Natural killer T cells promote collagen-induced arthritis in DBA/1 mice

The role of NKT cells in the pathogenesis of collagen-induced arthritis (CIA) remains unclear since most studies have used C57BL/6 (B6) mice, which are less susceptible to CIA than mice with a DBA/1 background. To clarify the immunological functions of NKT cells in CIA, it is necessary to analyze in detail the effects of NKT cell deficiency on CIA development in DBA/1 mice. The incidence and severity of CIA were significantly exacerbated in DBA/1CD1d^+/− mice as compared to DBA/1CD1d^−/− mice. In DBA/1CD1d^+/− mice, antigen-specific responses of B and T cells against CII were remarkably increased and inflammatory cytokine levels were also increased in vivo and in vitro. The number of IL-17-producing NKT cells significantly increased in DBA/1CD1d^+/− mice as the disease progressed. Our results clearly show that NKT cells are involved not only in accelerating the severity and incidence of CIA but also in perpetuating the disease progression.     

CD4 and CD8 T cells in susceptibility/protection to collagen-induced arthritis in HLA-DQ8-transgenic mice: implications for rheumatoid arthritis

To investigate the role of CD4 and CD8 T cells in arthritis, we generated transgenic mice deficient in CD4 and CD8 molecules expressing RA-susceptible gene HLA-DQ8. DQ8.CD4(-/-) mice were resistant to developing collagen-induced arthritis (CIA). However, DQ8.CD8(-/-) mice developed CIA with increased incidence and more severity than DQ8 mice. Both DQ8.CD8(-/-) and DQ8 mice produced rheumatoid factor. In addition, DQ8.CD8(-/-) mice produced antinuclear Abs. The B cell compartment and expression of DQ8 were normal in all the strains, although frequency of cells expressing DQ8 was less in CD4(-/-) mice. An increased frequency of CD3(+) double-negative (DN) T cells was found in DQ8.CD8(-/-) compared with DQ8.CD4(-/-) and DQ8 mice. These CD3(+) DN T cells produced high amounts of IL-10 in CD8-deficient mice. Analysis of cell division using a cell cycle tracking dye showed a higher rate of division of CD3(+) and CD3(+) DN T cells in DQ8.CD8(-/-) mice compared with DQ8.CD4(-/-) and DQ8 mice. Decreased apoptosis was seen in CIA-susceptible DQ8 and CD8-deficient mice, indicating a defect in activation-induced cell death. These observations suggest that CD4 cells are necessary for initiation of CIA in DQ8 mice. We hypothesize that CD8(+) T cells are not capable of initiating CIA in DQ8-transgenic mice but may have a regulatory/protective effect.     

Resistance to collagen-induced arthritis in SHPS-1 mutant mice

SHPS-1 is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Here we show that mice expressing a mutant form of SHPS-1 fail to develop type-II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans. Histological examinations of the arthritic paws from immunized wild-type mice revealed that cartilage was destroyed in association with marked mononuclear cell infiltration, while only mild cell infiltration was observed in immunized SHPS-1 mutant mice. Consistently, the serum levels of both IgG and IgG2a specific to CII and of IL-1β in immunized SHPS-1 mutant mice were markedly reduced compared with those apparent for wild-type mice. The CII-induced proliferation of, and production of cytokines by, T cells from immunized SHPS-1 mutant mice were reduced compared to wild-type cells. These results suggest that SHPS-1 is essential for development of CIA.     

Influence of complement C5 and V beta T cell receptor mutations on susceptibility to collagen-induced arthritis in mice

SWR/J mice are resistant to collagen-induced arthritis (CIA) despite having a susceptible H-2q haplotype. We have earlier demonstrated the possible role of the V beta TCR mutation of SWR in the resistance to CIA. To investigate the influence of the C5 deficiency of SWR in this resistance, crosses were made between SWR and A/J (C5 deficient, TCRwild, H-2a), and between SWR and C3H.A (C5 sufficient, TCRwild, H-2a). Upon immunization with bovine type II collagen in adjuvant, there was a similar incidence and severity of arthritis in H-2q-bearing mice in the back-crosses A x (SWR x A) ad C3H.A x (SWR x C3H.A). The absence of hemolytic complement was confirmed in the arthritic A x (SWR x A) back-cross mice by standard SRBC hemolytic assays. In addition C57L (H-2b) mice, which were C5 sufficient but had a V beta TCR deletion mutation similar to SWR, could not complement for CIA susceptibility in H-2q-bearing C57L x (SWR x C57L) back-crosses and in (C57L x SWR)F2 hybrids. These studies show that complement C5 does not play a significant role in CIA susceptibility, and further implicate the V beta TCR mutation in the resistance to CIA in SWR mice.     

Tracking of proinflammatory collagen-specific T cells in early and late collagen-induced arthritis in humanized mice

Rheumatoid arthritis is a chronic inflammatory disease associated with certain HLA-DR4 subtypes. The target autoantigen(s) is unknown, but type II collagen (CII) is a candidate, with a single immunodominant DR4-restricted 261-273 T cell epitope (CII(261-273)). In the present study, we have prepared HLA-DR4:CII(261-273) tetramers and analyzed peripheral blood, lymph node, and synovial fluid cells from DR4-transgenic mice with early and late collagen-induced arthritis to draw a fuller picture of the role of CII-reactive Th cells in disease development. Their frequencies increased approximately 20-fold in blood 1-2 wk postimmunization, and even more in acutely arthritic joints. Our data strongly suggest that CII-specific Th cells are necessary, but not sufficient for collagen-induced arthritis. The CII-specific Th cells displayed an activated proinflammatory Th1 phenotype, and their expansion correlated with onset and severity of arthritis and also with anti-CII Ab levels. Surprisingly, shortly after the first clinical signs of arthritis, activated HLA-DR4:CII tetramer(+) cells became undetectable in the synovial fluid and rare in the blood, but persisted in lymph nodes. Consequently, future human studies should focus on patients with early arthritis, and on their synovial cells, to re-evaluate the occurrence and pathogenic importance of CII-specific or other Th cells in rheumatoid arthritis.     

Extrathymic clonal deletion of self-reactive cells in athymic mice

The repertoire of T cells present in congenitally athymic mice was studied by flow cytometric analysis on populations of T cells expanded polyclonally in vitro. Athymic (BALB/c x C57BL/6)F1 mice have levels of potentially autoreactive V beta 3- and V beta 11-bearing T cells that are significantly higher than those of euthymic CB6F1 mice. Examination of potentially autoreactive cells in athymic AKR mice, however, yielded contrasting results. V beta 6+ cells, which are deleted intrathymically in normal AKR mice, are present in the repertoire of young (less than 6-wk-old) AKR nu/nu mice. Isolation of a cloned CD4+V beta 6+ cell line with Mls-1a reactivity from young AKR nu/nu mice indicates that the correlation between TCR usage and specificity is consistent with that described in euthymic mice and that this population contains autoreactive T cells that are not anergic. By 6 mo of age, however, cells expressing V beta 6 are no longer detectable. Inability to detect these cells is not simply caused by failure to expand these cells in culture, because freshly isolated populations from old nude mice exhibit the same selective absence of V beta 6-bearing cells. The data strongly suggest that extrathymic deletion, rather than clonal anergy, accounts for the apparent absence of autoreactive V beta 6-bearing cells in aged AKR nu/nu mice.     

Increased susceptibility to collagen-induced arthritis in female mice carrying congenic Cia40/Pregq2 fragments

Introduction

Collagen-induced arthritis (CIA) in mice is a commonly used experimental model for rheumatoid arthritis (RA). We have previously identified a significant quantitative trait locus denoted Cia40 on chromosome 11 that affects CIA in older female mice. This locus colocalizes with another locus, denoted Pregq2, known to affect reproductive success. The present study was performed to evaluate the role of the Cia40 locus in congenic B10.Q mice and to identify possible polymorphic candidate genes, which may also be relevant in the context of RA.

Methods

Congenic B10.Q mice carrying an NFR/N fragment surrounding the Cia40/Pregq2 loci were created by 10 generations of backcrossing (N10). The congenic mice were investigated in the CIA model, and the incidence and severity of arthritis as well as the serum levels of anti-collagen II (CII) antibodies were recorded.

Results

Significant effects on onset, incidence, severity, and anti-CII antibody titers were observed in female mice carrying a heterozygous congenic Cia40/Pregq2 fragment of NFR/N origin, containing one or more polymorphic genes. Congenic male mice did not show increased incidence of CIA, but males carrying a heterozygous fragment showed a significant increase in severity in comparison with wildtype B10.Q males (littermates).

Conclusion

The Cia40/Pregq2 locus at chromosome 11 contains one or more polymorphic genes of NFR/N origin that significantly influence both incidence and severity of CIA in heterozygous congenic mice of the B10.Q strain. The major polymorphic candidate genes for the effects on CIA are Cd79b, Abca8a, and Map2k6. The congenic fragment also contains polymorphic genes that affect reproductive behavior and reproductive success. The Sox9 gene, known to influence sex reversal, is a candidate gene for the reproductive phenotype.     

Type II collagen-reactive T cell clones from mice with collagen-induced arthritis

Certain strains of mice develop a symmetrical polyarthritis after immunization with type II collagen. The incidence of arthritis after such immunization is variable. To study the arthritogenic potential of T cells reactive with type II collagen, we isolated draining lymph node cells from mice that had developed arthritis after immunization with bovine type II collagen. From these immune lymph node cells we were able to clone T cells reactive with type II collagen. Two separate sets of T cell clones were isolated. The first set reacted with either native bovine or native chick type II collagen, but did not react with type I collagen. The second set of T cell clones reacted with bovine type II collagen, but did not respond to either native chick type II collagen or type I collagen. These clones will be tested for their influence on the development of arthritis in vivo.     

Isolation of T cell line capable of protecting mice against collagen-induced arthritis

A T cell line specific to human type II collagen (CII) was selected and propagated from DBA/1J mice immunized with human CII. The line cells were not reactive to type I or type III collagen of human origin, but they were cross-reactive to bovine, rat, and rabbit CII and they recognized both native and heat-denatured human CII. The cells were reactive to an N-terminal three-quarters fragment of human CII, produced by tadpole collagenase digestion of human CII, but not to a C-terminal one-quarter fragment of human CII. The cells showed Thy-1+, Lyt-1+, Lyt-2-, and L3T4+ phenotypes characteristic of T helper cells or delayed-type hypersensitive cells, determined by the immunofluorescence method. To clarify the role of T cells in the pathogenesis of collagen-induced arthritis, we inoculated this cell line into DBA/1J mice and found that they developed clinical arthritis, albeit at a low incidence. The cells attenuated by x-ray were capable of inducing resistance to the subsequent induction of collagen-induced arthritis of DBA/1J mice. The sera from mice protected by inoculation of the cell line exhibited anti-idiotypic antibody response against conventional and monoclonal anti-CII antibodies. Anti-T cell receptor response may be involved in the mechanism for the protective effect of the cell line against autoimmune murine arthritis.     

Complement deficiency ameliorates collagen-induced arthritis in mice

Collagen-induced arthritis (CIA) is an experimental animal model of human rheumatoid arthritis being characterized by synovitis and progressive destruction of cartilage and bone. CIA is induced by injection of heterologous or homologous collagen type II in a susceptible murine strain. DBA/1J mice deficient of complement factors C3 (C3(-/-)) and factor B (FB(-/-)) were generated to elucidate the role of the complement system in CIA. When immunized with bovine collagen type II emulsified in CFA, control mice developed severe arthritis and high CII-specific IgG Ab titers. In contrast, the C3(-/-) and FB(-/-) were highly resistant to CIA and displayed decreased CII-specific IgG Ab response. A repeated bovine collagen type II exposure 3 wk after the initial immunization led to an increase in the Ab response in all mice and triggered arthritis also in the complement-deficient mice. Although the arthritic score of the C3(-/-) mice was low, the arthritis in FB(-/-) mice ranked intermediate with regard to C3(-/-) and control mice. We conclude that complement activation by both the classical and the alternative pathway plays a deleterious role in CIA.     

The V beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice

Staphylococcal enterotoxin B is known to be a powerful T cell stimulant in mouse and man. In this paper we show that, for mice, this is because the protein in association with major histocompatibility complex class II molecules stimulates virtually all T cells bearing V beta 3 and V beta 8.1, 8.2, and 8.3, and few others. Neonatal mice given the enterotoxin eliminate all mature, and some immature, T cells bearing these V beta s, demonstrating that tolerance to exogenously administered antigen can be caused by clonal deletion of reactive T cells. The enterotoxin shares these superantigenic properties with known self-antigens in mice, Mls-1a and Mls-2a, and a B cell-derived product, a shared property that is unlikely to be coincidental or inconsequential.     

CD97 neutralisation increases resistance to collagen-induced arthritis in mice

Synovial tissue of rheumatoid arthritis (RA) patients is characterised by an influx and retention of CD97-positive inflammatory cells. The ligands of CD97, CD55, chondroitin sulfate B, and α5β1 (very late antigen [VLA]-5) are expressed abundantly in the synovial tissue predominantly on fibroblast-like synoviocytes, endothelium, and extracellular matrix. Based upon this expression pattern, we hypothesise CD97 expression to result in accumulation of inflammatory cells in the synovial tissue of RA patients. To determine the therapeutic effect of blocking CD97 in an animal model of RA, collagen-induced arthritis was induced in a total of 124 DBA/J1 mice. Treatment was started on day 21 (early disease) or on day 35 (longstanding disease) with the blocking hamster anti-mouse CD97 monoclonal antibody (mAb) 1B2, control hamster immunoglobulin, or NaCl, applied intraperitoneally three times a week. The paws were evaluated for clinical signs of arthritis and, in addition, examined by radiological and histological analysis. Mice receiving 0.5 mg CD97 mAb starting from day 21 had significantly less arthritis activity and hind paw swelling. Furthermore, joint damage and inflammation were reduced and granulocyte infiltration was decreased. When treatment was started on day 35, CD97 mAb treatment had similar effects, albeit less pronounced. The results support the notion that CD97 contributes to synovial inflammation and joint destruction in arthritis.     

T cell receptor alpha-chain influences reactivity to Mls-1 in V beta 8.1 transgenic mice.

Most, but not all, V beta 8.1+ T cells respond to M1s-1 and are clonally deleted in the thymus of M1s-1-expressing animals. To formally examine the role of the TCR alpha-chain in reactivity and tolerance to M1s-1, we have analyzed M1s-1 reactivity in a large panel of CD4+ hybridomas generated from TCR V beta 8.1 transgenic mice, that express an identical, potentially M1s-1-reactive beta-chain. The data show that the alpha-chain strongly influences the M1s-1 reactivity of the hybridomas and that the differences in reactivity had relevance for tolerance. Thus, V alpha 11+ hybridiomas were biased toward M1s-1 reactivity and V alpha 11+ T cells were correspondingly absent from the peripheral repertoire of M1s-1-expressing transgenic mice. V alpha 2+ hybridomas, on the other hand, were biased against M1s-1 reactivity, and V alpha 2+ T cells were correspondingly amplified in the M1s-1-expressing transgenic mice. Structural analysis of the alpha-chains revealed that the M1s-1 reactivity of the V alpha 11+ hybridomas segregated precisely with family member, such that V alpha 11.1+ hybridomas were M1s-1-reactive and V alpha 11.3+ hybridomas were not M1s-1-reactive. On the other hand, there was not a clear correlation between family member and M1s-1 reactivity in the V alpha 2+ hybridomas. The hybridomas also showed striking variation in their reactivity to staphylococcal enterotoxin B (SEB), and the SEB reactivity of the V alpha 11+ hybridomas correlated precisely with family member and with M1s-1 reactivity. In contrast, there was not a clear correlation with V alpha 2+ alpha-chain structure and SEB reactivity. Also, there was no correlation between M1s-1 reactivity and SEB reactivity in individual V alpha 2+ hybridomas, suggesting that the recognition of the two superantigens by the same TCR is not equivalent. Taken together, these data define a role for the TCR alpha-chain in superantigen reactivity and T cell tolerance, and provide a structural explanation for the different fates of M1s-1-reactive T cells in normal and transgenic mice.     

Liposome encapsulated aurothiomalate reduces collagen-induced arthritis in DBA/1J mice

Collagen-induced arthritis (CIA) generated in rats or mice has long been a model system for the study of rheumatoid arthritis in humans. In particular, this system has been used to study the mechanisms and effects of anti-arthritic drugs in the treatment of the disease. Sodium aurothiomalate (ATM) is an agent often used to treat rheumatoid arthritis in humans; however, it possesses inherent toxicities which limits its usefulness. Liposome-encapsulated drugs are currently being developed to minimize the toxicities associated with a variety of potentially beneficial drugs. We have chosen to encapsulate ATM into small unilamellar vesicles (SUVs) to determine whether greater efficacy would be achieved in treating CIA with SUV ATM as compared to using the free drug. SUVs were prepared from hydrogenated egg phosphatidylcholine and cholesterol. These SUVs were very stable. Vesicles stored at 4 degrees C lost only 0.09% of encapsulated ATM (SUV ATM) after 14 days and were able to reduce collagen-induced arthritis in these mice. Animals treated by i.m. injections of SUV ATM exhibited a 50% reduction in symptoms. More importantly, histological examination of knee joints of the affected animals verified that SUV ATM treatment prevented cellular infiltration of lymphocytes into the synovia of the collagen-sensitized mice. Conditioned media from spleen cell cultures was assayed for the presence of inflammatory lymphokines that might be affected by SUV ATM to account for the success in suppressing collagen-induced arthritis.     

The role of C5 and T-cell receptor Vb genes in susceptibility to collagen-induced arthritis

Collagen-induced arthritis (CIA) is a rodent arthritis model in which immunization with heterologous type II collagen induces an inflammatory polyarthritis. Susceptibility to the disease is mediated by major histocompatibility complex (MHC) genes as well as genes at other loci. Previous studies of the SWR/J mouse strain, which is resistant to CIA despite bearing the susceptible H-2 ^q haplotype, have suggested that this resistance is the result of a deletion of T-cell receptor (Tcr) Vb gene segments which is carried by this strain. Other studies have implicated a deficiency in complement component C5 as the cause for the resistance. In order to assess the relative importance of these two genes in susceptibility to CIA, and to provide an estimate of the number of independent genes involved in the disease, we analyzed 196 F₂ progeny of a (DBA/1 × SWR/J) cross for arthritis susceptibility, and expression of both C5 and Tcr genes. Thirty of the F₂ progeny developed arthritis. All of the arthritic mice had at least one copy of the wild-type C5 allele, while the Tcr-Vb haplotypes were distributed in Mendelian fashion. These results demonstrate that C5 sufficiency is an absolute requirement for CIA, but that Tcr-Vb genes located within the SWR deletion have little influence. Genetic analysis of the incidence rate suggests that there is polygenic control of susceptibility to CIA and that in addition to H-2, 5–6 other independent loci (including C5) may be involved.     

MHC class II derived recombinant T cell receptor ligands protect DBA/1LacJ mice from collagen-induced arthritis

We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the alpha1 and beta1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257-270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257-270 molecule could systemically reduce proinflammatory IL-17 and IFN-gamma production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257-270 molecule could also selectively inhibit IL-1beta, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.     

Streptococcus sanguis modulates type II collagen-induced arthritis in DBA/1J mice

Native type II collagen is tolerogenic when given orally or i.p. to DBA/1J mice and induces autoimmune arthritis when given s.c. in CFA. The tolerogenic epitope is contained in cyanogen bromide fragment 11 (CB11) and is structurally mimicked by PGEQGPK within the platelet aggregation-associated protein (PAAP) on Streptococcus sanguis. To learn whether S. sanguis modulates transmucosally the Ag-specific development of autoimmune arthritis, DBA/1J pups were given live S. sanguis, CB11, or type II collagen intragastrically. Feeding S. sanguis at 6 days postpartum delayed the onset of arthritis, and reduced the rate, final severity, and percentage of affected limbs. Next, PAAP(+) S. sanguis and type II collagen were tested for T cell cross-reactivity. T cells primed with the tolerogenic epitope of type II collagen proliferated more when incubated with PAAP(+) S. sanguis than with PAAP(-) Streptococcus gordonii or type II collagen, suggesting an Ag-specific transmucosal tolerogenic effect. In neonatal mice, therefore, bacterial surface Ags that mimic self can transmucosally stimulate Ag-specific inhibitory T cells. In adult mice immunized with type II collagen, these Ag-specific inhibitory T cells manifest later as attenuated arthritis. The PAAP(+) S. sanguis appear to activate adult memory, rather than naive, type II collagen-specific T cells, suggesting that systemic challenge with commensal self-mimicking microorganisms may perpetuate existing autoimmunity, but not initiate autorecognition.     

Bacterial lipopolysaccharide potentiates type II collagen-induced arthritis in mice

Collagen-induced arthritis (CIA) is an immunologically relevant animal model of human rheumatoid arthritis. Studies comparing the disease incidence in genetically susceptible male and female DBA/1LacJ mice demonstrated that under low density/low stress housing conditions, female mice had earlier onset (day 35) and higher disease incidence (25%) than the male mice (17% at day 49) when immunized with bovine type II collagen. A single subcutaneous or intraperitoneal injection of bacterial lipopolysaccharide (LPS) 17-24 days after collagen immunization greatly potentiated this standard CIA model in a dose related manner. 20-40 mug of LPS accelerated the onset of disease from day 35 to day 21 and exacerbated the clinical severity score from 0.27 to 2.00 at day 42. A similar administration of 6 mug of recombinant interleukin-beta produced a comparable potentiated CIA model. The acute phase protein, serum amyloid P (SAP), was elevated in the serum at day 26 to 440 mug ml(-1) for the LPS potentiated CIA mice compared to 65 mug ml(-1) in the non-potentiated immunized CIA mice. There was a significant correlation (r = 0.78) between SAP levels and disease expression in the LPS treated CIA mice. The rapidity and uniformity of disease expression in this LPS potentiated CIA model will allow more and different drugs to be evaluated with a smaller number of animals.     

Smoking and nicotine exposure delay development of collagen-induced arthritis in mice

Introduction  

Recent epidemiologic studies have implicated smoking as an environmental risk factor for the development of rheumatoid arthritis (RA). The aim of the present study is the evaluation of the role of cigarette smoke (CS) in the pathogenesis of collagen-induced arthritis in mice.