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1.  T cells that are naturally tolerant to cartilage-derived type II collagen are involved in the development of collagen-induced arthritis 
Arthritis Research  2000;2(4):315-326.
The immunodominant T-cell epitope that is involved in collagen-induced arthritis (CIA) is the glycosylated type II collagen (CII) peptide 256-270. In CII transgenic mice, which express the immunodominant CII 256-270 epitope in cartilage, the CII-specific T cells are characterized by a partially tolerant state with low proliferative activity in vitro, but with maintained effector functions, such as IFN-γ secretion and ability to provide B cell help. These mice were still susceptible to CIA. The response was mainly directed to the glycosylated form of the CII 256-270 peptide, rather than to the nonglycosylated peptide. Tolerance induction was rapid; transferred T cells encountered CII within a few days. CII immunization several weeks after thymectomy of the mice did not change their susceptibility to arthritis or the induction of partial T-cell tolerance, excluding a role for recent thymic emigrants. Thus, partially tolerant CII autoreactive T cells are maintained and are crucial for the development of CIA.
A discussion is ongoing regarding the possible role of cartilage-directed autoimmunity as a part of the pathogenesis of rheumatoid arthritis (RA). One possibility is that the association of RA with shared epitope-expressing DR molecules reflects a role for major histocompatibility complex (MHC) class II molecules as peptide receptors, and that the predilection of the inflammatory attack for the joint indicates a role for cartilage as a source of the antigenic peptides. A direct role for CII in the development of arthritis is apparent in the CIA model, in which a definite role for MHC class II molecules and a role for CII-derived peptides have been demonstrated [1,2,3]. Remarkably, it was found that the identified MHC class II molecule in the CIA model Aq has a structurally similar peptide binding pocket to that of the shared epitope, expressing DR4 molecules [4]. In fact, DR4 (DRB1*0401) and DR1 (DRB1*0101) transgenic mice are susceptible to CIA because of an immune response to a peptide that is almost identical to that which is involved in Aq-expressing mice [5,6]. They are both derived from position 260-273 of the CII molecule; the peptide binds to the Aqmolecule with isoleucine 260 in the P1 pocket, but with phenylalanine 263 in the P1 pocket of the DR4 and DR1 molecules.
Although these findings do not prove a role for CII in RA, they show that such recognition is possible and that there are structural similarities when comparing mouse with human. However, there are also strong arguments against such a possibility. First, arthritis can evolve without evidence for a cartilage-specific autoimmunity, as seen with various adjuvant-induced arthritis models [7,8] and in several observations using transgenic animals with aberrant immunity to ubiquitously expressed proteins [9,10,11]. Moreover, the MHC association in the adjuvant arthritis models correlates with severity of the disease rather than susceptibility [7,8], as has also been observed in RA [12]. Second, it has not been possible to identify the CII-reactive T cells from RA joints, or to achieve a strong and significant CII proliferative response from T cells derived from RA joints. Most recently these negative observations were corroborated using DR4+CII peptide tetramer reagents [13]. On the other hand, it has also been difficult to isolate autoreactive CII-specific T cells from CIA, and it can be anticipated that, even in the CIA model, T cells that are specific for CII will be hard to find in the joints [4].
We believe that the explanations for these observations in both experimental animals and humans are related to tolerance. The CIA model in the mouse is usually induced with heterologous CII, and is critically dependent on an immune response to the glycosylated CII peptide 256-270, which is bound to the MHC class II Aq molecule. In CII transgenic mice, expressing the heterologous (rat) form of the immunodominant CII 256-270 epitope in cartilage, we observed partial T-cell tolerance. This tolerance is characterized by a low proliferative activity, but with maintained effector functions such as production of IFN-γ and the ability to give help to B cells to produce anti-CII IgG antibodies [14]. Interestingly, these mice were susceptible to arthritis. However, a possibility was that T cells that had newly emerged from the thymus and that were not yet tolerized when the mice were immunized with CII led to the induction of arthritis. We have now addressed this possibility and found that induction of tolerance occurs within a few days, and that mice lacking recent thymic emigrants (ie thymectomized mice) display partially tolerant T cells and susceptibility to arthritis to the same extent as nonthymectomized mice. In addition we found that T cells that are reactive with the nonmodified peptides are relatively more affected by tolerance than T cells that are reactive with the more immunodominant glycosylated variants.
To investigate the possibility that T cells that are naturally tolerant to the cartilage protein CII are involved in the development of arthritis, and to exclude a role for nontolerized recent thymic T-cell emigrants in the development of arthritis.
Materials and methods:
A mutated mouse CII, expressing glutamic acid instead of aspartic acid at position 266, was expressed in a transgenic mouse called MMC (mutated mouse collagen) that has been described earlier [14]. The mice were thymectomized, or sham-operated, at 7 weeks of age and allowed to recover for 4 weeks before being immunized with rat CII in complete Freund's adjuvant. Arthritis development was recorded and sera analyzed for anti-CII IgG, IgG1 and IgG2a levels. To assay T-cell effector functions, other MMC and control mice were immunized in the hind footpads with rat CII in complete Freund's adjuvant, and the draining popliteal lymph nodes were taken 10 days later. The lymph node cells (LNCs) were used for proliferation assay, IFN-γ enzyme-linked immunosorbent assay (ELISA) and B-cell enzyme-linked immunospot (ELISPOT). For the proliferation assay, 106 cells were put in triplicate cultures in microtitre wells together with antigen and incubated for 72h before thymidine-labelling and harvesting 15-18h later. For IFN-γ ELISA analysis, supernatant from the proliferation plates was removed before harvesting and used in an ELISA to quantify the amount of IFN-γ produced [15]. B-cell ELISPOT was performed to enumerate the number of cells producing anti-CII IgG [16].
T-cell lines that were reactive towards rat CII were established by immunization with rat CII. An established T-cell line that was reactive with CII and specific for the CII 256-270 peptide was restimulated with freshly collected, irradiated, syngenic spleen cells and rat CII for 3 days followed by 2 weeks of IL-2 containing medium. Immediately before transfer, the cells were labelled with the cytoplasmic dye 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) [17]. Labelled cells (107) were injected intravenously into transgenic MMC mice and nontransgenic littermates. The mice were killed 4 days after cell transfer, and the concentration of CFSE-labelled cells was determined by flow cytometry.
Results and discussion:
To investigate whether and how quickly CII-reactive T cells will encounter CII in vivo, an established T-cell line that is reactive towards rat CII was labelled with the cytoplasmic dye CFSE and transferred into MMC-QD and control mice. Four days later the mice were killed, and it was found that MMC-transgenic mice had dramatically fewer CFSE-labelled cells in the spleen than did nontransgenic littermates (0.11% compared with 0.57%). Similarly, reduced numbers of CFSE-positive cells were observed in blood. This indicates that the T cells encountered the mutated CII that was present in the cartilage of MMC mice, but not in the nontransgenic littermates. Presumably, CII from cartilage is spread by antigen-presenting cells (APCs) to peripheral lymphoid organs. This observation also suggests that newly exported T cells from the thymus will be tolerized to CII in the periphery within less than 4 days.
To further investigate whether the MMC mice harbours naïve or tolerized T cells, the mice were immunized with CII at different time points after thymectomy that were well in excess of the times required for their encounter with CII. After 10 days, the response was analyzed in vitro towards both the nonglycosylated and the glycosylated CII 256-270 peptides as well as towards purified protein derivative. The galactosylated form of the peptide (Fig. 1) was used because this is the most immunodominant modification [18]. In contrast to control mice, LNCs from transgenic mice did not proliferate significantly towards the nonglycosylated peptide, indicating that these cells have been specifically tolerized, which is in accordance with earlier observations [14]. A reduced, but still significant proliferation was also observed toward the immunodominant glycosylated CII peptide. Most important, however, was that the proliferative response in the MMC mice did not decrease after thymectomy. Similarly, a significant IFN-γ production towards the glycosylated CII peptide was observed in the MMC mice. The response was somewhat reduced compared with that observed in nontransgenic littermates, and this was especially true for the response toward the nonglycosylated peptide. Again, no decrease in the MMC response by thymectomy was observed. Taken together, the T-cell response in transgenic mice was reduced in comparison with that in the nontransgenic littermates. Furthermore, the response in transgenic animals did not decrease by thymectomy (4 or 8 weeks before immunization), showing that autoreactive T cells are still maintained (and partially tolerized) with significant effector functions at least up to 8 weeks after thymectomy, excluding a exclusive role for recent thymic emigrants in the autoimmune response towards CII. To investigate whether thymectomized mice, lacking recent CII-specific thymic emigrants, were susceptible to CIA, mice were immunized with CII 4 weeks after thymectomy and were observed for arthritis development during the following 10 weeks. Clearly, the thymectomized MMC mice were susceptible to arthritis (five out of 18 developed arthritis; Fig. 2), and no significant differences in susceptibility between thymectomized and sham-operated mice, or between males and females, were seen. In accordance with earlier results [14], MMC transgenic mice had a significantly reduced susceptibility to arthritis as compared with the nontransgenic littermates (P < 0.0001 for arthritic scores, disease onset and incidence). All mice were bled at 35 days after immunization, and the total levels of anti-CII IgG were determined. Transgenic mice developed levels of anti-CII IgG significantly above background, but the antibody titres were lower than in nontransgenic littermates (P < 0.0001). No effect on the antibody levels by thymectomy was observed, nor did thethymectomy affect the distribution of IgG1 versus IgG2a titres,indicating that the observed tolerance is not associated with a shift from a T-helper-1- to a T-helper-2-like immune response. These findings show that T cells that are specific for a tissue-specific matrix protein, CII, are partially tolerized within a few days after thymus export and that these tolerized cells are maintained after thymectomy. Most important, mice that lack newly exported CII reactive T cells are still susceptible to CIA, suggesting that the partially tolerant T cells are involved in development of arthritis.
In the light of these data it is possible to explain some of the findings in RA. T-cell reactivity to CII has been shown in RA patients, but with a very weak proliferative activity [19,20]. This is fully compatible with observations in mouse and rat CIA when autologous CII, and not heterologous CII, are used for immunization. This is particularly true if the responses are recorded during the chronic phase of disease, in which the antigen-specific T-cell responses seem to be suppressed in both humans and experimental animals. These observations were confirmed in a recent report [21] in which it was shown that CII-reactive T-cell activity could be detected in RA patients if IFN-γ production but not proliferation was measured. In the present studies in mice the strongest response is seen towards post-translational modifications of the peptide. Because the T-cell contact points are the same whether the peptide is bound to DR4 or to Aq, it is fully possible that post-translational modifications of the peptide also plays a significant role in humans [22]. The fact that IgG antibodies specific for CII are found in many RA patients could be explained by maintained B-cell helper functions of CII-reactive T cells. In fact, it has been reported [23,24] that the occurrence of IgG antibodies to CII is associated with shared epitope DR4 molecules. These observations are thus compatible with a role for CII reactivity in RA. To avoid any confusion, it needs to be stressed that RA is a heterogeneous syndrome in which not only CII, but also other cartilage proteins and other mechanisms are of importance. Such a pathogenic heterogeneity is reflected by the multitude of experimental animal models that have demonstrated how many different pathways may lead to arthritis [25].
PMCID: PMC17814  PMID: 11056672
autoimmunity; rheumatoid arthritis; T lymphocytes; tolerance; transgenic
2.  Cia25 on rat chromosome 12 regulates severity of autoimmune arthritis induced with pristane and with collagen 
Annals of the Rheumatic Diseases  2007;66(7):952-957.
A genomewide scan in a DA×ACI F2 intercross studied for collagen‐induced arthritis (CIA) identified the severity quantitative trait locus Cia25 on rat chromosome 12. Cia25 co‐localises with loci regulating several forms of autoimmune diseases in rats, mice and humans, suggesting a common gene.
To characterise the effects of Cia25 on severity of arthritis in congenic rats.
DA.ACI(Cia25) congenic rats were constructed according to a genotype‐guided strategy, and tested for pristane‐induced arthritis (PIA) and CIA, induced with rat type II collagen (CII). A well‐established scoring system previously shown to correlate with histological damage, including cartilage and bone erosions, synovial hyperplasia and synovial inflammation, was used.
The introgression of ACI alleles at Cia25 into DA background, as in DA.ACI(Cia25) rats, was enough to significantly reduce arthritis severity by 60% in PIA and by 40% in CIA, both in males and females compared with DA rats of the same sex. Levels of IgG anti‐CII in male DA.ACI(Cia25) rats were 83% lower than in male DA. Levels of anti‐CII in females were not affected by the congenic interval.
Cia25 contains a gene that regulates disease severity in two distinct models of autoimmune arthritis. Although both genders were protected in arthritis studies, only male congenic rats had a dramatic reduction in levels of anti‐CII, suggesting the possibility of a second arthritis gene in this interval that operates via the regulation of autoantibodies in a sex‐specific manner. The identification of the gene(s) accounting for Cia25 is expected to generate novel prognostic biomarkers and targets for therapy.
PMCID: PMC1955106  PMID: 17329308
3.  Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis 
Arthritis Research  1999;1(1):81-91.
Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA.
Rheumatoid arthritis (RA) is associated with an increased production of a range of cytokines including tumour necrosis factor (TNF)-α and interleukin (IL)-1, which display potent proinflammatory actions that are thought to contribute to the pathogenesis of the disease. Although TNF-α seems to be the major cytokine in the inflammatory process, IL-1 is the key mediator with regard to cartilage and bone destruction. Apart from direct blockade of IL-1/TNF, regulation can be exerted at the level of modulatory cytokines such as IL-4 and IL-10. IL-4 is a pleiotropic T-cell derived cytokine that can exert either suppressive or stimulatory effects on different cell types, and was originally identified as a B-cell growth factor and regulator of humoral immune pathways. IL-4 is produced by activated CD4+ T cells and it promotes the maturation of Th2 cells. IL-4 stimulates proliferation, differentiation and activation of several cell types, including fibroblasts, endothelial cells and epithelial cells. IL-4 is also known to be a potent anti-inflammatory cytokine that acts by inhibiting the synthesis of proinflammatory cytokines such as IL-1, TNF-α, IL-6, IL-8 and IL-12 by macrophages and monocytes. Moreover, IL-4 stimulates the synthesis of several cytokine inhibitors such as interleukin-1 receptor antagonist (IL-1Ra), soluble IL-1-receptor type II and TNF receptors IL-4 suppresses metalloproteinase production and stimulates tissue inhibitor of metalloproteinase-1 production in human mononuclear phagocytes and cartilage explants, indicating a protective effect of IL-4 towards extracellular matrix degradation. Furthermore, IL-4 inhibits both osteoclast activity and survival, and thereby blocks bone resorption in vitro. Of great importance is that IL-4 could not be detected in synovial fluid or in tissues. This absence of IL-4 in the joint probably contributes to the disturbance in the Th1/Th2 balance in chronic RA.
Collagen-induced arthritis (CIA) is a widely used model of arthritis that displays several features of human RA. Recently it was demonstrated that the onset of CIA is under stringent control of IL-4 and IL-10. Furthermore, it was demonstrated that exposure to IL-4 during the immunization stage reduced onset and severity of CIA. However, after cessation of IL-4 treatment disease expression increased to control values.
Because it was reported that IL-4 suppresses several proinflammatory cytokines and matrix degrading enzymes and upregulates inhibitors of both cytokines and catabolic enzymes, we investigated the tissue protective effect of systemic IL-4 treatment using established murine CIA as a model. Potential synergy of low dosages of anti-inflammatory glucocorticosteroids and IL-4 was also evaluated.
DBA-1J/Bom mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected at day 28 after immunization and treated for days with IL-4, prednisolone, or combinations of prednisolone and IL-4. Arthritis score was monitored visually. Joint pathology was evaluated by histology, radiology and serum cartilage oligomeric matrix protein (COMP). In addition, serum levels of IL-1Ra and anticollagen antibodies were determined.
Treatment of established CIA with IL-4 (1 μg/day) resulted in suppression of disease activity as depicted in Figure 1. Of great interest is that, although 1 μg/day IL-4 had only a moderate effect on the inflammatory component of the disease activity, it strongly reduced cartilage pathology, as determined by histological examination (Fig. 1). Moreover, serum COMP levels were significantly reduced, confirming decreased cartilage involvement. In addition, both histological and radiological analysis showed that bone destruction was prevented (Fig. 1). Systemic IL-4 administration increased serum IL-1Ra levels and reduced anticollagen type II antibody levels. Treatment with low-dose IL-4 (0.1 μg/day) was ineffective in suppressing disease score, serum COMP or joint destruction. Synergistic suppression of both arthritis severity and COMP levels was noted when low-dose IL-4 was combined with prednisolone (0.05 mg/kg/day), however, which in itself was not effective.
In the present study, we demonstrate that systemic IL-4 treatment ameliorates disease progression of established CIA. Although clinical disease progression was only arrested and not reversed, clear protection against cartilage and bone destruction was noted. This is in accord with findings in both human RA and animal models of RA that show that inflammation and tissue destruction sometimes are uncoupled processes. Of great importance is that, although inflammation was still present, strong reduction in serum COMP was found after exposure to IL-4. This indicated that serum COMP levels reflected cartilage damage, although a limited contribution of the inflamed synovium cannot be excluded.
Increased serum IL-1Ra level (twofold) was found after systemic treatment with IL-4, but it is not likely that this could explain the suppression of CIA. We and others have reported that high dosages of IL-1Ra are needed for marked suppression of CIA. As reported previously, lower dosages of IL-4 did not reduce clinical disease severity of established CIA. Of importance is that combined treatment of low dosages of IL-4 and IL-10 appeared to have more potent anti-inflammatory effects, and markedly protected against cartilage destruction. Improved anti-inflammatory effect was achieved with IL-4/prednisolone treatment. In addition, synergistic effects were found for the reduction of cartilage and bone destruction. This indicates that systemic IL-4/prednisolone treatment may provide a cartilage and bone protective therapy for human RA.
Effects in mice of treatment with interleukin-4 or control on disease activity, cartilage damage and bone destruction. Mice were treated intraperitoneally for 7 days with either vehicle (control) or 1 μg/day interleukin-4 (IL-4). CIA, collagen-induced arthritis. *P < 0.05, versus control, by Mann-Whitney U test.
PMCID: PMC17779  PMID: 11056663
bone destruction; cartilage oligomeric matrix protein levels; collagen-induced arthritis; interleukin-4; prednisolone
4.  Cystatin C influences the autoimmune but not inflammatory response to cartilage type II collagen leading to chronic arthritis development 
Collagen-induced arthritis (CIA) is a mouse model for rheumatoid arthritis (RA) and is induced after immunization with type II collagen (CII). CIA, like RA, is an autoimmune disease leading to destruction of cartilage and joints, and both the priming and inflammatory phases have been suggested to be dependent on proteases. In particular, the cysteine proteases have been proposed to be detrimental to the arthritic process and even immunomodulatory. A natural inhibitor of cysteine proteases is cystatin C.
Cystatin C-deficient, sufficient and heterozygous mice were tested for onset, incidence and severity of CIA. The effect of cystatin C-deficiency was further dissected by testing the inflammatory effector phase of CIA; that is, collagen antibody-induced arthritis model and priming phase, that is, T cell response both in vivo and in vitro. In addition, in order to determine the importance of T cells and antigen-presenting cells (APCs), these cell populations were separated and in vitro T cell responses determined in a mixed co-culture system. Finally, flow cytometry was used in order to further characterize cell populations in cystatin C-deficient mice.
Here, we show that mice lacking cystatin C, develop arthritis at a higher incidence and an earlier onset than wild-type controls. Interestingly, when the inflammatory phase of CIA was examined independently from immune priming then cystatin C-deficiency did not enhance the arthritis profile. However, in line with the enhanced CIA, there was an increased T cell and B cell response as delayed-type hypersensitivity reaction and anti-CII antibody titers were elevated in the cystatin C-deficient mice after immunization. In addition, the ex vivo naïve APCs from cystatin C-deficient mice had a greater capacity to stimulate T cells. Interestingly, dendritic cells had a more activated phenotype in naïve cystatin C-deficient mice.
The lack of cystatin C enhances CIA and primarily affects in vivo priming of the immune system. Although the mechanism of this is still unknown, we show evidence for a more activated APC compartment, which would elevate the autoimmune response towards CII, thus resulting in an enhanced development of chronic arthritis.
PMCID: PMC3132044  PMID: 21443774
5.  Induction of IL-10-producing CD4+CD25+ T cells in animal model of collagen-induced arthritis by oral administration of type II collagen 
Arthritis Research & Therapy  2004;6(3):R213-R219.
Induction of oral tolerance has long been considered a promising approach to the treatment of chronic autoimmune diseases, including rheumatoid arthritis (RA). Oral administration of type II collagen (CII) has been proven to improve signs and symptoms in RA patients without troublesome toxicity. To investigate the mechanism of immune suppression mediated by orally administered antigen, we examined changes in serum IgG subtypes and T-cell proliferative responses to CII, and generation of IL-10-producing CD4+CD25+ T-cell subsets in an animal model of collagen-induced arthritis (CIA). We found that joint inflammation in CIA mice peaked at 5 weeks after primary immunization with CII, which was significantly less in mice tolerized by repeated oral feeding of CII before CIA induction. Mice that had been fed with CII also exhibited increased serum IgG1 and decreased serum IgG2a as compared with nontolerized CIA animals. The T-cell proliferative response to CII was suppressed in lymph nodes of tolerized mice also. Production of IL-10 and of transforming growth factor-β from mononuclear lymphocytes was increased in the tolerized animals, and CD4+ T cells isolated from tolerized mice did not respond with induction of IFN-γ when stimulated in vitro with CII. We also observed greater induction of IL-10-producing CD4+CD25+ subsets among CII-stimulated splenic T cells from tolerized mice. These data suggest that when these IL-10-producing CD4+CD25+ T cells encounter CII antigen in affected joints they become activated to exert an anti-inflammatory effect.
PMCID: PMC416445  PMID: 15142267
collagen-induced arthritis; IL-10; oral tolerance; type II collagen
6.  Grape Seed Proanthocyanidin Extract–Mediated Regulation of STAT3 Proteins Contributes to Treg Differentiation and Attenuates Inflammation in a Murine Model of Obesity-Associated Arthritis 
PLoS ONE  2013;8(11):e78843.
Grape seed proanthocyanidin extract (GSPE) is a natural flavonoid that exerts anti-inflammatory properties. Obesity is an inflammatory condition and inflammatory cells and their secretion of pro-inflammatory molecules contribute to the pathogenesis of obesity. Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by inflammation of joints lined by synovium. Previously, we demonstrated that obesity augmented arthritis severity in collagen induced arthritis (CIA), a murine model of human RA. Here, we investigated whether oral administration of GSPE showed antiobesity and anti-arthritic effects in high-fat diet-induced obese (DIO) mice and in obese CIA mice, respectively. The pathophysiologic mechanisms by which GSPE attenuates weight gain and arthritis severity in vivo were also investigated. In DIO mice, GSPE administration significantly inhibited weight gain, reduced fat infiltration in liver and improved serum lipid profiles. The antiobesity effect of GSPE was associated with increased populations of regulatory T (Treg) cells and those of decreased Th17 cells. Decrease of Th17 cells was associated with significant inhibition of their key transcriptional factors, pSTAT3Tyr705 and pSTAT3Ser727. On the contrary, GSPE-induced Treg induction was associated with enhanced pSTAT5 expression. To identify the anti-arthritis effects of GSPE, GSPE was given orally for 7 weeks after type II collagen immunization. GSPE treatment significantly attenuated the development of autoimmune arthritis in obese CIA model. In line with DIO mice, GSPE administration decreased Th17 cells and reciprocally increased Treg cells by regulating STAT proteins in autoimmune arthritis model. The expressions of pro-inflammatory cytokines and nitrotyrosine in synovium were significantly inhibited by GSPE treatment. Taken together, GSPE functions as a reciprocal regulator of T cell differentiation – suppression of Th17 cells and induction of Tregs in both DIO and obese CIA mice. GSPE may act as a therapeutic agent to treat immunologic diseases related with enhanced STAT3 activity such as metabolic disorders and autoimmune diseases.
PMCID: PMC3818494  PMID: 24223854
7.  Exacerbation of collagen induced arthritis by Fcγ receptor targeted collagen peptide due to enhanced inflammatory chemokine and cytokine production 
Antibodies specific for bovine type II collagen (CII) and Fcγ receptors play a major role in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Our aim was to clarify the mechanism of immune complex-mediated inflammation and modulation of the disease. CII pre-immunized DBA/1 mice were intravenously boosted with extravidin coupled biotinylated monomeric CII-peptide epitope (ARGLTGRPGDA) and its complexes with biotinylated FcγRII/III specific single chain Fv (scFv) fragment. Disease scores were monitored, antibody titers and cytokines were determined by ELISA, and binding of complexes was detected by flow cytometry and immune histochemistry. Cytokine and chemokine secretion was monitored by protein profiler microarray. When intravenously administered into collagen-primed DBA/1 mice, both CII-peptide and its complex with 2.4G2 scFv significantly accelerated CIA and increased the severity of the disease, whereas the monomeric peptide and monomeric 2.4G2 scFv had no effect. FcγRII/III targeted CII-peptide complexes bound to marginal zone macrophages and dendritic cells, and significantly elevated the synthesis of peptide-specific IgG2a. Furthermore, CII-peptide containing complexes augmented the in vivo secretion of cytokines, including IL-10, IL-12, IL-17, IL-23, and chemokines (CXCL13, MIP-1, MIP-2). These data indicate that complexes formed by the CII-peptide epitope aggravate CIA by inducing the secretion of chemokines and the IL-12/23 family of pro-inflammatory cytokines. Taken together, these results suggest that the in vivo emerging immune complexes formed with autoantigen(s) may trigger the IL-12/23 dependent pathways, escalating the inflammation in RA. Thus blockade of these cytokines may be beneficial to downregulate immune complex-induced inflammation in autoimmune arthritis.
PMCID: PMC3333823  PMID: 22532778
collagen-induced arthritis; Fc gamma receptor; immune complex; inflammation; targeting
8.  Statins accelerate the onset of collagen type II-induced arthritis in mice 
Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors) are effective in reducing the risk of cardiovascular morbidity and mortality in patients with hyperlipidemia, hypertension, or type II diabetes. Next to their cholesterol-lowering activity, statins have immunomodulatory properties. Based on these properties, we hypothesized that statin use may eventually lead to dysregulation of immune responses, possibly resulting in autoimmunity. We have recently shown in an observational study that statin use was associated with an increased risk of developing rheumatoid arthritis. Our objective was to investigate whether a causal relationship could be established for this finding.
The mouse collagen type II (CII)-induced arthritis (CIA) model was used, with immunization, challenge, and euthanasia at days 0, 21, and 42, respectively. Statins were given orally before (day -28 until day 21) or after (day 21 until day 42) CIA induction. Atorvastatin (0.2 mg/day) or pravastatin (0.8 mg/day) was administered. Arthritis was recorded three times a week. Serum anti-CII autoantibodies and cytokines in supernatants from Concanavalin-A-stimulated lymph node cells and CII-stimulated spleen cells were measured.
Statin administration accelerated arthritis onset and resulted in 100% arthritic animals, whereas only seven out of 12 nonstatin control animals developed arthritis. Atorvastatin administration after CIA induction resulted in earlier onset than atorvastatin administration before induction, or than pravastatin administration before or after induction. The arthritic score of animals given pravastatin before CIA induction was similar to that of the nonstatin controls, whereas the other groups that received statins showed higher arthritic scores. Atorvastatin administration, especially before CIA induction, increased anti-CII autoantibody production. IL-2 and IL-17 production by lymph node and spleen cells was higher in CIA animals than in PBS controls, but was not affected by statin administration. While IFNγ production was not affected by CIA induction, atorvastatin administration before CIA induction increased the production of this cytokine.
These data support previous results from our observational studies, indicating a role for statins in the induction of autoimmunity.
PMCID: PMC3446464  PMID: 22537858
9.  Comparative analysis of collagen type II-specific immune responses during development of collagen-induced arthritis in two B10 mouse strains 
Arthritis Research & Therapy  2012;14(6):R237.
Immune responses against collagen type II (CII) are crucial for the development of collagen-induced arthritis (CIA). The aim of the present study was to evaluate and compare the CII-directed T cell and antibody specificity at different time points in the course of CIA using two mouse strains on the B10 genetic background - B10.Q, expressing Aq MHC class II molecules, and B10.DR4.Ncf1*/*, expressing human rheumatoid arthritis-associated MHC II DR4 molecules (DRA*0101/DRB*0401).
B10.Q and B10.DR4.Ncf1*/* mice were immunized with CII emulsified in adjuvant and development of CIA was assessed. T cells from draining lymph nodes were restimulated in vitro with CII peptides and interferon-gamma (IFN-γ) levels in culture supernatants were evaluated by ELISA. CII-specific antibody levels in serum samples were measured by ELISA.
At four different CIA time points we analyzed T cell specificity to the immunodominant CII epitope 259-273 (CII259-273) and several posttranslationally modified forms of CII259-273 as well as antibody responses to three B cell immunodominant epitopes on CII (C1, U1, J1). Our data show that CII-specific T and B cell responses increase dramatically after disease onset in both strains and are sustained during the disease course. Concerning anti-CII antibody fine specificity, during all investigated stages of CIA the B10.Q mice responded predominantly to the C1 epitope, whereas the B10.DR4.Ncf1*/* mice also recognized the U1 epitope. In the established disease phase, T cell reactivity toward the galactosylated CII259-273 peptide was similar between the DR4- and the Aq-expressing strains whereas the response to the non-modified CII peptide was dramatically enhanced in the DR4 mice compared with the B10.Q. In addition, we show that the difference in the transgenic DR4-restricted T cell specificity to CII259-273 is not dependent on the degree of glycosylation of the collagen used for immunization.
The present study provides important evaluation of CII-specific immune responses at different phases during CIA development as well as a comparative analysis between two CIA mouse models. We indicate significant differences in CII T cell and antibody specificities between the two strains and highlight a need for improved humanized B10.DR4 mouse model for rheumatoid arthritis.
PMCID: PMC3674594  PMID: 23116329
10.  The role of lipopolysaccharide injected systemically in the reactivation of collagen-induced arthritis in mice 
British Journal of Pharmacology  2000;129(7):1309-1314.
We investigated the role of bacterial lipopolysaccharide (LPS) in the reactivation of autoimmune disease by using collagen-induced arthritis (CIA) in mice in which autoimmunity to the joint cartilage component type II collagen (CII) was involved.CIA was induced by immunization with CII emulsified with complete Freund's adjuvant at the base of the tail (day 0) followed by a booster injection on day 21. Varying doses of LPS from E. coli were i.p. injected on day 50.Arthritis began to develop on day 25 after immunization with CII and reached a peak on day 35. Thereafter, arthritis subsided gradually but moderate joint inflammation was still observed on day 50. An i.p. injection of LPS on day 50 markedly reactivated arthritis on a dose-related fashion. Histologically, on day 55, there were marked oedema of synovium which had proliferated by the day of LPS injection, new formation of fibrin, and intense infiltration of neutrophils accompanied with a large number of mononuclear cells. The reactivation of CIA by LPS was associated with increases in anti-CII IgG and IgG2a antibodies as well as various cytokines including IL-12, IFN-γ, IL-1β, and TNF-α. LPS from S. enteritidis, S. typhimurium, and K. neumoniae and its component, lipid A from E. coli also reactivated the disease. Polymyxin B sulphate suppressed LPS- or lipid A-induced reactivation of CIA.These results suggest that LPS may play an important role in the reactivation of autoimmune joint inflammatory diseases such as rheumatoid arthritis in humans.
PMCID: PMC1571961  PMID: 10742285
Lipopolysaccharide; collagen-induced arthritis; cytokines; autoimmune disease; rheumatoid arthritis
11.  Involvement of RORγt-overexpressing T cells in the development of autoimmune arthritis in mice 
Differentiation of T helper 17 cells is dependent on the expression of transcription retinoid-related orphan receptor gamma t (RORγt). The purpose of our study is to determine the role of RORγt expression in T cells on the development of collagen-induced arthritis (CIA).
CIA was induced in C57BL/6 and T cell-specific RORγt transgenic (RORγt Tg) mice. At day 10 post-1st-immunization, lymph node (LN) cells were cultured with type II collagen (CII), and the expression levels of various cytokines and transcription factors on CD4+ T cells were measured. Total cells or CD4+ cells of draining LN were harvested from each mouse group after CII-immunization and transferred into C57BL/6 mice, and then CIA was induced in recipient mice. The expression levels of RORγt and other surface antigens, and the production of cytokines were analyzed in forkhead box P3 (Foxp3)+ regulatory T (Treg) cells. Foxp3+ Treg cells were analyzed for suppressive activity against proliferation of effector CD4+ T cells. Interlukin (IL)-10 neutralizing antibody was administrated in the course of CIA.
CIA was significantly suppressed in RORγt Tg mice compared with C57BL/6 mice. RORγt expression and IL-17 production were significantly higher in CII-reactive CD4+ T cells from RORγt Tg mice. Arthritis was significantly attenuated in C57BL/6 mice recipient of cells from RORγt Tg mice. Most of Foxp3+ Treg cells expressed RORγt, produced IL-10 but not IL-17, and overexpressed CC chemokine receptor 6 (CCR6) and surface antigens related to the suppressive activity of Foxp3+ Treg cells in RORγt Tg mice. In vitro suppression assay demonstrated significant augmentation of the suppressive capacity of Foxp3+ Treg cells in RORγt Tg mice. CIA was exacerbated in both C57BL/6 mice and RORγt Tg mice by the treatment of anti-IL-10 antibody.
Our results indicated that RORγt overexpression in T cells protected against the development of CIA. The protective effects were mediated, at least in part, through the anti-inflammatory effects including high production of IL-10 of RORγt+Foxp3+ Treg cells.
PMCID: PMC4436146  PMID: 25928901
12.  A Mouse Model of Adoptive Immunotherapeutic Targeting of Autoimmune Arthritis Using Allo-Tolerogenic Dendritic Cells  
PLoS ONE  2013;8(10):e77729.
Tolerogenic dendritic cells (tDCs) are immunosuppressive cells with potent tolerogenic ability and are promising immunotherapeutic tools for treating rheumatoid arthritis (RA). However, it is currently unknown whether allogeneic tDCs (allo-tDCs) induce tolerance in RA, and whether the numbers of adoptively transferred allo-tDCs, or the requirement for pulsing with relevant auto-antigens are important.
tDCs were derived from bone marrow precursors of C57BL/B6 mice, which were induced in vitro by GM-CSF, IL-10 and TGF-β1. Collagen-induced arthritis (CIA) was modeled in D1 mice by immunization with type II collagen (CII) to test the therapeutic ability of allo-tDCs against CIA. Clinical and histopathologic scores, arthritic incidence, cytokine and anti-CII antibody secretion, and CD4+Th subsets were analyzed.
tDCs were characterized in vitro by a stable immature phonotype and a potent immunosuppressive ability. Following adoptive transfer of low doses (5×105) of CII-loaded allo-tDCs, a remarkable anti-arthritic activity, improved clinical scores and histological end-points were found. Serological levels of inflammatory cytokines and anti-CII antibodies were also significantly lower in CIA mice treated with CII-pulsed allo-tDCs as compared with allo-tDCs. Moreover, treatment with allo-tDCs altered the proportion of Treg/Th17 cells.
These findings suggested that allo-tDCs, especially following antigen loading, reduced the severity of CIA in a dose-dependent manner. The dampening of CIA was associated with modulated cytokine secretion, Treg/Th17 polarization and inhibition of anti-CII secretion. This study highlights the potential therapeutic utility of allo-tDCs in autoimmune arthritis and should facilitate the future design of allo-tDC immunotherapeutic strategies against RA.
PMCID: PMC3812020  PMID: 24204938
13.  Helminth Antigens Enable CpG-Activated Dendritic Cells to Inhibit the Symptoms of Collagen-induced Arthritis through Foxp3+ Regulatory T Cells 
PLoS ONE  2012;7(7):e40356.
Dendritic cells (DC) have the potential to control the outcome of autoimmunity by modulating the immune response. In this study, we tested the ability of Fasciola hepatica total extract (TE) to induce tolerogenic properties in CpG-ODN (CpG) maturated DC, to then evaluate the therapeutic potential of these cells to diminish the inflammatory response in collagen induced arthritis (CIA). DBA/1J mice were injected with TE plus CpG treated DC (T/C-DC) pulsed with bovine collagen II (CII) between two immunizations with CII and clinical scores CIA were determined. The levels of CII-specific IgG2 and IgG1 in sera, the histological analyses in the joints, the cytokine profile in the draining lymph node (DLN) cells and in the joints, and the number, and functionality of CD4+CD25+Foxp3+ T cells (Treg) were evaluated. Vaccination of mice with CII pulsed T/C-DC diminished the severity and incidence of CIA symptoms and the production of the inflammatory cytokine, while induced the production of anti-inflammatory cytokines. The therapeutic effect was mediated by Treg cells, since the adoptive transfer of CD4+CD25+ T cells, inhibited the inflammatory symptoms in CIA. The in vitro blockage of TGF-β in cultures of DLN cells plus CII pulsed T/C-DC inhibited the expansion of Treg cells. Vaccination with CII pulsed T/C-DC seems to be a very efficient approach to diminish exacerbated immune response in CIA, by inducing the development of Treg cells, and it is therefore an interesting candidate for a cell-based therapy for rheumatoid arthritis (RA).
PMCID: PMC3405066  PMID: 22848374
14.  Impact of Schistosoma japonicum Infection on Collagen-Induced Arthritis in DBA/1 Mice: A Murine Model of Human Rheumatoid Arthritis 
PLoS ONE  2011;6(8):e23453.
The hygiene hypothesis suggests that helminth infections prevent a range of autoimmune diseases.
Methodology/Principal Findings
To investigate the effects of S. japonicum infection on collagen-induced arthritis (CIA), male DBA/1 mice were challenged with unisexual or bisexual S. japonicum cercariae two weeks prior to bovine type II collagen (CII) immunization or at the onset of CIA. S. japonicum infection prior to CII immunization significantly reduced the severity of CIA. ELISA (enzyme linked immunosorbent assay) showed that the levels of anti-CII IgG and IgG2a were reduced in prior schistosome-infected mice, while anti-CII IgG1 was elevated. Splenocyte proliferation against both polyclonal and antigen-specific stimuli was reduced by prior schistosome infection as measured by tritiated thymidine incorporation (3H-TdR). Cytokine profiles and CD4+ T cells subpopulation analysis by ELISA and flow cytometry (FCM) demonstrated that prior schistosome infection resulted in a significant down-regulation of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β and IL-6) and Th1 cells, together with up-regulation of the anti-inflammatory cytokine IL-10 and Th2 cells. Interestingly, the expansion of Treg cells and the reduction of Th17 cells were only observed in bisexually infected mice. In addition, prior schistosome infection notably reduced the expression of pro-inflammatory cytokines and receptor activator of NF-κB ligand (RANKL) in the inflamed joint. However, the disease was exacerbated at one week after infection when established CIA mice were challenged with bisexual cercariae.
Our data provide direct evidence that the Th2 response evoked by prior S. japonicum infection can suppress the Th1 response and pro-inflammatory mediator and that bisexual infection with egg-laying up-regulates the Treg response and down-regulates the Th17 response, resulting in an amelioration of autoimmune arthritis. The beneficial effects might depend on the establishment of a Th2-dominant response rather than the presence of the eggs. Our results suggest that anti-inflammatory molecules from the parasite could treat autoimmune diseases.
PMCID: PMC3152573  PMID: 21858123
15.  Anti-Inflammatory Effects of Recombinant Human PDCD5 (rhPDCD5) in a Rat Collagen-Induced Model of Arthritis 
Inflammation  2014;38(1):70-78.
Programmed cell death 5 (PDCD5) was first identified as a gene upregulated in cells undergoing apoptosis. We recently demonstrated the inhibitory effect of PDCD5 on experimentally induced autoimmune encephalomyelitis. In this study, we investigated the anti-inflammatory effects of recombinant human PDCD5 (rhPDCD5) in a rat collagen-induced arthritis (CIA) model. We find that vaccination of collagen II (CII) induced CIA rats with rhPDCD5 significantly delayed the occurrence and reduced the severity of CIA rats. rhPDCD5 also restored the loss of Foxp3+ regulatory T (Treg) cells and decreased the population of Th1 and Th17 in CIA rats. Simultaneously, rhPDCD5 treatment suppressed the production of pro-inflammatory cytokines (interleukin (IL)-6, IL-17A, tumor necrosis factor-α (TNF-α), and interferon gamma (IFN-γ)) and increased the secretion of anti-inflammatory cytokines (transforming growth factor beta 1 (TGF-β1) and IL-10) in CIA rats. In addition, rhPDCD5 inhibited the ability of CII to induce proliferation of splenocytes and lymph node cells (LNCs) and promoted the CII-activated CD4+ cell apoptosis. These results of rhPDCD5-treated CIA rats were similar with those of recombinant human TNF-α receptor IgG Fc (rhTNFR:Fc). Thus, to our knowledge, we provide the first evidence that rhPDCD5 may be an efficient approach to diminishing exacerbated immune responses in CIA, indicating its therapeutic potential in the treatment of rheumatoid arthritis and other autoimmune diseases.
PMCID: PMC4312386  PMID: 25178696
PDCD5; regulatory T cells; collagen-induced arthritis; Th17; Th1
16.  IL-23 Dependent and Independent Stages of Experimental Arthritis: No Clinical Effect of Therapeutic IL-23p19 Inhibition in Collagen-induced Arthritis 
PLoS ONE  2013;8(2):e57553.
IL-23p19 deficient mice have revealed a critical role of IL-23 in the development of experimental autoimmune diseases, such as collagen-induced arthritis (CIA). Neutralizing IL-23 after onset of CIA in rats has been shown to reduce paw volume, but the effect on synovial inflammation and the immunological autoimmune response is not clear. In this study, we examined the role of IL-23 at different stages of CIA and during T cell memory mediated flare-up arthritis with focus on changes in B cell activity and Th1/Th17 modulation. Anti-IL-23p19 antibody (anti-IL23p19) treatment, starting 15 days after the type II collagen (CII)-immunization but before clinical signs of disease onset, significantly suppressed the severity of CIA. This was accompanied with significantly lower CII-specific IgG1 levels and lower IgG2a levels in the anti-IL-23p19 treated mice compared to the control group. Importantly, neutralizing IL-23 after the first signs of CIA did not ameliorate the disease. This was in contrast to arthritic mice that underwent an arthritis flare-up since a significantly lower disease score was observed in the IL-23p19 treated mice compared to the control group, accompanied by lower synovial IL-17A and IL-22 expression in the knee joints of these mice. These data show IL-23-dependent and IL-23-independent stages during autoimmune CIA. Furthermore, the memory T cell mediated flare-up arthritis is IL-23-mediated. These data suggest that specific neutralization of IL-23p19 after onset of autoimmune arthritis may not be beneficial as a therapeutic therapy for patients with rheumatoid arthritis (RA). However, T cell mediated arthritis relapses in patients with RA might be controlled by anti-IL-23p19 treatment.
PMCID: PMC3585376  PMID: 23469022
17.  Consumption of Hydrolyzable Tannins Rich Pomegranate Extract (POMx) Suppresses Inflammation and Joint Damage In Rheumatoid Arthritis 
Although consumption of dietary supplements containing pomegranate extract by arthritis patients is on the rise, efficacy of such preparations in suppressing joint inflammation and damage is not known. The present study was designed to evaluate a standardized preparation of pomegranate extract (POMx) using collagen-induced arthritis in mice (CIA)-a widely used animal model of rheumatoid arthritis (RA).
CIA susceptible DBA/1 mice were fed POMx by gavage before and after immunization with chicken type-II collagen (CII). Severity of clinical arthritis was scored using a visual scoring system. Arthritic joints were analyzed by histopathology and graded. Lysates were generated from mouse joints and the levels of anti-type-II collagen IgG and inflammatory cytokines IL-1β, IL-6 and TNF-α were quantified by ELISA. Effect of POMx on LPS-induced NO production was determined by Griess reaction and MAP kinase activation was studied by Western immunoblotting in mouse macrophages.
Consumption of POMx potently delayed the onset and reduced the incidence of CIA in mice. Severity of arthritis was also significantly lower in POMx -fed animals. Histopathology of the arthritic joints from POMx-fed mice demonstrated reduced joint infiltration by the inflammatory cells and the destruction of bone and cartilage were alleviated. Levels of IL-6 were significantly decreased in the joints of POMx-fed mice with CIA. In mouse macrophages, POMx abrogated multiple signal transduction pathways and downstream mediators implicated in RA pathogenesis.
Our studies suggest that inhibition of a spectrum of signal transduction pathways and the downstream pathogenic cellular response by POMx or compounds derived from it may be a useful approach for the prevention of onset and severity of inflammatory arthritis.
PMCID: PMC2577876  PMID: 18490140
Pomegranate; Collagen-induced arthritis; Rheumatoid Arthritis; IL-6; TNF-α
18.  Deletion of Fcγ Receptor IIB Renders H-2b Mice Susceptible to Collagen-induced Arthritis  
Autoimmune diseases, like rheumatoid arthritis, result from a dysregulation of the immune response culminating in hyperactivation of effector cells leading to immune-mediated injury. To maintain an appropriate immune response and prevent the emergence of autoimmune disease, activation signals must be regulated by inhibitory pathways. Biochemical and genetic studies indicate that the type IIB low-affinity receptor for immunoglobulin (Ig)G (FcγRIIB) inhibits cellular activation triggered through antibody or immune complexes and may be an important component in preventing the emergence of autoimmunity. To investigate the role of FcγRIIB in the development of type II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans, we have examined its contribution in determining the susceptibility to CIA in the nonpermissive H-2b haplotype. H-2b mice immunized with bovine CII do not develop appreciable disease. In contrast, immunization of the FcγRIIB-deficient, H-2b mice with bovine CII induced CIA at an incidence of 42.2%. The maximal arthritis index of the FcγRIIB-deficient mice developing CIA (6.9 ± 3.6) was comparable to that of DBA/1 mice (8.6 ± 1.9), an H-2q strain susceptible for CIA induction. IgG1, IgG2a, and IgG2b antibody responses against CII were elevated in the FcγRIIB-deficient animals, especially in those mice showing arthritis, but less pronounced than DBA/1 mice. Histological examinations of the arthritic paws from FcγRIIB-deficient mice revealed that cartilage was destroyed and bone was focally eroded in association with marked lymphocyte and monocyte/macrophage infiltration, very similar to the pathologic findings observed in DBA/1 mice. These results indicate that a nonpermissive H-2b haplotype can be rendered permissive to CIA induction through deletion of FcγRIIB, suggesting that FcγRIIB plays a critical role in suppressing the induction of CIA.
PMCID: PMC1887699  PMID: 9874575
collagen-induced arthritis; autoimmunity; Fc receptor; gene targeting; macrophage
19.  TNF receptor gene therapy results in suppression of IgG2a anticollagen antibody in collagen induced arthritis 
Annals of the Rheumatic Diseases  2003;62(8):707-714.
Background: Therapeutic strategies to block tumour necrosis factor α (TNFα) activity in experimental autoimmune arthritis models and rheumatoid arthritis (RA) have proved highly successful, and provide sustained beneficial effects.
Objective: To examine whether TNFα inhibition has immunological activity beyond the reduction of inflammation in collagen induced arthritis (CIA), an established experimental model of RA.
Methods: Arthritic DBA/1 mice received single periarticular injections of retroviral constructs encoding human TNF receptor (TNF-R) into the affected arthritic paw, at the onset of arthritis. Severity of arthritis, antibodies to collagen type II (CII), and extent of pathological joint damage of arthritic paws were compared between TNF-R and media treated (control) animals 3, 7, 14, 21, and 49 days after disease onset.
Results: Severity of CIA was significantly decreased in TNF-R treated animals compared with controls, 14–34 days after disease onset. Joint destruction was reduced in TNF-R injected joints and in the uninjected contralateral and ipsilateral paws of TNF-R treated animals. Seven days after disease onset, TNF-R treated mice had lower levels of inflammatory Th1 driven IgG2a antibodies to CII (p<0.05) than controls. This altered the anticollagen IgG2a:IgG1 ratio towards Th2 driven IgG1.
Conclusions: Local TNF-R gene therapy in CIA appears to have systemic effects on the anti-CII antibodies. The overall influence of TNF-R gene therapy is that it inhibits the progression of CIA mainly by suppressing the inflammatory Th1 response rather than by stimulating a Th2 response. Therefore, periarticular TNF-R gene therapy may have excellent therapeutic potential in RA.
PMCID: PMC1754640  PMID: 12860724
20.  Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4 
Arthritis Research  2000;2(4):293-302.
To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9].
In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset.
Materials and methods:
Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index.
Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.
PMCID: PMC17812  PMID: 11056670
adenoviral vectors; collagen-induced arthritis; gene therapy; IL-4; IL-10; rheumatoid arthritis
21.  Enhanced expression of genes involved in coagulation and fibrinolysis in murine arthritis 
Arthritis Research  2000;2(6):504-512.
We have analyzed the pattern of procoagulant and fibrinolytic gene expression in affected joints during the course of arthritis in two murine models. In both models, we found an increased expression of tissue factor, tissue factor pathway inhibitor, urokinase plasminogen activator, and plasminogen activator inhibitor 1, as well as thrombin receptor. The observed pattern of gene expression tended to favor procoagulant activity, and this pattern was confirmed by functional assays. These alterations would account for persistence of fibrin within the inflamed joint, as is seen in rheumatoid arthritis.
Accumulation of fibrin in the joints remains one of the most striking histopathological features of rheumatoid arthritis (RA). Recently, we have provided evidence of the deleterious role of synovial fibrin deposition in arthritic joints in antigen-induced arthritis (AIA), a well-established murine model of RA.
A local imbalance between fibrin formation and fibrin dissolution may result in fibrin deposition in the joints.
On the one hand, fibrin formation is mainly initiated by tissue factor (TF), a transmembrane protein serving as a receptor for factor VII. Under normal conditions, TF expression and activity are tightly regulated. Constitutive TF expression is restricted to perivascular and epithelial cells, and the catalytic activity of the TF/VIIa complex can be inhibited by tissue factor pathway inhibitor (TFPI). Pathological conditions can perturb the cell-type-restricted pattern of TF expression. In particular, recent reports have shown that transcriptional activation of TF can be mediated by molecular mechanisms involving induction of the early growth response gene 1 (EGR1) or of the protease-activated receptor (PAR1) or vascular endothelial growth factor (VEGF) genes.
On the other hand, fibrin degradation is mediated primarily by plasmin, which is the active form of the zymogen plasminogen. Conversion of plasminogen to plasmin is under the control of serine protease plasminogen activators, such as the urokinase plasminogen activator (uPA), and their inhibitors, such as the plasminogen activator inhibitor (PAI-1).
We hypothesized that the deposition of fibrin in the joints may result from an imbalance in the local expression of key genes involved in coagulation and fibrinolytic pathways. To test this hypothesis, we investigated mRNA levels in arthritic versus nonarthritic joint tissues from two murine models of RA: AIA and collagen-induced arthritis (CIA). Genes that are directly implicated in coagulation (TF, TFPI) and fibrinolysis (UPA, PAI1), and other genes that may influence the expression of TF (EGR1, PAR1, VEGF), were investigated using a novel multiprobe RNase protection assay (RPA). Furthermore, we evaluated coagulation activity in arthritic and nonarthritic mice.
Mice with AIA or CIA were sacrificed at different time points: 2, 4, and 16 h and 3, 7, and 14 d after intra-articular antigen injection for AIA; 42 d after the first immunization for CIA. Total RNA was prepared from arthritic and nonarthritic knees for AIA, or arthritic and nonarthritic hind paws for CIA. Messenger RNA (mRNA) levels of the genes described above were determined by RPA and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. Coagulation assays were performed on joint tissue extracts and concentrations of thrombin-antithrombin III (TAT) complex were measured in plasma.
In AIA, all the genes studied except VEGF were upmodulated as early as 2 h. PAR1, TFPI, EGR1, and UPA expression decreased to control levels by 16 h, whereas the expression of TF and PAI1 remained elevated. At later times, only TF, PAI1, and UPA showed sustained overexpression. In CIA, gene expression was assayed at only one time point (42 d after immunization) and all genes showed higher mRNA levels in the affected paws than in control paws. In AIA mice, procoagulant activity and TF activity were significantly increased in arthritic joints, and in CIA mice, plasma TAT levels were significantly enhanced.
Fibrin deposition in synovia is prominent in both RA and experimental arthritis, suggesting that this protein may play a role in the pathogenesis of chronic inflammation. In this study, we have tried to shed some light on the molecular mechanisms leading to extravascular fibrin deposition, using two well-established mouse models of RA: AIA and CIA. The kinetics of gene expression was first analyzed in mice with AIA, because this model allows for an accurate, temporally controlled sampling of synovial inflammation. We then extended our observations by analyzing one time point in CIA, 42 d after immunization, when chronic inflammation is present. We found that in both models, coagulation and fibrinolysis in arthritic joints were significantly increased, and that the most significant increases were in TF and PAI-1.
Although the molecular mechanism or mechanisms responsible for the transcriptional changes observed are not completely understood, the increases in TF, PAI-1, and uPA are probably due to the production of proinflammatory cytokines such as IL-1 and TGF-α. These cytokines, whose presence in the inflamed synovium is well documented, are known to induce these genes through the activation of nuclear factor κB (NF-κB), a transcription factor. TF induction is also under the control of a proximal enhancer containing a binding site for the inducible transcription factor EGR1. Indeed, the early rise of EGR1 expression in AIA is consistent with its classification as immediate-early gene and may be responsible for the induction of early expression of TF. Early TF stimulation in AIA can also be accounted for by the transient overexpression of PAR1. Contrary to what has been shown in RA, VEGF expression remained essentially unchanged throughout the progression of AIA, probably reflecting a peculiarity of this murine model.
The alteration of the patterns of gene expression was accompanied by increased functional coagulation activity, which was more marked in AIA than in CIA.
Prominent fibrin deposition in two different animal models of RA – AIA and CIA – can be attributed to modulations in key regulatory genes for coagulation and fibrinolysis.
PMCID: PMC17822  PMID: 11056680
arthritis; coagulation; fibrinolysis; mice; RNase protection
22.  Bifunctional Peptide Inhibitors Suppress Interleukin-6 Proliferation and Ameliorates Murine Collagen-Induced Arthritis 
The objective of this study is to evaluate the efficacy and potential mechanism of action of type-II collagen bifunctional peptide inhibitor (CII-BPI) molecules in suppressing rheumatoid arthritis in the collagen-induced arthritis (CIA) mouse model. CII-BPI molecules (CII-BPI-1, CII-BPI-2, and CII-BPI-3) were formed through conjugation between an antigenic peptide derived from type-II collagen and a cell adhesion peptide LABL (CD11a237-246) from the I-domain of LFA-1 via a linker molecule. The hypothesis is that the CII-BPI molecules simultaneously bind to MHC-II and ICAM-1 on the surface of APC and block maturation of the immunological synapse. As a result, the differentiation of naïve T cells is altered from inflammatory to regulatory and/or suppressor T cells. The efficacies of CII-BPI molecules were evaluated upon intravenous injections in CIA mice. Results showed that CII-BPI-1 and CIIBPI-2 suppressed the joint inflammations in CIA mice in a dose-dependent manner and were more potent than the respective antigenic peptides alone. CII-BPI-3 was not as efficacious as CII-BPI-1 and CII-BPI-2. Significantly less joint damage was observed in CII-BPI-2 and CII-2 treated mice than in the control. The production of IL-6 was significantly lower at the peak of disease in mice treated with CII-BPI-2 compared to those treated with CII-2 and control. In conclusion, this is the first proof-of-concept study showing that BPI molecules can be used to suppress RA and may be a potential therapeutic strategy for the treatment of rheumatoid arthritis.
PMCID: PMC4524745  PMID: 26251760
Autoimmunity; Rheumatoid arthritis; Bifunctional peptide inhibitors; T cells; Interleukin-6
23.  Tumour necrosis factor receptor gene therapy affects cellular immune responses in collagen induced arthritis in mice 
Annals of the Rheumatic Diseases  2005;64(11):1550-1556.
Background: Collagen induced arthritis (CIA) is an animal model of rheumatoid arthritis (RA) amenable to immunotherapy directed against tumour necrosis factor α (TNFα).
Objective: To evaluate whether local TNF receptor (TNF-R) gene therapy in DBA/1 mice exerts an influence beyond anti-inflammatory effects. Two measures of CIA pathogenesis were investigated—namely, immunity to collagen II (CII) 245–270 peptide (the major immunodominant epitope within bovine CII) and the preferential activation of T cell Vß8.2 variable region receptors in arthritic DBA/1 mice.
Methods: DBA/1 mice received single periarticular injections of media or retroviral vectors containing LacZ or human TNF-R into affected arthritic paws at disease onset. Disease severity was monitored, immune responses towards the immunodominant bovine CII 245–270 and subdominant CII 334–360 peptide epitopes were assessed by ELISA, and T cell Vß usage was analysed by real time polymerase chain reaction for the LacZ transduced, TNF-R, and viral-free media treated control animals. The therapeutic influence of TNF-R gene transduction was compared with other groups at different times after treatment.
Results: Reduced disease severity was seen 15–35 days after treatment, with a concomitant increase in immunity towards the subdominant CII 334–360 peptide epitope rather than the immunodominant CII 245–270 peptide in TNF-R treated animals. Early in the disease, TNF-R treated animals demonstrated a reduction of bias towards the otherwise predominant Vß8.2 T cell subset.
Conclusions: TNF-R gene therapy influences cellular immunity in CIA, leading to overall disease amelioration, thus suggesting that TNF inhibition may have therapeutic potential beyond the control of inflammation in RA.
PMCID: PMC1755256  PMID: 15860512
24.  Porphyromonas gingivalis oral infection exacerbates the development and severity of collagen-induced arthritis 
Arthritis Research & Therapy  2013;15(6):R186.
Clinical studies suggest a direct influence of periodontal disease (PD) on serum inflammatory markers and disease assessment of patients with established rheumatoid arthritis (RA). However, the influence of PD on arthritis development remains unclear. This investigation was undertaken to determine the contribution of chronic PD to immune activation and development of joint inflammation using the collagen-induced arthritis (CIA) model.
DBA1/J mice orally infected with Porphyromonas gingivalis were administered with collagen II (CII) emulsified in complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (IFA) to induce arthritis. Arthritis development was assessed by visual scoring of paw swelling, caliper measurement of the paws, mRNA expression, paw micro-computed tomography (micro-CT) analysis, histology, and tartrate resistant acid phosphatase for osteoclast detection (TRAP)-positive immunohistochemistry. Serum and reactivated splenocytes were evaluated for cytokine expression.
Mice induced for PD and/or arthritis developed periodontal disease, shown by decreased alveolar bone and alteration of mRNA expression in gingival tissues and submandibular lymph nodes compared to vehicle. P. gingivalis oral infection increased paw swelling and osteoclast numbers in mice immunized with CFA/CII. Arthritis incidence and severity were increased by P. gingivalis in mice that received IFA/CII immunizations. Increased synovitis, bone erosions, and osteoclast numbers in the paws were observed following IFA/CII immunizations in mice infected with P gingivalis. Furthermore, cytokine analysis showed a trend toward increased serum Th17/Th1 ratios when P. gingivalis infection was present in mice receiving either CFA/CII or IFA/CII immunizations. Significant cytokine increases induced by P. gingivalis oral infection were mostly associated to Th17-related cytokines of reactivated splenic cells, including IL-1β, IL-6, and IL-22 in the CFA/CII group and IL-1β, tumor necrosis factor-α, transforming growth factor-β, IL-6 and IL-23 in the IFA/CII group.
Chronic P. gingivalis oral infection prior to arthritis induction increases the immune system activation favoring Th17 cell responses, and ultimately accelerating arthritis development. These results suggest that chronic oral infection may influence RA development mainly through activation of Th17-related pathways.
PMCID: PMC3979094  PMID: 24456966
25.  Attenuation of Collagen-Induced Arthritis in Mice by Salmon Proteoglycan 
BioMed Research International  2014;2014:406453.
Rheumatoid arthritis (RA) is a serious autoimmune disease caused by chronic inflammation of connective tissues. The basic principle of RA treatment is aimed to reduce joint inflammation. Our previous studies demonstrated that salmon cartilage proteoglycan (PG) suppresses excess inflammation in different mouse inflammatory diseases. In this study, we investigated the prophylactic effect of PG on the progression of RA using an experimental mouse model, collagen-induced arthritis (CIA). Clinical and histological severity of CIA was attenuated by daily oral administration of PG. In the joints of PG-administered mice, infiltration of macrophages and neutrophils and also osteoclast accumulation were limited. In comparison to nonadministered mice, anti-collagen antibodies in the sera of PG-administered mice did not alter. On the other hand, local expression of interleukin-17A (IL-17A), IL-6, IL-1β, interferon-γ (IFN-γ), C-C chemokine ligand 2 (CCL2), C-X-C chemokine ligand 1 (CXCL1), and CXCL2 in the joints of PG-administered mice decreased. Moreover, in the response of type II collagen- (CII-) restimulation ex vivo, IL-17A and IFN-γ production by splenocytes from PG-administered mice was less than that of control mice. These data suggested that daily ingested PG attenuated CIA pathogenesis by modulating immune response of splenocytes to CII stimulation and local production inflammatory cytokines and chemokines in the joints.
PMCID: PMC4054874  PMID: 25032213

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