Membrane-anchored CD40 is processed by the tumor necrosis factor-alpha-converting enzyme. Implications for CD40 signaling

The soluble form of CD40 (sCD40), which co-exists with the membrane-anchored form (mCD40), is a natural antagonist of mCD40/CD154 interaction. However, the mechanism leading to the production of sCD40 has never been investigated. Here, we show that the engagement of mCD40 on the surface of B lymphocytes by anti-CD40 antibody led to enhanced sCD40 release associated with decreased amounts of mCD40. This sCD40 production was not affected by vesicular traffic inhibitors but was completely blocked by a broad-spectrum synthetic metalloproteinase (MP) inhibitor (GM6001) or a membrane-anchored MP-specific inhibitor (dec-RVKR-cmk). Recombinant MP disintegrin tumor necrosis factor-alpha converting enzyme (TACE) cleaved the purified CD40 ectodomain/Fc chimeric protein in vitro, giving rise to an sCD40 form similar to that shed from B cell cultures. Moreover, spontaneous production of sCD40 by mCD40-transfected human embryonic kidney cells (constitutively expressing TACE) was enhanced by the overexpression of TACE and abrogated by co-transfection with a dominant-negative TACE mutant. These results provide strong evidence that sCD40 production is an active process regulated by the engagement of mCD40 and its proteolytic cleavage by TACE or a related MP disintegrin. Given the antagonistic activity of sCD40 on the CD40/CD154 interaction, this shedding mechanism might represent an important negative feedback control of CD40 functions.     

A novel mechanism for TNFR-associated factor 6-dependent CD40 signaling

Members of the TNFR family play critical roles in the regulation of the immune system. One member of the family critical for efficient activation of T-dependent humoral immune responses is CD40, a cell surface protein expressed by B cells and other APC. The cytoplasmic domain of CD40 interacts with several members of the TNFR-associated factor (TRAF) family, which link CD40 to intracellular signaling pathways. TRAF2 and 6 appear to play particularly important roles in CD40 signaling. Previous studies suggest that the two molecules have certain overlapping roles in signaling, but that unique roles for each molecule also exist. To better define the roles of TRAF2 and TRAF6 in CD40 signaling, we used somatic cell gene targeting to generate TRAF-deficient mouse B cell lines. A20.2J cells deficient in TRAF6 exhibit marked defects in CD40-mediated JNK activation and the up-regulation of CD80. Our previous experiments with TRAF2-deficient B cell lines suggest that TRAF6 and TRAF2 may have redundant roles in CD40-mediated NF-kappaB activation. Consistent with this hypothesis, we found CD40-mediated activation of NF-kappaB intact in TRAF6-deficient cells and defective in cells lacking both TRAF2 and TRAF6. Interestingly, we found that TRAF6 mutants defective in CD40 binding were able to restore CD40-mediated JNK activation and CD80 up-regulation in TRAF6-deficient cells, indicating that TRAF6 may be able to contribute to certain CD40 signals without directly binding CD40.     

Recruitment of CD40 and tumor necrosis factor receptor-associated factors 2 and 3 to membrane microdomains during CD40 signaling

Signals delivered to antigen-presenting cells through CD40 are critical for the activation of immune responses. Intracellular tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are key elements of the signal transduction pathways of many TNF receptor family members, including CD40. We show for the first time that engagement of CD40 in intact B cells induces the rapid translocation of TRAF2 from the cytoplasm to the plasma membrane. We found that CD40 engagement also results in its recruitment, together with TRAF2 and TRAF3, to membrane microdomains, regions of the plasma membrane enriched in signaling molecules such as the Src family kinases. Using a membrane-permeable chelator of zinc or a mutant TRAF2 molecule, we show that the putative zinc-binding domains of TRAFs contribute to their recruitment to microdomains and to the downstream activation of c-Jun N-terminal kinase. We suggest that the zinc RING and zinc finger domains of TRAFs are required for communication between CD40 and microdomain-associated signaling molecules and may serve a similar role in the signal transduction pathways of other TNF receptor family members.     

Association between IL-6 and CD40 signaling. IL-6 induces phosphorylation of CD40 receptors.

CD40 mAb at subsaturating doses inhibit the growth of transformants of the M12 murine cell line expressing intact full length CD40 molecules (M12/CD40+ cells) but do not inhibit the growth of two M12 transformants expressing either a mutant CD40 cDNA missing most of the cytoplasmic tail (CD40/tailless) or a mutant cDNA with a substitution at residue 234 (CD40/234A, Ala for Thr). Using these transformants, we tested a panel of cytokines for the ability to mimic CD40 mAb. rIL-6 behaved like CD40 mAb and inhibited the growth of M12/CD40+ cells but not of CD40/tailless or CD40/234A mutants. The effect of IL-6 on M12/CD40+ cells not only required intact CD40 including threonine 234 but also was specific because IL-6 mAb blocked the inhibitory activity. The M12/CD40+ cells responsive to IL-6 expressed greater than 300,000 CD40 molecules/cells but, like M12/CD40-controls, expressed only small numbers (less than 50/cell) of high affinity IL-6R, indicating that CD40 is not a receptor for IL-6. Nevertheless, IL-6 utilizes intact CD40 efficiently when it signals these cells: treatment of M12/CD40+ cells with IL-6 induced increased phosphorylation of CD40. Conversely, triggering CD40 on M12/CD40+ cells leads to IL-6 production. Similar effects were evident in human CD40+ B cells: IL-6 increased the phosphorylation of CD40 in the IL-6-responsive cell line, CESS, and CD40 mAb induced IL-6 production in activated human B cells. Thus, CD40 may function to receive and regulate IL-6-dependent signals in B cells.     

CD40 signaling and Alzheimer's disease pathogenesis

The interaction between CD40 and its cognate ligand, CD40 ligand, is a primary regulator of the peripheral immune response, including modulation of T lymphocyte activation, B lymphocyte differentiation and antibody secretion, and innate immune cell activation, maturation, and survival. Recently, we and others have identified CD40 expression on a variety of CNS cells, including endothelial cells, smooth muscle cells, astroglia and microglia, and have found that, on many of these cells, CD40 expression is enhanced by pro-inflammatory stimuli. Importantly, the CD40–CD40 ligand interaction on microglia triggers a series of intracellular signaling events that are discussed, beginning with Src-family kinase activation and culminating in microglial activation as evidenced by tumor necrosis factor- secretion. Based on the involvement of microglial activation and brain inflammation in Alzheimer's disease pathogenesis, we have investigated co-stimulation of microglia, smooth muscle, and endothelial cells with CD40 ligand in the presence of low doses of freshly solubilized amyloid-β peptides. Data reviewed herein show that CD40 ligand and amyloid-β act synergistically to promote pro-inflammatory responses by these cells, including secretion of interleukin-1β by endothelial cells and tumor necrosis factor- by microglia. As these cytokines have been implicated in neuronal injury, a comprehensive model of pro-inflammatory CD40 ligand and amyloid-β initiated Alzheimer's disease pathogenesis (mediated by multiple CNS cells) is proposed.     

CD40 engagement on synovial fibroblast up-regulates production of vascular endothelial growth factor

We tested the impact of CD40 engagement on the production of vascular endothelial growth factor (VEGF) from rheumatoid synovial fibroblasts. Fibroblast-like synovial cells (FLS) were prepared from the synovial tissues of rheumatoid arthritis patients and cultured in the presence of CD40 ligand-transfected (CD40L+) L cells. VEGF levels were determined in the culture supernatants by ELISA. Stimulation of FLS by CD40L+ L cells increased the production of VEGF by 4.1-fold over the constitutive levels of unstimulated FLS. The CD40L on activated T cells from rheumatoid synovial fluid also up-regulated VEGF production from FLS. Neither indomethacin nor Abs to IL-1beta, TNF-alpha, and TGF-beta did affect CD40L-induced VEGF production. Stimulation of FLS with TNF-alpha, IL-1beta, and TGF-beta increased VEGF production by 1.6-, 2.0-, and 5.2-fold, respectively, and displayed an additive effect on the production of VEGF by CD40L. VEGF mRNA expression was also up-regulated by the stimulation of FLS with membranes from the CD40L+ L cells. Dexamethasone completely abrogated CD40L-induced VEGF production. In addition, pyrrolidine dithiocarbamate partially down-regulated CD40L-induced VEGF production, showing that the NF-kappaB pathway was partly involved in the signaling of CD40L leading to VEGF production. Collectively, these results suggest that the interaction between CD40 on synovial fibroblasts and CD40L expressed on activated T lymphocytes may be directly involved in the neovascularization in rheumatoid synovitis by enhancing the production of VEGF.     

Intrinsic AHR in IL-5 transgenic mice is dependent on CD4(+) cells and CD49d-mediated signaling

Overexpression of interleukin (IL)-5 by the airway epithelium in mice using the rat CC10 promoter (NJ.1726 line) leads to several histopathologies characteristic of human asthma, including airway hyperreactivity (AHR). We investigated the contribution of B and T cells, as well as CD4 expression, to the development of AHR in IL-5 transgenic mice. NJ.1726 mice on a T cell or CD4 knockout background, but not on a B cell knockout background, lost intrinsic AHR. These effects occurred without decreases in IL-5 or eosinophils. We further investigated the contribution of alpha(4)-integrin signaling to the development of AHR in IL-5 transgenic mice through the administration of anti-CD49d (alpha(4)-integrin) antibody (PS/2). Administration of PS/2 resulted in immediate (16-h) inhibition of AHR. The inhibition of AHR was not associated with a decrease in airway eosinophils. These studies demonstrate that, despite the presence of increased levels of IL-5 and eosinophils in the lungs of NJ.1726 mice, CD4(+) cells and alpha(4)-integrin signaling are necessary for the intrinsic AHR that develops in IL-5 transgenic mice.     

Requirement of oxidation-dependent CD40 homodimers for CD154/CD40 bidirectional signaling

It is well established that the CD154/CD40 interaction is required for T cell-dependent B cell differentiation and maturation. However, the early molecular and structural mechanisms that orchestrate CD154 and CD40 signaling at the T cell/APC contact site are not well understood. We demonstrated that CD40 engagement induces the formation of disulfide-linked (dl) CD40 homodimers that predominantly associate with detergent-resistant membrane microdomains. Mutagenesis and biochemical analyses revealed that (a) the integrity of the detergent-resistant membranes is necessary for dl-CD40 homodimer formation, (b) the cytoplasmic Cys(238) of CD40 is the target for the de novo disulfide oxidation induced by receptor oligomerization, and (c) dl-CD40 homodimer formation is required for CD40-induced interleukin-8 secretion. Stimulation of CD154-positive T cells with staphylococcal enterotoxin E superantigen that mimics nominal antigen in initiating cognate T cell/APC interaction revealed that dl-CD40 homodimer formation is required for interleukin-2 production by T cells. These findings indicate that dl-CD40 homodimer formation has a physiological role in regulating bidirectional signaling.     

CD40 signaling in human dendritic cells is initiated within membrane rafts

Despite CD40's role in stimulating dendritic cells (DCs) for efficient specific T-cell stimulation, its signal transduction components in DCs are still poorly documented. We show that CD40 receptors on human monocyte-derived DCs associate with sphingolipid- and cholesterol-rich plasma membrane microdomains, termed membrane rafts. Following engagement, CD40 utilizes membrane raft-associated Lyn Src family kinase, and possibly other raft-associated Src family kinases, to initiate tyrosine phosphorylation of intracellular substrates. CD40 engagement also leads to a membrane raft-restricted recruitment of tumor necrosis factor (TNF) receptor-associated factor (TRAF) 3 and, to a lesser extent, TRAF2, to CD40's cytoplasmic tail. Thus, the membrane raft structure plays an integral role in proximal events of CD40 signaling in DCs. We demonstrate that stimulation of Src family kinase within membrane rafts initiates a pathway implicating ERK activation, which leads to interleukin (IL)-1alpha/beta and IL-1Ra mRNA production and contributes to p38-dependent IL-12 mRNA production. These results provide the first evidence that membrane rafts play a critical role in initiation of CD40 signaling in DCs, and delineate the outcome of CD40-mediated pathways on cytokine production.     

CD40-40L signaling in vascular inflammation

Ligation of CD40 in circulating cells or in the vessel wall may promote mononuclear cell recruitment, participate in the weakening of the plaque, and contribute to thrombosis. This process appears to be redox-sensitive, but the precise signaling mechanism by which the interaction between CD40L and its receptor CD40 mediates inflammatory secretion is unclear. Our previous studies have shown that the CD40-CD40L interaction modulates release of reactive oxygen species (ROS) and the current findings demonstrate that in endothelial cells CD40L dose dependently induces intracellular CD40L and MCP1 release in a redox sensitive manner. Pharmacological inhibition of phosphatidylinositol 3-kinase and p38 MAPK as well as adenovirus-mediated inactivation of Akt and p38 MAPK inhibited CD40L effects on endothelial cells. Akt, in particular, appeared to mediate CD40L-induced CD40L synthesis and MCP1 release by endothelial cells in a redox sensitive manner via NFkappaB activation. In addition, using confocal microscopy, exogenous addition of recombinant CD40L or adenoviral mediated CD40L overexpression was found to stimulate nuclear translocation of NFkappaB, which was further augmented by Akt overexpression and inhibited by Akt inactivation. These data support a mechanism whereby redox-sensitive CD40-CD40L interactions induce activation of Akt and p38 MAPK, leading to stimulation of NFkappaB and enhanced synthesis of CD40L and MCP1. Increased CD40L and MCP1 may contribute to the adherence of CD40-positive cells, such as platelets and monocytes, to the vessel wall modulating atherothrombosis.     

The development of functional CD8 T cell memory after Listeria monocytogenes infection is not dependent on CD40

The immunologic requirements for generating long-lived protective CD8 T cell memory remain unclear. Memory CD8 populations generated in the absence of CD4 Th cells reportedly have functional defects, and at least a subset of CD8 T cells transiently express CD40 after activation, suggesting that direct CD4-CD8 T cell interactions through CD40 may influence the magnitude and functional quality of memory CD8 populations. To ascertain the role of CD40 in such direct T cell interactions, we investigated CD8 T cell responses in CD40-/- mice after infection with Listeria monocytogenes, an intracellular bacterium that induces APC activation and thus priming of CD8 T cells independently of CD4 Th cell help through CD40. In this study we show that memory CD8 T cells generated in CD40-deficient mice show in vivo cytotoxicity and cytokine production equivalent to CD8 memory T cells from wild-type mice. Upon secondary Listeria infection, CD40-/- memory CD8 T cells expand to greater numbers than seen in wild-type mice. These results indicate that CD40 ligation on CD8 T cells, although reportedly a part of CD8 T cell memory development in an H-Y-directed response, is not needed for the development of functional memory CD8 T cell populations after Listeria infection.     

Molecular characterization of CD40 signaling intermediates

Signal transduction through the CD40 receptor is initiated by binding of its trimeric ligand and propagated by interactions of tumor necrosis factor receptor-associated factor (TRAF) proteins with the multimerized CD40 cytoplasmic domain. Using defined multimeric constructs of the CD40 cytoplasmic domain expressed as either soluble or myristoylated proteins, we have addressed the extent of receptor multimerization needed to initiate signal transduction and identified components of CD40 signaling complexes. Signal transduction in human embryonic kidney 293 cells, measured by nuclear factor kappaB activation, was observed in cells expressing soluble trimeric CD40 cytoplasmic domain and to a lesser extent in cells expressing dimeric CD40 cytoplasmic domain. Nuclear factor kappaB activation was strongest in cells expressing myristoylated trimeric CD40 cytoplasmic domain. Signal transduction through trimeric CD40 cytoplasmic domains with various point mutations in the TRAF binding sites was similar to signal transduction through analogous full-length receptors. Transiently expressed soluble trimeric CD40 cytoplasmic domain was isolated as complexes that contained TRAF2, TRAF3, TRAF5, TRAF6, and the inhibitor of apoptosis protein (c-IAP1). Association of c-IAP1 with the CD40 cytoplasmic domain complex was indirect and dependent on the presence of an intact TRAF1/2/3 binding site. These results suggest that extracellular ligation of CD40 can be bypassed and that soluble trimerized CD40 complexes can be isolated and used to identify components that link CD40 with signaling pathways.     

OX40-mediated memory T cell generation is TNF receptor-associated factor 2 dependent

Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adapter protein that associates with the cytoplasmic tail of OX40, may play a critical role in OX40-mediated signal transduction. To investigate the in vivo role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we bred OVA-specific TCR transgenic mice to TRAF2 dominant-negative (TRAF2 DN) mice. Following Ag stimulation and OX40 engagement of TRAF2 DN T cells in vivo, the number of long-lived OVA-specific T cells and effector T cell function was dramatically reduced when compared with wild-type T cells. We also demonstrate that CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF2 DN defect was partially overcome by CTLA-4 blockade in vivo. The data provide evidence that TRAF2 is linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.     

beta-Chemokine production in CD40L-stimulated monocyte-derived macrophages requires activation of MAPK signaling pathways

CD40 ligand is a cell surface molecule on CD4(+) T cells that interacts with its receptor, CD40, on antigen presenting cells to mediate humoral and cellular immune responses. Our previous studies demonstrated that a trimeric soluble form of CD40L (CD40LT) activates macrophages to produce beta-chemokines and decrease CCR5 and CD4 cell surface expression, thus inducing resistance to HIV-1 infection. However, the mechanism(s) by which CD40LT mediates these effects in primary macrophages remains unclear. In this report, we demonstrate that CD40LT induces synthesis of beta-chemokines through the activation of MAPK signaling pathways. Treatment of macrophages with CD40LT results in a rapid activation of p38 and ERK1/2 mitogen-activated protein kinases. Inhibitors of these MAPKs blocked beta-chemokine production, while protein kinase A and C inhibitors had little or no effect. We also provide evidence that CD40LT stimulates beta-chemokine production directly, as well as indirectly via a TNF-alpha-dependent mechanism. At the early time points, CD40LT directly stimulated beta-chemokine production, whereas at later time points the effect was mediated to some extent by TNF-alpha. In conclusion, our results suggest that CD40-CD40L interactions are important for the activation of monocyte-derived macrophage antiviral response affecting both viral replication and the recruitment of immune cells.     

Methacholine-induced airway hyperresponsiveness is dependent on Galphaq signaling

Airway function in health and disease as well as in response to bronchospastic stimuli (i.e., irritants, allergens, and inflammatory mediators) is controlled, in part, by cholinergic muscarinic receptor regulation of smooth muscle. In particular, the dependence of airway smooth muscle contraction/relaxation on heterotrimeric G protein-coupled receptor signaling suggests that these events underlie the responses regulating airway function. Galphaq-containing G proteins are proposed to be a prominent signaling pathway, and the availability of knockout mice deficient of this subunit has allowed for an investigation of its potential role in airway function. Airway responses in Galphaq-deficient mice (activities assessed by both tracheal tension and in vivo lung function measurements) were attenuated relative to wild-type controls. Moreover, ovalbumin sensitization/aerosol challenge of Galphaq-deficient mice also failed to elicit an allergen-induced increase in airway reactivity to methacholine. These findings indicate that cholinergic receptor-mediated responses are dependent on Galphaq-mediated signaling events and identify Galphaq as a potential target of preventative/intervening therapies for lung dysfunction.     

Interleukin-1 signaling is dependent on free thiols

Stimulation of the Interleukin-1 receptor type I (IL-1-RI) with IL-1 activates an associated serine/threonine kinase, IRAK, which phosphorylates downstream targets, resulting in NFkappaB activation. The signaling cascade is accompanied by oxidative processes and contains putative targets for redox regulation. Preincubation of the murine T cell line EL-4 and the human umbilical cord vein endothelial cell line ECV 304 with thiol modifying compounds like diamide, menadione or phenylarsine oxide inhibited the IL-1-induced phosphorylation of an endogenous substrate with a molecular mass of 60 kD. In the endothelial cell line, a second target of about 85 kD was phosphorylated after IL-1 stimulation, which was also inhibited by thiol modification. These data suggest that IL-1 signal transduction depends on free thiols which might be targets for redox regulation not only in lymphocytes, but also in endothelial cells.     

IL-12p70 production by Leishmania major-harboring human dendritic cells is a CD40/CD40 ligand-dependent process

Leishmaniasis, a vector-borne parasitic disease, is transmitted during a sandfly blood meal as the parasite is delivered into the dermis. The parasite displays a unique immune evasion mechanism: prevention of IL-12 production within its host cell, the macrophage (i.e., where it differentiates and multiplies). Given the close proximity of skin dendritic cells (DC) to the site of parasite delivery, their critical role in initiating immune responses and the self-healing nature of Leishmania major (Lm) infection, we examined the interaction between myeloid-derived human DC and Lm metacyclic promastigotes (infectious-stage parasites) to model the early "natural" events of infection. We found that DC can take up Lm and, after this internalization, undergo changes in surface phenotype suggesting "maturation". Despite the intracellular location of the parasite and resultant up-regulation of costimulatory and class II molecules, there was no detectable cytokine release by these Lm-harboring DC. However, using intracellular staining and flow cytometry to analyze cytokine production at the single-cell level, we found that Lm-harboring DC, but not monocytes, produce large amounts of IL-12p70 in a CD40 ligand (CD40L)-dependent manner. Finally, DC generated from mononuclear cells from patients with cutaneous leishmaniasis (Lm), once loaded with live metacyclic promastigotes, were found to reactivate autologous primed T lymphocytes and induce a CD40L-dependent IFN-gamma response. Our results link the required CD40/CD40L interactions for healing with DC-derived IL-12p70 production and provide a mechanism to explain the genesis of a protective T cell-mediated response in the face of local immune evasion within the macrophage at the site of Leishmania delivery.     

Human orbital fibroblasts are activated through CD40 to induce proinflammatory cytokine production

CD40 is an important signaling and activation antigen found oncertain bone marrow-derived cells. Recently, CD40 has also been shownto be expressed by nonhematopoietic cells, including certain humanfibroblasts, but not others. Little is known about the function of CD40on fibroblasts. The current study investigates the hypothesis that CD40is expressed on orbital fibroblasts and represents a pathway forinteraction between these fibroblasts and CD40 ligand-expressing cells,such as T lymphocytes and mast cells. We report here that orbitalconnective tissue fibroblasts, obtained from normal donors and frompatients with severe thyroid-associated ophthalmopathy (TAO), expressfunctional CD40. CD40 is upregulated ~10-fold by interferon- (500 U/ml) treatment for 72 h. These fibroblasts become activated throughtriggering of CD40 with CD40 ligand (CD40L). This is evidenced bynuclear translocation of nuclear factor-B and induction of theproinflammatory and chemoattractant cytokines interleukin-6 andinterleukin-8, respectively. These data support the concept thatcognate interactions between orbital fibroblasts and infiltrating Tlymphocytes, via the CD40-CD40L pathway, may promote the tissueremodeling observed in TAO and other inflammatory diseases of theorbit. Disruption of the CD40-CD40L interaction may represent atherapeutic intervention to reduce the inflammatory components of TAO,which remains a vexing clinical problem.

     

Synoviocyte-mediated expansion of inflammatory T cells in rheumatoid synovitis is dependent on CD47-thrombospondin 1 interaction

Fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis elicit spontaneous proliferation of autologous T cells in an HLA-DR and CD47 costimulation-dependent manner. T cell costimulation through CD47 is attributed to specific interaction with thrombospondin-1 (TSP1), a CD47 ligand displayed on FLS. CD47 binding by FLS has broad biological impact that includes adhesion and the triggering of specific costimulatory signals. TSP1(+) FLS are highly adhesive to T cells and support their aggregation and growth in situ. Long-term cultures of T cells and FLS form heterotypic foci that are amenable to propagation without exogenous growth factors. T cell adhesion and aggregate formation on TSP1(+) FLS substrates are inhibited by CD47-binding peptides. In contrast, FLS from arthroscopy controls lack adhesive or T cell growth-promoting activities. CD47 stimulation transduces a costimulatory signal different from that of CD28, producing a gene expression profile that included induction of ferritin L chain, a component of the inflammatory response. Ferritin L chain augments CD3-induced proliferation of T cells. Collectively, these results demonstrate the active role of FLS in the recruitment, activation, and expansion of T cells in a CD47-dependent manner. Because TSP1 is abundantly expressed in the rheumatoid synovium, CD47-TSP1 interaction is proposed to be a key component of an FLS/T cell regulatory circuit that perpetuates the inflammatory process in the rheumatoid joint.