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.

     

Proinflammatory action of the antiinflammatory drug infliximab in tumor necrosis factor receptor–associated periodic syndrome

Objective

Tumor necrosis factor receptor (TNFR)–associated periodic syndrome (TRAPS) is an autosomal‐dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene. Unlike other autoinflammatory diseases in which anti‐TNF therapy is largely a successful treatment option, therapy with the anti‐TNF drug infliximab is often ineffective in patients with TRAPS. Moreover, in certain cases, infliximab actually triggers severe episodes of inflammation. The aim of this study was to elucidate the mechanisms underlying such a reaction.

Methods

Peripheral blood mononuclear cells (PBMCs) were obtained from patients with TRAPS. Both caspase 3 activity and NF‐κB subunit activity were determined by enzyme‐linked immunosorbent assay. Cytokine secretion was assessed using a specific customized human multiplex bead immunoassay kit.

Results

Unlike findings in controls, cells from a family of 9 patients, all of whom carried the T50M mutation in TNFRSF1A, failed to respond to infliximab through proapoptotic induction of caspase 3 activity. Instead, we observed enhanced antiapoptotic c‐Rel subunit activity, accompanied by a significant increase in secretion of the proinflammatory cytokines interleukin‐ 1β (IL‐1β), IL‐1 receptor, IL‐6, IL‐8, and IL‐12.

Conclusion

Altered extracellular conformation of TNFRI, resulting from the T50M mutation in TNFRSF1A, results in failure of PBMCs to induce an apoptotic response to infliximab. We hypothesize that failure to shed infliximab‐bound TNF/TNFRI from the cell surface of cells from patients with the T50M mutation triggers c‐Rel activation, and that this leads to a marked increase in cytokine secretion and an increased proinflammatory response. In light of these findings, we strongly advise caution when prescribing infliximab as anti‐TNF therapy to patients with TRAPS.

     

1,25‐dihydroxyvitamin D3 modulates Th17 polarization and interleukin‐22 expression by memory T cells from patients with early rheumatoid arthritis

Objective

To examine the immunologic mechanism by which 1,25‐dihydroxyvitamin D₃ (1,25[OH]₂D₃) may prevent corticosteroid‐induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology.

Methods

Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO− (non‐memory) T cells separated by fluorescence‐activated cell sorting (FACS) from treatment‐naive patients with early RA were stimulated with anti‐CD3/anti‐CD28 in the absence or presence of various concentrations of 1,25(OH)₂D₃, dexamethasone (DEX), and 1,25(OH)₂D₃ and DEX combined. Levels of T cell cytokines were determined by enzyme‐linked immunosorbent assay and flow cytometry.

Results

The presence of 1,25(OH)₂D₃ reduced interleukin‐17A (IL‐17A) and interferon‐γ levels and increased IL‐4 levels in stimulated PBMCs from treatment‐naive patients with early RA. In addition, 1,25(OH)₂D₃ had favorable effects on tumor necrosis factor α (TNFα):IL‐4 and IL‐17A:IL‐4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL‐17A– and IL‐22–expressing CD4+ T cells and IL‐17A–expressing memory T cells were observed in PBMCs from treatment‐naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO− cells between these 2 groups. Interestingly, 1,25(OH)₂D₃, in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL‐17A, IL‐17F, TNFα, and IL‐22 production by memory T cells sorted by FACS from patients with early RA.

Conclusion

These data indicate that 1,25(OH)₂D₃ may contribute its bone‐sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL‐4.

     

Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis

Objective

To analyze the frequency, surface phenotype, and cytokine secretion of CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) compared with both healthy control subjects and patients with rheumatoid arthritis (RA).

Methods

Eight‐color flow cytometry was used to analyze the surface phenotype and cytokine production of PBMCs from 20 patients with AS, 12 patients with RA, and 16 healthy control subjects, following stimulation ex vivo with phorbol myristate acetate and ionomycin for 5 hours. Secretion of interleukin‐17 (IL‐17) by PBMCs was measured by enzyme‐linked immunosorbent assay, following stimulation with anti‐CD3/CD28 for 4 days.

Results

The percentages of IL‐17–positive CD4+ T cells and IL‐22–positive CD4+ T cells were increased in the PBMCs of both patients with AS and patients with RA compared with healthy control subjects, whereas there were no differences in the percentages of interferon‐γ (IFNγ)–positive or IL‐10–positive CD4+ T cells. Likewise, concentrations of IL‐17 in supernatants from patients with AS were significantly higher compared with those from healthy control subjects. In patients with RA, the concentrations of IL‐17 were increased but not significantly. There was a correlation between the percentages of IL‐17–positive CD4+ T cells detected in PBMCs and the amounts of IL‐17 in culture supernatants (r = 0.414, P = 0.0034). All IL‐17–producing cells were CD4+CD45RO+; most expressed both CCR6 and CCR4, but only 50% expressed the IL‐23 receptor (IL‐23R). Nevertheless, there was a positive relationship between the percentage of IL‐23R–positive CD4+ T cells and the frequency of IL‐17–positive CD4+ T cells or IL‐22–positive CD4+ T cells (r = 0.57, P < 0.0001 and r = 0.46, P = 0.001, respectively). A significant proportion of cells that produced IL‐17 also produced IL‐22 and IFNγ, but none produced IL‐10.

Conclusion

The frequencies of IL‐17–positive and IL‐22–positive CD4+ T cells were increased in PBMCs from patients with AS and patients with RA, resulting in secretion of higher quantities of IL‐17 by PBMCs following stimulation. These data support the hypothesis that Th17 cells, particularly when present in excess of IL‐10–producing cells, are involved in the pathogenesis of inflammatory arthritis.

     

Inhibition of antigen‐presenting cell function and stimulation of human peripheral blood mononuclear cells to express an antiinflammatory cytokine profile by the stress protein BiP: Relevance to the treatment of inflammatory arthritis

Objective

The stress protein and endoplasmic reticulum chaperone, immunoglobulin binding protein (BiP), is an autoantigen in rheumatoid arthritis (RA). Stress proteins, however, may have extracellular functions, mediated via cell surface receptors, that may include immunomodulatory functions. We sought to determine whether cell‐free BiP is present in the synovial fluid (SF) of patients with RA and to further investigate the possible extracellular antiinflammatory and immunomodulatory properties of BiP in peripheral blood mononuclear cells (PBMCs) in vitro.

Methods

The presence of BiP in SF was established by Western blotting. PBMCs were stimulated with exogenous recombinant human BiP, and cytokine production and cell proliferation were measured in the presence and absence of cell signaling inhibitors or neutralizing anti–interleukin‐10 (anti–IL‐10) monoclonal antibody. Cytokine levels were quantified by enzyme‐linked immunosorbent assay, cell proliferation by tritiated thymidine uptake, and cell surface molecule expression by flow cytometry.

Results

PBMCs responded to BiP with secretion of an antiinflammatory profile of cytokines. Although BiP stimulated the early production of tumor necrosis factor α (TNFα), the major cytokine induced was IL‐10. Soluble TNF receptor II and IL‐1 receptor antagonist secretion was also increased. Addition of SB203580, the MAPK p38 pathway inhibitor, partially inhibited the production of IL‐10 and TNFα, whereas they were unaffected by the MAPK ERK‐1/2 inhibitor PD98059. BiP also inhibited the recall antigen response by PBMCs to tuberculin purified protein derivative. Further investigation showed that incubation of monocytes in the presence of either BiP or IL‐10 down‐regulated CD86 and HLA–DR expression. The effect observed with IL‐10 was transient compared with the long‐lasting reduction induced by BiP.

Conclusion

Extracellular BiP may stimulate immunomodulatory and antiinflammatory pathways, which are only partly due to the production of IL‐10. These properties may be of relevance for the treatment of diseases such as RA.

     

In vitro cytokine production and proliferation of T cells from patients with anti‐proteinase 3‐ and antimyeloperoxidase‐associated vasculitis,in response to proteinase 3 and myeloperoxidase

Objective

To investigate in vitro proliferative responses of CD4+ T cells and generation of specific cytokines induced by stimulation of peripheral blood mononuclear cells (PBMCs) from patients with antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis with the autoantigens proteinase 3 (PR3) and myeloperoxidase (MPO).

Methods

PBMCs from vasculitis patients with PR3 ANCA or MPO ANCA and from healthy controls were stimulated for 7 days with PR3, MPO, or control stimuli. Proliferation of CD4+ T cells was assessed by flow cytometry, using the proliferation marker Ki‐67. Levels of the pro‐proliferative cytokines interleukin‐2 (IL‐2) and IL‐6 and of the Th1 and Th2 cytokines interferon‐γ (IFNγ) and IL‐10 in culture supernatants were determined.

Results

PR3 and MPO induced proliferative responses in CD4+ T cells from individual patients with ANCA‐associated vasculitides and healthy controls in vitro. Neither PR3 nor MPO elicited significant IL‐2 production. Levels of IL‐6 were highest after stimulation with PR3 but low after stimulation with MPO, independent of study group. Stimulation with PR3, and to a lesser extent with MPO, induced a Th2 cytokine milieu, characterized by high production of IL‐6 and IL‐10 and low production of IFNγ in patients and controls.

Conclusion

PR3 and MPO promote proliferation of CD4+ T cells from patients with ANCA‐associated vasculitides, but also cross‐stimulate T cells from healthy individuals. Strong IL‐10 production elicited by PR3 in vitro may act as an inhibitory signal for T cell proliferation and may have an important immunoregulatory function in vivo.

     

ANTI‐CD3 therapy expands the numbers of CD4+ and CD8+ treg cells and induces sustained amelioration of collagen‐induced arthritis

Objective

To assess the therapeutic potential of anti‐CD3 monoclonal antibodies (mAb) for rheumatoid arthritis, using collagen‐induced arthritis as an animal model.

Methods

Arthritis was induced in DBA/1 mice by immunization with type II collagen. After disease onset, a single injection of anti‐CD3 mAb (20 μg/mouse) was administered, and arthritis severity was monitored over a 10‐day period.

Results

Anti‐CD3 mAb treatment resulted in a sustained reduction in disease activity, which was associated with an increase in the proportion of naturally occurring CD4+CD25+FoxP3+ regulatory T (Treg) cells and the generation of a population of CD8+CD25+FoxP3+ Treg cells. Anti‐CD3 mAb treatment did not alter the capacity of CD4+ Treg cells to suppress effector T cell proliferation and interferon‐γ (IFNγ) production in vitro. However, CD4+ Treg cells from both anti‐CD3 mAb–treated and control mice were unable to suppress interleukin‐17 (IL‐17) production. In contrast, CD8+ Treg cells induced by anti‐CD3 therapy suppressed IL‐17 production as well as CD4+ T cell proliferation and IFNγ production.

Conclusion

These results show that anti‐CD3 mAb treatment has important therapeutic potential for rheumatoid arthritis and has the capacity to generate antiarthritic CD8+ Treg cells and expand the relative numbers of CD4+ Treg cells.

     

A role for geranylgeranylation in interleukin‐1β secretion

Objective

Mevalonate kinase deficiency (MKD) is an autosomal‐recessive disorder characterized by recurring episodes of inflammation. MK catalyzes the phosphorylation of mevalonic acid, which is an early step in isoprenoid biosynthesis. The goal of our study was to determine whether a temporary shortage of certain isoprenoid end products and/or the accumulation of mevalonic acid is the cause of interleukin‐1β (IL‐1β) secretion in MKD.

Methods

We studied the effect of the addition of intermediate metabolites and inhibitors of the isoprenoid biosynthesis pathway on IL‐1β secretion by peripheral blood mononuclear cells (PBMCs) of patients with MKD and healthy controls.

Results

Inhibition of enzymes involved in geranylgeranyl pyrophosphate (GGPP) synthesis or geranylgeranylation of proteins led to a marked increase of lipopolysaccharide‐stimulated IL‐1β secretion in PBMCs of control subjects. Furthermore, the increased IL‐1β secretion by PBMCs of patients with MKD was reversed by supplementation with GGPP as well as with mevalonic acid. IL‐1β secretion was increased only when control PBMCs were incubated with excessive amounts of mevalonic acid. Finally, a reduction in IL‐1β secretion by MKD PBMCs was also observed when sterol biosynthesis was inhibited, favoring nonsterol isoprenoid biosynthesis.

Conclusion

Our results indicate that a shortage of geranylgeranylated proteins, rather than an excess of mevalonate, is likely to cause increased IL‐1β secretion by PBMCs of patients with MKD.

     

Better preserved immune responses after immunization with rgp 160 in HIV-1 infected patients treated with highly active antiretroviral therapy than in untreated patients with similar CD4 levels during at 2 years' follow-up

Objective

To study the impact of effective highly active antiretroviral therapy (HAART) on the preservation of long‐term CD4 memory cells induced by vaccines in HIV‐1‐infected patients.

Methods

Thirty HIV‐1‐positive patients on HAART with undetectable viral load were randomized into three groups: 10 received HIV‐1 rgp160 vaccine, 10 received tetanus vaccine and 10 patients were not immunized. As controls, 10 HIV‐negative volunteers were immunized with tetanus vaccine. The results were compared with an rgp160 vaccine study performed before the era of HAART on patients with comparable CD4 levels. All patients were monitored for 2 years for T‐cell proliferative responses, T‐cell subset levels, serum IgG and viral load.

Results

After 1 year all patients immunized with rgp160 had strong T‐cell responses to the rgp160 antigen. After 2 years this response was preserved in the HAART‐treated group, but not in the rgp160 immunized non‐HAART group, despite comparable CD4 levels. Recall effects of the CD4‐specific responses towards other antigens were seen in the rgp160‐immunized HAART group.

Conclusion

Immunization with rpg 160 leads to positive effects on HIV‐specific T‐cell proliferative responses in patients both with and without HAART. Immune responses after immunization is better preserved in HAART‐treated patients who have low viral amounts than in individuals with high viral load and no HAART despite comparable CD4 levels during 2 years' follow‐up. Interruption of HAART with return of a high viral load might thus have negative effects on T‐cell functions in the long term, even if CD4 levels are kept at clinically acceptable levels.

     

Interleukin‐17A induction of angiogenesis,cell migration,and cytoskeletal rearrangement

Objective

To examine the ability of interleukin‐17A (IL‐17A) to stimulate angiogenesis, cell migration, and cytoskeletal rearrangement.

Methods

The effect of IL‐17A on microvascular tube formation and extracellular matrix invasion by human dermal endothelial cells (HDECs) was assessed using Matrigel matrix and Transwell Matrigel invasion chambers. IL‐17A–induced growth‐related oncogene α (GROα) and monocyte chemotactic protein 1 (MCP‐1) production in rheumatoid arthritis synovial fibroblasts (RASFs) and HDECs was measured by enzyme‐linked immunosorbent assay. IL‐17A–induced migration was assessed using peripheral blood mononuclear cell (PBMC) migration assays and wound‐repair scratch assays, with or without anti‐GROα and anti–MCP‐1 antibodies. Binding of β1 integrin receptors was assessed using integrin binding assays. Cytoskeletal assembly/disassembly in RASFs and HDECs were assessed by immunofluorescence staining for F‐actin. IL‐17A–induced cell migration and cytoskeletal disassembly were assessed in the presence of a Rac1 inhibitor (NSC23766). Rac1 activation following IL‐17 stimulation in the presence or absence of anti‐GROα, anti–MCP‐1, or IgG control was assessed by Rac GTPase pull‐down assays and Western blotting.

Results

IL‐17A significantly up‐regulated angiogenesis and endothelial cell invasion. It significantly induced GROα and MCP‐1 expression in RASFs. Migration of PBMCs, RASFs, and HDECs was induced by IL‐17A; these effects were blocked by anti‐GROα or anti–MCP‐1 antibodies. IL‐17A significantly up‐regulated β1 integrin receptor binding and induced cytoskeletal disassembly in RASFs and HDECs. Rac1 activation was directly induced by IL‐17A. IL‐17A–induced wound repair and actin rearrangement were inhibited by a pharmacologic inhibitor of Rac1 (NSC23766). Anti‐GROα or anti–MCP‐1 antibodies had no effect on IL‐17A–induced Rac1 activation.

Conclusion

IL‐17A induces angiogenesis, cell migration, and cell invasion, all of which are key processes in the pathogenesis of rheumatoid arthritis and ones that are mediated in part through chemokine‐ and cytoskeleton‐dependent pathways.

     

The spontaneous remission of juvenile idiopathic arthritis is characterized by CD30+ T cells directed to human heat‐shock protein 60 capable of producing the regulatory cytokine interleukin‐10

Objective

To test the hypothesis that T cell reactivity to self heat‐shock protein 60 (Hsp60) in patients with remitting juvenile idiopathic arthritis (JIA) is part of an antiinflammatory, regulatory mechanism.

Methods

Using peripheral blood–derived mononuclear cells (PBMCs) and synovial fluid–derived mononuclear cells (SFMCs) obtained from patients with JIA, we analyzed the expression of CD30 and the induction of regulatory cytokines in response to human and mycobacterial Hsp60.

Results

In oligoarticular JIA patients, in vitro activation of PBMCs and SFMCs with Hsp60 induced a high expression of CD30 on CD4+, activated (HLA–DR–positive), memory (CD45RO+) T cells. The expression of CD30 induced by human Hsp60 was much higher than that induced by mycobacterial Hsp60. In oligoarticular JIA patients with active disease, the expression of CD30 in response to human Hsp60 was paralleled by a high interleukin‐10 (IL‐10):interferon‐γ (IFNγ) ratio. In addition, restimulated human Hsp60–specific T cell lines from oligoarticular JIA patients showed a high production of IL‐10 and a low production of IFNγ. In contrast, PBMCs and SFMCs from polyarticular JIA patients responded to human Hsp60 with virtually no expression of CD30 and a low IL‐10:IFNγ ratio.

Conclusion

The results show that T cells responding to human Hsp60 in oligoarticular JIA patients express CD30, and during active phases of the disease, these T cells have a cytokine profile with a high IL‐10:IFNγ ratio. These findings suggest that in oligoarticular JIA patients, human Hsp60–specific CD4+ cells have a regulatory function and contribute to disease remission.

     

Blockade of the interleukin‐7 receptor inhibits collagen‐induced arthritis and is associated with reduction of T cell activity and proinflammatory mediators

Objective

To study the effects of interleukin‐7 receptor α‐chain (IL‐7Rα) blockade on collagen‐induced arthritis (CIA) and to investigate the effects on T cell numbers, T cell activity, and levels of proinflammatory mediators.

Methods

We studied the effect of anti–IL‐7Rα antibody treatment on inflammation and joint destruction in CIA in mice. Numbers of thymocytes, splenocytes, T cell subsets, B cells, macrophages, and dendritic cells were assessed. Cytokines indicative of Th1, Th2, and Th17 activity and several proinflammatory mediators were assessed by multianalyte profiling in paw lysates. In addition, T cell–associated cytokines were measured in supernatants of lymph node cell cultures.

Results

Anti–IL‐7Rα treatment significantly reduced clinical arthritis severity in association with reduced radiographic joint damage. Both thymic and splenic cellularity were reduced after treatment with anti–IL‐7Rα. IL‐7Rα blockade specifically reduced the total number of cells as well as numbers of naive, memory, CD4+, and CD8+ T cells from the spleen and significantly reduced T cell–associated cytokines (interferon‐γ, IL‐5, and IL‐17). IL‐7Rα blockade also decreased local levels of proinflammatory cytokines and factors associated with tissue destruction, including tumor necrosis factor α, IL‐1β, IL‐6, matrix metalloproteinase 9, and RANKL. IL‐7Rα blockade did not significantly affect B cells, macrophages, and dendritic cells. B cell activity, indicated by serum anticollagen IgG antibodies, was not significantly altered.

Conclusion

Blockade of IL‐7Rα potently inhibited joint inflammation and destruction in association with specific reductions of T cell numbers, T cell–associated cytokines, and numerous mediators that induce inflammation and tissue destruction. This study demonstrates an important role of IL‐7R–driven immunity in experimental arthritis and indicates the therapeutic potential of IL‐7Rα blockade in human arthritic conditions.

     

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.

     

Inhibition of interleukin‐6 receptor directly blocks osteoclast formation in vitro and in vivo

Objective

To investigate the efficacy of a murine anti–interleukin‐6 receptor (anti–IL‐6R) antibody in directly blocking tumor necrosis factor (TNF)– and RANKL‐mediated osteoclastogenesis in vitro and in vivo.

Methods

The efficacy of a murine antibody against IL‐6R in blocking osteoclast differentiation of mononuclear cells stimulated with RANKL was tested. In addition, arthritic human TNFα–transgenic mice were treated with anti–IL‐6R antibody, and osteoclast formation and bone erosion were assessed in arthritic paws.

Results

Blockade of IL‐6R dose dependently reduced osteoclast differentiation and bone resorption in monocyte cultures stimulated with RANKL or RANKL plus TNF. In human TNFα–transgenic mice, IL‐6R blockade did not inhibit joint inflammation, but it strongly reduced osteoclast formation in inflamed joints as well as bone erosion in vivo. Neither the cell influx into joints nor the synovial expression of IL‐6 and RANKL changed with IL‐6R blockade, while the synovial expression of IL‐1 was significantly reduced. In contrast, TNF‐mediated systemic bone loss was not inhibited by IL‐6R blockade.

Conclusion

These data suggest that blockade of IL‐6R directly affects osteoclast formation in vitro and in vivo, suggesting a direct and specific effect of anti–IL‐6R therapy on osteoclasts independently of its antiinflammatory effects. This effect adds significantly to the structure‐sparing potential of pharmacologic blockade of IL‐6R in arthritis.

     

Immunoregulatory effects of allogeneic mixed chimerism induced by nonmyeloablative bone marrow transplantation on chronic inflammatory arthritis and autoimmunity in interleukin‐1 receptor antagonist–deficient mice

Objective

To investigate the immunoregulatory effects of allogeneic mixed chimerism induced by T cell–depleted, nonmyeloablative bone marrow transplantation (BMT) on chronic inflammatory arthritis and autoimmunity in mice deficient in interleukin‐1 receptor antagonist (IL‐1Ra).

Methods

IL‐1Ra^−/− mice (H‐2K^d) were treated with antibody to asialoganglioside G_M1 (anti–natural killer cell), total body irradiation (500 cGy), and T cell–depleted, nonmyeloablative BMT derived from C57BL/6 mice (H‐2K^b). Engraftment and chimerism were evaluated in peripheral blood, lymph nodes, and spleen by multicolor flow cytometry. The severity of arthritis was evaluated by clinical scoring and histopathologic assessment. Levels of IgG1 and IgG2a subtypes of anti–type II collagen (anti‐CII) antibodies were measured in serum samples. After T cells were stimulated with CII, ovalbumin, and phytohemagglutinin, T cell proliferative responses and levels of cytokine production (interferon‐γ [IFNγ], tumor necrosis factor α [TNFα], interleukin‐10 [IL‐10], and IL‐17) were assayed in culture supernatants.

Results

All IL‐1Ra^−/− mice receiving BMT showed marked improvement in arthritis within 3 weeks, as well as successful induction of mixed chimerism. These mice showed higher levels of IgG1, and lower levels of IgG2a anti‐CII antibodies and weaker T cell proliferative responses than did mice in the control groups (either no treatment or conditioning alone without bone marrow rescue). In mixed chimeras, the levels of IFNγ, TNFα, and IL‐17 produced from CII‐stimulated T cells were significantly suppressed and IL‐10 production was significantly higher as compared with controls.

Conclusion

The introduction of allogeneic mixed chimerism showed a strong immunoregulatory potential to correct established chronic inflammatory arthritis and autoimmunity originating from a dysregulated proinflammatory cytokine network.

     

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.