A shift in the balance of inhibitory and activating Fcγ receptors on monocytes toward the inhibitory Fcγ receptor IIb is associated with prevention of monocyte activation in rheumatoid arthritis

Objective

To assess surface expression of the inhibitory receptor for IgG (Fcγ receptor IIb [FcγRIIb]) in relation to activating FcγR on monocyte/macrophages from patients with rheumatoid arthritis (RA) and healthy controls and to study the influence of proinflammatory and antiinflammatory cytokines on the balance of inhibitory and activating FcγR.

Methods

Using a combination of genotyping and phenotyping, surface expression of FcγRIIb on monocytes from healthy control subjects and RA patients was demonstrated. Expression of FcγR on CD14+ monocytes was assessed by flow cytometry. Regulation of inhibitory and activating FcγR on monocytes by proinflammatory (interferon‐γ [IFNγ], tumor necrosis factor α [TNFα]) and antiinflammatory (interleukin‐4 [IL‐4], IL‐10) cytokines was studied. A functional change in cytokine‐modulated monocytes was assessed in secondary cultures by their ability to produce TNFα upon FcγR crosslinking by IgG.

Results

Monocytes from healthy controls and RA patients expressed FcγRIIb at similar levels, in contrast to the higher levels of activating FcγRI and FcγRIIa in RA patients. The regulation of FcγR expression was comparable for patients and controls. IFNγ selectively up‐regulated FcγRI. TNFα down‐regulated expression of FcγRIIb and the activating FcγR, whereas IL‐10 up‐regulated expression of monocytic FcγRIIb and all activating FcγR. Increased or sustained levels of activating over inhibitory FcγR induced by IFNγ, TNFα, and IL‐10 alone were associated with enhanced IgG‐triggered TNFα production. In contrast, IL‐4 and, more specifically, IL‐4 plus IL‐10 altered the FcγR balance in favor of FcγRIIb and completely prevented IgG‐triggered TNFα production.

Conclusion

The altered balance of FcγR in favor of activating receptors in RA may contribute to increased activation of monocyte/macrophages. A change in the FcγR balance toward the inhibitory FcγRIIb may offer a novel treatment strategy for preventing the pleiotropic activity of FcγR‐triggered macrophages.

     

Salmonella septicemia in rheumatoid arthritis patients receiving anti–tumor necrosis factor therapy: Association with decreased interferon‐γ production and toll‐like receptor 4 expression

Objective

Patients treated with antibodies to tumor necrosis factor α (TNFα) have an increased susceptibility to intracellular infections. We describe 2 patients with rheumatoid arthritis (RA) who developed Salmonella septicemia during anti‐TNF treatment. The aim of this study was to identify the mechanisms involved in the increased susceptibility of anti‐TNF–treated patients to intracellular microorganisms.

Methods

We evaluated an additional 6 RA patients receiving anti‐TNF antibodies, 5 RA patients not receiving anti‐TNF therapy, and 6 age‐ and sex‐matched healthy volunteers. The in vitro production of cytokines (interleukin‐1β [IL‐1β], IL‐6, interferon‐γ [IFNγ], and IL‐10) upon bacterial stimulation of whole blood and the expression of Toll‐like receptor 4 (TLR‐4) on dendritic cells from RA patients treated with infliximab, RA patients not treated with infliximab, and healthy controls were compared.

Results

Stimulation with heat‐killed Salmonella typhimurium or Candida albicans led to a significantly decreased production of IFNγ, but not to a decreased production of IL‐10, IL‐β, or IL‐6, in anti‐TNF–treated RA patients compared with RA patients who were not receiving anti‐TNF antibodies and compared with healthy controls. TNF‐blocking treatment ex vivo significantly inhibited TLR‐4 expression on dendritic cells from RA patients and healthy controls.

Conclusion

Since recognition of microorganisms by TLR‐4 and activation of phagocytes by IFNγ are essential mechanisms for the defense against intracellular and fungal pathogens, we propose that this pathway is crucial for the increased susceptibility to these microorganisms in patients receiving anti‐TNF therapy.

     

Immune complexes containing citrullinated fibrinogen costimulate macrophages via Toll‐like receptor 4 and Fcγ receptor

Objective

Rheumatoid arthritis (RA) is associated with the presence of anti–citrullinated protein antibodies (ACPAs). Nearly two‐thirds of patients with ACPA‐positive RA have immune complexes that contain citrullinated fibrinogen, and these citrullinated fibrinogen–containing immune complexes (cFb‐IC) can exacerbate disease in murine models of RA; however, the exact role of such ACPA ICs in RA pathogenesis has remained elusive. We undertook the present study to investigate a novel mechanism by which ACPAs specifically targeting citrullinated fibrinogen may directly stimulate macrophage tumor necrosis factor (TNF) production.

Methods

Murine or human macrophages were stimulated with native fibrinogen (nFb), cFb, or in vitro–generated nFb‐IC or cFb‐IC, and TNF production was measured by enzyme‐linked immunosorbent assay. ICs were generated with either polyclonal anti‐Fb antibodies or pooled IgG from patients with ACPA‐positive RA. To evaluate the role of the Toll‐like receptor 4 (TLR‐4)/myeloid differentiation protein (MyD88) pathway and the Fcγ receptor (FcγR) pathway in the induction of TNF by Fb and Fb‐IC, parallel experiments were performed using 1) TLR‐4–deficient or MyD88‐deficient macrophages, and 2) inhibitors of TLR‐4 or FcγR.

Results

Citrullinated Fb stimulated macrophage TNF production more potently than did native Fb. Incorporation of cFb into ICs augmented its ability to stimulate TNF production by macrophages. Stimulation of TNF by cFb was dependent on TLR‐4 and MyD88, while stimulation by cFb‐IC was dependent on both TLR‐4/MyD88 and FcγR.

Conclusion

We demonstrated that cFb‐IC can costimulate macrophages via dual engagement of TLR‐4 and FcγR, resulting in the synergistic induction of TNF production. Our findings suggest a potential role of citrullination in increasing the potency of an endogenous innate immune ligand and provide insight into the mechanism by which anticitrulline autoimmunity may contribute to the onset and propagation of inflammation in RA.

     

Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45ro+CD25+CD127low regulatory T cells

Objective

Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127^low FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin‐17 (IL‐17)–producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function.

Methods

The presence and phenotype of CD4+CD45RO+CD25+CD127^low T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence‐activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti‐CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme‐linked immunosorbent assay, and proliferation assays.

Results

In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127^low Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro–activated monocytes induced an increase in the percentage of IL‐17–positive, interferon‐γ (IFNγ)–positive, and tumor necrosis factor α (TNFα)–positive Treg cells as well as IL‐10–positive Treg cells. The observed increase in IL‐17–positive and IFNγ‐positive Treg cells was driven by monocyte‐derived IL‐1β, IL‐6, and TNFα and was mediated by both CD14+CD16− and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL‐17 production.

Conclusion

Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.

     

Proinflammatory mediator–induced reversal of CD4+,CD25+ regulatory T cell–mediated suppression in rheumatoid arthritis

Objective

We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg–mediated suppression.

Methods

Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme‐linked immunosorbent assay. Magnetically sorted CD4+,CD25– and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti‐CD3 monoclonal antibody (mAb) and autologous antigen‐presenting cells, in the absence or presence of anti‐CD28 mAb or the proinflammatory cytokines interleukin‐6 (IL‐6), tumor necrosis factor α (TNFα), or IL‐7.

Results

Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB‐derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti‐CD28 mAb to cocultures of CD4+,CD25– and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg–mediated suppression in both PB and SF. Furthermore, IL‐7 and, to a limited extent, TNFα, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg–mediated suppression. In contrast, IL‐6 did not influence Treg‐mediated suppression.

Conclusion

Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.

     

Anti–ribosomal P protein antibody in human systemic lupus erythematosus up‐regulates the expression of proinflammatory cytokines by human peripheral blood monocytes

Objective

Autoantibodies to ribosomal P proteins (anti‐P antibodies) are detected in 12–16% of patients with systemic lupus erythematosus (SLE) and have been found to be associated with some manifestations of the disease, including lupus psychosis and hepatitis. Recent studies have disclosed that anti‐P antibodies react with activated T cells but not with B cells, suggesting possible direct effects of anti‐P antibodies on immune regulation. The present study was designed to explore the presence of the epitope recognized by anti‐P antibodies on human peripheral blood monocytes.

Methods

Highly purified peripheral blood monocytes obtained from healthy donors were cultured with or without interferon‐γ (IFNγ) in the presence of either anti‐P antibodies purified by affinity chromatography from the sera of patients with SLE or control IgG.

Results

Flow cytometry analysis disclosed that fresh (day 0) monocytes did not express the ribosomal P epitope, whereas expression of the ribosomal P epitope was induced on annexin V–negative monocytes after activation through plastic adherence for 48 hours. More important, anti‐P antibodies (compared with normal IgG or IgG from SLE patients devoid of anti‐P antibodies) enhanced the production of tumor necrosis factor α (TNFα) and interleukin‐6 (IL‐6) by activated monocytes. Accordingly, anti‐P antibodies also up‐regulated the expression of TNFα and IL‐6 messenger RNA in activated monocytes. Of note, F(ab′)₂ fragments of anti‐P antibodies, which do not result in Fcγ receptor (FcγR) crosslinking, also effectively up‐regulated the expression of TNFα and IL‐6.

Conclusion

These results indicate that human peripheral blood monocytes express the ribosomal P epitope upon activation, irrespective of induction of apoptosis. Moreover, the data suggest that anti‐P antibodies might modify a variety of inflammatory responses through up‐regulation of the expression of proinflammatory cytokines in monocytes, in a manner that does not involve FcγR crosslinking.

     

Outside‐to‐inside signaling through transmembrane tumor necrosis factor reverses pathologic interleukin‐1β production and deficient apoptosis of rheumatoid arthritis monocytes

Objective

Monocytes are a major source of proinflammatory cytokines in rheumatoid arthritis (RA), and inhibitors of monocytic cytokines are highly efficient agents for treatment of the disease. The aim of this study was to analyze the effects of a therapeutic anti–tumor necrosis factor α (anti‐TNFα) antibody on monocytes from patients with RA and healthy control subjects.

Methods

Peripheral blood monocytes from patients with RA and healthy control subjects were incubated in the presence of anti‐TNFα antibody or IgG. Annexin V staining, caspase activation, poly(ADP‐ribose) polymerase cleavage, and DNA staining with propidium iodide were used to analyze apoptosis. The signaling events elicited in monocytes by infliximab were analyzed by Western blotting and electromobility shift assay.

Results

Peripheral blood monocytes from patients with RA were characterized by increased expression of transmembrane TNFα, spontaneous in vitro production of interleukin‐1β (IL‐1β), and a decreased rate of spontaneous ex vivo apoptosis. Incubation with infliximab induced significantly increased apoptosis in monocytes from patients with RA but not in monocytes from healthy control subjects. This apoptosis was triggered by reverse signaling of transmembrane TNF after ligation by infliximab and was independent of caspase activation. Instead, transmembrane TNF reverse signaling inhibited the constitutive NF‐κB activation in RA monocytes, suppressed IL‐1β secretion, and normalized spontaneous in vitro apoptosis. This normalization was reversible by the addition of exogenous IL‐1β.

Conclusion

This study demonstrates that outside‐to‐inside signaling through transmembrane TNF after ligation by infliximab inhibits constitutive NF‐κB activation and suppresses spontaneous IL‐1β production by monocytes from patients with RA. Besides the induction of monocyte apoptosis, this inhibition could also contribute to the therapeutic effects observed during treatment with TNFα inhibitors.

     

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.

     

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.

     

Expression of Toll‐like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin‐12 and interleukin‐18 via interferon‐γ

Objective

To study the expression of Toll‐like receptor 2 (TLR‐2) and TLR‐4 and its association with proinflammatory cytokines in synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals.

Methods

Synovial tissue specimens from 29 RA patients were stained for TLR‐2, TLR‐4, and proinflammatory cytokines (interleukin‐1β [IL‐1β], IL‐12, IL‐17, IL‐18, and tumor necrosis factor α [TNFα]). The expression of TLR‐2, TLR‐4, and cytokines as well as the degree of inflammation in synovial tissue were compared between patients with RA, patients with OA (n = 5), and healthy individuals (n = 3). Peripheral blood mononuclear cells (PBMCs) were incubated with IL‐12 and IL‐18, and TLR expression was assessed using fluorescence‐activated cell sorter analysis. Production of TNFα and IL‐6 was measured using Luminex bead array technology.

Results

In RA synovial tissue, the expression of TLR‐2 was slightly higher than that of TLR‐4. Interestingly, both TLR‐2 and TLR‐4 were expressed at higher levels in moderately inflamed synovium, as compared with synovial tissue with no or severe inflammation. TLR expression in both the lining and the sublining was associated with the presence of IL‐12 and IL‐18, but no other cytokines, in the lining. The expression of both TLRs was low in synovial tissue from OA patients and healthy donors. Stimulation of PBMCs with IL‐12 and IL‐18 resulted in increased expression of both TLR‐2 and TLR‐4; this could be blocked with anti–interferon‐γ (anti‐IFNγ) antibodies, suggesting a role for IFNγ. Lipopolysaccharide‐ or lipoteichoic acid–mediated triggering of PBMCs incubated with IL‐12/IL‐18 or IFNγ led to an increased production of both TNFα and IL‐6, indicating the functionality of TLR‐2 and TLR‐4.

Conclusion

TLR‐2 and TLR‐4 are expressed in synovial tissue of patients with clinically active disease and are associated with the levels of both IL‐12 and IL‐18. The synergistic effect of IL‐12 and IL‐18 on T cell IFNγ production seems to regulate expression of TLR‐2 and TLR‐4 in the synovial tissue of RA patients.

     

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.

     

Increased macrophage activation mediated through toll‐like receptors in rheumatoid arthritis

Objective

Macrophages are the major source of inflammation mediators that are important in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to analyze macrophages obtained from the joints of RA patients in order to characterize the expression of Toll‐like receptor 2 (TLR‐2) and TLR‐4 and the responses to TLR ligation.

Methods

Cells were isolated from the synovial fluid (SF) of RA patients or patients with other forms of inflammatory arthritis. Cell surface TLR‐2 and TLR‐4 expression and intracellular tumor necrosis factor α (TNFα) and interleukin‐8 (IL‐8) expression by CD14+ macrophages were determined by flow cytometry. Peptidoglycan (PG) and lipopolysaccharide (LPS) were used as ligands for TLR‐2 and TLR‐4, respectively.

Results

The expression of TLR‐2 and TLR‐4 was increased on CD14+ macrophages from the joints of RA patients compared with that on control in vitro–differentiated macrophages or control peripheral blood monocytes. Neither TLR‐2 expression nor TLR‐4 expression differed between RA and other forms of inflammatory arthritis. However, PG‐ and LPS‐induced TNFα expression and IL‐8 expression were greater with RA SF macrophages than with those obtained from the joints of patients with other forms of inflammatory arthritis or with control macrophages. PG‐induced TNFα expression and IL‐8 expression were highly correlated with TLR‐2 expression in normal macrophages, but not with that in macrophages obtained from joints of RA patients or patients with other forms of inflammatory arthritis.

Conclusion

TLR‐2 and TLR‐4 ligation resulted in increased activation of RA synovial macrophages compared with those from patients with other forms of inflammatory arthritis or compared with control macrophages. Factors other than the level of TLR‐2 and TLR‐4 expression contributed to the increased activation of RA SF macrophages. These observations support the notion of a potential role for activation through TLR‐2 and TLR‐4 in the inflammation and joint destruction of RA.

     

Activation of human synovial mast cells from rheumatoid arthritis or osteoarthritis patients in response to aggregated IgG through Fcγ receptor I and Fcγ receptor II

Objective

Substantial evidence suggests that human synovial mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). A plausible pathway for the activation of synovial MCs is through IgG receptors, given the prevalence of circulating IgG isotype autoantibodies and synovial immune complexes in patients with RA. However, IgG receptor expression on human synovial MCs remains uncharacterized. The aim of this study was to identify which IgG receptor(s) on synovial MCs are responsible for MC activation in immune complexes.

Methods

Synovial tissue specimens were obtained from patients with RA or patients with osteoarthritis (OA) who were undergoing joint replacement surgery, and synovial MCs were enzymatically dispersed. Cultured synovium‐derived MCs were generated by culturing synovial cells with stem cell factor, and receptor expression was analyzed using fluorescence‐activated cell sorting. Mediators released from MCs were measured using enzyme immunoassays or enzyme‐linked immunosorbent assays.

Results

Primary synovial MCs and cultured synovium‐derived MCs obtained from both patients with RA and patients with OA expressed Fcε receptor I (FcεRI), FcγRI, and FcγRII but not FcγRIII. Cultured synovium‐derived MCs induced degranulation and the production of prostaglandin D₂ and tumor necrosis factor α (TNFα) through FcγRI. The aggregation of FcγRII caused histamine release from cultured MCs but not from primary MCs. Histamine release induced by aggregated IgG was significantly inhibited by neutralizing anti‐FcγRI monoclonal antibody and anti‐FcγRII monoclonal antibody.

Conclusion

With regard to the FcR expression profile, synovial MCs from patients with RA and patients with OA were similar. FcγRI was responsible for producing abundant TNFα from synovial MCs in response to aggregated IgG. Immune complexes may activate synovial MCs through FcγRI and FcγRII.

     

Inhibition of synovial hyperplasia,rheumatoid T cell activation,and experimental arthritis in mice by sulforaphane,a naturally occurring isothiocyanate

Objective

To investigate whether sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables such as broccoli, regulates synoviocyte hyperplasia and T cell activation in rheumatoid arthritis (RA).

Methods

Synoviocyte survival was assessed by MTT assay. The levels of Bcl‐2, Bax, p53, and pAkt were determined by Western blot analysis. Cytokine concentrations in culture supernatants from mononuclear cells were analyzed by enzyme‐linked immunosorbent assay. The in vivo effects of SFN were examined in mice with experimentally induced arthritis.

Results

SFN induced synoviocyte apoptosis by modulating the expression of Bcl‐2/Bax, p53, and pAkt. In addition, nonapoptotic doses of SFN inhibited T cell proliferation and the production of interleukin‐17 (IL‐17) and tumor necrosis factor α (TNFα) by RA CD4+ T cells stimulated with anti‐CD3 antibody. Anti‐CD3 antibody–induced increases in the expression of retinoic acid–related orphan receptor γt and T‐bet were also repressed by SFN. Moreover, the intraperitoneal administration of SFN to mice suppressed the clinical severity of arthritis induced by injection of type II collagen (CII), the anti‐CII antibody levels, and the T cell responses to CII. The production of IL‐17, TNFα, IL‐6, and interferon‐γ by lymph node cells and spleen cells from these mice was markedly reduced by treatment with SFN. Anti‐CII antibody–induced arthritis in mice was also alleviated by SFN injection.

Conclusion

SFN was found to inhibit synovial hyperplasia, activated T cell proliferation, and the production of IL‐17 and TNFα by rheumatoid T cells in vitro. The antiarthritic and immune regulatory effects of SFN, which were confirmed in vivo, suggest that SFN may offer a possible treatment option for RA.

     

Macrophage subpopulations in rheumatoid synovium: Reduced CD163 expression in CD4+ T lymphocyte–rich microenvironments

Objective

The cell surface glycoprotein CD163 is a member of the cysteine‐rich scavenger receptor family, highly specific for leukocytes of the mononuclear phagocyte lineage. In vitro, it is induced by glucocorticoids, interleukin‐6 (IL‐6), and IL‐10 and down‐regulated by interferon‐γ (IFNγ), indicating that it has a role in antiinflammatory or other immunomodulatory pathways. We assessed CD163 expression in microenvironments within rheumatoid arthritis (RA) synovium to clarify the relationships among CD4+ T lymphocytes, IFNγ, and macrophage function in RA.

Methods

Double immunofluorescence and serial immunoenzymatic studies were performed on normal, osteoarthritic, and RA synovium and tonsil with antibodies to CD163, CD45, CD68, CD14, CD3, CD4, CD8, CD19, and IFNγ.

Results

CD163 was observed on all CD14+ cells in synovium and tonsil with the exception of cells within larger T lymphocyte clusters in synovium and within tonsillar follicles. All brightly CD14+ cells in or around vessel walls (interpreted as immigrant monocytes) were CD163+. CD163 labeled fewer cells than did CD68 in synovial intima, but all CD45+ intimal cells were CD163+. CD4+,IFNγ+ T lymphocytes in RA synovium were chiefly localized within clusters containing CD68+, CD163− cells.

Conclusion

Within RA synovium, CD163 has major advantages as a macrophage marker and does not appear to be restricted to “mature” macrophages. CD163 discriminates between synovial macrophages and synovial intimal fibroblasts, which also stain positively for CD68 in diseased tissue.