Plasmid encoding interleukin‐4 in the amelioration of murine collagen‐induced arthritis

Objective

To evaluate the therapeutic effect of the administration of plasmid encoding interleukin‐4 (IL‐4) via gene‐gun delivery and via intradermal injection on collagen‐induced arthritis (CIA).

Methods

IL‐4 plasmid was administered by gene‐gun delivery and intradermal injection to DBA/1 mice immunized with type II collagen (CII). The therapeutic effect on the development of CIA was evaluated clinically with a visual scoring method for arthritis and serologically by enzyme‐linked immunosorbent assays and polymerase chain reaction.

Results

Treatment with IL‐4–expressing plasmid significantly reduced the incidence and severity of CIA, including a reduction in the anti‐CII antibody level. In particular, gene‐gun delivery had a higher immunosuppressive effect on CIA compared with intradermal injection. As shown by in vitro stimulation assay, the spleen cells from mice immunized with CII and treated with IL‐4 plasmid via gene gun exhibited higher Th2 cytokine responses compared with cells treated with control plasmid after in vitro stimulation with CII.

Conclusion

The results of this study suggest that treatment with IL‐4 plasmid may constitute a new clinical use of cytokine gene therapy for rheumatoid arthritis.

     

Interferon‐γ regulates susceptibility to collagen‐induced arthritis through suppression of interleukin‐17

Objective

The enhanced expression of experimental arthritis in the absence of interferon‐γ (IFNγ) suggests that IFNγ suppresses arthritis. Interleukin‐17 (IL‐17) is a pivotal T cell cytokine in arthritis, and in vitro studies have indicated that IFNγ suppresses IL‐17 production. We undertook this study to test the hypothesis that resistance to collagen‐induced arthritis (CIA) in C57BL/6 (B6) mice is regulated by IFNγ‐mediated suppression of IL‐17.

Methods

Wild‐type (WT) B6 mice, IFNγ‐knockout (KO) B6 mice, and DBA/1 mice were immunized with type II collagen (CII) in Freund's complete adjuvant (CFA). Lymphocytes from immunized mice were analyzed for cytokine production ex vivo by intracellular staining or restimulation with CII and enzyme‐linked immunosorbent assays. In vivo blockade of IL‐17 was achieved with an anti–IL‐17 monoclonal antibody (mAb).

Results

CII restimulation of T cells from CII/CFA‐immunized mice resulted in an ∼5‐fold increase in IL‐17 production in IFNγ‐KO B6 mice compared with WT B6 mice. Neutralization of IFNγ increased IL‐17 production in WT B6 mice, and neutralization of IL‐4 had a synergistic effect. Interestingly, the prototypical CIA‐susceptible strain DBA/1 also demonstrated a high IL‐17 and a low IFNγ cytokine profile compared with WT B6 mice. Administration of the anti–IL‐17 mAb attenuated arthritis in DBA/1 mice and almost completely prevented expression of arthritis in IFNγ‐KO B6 mice.

Conclusion

These results indicate that sensitivity of IFNγ‐deficient B6 mice to CIA is associated with high IL‐17 production and that this cytokine is required for expression of arthritis in this strain.

     

Murine tumor necrosis factor α–induced adipose‐related protein (tumor necrosis factor α–induced protein 9) deficiency leads to arthritis via interleukin‐6 overproduction with enhanced NF‐κB,STAT‐3 signaling,and dysregulated apoptosis of macrophages

Objective

To elucidate the role of tumor necrosis factor α–induced adipose‐related protein (TIARP; or tumor necrosis factor α–induced protein 9 [TNFAIP‐9]) in the development and pathogenesis of arthritis.

Methods

We generated TIARP‐deficient (TIARP^−/−) mice and investigated several organs in aged mice. Peritoneal macrophages were collected and cultured with lipopolysaccharide (LPS) and TNFα, and then the production of cytokines and subsequent NF‐κB signal transduction were analyzed. We also examined the susceptibility of young TIARP^−/− mice to collagen‐induced arthritis (CIA). Draining lymph nodes and splenocytes were isolated and cultured, and serum levels of anti–type II collagen (anti‐CII) antibodies, interleukin‐6 (IL‐6), and TNFα on day 60 were measured. We further investigated the effects of anti–IL‐6 receptor monoclonal antibody (mAb) on the development of arthritis in TIARP^−/− mice. IL‐6/STAT‐3 signaling was also analyzed using TIARP^−/− macrophages.

Results

TIARP^−/− mice developed spontaneous enthesitis and synovitis, had high serum levels of IL‐6, had increased CD11b+ cell counts in the spleen, and showed enhanced LPS‐ and TNFα‐induced IL‐6 expression in macrophages. Sustained degradation of IκBα with dysregulated apoptosis was also noted in TIARP^−/− macrophages. CIA was clearly exacerbated in TIARP^−/− mice, accompanied by marked neutrophil and macrophage infiltration in joints. The levels of anti‐CII antibodies in serum were unchanged, whereas autoreactive Th1 cell and Th17 cell responses were higher in TIARP^−/− mice. Treatment with anti–IL‐6 receptor mAb prevented the development of CIA in TIARP^−/− mice, and TIARP^−/− macrophages showed increased IL‐6–induced STAT‐3 phosphorylation.

Conclusion

These findings suggest that TIARP acts as a negative regulator of arthritis by suppressing IL‐6 production, its signaling and TNFα‐induced NF‐κB signaling, resulting in enhanced apoptosis in macrophages.

     

Proinflammatory role of the Th17 cytokine interleukin‐22 in collagen‐induced arthritis in C57BL/6 mice

Objective

To investigate the role of interleukin‐22 (IL‐22) in collagen‐induced arthritis (CIA), an animal model of rheumatoid arthritis.

Methods

C57BL/6 mice were immunized with type II collagen (CII) in Freund's incomplete adjuvant with added Mycobacterium tuberculosis, and levels of IL‐22 and its specific receptor, IL‐22 receptor type I (IL‐22RI), were measured in sera and tissue by enzyme‐linked immunosorbent assay and real‐time quantitative polymerase chain reaction analysis. Clinical and histologic signs of arthritis were recorded and compared with those in C57BL/6 mice deficient in the IL‐22 gene (IL‐22^–/–). Humoral and cellular immune responses against CII were analyzed. In vitro osteoclastogenesis assays were performed on splenocytes.

Results

Upon immunization with CII in Freund's incomplete adjuvant plus heat‐killed Mycobacterium tuberculosis, sera from C57BL/6 mice were found to contain high levels of IL‐22, and the specific IL‐22RI was expressed in lymphoid tissue, including splenocytes. IL‐22^–/– mice were less susceptible to CIA than were wild‐type mice, as evidenced by their decreased incidence of arthritis and decreased pannus formation. Remarkably, the less severe form of arthritis in IL‐22^–/– mice was associated with increased production of CII‐specific and total IgG antibodies, whereas cellular CII responses were unchanged. In vitro, IL‐22 was found to promote osteoclastogenesis, a process that might contribute to its proinflammatory activity in CIA.

Conclusion

Endogenous IL‐22 plays a proinflammatory role in CIA in C57BL/6 mice. Our data also indicate that IL‐22 promotes osteoclastogenesis and regulates antibody production.

     

Reduction of the incidence and severity of collagen‐induced arthritis by constitutive Nur77 expression in the T cell lineage

Objective

To test the hypothesis that constitutive expression of Nur77 in the T cell lineage will suppress the development and pathogenesis of collagen‐induced arthritis (CIA) and to understand the mechanisms by which Nur77 overexpression influences the arthritogenic response to type II collagen (CII).

Methods

Nur77‐transgenic and wild‐type C57BL/6 mice were immunized with CII and were monitored for the development and severity of arthritis. Pathologic changes were examined by histology and radiography. The effects of Nur77 overexpression on immune responses were evaluated by cytokine production in vitro and serum levels of CII‐specific antibodies. Sensitivity of T cells to apoptosis induction was analyzed in vitro following stimulation with anti‐CD3 monoclonal antibody or glucocorticoid.

Results

The incidence and severity of CIA was significantly decreased in Nur77‐transgenic mice compared with wild‐type controls. Attenuation of the disease was associated with increased apoptosis induction in transgenic T cells and decreased production of CII‐specific IgG2a antibodies in transgenic mice. Overexpression of Nur77 in the T cell compartment did not affect Th1/Th2 cytokine production or balance.

Conclusion

Nur77 overexpression in the T cell lineage attenuates the development and progression of CIA, probably by promoting activation‐induced T cell apoptosis and by inhibiting CII‐specific antibody production.

     

The involvement of Vα14 natural killer T cells in the pathogenesis of arthritis in murine models

Objective

To examine the physiologic role of natural killer T (NKT) cells bearing V_α14 T cell receptor (TCR) in the pathogenesis of collagen‐induced arthritis (CIA) and antibody‐induced arthritis in mice.

Methods

NKT cells were stained with α‐galactosylceramide–loaded CD1 dimer, and then assessed using flow cytometry. CIA was induced in mice by immunization on days 0 and 21 with type II collagen (CII) emulsified with an equal volume of Freund's complete adjuvant. Anti‐CII antibodies were measured by enzyme‐linked immunosorbent assay. For antibody‐induced arthritis, mice were injected with anti‐CII monoclonal antibodies (mAb) followed by lipopolysaccharide, or with serum from KRN TCR–transgenic mice crossed with nonobese diabetic mice (K/BxN). The severity of arthritis was monitored with a macroscopic scoring system.

Results

The number of NKT cells increased in the liver at the peak of the clinical course of CIA. Administration of anti‐CD1 mAb inhibited development of CIA. The severity of CIA in NKT cell–deficient mice was reduced compared with that in wild‐type mice. The IgG1:IgG2a ratio of anti‐CII was elevated and production of interleukin‐10 from draining lymph node cells was increased in NKT cell–deficient mice. NKT cell–deficient mice were significantly less susceptible to antibody‐induced arthritis.

Conclusion

NKT cells contribute to the pathogenesis of arthritis by enhancing autoantibody‐mediated inflammation. NKT cells also contribute to the disease process in a deleterious way, due, at least in part, to the alteration of the Th1/Th2 balance in T cell response to CII.

     

Role of placenta growth factor and its receptor flt‐1 in rheumatoid inflammation: A link between angiogenesis and inflammation

Objective

To investigate the direct effects of placenta growth factor (PlGF) and its specific receptor, flt‐1, which are known to mediate angiogenesis, on the inflammatory process of rheumatoid arthritis (RA).

Methods

Expression of PlGF and flt‐1 in the synovial tissue of RA patients was examined using immunohistochemistry. Enzyme‐linked immunosorbent assay was used to determine the concentrations of PlGF, tumor necrosis factor α (TNFα), and interleukin‐6 (IL‐6) in culture supernatants of either mononuclear cells or synoviocytes. The flt‐1 expression level in mononuclear cells was analyzed by flow cytometry. Experimental arthritis was induced in mice either by immunization with type II collagen (CII) or by injection of anti‐CII antibody.

Results

PlGF was highly expressed in the synovium of RA patients, and its primary source was fibroblast‐like synoviocytes (FLS). When stimulated with IL‐1β, FLS from RA patients produced higher amounts of PlGF than did FLS from patients with osteoarthritis. Exogenous PlGF specifically increased the production of TNFα and IL‐6 in mononuclear cells from RA patients (but not those from healthy controls) via a calcineurin‐dependent pathway. The response to PlGF was associated with increased expression of flt‐1 on RA monocytes, which could be induced by IL‐1β and TNFα. A novel anti–flt‐1 hexapeptide, GNQWFI, abrogated the PlGF‐induced increase in TNFα and IL‐6 production, and also suppressed CII‐induced arthritis and serum IL‐6 concentrations in mice. Moreover, genetic ablation of PlGF prevented the development of anti‐CII antibody–induced arthritis in mice.

Conclusion

Our data suggest that enhanced expression of PlGF and flt‐1 may contribute to rheumatoid inflammation by triggering production of proinflammatory cytokines. The use of the novel anti–flt‐1 peptide, GNQWFI, may be an effective strategy for the treatment of RA.

     

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.

     

Genetic control of antibody production during collagen‐induced arthritis development in heterogeneous stock mice

Objective

To identify genetic factors driving pathogenic autoantibody formation in collagen‐induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA), in order to better understand the etiology of RA and identify possible new avenues for therapeutic intervention.

Methods

We performed a genome‐wide analysis of quantitative trait loci controlling autoantibody to type II collagen (anti‐CII), anti–citrullinated protein antibody (ACPA), and rheumatoid factor (RF). To identify loci controlling autoantibody production, we induced CIA in a heterogeneous stock–derived mouse cohort, with contribution of 8 inbred mouse strains backcrossed to C57BL/10.Q. Serum samples were collected from 1,640 mice before arthritis onset and at the peak of the disease. Antibody concentrations were measured by standard enzyme‐linked immunosorbent assay, and linkage analysis was performed using a linear regression–based method.

Results

We identified loci controlling formation of anti‐CII of different IgG isotypes (IgG1, IgG3), antibodies to major CII epitopes (C1, J1, U1), antibodies to a citrullinated CII peptide (citC1), and RF. The anti‐CII, ACPA, and RF responses were all found to be controlled by distinct genes, one of the most important loci being the immunoglobulin heavy chain locus.

Conclusion

This comprehensive genetic analysis of autoantibody formation in CIA demonstrates an association not only of anti‐CII, but interestingly also of ACPA and RF, with arthritis development in mice. These results underscore the importance of non–major histocompatibility complex genes in controlling the formation of clinically relevant autoantibodies.

     

Treatment of murine collagen‐induced arthritis by the stress protein BiP via interleukin‐4–producing regulatory T cells: A novel function for an ancient protein

Objective

Following the demonstration that the stress protein, BiP, prevented induction of collagen‐induced arthritis (CIA) in HLA–DRB*0101^+/+ (HLA–DR1^+/+) mice, we investigated the immunotherapeutic ability of BiP to suppress disease during the active phase of CIA in HLA–DR1^+/+ and DBA/1 mice.

Methods

BiP was administered either subcutaneously or intravenously to DBA/1, HLA–DR1^+/+, or interleukin‐4 (IL‐4)–knockout mice at the onset of arthritis. Immune cells were used in adoptive transfer studies or were restimulated in culture with BiP or type II collagen (CII). Proliferation and cytokine release were measured. In addition, serum anti‐CII antibodies were measured by enzyme‐linked immunosorbent assay. Disease progression was scored using a visual analog scale.

Results

BiP was successful in suppressing established CIA in HLA–DR1^+/+ and DBA/1 mice. Serum levels of anticollagen IgG antibodies were reduced in BiP‐treated mice. T cells from BiP‐immunized mice produced Th2 cytokines, in particular, IL‐4. Treatment with BiP was also shown to increase the production of CII‐specific IL‐5, IL‐10, and interferon‐γ at the termination of the study. Development of severe CIA was prevented by the intravenous transfer of BiP‐specific cells at the time of CIA induction in HLA–DR1^+/+ mice or by transferring BiP‐specific cells to DBA/1 mice at the onset of disease. BiP failed to ameliorate the development of CIA in IL‐4^−/−, HLA–DR1^+/+ mice.

Conclusion

These novel results show that BiP can suppress active CIA by the induction of regulatory cells that act predominantly via IL‐4. Thus, BiP is a potential immunotherapeutic agent for the treatment of patients with rheumatoid arthritis.

     

Arthritis in mice that are deficient in interleukin‐1 receptor antagonist is dependent on genetic background

Objective

To determine the effect of deletion of interleukin‐1 receptor antagonist (IL‐1Ra) protein in an animal model of rheumatoid arthritis.

Methods

BALB/c mice deficient in IL‐1Ra (IL‐1Ra^−/−) were bred with collagen‐induced arthritis (CIA)–susceptible DBA/1 mice and B10 mice transgenic for HLA–DRB1*0101 (B10.DR1). After generation of IL‐1Ra^−/− mice on the DBA/1 and B10.DR1 backgrounds, the mice were observed for the development of spontaneous arthritis and immunized for induction of CIA.

Results

We found that although BALB/c mice deficient in IL‐1Ra (BALB/c^−/−) spontaneously developed chronic inflammatory arthritis, DBA/1 IL‐1Ra–deficient (DBA/1^−/−) and B10.DR1 IL‐1Ra–deficient (B10.DR1^−/−) mice did not. Splenocytes from BALB/c^−/− mice produced elevated levels of IL‐2, IL‐4, IL‐6, IL‐10, IL‐17, and granulocyte–macrophage colony‐stimulating factor in response to anti‐CD3 stimulation. After immunization with type II collagen (CII), DBA/1^−/− and B10.DR1^−/− mice had a significantly earlier onset of CIA, and with increased severity compared with IL‐1Ra^+/+ mice. Immunization of BALB/c^−/− mice with CII did not aggravate spontaneous arthritis. All of the immunized mice developed antibodies to CII that correlated with arthritis severity. Levels of antibody to CII in the BALB/c^−/− strain were relatively low.

Conclusion

These data indicate that the spontaneous arthritis of IL‐1Ra deficiency is highly dependent on non–major histocompatibility complex genes and that autoimmunity to CII is not the major disease‐inducing event. Class II immune response genes are more important for the regulation of CIA, and although these 2 models of arthritis share many pathogenic mechanisms, they also have significant differences.

     

Interleukin‐1 and tumor necrosis factor α blockade treatment of experimental polymyositis in mice

Objective

Histologic studies of the muscles suggest that cytokines are involved in inflammatory myopathy. The therapeutic effects of cytokine blockade are controversial, with anecdotal reports of clinical efficacy. The aim of this study was to discern the significance of interleukin‐1 (IL‐1) and tumor necrosis factor α (TNFα) as therapeutic targets in polymyositis (PM) by studying their involvement and the effects of their blockade in C protein–induced myositis (CIM), a murine model of PM.

Methods

C57BL/6 mice were immunized with recombinant skeletal C protein fragments to induce CIM. The expression of IL‐1 and TNFα in the muscles of mice with CIM was detected using immunohistochemical and real‐time polymerase chain reaction analyses. After the onset of myositis, the mice with CIM were treated with recombinant IL‐1 receptor antagonist (IL‐1Ra), anti–IL‐1R monoclonal antibody, recombinant TNF receptor (p75)–fusion protein (TNFR‐Fc), or anti‐TNFα monoclonal antibody. The muscles were examined histologically for the severity of myositis.

Results

IL‐1α– and TNFα‐positive macrophages were observed in the muscle tissue of mice with CIM as early as 7 days after immunization. IL‐1α, IL‐1β, and TNFα expression in the muscles increased as the severity of myositis peaked, at both the messenger RNA and protein levels. Continuous subcutaneous delivery of IL‐1Ra resulted in suppression of established CIM. Intermittent delivery (1‐day intervals) of anti–IL‐1R monoclonal antibody suppressed myositis, while intermittent delivery of IL‐1Ra did not suppress myositis. Treatment with anti‐TNFα monoclonal antibody and with TNFR‐Fc also reduced the severity of CIM.

Conclusion

IL‐1 and TNF blockade ameliorated CIM after disease onset and should potentially be a new strategy for the treatment of inflammatory myopathy. As IL‐1 blockade, treatment with anti–IL‐1R monoclonal antibody appeared more feasible than the other approaches.

     

CXCL13 neutralization reduces the severity of collagen‐induced arthritis

Objective

To investigate the role of CXCL13 in the development and pathogenesis of collagen‐induced arthritis (CIA), and to determine the mechanisms involved in the modulation of arthritogenic response by CXCL13 neutralization.

Methods

Mice were immunized with type II collagen (CII) and treated with anti‐CXCL13 or control antibodies during boosting. Mice were monitored for the development and severity of arthritis. The effects of CXCL13 neutralization on immune response to CII were evaluated by cytokine production by CII‐specific T cells and CII‐specific antibody production. Follicular response in the spleen and in synovial tissue was determined by in situ immunohistology.

Results

Mice receiving neutralizing antibodies to CXCL13 developed significantly less severe arthritis compared with mice injected with phosphate buffered saline or control antibodies. Follicular response both in the spleen and in synovial tissue was inhibited by anti‐CXCL13 treatment. Injection with anti‐CXCL13 antibodies did not significantly affect antigen‐specific recall lymphocyte proliferation or type 1 cytokine production in vitro. Antibody response specific to CII was not inhibited by anti‐CXCL13 treatment. However, anti‐CXCL13 treatment induced significantly higher levels of interleukin‐10 production after in vitro CII stimulation.

Conclusion

Neutralization of CXCL13 inhibits the development of CIA and reduces follicular response in both lymphoid and nonlymphoid tissues. These findings may have important implications regarding the pathogenesis and treatment of autoimmune 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 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.

     

Treatment with a neutralizing anti‐murine interleukin‐17 antibody after the onset of collagen‐induced arthritis reduces joint inflammation,cartilage destruction,and bone erosion

Objective

Interleukin‐17 (IL‐17) is a proinflammatory cytokine that is expressed in the synovium of rheumatoid arthritis (RA) patients. This T cell cytokine is implicated in the initiation phase of arthritis. However, the role of IL‐17 during the effector phase of arthritis has still not been identified; this was the objective of the present study.

Methods

Mice with collagen‐induced arthritis (CIA) were treated with polyclonal rabbit anti‐murine IL‐17 (anti–IL‐17) antibody–positive serum or normal rabbit serum after the first signs of arthritis. In addition, during a later stage of CIA mice were selected and treated with anti–IL‐17 antibody or control serum. Arthritis was monitored visually, and joint pathology was examined radiologically and histologically. Systemic IL‐6 levels were measured by enzyme‐linked immunosorbent assay, and local synovial IL‐1 and receptor activator of NF‐κB ligand (RANKL) expression was analyzed using specific immunohistochemistry.

Results

Treatment with a neutralizing anti–IL‐17 antibody after the onset of CIA significantly reduced the severity of CIA. Radiographic analysis revealed marked suppression of joint damage in the knee and ankle joints. Histologic analysis confirmed the suppression of joint inflammation and showed prevention of cartilage and bone destruction after anti–IL‐17 antibody therapy. Systemic IL‐6 levels were significantly reduced after anti–IL‐17 antibody treatment. Moreover, fewer IL‐1β–positive and RANKL‐positive cells were detected in the synovium after treatment with neutralizing IL‐17. Interestingly, initiation of anti–IL‐17 antibody therapy during a later stage of CIA, using mice with higher clinical arthritis scores, still significantly slowed the progression of the disease.

Conclusion

IL‐17 plays a role in early stages of arthritis, but also later during disease progression. Systemic IL‐6 was reduced and fewer synovial IL‐1–positive and RANKL‐positive cells were detected after neutralizing endogenous IL‐17 treatment, suggesting both IL‐1–dependent and IL‐1–independent mechanisms of action. Our data strongly indicate that IL‐17 neutralization could provide an additional therapeutic strategy for RA, particularly in situations in which elevated IL‐17 may attenuate the response to anti–tumor necrosis factor/anti–IL‐1 therapy.

     

Escherichia coli heat‐labile enterotoxin B subunit prevents autoimmune arthritis through induction of regulatory CD4+ T cells

Objective

The receptor‐binding B subunit of Escherichia coli heat‐labile enterotoxin (EtxB) is a highly stable, nontoxic protein that is capable of modulating immune responses. This study was conducted to determine whether mucosal administration of EtxB can block collagen‐induced arthritis (CIA) and to investigate the mechanisms involved.

Methods

Clinical arthritis in DBA/1 mice was monitored following mucosal administration of EtxB on 4 occasions. The dependence of disease prevention on receptor binding by EtxB and the associated alterations to the immune response to type II collagen (CII) were assessed. Adoptive transfer experiments and lymph node cell cocultures were used to investigate the underlying mechanisms.

Results

Both intranasal and intragastric delivery of EtxB were effective in preventing CIA; a 1‐μg dose of EtxB was protective after intranasal administration. A non–receptor‐binding mutant of EtxB failed to prevent disease. Intranasal EtxB lowered both the incidence and severity of arthritis when given either at the time of disease induction or 25 days later. EtxB markedly reduced levels of anti‐CII IgG2a antibodies and interferon‐γ (IFNγ) production while not affecting levels of IgG1, interleukin‐4 (IL‐4), or IL‐10. Disease protection could be transferred by CD4+ T cells from treated mice, an effect that was abrogated upon depletion of the CD25+ population. In addition, CD4+CD25+ T cells from treated mice were able to suppress anti‐CII IFNγ production by CII‐primed lymph node cells.

Conclusion

Mucosal administration of EtxB can be used to prevent or treat CIA. Modulation of the anti‐CII immune response by EtxB is associated with a reduction in Th1 cell reactivity without a concomitant shift toward Th2. Instead, EtxB mediates its effects through enhancing the activity of a population of CD4+ regulatory T cells.

     

A tumor necrosis factor receptor loop peptide mimic inhibits bone destruction to the same extent as anti–tumor necrosis factor monoclonal antibody in murine collagen‐induced arthritis

Objective

The cyclic peptide WP9QY (YCWSQYLCY) was designed to mimic the most critical tumor necrosis factor α (TNFα) recognition loop on TNF receptor I, and it prevents interactions of TNFα with its receptor. We undertook this study to compare the effects of the WP9QY peptide on collagen‐induced arthritis (CIA) in mice with those of anti‐TNFα monoclonal antibody.

Methods

CIA was induced by primary and secondary immunizations. Osmotic minipumps were implanted in the backs of all mice on the day of the booster injection (day 21), and vehicle, anti‐TNF antibody (4 mg/kg/day), or WP9QY peptide (2 mg/kg/day or 4 mg/kg/day) was continuously infused until the mice were killed (day 40). Thereafter, clinical, radiographic, and histologic assessments were performed.

Results

WP9QY treatment inhibited CIA‐induced increases in the arthritis score, but onset of disease was not delayed by the peptide. The inhibitory effect of WP9QY on inflammation was definitely weaker than that of anti‐TNF antibody. Microfocal computed tomography analyses, however, revealed that WP9QY blocked CIA‐induced bone destruction at the knee joints to the same extent as did anti‐TNF antibody. In addition, WP9QY inhibited synovial pannus infiltration and reduced osteoclast number. Furthermore, inhibition of CIA‐induced systemic bone loss by WP9QY was more apparent than that by anti‐TNF antibody.

Conclusion

The TNFα antagonist WP9QY would be a useful template for the development of small molecular inhibitors to prevent both inflammatory bone destruction and systemic bone loss in rheumatoid arthritis.

     

Interleukin‐20 antibody is a potential therapeutic agent for experimental arthritis

Objective

Interleukin‐20 (IL‐20) is a proinflammatory cytokine involved in the pathogenesis of rheumatoid arthritis (RA). We investigated whether anti–IL‐20 antibody treatment would modulate the severity of the disease in a collagen‐induced arthritis (CIA) rat model.

Methods

We generated a CIA model by immunizing rats with bovine type II collagen. Rats with CIA were treated subcutaneously with anti–IL‐20 antibody 7E, with the tumor necrosis factor (TNF) blocker etanercept, or with 7E in combination with etanercept. Arthritis severity was determined according to the hind paw thickness, arthritis severity score, degree of cartilage damage, bone mineral density, and cytokine production, which were evaluated using radiologic scans, microfocal computed tomography, and enzyme‐linked immunosorbent assay. To analyze gene regulation by IL‐20, rat synovial fibroblasts (SFs) were isolated and analyzed for the expression of RANKL, IL‐17, and TNFα. We also used real‐time quantitative polymerase chain reaction analysis and flow cytometry to determine IL‐20–regulated RANKL in mouse osteoblastic MC3T3‐E1 cells and Th17 cells.

Results

In vivo, treatment with 7E alone or in combination with etanercept significantly reduced the severity of arthritis by decreasing the hind paw thickness and swelling, preventing cartilage damage and bone loss, and reducing the expression of IL‐20, IL‐1β, IL‐6, RANKL, and matrix metalloproteinases (MMPs) in synovial tissue. In vitro, IL‐20 induced TNFα expression in SFs from rats with CIA. IL‐20 markedly induced RANKL production in SFs, osteoblasts, and Th17 cells.

Conclusion

Selectively blocking IL‐20 inhibited inflammation and bone loss in rats with CIA. Treatment with 7E combined with etanercept protected rats from CIA better than treatment with etanercept alone. Our findings provide evidence that IL‐20 is a novel target and that 7E may be a potential therapeutic agent for RA.