritis or healthy controls. In addition, significantly higher levels of total G6PI protein concentration (including both enzymatically active and inactive forms) were present in sera of RA patients vs. those with other immune-based or non-immune-based inflammatory arthritis.G6PI in sera and SF were present both as G6PI-containing immune complexes and as free G6PI, with the majority of free G6PI existing as tetramers with lesser amounts of dimers and monomers. Levels of G6PI enzymatic activity in the sera of most immune-based inflammatory arthritis patients are elevated and may reflect ongoing inflammation and cell destruction. The high serum levels of enzymatically inactive forms of G6PI in RA relative to those in other arthritic diseases are partially due to G6PI-containing immune complexes, a portion of which also contains C1q. Overall, our study supports the notion that elevated G6PI levels present in patients with immune-based inflammatory arthritis may contribute to elevated levels of anti-G6PI Abs and G6PI/anti-G6PI immune complexes. This, in turn, may trigger production of proinflammatory cytokines and perpetuate the inflammatory process.     

Effector T cells in rheumatoid arthritis: lessons from animal models

The development of an immune response to self antigens drives naive T cells to differentiate into subsets of CD8(+) and CD4(+) effector cells including T(H)1, T(H)2, cells and the more recently described T(H)17, and regulatory T cells (T(reg)). Rheumatoid arthritis is an autoimmune disease that engages an uncontrolled influx of inflammatory cells to the joints, eventually leading to joint damage. The role that effector T cells play in the local or systemic maintenance of, or protection against, inflammation and subsequent joint damage is now becoming better understoodthrough the use of animal models. In this review, we will explore the different animal models of RA, and their contribution to elucidating the role that effector T cells play in the regulation, induction, and maintenance of inflammatory joint disease. This understanding will aid in the design of more effective therapeutic strategies for rheumatoid arthritis and other autoimmune disorders.     

Preventing and curing citrulline-induced autoimmune arthritis in a humanized mouse model using a Th2-polarizing iNKT cell agonist

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non-polymorphic CD1d molecules. They are capable of rapidly releasing pro- and/or anti-inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2-polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline-induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen-experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells. Interestingly, administering the Th1-promoting iNKT cell glycolipid ligand α-C-galactosylceramide into HLA-DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline-induced arthritis. Therefore, our findings indicate a role for Th1-mediated immunopathology in citrulline-induced arthritis and provide the first evidence that iNKT cell manipulation by Th2-skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA.     

Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule

Rheumatoid arthritis (RA) is genetically associated with MHC class II molecules that contain the shared epitope. These MHC molecules may participate in disease pathogenesis by selectively binding arthritogenic peptides for presentation to autoreactive CD4(+) T cells. The nature of the arthritogenic Ag is not known, but recent work has identified posttranslationally modified proteins containing citrulline (deiminated arginine) as specific targets of the IgG Ab response in RA patients. To understand how citrulline might evoke an autoimmune reaction, we have studied T cell responses to citrulline-containing peptides in HLA-DRB1*0401 transgenic (DR4-IE tg) mice. In this study, we demonstrate that the conversion of arginine to citrulline at the peptide side-chain position interacting with the shared epitope significantly increases peptide-MHC affinity and leads to the activation CD4(+) T cells in DR4-IE tg mice. These results reveal how DRB1 alleles with the shared epitope could initiate an autoimmune response to citrullinated self-Ags in RA patients.     

Immunization with an immunodominant self-peptide derived from glucose-6-phosphate isomerase induces arthritis in DBA/1 mice

Introduction  

T-helper (Th) lymphocytes are critically required for the pathogenesis of glucose-6-phosphate isomerase (G6PI)-induced arthritis, but neither the G6PI epitopes recognized by arthritogenic T cells nor their pathogenic effector functions have been fully elucidated to date. We aimed at identifying arthritogenic G6PI peptides.     

Dendritic cells and the promise of antigen-specific therapy in rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory disease resulting from an autoimmune response to self-antigens, leading to inflammation of synovial tissue of joints and subsequent cartilage and bone erosion. Current disease-modifying anti-rheumatic drugs and biologic inhibitors of TNF, IL-6, T cells and B cells block inflammation nonspecifically, which may lead to adverse effects, including infection. They do not generally induce long-term drug-free remission or restoration of immune tolerance to self-antigens, and lifelong treatment is usual. The development of antigen-specific strategies in RA has so far been limited by insufficient knowledge of autoantigens, of the autoimmune pathogenesis of RA and of the mechanisms of immune tolerance in man. Effective tolerance-inducing antigen-specific immunotherapeutic strategies hold promise of greater specificity, of lower toxicity and of a longer-term solution for controlling or even preventing RA. This paper reviews current understanding of autoantigens and their relationship to immunopathogenesis of RA, and emerging therapeutics that aim to leverage normal tolerance mechanisms for implementation of antigen-specific therapy in RA.     

Type II collagen-reactive T cell clones from mice with collagen-induced arthritis

Certain strains of mice develop a symmetrical polyarthritis after immunization with type II collagen. The incidence of arthritis after such immunization is variable. To study the arthritogenic potential of T cells reactive with type II collagen, we isolated draining lymph node cells from mice that had developed arthritis after immunization with bovine type II collagen. From these immune lymph node cells we were able to clone T cells reactive with type II collagen. Two separate sets of T cell clones were isolated. The first set reacted with either native bovine or native chick type II collagen, but did not react with type I collagen. The second set of T cell clones reacted with bovine type II collagen, but did not respond to either native chick type II collagen or type I collagen. These clones will be tested for their influence on the development of arthritis in vivo.     

The involvement of Toll‐like receptor 9 in the pathogenesis of erosive autoimmune arthritis

Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll‐like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell‐dependent phase of inflammatory arthritis. In rats with pristane‐induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL‐6, α‐1‐acid‐glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9^?/? mice, streptococcal cell wall (SCW)‐induced arthritis was reduced in the T cell‐dependent phase, whereas T cell‐independent serum‐transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell‐dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis.     

Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis.

Neutrophils are prominent participants in the joint inflammation of human rheumatoid arthritis (RA) patients, but the extent of their role in the inductive phase of joint inflammation is unknown. In the K/BxN mouse RA model, transfer of autoreactive Ig from the K/BxN mouse into mice induces a rapid and profound joint-specific inflammatory response reminiscent of human RA. We observed that after K/BxN serum transfer, the earliest clinical signs of inflammation in the ankle joint correlated with the presence of neutrophils in the synovial regions of recipient mouse ankle joints. In this study, we investigated the role of neutrophils in the early inflammatory response to transferred arthritogenic serum from the K/BxN transgenic mouse. Mice were treated with a neutrophil-depleting mAb before and following transfer of arthritogenic serum and scored for clinical indications of inflammation and severity of swelling in ankle joints and front paws. In the absence of neutrophils, mice were completely resistant to the inflammatory effects of K/BxN serum. Importantly, depletion of neutrophils in diseased recipient mice up to 5 days after serum transfer reversed the inflammatory reaction in the joints. Transfer of serum into mice deficient in the generation of nitrogen or oxygen radicals (inducible NO synthase 2 or gp91(phox) genes, respectively) gave normal inflammatory responses, indicating that neither pathway is essential for disease induction. These studies have identified a critical role for neutrophils in initiating and maintaining inflammatory processes in the joint.     

Induction of a B-cell-dependent chronic arthritis with glucose-6-phosphate isomerase

Antibodies specific for glucose-6-phosphate isomerase (G6PI) from T-cell receptor transgenic K/BxN mice are known to induce arthritis in mice, and immunization of DBA/1 mice with G6PI led to acute arthritis without permanent deformation of their joints. Because rheumatoid arthritis is a chronic disease, we set out to identify the capacity of G6PI to induce chronic arthritis in mice. Immunization with recombinant human G6PI induced a chronically active arthritis in mice with a C3H genomic background, whereas the DBA/1 background allowed only acute arthritis and the C57BL/10 background permitted no or very mild arthritis. The disease was associated with the major histocompatibility region sharing an allelic association similar to that of collagen-induced arthritis (i.e. q > p > r). All strains developed a strong antibody response to G6PI that correlated only in the C3H.NB strain with arthritis severity. Similarly, a weak response to type II collagen in a few mice was observed, which was associated with arthritis in C3H.NB mice. Mice on the C3H background also developed ankylosing spondylitis in the vertebrae of the tail. Both C3H.Q and B10.Q mice deficient for B cells were resistant to arthritis. We conclude that G6PI has the ability to induce a chronic arthritis, which is MHC associated and B-cell dependent. Thus, there are striking similarities between this and the collagen-induced arthritis model.     

Suppression of collagen‐induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of type II collagen

Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We previously reported that altered peptide ligands (APLs) of type II collagen (CII256‐271) suppress the development of collagen‐induced arthritis (CIA). In this study, we generated transgenic rice expressing CII256‐271 and APL6 contained in fusion proteins with the rice storage protein glutelin in the seed endosperm. These transgene products successfully and stably accumulated at high levels (7–24 mg/g seeds) in protein storage vacuoles (PB‐II) of mature seeds. We examined the efficacy of these transgenic rice seeds by performing oral administration of the seeds to CIA model mice that had been immunized with CII. Treatment with APL6 transgenic rice for 14 days significantly inhibited the development of arthritis (based on clinical score) and delayed disease onset during the early phase of arthritis. These effects were mediated by the induction of IL‐10 from CD4⁺ CD25^? T cells against CII antigen in splenocytes and inguinal lymph nodes (iLNs), and treatment of APL had no effect on the production of IFN‐γ, IL‐17, IL‐2 or Foxp3⁺ Treg cells. These findings suggest that abnormal immune suppressive mechanisms are involved in the therapeutic effect of rice‐based oral vaccine expressing high levels of APLs of type II collagen on the autoimmune disease CIA, suggesting that the seed‐based mucosal vaccine against CIA functions via a unique mechanism.     

The interplay between inflammation and metabolism in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by extensive synovitis resulting in erosions of articular cartilage and marginal bone that lead to joint destruction. The autoimmune process in RA depends on the activation of immune cells, which use intracellular kinases to respond to external stimuli such as cytokines, immune complexes, and antigens. An intricate cytokine network participates in inflammation and in perpetuation of disease by positive feedback loops promoting systemic disorder. The widespread systemic effects mediated by pro-inflammatory cytokines in RA impact on metabolism and in particular in lymphocyte metabolism. Moreover, RA pathobiology seems to share some common pathways with atherosclerosis, including endothelial dysfunction that is related to underlying chronic inflammation. The extent of the metabolic changes and the types of metabolites seen may be good markers of cytokine-mediated inflammatory processes in RA. Altered metabolic fingerprints may be useful in predicting the development of RA in patients with early arthritis as well as in the evaluation of the treatment response. Evidence supports the role of metabolomic analysis as a novel and nontargeted approach for identifying potential biomarkers and for improving the clinical and therapeutical management of patients with chronic inflammatory diseases. Here, we review the metabolic changes occurring in the pathogenesis of RA as well as the implication of the metabolic features in the treatment response.     

IL-6 in autoimmune disease and chronic inflammatory proliferative disease

Interleukin 6 (IL-6), which was originally identified as a B-cell differentiation factor, is now known to be a multifunctional cytokine that regulates the immune response, hematopoiesis, the acute phase response, and inflammation. Deregulation of IL-6 production is implicated in the pathology of several disease processes. The expression of constitutively high levels of IL-6 in transgenic mice results in fatal plasmacytosis, which has been implicated in human multiple myeloma. Increased IL-6 levels are also observed in several diseases, including rheumatoid arthritis (RA), systemic-onset juvenile chronic arthritis (JCA), osteoporosis, and psoriasis. IL-6 is critically involved in experimentally induced autoimmune disease, such as antigen-induced arthritis (AIA), and experimental allergic encephalomyelitis. All these clinical data and animal models suggest that IL-6 plays critical roles in the pathogenesis of autoimmune diseases. Here we review the evidence for the involvement of IL-6 in the pathophysiology of autoimmune diseases and chronic inflammatory proliferative diseases (CIPD) and discuss the possible molecular mechanisms of its involvement.     

The human T cell leukemia virus type I-tax gene is responsible for the development of both inflammatory polyarthropathy resembling rheumatoid arthritis and noninflammatory ankylotic arthropathy in transgenic mice

We previously reported that inflammatory arthropathy resembling rheumatoid arthritis (RA) develops among transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR region of human T cell leukemia virus type I (LTR-pX-Tg mice). Because four genes are encoded in this region, we produced transgenic mice that only express the tax gene to examine its role in the development of arthritis. Transgenic mice were produced by constructing DNAs that express the tax gene alone under the control of either its own LTR or CD4 enhancer/promoter and by microinjecting them into C3H/HeN-fertilized ova. We produced seven transgenic mice carrying the LTR-tax gene and nine mice carrying the CD4-tax and found that one of the LTR-tax-Tg mice and five of CD4-tax-Tg mice developed RA-like inflammatory arthropathy similar to LTR-pX-Tg mice, indicating that the tax gene is arthritogenic. On the other hand, the other two LTR-tax-Tg mice had ankylotic changes caused by new bone formation without inflammation. In these ankylotic mice, tax mRNA, inflammatory cytokine mRNA, and autoantibody levels except for TGF-beta1 level were lower than those in LTR-pX- or CD4-tax-Tg mice. These results show that Tax is responsible for the development of inflammatory arthropathy resembling RA and that this protein also causes ankylotic arthropathy.     

IL-9, a local growth factor for synovial T cells in inflammatory arthritis

ObjectiveThe regulatory role of the T_h9 cells along with its signature cytokine IL-9 in human immune system and its aberrant activation in autoimmune diseases is currently under investigation. We are reporting the functional significance of IL-9 in the pathogenesis of autoimmune inflammatory arthritis.MethodsCD3⁺ T cells were obtained from peripheral blood (PB) and synovial fluid (SF) of psoriatic arthritis (PsA), rheumatoid arthritis (RA), and osteoarthritis (OA) patients. MTT, FACS based CFSE dilution assay and apoptosis assay (Annexin-V) were performed to determine the pro-growth/survival effect of human recombinant IL-9 on activated CD3⁺ T cells. Immunoblots were performed to determine the signaling proteins responsible for the progrowth/survival effect of IL-9.ResultsSF of PsA and RA was enriched with IL-9 producing CD3⁺ T cells compared to the SF in OA. IL-9 level measured by ELISA was significantly elevated in PsA and RA patients compared to SF in OA (<.001). Activated T cells of PsA and RA had higher levels of IL-9 receptors. IL-9 promoted proliferation and survival of the CD3⁺ T cells of PB and SF of PsA and RA and compared to untreated (media) controls (p < .005, t-test). IL-9 induced proliferation of T cells was dependent on PI3K/Akt/mTOR signaling pathway.ConclusionIL-9 is functionally active, and is a pro-growth/survival factor for the localized pathologic T cells in the synovium of inflammatory arthritis. The pro-growth/survival effect is mediated by the activation of mTOR kinase cascade. To our knowledge, this is the first report of a functional role of IL-9 in human autoimmune arthritis.     

Lessons from animal models of arthritis over the past decade

This review summarizes the major developments in animal models of arthritis in the past decade. It focuses on novel transgenic models, addresses the involvement of cytokines and discusses novel findings in cartilage and bone erosion. It is clear that interest has been raised in the direct arthritogenic role of autoantibodies, apart from T cell involvement, and their interaction with cells through Fcgamma receptors. In addition, a role for IL-6 and IL-17 and Th17 cells seems apparent in most T cell-driven arthritis models, with environmental triggering through Toll-like receptors contributing to this process. Further insights into enzymes involved in cartilage proteoglycan loss in arthritis, as well as mediators regulating bone erosion and bone apposition, have been gained.     

Pathogenic antibody recognition of cartilage

Antibodies against cartilage proteins are highly prevalent in the sera and synovial fluids of rheumatoid arthritis (RA) patients and also precede disease induction in various spontaneous and induced animal models of arthritis. These antibodies play an important role in the induction and perpetuation of the clinical disease. Antibodies binding to cartilage protein(s), especially the major articular cartilage protein, collagen type II (CII) can induce, in naive mice, an acute form of arthritis that can substantially destroy the cartilage and bone architecture. More importantly, these anti-CII antibodies can also directly cause the destruction of the target tissue preceding and independently of disease development and in the absence of any other pathogenic inflammatory factors or the action of immune cells. Alternatively, antibodies to citrullinated protein antigens and rheumatoid factor are well-validated prognostic and diagnostic markers of severe erosive RA, although their arthritogenic potential is questioned. Recently, we have found that the monoclonal antibodies to citrulline-modified cartilage protein can bind cartilage and synovial tissue and mediate arthritis in mice. Similarly, one of the pathogenic anti-CII monoclonal antibodies has rheumatoid-factor-like activity, suggesting a disease-inducing role for these commonly prevalent antibodies in RA patients. Interestingly, recent findings have also shown that the enzymatic cleavage or modification of pathogenic IgG antibodies protects the cartilage surface, thereby opening up new therapeutic possibilities for protecting the cartilage from inflammatory damage.