Glycoprotein 96 perpetuates the persistent inflammation of rheumatoid arthritis

Objective

The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. The aim of this study was to determine the contribution of endogenous gp96 in Toll‐like receptor (TLR)–mediated macrophage activation in RA.

Methods

RA synovial fluid was used to activate macrophages and HEK–TLR‐2 and HEK–TLR‐4 cells. Neutralizing antibodies to TLR‐2, TLR‐4, and gp96 were used to inhibit activation. RA synovial fluid macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of tumor necrosis factor α (TNFα) or interleukin‐8 messenger RNA. Arthritis was induced in mice by K/BxN serum transfer. The expression of gp96 was determined by immunoblot analysis, enzyme‐linked immunosorbent assay, and immunohistochemistry. Arthritis was treated with neutralizing anti‐gp96 antiserum or control serum.

Results

RA synovial fluid induced the activation of macrophages and HEK–TLR‐2 and HEK–TLR‐4 cells. RA synovial fluid–induced macrophage and HEK–TLR‐2 activation was suppressed by neutralizing anti‐gp96 antibodies only in the presence of high (>800 ng/ml) rather than low (<400 ng/ml) concentrations of gp96. Neutralization of RA synovial fluid macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting the role of gp96 in RA, joint tissue gp96 expression was induced in mice with the K/BxN serum–induced arthritis, and neutralizing antibodies to gp96 ameliorated joint inflammation, as determined by clinical and histologic examination.

Conclusion

These observations support the notion that gp96 plays a role as an endogenous TLR‐2 ligand in RA and identify the TLR‐2 pathway as a therapeutic target.

     

The safety and efficacy of a JAK inhibitor in patients with active rheumatoid arthritis: Results of a double‐blind,placebo‐controlled phase IIa trial of three dosage levels of CP‐690,550 versus placebo

Objective

To determine the efficacy, safety, and tolerability of 3 different dosages of CP‐690,550, a potent, orally active JAK inhibitor, in patients with active rheumatoid arthritis (RA) in whom methotrexate, etanercept, infliximab, or adalimumab caused an inadequate or toxic response.

Methods

Patients (n = 264) were randomized equally to receive placebo, 5 mg of CP‐690,550, 15 mg of CP‐690,550, or 30 mg of CP‐690,550 twice daily for 6 weeks, and were followed up for an additional 6 weeks after treatment. The primary efficacy end point was the American College of Rheumatology 20% improvement criteria (ACR20) response rate at 6 weeks.

Results

By week 6, the ACR20 response rates were 70.5%, 81.2%, and 76.8% in the 5 mg, 15 mg, and 30 mg twice daily groups, respectively, compared with 29.2% in the placebo group (P < 0.001). Improvements in disease activity in CP‐690,550–treated patients compared with placebo were seen in all treatment groups as early as week 1. ACR50 and ACR70 response rates significantly improved in all treatment groups by week 4. The most common adverse events reported were headache and nausea. The infection rate in both the 15 mg twice daily group and the 30 mg twice daily group was 30.4% (versus 26.2% in the placebo group). No opportunistic infections or deaths occurred. Increases in mean low‐density lipoprotein cholesterol and high‐density lipoprotein cholesterol levels, and increases in mean serum creatinine level (0.04–0.06 mg/dl) were seen in all CP‐690,550 treatment arms.

Conclusion

Our findings indicate that CP‐690,550 is efficacious in the treatment of RA, resulting in rapid, statistically significant, and clinically meaningful reductions in the signs and symptoms of RA. Further studies of CP‐690,550 in RA are warranted.

     

Increased expression of Fcγ receptors II and III on macrophages of rheumatoid arthritis patients results in higher production of tumor necrosis factor α and matrix metalloproteinase

Objective

To evaluate Fcγ receptor (FcγR) expression on synovial macrophages from rheumatoid arthritis (RA) patients and to determine whether this expression correlates with the production of the proinflammatory cytokines tumor necrosis factor α (TNFα), interleukin‐1β (IL‐1β), IL‐12, and matrix metalloproteinase 1 (MMP‐1). We also sought to determine whether mature macrophages from RA patients express aberrant levels of FcγRI, FcγRII, and FcγRIII, and to determine the production of inflammatory mediators after immune complex (IC) stimulation.

Methods

Immunohistochemistry was performed on cryostat sections of synovial biopsy specimens obtained from 27 RA patients and 5 controls. FcγR I, II, and III were detected, as well as the proinflammatory mediators IL‐1, TNFα, IL‐12, and MMP‐1. Monocytes were isolated from the blood of 10 RA patients and 10 healthy controls and cultured for 7 days with macrophage colony‐stimulating factor to obtain macrophages. Using fluorescence‐activated cell sorting, the expression of FcγRI, FcγRII, and FcγRIII was determined. On day 7, macrophages were stimulated with heat‐aggregated gamma globulins (HAGGs) for 24 hours. Production of cytokines was measured using enzyme‐linked immunosorbent assay, and production of gelatinases/collagenases was measured by degradation of fluorescent gelatin.

Results

Immunohistochemistry showed higher FcγRII and FcγRIII expression in RA synovium than in controls. FcγRII and FcγRIII, but not FcγRI, were highly correlated with the number of synovial macrophages. Consistent with this, TNFα expression correlated positively with FcγRIII expression. Moreover, MMP‐1 expression strongly correlated with FcγR I, II, and III expression. Mature macrophages from RA patients showed significantly enhanced expression of FcγRII and FcγRIII compared with controls. Twenty‐four hours after stimulation of RA macrophages with HAGGs, significantly higher production of TNFα and gelatinase/collagenase was measured.

Conclusion

RA synovium and mature RA macrophages express significantly elevated levels of FcγRII and FcγRIII, resulting in much higher production of TNFα and gelatinase/collagenase after IC stimulation. These data suggest that disturbed expression of FcγR on mature synovial macrophages is involved in the pathology of RA.

     

Galectin 3 induces a distinctive pattern of cytokine and chemokine production in rheumatoid synovial fibroblasts via selective signaling pathways

Objective

High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts.

Methods

Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor α (TNFα), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme‐linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF‐κB activation assay.

Results

Galectin 3 induced secretion of interleukin‐6 (IL‐6), granulocyte–macrophage colony‐stimulating factor, CXCL8, and MMP‐3 in both synovial and skin fibroblasts. By contrast, galectin 3–induced secretion of TNFα, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFα blockade ruled out autocrine TNFα‐stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL‐6 production, but phosphatidylinositol 3‐kinase (PI 3‐kinase) was required for selective CCL5 induction. NF‐κB activation was required for production of both IL‐6 and CCL5.

Conclusion

Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell–recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3‐kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.

     

Rheumatoid arthritis synovial fluid macrophages express decreased tumor necrosis factor–related apoptosis‐inducing ligand R2 and increased decoy receptor tumor necrosis factor–related apoptosis‐inducing ligand R3

Objective

To characterize the expression pattern of tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) and its cognate receptors (TRAIL R1, R2, R3, and R4) on rheumatoid arthritis (RA) synovial fluid (SF) lymphocytes and monocyte/macrophages and on cultured RA synovial fibroblasts.

Methods

The expression of TRAIL and TRAIL receptors on RA SF lymphocytes and monocyte/macrophages, normal macrophages, and RA synovial fibroblasts was examined by flow cytometry with previously characterized monoclonal antibodies. The ability of adenoviral‐mediated delivery of TRAIL to induce macrophage or RA synovial fibroblast apoptosis was examined by flow cytometry.

Results

By flow cytometry, neither TRAIL nor its cognate receptors was detectable on RA SF lymphocytes or RA synovial fibroblasts. In contrast, RA SF macrophages expressed TRAIL R3, a decoy receptor (P < 0.01 versus isotype control), but not TRAIL, or TRAIL R1, R2, or R4. Normal peripheral blood–derived monocyte‐differentiated macrophages expressed TRAIL R2 (P < 0.01), but not TRAIL or the other TRAIL receptors. Adenoviral‐mediated delivery of TRAIL had no effect on the survival of normal macrophages or RA synovial fibroblasts but readily induced apoptosis in the prostate cancer cell line (PC‐3) that expressed TRAIL R1 and R2.

Conclusion

TRAIL R1 and R2, which are required for signal transmission by TRAIL, were not detected on RA SF lymphocytes, macrophages, or synovial fibroblasts. These observations do not support a potential therapeutic role for TRAIL in RA.

     

Effectiveness of anti–folate receptor β antibody conjugated with truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages

Objective

To define the distribution of folate receptor β (FRβ)–expressing cells in various tissues, including rheumatoid arthritis (RA) synovial tissues, and to verify the effects of an immunotoxin composed of an anti‐FRβ monoclonal antibody (mAb) and truncated Pseudomonas exotoxin A (PEA) on apoptosis and tumor necrosis factor α (TNFα) production by adherent synovial mononuclear cells from RA patients.

Methods

Anti‐FRβ mAb were produced by immunizing mice with FRβ‐transfected murine pre–B cells. The distribution of the FRβ antigen was examined by immunohistochemical analysis using anti‐FRβ mAb and macrophage‐specific anti‐CD163 mAb. Anti‐FRβ mAb was chemically crosslinked with truncated PEA. FRβ‐expressing macrophages were produced by the transfection of adenovirus vector containing the FRβ gene. Apoptotic cells were detected by staining with propidium iodide. TNFα was measured by enzyme‐linked immunosorbent assay.

Results

FRβ‐expressing cells were not present in peripheral blood leukocytes and their activated cells. In all of the tissues examined, most FRβ‐expressing cells were CD163+. The immunotoxin significantly induced the apoptosis of FRβ‐transfected macrophages and adherent RA synovial mononuclear cells and inhibited TNFα production by adherent RA synovial mononuclear cells.

Conclusion

We demonstrated the limited distribution of FRβ‐expressing cells in various tissues. The immunotoxin targeting FRβ‐expressing cells will provide a therapeutic tool for rheumatoid synovitis.

     

Heterogeneous requirement of IκB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: Implications for therapy

Objective

To investigate the potential role of IκB kinase 1 (IKK‐1) and IKK‐2 in the regulation of nuclear factor κB (NF‐κB) activation and the expression of tumor necrosis factor α (TNFα), as well as interleukin‐1β (IL‐1β), IL‐6, IL‐8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), in rheumatoid arthritis (RA).

Methods

Recombinant adenoviruses expressing β‐galactosidase, dominant‐negative IKK‐1 and IKK‐2, or IκBα were used to infect ex vivo RA synovial membrane cultures and synovial fibroblasts obtained from patients with RA undergoing joint replacement surgery, or human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), and monocyte‐derived macrophages from healthy volunteers. Then, their effect on the spontaneous or stimulus‐induced release of inflammatory cytokines, VEGF, and MMPs from RA synovial membrane cells was examined.

Results

IKK‐2 was not required for lipopolysaccharide (LPS)–induced NF‐κB activation or TNFα, IL‐6, or IL‐8 production in macrophages, but was essential for this process in response to CD40 ligand, TNFα, and IL‐1. In synovial fibroblasts, dermal fibroblasts, and HUVECs, IKK‐2 was also required for LPS‐induced NF‐κB activation and IL‐6 or IL‐8 production. In RA synovial membrane cells, IKK‐2 inhibition had no effect on spontaneous TNFα production but significantly reduced IL‐1β, IL‐6, IL‐8, VEGF, and MMPs 1, 2, 3, and 13.

Conclusion

Our study demonstrates that IKK‐2 is not essential for TNFα production in RA. However, because IKK‐2 regulates the expression of other inflammatory cytokines (IL‐1β, IL‐6, and IL‐8), VEGF, and MMPs 1, 2, 3, and 13, which are involved in the inflammatory, angiogenic, and destructive processes in the RA joint, it may still be a good therapeutic target.

     

Blocking ERK‐1/2 reduces tumor necrosis factor α–induced interleukin‐18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin‐18 binding protein A

Objective

To examine the mechanism of regulation of interleukin‐18 (IL‐18) bioactivity by IL‐18 binding protein (IL‐18BP) induction.

Methods

Levels of IL‐18 and IL‐18BPa in synovial fluid samples from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) were determined by enzyme‐linked immunosorbent assays (ELISAs), followed by calculation of free IL‐18. IL‐18 and IL‐18BPa synthesis in RA synovial fibroblasts that had been treated with proinflammatory and antiinflammatory cytokines were assessed by quantitative real‐time polymerase chain reaction and ELISA, respectively, followed by IL‐18 bioactivity determination using KG‐1 cells. Chemical signaling inhibitors were used for determination of the signal transduction pathways involved in IL‐18BPa/IL‐18 regulation. Tumor necrosis factor α (TNFα)–induced caspase 1 activity was determined by a colorimetric assay.

Results

IL‐18BPa was lower in RA synovial fluid than in OA synovial fluid (P < 0.05; n = 8), and free IL‐18 was higher in RA synovial fluid than in OA synovial fluid. TNFα induced RA synovial fibroblast IL‐18BPa and IL‐18 in a time‐dependent manner (P < 0.05). Evaluation of signaling pathways suggested that TNFα induced IL‐18 production through the ERK‐1/2, protein kinase Cδ (PKCδ), and Src pathways, whereas IL‐18BPa synthesis was mediated through the NFκB, PKC, Src, and JNK pathways. Furthermore, addition of exogenous IL‐18BPa‐Fc reduced the RA synovial fibroblast phosphorylation of ERK‐1/2 induced by TNFα.

Conclusion

These results suggest that IL‐18BPa reduces IL‐18 bioactivity induced by TNFα, by regulating the ERK‐1/2 pathway in RA synovial fibroblasts. Targeting IL‐18 bioactivity by induction or addition of IL‐18BPa may provide another therapeutic option in the management of RA.

     

Monoclonal anti‐CD8 therapy induces disease amelioration in the K/BxN mouse model of spontaneous chronic polyarthritis

Objective

CD8+ T cells are part of the T cell pool infiltrating the synovium in rheumatoid arthritis (RA). However, their role in the pathogenesis of RA has not been fully delineated. Using the K/BxN mouse model of spontaneous chronic arthritis, which shares many similarities with RA, we studied the potential of CD8+ T cell depletion with monoclonal antibodies (mAb) to stop and reverse the progression of experimental arthritis.

Methods

CD8+ T cells from the blood and articular infiltrate of K/BxN mice were characterized for cell surface phenotypic markers and for cytokine production. Additionally, mice were treated with specific anti‐CD8 mAb (YTS105 and YTS169.4), with and without thymectomy.

Results

CD8+ T cells from the peripheral blood and joints of K/BxN mice were mainly CD69+ and CD62L−CD27+ T cells expressing proinflammatory cytokines (interferon‐γ [IFNγ], tumor necrosis factor α [TNFα], interleukin‐17a [IL‐17A], and IL‐4), and granzyme B. In mice receiving anti‐CD8 mAb, the arthritis score improved 5 days after treatment. Recovery of the CD8+ T cells was associated with a new increase in the arthritis score after 20 days. In thymectomized and anti‐CD8 mAb–treated mice, the arthritis score improved permanently. Histologic analysis showed an absence of inflammatory infiltrate in the anti‐CD8 mAb–treated mice. In anti‐CD8 mAb–treated mice, the serologic levels of TNFα, IFNγ, IL‐6, and IL‐5 normalized. The levels of the disease‐related anti–glucose‐6‐phosphate isomerase antibodies did not change.

Conclusion

These results indicate that synovial activated effector CD8+ T cells locally synthesize proinflammatory cytokines (IFNγ, TNFα, IL‐17, IL‐6) and granzyme B in the arthritic joint, thus playing a pivotal role in maintaining chronic synovitis in the K/BxN mouse model of arthritis.

     

Cyclin‐dependent kinase inhibitor p21, via its C‐terminal domain,is essential for resolution of murine inflammatory arthritis

Objective

The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin‐dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21‐mediated suppression of experimental inflammatory arthritis.

Methods

Experimental arthritis was induced in wild‐type or p21^−/− (C57BL/6) mice, using the K/BxN serum–transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide‐induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex‐based assay, flow cytometry, or enzyme‐linked immunosorbent assay.

Results

Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21^−/− mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21‐intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages.

Conclusion

These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.

     

Expression of hypoxia‐inducible factor 1α by macrophages in the rheumatoid synovium: Implications for targeting of therapeutic genes to the inflamed joint

Objective

To determine if the rheumatoid synovium is a suitable target for hypoxia‐regulated gene therapy.

Methods

Sequential sections of wax‐embedded synovial membrane samples were obtained from 10 patients with rheumatoid arthritis (RA), 10 with primary osteoarthritis (OA), and from 6 healthy controls. Membrane sections from each patient were immunostained for hypoxia‐inducible factor 1α (HIF‐1α) and CD68 (a pan–macrophage marker).

Results

HIF‐1α was expressed abundantly by macrophages in most rheumatoid synovia, predominantly close to the intimal layer but also in the subintimal zone. There was markedly lower expression of HIF‐1α in OA synovia, and it was absent from all of the healthy synovia.

Conclusion

These observations indicate that macrophages transduced with a therapeutic gene under the control of a hypoxia‐inducible promoter could be administered to RA patients systemically. Migration of these cells to synovial tissue would result in the transgene being switched on in diseased joints but not in healthy tissues.

     

Dysregulation of interleukin‐10–dependent gene expression in rheumatoid arthritis synovial macrophages

Objective

The inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin‐1 (IL‐1) are produced by activated macrophages, are key mediators of pathogenesis, and are validated therapeutic targets in rheumatoid arthritis (RA) and seronegative spondylarthritis (SpA). IL‐10 is a potent antiinflammatory cytokine that suppresses macrophage TNFα and IL‐1 production, yet is not effective in suppressing inflammatory arthritis. To gain insight into IL‐10 responses in inflammatory arthritis, we used microarray analysis to determine the patterns of IL‐10–inducible gene expression in freshly isolated RA and seronegative SpA synovial macrophages.

Methods

Macrophages from the synovial fluid of 5 patients with RA and 3 with seronegative SpA (2 with psoriatic arthritis and 1 with ankylosing spondylitis) were isolated by positive selection and stimulated ex vivo with IL‐10 or interferon‐γ (IFNγ). Gene expression was analyzed using Affymetrix microarrays and protocols. Real‐time polymerase chain reaction was used to confirm changes in gene expression.

Results

The number of genes induced by IL‐10 in arthritic macrophages was markedly smaller than that induced in control macrophages, and the strength of induction was lower in arthritic macrophages for most genes. The residual response of arthritic macrophages to IL‐10 stimulation was qualitatively altered, such that IL‐10 preferentially increased expression of IFNγ‐inducible genes. In contrast, arthritic macrophages expressed many IFNγ‐inducible genes prior to stimulation, and their response to IFNγ remained mostly intact.

Conclusion

These results demonstrate that IL‐10 responses are dysregulated in RA synovial macrophages. An altered biologic response to IL‐10, with attenuation of its antiinflammatory function and a concomitant retention of IFNγ‐like activating functions, provides a basis for the lack of efficacy of IL‐10 in suppressing inflammatory arthritis.

     

Selective elimination of synovial inflammatory macrophages in rheumatoid arthritis by an Fcγ receptor I–directed immunotoxin

Objective

To determine whether monocyte/macrophages from rheumatoid arthritis (RA) patients can be selectively eliminated by a toxin‐conjugated antibody CD64–ricin A (CD64‐RiA) directed toward the high‐affinity receptor for IgG (FcγRI), exploiting the capacity of FcγRI to efficiently endocytose antibody which it has bound.

Methods

Mononuclear cells from peripheral blood (PB) and synovial fluid (SF) obtained from RA patients were cultured in the presence of CD64‐RiA. Cell death of monocyte/macrophages was measured by phenotypic changes (light‐scatter patterns and CD14 and FcγRI expression) and apoptosis (nuclear DNA fragmentation). We then tested whether CD64‐RiA–induced cell death of macrophages affected their capacity to stimulate antigen‐induced lymphocyte proliferation and to secrete cytokines. Additionally, the capacity of CD64‐RiA to inhibit proinflammatory activity and cartilage degradation by RA synovial tissue explants was evaluated.

Results

Inflammatory macrophages from RA SF expressed elevated levels of FcγRI and were selectively eliminated by CD64‐RiA via apoptotic cell death. Monocyte/macrophages from RA PB, which had lower levels of FcγRI expression, were much less affected. Induction of SF macrophage apoptosis was associated with efficient inhibition of antigen‐induced lymphocyte proliferation and a reduction in tumor necrosis factor α (TNFα) release. Consistent with these effects on SF macrophages, CD64‐RiA also inhibited TNFα production, interleukin‐1β production, and cartilage‐degrading activity of RA synovial tissue explants.

Conclusion

Together, these data underscore the crucial role of synovial macrophages in RA joint inflammation and indicate that selective elimination of these cells through FcγRI‐directed immunotoxins could be a novel approach to the treatment of RA.

     

Evidence that anti–tumor necrosis factor therapy with both etanercept and infliximab induces apoptosis in macrophages,but not lymphocytes,in rheumatoid arthritis joints: Extended report

Objective

Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF) antagonists is highly effective, but their mechanisms of action are not completely clear. Since anti‐TNF therapy induces a decrease in synovial cellularity, this study focused on the modulation of RA synovial apoptosis following treatment with either soluble TNF receptor (etanercept) or TNF chimeric monoclonal antibody (infliximab).

Methods

Apoptosis (TUNEL and active caspase 3 staining) and cell surface markers were evaluated by immunohistochemistry in synovial biopsy samples obtained before and after 8 weeks of treatment with etanercept (12 patients) or infliximab (9 patients). We also determined by flow cytometry the in vitro effect of etanercept and infliximab on apoptosis of RA mononuclear cells derived from the synovial fluid (SF) and peripheral blood (PB).

Results

Eight weeks of treatment with etanercept and with infliximab significantly increased synovial apoptosis. This change was accompanied by a significant decrease in the synovial monocyte/macrophage population. The decrease in lymphocyte numbers did not reach statistical significance. In vitro, 24 hours of incubation with either etanercept or infliximab induced apoptosis of the SF monocyte/macrophage population. PB monocyte/macrophages were less susceptible to anti‐TNF–mediated apoptosis. No changes in the rate of apoptosis were observed in the lymphocyte population derived from either SF or PB.

Conclusion

In RA patients, both etanercept and infliximab are able to induce cell type–specific apoptosis in the monocyte/macrophage population. This suggests a potential pathway that would account for the diminished synovial inflammation and the decreased numbers of synovial macrophages evident after TNF blockade.

     

Increased intraarticular interleukin‐7 in rheumatoid arthritis patients stimulates cell contact–dependent activation of CD4+ T cells and macrophages

Objective

To determine the level of intraarticular expression of interleukin‐7 (IL‐7) in patients with rheumatoid arthritis (RA) and to investigate the mechanisms by which IL‐7 facilitates activation of CD4+ T cells and monocyte/macrophages in RA.

Methods

IL‐7 levels were measured in synovial fluid obtained from patients with RA and patients with osteoarthritis (OA). Immunohistologic analysis was used to assess the expression of IL‐7 in synovial tissue from patients with RA. Proliferation and activation markers were determined in order to measure the effect of IL‐7 on mononuclear cells, isolated CD4+ T cells, and monocyte/macrophages from the peripheral blood and synovial fluid. Cocultures of CD4+ T cells and monocytic cells in the absence or presence of a semipermeable membrane were performed to assess the extent to which IL‐7 induces its effects, either contact dependently or via soluble mediators.

Results

IL‐7 levels were increased in synovial fluid from patients with RA compared with the levels in synovial fluid from patients with OA. Macrophages, fibroblasts, and endothelial cells in the joint lining tissue expressed abundant IL‐7. In vitro, synovial fluid CD4+ T cells and macrophages were hyperresponsive to IL‐7 when compared with peripheral blood cells. Furthermore, IL‐7 enhanced cell contact–dependent activation of CD4+ T cells and monocyte/macrophages.

Conclusion

The abundant intraarticular expression of IL‐7 and the stimulation by IL‐7 of contact‐dependent activation of CD4+ T cells and monocytic cells indicate that this cytokine plays an important proinflammatory role in RA synovitis. Further identification of IL‐7–induced pathways may improve understanding of the important interactive role of CD4+ T cells and monocytic cells in RA.