Increased resistance to collagen-induced arthritis in CD44-deficient DBA/1 mice.

OBJECTIVE: To study the role of CD44, the principal hyaluronan (HA) receptor, in experimental arthritis. METHODS: We generated CD44 gene deficiency in arthritis-susceptible DBA/1LacJ mice to study the role of CD44 directly in collagen-induced arthritis (CIA). Wild-type and CD44-deficient mice were immunized with chicken type II collagen, and the onset and severity of CIA were monitored up to day 64. The immune status of immunized mice was determined at the end of the experiments. Cell transfer experiments were performed to monitor lymphocyte traffic to the inflamed joints. RESULTS: Mice homozygous for the CD44 mutation developed normally and showed no phenotypic defects. Although they showed a normal response to immunization with type II collagen and had Th1/Th2 ratios comparable with those in wild-type animals, CD44-deficient mice exhibited significant reductions in both the incidence and severity of CIA. This was accompanied by altered serum levels of HA, reduced expression of L-selectin, and a delayed entry of intravenously injected CD44-deficient donor lymphocytes into the arthritic joints of recipient mice. CONCLUSION: While CD44 is not essential for morphogenesis and autoimmunity, this cell surface receptor seems to play an important role in the development of arthritis, most likely by directing leukocyte traffic to the site of inflammation.     

Reduced susceptibility to collagen‐induced arthritis in DBA/1J mice expressing the TSG‐6 transgene

Objective

Expression of TSG‐6 (tumor necrosis factor–stimulated gene 6) is induced by proinflammatory cytokines. This study was undertaken to examine the effects of local expression of TSG‐6 in arthritic joints of TSG‐6 transgenic mice, in the collagen‐induced arthritis (CIA) model.

Methods

We generated transgenic mice that harbored the TSG‐6 gene under the control of the T cell–specific lck promoter. Arthritis was induced by immunization with bovine type II collagen (CII), and its progression was monitored based on the incidence of arthritis, the arthritis index, and footpad swelling. Anti‐CII antibodies and cytokine production were determined by enzyme‐linked immunosorbent assay. Gene expression arrays were used to compare gene expression profiles of transgenic and control mice at various stages of CIA.

Results

TSG‐6 was expressed in limbs of transgenic mice after immunization with CII, while its expression in nontransgenic animals was insignificant. The incidence of CIA was reduced in TSG‐6 transgenic animals, its onset delayed, and all parameters of clinical arthritis significantly reduced. However, the immune response against CII was not significantly inhibited in TSG‐6 transgenic mice.

Conclusion

TSG‐6 expression has been demonstrated in patients with rheumatoid and other forms of arthritis. Our data show that local expression of TSG‐6 at sites of inflammation results in potent inhibition of inflammation and joint destruction in a model of autoimmune arthritis in mice. Therefore, it is likely that TSG‐6 plays a similar modulatory role in human rheumatoid arthritis and related diseases and may have potential for the treatment of autoimmune arthritis in humans.

     

Enhancement of collagen‐induced arthritis in mice genetically deficient in extracellular superoxide dismutase

Objective

To examine the influence of superoxide on the severity of collagen‐induced arthritis (CIA) in mice.

Methods

CIA was induced in DBA/1J mice lacking the extracellular superoxide dismutase (EC‐SOD) gene (knockout [KO]) and in normal DBA/1J mice (wild‐type [WT]).

Results

The clinical disease activity score was significantly higher in EC‐SOD–KO mice than in WT mice between days 36 and 53, and the histologic scores for joint damage on day 53 increased 2‐fold or more in the EC‐SOD–KO mice. There were no significant differences between the 2 groups of mice in proliferation indices of spleen or lymph node cells in vitro after stimulation with type II collagen. Although both IgG1 and IgG2a anticollagen antibody levels increased in both groups of mice between days 21 and 53, there were no significant differences between the 2 groups. Lipopolysaccharide‐stimulated spleen cells from EC‐SOD–KO mice produced greater levels of tumor necrosis factor α (TNFα) over 48 hours in culture compared with cells from WT mice. Increased steady‐state levels of messenger RNA (mRNA) for interferon‐γ (IFNγ), TNFα, and interleukin‐1β (IL‐1β), and lower levels of IL‐1 receptor antagonist (IL‐1Ra) mRNA were present in the joints of the EC‐SOD–KO mice compared with the WT mice.

Conclusion

The absence of EC‐SOD leads to more severe CIA, which may be accompanied by enhanced production of the proinflammatory cytokines IFNγ, TNFα, and IL‐1β, and decreased production of the antiinflammatory cytokine IL‐1Ra in the joints.

     

Apolipoprotein E–deficient mice are resistant to the development of collagen‐induced arthritis

Objective

To determine whether elevated serum lipid levels resulting from feeding animals a high‐fat diet can affect the inflammatory process in C57BL/6 (B6) wild‐type (WT) and B6 ApoE^−/− mouse models of collagen‐induced arthritis (CIA).

Methods

Male B6 WT or ApoE^−/− mice were fed either a normal chow diet or a high‐fat diet. CIA was induced in mice at 12 weeks of age using type II chicken collagen, Freund's complete adjuvant, and, on occasion, a lipopolysaccharide boost. Expression levels of autoantibodies and cytokines were measured using enzyme‐linked immunosorbent assay and multiplex assay, respectively.

Results

Whereas B6 WT mice developed severe articular inflammation after collagen immunization, ApoE^−/− mice developed no clinical or histologic evidence of disease regardless of whether they had been fed a high‐fat diet or a normal chow diet. The fact that arthritis was not present in ApoE^−/− mice did not result from inadequate production of serum IgG2a collagen antibodies, since levels observed in ApoE^−/− mice were similar to those observed in arthritic B6 WT control mice. Critically, development of atherosclerosis in ApoE^−/− mice was not affected by the CIA protocol.

Conclusion

Our findings suggest that ApoE^−/− mice are resistant to the development of CIA. Intriguingly, induction of host autoimmunity in the absence of articular inflammation had no effect on atherosclerosis progression, suggesting that articular inflammatory load may be a critical risk factor in vascular pathology.

     

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.

     

Stromal cells and osteoclasts are responsible for exacerbated collagen‐induced arthritis in interferon‐β–deficient mice

Objective

Clinical trials using interferon‐β (IFNβ) in the treatment of rheumatoid arthritis have shown conflicting results. We undertook this study to understand the mechanisms of IFNβ in arthritis at a physiologic level.

Methods

Collagen‐induced arthritis (CIA) was induced in IFNβ‐deficient and control mice. The role of IFNβ was investigated in both the priming and effector phases of the disease. The effect of IFNβ deficiency on synovial cells, macrophages, and fibroblasts from preimmunized mice was analyzed by flow cytometry, immunohistochemistry, and enzyme‐linked immunosorbent assay. Differences in osteoclast maturation were determined in situ by histology of arthritic and naive paws and by in vitro maturation studies of naive bone marrow cells. The importance of IFNβ‐producing fibroblasts was determined by transfering fibroblasts into mice at the time of CIA immunization.

Results

Mice lacking IFNβ had a prolonged disease with a higher incidence compared with control mice. IFNβ deficiency was found to influence the effector phase, but not the priming phase, of arthritis. Compared with control mice, IFNβ‐deficient mice had greater infiltration of CD11b+ cells and greater production of tumor necrosis factor α in vivo, and their macrophages and fibroblasts were both more activated in vitro. Moreover, IFNβ‐deficient mice generated a greater number of osteoclasts in vitro, and mice immunized to induce arthritis, but not naive mice, had a greater number of osteoclasts in vivo compared with control mice. Importantly, IFNβ‐competent fibroblasts were able to ameliorate arthritis in IFNβ‐deficient recipients.

Conclusion

Our data indicate that IFNβ is involved in regulating the activation state of osteoclasts and stromal cells, including macrophages and fibroblasts, but that it has little effect on T cells.

     

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.

     

Suppression of collagen‐induced arthritis in growth arrest and DNA damage–inducible protein 45β–deficient mice

Objective

Growth arrest and DNA damage–inducible protein 45β (GADD45β) is involved in stress responses, cell cycle regulation, and oncogenesis. Previous studies demonstrated that GADD45β deficiency exacerbates K/BxN serum–induced arthritis and experimental allergic encephalomyelitis (EAE) in mice, indicating that GADD45β plays a suppressive role in innate and adaptive immune responses. To further understand how GADD45β regulates autoimmunity, we evaluated collagen‐induced arthritis in GADD45β^–/– mice.

Methods

Wild‐type (WT) and GADD45β^–/– DBA/1 mice were immunized with bovine type II collagen (CII). Serum anticollagen antibody levels were quantified by enzyme‐linked immunosorbent assay. Expression of cytokines and matrix metalloproteinases in the joint and spleen was determined by quantitative polymerase chain reaction. The in vitro T cell cytokine response to CII was measured by multiplex analysis. CD4+CD25+ Treg cells and Th17 cells were quantified using flow cytometry.

Results

GADD45β^–/– mice showed significantly lower arthritis severity and joint destruction compared with WT mice. MMP‐3 and MMP‐13 expression was also markedly reduced in GADD45β^–/– mice. However, serum anti‐CII antibody levels were similar in both groups. FoxP3 and interleukin‐10 (IL‐10) expression was increased 2–3‐fold in splenocytes from arthritic GADD45β^–/– mice compared with those from WT mice. Flow cytometric analysis showed greater numbers of CD4+CD25+ Treg cells in the spleen of GADD45β^–/– mice than in the spleen of WT mice. In vitro studies showed that interferon‐γ and IL‐17 production by T cells was significantly decreased in GADD45β^–/– mice.

Conclusion

Unlike passive K/BxN arthritis and EAE, GADD45β deficiency in CIA was associated with lower arthritis severity, elevated IL‐10 expression, decreased IL‐17 production, and increased numbers of Treg cells. The data suggest that GADD45β plays a complex role in regulating adaptive immunity and, depending on the model, either enhances or suppresses inflammation.

     

CD44 expression on chondrocytes in knees of DBA/1 mice with type II collagen-induced arthritis.

OBJECTIVE: To investigate aspects of CD44 expression in the cartilage destruction of the knees of mice with collagen-induced arthritis. METHODS: DBA/1 mice were immunized with bovine type II collagen emulsified with Freund's incomplete adjuvant. Histological changes in the knees were evaluated in sections stained with hematoxylin and eosin, toluidine blue, and immunostaining using anti-CD44 monoclonal antibodies. RESULTS: CD44 expression was observed in the synovial lining and articular chondrocytes. A total of 10/10 knees had CD44-positive synovial lining cells at 6 weeks after immunization, which was higher than that at 4 weeks (7 of 10 knees, p < 0.05). At 4 weeks 3 of 10 knees with CD44-positive chondrocytes were observed, and 7 of 10 knees at 6 weeks after immunization. The number of knees at 6 weeks was higher than that at 4 weeks (p < 0.01). Nine of 10 knees showed loss of metachromasia on toluidine blue staining at 6 weeks after immunization, which was higher than the number at 4 weeks. The grade of metachromasia loss and the length of time after immunization were significantly correlated (p < 0.05, Spearman rank correlation; p = 0.46). CONCLUSION: These findings suggest that CD44 may play some role in the early stage of cartilage destruction in DBA/1 mice immunized with type II collagen.     

Type ii collagen—induced arthritis in mice

The passive transfer of high levels of anti-type II collagen antibody from mice with type II collagen-induced arthritis may transfer arthritis to syngeneic recipient animals. However, the transfer of either arthritogenic or sub-arthritogenic doses of polyclonal anti-type II collagen antibody, or non-arthritogenic monoclonal anti-type II collagen antibody may protect mice against the subsequent induction of arthritis by type II collagen. A protective effect was also observed in mice given free native type II collagen intravenously before the administration of collagen in an arthritogenic protocol.     

Stimulation of nicotinic acetylcholine receptors attenuates collagen‐induced arthritis in mice

Objective

The parasympathetic nervous system, through the vagus nerve, can down‐regulate inflammation in vivo by decreasing the release of cytokines, including tumor necrosis factor α (TNFα), by activated macrophages. The vagus nerve may exert antiinflammatory actions via a specific effect of its principal neurotransmitter, acetylcholine, on the α7 subunit of nicotinic acetylcholine receptors (α7nAChR) on macrophages. The present study was undertaken to obtain insight into the role of the cholinergic antiinflammatory pathway in arthritis.

Methods

To inhibit the cholinergic antiinflammatory pathway, mice were subjected to unilateral cervical vagotomy or sham surgery, after which arthritis was induced with type II collagen. In a separate study, nicotine was added to the drinking water of mice with collagen‐induced arthritis (CIA). In addition, we investigated the effects of intraperitoneally (IP)–injected nicotine and the specific α7nAChR agonist AR‐R17779.

Results

Clinical arthritis was exacerbated by vagotomy and ameliorated by oral nicotine administration. Moreover, oral nicotine inhibited bone degradation and reduced TNFα expression in synovial tissue. Both IP‐injected nicotine and AR‐R17779 ameliorated clinical arthritis and reduced synovial inflammation. This was accompanied by a reduction of TNFα levels in both plasma and synovial tissue. The effect of AR‐R17779 was more potent compared with that of nicotine and was associated with delayed onset of the disease as well as with protection against joint destruction.

Conclusion

These data provide the first evidence of a role of the cholinergic antiinflammatory pathway in the murine CIA model of rheumatoid arthritis.

     

Suppression of immune responses in collagen‐induced arthritis by a rationally designed CD80‐binding peptide agent

Objective

The CD80/CD86–CD28/CD152 costimulatory pathways transmit signals for CD4+ T cell activation and suppression and are critically involved in the pathogenesis of rheumatoid arthritis (RA). A significant number of CD4+ T cells and macrophages in the rheumatoid synovium express elevated levels of CD80, increasing the potential for costimulation in trans of naive T cells. To determine the effect of blockade of this costimulatory axis in RA, we designed novel CD80‐binding peptides and evaluated their therapeutic potential in collagen‐induced arthritis (CIA), an animal model of RA.

Methods

The conserved MYPPPY motif of CD152 adopts a polyproline type II (PPII) helical conformation in the CD80–CD152 complex. The pairing preferences of the critical residues at the CD80–CD152 interface and their propensity to form PPII helices were integrated to design peptides with optimum PPII helical content that selectively block CD80–receptor interactions. The clinical efficacy was tested in DBA/1LacJ mice that were administered the CD80 blocking agents, called CD80‐binding competitive antagonist peptides (CD80‐CAPs), at the time of immunization with bovine type II collagen or 3 weeks after immunization.

Results

A single administration of select CD80‐CAPs significantly reduced the clinical, radiologic, and histologic disease severity in CIA. Importantly, administration of CD80‐CAPs during activated immune response significantly suppressed disease development by reducing mononuclear cell infiltration in the joints and mediating peripheral deletion of activated CD4+ T cells.

Conclusion

A rationally designed CD80‐binding peptide both prevents and suppresses CIA, suggesting a potential application in RA. Apoptosis of activated CD4+ T cells following in vivo blockade suggests that the effects of CD80‐CAPs may be long‐lasting.

     

Identification of new quantitative trait loci in mice with collagen‐induced arthritis

Objective

Collagen‐induced arthritis (CIA) in the mouse is one of the most widely used autoimmune experimental models, with many features similar to rheumatoid arthritis. This study sought to identify potential genetic regulatory mechanisms of CIA in major histocompatibility complex–matched (H2‐q) F₂ hybrid mice.

Methods

We used 126 polymorphic markers to perform simple sequence‐length polymorphism analysis on 290 F₂ hybrids of arthritis‐susceptible (DBA/1J) and arthritis‐resistant (FVB/N) inbred mouse strains. The major clinical traits (disease severity and onset) were assessed, and serum antibodies specific to type II collagen (CII) were determined by enzyme‐linked immunosorbent assay in 270 F₂ mice. Lymph nodes from 94 F₂ mice were used to test the ratio of CD4 to CD8 by fluorescence‐activated cell sorter analysis, and cell proliferation was determined by XTT test.

Results

Two quantitative trait loci (QTLs) identified in previous studies were confirmed; these were severity‐controlling Cia2 and onset‐controlling Cia4 on chromosome 2. Moreover, we identified 5 new QTLs, 1 for CII‐specific IgG2a antibodies on chromosome 5, 2 controlling the CII‐specific IgG1 antibody response on chromosomes 10 and 13, 1 for the CD4:CD8 ratio on chromosome 2, and 1 for cell proliferation (measured by XTT test) on chromosome 16. Complement component C5 was identified as the probable main candidate gene for the QTLs Cia2 and Cia4. F₂ mice carrying a 2‐basepair deletion of C5, the FVB/N allele, had low incidence and less severe disease as compared with those carrying the DBA/1J allele.

Conclusion

This genome scan provides additional evidence confirming the role of C5 as a probable candidate gene for Cia2 and Cia4 loci, and identifies new QTLs controlling new traits in autoimmune arthritis.

     

Enhancement of collagen‐induced arthritis in female mice by estrogen receptor blockage

Objective

To determine whether estrogen‐mediated suppression of collagen‐induced arthritis (CIA) in mice acts via the nuclear estrogen receptors (ERs).

Methods

CIA was induced in noncastrated normal (B10.Q × DBA/1)F₁ (QD) female mice. The mice were treated with the ER antagonist ICI 182,780, which binds to both ERα and ERβ, either on days 2, 6, 10, and 14 or on days 14, 18, 22, and 26 after type II collagen (CII) immunization. The effects of treatment and development of arthritis were correlated with the estrus cycle by inspection of vaginal smears (VS). By a combination of treatments with both estriol (E₃) and ICI 182,780 during the time of expected onset of CIA in castrated QD female mice, the protective effect of E₃ in CIA was analyzed.

Results

Treatment with ICI 182,780 of QD female mice immunized with CII triggered an earlier onset of arthritis during the period when the estrus cycle was blocked. The arthritis‐modulating effect of ICI 182,780 was even obtained at doses that were insufficient to block estrus cycling, as observed in the VS response. E₃ is an estrogen with low estrogenic potency but with a relatively potent antiarthritis effect. Doses of ICI 182,780 that were suboptimal for blocking estrus cycling blocked the E₃‐mediated suppression of CIA in castrated female mice.

Conclusion

These findings show that estrogen‐induced suppression of CIA is mediated via the nuclear ERs and is operating at physiologic, possibly even subphysiologic, levels of estrogens.

     

A comparative genetic analysis between collagen‐induced arthritis and pristane‐induced arthritis

Objective

To compare the genetic regulation of collagen‐induced arthritis (CIA) with that of pristane‐induced arthritis (PIA) in rats.

Methods

A genome‐wide linkage analysis of an (E3 × DA)DA backcross of rats with CIA (n = 364 male rats; the same strain combinations as previously used to determine the genetic control of PIA) was performed. The strongest loci in both CIA and PIA (i.e., Cia12/Pia4 and Cia13/Pia7) were isolated in congenic strains. Susceptibility in both congenic strains was tested in rats with CIA and in rats with PIA.

Results

We found a striking, although not complete, similarity of the arthritis‐controlling loci in CIA and in PIA, as well as the previously defined loci associated with cartilage destruction, antibody production, and the acute‐phase response. All major PIA quantitative trait loci (QTLs) identified in early severe arthritis were also strong regulators of CIA. The 2 strongest QTLs, Cia12/Pia4 on chromosome 12 and Cia13/Pia7 on chromosome 4, were also analyzed in congenic strains with DA or E3 as the background genome. Consistent with the results of linkage analysis, the congenic strain experiments showed that the chromosome 4 locus was more penetrant in CIA than in PIA, while the chromosome 12 locus almost completely dominated the control of PIA severity.

Conclusion

The underlying genetic control of CIA was found to have many, but not all, pathogenic mechanisms in common with PIA, despite the use of a cartilage‐specific antigen (type II collagen) to induce CIA but not PIA.