Rheumatoid arthritis: regulation of synovial inflammation

Rheumatoid arthritis (RA) is a systemic, inflammatory autoimmune disorder that presents as a symmetric polyarthritis associated with swelling and pain in multiple joints, often initially occurring in the joints of the hands and feet. Articular inflammation causes activation and proliferation of the synovial lining, expression of inflammatory cytokines, chemokine-mediated recruitment of additional inflammatory cells, as well as B cell activation with autoantibody production. A vicious cycle of altered cytokine and signal transduction pathways and inhibition of programmed cell death contribute to synoviocyte and osteoclast mediated cartilage and bone destruction. A combination of targeted interventions at various stages in the pathogenesis of RA will likely be required to control symptoms in certain patients with this complex and potentially disabling disease. The regulation of rheumatoid synovial inflammation will be reviewed, followed by a brief summary of the therapeutic implications of these advances, including strategies targeting key cytokines, signal transduction molecules, co-stimulatory molecules, B cells, chemokines, and adhesion molecules.     

B cells in rheumatoid synovitis

In rheumatoid arthritis, T cells, B cells, macrophages, and dendritic cells invade the synovial membranes, establishing complex microstructures that promote inflammatory/tissue destructive lesions. B cell involvement has been considered to be limited to autoantibody production. However, recent studies suggest that B cells support rheumatoid disease through other mechanisms. A critical element of rheumatoid synovitis is the process of ectopic lymphoid neogenesis, with highly efficient lymphoid architectures established in a nonlymphoid tissue site. Rheumatoid synovitis recapitulates the pathways of lymph node formation, and B cells play a key role in this process. Furthermore, studies of rheumatoid lesions implanted in immunodeficient mice suggest that T cell activation in synovitis is B cell dependent, indicating the role played by B cells in presenting antigens and providing survival signals.     

Recent developments in the immunobiology of rheumatoid arthritis

Progress into the understanding of immunopathology in rheumatoid arthritis is reviewed in the present article with regard to pro-inflammatory cytokine production, cell activation and recruitment, and osteoclastogenesis. Studies highlight the potential importance of T helper 17 cells and regulatory T cells in driving and suppressing inflammation in rheumatoid arthritis, respectively, and highlight other potential T-cell therapeutic targets. The genetic associations of the HLA shared epitope alleles with antibodies to citrullinated peptides in rheumatoid arthritis patients indicate that T cells are providing help to B cells to produce autoantibodies, and there is increasing evidence that these autoantibodies are pathogenic in rheumatoid arthritis.     

Lymphoid chemokine B cell-attracting chemokine-1 (CXCL13) is expressed in germinal center of ectopic lymphoid follicles within the synovium of chronic arthritis patients

A unique feature in inflammatory tissue of rheumatoid arthritis (RA) is the formation of ectopic lymphoid aggregates with germinal center (GC)-like structures that can be considered to contribute to the pathogenesis of RA, because local production of the autoantibody, rheumatoid factor, is thought to be a causative factor in tissue damage. However, the factors governing the formation of GC in RA are presently unknown. To begin to address this, the expression of B cell attracting chemokine (BCA-1) (CXCL13), a potent chemoattractant of B cells, was examined in the synovium of patients with RA or with osteoarthritis (OA). Expression of BCA-1 mRNA was detected in all RA samples, but in only one of five OA samples. Lymphoid follicles were observed in four of seven RA samples and in two of eight OA samples, and in most of them BCA-1 protein was detected in GC. BCA-1 was not detected in tissues lacking lymphoid follicles. Notably, BCA-1 was detected predominantly in follicular dendritic cells in GC. CD20-positive B cells were aggregated in regions of BCA-1 expression, but not T cells or macrophages. These data suggest that BCA-1 produced by follicular dendritic cells may attract B cells and contribute to the formation of GC-like structures in chronic arthritis.     

Mesenchymal stromal cells. Nurse-like cells reside in the synovial tissue and bone marrow in rheumatoid arthritis

A major question concerning the immunopathology of rheumatoid arthritis is why the disease is localized to particular joints. A possible explanation could be the presence within the synovium of cells that foster inflammation or easy accessibility of the synovium to migratory disease enhancing cells. Within both the bone marrow and the synovium, fibroblastic stromal cells play an important role in supporting the differentiation and survival of normal cells, and also contribute to the pathologic processes. Among fibroblastic stromal cells in synovial tissue and bone marrow, nurse-like cells are a unique population having the specific capacity to promote pseudoemperipolesis (adhesion and holding beneath) of lymphocytes, and also the ability to promote the growth and function of some populations of lymphocytes and monocytes. Nurse-like cells could therefore contribute to the immunopathogenesis of rheumatoid arthritis, and may contribute to the localization of inflammation within specific joints. The present review considers the evidence that supports these possibilities.     

Substance P, neurokinin A, and neurokinin B induce generation of IL-1-like activity in P388D1 cells. Possible relevance to arthritic disease

Near nanomolar concentrations of substance P induce production of IL-1 or an IL-1-like activity in the mouse macrophage cell line P388D1. Moreover, this could be accomplished with the carboxyl-terminal octapeptide substance P4-11, and could be inhibited with the substance P antagonist [D-Pro2, D-Trp7,9]-substance P. Two other mammalian neurokinins, neurokinin A and neurokinin B, were also found to induce secretion of IL-1-like activity in P388D1 cells. These findings suggest that activation of immune cells by neuromodulators can contribute to the maintenance of the chronic inflammatory state and the immunopathology observed in arthritic disease mediated by IL-1. The results also suggest that one approach to the treatment of rheumatoid arthritis might be to attempt to inhibit the local effects of immuno-modulatory neuropeptides, specifically the neurokinins, in affected joints.     

A role for complement in antibody-mediated inflammation: C5-deficient DBA/1 mice are resistant to collagen-induced arthritis

Collagen-induced arthritis (CIA) represents an animal model of autoimmune polyarthritis with significant similarities to human rheumatoid arthritis that can be induced upon immunization with native type II collagen. As in rheumatoid arthritis, both cellular and humoral immune mechanisms contribute to disease pathogenesis. Genotypic studies have identified at least six genetic loci contributing to arthritis susceptibility, including the class II MHC. We have examined the mechanism of Ab-mediated inflammation in CIA joints, specifically the role of complement activation, by deriving a line of mice from the highly CIA-susceptible DBA/1LacJ strain that are congenic for deficiency of the C5 complement component. We show that such C5-deficient DBA/1LacJ animals mount normal cellular and humoral immune responses to native type II collagen, with the activation of collagen-specific TNF-alpha-producing T cells in the periphery and substantial intra-articular deposition of complement-fixing IgG Abs. Nevertheless, these C5-deficient mice are highly resistant to the induction of CIA. These data provide evidence for an important role of complement in Ab-triggered inflammation and in the pathogenesis of autoimmune arthritis.     

Specific Btk inhibition suppresses B cell- and myeloid cell-mediated arthritis

Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.     

Expression of Cell Adhesion Molecules and Cytokines in Murine Antigen-Induced Arthritis

Adhesion molecules and cytokines are important in chronic inflammatory conditions such as rheumatoid arthritis (RA) by virtue of their role in cell activation and emigration. Using immunohistochemical techniques we studied the expression of adhesion molecules and cytokines in cryopreserved sections of murine knee joint in the course of antigen-induced arthritis, an animal model of human RA. Various adhesion molecules and cytokines are expressed in the arthritic joint tissue. LFA-I, Mac-1, CD44, ICAM-I and P-selectin were strongly expressed in the acute phase and to a lesser degree in the chronic phase of arthritis. VLA-4 and VCAM-I appeared to be moderately expressed on day 1, L-selectin between days 1 and 3. LFA-I, Mac-I, CD44, a4-integrin, ICAM-I and the selectins were found expressed on cells of the synovial infiltrate, LFA-1, Mac-1 and ICAM-I on the synovial lining layer, and VCAM-I and P-selectin on endothelial cells. Expression of E-selectin could be demonstrated throughout the experiment at a low level in cells of the acute cell infiltrate. Cytokines, especially IL-2, IL-4, IL-6, TNF, and IFN-7, were heavily expressed during the acute phase of arthritis in cellular infiltrate. Taken together these data demonstrate that cytokines and their activation of adhesion molecules contribute to cell infiltration and activation during the initial phase of arthritis and to the induction and progression of tissue destruction in arthritic joints. These molecules might be potential targets for novel therapeutic strategies in inflammatory and arthritic disorders.     

Interaction between synovial inflammatory tissue and bone marrow in rheumatoid arthritis

Rheumatoid arthritis (RA) leads to destruction of cartilage and bone. Whether rheumatoid arthritis also affects the adjacent bone marrow is less clear. In this study, we investigated subcortical bone marrow changes in joints from patients with RA. We describe penetration of the cortical barrier by synovial inflammatory tissue, invasion into the bone marrow cavity and formation of mononuclear cell aggregates with B cells as the predominant cell phenotype. B cells expressed common B cell markers, such as CD20, CD45RA, and CD79a, and were mature B cells, as indicated by CD27 expression. Plasma cells were also present and were enriched in the regions between aggregates and inflammatory tissue. Moreover, molecules for B cell chemoattraction, such as BCA-1 and CCL-21, homing, mucosal addressin cell adhesion molecule-1 and survival, BAFF, were expressed. Endosteal bone next to subcortical bone marrow aggregates showed an accumulation of osteoblasts and osteoid deposition. In summary, we show that synovial inflammatory tissue can reach the adjacent bone marrow by fully breaking the cortical barrier, which results in formation of B cell-rich aggregates as well as increased formation of new bone. This suggests that bone marrow is an additional compartment in the disease process of RA.     

NF-κB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor

Inhibition of NF-κB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-κB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-κB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-κB inhibitors to rheumatoid arthritis therapy.     

CD23+ CD21(high) CD1d(high) B cells in inflamed lymph nodes are a locally differentiated population with increased antigen capture and activation potential

CD23(+)CD21(high)CD1d(high) B cells in inflamed nodes (Bin cells) accumulate in the lymph nodes (LNs) draining inflamed joints of the TNF-α-transgenic mouse model of rheumatoid arthritis and are primarily involved in the significant histological and functional LN alterations that accompany disease exacerbation in this strain. In this study, we investigate the origin and function of Bin cells. We show that adoptively transferred GFP(+) sorted mature follicular B (FoB) cells home preferentially to inflamed LNs of TNF-α-transgenic mice where they rapidly differentiate into Bin cells, with a close correlation with the endogenous Bin fraction. Bin cells are also induced in wild-type LNs after immunization with T-dependent Ags and display a germinal center phenotype at higher rates compared with FoB cells. Furthermore, we show that Bin cells can capture and process Ag-immune complexes in a CD21-dependent manner more efficiently than can FoB cells, and they express greater levels of MHC class II and costimulatory Ags CD80 and CD86. We propose that Bin cells are a previously unrecognized inflammation-induced B cell population with increased Ag capture and activation potential, which may facilitate normal immune responses but may contribute to autoimmunity when chronic inflammation causes their accumulation and persistence in affected LNs.     

B cell depletion reduces the number of autoreactive T helper cells and prevents glucose-6-phosphate isomerase-induced arthritis

The therapeutic benefit of B cell depletion in patients with rheumatoid arthritis has provided proof of concept that B cells are relevant for the pathogenesis of arthritis. It remains unknown which B cell effector functions contribute to the induction or chronification of arthritis. We studied the clinical and immunological effects of B cell depletion in glucose-6-phosphate isomerase-induced arthritis. We targeted CD22 to deplete B cells. Mice were depleted of B cells before or after immunization with glucose-6-phosphate isomerase (G6PI). The clinical and histological effects were studied. G6PI-specific antibody responses were measured by ELISA. G6PI-specific T helper (Th) cell responses were assayed by polychromatic flow cytometry. B cell depletion prior to G6PI-immunization prevented arthritis. B cell depletion after immunization ameliorated arthritis, whereas B cell depletion in arthritic mice was ineffective. Transfer of antibodies from arthritic mice into B cell depleted recipients did not reconstitute arthritis. B cell depleted mice harbored much fewer G6PI-specific Th cells than control animals. B cell depletion prevents but does not cure G6PI-induced arthritis. Arthritis prevention upon B cell depletion is associated with a drastic reduction in the number of G6PI-specific effector Th cells.     

Suppression of proteoglycan-induced arthritis by anti-CD20 B Cell depletion therapy is mediated by reduction in autoantibodies and CD4+ T cell reactivity

B cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) since the discovery of RA as an autoimmune disease. There is renewed interest in B cells in RA based on the clinical efficacy of B cell depletion therapy in RA patients. Although, reduced titers of rheumatoid factor and anti-cyclic citrullinated peptide Abs are recorded, the mechanisms that convey clinical improvement are incompletely understood. In the proteoglycan-induced arthritis (PGIA) mouse model of RA, we reported that Ag-specific B cells have two important functions in the development of arthritis. PG-specific B cells are required as autoantibody-producing cells as well as Ag-specific APCs. Herein we report on the effects of anti-CD20 mAb B cell depletion therapy in PGIA. Mice were sensitized to PG and treated with anti-CD20 Ab at a time when PG-specific autoantibodies and T cell activation were evident but before acute arthritis. In mice treated with anti-CD20 mAb, development of arthritis was significantly reduced in comparison to control mAb-treated mice. B cell depletion reduced the PG-specific autoantibody response. Furthermore, there was a significant reduction in the PG-specific CD4(+) T cell recall response as well as significantly fewer PG-specific CD4(+) T cells producing IFN-gamma and IL-17, but not IL-4. The reduction in PG-specific T cells was confirmed by the inability of CD4(+) T cells from B cell-depleted mice to adoptively transfer disease into SCID mice. Overall, B cell depletion during PGIA significantly reduced disease and inhibited both autoreactive B cell and T cell function.     

Chemokine receptor expression and functional effects of chemokines on B cells: implication in the pathogenesis of rheumatoid arthritis

Introduction  

Accumulation of B cells in the rheumatoid arthritis (RA) synovium has been reported, and it has been thought that these cells might contribute to the pathogenesis of RA by antigen presentation, autoantibody production, and/or inflammatory cytokine production. Chemokines could enhance the accumulation of B cells in the synovium. The aims of this study were to determine chemokine receptor expression by B cells both in the peripheral blood of normal donors and subjects with RA, and at the inflammatory site in RA, and the effects of chemokines on B cell activation.     

Expression of CD80/86 on B cells is essential for autoreactive T cell activation and the development of arthritis

Depletion of B cells in rheumatoid arthritis is therapeutically efficacious. Yet, the mechanism by which B cells participate in the inflammatory process is unclear. We previously demonstrated that Ag-specific B cells have two important functions in the development of arthritis in a murine model of rheumatoid arthritis, proteoglycan (PG)-induced arthritis (PGIA). PG-specific B cells function as autoantibody-producing cells and as APCs that activate PG-specific T cells. Moreover, the costimulatory molecule CD86 is up-regulated on PG-specific B cells in response to stimulation with PG. To address the requirement for CD80/CD86 expression on B cells in the development of PGIA, we generated mixed bone marrow chimeras in which CD80/CD86 is specifically deleted on B cells and not on other APC populations. Chimeras with a specific deficiency in CD80/CD86 expression on B cells are resistant to the induction of PGIA. The concentration of PG-specific autoantibody is similar in mice sufficient or deficient for CD80/86-expressing B cells, which indicates that resistance to PGIA is not due to the suppression of PG-specific autoantibody production. CD80/86-deficient B cells failed to effectively activate PG-specific autoreactive T cells as indicated by the failure of T cells from PG-immunized CD80/86-deficient B cell chimeras to transfer arthritis into SCID mice. In vitro secondary recall responses to PG are also dependent on CD80/86-expressing B cells. These results demonstrate that a CD80/86:CD28 costimulatory interaction between B cells and T cells is required for autoreactive T cell activation and the induction of arthritis but not for B cell autoantibody production.     

T cell activation in rheumatoid synovium is B cell dependent

Rheumatoid arthritis results from a T cell-driven inflammation in the synovial membrane that is frequently associated with the formation of tertiary lymphoid structures. The significance of this extranodal lymphoid neogenesis is unknown. Microdissection was used to isolate CD4 T cells residing in synovial tissue T cell/B cell follicles. CD4 T cells with identical TCR sequences were represented in independent, nonadjacent follicles, suggesting recognition of the same Ag in different germinal centers. When adoptively transferred into rheumatoid arthritis synovium-SCID mouse chimeras, these CD4 T cell clones enhanced the production of IFN-gamma, IL-1beta, and TNF-alpha. In vivo activity of adoptively transferred CD4 T cells required matching of HLA-DRB1 alleles and also the presence of T cell/B cell follicles. HLA-DRB1-matched synovial tissues that were infiltrated by T cells, macrophages, and dendritic cells, but that lacked B cells, did not support the activation of adoptively transferred CD4 T cell clones, raising the possibility that B cells provided a critical function in T cell activation or harbored the relevant Ag. Dependence of T cell activation on B cells was confirmed in B cell depletion studies. Treatment of chimeric mice with anti-CD20 mAb inhibited the production of IFN-gamma and IL-1beta, indicating that APCs other than B cells could not substitute in maintaining T cell activation. The central role of B cells in synovial inflammation identifies them as excellent targets for immunosuppressive therapy.     

Altered dynamics of Kv1.3 channel compartmentalization in the immunological synapse in systemic lupus erythematosus

Aberrant T cell responses during T cell activation and immunological synapse (IS) formation have been described in systemic lupus erythematosus (SLE). Kv1.3 potassium channels are expressed in T cells where they compartmentalize at the IS and play a key role in T cell activation by modulating Ca(2+) influx. Although Kv1.3 channels have such an important role in T cell function, their potential involvement in the etiology and progression of SLE remains unknown. This study compares the K channel phenotype and the dynamics of Kv1.3 compartmentalization in the IS of normal and SLE human T cells. IS formation was induced by 1-30 min exposure to either anti-CD3/CD28 Ab-coated beads or EBV-infected B cells. We found that although the level of Kv1.3 channel expression and their activity in SLE T cells is similar to normal resting T cells, the kinetics of Kv1.3 compartmentalization in the IS are markedly different. In healthy resting T cells, Kv1.3 channels are progressively recruited and maintained in the IS for at least 30 min from synapse formation. In contrast, SLE, but not rheumatoid arthritis, T cells show faster kinetics with maximum Kv1.3 recruitment at 1 min and movement out of the IS by 15 min after activation. These kinetics resemble preactivated healthy T cells, but the K channel phenotype of SLE T cells is identical to resting T cells, where Kv1.3 constitutes the dominant K conductance. The defective temporal and spatial Kv1.3 distribution that we observed may contribute to the abnormal functions of SLE T cells.     

Endothelial cell phenotypes in the rheumatoid synovium: activated,angiogenic, apoptotic and leaky

Endothelial cells are active participants in chronic inflammatory diseases. These cells undergo phenotypic changes that can be characterised as activated, angiogenic, apoptotic and leaky. In the present review, these phenotypes are described in the context of human rheumatoid arthritis as the disease example. Endothelial cells become activated in rheumatoid arthritis pathophysiology, expressing adhesion molecules and presenting chemokines, leading to leukocyte migration from the blood into the tissue. Endothelial cell permeability increases, leading to oedema formation and swelling of the joints. These cells proliferate as part of the angiogenic response and there is also a net increase in the turnover of endothelial cells since the number of apoptotic endothelial cells increases. The endothelium expresses various cytokines, cytokine receptors and proteases that are involved in angiogenesis, proliferation and tissue degradation. Associated with these mechanisms is a change in the spectrum of genes expressed, some of which are relatively endothelial specific and others are widely expressed by other cells in the synovium. Better knowledge of molecular and functional changes occurring in endothelial cells during chronic inflammation may lead to the development of endothelium-targeted therapies for rheumatoid arthritis and other chronic inflammatory diseases.     

Microparticles stimulate angiogenesis by inducing ELR+ CXC‐chemokines in synovial fibroblasts

Microparticles (MPs) are small membrane‐vesicles that accumulate in the synovial fluids of patients with rheumatoid arthritis (RA). In the arthritic joints, MPs induce a pro‐inflammatory and invasive phenotype in synovial fibroblasts (SFs). The present study investigated whether activation of SFs by MPs stimulates angiogenesis in the inflamed joints of patients with RA. MPs were isolated from Jurkat cells and U937 cells by differential centrifugation. SFs were co‐cultured with increasing numbers of MPs. The effects of supernatants from co‐cultures on endothelial cells were studied in vitro and in vivo using MTT assays, annexin V and propidium iodide staining, trans‐well migration assays and modified matrigel pouch assays. MPs strongly induced the expression of the pro‐angiogenic ELR⁺ chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in RASFs. Other vascular growth factors were not induced. Supernatants from co‐cultures enhanced the migration of endothelial cells, which could be blocked by neutralizing antibodies against ELR⁺ chemokines. Consistent with the specific induction of ELR⁺ chemokines, proliferation and viability of endothelial cells were not affected by the supernatants. In the in vivo bio‐chamber assay, supernatants from RASFs co‐cultured with MPs stimulated angiogenesis with a significant increase of vessels infiltrating into the matrigel chamber. We demonstrated that MPs activate RASFs to release pro‐angiogenic ELR⁺ chemokines. These pro‐angiogenic mediators enhance migration of endothelial cells and stimulate the formation of new vessels. Our data suggest that MPs may contribute to the hypervascularization of inflamed joints in patients with rheumatoid arthritis.