Understanding the pathogenesis of joint inflammation and destruction in rheumatoid arthritis involves dissection of the cellular and molecular interactions that occur in synovial tissue. Development of effective targeted therapies has been based on progress in achieving such insights. Safer and more specific approaches to treatment could flow from discovery of cell-cell interaction pathways that are specific to inflammation of the joint and less important in the defense against systemic infection. This article highlights selected cell-cell interactions in rheumatoid arthritis synovium that may be worthy of evaluation as future therapeutic targets.
Although the cause of rheumatoid arthritis (RA) remains unknown, insights into the pathogenesis of RA have been achieved by careful study of inflammatory, immune, and tissue-destructive processes that take place in synovial tissue. Successful approaches have included ex vivo analysis of RA synovium, experiments in animal model systems, and use of cultured cell lines, especially fibroblast-like synoviocytes, derived from patients’ synovial tissue. These insights have led to remarkable advances in the treatment of RA and other diseases, such as the use of tumor necrosis factor (TNF)-blocking biologics.
None of the molecular targets of medications currently used in the treatment of RA are expressed uniquely in synovial tissue. Instead, all of them are of great importance in host defense as well. Recent research is revealing important molecules and pathways pertinent to joint inflammation and damage that may be less central to host defense compared with currently targeted molecules. A conceptual framework for such investigations is the realization that although RA synovium can display some features of an immune organ and is justifiably regarded as a tertiary lymphoid structure, it also contains cells that are distinct from those found in primary or secondary lymphoid tissue, namely, the intrinsic structural cells of the joint such as fibroblast-like synoviocytes.
It is clear that no one cell type explains the pathologic behavior of RA synovial tissue. It is the interactions between these cells that define the disease. The 3 most abundant cell populations in RA synovium are the monocyte/macrophage synoviocytes (type A), the fibroblast-like synoviocytes (FLS) (type B), and T lymphocytes (which are strikingly heterogeneous). Other critically important cells of the RA synovium include B lymphocytes, plasma cells, dendritic cells, mast cells, endothelial cells, osteoclasts, and adjacent chondrocytes. These various cell types can interact in 2 general ways: first through secreted mediators, notably inflammatory cytokines such as TNF, interleukin (IL)-6, IL-17, and many others; and second through direct cell-cell contact that is mediated by cell surface receptors and ligands, including some membrane-anchored cytokines.
This article focuses on selected cell-cell interactions that may be important in the pathogenesis of RA. It is hoped that the relative molecular specificity of some of these interactions for events in the joint compared with the systemic immune response will provide more specific targets ( Table 1 ) for a new generation of biologic and nonbiologic therapeutics.
| Cell Types | Potential New Molecular Targets |
|---|---|
| FLS-FLS | Cadherin-11, fractalkine |
| FLS-T | B7-H3, IL-15 |
| T-APC | OX40, CCL20, IL-7 |
| B-T | CXCL13, ICOS, OX40, BAFF |
| B-FLS | BAFF, osteopontin |
| Leucocyte-endothelial | Le y /H |
Homotypic and autocrine interactions of FLS
Cadherin-11
During the course of RA, the cells of the synovial lining undergo extensive hyperplasia to form the synovial pannus that invades and destroys cartilage and bone. Recent discoveries have highlighted a critical role for cadherin-11 in these events. Cadherin-11 has been identified as a strongly expressed intercellular adhesion molecule on human and mouse FLS. Transfection of L cells with cadherin-11 led to the formation of sheet-like structures with an organization similar to that seen in synovial lining ; furthermore, cadherin-11 localized to cell-to-cell junctions between FLS. Joints in cadherin-11 null mice had an underdeveloped synovial lining and decreased extracellular matrix. These data indicate that cadherin-11 plays a vital role in the formation of the synovial lining layer by mediating FLS/FLS connections.
Cadherin-11 staining correlated strongly with cellular infiltration of macrophages and T lymphocytes in RA synovium. Cadherin-11 staining also correlated with erythrocyte sedimentation rate and C-reactive protein level although not as strongly. Cadherin-11 expression in synovium is not specific to RA, because cadherin-11 staining on synovial biopsies was similar in inflamed joints from RA, osteoarthritis (OA), and psoriatic arthritis (PsA). There was also a positive correlation between cadherin-11 staining of lung tissue from patients with RA-associated interstitial pneumonitis (IP) and CD4 + T-cell infiltration of the lung. Cadherin-11 null mice showed an average of 50% reduction in clinical arthritis activity in the K/BxN serum transfer model. Cadherin-11 has been explored as a possible therapeutic target using the same mouse serum transfer model. Cadherin-11-Fc and an anticadherin-11 mAb ameliorated clinical arthritis when administered with arthritogenic serum. More significant for potential treatment of human disease, anticadherin-11 mAb ameliorated established arthritis in a modified K/BxN serum transfer model.
Fractalkine and its Receptor
Fractalkine (FKN) is a potent chemoattractant and adhesion molecule that is found in increased levels in RA synovium. RA FLS secrete FKN and express its receptor, CX 3 CR1. Soluble FKN induced proliferation of FLS that was blocked by addition of anti-CX 3 CR1. Even in the absence of sFKN, the antibody was able to decrease FLS proliferation, revealing an autocrine growth loop. FKN also induced migration of RA FLS and caused significant reorganization of F-actin within FLS. It is likely that FKN could act in an autocrine fashion to aid in pannus invasion of the bone and cartilage through FLS growth and migration.
FLS/T cell interactions
In RA, FLS and other cells produce chemokines that attract T cells to the joint. FLS and T cells then interact in the synovium through secreted factors and direct cell-to-cell interactions, resulting in activation of both cell types. FLS proliferate when cocultured with CD4 + T cells, especially when RA T cells are used.
Synovial T cells in RA patients include an expanded population of CD4 + CD28 − cells and this subset of cells in particular greatly enhances FLS proliferation. CD4 + CD28 − cells, which are sometimes considered to be senescent, aberrantly express CX 3 CR1, and anti-CX 3 CR1 decreases the FLS growth-promoting activity of these cells without a significant effect on CD4 + CD28 + T cells. Stimulation of CD4 + CD28 − T cells by FKN through CX 3 CR1 increases TNFα production, and TNFα can then act on FLS to increase growth, FKN secretion, and CX 3 CR1 expression. These data suggest an important relationship between TNFα and FKN/CX 3 CR1 in FLS/T cell interactions in the RA joint, in which production and action of these molecules occur in linked paracrine and autocrine loops involving T cells and FLS.
IL-15
IL-15 is constitutively expressed on FLS and is a potent T-cell growth factor that can cause activation/proliferation of effector T cells (T eff ) and regulatory T cells (T reg ) in T-cell/FLS cocultures. Moreover, IL-15 can decrease apoptosis of various cell types including FLS and T cells. The IL-15 receptor is a trimer (IL-15Rα,β,γ c ) and subunits of IL-15R are expressed by various cell types, including FLS and T cells. IL-15 can function as a secreted or membrane-bound cytokine, with signaling similar to other cytokines through the full trimeric IL-15R, or through dimeric IL-15Rβγ c receptor ( cis presentation). IL-15 can also signal through a unique trans signaling form, in which distinct subunits of IL-15R are expressed on the surface of interacting cells. IL-15 can be recycled by the cell and presented on the cell surface by IL-15Rα, a pathway that allows for persistence of an IL-15 signal even when soluble IL-15 is no longer available. In cultures containing T eff and T reg cocultured with RA FLS, proliferation and function of both T cell subsets were stimulated, with a net proinflammatory effect. These effects were not observed when OA FLS or dermal fibroblasts were used and were dependent on cell-to-cell contact. Neutralizing IL-15 during T-cell/FLS cocultures significantly attenuated the proliferation of T eff and T reg , T eff production of TNFα and IFNγ, and T reg inhibition of T eff . The overall proinflammatory effect of IL-15 makes it a potential target for RA therapy. A proof-of-concept study has been conducted using a human immunoglobulin (Ig)G1 anti-IL-15 monoclonal antibody in RA. This antibody suppressed effects of IL-15 in vitro, and showed meaningful efficacy in phase I and II trials.
FLS as Antigen-presenting Cells
FLS can also act as antigen-presenting cells (APCs) in the initiation of T-lymphocyte responses. For example, FLS induced secretion of IL-2 by class II MHC-restricted CD4 + T-cell hybridomas specific for arthritogenic autoantigens, specifically human cartilage gp-39 (HC gp-39) and human type II collagen (CII). The T-cell hybridomas in these experiments were developed from HLA-DR4 transgenic mice, and therefore respond to peptide antigens that are loaded onto and presented by HLA-DR4. Activation of the hybridomas required reexpression of class II on the FLS, which occurs in vivo in RA and is reinduced in vitro by IFNγ. The T-cell response to peptide antigen presented by FLS was exquisitely MHC-restricted, identical to experiments in which professional APCs were used, and blocking antibodies to human class II MHC or murine CD4 prevented IL-2 production. Because T-cell hybridomas do not require a second signal to respond to peptide antigen, this system was not useful for defining potential costimulatory ligands on FLS. FLS do not express the classic APC costimulatory molecules CD80 or CD86 (B7-1, B7-2) at functionally relevant levels.
B7-H3
Of the molecules that belong to the B7 family, B7-H3 is expressed strongly and constitutively on FLS in vitro. Moreover, immunostaining of RA synovium showed broad B7-H3 expression nearly identical in distribution to the FLS marker cadherin-11. Dual-color immunohistochemical analysis showed CD3 + T cells in close proximity to FLS expressing B7-H3. Furthermore, in coculture experiments, B7-H3 localized to the contact point between FLS and cytokine-activated T cells (Tck) or during FLS presentation of superantigen to T cells. Previous studies had indicated that B7-H3 can have either stimulatory or inhibitory effects on T cells, and results with FLS/T-cell cocultures were consistent with a dual role for B7-H3. RNAi knockdown of B7-H3 in FLS decreased production of TNFα, IFNγ, and IL-2 by cocultured Tck but increased production of these cytokines by resting T cells. The T-cell ligand or ligands for B7-H3 have not yet been defined. B7-H3, in contrast to B7-1 and B7-2, is expressed on human solid tumors, in part controlled by the microRNA miR-29. In early-phase human clinical trials, the B7-H3–specific mAb 8H9 was reported to prolong survival in patients with solid tumors and central nervous system metastasis. Further trials of anti-B7-H3 in cancer could yield safety information pertinent to consideration of therapeutic trials of this antibody in RA.
FLS/T cell interactions
In RA, FLS and other cells produce chemokines that attract T cells to the joint. FLS and T cells then interact in the synovium through secreted factors and direct cell-to-cell interactions, resulting in activation of both cell types. FLS proliferate when cocultured with CD4 + T cells, especially when RA T cells are used.
Synovial T cells in RA patients include an expanded population of CD4 + CD28 − cells and this subset of cells in particular greatly enhances FLS proliferation. CD4 + CD28 − cells, which are sometimes considered to be senescent, aberrantly express CX 3 CR1, and anti-CX 3 CR1 decreases the FLS growth-promoting activity of these cells without a significant effect on CD4 + CD28 + T cells. Stimulation of CD4 + CD28 − T cells by FKN through CX 3 CR1 increases TNFα production, and TNFα can then act on FLS to increase growth, FKN secretion, and CX 3 CR1 expression. These data suggest an important relationship between TNFα and FKN/CX 3 CR1 in FLS/T cell interactions in the RA joint, in which production and action of these molecules occur in linked paracrine and autocrine loops involving T cells and FLS.
IL-15
IL-15 is constitutively expressed on FLS and is a potent T-cell growth factor that can cause activation/proliferation of effector T cells (T eff ) and regulatory T cells (T reg ) in T-cell/FLS cocultures. Moreover, IL-15 can decrease apoptosis of various cell types including FLS and T cells. The IL-15 receptor is a trimer (IL-15Rα,β,γ c ) and subunits of IL-15R are expressed by various cell types, including FLS and T cells. IL-15 can function as a secreted or membrane-bound cytokine, with signaling similar to other cytokines through the full trimeric IL-15R, or through dimeric IL-15Rβγ c receptor ( cis presentation). IL-15 can also signal through a unique trans signaling form, in which distinct subunits of IL-15R are expressed on the surface of interacting cells. IL-15 can be recycled by the cell and presented on the cell surface by IL-15Rα, a pathway that allows for persistence of an IL-15 signal even when soluble IL-15 is no longer available. In cultures containing T eff and T reg cocultured with RA FLS, proliferation and function of both T cell subsets were stimulated, with a net proinflammatory effect. These effects were not observed when OA FLS or dermal fibroblasts were used and were dependent on cell-to-cell contact. Neutralizing IL-15 during T-cell/FLS cocultures significantly attenuated the proliferation of T eff and T reg , T eff production of TNFα and IFNγ, and T reg inhibition of T eff . The overall proinflammatory effect of IL-15 makes it a potential target for RA therapy. A proof-of-concept study has been conducted using a human immunoglobulin (Ig)G1 anti-IL-15 monoclonal antibody in RA. This antibody suppressed effects of IL-15 in vitro, and showed meaningful efficacy in phase I and II trials.
FLS as Antigen-presenting Cells
FLS can also act as antigen-presenting cells (APCs) in the initiation of T-lymphocyte responses. For example, FLS induced secretion of IL-2 by class II MHC-restricted CD4 + T-cell hybridomas specific for arthritogenic autoantigens, specifically human cartilage gp-39 (HC gp-39) and human type II collagen (CII). The T-cell hybridomas in these experiments were developed from HLA-DR4 transgenic mice, and therefore respond to peptide antigens that are loaded onto and presented by HLA-DR4. Activation of the hybridomas required reexpression of class II on the FLS, which occurs in vivo in RA and is reinduced in vitro by IFNγ. The T-cell response to peptide antigen presented by FLS was exquisitely MHC-restricted, identical to experiments in which professional APCs were used, and blocking antibodies to human class II MHC or murine CD4 prevented IL-2 production. Because T-cell hybridomas do not require a second signal to respond to peptide antigen, this system was not useful for defining potential costimulatory ligands on FLS. FLS do not express the classic APC costimulatory molecules CD80 or CD86 (B7-1, B7-2) at functionally relevant levels.
B7-H3
Of the molecules that belong to the B7 family, B7-H3 is expressed strongly and constitutively on FLS in vitro. Moreover, immunostaining of RA synovium showed broad B7-H3 expression nearly identical in distribution to the FLS marker cadherin-11. Dual-color immunohistochemical analysis showed CD3 + T cells in close proximity to FLS expressing B7-H3. Furthermore, in coculture experiments, B7-H3 localized to the contact point between FLS and cytokine-activated T cells (Tck) or during FLS presentation of superantigen to T cells. Previous studies had indicated that B7-H3 can have either stimulatory or inhibitory effects on T cells, and results with FLS/T-cell cocultures were consistent with a dual role for B7-H3. RNAi knockdown of B7-H3 in FLS decreased production of TNFα, IFNγ, and IL-2 by cocultured Tck but increased production of these cytokines by resting T cells. The T-cell ligand or ligands for B7-H3 have not yet been defined. B7-H3, in contrast to B7-1 and B7-2, is expressed on human solid tumors, in part controlled by the microRNA miR-29. In early-phase human clinical trials, the B7-H3–specific mAb 8H9 was reported to prolong survival in patients with solid tumors and central nervous system metastasis. Further trials of anti-B7-H3 in cancer could yield safety information pertinent to consideration of therapeutic trials of this antibody in RA.
T cells and APCs in the RA synovium
The following sections highlight examples of selected cytokine-mediated and cognate cell-cell contact-driven interactions between T cells and synovial APCs that are current or potential therapeutic targets for the treatment of RA. Interruption of T-cell costimulation between T cells and APCs in the synovium is a worthwhile approach given the clinical success of blocking T-cell costimulation with CTLA-4Ig in RA. Resting T cells require at least 2 signals to differentiate into effector T cells: the first signal through engagement of the T-cell antigen receptor by the antigen-MHC complex on the APC, and the second signal by engagement of costimulatory molecules such as CD28, on T cells by ligands such as CD80/86 on APCs. Effector T-cell differentiation leads to the expression of additional surface molecules. These inducible structures may have stimulatory (ICOS, OX40) or inhibitory (CTLA-4) potential.
CTLA-4 and Indoleamine Dioxygenase
CD28 is the prototypic T-cell receptor (TCR) for costimulatory signals. The ligation of CD28 by CD80/86 (B7-1 and B7-2) sends activating signals into the T cell and the APC. CTLA-4 (CD152) is up-regulated on activated T cells and binds to the CD28 ligands CD80/86. The ligation of CD80/86 by CTLA-4 sends inhibitory signals directly into the T cell. The ligation of CD80/86 by CTLA-4 can also deliver regulatory signals to the APC. The interaction of CD80/86 with CTLA-4 leads to the induction of indoleamine dioxygenase (IDO) in APCs. IDO is believed to be critical in inducing anergy in T cells, because IDO depletes tryptophan that is necessary for T-cell activation. Blocking the activity of IDO in a mouse model of arthritis led to increased severity of arthritis and accumulation of Th1 and Th17 cells in the inflamed joints. Conversely, administration of l -kynurenine, a metabolite of l -tryptophan, resulted in amelioration of arthritis. These findings suggest manipulation of tryptophan degradation as a therapeutic target in RA.
OX40/OX40L
OX40 (CD134) is predominantly expressed on activated CD4 and CD8 T cells following stimulation via TCR and CD28. Proinflammatory cytokines, IL-1, IL-2, and TNFα can further augment the expression of OX40. The ligand of OX40 and OX40L (CD252) is expressed on APCs including dendritic cells, B cells, and macrophages. OX40L is induced on APCs after stimulation via CD40. There is bidirectional activation of T cells and APCs via the OX40/OX40L pathway. Stimulation of OX40 on T cells induces proliferation and cytokine secretion, and signaling via OX40L into APCs induces the secretion of proinflammatory cytokines by APCs.
In RA, there is increased expression of OX40 on peripheral blood CD4 T cells with a trend toward a positive correlation with serum C-reactive protein levels. OX40 is expressed on CD4- and CD8-positive synovial T cells, more so on CD4-positive T cells, and levels of expression correlate with disease activity. OX40 and OX40L expressing cells are also present in the RA synovium. Although there is increased expression of OX40 and OX40L in the RA synovium, the role of OX40/OX40L in mediating immune events in RA is unclear. However, preclinical studies in animal models blocking the interaction of OX40/OX40L have shown promise.
CCL20
Chemokines mediate inflammatory responses by stimulating the recruitment of leucocytes. The chemokine CCL20 (MIP-3α) and its receptor CCR6 play a role in the migration of different cell types to the RA synovium. CCL20 is a CC-chemokine expressed on macrophages, dendritic cells, and lymphocytes. CCR6, the receptor for CCL20, is expressed on Th17 cells, immature dendritic cells, and B cells. CCL20 is chemotactic for Th17 cells and dendritic cells, which express CCR6. Th17 cells secrete CCL20 and recruit other CCR6-expressing Th17 cells to the site of Th17 cell-mediated damage. Increased levels of CCL20 have been observed in the synovial fluid of RA patients compared with OA patients, and protein concentrations of CCL20 are increased in the peripheral blood of patients with RA. Expression of CCL20 is induced in FLS by the synergistic interaction of proinflammatory cytokines, including TNFα, IL-1β and IL-17, and cytokine-stimulated FLS can recruit mononuclear cells in a CCL20/CC6–dependent manner. The results suggest that CCL20 produced by FLS recruits monocytes and Th17 T cells to the synovium and is an important chemokine in the pathogenesis of RA. Moreover, blockade of CCL20 binding to CCR6 with a neutralizing antibody was effective in treating arthritis in a T-cell transfer model in mice. Further supporting the role of CCL20 as an important chemokine in RA, a recent study demonstrated that treatment with infliximab, etanercept, or tocilizumab reduced serum levels of CCL20 in patients with RA.
IL-7
IL-7 is a member of the IL-2 family. IL-7 is associated with endothelial cells, FLS, and macrophages in the RA synovium and colocalizes with deposits of extracellular matrix collagen IV. IL-7R is composed of IL-7Rα and IL-2Rγ and is expressed on CD4 + and CD8 + T cells, NK T cells, and monocytes. IL-7 levels are increased in RA compared with OA synovial fluid. Serum IL-7 levels correlate with disease activity in RA. In RA patients who are poor responders to anti-TNF therapy, persistently increased serum levels of IL-7 are seen. Moreover, reduced levels of serum IL-7 were observed in patients with early RA treated with methotrexate, and the reduction correlated with disease suppression.
Within RA synovial biopsies, samples with lymphoid follicles demonstrated consistent IL-7 staining, and gene expression analysis of RA synovial samples revealed increased expression of genes involved in IL-7 signal transduction. IL-7 may be responsible for generation of tertiary lymphoid follicles observed in RA synovium, because IL7 is known to be crucial for the development of lymphoid tissue. Enhanced expression of IL-7Rα and IL-7 in patients with RA may contribute to joint inflammation by activating T cells, B cells, and macrophages because treatment with soluble human IL-7Rα inhibited IL-7R–mediated immune activation in vitro. These research findings suggest that IL-7 and its receptor are potential targets for immune modulation in RA therapy.
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