Constitutive expression of BCL‐XL in the T lineage attenuates collagen‐induced arthritis in Bcl‐XL transgenic mice

Objective

To determine if inhibition of T cell apoptosis through constitutive expression of Bcl‐X _L in the T lineage influences inflammatory arthritis in the mouse collagen‐induced arthritis (CIA) model.

Methods

The incidence and severity of arthritis were quantified in Bcl‐X _L transgenic mice and nontransgenic littermates after immunization with type II collagen (CII). To correlate T cell responses with disease phenotype, antigen‐specific T cell proliferation was measured by ³H‐thymidine incorporation. Apoptosis and cell cycle progression were analyzed by flow cytometry using propidium iodide. Production of CII‐specific interferon‐γ (IFNγ), interleukin‐5 (IL‐5), and IL‐10 was determined by enzyme‐linked immunosorbent assay.

Results

Disease severity in CIA was significantly attenuated in Bcl‐X _L transgenic mice compared with their nontransgenic littermates. Inhibition of CIA was associated with decreased T cell apoptosis, delayed cell cycle progression, and reduced IFNγ production.

Conclusion

Rather than promoting inflammation, inhibition of apoptosis by expression of the Bcl‐X _L protein in the T lineage attenuates disease progression in CIA, probably through inhibition of IFNγ production.

     

CD28‐dependent differentiation into the effector/memory phenotype is essential for induction of arthritis in interleukin‐1 receptor antagonist–deficient mice

Objective

Interleukin‐1 receptor antagonist (IL‐1Ra)–deficient mice on a BALB/c background spontaneously develop a chronic inflammatory polyarthropathy closely resembling that of rheumatoid arthritis in humans. To elucidate the role of CD28 costimulatory signals in the development of this disease, we studied IL‐1Ra/CD28–double‐deficient mice.

Methods

We crossed IL‐1Ra–deficient mice with CD28–deficient mice and observed the incidence and severity of arthritis. To investigate functions of IL‐1Ra/CD28–double‐deficient T cells, cells were stimulated with CD3 monoclonal antibody or allogeneic antigen‐presenting cells (APCs) and their proliferative responses and levels of cytokine production were measured.

Results

Disease severity was lower in IL‐1Ra/CD28–double‐deficient mice than in mice that were deficient only in IL‐1Ra, although incidence of arthritis was not affected by the presence or absence of CD28. When pathogenic IL‐1Ra–KO T cells were transferred into nude mice, severe arthritis developed. Even though T cells from double‐deficient mice showed the same diminished proliferative capacity as was seen in T cells from CD28–single‐deficient animals, nude mice into which double‐deficient T cells were transferred never developed arthritis.

Conclusion

These findings indicate that IL‐1Ra/CD28–double‐deficient T cells can be activated by IL‐1Ra–deficient activated APCs, resulting in induction of arthritis; however, these T cells did not induce the disease under normal conditions, because they did not differentiate into effector/memory phenotype.

     

Suppressive oligonucleotides protect against collagen‐induced arthritis in mice

Objective

To examine whether systemic administration of oligonucleotides (ODNs), known to inhibit the production of proinflammatory cytokines, alters host susceptibility to collagen‐induced arthritis (CIA), a murine model of rheumatoid arthritis (RA).

Methods

CIA was induced by injecting DBA/1 mice with type II collagen (CII) in Freund's complete adjuvant, followed 3 weeks later by CII in Freund's incomplete adjuvant. The effect of suppressive ODNs on the incidence and severity of disease was monitored, as were immune correlates of CIA.

Results

Suppressive ODNs administered during the inductive phase of CIA significantly reduced the incidence and severity of arthritis. Treatment with suppressive ODNs significantly decreased serum titers of pathogenic IgG anti‐CII autoantibodies and interferon‐γ production by collagen‐reactive T cells.

Conclusion

Suppressive ODNs may be of therapeutic value in the treatment of RA, and potentially other autoimmune diseases.

     

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.

     

Protective effect of RC‐3095, an antagonist of the gastrin‐releasing peptide receptor,in experimental arthritis

Objective

To evaluate the antiinflammatory effects of RC‐3095 in 2 experimental models of arthritis, collagen‐induced arthritis (CIA) and antigen‐induced arthritis (AIA), and to determine the mechanisms of action involved.

Methods

RC‐3095 was administered daily to mice with CIA and mice with AIA, after induction of disease with methylated bovine serum albumin. Disease incidence and severity were assessed using a clinical index and evaluation of histologic features, respectively. In mice with CIA, gastrin‐releasing peptide receptor (GRPR) was detected by immunohistochemical analysis, while in mice with AIA, migration of neutrophils, presence of glycosaminoglycans, and lymphocyte proliferation, determined using the MTT assay, were assessed. Expression of cytokines interleukin‐17 (IL‐17), IL‐1β, and tumor necrosis factor α (TNFα) was evaluated in all mouse knees using enzyme‐linked immunosorbent assay. Treg cell production was assessed by flow cytometry in the joints of mice with AIA.

Results

In mice with AIA, administration of RC‐3095 reduced neutrophil migration, mechanical hypernociception, and proteoglycan loss. These findings were associated with inhibition of the levels of all 3 proinflammatory cytokines, decreased lymphocyte proliferation, and increased Treg cell numbers. In the CIA model, treatment with RC‐3095 led to a significant reduction in arthritis clinical scores and the severity of disease determined histologically. Synovial inflammation, synovial hyperplasia, pannus formation, and extensive erosive changes were all dramatically reduced in the arthritic mice treated with RC‐3095. Furthermore, arthritic mice treated with RC‐3095 showed a significant reduction in the concentrations of IL‐17, IL‐1β, and TNFα, and showed a diminished expression of GRPR.

Conclusion

These findings suggest that the GRP pathway has a significant role in chronic arthritis, and its inhibition can be explored as a possible therapeutic strategy in rheumatoid arthritis.

     

Down‐regulation of Th1‐mediated pathology in experimental arthritis by stimulation of the Th2 arm of the immune response

Objective

To assess whether systemic administration of recombinant interferon‐γ (rIFNγ), a proinflammatory cytokine that influences the differentiation of naive T cells into Th1 cells, promotes the induction of arthritis in DBA/1 mice immunized with type II collagen (CII) in Freund's incomplete adjuvant (IFA) and to determine the antiarthritic effect of treatment with CII in IFA.

Methods

DBA/1 mice were immunized with CII in IFA and injected intraperitoneally with rIFNγ (8,000 units/mouse/day) or with recombinant interleukin‐12 (rIL‐12; 100 ng/mouse/day). In another experiment, mice were immunized with CII in Freund's complete adjuvant (CFA) and treated with a single dose of CII in IFA on the day of immunization or on the day of disease onset. Mice were monitored to assess the effect of the treatment on the severity of disease. Th1/Th2 cytokines and anti‐CII antibodies were measured by enzyme‐linked immunosorbent assay.

Results

The administration of rIL‐12 or rIFNγ to mice immunized with CII in IFA restored the Th1 response and resulted in the development of arthritis. We then determined whether immunization with CII in IFA had the capacity to prevent and/or ameliorate collagen‐induced arthritis. A single intraperitoneal injection of CII in IFA prevented arthritis when given at the time of immunization with CII in CFA and reduced disease severity when given at the time of arthritis onset. The administration of CII in IFA resulted in a profound down‐regulation of IFNγ production and an up‐regulation of IL‐10 in cultures of draining lymph node cells.

Conclusion

These findings demonstrate that it is possible to deflect an ongoing pathogenic Th1 response to an antigen by reimmunization of the same antigen with a Th2‐polarizing adjuvant.

     

Acute CD4+ T lymphocyte–dependent interleukin‐1–driven arthritis selectively requires interleukin‐2 and interleukin‐4, joint macrophages,granulocyte–macrophage colony‐stimulating factor,interleukin‐6, and leukemia inhibitory factor

Objective

To further investigate the effects of interleukin‐1 (IL‐1) in immune‐mediated joint inflammation, we examined the role of IL‐2, Th1 interferon‐γ (IFNγ), and Th2 (IL‐4) cytokines, joint macrophages, and macrophage‐derived cytokines (IL‐12 p40, IL‐6, leukemia inhibitory factor [LIF], oncostatin M [OSM], and granulocyte–macrophage colony‐stimulating factor [GM‐CSF]) in a CD4+ T lymphocyte–dependent model of acute arthritis.

Methods

Methylated bovine serum albumin (mBSA)/IL‐1–induced arthritis was elicited in wild‐type, gene‐knockout, and monoclonal antibody–treated mice. Synovial lining macrophages were selectively depleted by intraarticular injection of clodronate liposomes prior to disease induction. The severity of arthritis was assessed histologically.

Results

Mice deficient in IL‐2 were almost completely protected from arthritis, and neutralization of IL‐4 reduced the severity of disease. In contrast, arthritis severity and resolution appeared to be independent of IFNγ. Synovial lining macrophage depletion markedly reduced arthritis severity. IL‐6 or LIF deficiency was only modestly protective, although as previously reported, GM‐CSF deficiency conferred profound disease resistance. IL‐12 p40–deficient mice (which lack IL‐12 and IL‐23) and OSM receptor–deficient mice were susceptible to mBSA/IL‐1–induced arthritis.

Conclusion

Acute mBSA/IL‐1–induced arthritis is dependent on IL‐2 and IL‐4, but not IFNγ. In vivo, the Th1/Th2 paradigm may be distorted by the presence of macrophage‐derived cytokines such as IL‐1. Synovial lining macrophages are essential in mBSA/IL‐1–induced arthritis. However, the requirement for macrophage‐derived cytokines is selective; that is, IL‐6, LIF, and especially GM‐CSF are necessary, but IL‐12, IL‐23, and OSM are dispensable. IL‐1 may therefore influence both adaptive and innate immune mechanisms in acute inflammatory arthritis.

     

Immunogenicity of recombinant type IX collagen in murine collagen‐induced arthritis

Objective

Past attempts to isolate type IX collagen (CIX) from cartilage using limited proteolysis yielded partially degraded material. Recent application of recombinant technology, however, has allowed the preparation of intact native CIX. We used the murine collagen‐induced arthritis model to characterize the immunologic properties of recombinant human CIX (rCIX) produced using a baculovirus expression system.

Methods

A panel of B10 congenic mice was immunized with rCIX emulsified with Freund's complete adjuvant (CFA). The ability of the rCIX to induce tolerance and suppress arthritis was determined by administration intravenously or orally before challenge with CII/CFA.

Results

None of the mice immunized with rCIX developed overt arthritis, although 2 of 5 HLA–DR1 transgenic mice developed limited digital erythema and swelling. Recombinant CIX administered by either route effectively induced suppression of arthritis, although the suppression was less pronounced than that induced with CII. Immune responses to CIX and CII were specific, suggesting that bystander suppression, rather than cross‐reactivity with CII, was instrumental in suppressing arthritis.

Conclusion

These data show that CIX down‐regulates arthritis in mice while having no associated risk of inducing arthritis.

     

Targeting of Gr‐1+,CCR2+ monocytes in collagen‐induced arthritis

Objective

The chemokine receptor CCR2 is highly expressed on monocytes and considered a promising target for treatment of rheumatoid arthritis. However, blockade of CCR2 with a monoclonal antibody (mAb) during progression of collagen‐induced arthritis results in a massive aggravation of the disease. In this study we investigated why CCR2 antibodies have proinflammatory effects, how these effects can be avoided, and whether CCR2+ monocytes are useful targets in the treatment of arthritis.

Methods

Arthritis was induced in DBA/1 mice by immunization with type II collagen. Mice were treated with mAb against CCR2 (MC‐21), IgE, or isotype control antibodies at various time points. Activation of basophils and depletion of monocyte subsets were determined by fluorescence‐activated cell sorter analysis and enzyme‐linked immunosorbent assay.

Results

Crosslinkage of CCR2 activated basophils to release interleukin‐6 (IL‐6) and IL‐4. In vivo, IL‐6 release occurred only after exposure to high doses of MC‐21, whereas application of low doses of the mAb circumvented the release of IL‐6. Regardless of the dose level used, the antibody MC‐21 efficiently depleted Gr‐1+,CCR2+ monocytes from the synovial tissue, peripheral blood, and spleen of DBA/1 mice. Activation of basophils with high doses of MC‐21 or with antibodies against IgE resulted in a marked aggravation of collagen‐induced arthritis and an increased release of IL‐6. In contrast, low‐dose treatment with MC‐21 in this therapeutic setting had no effect on IL‐6 and led to marked improvement of arthritis.

Conclusion

These results show that depletion of CCR2+ monocytes may prove to be a therapeutic option in inflammatory arthritis, as long as the dose‐dependent proinflammatory effects of CCR2 mAb are taken into account.

     

Validity of self‐administered quality of well‐being scale in musculoskeletal disease

Objective

To evaluate the self‐administered Quality of Well‐Being (QWB‐SA) Scale for patients with rheumatic diseases.

Methods

Family medicine patients (n = 562) and rheumatology patients (n = 334) were assessed using the following tools: QWB‐SA, Health Assessment Questionnaire (HAQ), Arthritis Impact Measurement Scales (AIMS), and Rapid Assessment of Disease Activity in Rheumatology (RADAR).

Results

Patients with arthritis had significantly lower QWB‐SA scores and significantly higher HAQ scores than family medicine patients with and without adjustment for covariates. The QWB‐SA was significantly associated with quartiles from the RADAR, AIMS, and HAQ, providing evidence for the validity of the generic measure in patients with arthritis. Discriminant function analysis was used to create an arthritis‐specific scoring system for the QWB‐SA. Analyses demonstrated systematic relationships between the Quality of Well‐Being arthritis composite and the disease‐specific RADAR, AIMS, and HAQ.

Conclusions

Evidence supports the validity of the QWB‐SA for patients with rheumatic diseases. QWB‐SA items can be used to calculate an arthritis‐specific score. The QWB‐SA can be used to gain generic information for cost‐utility analysis and disease‐specific outcomes information for patients with arthritis.

     

The beneficial effect of interleukin‐12 on arthritis induced by group B streptococci is mediated by interferon‐γ and interleukin‐10 production

Objective

To assess the effect of interleukin‐12 (IL‐12) administration on the evolution of systemic infection and septic arthritis induced by group B streptococci (GBS) in mice.

Methods

CD1 mice were inoculated intravenously with arthritogenic strain 1/82 of type IV GBS. Exogenous murine IL‐12 was administered intraperitoneally 18 hours or 5 days after infection with 1 × 10⁷ GBS, at doses ranging from 0.5 to 2.5 μg per mouse. Mice were monitored daily for survival and for signs of arthritis. In a subsequent set of experiments, mice were killed at selected times for examination of bacterial clearance, histopathologic changes in the joints, and cytokine production.

Results

IL‐12 administration before the onset of clinical signs had a beneficial effect on GBS‐induced arthritis and was clearly dose‐dependent. The 2.5‐μg dose per mouse totally prevented death from GBS‐induced arthritis. The decrease in pathology was associated with a reduction of the bacterial burden and a change in the cytokine profile. In particular, systemic and joint levels of interferon‐γ (IFNγ) and IL‐10 significantly increased in mice treated with IL‐12, whereas a decrease in IL‐6 and IL‐1β production was observed. The beneficial effects of IL‐12, in terms of the incidence and severity of articular lesions, were reversed by coadministration of anti‐IFNγ or anti–IL‐10–neutralizing antibodies.

Conclusion

The findings of this study demonstrate that IL‐12 is important in controlling the cytokine production that leads to the evolution of GBS‐induced experimental arthritis. The amelioration of articular lesions is mostly attributable to IL‐12–induced IFNγ, but with a relevant participation of IL‐12–induced IL‐10.

     

Chronic relapsing homologous collagen‐induced arthritis in DBA/1 mice as a model for testing disease‐modifying and remission‐inducing therapies

Objective

To test whether the chronic relapsing arthritis induced by immunizing DBA/1 mice with homologous type II collagen is a valuable model for testing disease‐modifying antiarthritic drugs.

Methods

Six‐week‐old male DBA/1 mice were immunized with murine type II collagen in Freund's complete adjuvant, resulting in a chronic relapsing polyarthritis in >80% of the mice 4 weeks after immunization. At the onset of clinical arthritis, mice were treated for 4 weeks with different treatments, including anti–tumor necrosis factor (anti‐TNF) and anti–interleukin‐12 (anti–IL‐12) antibodies, salbutamol, or indomethacin. Alternatively, treatment was administered as a pulse at the beginning of clinical arthritis. Pulse treatments tested included anti‐CD3 in combination with anti‐TNF, anti‐TNF alone, and anti‐CD4, either alone or in combination with anti‐TNF. After 4 weeks of arthritis, mice were killed and hind paws were assessed histologically for joint damage.

Results

Anti‐TNF and salbutamol both suppressed clinical arthritis more effectively than indomethacin and, moreover, protected the joints from damage, whereas indomethacin did not. Anti–IL‐12 treatment initiated after the onset of clinical symptoms accelerated disease. Pulse therapy with anti‐CD3 plus anti‐TNF was found to induce remission, clinically as well as histologically, whereas a pulse with either anti‐CD4, anti‐TNF, or the combination of anti‐CD4 plus anti‐TNF was less effective.

Conclusion

Chronic relapsing homologous collagen‐induced arthritis is a valuable model for identifying remission‐inducing antiarthritic drugs and has predictive value with respect to their joint‐protective potency.

     

Gamma/delta T cells are the predominant source of interleukin‐17 in affected joints in collagen‐induced arthritis,but not in rheumatoid arthritis

Objective

Although interleukin‐17 (IL‐17)–producing γ/δ T cells were reported to play pathogenic roles in collagen‐induced arthritis (CIA), their characteristics remain unknown. The aim of this study was to clarify whether γ/δ T cells or CD4+ T cells are the predominant IL‐17–producing cells, and to determine what stimulates γ/δ T cells to secret IL‐17 in mice with CIA. The involvement of IL‐17–producing γ/δ T cells in SKG mice with autoimmune arthritis and patients with rheumatoid arthritis (RA) was also investigated.

Methods

IL‐17–producing cells in the affected joints of mice with CIA were counted by intracellular cytokine staining during 6 distinct disease phases, and these cells were stimulated with various combinations of cytokines or specific antigens to determine the signaling requirements. Similar studies were performed using SKG mice with arthritis and patients with RA.

Results

Gamma/delta T cells were the predominant population in IL‐17–producing cells in the swollen joints of mice with CIA, and the absolute numbers of these cells increased in parallel with disease activity. IL‐17–producing γ/δ T cells expressed CC chemokine receptor 6, were maintained by IL‐23 but not by type II collagen in vitro, and were induced antigen independently in vivo. Furthermore, IL‐17 production by γ/δ T cells was induced by IL‐1β plus IL‐23 independently of T cell receptor. In contrast to what was observed in mice with CIA, IL‐17–producing γ/δ T cells were nearly absent in the affected joints of SKG mice and patients with RA, and Th1 cells were predominant in the joints of patients with RA.

Conclusion

Gamma/delta T cells were antigen independently stimulated by inflammation at affected joints and produced enhanced amounts of IL‐17 to exacerbate arthritis in mice with CIA but not in SKG mice with arthritis or patients with RA.

     

Arthritis beliefs and self‐care in an urban American Indian population

Objective

To describe beliefs and self‐care strategies of American Indians with chronic arthritis joint pain.

Method

In‐depth interviews were conducted with a convenience sample of urban‐dwelling American Indians (n = 56) concerning self‐care and beliefs about arthritis; objective measures of arthritis disease activity were obtained through standardized interview protocols.

Results

Joint pain was not generally assumed to be arthritis nor directly related to aging. Belief that chronic pain affecting multiple joints was a serious and unexpected condition oriented American Indians' decisions to seek medical attention. However, verbal communications about pain may be subtle or under emphasized. Few coping strategies were used to control either chronic or episodic pain.

Conclusions

Chronic arthritis pain may not be optimally managed in this population. Cultural assessment should recognize that American Indian patients may understate serious symptoms. Community educational interventions should target this population to enhance self‐care, pain management, and communication of arthritis symptoms to physicians.

     

Reduction of CpG‐induced arthritis by suppressive oligodeoxynucleotides

Objective

Bacterial DNA contains immunostimulatory CpG motifs that cause inflammation when injected into the knee joints of normal mice. We examined whether synthetic oligodeoxynucleotides (ODN) that suppress CpG‐induced immune responses prevent CpG‐induced arthritis.

Methods

CpG, suppressive, and/or control ODN were injected into the knees of BALB/c mice. Joint swelling and inflammation were evaluated by physical measurement, by histologic analysis of joint tissue, and by magnetic resonance imaging.

Results

Immunostimulatory CpG DNA induced local arthritis, characterized by swelling of the knee joints, the presence of inflammatory cell infiltrates, the perivascular accumulation of mononuclear cells, and hyperplasia of the synovial lining. Administering suppressive (but not control) ODN reduced the manifestations and severity of arthritis up to 80%.

Conclusion

Suppressive ODN may be useful for the prevention or treatment of arthritis induced by bacterial DNA.

     

Angiotensin receptor blockers suppress antigen‐specific T cell responses and ameliorate collagen‐induced arthritis in mice

Objective

The renin–angiotensin system plays an important role in the regulation of cardiovascular, renal, and endocrine functions. Recent studies have demonstrated that angiotensin II has proinflammatory effects that may contribute to the pathogenesis of immune‐mediated diseases. We used the collagen‐induced arthritis (CIA) model to investigate the influence of angiotensin II receptor blockers (ARBs) on antigen‐specific immune responses and determine whether ARBs have preventive or therapeutic effects on the development of arthritis.

Methods

We administered ARBs (olmesartan, candesartan, and telmisartan) to mice and evaluated antigen‐specific T cell proliferation and cytokine production following immunization with ovalbumin (OVA) or type II collagen in Freund's complete adjuvant (CFA) or aluminum hydroxide (alum). Next, we induced CIA in DBA/1 mice and administered olmesartan. The severity and incidence of arthritis were scored according to clinical manifestations, and joint tissue sections were examined histopathologically.

Results

ARBs severely suppressed lymphocyte proliferation and interferon‐γ production in mice immunized with OVA or type II collagen in CFA. Olmesartan also suppressed lymphocyte proliferation in mice immunized with ovalbumin in alum. In the CIA model, olmesartan reduced the mean arthritis score and the incidence of severe arthritis, even when it was administered only after disease onset. Histopathologic findings for joint destruction were improved in olmesartan‐treated mice.

Conclusion

ARBs suppressed antigen‐specific immune responses for Th1 and Th2 in vivo. Furthermore, olmesartan suppressed the development of severe arthritis and joint destruction in the CIA model. These findings suggest that ARBs may have therapeutic potential in rheumatoid arthritis.