Single Immunization of Newborn Mice with Heterologous Type-II Collagen Induces Arthritic Disease

Collagen-induced arthritis (CIA) is a widely accepted model of autoimmune disease with significant similarities to rheumatoid arthritis in humans. CIA is provoked in susceptible strains upon immunization of adult mice with native type-II collagen in complete Freund's adjuvant (CFA). Neonatal exposure to antigen is supposed to result in T cell clone deletion and induction of tolerance. Here we report that the neonatal injection of bovine type-II collagen (bCII) to ICR (CD-2) mice triggers the development of autoimmune chronic joint inflammation. Compared with standard CIA significant joint swelling was not observed and anti-collagen antibodies were not detected if the second challenge with the antigen was not supplied. Histopathologic examination of the joints showed cell infiltration, synovial hyperplasia and at the later period bone destruction. Mice immunized as neonates expressed Ag-specific proliferative response and delayed type hypersensitivity (DTH) reaction to bCII.     

Ethinyl estradiol treats collagen-induced arthritis in DBA/1LacJ mice by inhibiting the production of TNF-alpha and IL-1beta

We previously demonstrated the therapeutic effects of ethinyl estradiol (EE), an orally active estrogen and a component of birth control pills, in encephalitogenic autoimmune encephalomyelitis (EAE). In this study, we report the effectiveness of EE in treating collagen-induced arthritis (CIA) induced with bovine type II collagen (bCII) in DBA/1LacJ mice, a CIA susceptible strain. Both low and high doses of EE notably suppressed clinical and histological signs of CIA in a dose-dependent manner compared to vehicle-treated controls. Oral treatment with EE decreased proliferation and secretion of pro-inflammatory factors, TNF-alpha IFN-gamma, MCP-1 and IL-6 by bCII peptide-specific T cells, production of bCII-specific IgG2a antibodies, and mRNA for cytokines, chemokines and chemokine receptors in joint tissue. This is the first report demonstrating effective treatment of joint inflammation and clinical signs of CIA with orally administered ethinyl estradiol, thus supporting its possible clinical use for treating rheumatoid arthritis in humans.     

Vaccination with an immunodominant peptide of bovine type II collagen induces an anti-TCR response, and modulates the onset and severity of collagen-induced arthritis

T cell responses directed toward TCR-derived peptides have been shown to be an important regulatory mechanism of protection against autoimmunity. Here, we show that a naturally induced TCR-directed immune response can delay the onset of collagen-induced arthritis (CIA), an animal model of autoimmune rheumatoid arthritis in humans. DBA/1 mice were pretreated with an immunodominant peptide, p245-270, from bovine type II collagen (bCII) and were subsequently immunized with whole bCII for the induction of arthritis. The results showed that preactivation of p245-270-reactive cells delayed the onset and reduced the severity of CIA, compared with animals in the control group. Interestingly, the serum antibody response to bCII and the bCII-specific cytokine were not affected under these conditions. This result indicates that the observed protection was neither directly due to a lower antibody response nor due to the immune deviation of the anti-bCII T cell response. Furthermore, immunization with p245-270, but not bCII, induced a strong response to the B5 peptide, an immunodominant region of the TCR V(beta)8.2 (amino acids 76-101) that binds very strongly to I-A(q). These data suggest that at a critical phase in the loss of self-tolerance, an effective anti-TCR response, induced naturally, can regulate the pathogenic autoimmune response and thus may provide protection against autoimmunity.     

T cell lines generated with type II collagen proliferate in an autologous mixed lymphocyte response.

Collagen-induced arthritis (CIA) is a T cell-dependent disease induced in susceptible rodents by immunizing with bovine type II collagen (bCII). In order to study T cell responses, a programme to generate bCII-specific T cell lines from arthritic rats was initiated. Lymph node cells from bCII-immune WA/KIR/kcl rats were cultured with bCII in vitro, and the T cells were isolated and restimulated with bCII-pulsed antigen presenting cells (APC) (thymus cells or splenic low density cells). However, T cells, generated initially to bCII, subsequently proliferated upon co-culture with syngeneic APC even in the absence of bCII. This suggests that exposure to bCII resulted in the activation of a population of self-reactive T cells which proliferate in an autologous mixed lymphocyte response. In contrast, short-term T cell lines generated to ovalbumin, heat-denatured bCII and the collagen peptide bCII(184-198) proliferated in response to specific antigen-pulsed APC without demonstrating self-reactivity. Since denatured bCII and bCII(184-198) peptide are not arthritogenic and failed to generate self reactivity in vitro, this suggests that the native triple helical conformation of bCII is required for stimulating autoreactive T cell responses.     

Antigen presentation of Type II collagen in rats

Collagen-induced arthritis (CIA) is a T-cell dependent disease of rats which follows immunization with bovine type II collagen (bCII). Susceptibility to CIA is linked to the genes encoding the major histocompatibility complex (MHC), suggesting that antigen presentation is important in disease pathogenesis. Antigen-presenting cells (APC) (macrophages, dendritic cells (DC) and B cells) were prepared from WA/KIR/KCL rats and presentation of antigen, in the form of native protein (bCII) or synthetic peptide (bCII:184-198), was assessed in T-cell proliferation assays. Whilst macrophages inhibited proliferative responses to bCII, splenic or thymic low density cells, enriched for DC, presented both bCII and bCII(184-198) peptide. However, bone marrow-derived DC, which stimulated T-cell responses to OVA, failed to present bCII, suggesting differences in processing of these two antigens. B-cell depletion from lymph node cells abrogated the proliferative response to bCII and reconstitution of a T-cell population with B cells restored the proliferative response, indicating that B cells are important for stimulating T-cell responses to bCII. B cells play a critical role in CIA by producing pathogenic anti-bCII antibodies, and we propose that B cells are also important APC which present bCII to CD4+ T cells.     

Autoreactivity in collagen-induced arthritis of rats: a potential role for T cell responses to self MHC peptides

Collagen-induced arthritis (CIA) is a chronic inflammatory arthropathy of rats which follows immunization with bovine type II collagen (bCII). T cell lines generated from arthritic rats have been shown to be self-reactive and proliferate in an autologous MLR, which is MHC-dependent. However, the peptides which drive this autoreactive response remain to be elucidated. T cell lines, generated initially to bCII, were cultured with synthetic peptides representing potential autoreactive self epitopes. C1q-c(50-64) peptide, which demonstrates sequence homology to the bCII(184-198) peptide, failed to stimulate T cell proliferation suggesting that the autologous MLR was not due to antigen cross-reactivity with this self peptide. In contrast, several peptides from the amino-terminal region of the RT1D(u) MHC class II molecule stimulated proliferative responses. These results suggest that immunization with bCII leads to activation of a population of autoreactive T cells which respond in an autologous MLR, and that this response could be due, in part, to T cell reactivity to self MHC peptides.     

A synthetic androstene analogue inhibits collagen-induced arthritis in the mouse

Dehydroepiandrosterone (DHEA), a precursor of immune-regulating hormones (IRH) including the androstenes, has attracted much interest over the last several decades because of its many antiaging, metabolic, and immune modulating effects. 5-Androstene-16alpha fluoro-17-one (fluasterone, also known as HE2500) is a synthetic androstene analogue that retains anti-inflammatory, antiproliferative, and immune-regulating activities of the parent molecule, but is nontoxic and practically devoid of androgenic or estrogenic side effects. In the present studies, we tested the ability of fluasterone to limit disease in the DBA mouse model of collagen-induced arthritis (CIA). We found that mice receiving injections of fluasterone displayed significant delay in onset, decrease in CIA peak score, and significant decrease of the daily mean clinical score. Benefit was associated with significant decreases in (1). bovine type II collagen (bCII)-specific IgG(1) and IgG(2a) antibody levels in serum; (2). production of TNF-alpha, IL-6, IFN-gamma, but not IL-10; (3). lymphocyte proliferative response to bCII protein; and (4). joint inflammation, erosion, and synovial proliferation as judged by histological analysis. This is the first study to report that an IRH can ameliorate ongoing disease in a CIA mouse model with relevance to RA and to correlate that finding with decreases in pro-inflammatory cytokines.     

Candida albicans cell-wall fraction exacerbates collagen-induced arthritis in mice

The immune system generates a specific response against most pathogens, while developing tolerance to self-antigens. Commensal micro-organisms can express molecular structures that mimic self-epitopes. During acute infection, such pathogen may activate self-reactive T-cell clones promoting autoimmunity. In the present study, a beta-mercaptoethanol cell-wall fraction (MF) from Candida albicans was injected into the paw of naive ICR and BALB/c mice and into the paw of ICR mice with bovine collagen type II-induced arthritis (CIA). Development of inflammation was monitored for 6 weeks. MF provoked a stable swelling and histopathologic changes in the injected joint, with a predominance of T-helper 1 cytokines in ICR mice. In BALB/c strain, a swelling was observed only in the early period, with no evidence of joint pathology. Injection of the MF fraction exacerbated the disease in ICR mice with CIA, and this was associated with the elevation of interferon-gamma and anti-bovine type II collagen (bCII) immunoglobulin G2a antibodies. These results indicate that component(s) in the MF fraction cross-react with bCII-specific cells.     

Neutrophil extracellular traps exacerbate Th1‐mediated autoimmune responses in rheumatoid arthritis by promoting DC maturation

Aberrant formation of neutrophil extracellular traps (NETs) is a key feature in rheumatoid arthritis (RA) and plays a pivotal role in disease pathogenesis. However, the mechanism through which NETs shape the autoimmune response in RA remains elusive. In this study, we demonstrate that inhibition of peptidylarginine deiminases activity in collagen‐induced arthritis (CIA) mouse model significantly reduces NET formation, attenuates clinical disease activity, and prevents joint destruction. Importantly, peptidylarginine deiminase 4 blocking markedly reduces the frequency of collagen‐specific IFN‐γ‐producing T helper 1 (Th1) cells in the draining lymph nodes of immunized mice. Exposure of dendritic cells (DCs) to CIA‐derived NETs induces DC maturation characterized by significant upregulation of costimulatory molecules, as well as elevated secretion of IL‐6. Moreover, CIA‐NET‐treated DCs promote the induction of antigen‐specific Th1 cells in vitro. Finally, NETs from RA patients show an increased potential to induce the maturation of DCs from healthy individuals, corroborating the findings obtained in CIA mouse model. Collectively, our findings delineate an important role of NETs in the induction and expansion of Th1 pathogenic cells in CIA through maturation of DCs and reveal a novel role of NETs in shaping the RA‐autoimmune response that could be exploited therapeutically.     

The plasminogen activator/plasmin system is essential for development of the joint inflammatory phase of collagen type II-induced arthritis

The plasminogen activator (PA) system has been proposed to have important roles in rheumatoid arthritis. Here we have used the autoimmune collagen type II (CII)-induced arthritis (CIA) model and mice deficient for urokinase-type PA (uPA) or plasminogen to investigate the role of the PA system for development of arthritis. Our data revealed that uPA-deficient mice have a lower severity and incidence of CIA than wild-type mice. Furthermore, although >80% of wild-type control mice developed CIA, we found that none of the 50 plasminogen-deficient littermates that were tested developed CIA within a 40-day period. Antibody generation after CII immunization as well as the binding of labeled anti-CII antibodies to the surface of cartilage were similar in wild-type and plasminogen-deficient mice. No sign of inflammation was seen when plasminogen-deficient mice were injected with a mixture of monoclonal antibodies against CII. However, after daily injections of human plasminogen, these mice developed arthritis within 5 days. Our finding that infiltration of inflammatory cells into the synovial joints was impaired in plasminogen-deficient mice suggests that uPA and plasminogen are important mediators of joint inflammation. Active plasmin is therefore essential for the induction of pathological inflammatory joint destruction in CIA.     

Cysteine proteases in Langerhans cells limits presentation of cartilage derived type II collagen for autoreactive T cells

Development of type-II collagen (CII)-induced arthritis (CIA) is dependent on activation of CII-reactive T cells. Dendritic cells (DCs) are believed to play a crucial role in antigen-specific priming of T cells but it is still unclear how the CII-reactive T cells are primed since Langerhans cells (LCs) are poor antigen-presenting cells for CII. In the present study we show that LCs, treated with cysteine protease inhibitors, are able to process and present CII to T-cell hybridomas specific for the immune-dominant glycosylated 259-270 peptide bound to the MHC class II molecule A(q). Interestingly, the self (mouse) CII peptide could also now be efficiently presented. The poor presentation by LCs is a peptide-specific effect, since both bovine CII (bCII) (presenting a different peptide on H-2(r)) and ovalbumin could be efficiently presented, and blockage of cysteine proteases did not enhance antigen presentation. The enhanced CII-presentation by cysteine protease inhibition is seen mainly in LCs and not in antigen-primed B cells or macrophages. B cell and macrophage presentation of CII occur even without protease inhibition and are only to a minor extent influenced by cysteine protease inhibition. These data suggest that a LC deficiency in processing of the immune-dominant CII epitope in both CIA and RA may limit the exposure of this self-antigen to T cells, but that presentation can be overcome by modulation of the peptide proteolysis during CII processing.     

To B or not to B: role of B cells in pathogenesis of arthritis in HLA transgenic mice

Population studies have shown that amongst all the genetic factors linked with autoimmune disease development, MHC class II genes are the most significant. Experimental autoimmune arthritis resembling human rheumatoid arthritis (RA) can be induced in susceptible strains of mice following immunization with type II collagen (CIA). We generated transgenic mice lacking endogenous class II molecules and expressing various HLA genes including RA-associated, HLA-DRB1*0401 and HLA-DQ8, and RA-resistant, DRB1*0402, genes. The HLA molecules in these mice are expressed on the cell surface and can positively select CD4+ T cells expressing various Vβ T cell receptors. Endogenous class II invariant chain is required for proper functioning of the class II transgene. Arthritis development in transgenic mice is CD4+ and B cells dependent. Studies in humanized mice showed that B cells are required as antigen presenting cells in addition to antibody producing cells for the development of CIA. The transgenic mice expressing *0401 and *0401/DQ8 genes developed sex-biased arthritis with predominantly females being affected, similar to that of human RA. Further, the transgenic mice produced autoantibodies like rheumatoid factor and anti-cyclic antibodies. Antigen presentation by B cells leads to a sex-specific immune response in DRB1*0401 mice suggesting a role of B cells and HLA-DR in rendering susceptibility to develop arthritis in females.     

Intra-articular IL-10 gene transfer regulates the expression of collagen-induced arthritis (CIA) in the knee and ipsilateral paw

We studied the effects of local IL-10 application, introduced by a recombinant human type 5 adenovirus vector, in the mouse knee joint during the early phase of CIA. One intra-articular injection with the IL-10-expressing virus (Ad5E1mIL-10) caused substantial over-expression of IL-10 in the mouse knee joint, using virus dosages which did not induce distracting inflammation. High expression of IL-10 was noted for a few days, being maximal at day 1. One intra-articular injection of Ad5E1mIL-10 in the knee joints of collagen type II (CII)-immunized mice, before onset of CIA was noted, reduced the incidence of collagen arthritis in that knee. Of high interest, the protective effect of local IL-10 expression by Ad5E1mIL-10 was not restricted to the knee joint alone. The arthritis incidence in the ipsilateral paw was highly suppressed. In contrast, local IL-10 over-expression was not effective when treatment was started after onset of CIA. Further analysis in the acute streptococcal cell wall-induced arthritis model revealed that local IL-10 over-expression markedly suppressed the production of tumour necrosis factor-alpha (TNF-alpha) and IL-1alpha, but had no significant effect on IL-1beta and IL-12 production in the inflamed synovium. These data indicate that local over-expression of IL-10 in the knee joint of mice regulates the expression of collagen arthritis, probably through down-regulation of TNF-alpha.     

LFA-1 decreases the antigen dose for T cell activation in vivo

Leukocyte adhesion molecule leukocyte function-associated antigen (LFA)-1 not only mediates intercellular binding but also delivers co-stimulatory signals in T cells. LFA-1 has been shown to decrease the threshold of TCR signal and an antigen dose required for T cell activation and proliferation in vitro. However, physiological significance of the role of LFA-1 in TCR signal has remained unclear. We examined whether LFA-1 decreased the antigen dose for T cell activation in vivo. We showed here that, although collagen-induced arthritis (CIA) could not be induced by immunization and challenge with a standard amount of type-II collagen in LFA-1-deficient mice, a higher dose of the antigen did induce CIA in the absence of LFA-1. We also showed that CD4+ T cells could be primed by immunization with a high, but not low, dose of ovalbumin antigen in LFA-1-deficient mice. These results suggest that LFA-1 decreases the threshold of TCR signal for T cell activation in vivo as well as in vitro. Further studies using TCR-transgenic LFA-1-deficient mice showed that LFA-1 cooperated with TCR in sustained Erk1/2 phosphorylation. Moreover, TCR could induce sustained Erk1/2 phosphorylation in the absence of LFA-1 when T cells were stimulated with a high, but not low, dose of antigen, suggesting that LFA-1 may cooperate with TCR in sustaining Erk1/2 phosphorylation.     

Complement receptor CR2/CR1 deficiency protects mice from collagen-induced arthritis and associates with reduced autoantibodies to type II collagen and citrullinated antigens

Collagen-induced arthritis (CIA), a model of autoimmune inflammatory arthritis, depends upon complement activation and effective B cell responses. To determine the importance of complement receptors CR2/CR1 in CIA, the Cr2−/− genotype was backcrossed onto the DBA/1j strain. CIA was induced by immunization with bovine type II collagen in CFA on days 0 and 21. Cr2−/− mice demonstrated a significantly diminished arthritis severity, decreased antibodies to bovine and murine collagen, and a significant reduction in antibodies to citrullinated antigens. Autoantibodies to citrullinated antigens have been shown to amplify anti-type II collagen passive transfer arthritis. To test the hypothesis that that simple replacement of such antibodies might re-establish severe disease in Cr2−/− mice, monoclonal antibodies to citrullinated antigens were administered to mice during the disease course. Although citrullinated antigens targeted by these antibodies were present within the joints of all mice, addition of these monoclonal antibodies increased disease severity only in Cr2+/+ mice. Taken together, these data suggest that CR2/CR1 are required to develop robust autoimmunity in the CIA model and that amplification of arthritis by antibodies to citrullinated antigens depends on factor(s) absent in arthritic Cr2−/− mice.     

Induction of transient arthritis by the adoptive transfer of a collagen II specific Th1 clone to HLA-DR4 (B1*0401) transgenic mice

Collagen II arthritis (CIA) represents an animal model of human RA that can be induced in DBA/1J (H-2(q)) but not in C57BL/6 mice (H-2(b)). A vigorous CII specific CD4 Th1-cell response but not IgG2 anti-CII antibody or CIA could be induced in C57BL/6 mice made transgenic for the RA shared epitope DR4 (B1*0401). We developed CD4 Th1-cell clones specific for CII from these transgenic (tg) mice in order to determine if the adoptive transfer of these clones into syngeneic tg C57BL/6 recipients could induce CIA. Three bovine CII specific (bCII) CD4 Th1-cell clones and one T-cell line specific for an immunodominant region of bCII (p261-273) were generated. Among these only one clone that could up-regulate anti-CII, IgG2 antibody in the recipient mice was able to induce transient arthritis. However, this level of IgG2 anti-CII antibody was only one-third of that seen in CII immunized DBA/1J mice that develop persistent arthritis. These results confirm our previous observations that the induction of CIA requires a sustained IgG2 antibody response to CII, an effect difficult to achieve even in DR4 (B1*0401) tg mice reconstituted with CD4 Th1 cells. This suggests that a rate limiting step in the development of human RA among those individuals expressing the RA shared epitope may be the requirement to generate sustained levels of complement fixing antibody to arthritogenic antigens.     

组蛋白去乙酰化酶抑制剂TSA对小鼠胶原诱导性关节炎的治疗作用

目的 研究组蛋白去乙酰酶化抑制剂制滴菌素A(TSA)对小鼠胶原诱导性关节炎的作用,初步探讨其作用机制.方法 将雄性DBA/1小鼠随机分为胶原诱导性关节炎(CIA)模型组、预防性给药组、治疗性给药组、正常组.免疫后第35天处死小鼠,检测小鼠足爪炎症的肿胀度,病理切片观察炎症变化,MTT法检测脾淋巴细胞对胶原的增殖反应,实时荧光定量PCR检测TSA对小鼠脾细胞IFN-γ、IL-4 mRNA表达的影响,ELISA检测IFN-γ、IL-4的含量.结果 CIA模型组关节炎评分、对CⅡ的增殖反应、IFN-γ水平均高于正常对照组;给药组关节炎评分、对CⅡ的增殖反应低于CIA模型组;给药组IFN-γ水平低于CIA组,但IL-4水平高于CIA组.结论 TSA对CIA具有抑制作用,其可能机制是抑制T细胞的活化、增殖,调节Th1/Th2的失衡状态.     

The influence of HLA-DR4 (0401) on the immune response to type II collagen and the development of collagen induced arthritis in mice

Rheumatoid arthritis (RA) is an autoimmune disease that is genetically associated with the MHC class II molecule HLA-DRbeta1*0401 (DR4). In order to determine if this MHC can influence the immune response to the candidate autoantigen type II collagen (CII), we have studied collagen induced arthritis (CIA) resistant C57BL/6 mice, made transgenic (Tg) for human DR4. These DR4 Tg mice exhibited a strong T cell proliferative response to CII and its DR4 restricted peptide p261-273 after immunization with these antigens that was not seen in the C57BL/6 wild type mice. DR4 Tg mice also exhibited an increase in IFN-gamma production in response to CII, indicating the activation of Th1 cells. While these Tg mice produced IgM anti-CII antibodies, they failed to produce a detectable level of IgG2a (Th1 type) anti-bCII antibody and did not develop CIA. This study shows that a Th1 type T cell response to CII can be established in CIA non-susceptible mice by introducing the human transgene, DR4. This T cell response, however, is not sufficient to induce an antibody isotype switch to IgG2a, nor is it sufficient for the induction of CIA. These results may help to explain why many individuals expressing HLA-DRbeta1*0401 do not develop RA.     

The role of the B cells in the adoptive transfer of collagen-induced arthritis from DBA/1 (H-2q) to SCID (H−2d) mice

Collagen-induced arthritis (CIA) can be transferred from DBA/1 to SCID mice when native type II collagen (CII) is administered together with spleen cells, arthritis appearing some 14 days after cell transfer. In the present study, we demonstrate that both donor T- and B-lymphocyte populations play a role in this model, and that arthritis arises in SCID recipients of either murine or bovine native CII. Furthermore, the requirement for administration of soluble native CII can be replaced by subarthritogenic doses of serum from Wistar rats with CIA. In this case a fully developed arthritis appears as early as 2 days after cell transfer. However, protein G-purified IgG from CIA rat serum together with splenocytes from arthritic DBA/1 mice does not transfer arthritis. A key role of B cells in this model appears to be the production of a humoral arthritogenic factor since arthritis can be successfully transferred to SCID mice by CIA rat serum administered together with a B cell-depleted splenocyte population consisting of T cells and donor-histocompatible antigen-presenting cells. By contrast, transfer of disease cannot be achieved by co-administration of CIA rat serum and purified donor T cells, indicating that the presence of donor antigen-presenting cells is a requirement for adoptive transfer of arthritis. We propose that joint damage initiated by arthritogenic product(s) of the B cell lineage releases soluble antigens that are presented to T cells which perpetuate the disease. The finding that arthritis can be generated in SCID recipients of CIA rat serum together with splenocytes from non-arthritic DBA/1 mice immunized with denatured CII supports the hypothesis that T cells with specificity for denatured joint components perpetuate disease initiated by humoral factors.     

Autologous Mixed Lymphocyte Responses in Experimentally-induced Arthritis of the Lewis Rat

Lewis rats develop immune-mediated arthritis following injection with a variety of agents including bovine type II collagen (bCII), mycobacteria, muramyl dipeptide and CP20961. Since susceptibility to experimentally-induced arthritis has been linked to the genes encoding the major histocompatibility complex, it is hypothesized that antigen presentation to autoreactive T-cells is a critical event in the pathogenesis of disease. T-cells, isolated from Lewis rats immunized with bCII or mycobacteria, were co-cultured with splenic or thymic antigen presenting cells (APC) and proliferative responses to antigen were assessed by 3 H-thymidine incorporation. T-cell proliferation was observed upon culture with APC without requiring the addition of antigen. T-cells from rats injected with non-immunogenic adjuvants also demonstrated an increased autologous MLR compared to T-cells from non-injected animals. In contrast, T-cells from animals immunized with non-arthritogenic antigens, including ovalbumin or tetanus toxoid, proliferated only when co-cultured with specific antigen-pulsed APC. These results suggest that immunization with arthritogens activates a population of self-reactive T-cells, which respond in an autologous MLR. We propose that these autoreactive T-cells recognize endogenously-derived self peptides rather than peptides derived from a joint autoantigen.