Prevention of experimental autoimmune arthritis with a peptide fragment of type II collagen

Collagen arthritis is induced in inbred rats with the injection of native type II collagen. The pathogenesis of this experimental autoimmune disease is T cell dependent. This study demonstrates that collagen-specific Tcells, derived from pathogenic and nonpathogenic rat Tcell lines, both recognize the same peptide epitope. The epitope, consisting of amino acids 58–73 of cyanogen bromide fragment 11 of type II collagen, was as effective as whole collagen in stimulating a panel of collagen-specific rat/mouse Tcell hybridomas. This peptide may, therefore, constitute a dominant epitope for CD4⁺ rat Tcells in their response to type II collagen. Administration of the peptide to either neonatal or adult rats prevented the subsequent induction of experimental arthritis with whole collagen, demonstrating that the in vivo response to this dominant epitope is, therefore, relevant in the pathogenesis of arthritis. Despite its ability to prevent collagen-induced arthritis, administration of this peptide in incomplete Freund's adjuvant intradermally did not induce disease.     

Polyarthritis in MRL-lpr/lpr mice: mouse type II collagen is antigenic but not arthritogenic

In addition to a lupus-like syndrome and massive T cell proliferation, MRL-lpr/lpr(MRL/l) mice develop an arthritic process very similar serologically and histologically to human rheumatoid arthritis (RA). Recently, we have developed in DBA/1 mice an experimental model of autoimmune arthritis (EAA) which shares clinical features with RA, by injecting homologous type II collagen (CII). In order to investigate the possible relationship between the spontaneous polyarthritis of MRL/l mice and collagen induced EAA, we immunized MRL/l mice with mouse (M) CII. Our findings revealed that the injection of 100 micrograms M-CII in young or old MRL/l mice did not modify the articular pathology which spontaneously develops in non-injected mice. Circulating autoantibodies to native M-CII were found in the sera of immunized young mice but were not detected in non injected or immunized old mice. Conversely, denatured alpha 1 (II) chains or CB peptides derived from M-CII were recognized by most of the MRL/l sera whether mice had been immunized or not. The incidence of positive sera as well as the intensity of the response evaluated by Western blot analysis increased with the age of the mice. Taken together, our data suggest that, even if the injection of homologous CII in MRL/l mice may accelerate the onset of joint pathology, the spontaneous disease arises independently of an autoimmune response against native CII.     

Identification and characterization of a major tolerogenic T-cell epitope of type II collagen that suppresses arthritis in B10.RIII mice.

Tolerization of B10.RIII mice (H-2r) with intravenously injected type II collagen (CII) renders the animals resistant to induction of collagen-induced arthritis (CIA). In order to clarify H-2r-restricted T-cell responses that modulate CIA, we have analysed the T-cell proliferative response of B10.RIII mice against cyanogen bromide (CB) peptides of CII, and detected the strongest response to alpha 1(II)-CB10 (CII 552-897). A panel of chemically synthesized overlapping peptide homologues was used to deduce the minimum structure of this determinant which was found to be CII 610-618. A 15-residue synthetic peptide flanking this region, CII 607-621, was found to effectively suppress arthritis when administered as a tolerogen. Collectively, these data identify the structural component within alpha 1(II)-CB10 which is capable of inducing tolerance in B10.RIII mice. A similar approach to the treatment of autoimmune arthritis, involving the institution of self-tolerance, has potential applicability to human rheumatoid arthritis.     

Immunogenetics of type II collagen autoimmunity and susceptibility to collagen arthritis.

The MHC restriction of the antibody response and development of arthritis after immunization with autologous or heterologous type II collagens in mice have been investigated. Mice from three different H-2q-carrying strains (DBA/1, NFR/N and B10.G) with different non-MHC genes, as well as B10-congenic strains carrying wild type H-2q-related or H-2r haplotypes, were susceptible for collagen arthritis. All strains tested developed an antibody response cross-reacting with autologous type II collagen after immunization with heterologous type II collagen; H-2q predisposes for a high response against chick, rat and bovine type II collagen, H-2r and H-2b for a high response against bovine type II collagen. Only mouse strains with H-2q, H-2r, H-2w3 or H-2w17 were responders to mouse type II collagen, and only these strains developed arthritis after immunization with heterologous or autologous type II collagens. These findings indicate that the ability to mount an immune response against autologous type II collagen is a prerequisite for the susceptibility to collagen arthritis. A cross-reactive autoimmune response after immunization with various heterologous type II collagen may enhance further the development of arthritis.     

Studies on type II collagen induced arthritis in mice

A consistent and reproducible polyarthritis was induced in mice by immunizing them with type II collagen in Complete Freunds adjuvant (CFA) and Bacillus Calmette-Guerin (BCG) vaccine. Several inbred strains of mice were investigated for the ability to develop collagen induced arthritis (CIA). DBA/1 mice (H-2q) produced the highest incidence and the most severe arthritis of all the strains examined. Viable BCG vaccine was essential for the induction of a reproducible disease in this strain. The effects of some anti-inflammatory and anti-rheumatic compounds were examined on the developing and established lesions of CIA. These effects were determined by assessing the paw inflammation using a subjective scoring system and measuring foot weight. Furthermore, levels of serum amyloid P component (SAP) were also determined.Benoxaprofen, cyclophosphamide, indomethacin and prednisolone inhibited the paw inflammation in the developing disease whilst the anti-rheumatic compounds auranofin and D-penicillamine exacerbated the paw inflammation. Cyclophosphamide and prednisolone inhibited the established lesions but only prednisolone prevented the development of further lesions in the established disease. The SAP levels in the prednisolone treated group were also reduced. Auranofin treatment exacerbated the inflammation of both the established and the developing lesions in the same animal. D-penicillamine was inactive in the established disease.     

A pH-induced modification of CII increases its arthritogenic properties

Immunoreactivity to collagen type II (CII) has been implicated in the pathogenesis of rheumatoid arthritis. Patients have been described to have an acidic pH in their inflamed synovial tissue. It is known that protein structures are modified by environmental pH, thus it is plausible that changes in synovial pH could affect the conformation of proteins like CII. Posttranslational modifications could alter the biophysical properties of cartilage proteins leading to autoimmunity. In this study we investigated if arthritogenicity of CII was affected by changes in pH, and if so, this could be correlated to altered protein conformation. Immunisation with CII at neutral pH induced a milder disease than did CII at acidic pH. All animals elicited a humoral response to CII, although with a significantly higher IgG1/IgG2b-ratio in the pH 7.4 group. Analysis by circular dichroism and electron microscopy indicated less fibrillation of CII at low pH as compared to neutral pH. Our results suggest that CII is more immunogenic and arthritogenic in an acidic environment than in a neutral environment. We can correlate these findings to pH-induced conformational changes of CII. Hence, self-tolerance to CII might be affected by changes in pH leading to altered and increased arthritogenicity.     

Molecular characterisation of type II collagen from chick sternal cartilage and its anti-rheumatoid arthritis activity

Type II collagen (CII) was purified from chick sternal cartilage using a combination of pepsin digestion, NaCl precipitation and ion exchange chromatography. Following, the effect of purified CII on the collagen-induced rat arthritis (CIA) model was investigated. Circular dichroism spectral and atomic force microscopy analysis indicated that the purified CII retained its intermolecular cross-links during the preparation process. Compared with the control group, oral administration of 600 µg/kg CII over a period of 35 days markedly decreased the index of arthritis (30.98%) and suppressed paw swelling (20.28%) in CIA rats. Furthermore, CII treatment also dose-dependently reduced the serum level of anti-CII antibody and inhibited the over-production of inflammatory cytokines levels (tumour necrosis factor α, interleukin 1 β and interferon γ) in CIA rats. In conclusion, CII extracted from chick sternal cartilage possesses anti-rheumatoid arthritis activity, which may be a result of its regulation of the humoral and cellular immune systems.     

Prevention of collagen-induced arthritis (CIA) by treatment with polyethylene glycol-conjugated type II collagen; distinct tolerogenic property of the conjugated collagen from the native one

Administration of a soluble protein into animals prior to challenge immunization induces immunological tolerance which is specific for the protein. In addition, chemical modification of proteins with polyethylene glycol (PEG) has been reported to convert the immunogenic proteins to become tolerogenic. However, differences in tolerogenic properties between PEG-modified proteins and the native counterparts have never been analysed. The ability of PEG-conjugated type II collagen (PEG-CII) to attenuate CIA, an animal model for rheumatoid arthritis, was compared with the native unconjugated CII. Groups of DBA/1 J mice were treated weekly with i.p. injections with PEG-CII, native CII, or vehicle alone for 3 weeks, before they were challenged with CII in adjuvants. The induction of tolerance was confirmed in both PEG-CII- and CII-pretreated mice when suppression of lymph node T cell proliferation in response to CII was noted. The degrees of suppression of T cell proliferation were comparable between the two pretreated groups. However, induction of arthritis and production of IgG anti-CII antibody were more markedly suppressed in PEG-CII-pretreated mice than in native CII-pretreated mice, although the severity of arthritis and antibody levels in the latter group were also lower than in control mice. IgG2a and IgG2b antibody levels were equally suppressed in the two pretreated groups, whereas the IgG1 level was significantly lower in the PEG-CII-pretreated group than in the native CII-pretreated group. The results provide the first evidence that attachment of PEG to CII renders the protein more tolerogenic.     

Immunity to type IX collagen in rodents: a study of type IX collagen for autoimmune and arthritogenic activities

Type IX collagen (CIX), a cartilage-specific glycoprotein, constitutes ≤ 10% of cartilage collagen. To ascertain whether CIX can induce arthritis as shown for type II and XI collagen (CII and CXI), outbred rats were sensitized with bovine, chick and human CIX; inbred rats, mice, and guinea pigs were sensitized with bovine CIX. Mice and guinea pigs proved resistant to arthritis, as did rats sensitized with CIX/Freund's incomplete adjuvant (FIA). Arthritis was seen in rats when 100 μg of Mycobacterium tuberculosis (Mtb) were added to FIA, but seldom with smaller doses of Mtb, suggesting the arthritis was adjuvant-induced. High levels of antibodies to rat CIX, containing complement-fixing subclasses, were detected in rat sera in addition to DTH and lymphocyte proliferation responses to rat CIX. Given the potential for CIX-induced disease, CIX-sensitized rats were injected intraperitoneally with lipopolysaccharide (LPS) to stimulate proinflammatory cytokine release, and intra-articularly with rat CIX to stimulate arthritis. LPS stimulation was ineffective; however, intra-articularly injected CIX produced transient synovitis. When rats with stable adjuvant arthritis were sensitized with CIX/FIA, significant increases in paw volume were measured compared with controls given CI/FIA. Immunohistochemical studies of actively and passively sensitized rats revealed deposits of CIX antibody, but not C3, at the joint margins where proteoglycan staining was weak. Together, these findings suggest that autoimmunity to CIX, in contrast to CII and CXI, is not directly pathogenic but may contribute to joint injury provided arthritis is initiated by an independent disease process.     

Collagen antibody-induced arthritis in mice: Development of a new arthritogenic 5-clone cocktail of monoclonal anti-type II collagen antibodies

A cocktail of 4 monoclonal anti-type II collagen antibodies recognizing conserved epitopes located within the CB11 fragment (CII 124-402) of type II collagen is currently used as an arthritogenic antibody preparation for inducing collagen antibody induced arthritis (CAIA). In order to increase the arthritogenicity of this cocktail, we have developed 7 new monoclonal antibodies to anti-type II collagen from spleen cells of DBA/1J mice immunized with bovine type II collagen, and tested for their additional effect on the arthritogenicity over that of the current 4-clone cocktail. Three of the clones (CII-3, -5 and -6) bind to the LyC1 (CII 124-290) peptide of CB11 and 1 (CII-7) of the clones binds to CB9.7 (CII 898-1020), and highly cross-reacted with other species of type II collagen. This indicates that these clones recognize conserved epitopes within type II collagen, including mouse type II collagen. On the other hand, 2 other clones (CII-1 and -4) directed against CB9.7 and 1 clone (CII-2) against CB8 (CII 403-551) were less reactive with other species of type II collagen.The arthritogenicity of the current 4-clone cocktail was significantly increased by addition of a fifth clone, CII-3. No effects were observed with other clones. The arthritogenicity of this new 5-clone cocktail was 2-fold greater than the current 4-clone cocktail in all strains of mice tested: the CIA-responder strain DBA/1J, the CIA-resistant BALB/c (H-2^d), the T-cell deficient C.B-17/l scid/scid and the CAIA-low responder C57BL/6 (H-2^b) strain. These results clearly indicated the importance of epitope specificity of arthritogenicity of autoantibodies to type II collagen. Due to its enhanced arthritogenicity, this 5-clone cocktail is capable of inducing a more consistent and severe arthritis with lower doses compared to the current 4-clone cocktail, and will provide an effective new reagent for inducing arthritis in various strains of CAIA low responder mice.     

Assessment of drugs for activity in established type II collagen arthritis

Arthritis was induced in a proportion of rats sensitized with type II collagen and Freund's incomplete adjuvant. Rats which developed arthritis had significantly higher antibody titres and significantly greater delayed hypersensitivity responses to type II collagen than rats which did not develop arthritis. Anti-inflammatory and anti-rheumatic drugs were evaluated against type II collagen arthritis. Dexamethasone reduced inflammatory swelling, reduced both the antibody titre and delayed hypersensitivity to type II collagen and exerted joint protection. Indomethacin reduced inflammatory swelling. Azathioprine, Clozic, Levamisole,D-Penicilamine and Sulphapyridine did not exert significant beneficial activity in this test.     

Pattern of humoral reactivity to type II collagen in rheumatoid arthritis

Humoral immunity directed against type II collagen (CII) is a common although not specific feature of rheumatoid arthritis (RA). We have shown that 10 to 15% of the sera either from RA patients (n = 88) or from healthy controls (n = 149) reacted with native human CII. Conversely, autoantibodies to the alpha-1 (II) chains were significantly more frequent in the RA group (26.1% versus 6.0%, P<0.001), suggestingthatdenaturedCII may bean autoantigenin RA. Thus, human CII was cleaved with cyanogen bromide (CB), and immunoblotting techniques were performed on 19 RA and 21 normal sera. Among the four major CB peptides, CB10 and CB11 were recognized by most of the sera tested without distinction between normal or RA sera. Inhibition experiments using an ELISA have shown that: (i) antibodies to the native CII molecule did not cross-react with those recognizing the CB peptides, and vice-versa; (ii) the binding of the sera to native CII was partially inhibited by pre-incubation with alpha-1 (II) chains, and vice-versa; (iii) pre-incubation of the sera with CB peptides partially blocked the binding to alpha-1 (II) chains, whereas pre-incubation of the sera with alpha-1 (II) chains totally inhibited the reactivity against CB peptides; and (iv) a substantial proportion of the epitopes recognized by anti-CII autoantibodies was neither species specific nor type specific. Taken together, these findings reveal the existence of several populations of anti-CII autoantibodies: some antibodies react exclusively with conformational determinants of the CII molecule, and others are directed towards linear structures of alpha-1 (II) chains.     

Denaturation of type II collagen in articular cartilage in experimental murine arthritis. Evidence for collagen degradation in both reversible and irreversible cartilage damage

Degradation of type II collagen is thought to be a key step in the destruction of articular cartilage in patients with rheumatoid arthritis or osteoarthritis. The aim of this study was to investigate whether type II collagen degradation is associated with cartilage destruction. Type II collagen degradation was studied in two murine arthritis models, zymosan-induced arthritis (ZIA), which develops reversible articular cartilage damage based on proteoglycan analysis, and antigen-induced arthritis (AIA), in which there is irreversible damage to the cartilage. Type II collagen degradation was assayed immunohistochemically using the COL2-3/4m antibody which recognizes denatured type II collagen, such as is produced by collagenase cleavage. In both models, degradation of type II collagen was observed in the non-calcified articular cartilage of arthritic but not of control knees. In the patella-femoral compartment, collagen denaturation started to increase on day 3 (ZIA) and day 7 (AIA) and remained high on day 14. In contrast, in the tibia-femoral compartment, type II collagen breakdown was not increased before 14 days in either model. By 28 days, collagen denaturation was strongly reduced in the patella-femoral compartment in the ZIA model, but persisted in the tibia-femoral compartment in both models. In conclusion, increased type II collagen degradation was found in articular cartilage of both ZIA and AIA animals. Since ZIA does not develop irreversible cartilage destruction, this indicates that cartilage may have the ability to withstand a limited degree of type II collagen degradation without developing irreversible damage. Copyright © 1999 John Wiley & Sons, Ltd.     

Enhancing effects of tilorone on collagen arthritis and humoral immune response to type II collagen

The effect of tilorone, which is known to suppress adjuvant arthritis, on the induction of collagen arthritis in rats was investigated. Combined data of the present experiments show that all of the tilorone-treated rats except one in the lowest dosage group developed arthritis but that the incidence of arthritis in the tilorone-treated groups was not significantly different from that of the control group. The results also show that the two higher dosages (12.5 and 25 mg/kg/day) of tilorone caused a significant increase in the severity of collagen arthritis. Humoral immune response to type II collagen was significantly augmented in these two higher dosage groups; however, delayed-type hypersensitivity response to type II collagen was suppressed while tilorone was administered continuously. In addition, treatment with tilorone caused a significant increase in the concentration of anticollagen IgG extractable from the joint tissue. Anticollagen IgG subclass analysis revealed that the major subclass was IgG2a in both the serum and paw extract, with minor amounts of IgG2b, IgG2c, and IgG1. The response of all these subclasses was almost equally activated by tilorone treatment.     

Adjuvant arthritis pretreatment with type II collagen and Mycobacterium butyricum.

A treatment previous to adjuvant arthritis induction has been performed with type II collagen (CII) or Mycobacterium butyricum (Mb), which is the inducer of the pathology. Pretreatment was administered in two different ways: a) subcutaneously or intradermally 14 days before arthritis induction, and b) intravenously 3 days before induction. In order to relate the change in inflammation to the corresponding antigen immune response, serum antibodies and delayed type hypersensitivity (DTH) against CII or Mb were studied. Pretreatment with s.c. CII 14 days before induction produced slight protection against arthritis and significantly delayed its onset; systemic inflammation showed good positive correlation with anti-CII antibodies. The CII administered i.v. 3 days before arthritic challenge did not significantly modify the inflammatory process. The use of i.d. subarthritogenic doses of Mb 14 days before induction protected a high percentage of the animals from the posterior arthritic challenge; this protection was accompanied by high anti-Mb antibody titers and DTH reaction. When Mb was given i.v. 3 days before induction, a partial protection of inflammation was observed; arthritis was milder and its onset was delayed. These changes were accompanied by reduced humoral and cellular response to Mb.     

Adjuvant polyarthritis. VIII. Differences in immunopathogenesis between type II collagen arthritis and adjuvant arthritis

The severity of type II collagen-induced arthritis was found to correlate with the serum tiers of anti-type II collagen antibody, but not with cell-mediated immunity to type II collagen. In contrast, no significant levels of either the humoral or the cell-mediated immunity to type II collagen were found in ras with Freund's complete adjuvant-induced arthritis. Pre-treatment of young rats with an oily preparation of type II collagen prevented the development of arthritis in these animals in response to a subsequent injection of oily preparation of type II collagen, but had no effect on the development of arthritis in response to a subsequent injection of Freund's complete adjuvant. It is concluded that while an immune response directed toward the injected type II collagen is responsible for the development of type II collagen arthritis, it does not play an important role in the induction of Freund's adjuvant-induced arthritis.This work was supported by US Public Health Service, National Institutes of Health Grant AM 26571.     

Suppression of adjuvant arthritis by antibodies specific for collagen type II

Immunization of Lewis rats with an alum flocculate of collagen type II, prior to the induction of arthritis by an intradermal injection of Mycobacterium butyricum in oil, caused reduced arthritic responses when compared with non-pretreated control animals. The majority of collagen-immunized animals that expressed diminished disease possessed low levels of serum antibody to collagen type II whereas most rats with undiminished disease did not. Passive immunization of Lewis rats with antibody to collagen type II also effectively reduced the severity of adjuvant-induced arthritis. Two possible mechanisms that might explain suppression of this disease by antibodies to collagen type II are discussed.     

An altered peptide ligand of type II collagen suppresses autoimmune arthritis

On the basis of the hypothesis that immunity to type II collagen (CII) contributes to joint inflammation, our goal is to develop an immunotherapy capable of selectively blocking immunity to a particular autoantigen without interfering with the beneficial functions of the immune system. CII is the major protein component of articular cartilage and autoimmunity to CII is strongly associated with rheumatoid arthritis in man. Our laboratory has previously identified a region of type II collagen (CII), CII245-270 that contains a prominent T-cell epitope in the immune response to CII. Residues critical to the I-Aq-restricted presentation of this determinant have been characterized. When synthetic analog peptides were developed that contain site-directed substitutions in critical positions, we found that that CII245-270 (A260, B261, N263) (A9), profoundly suppressed collagen-induced arthritis. When DBA/1 mice were coimmunized with CII and the analog peptide, the incidence and severity of arthritis was greatly reduced concordant with the humoral immune responses to CII. Moreover, the suppression could be transferred with A9-immune spleen cells and was accompanied by a Th2-type cytokine profile. When we compared T-cell signals in response to A9 to those of wild-type (WT) peptide, we found that APCs prepulsed with WT peptide induced strong phosphorylation of both TCR zeta chain and Zap-70, while A9 did not. Since T cells clearly respond to A9 with cytokine secretion, we hypothesize that A9 induces an alternate signaling pathway and we speculate that this pathway involves phosphorylation of Syk, a kinase ordinarily utilized by B cells. Activation of this alternative pathway is a novel observation and may represent an important means by which the phenotype of the responding T cell is altered. Elucidation of the mechanism by which A9 prevents arthritis may lead to development of novel immunotherapeutic approaches to antigen specific treatment of autoimmunity.     

口服Ⅱ型胶原蛋白诱导免疫耐受治疗佐剂关节炎的研究

目的： 观察口服Ⅱ型胶原蛋白（ｔｙｐｅＩＩｃｏｌｌａｇｅｎ, ＣＩＩ） 对大鼠佐剂关节炎（ａｄｊｕｖａｎｔａｒｔｈｒｉｔｉｓ, ＡＡ） 的治疗作用。方法： 采用酶消化法, 提取牛软骨ＣＩＩ, 经口插管灌注将ＣＩＩ给予佐剂关节炎ＳＤ大鼠。结果： 口服ＣＩＩ可推迟ＡＡ的发病, 降低发病率, 明显减轻病变关节的炎症反应和使病程缩短。结论： 口服ＣＩＩ可诱导抗原特异性免疫耐受并对淋巴细胞的增殖反应有抑制作用, 而且此种免疫耐受作用可以剂量依赖的方式通过淋巴细胞转移。     

Oestrogen exhibits type II collagen protective effects and attenuates collagen-induced arthritis in rats

As anti-inflammatory treatments used in rheumatoid arthritis, such as glucocorticoids, often result in secondary detrimental effects on bone health, the objective of this study was to investigate the effects of oestrogen therapy (ET) on the development and activity of collagen-induced arthritis (CIA) in rats, with a focus on assessment of chondroprotective effects using biomarkers of type II collagen degradation. Forty female Lewis rats were allocated into four intervention groups: (i) control + vehicle; (ii) CIA + vehicle; (iii) CIA + ET; and (iv) CIA + prednisolone. During the 28-day intervention period we monitored body weight, time-point of disease onset, incidence of manifest disease and paw volume. Levels of the type II collagen degradation marker (CTX-II) were measured in serum. At euthanasia, hind paws were isolated, extracted for proteins and measured for the concentration of CTX-II. Matrix metalloproteinase (MMP) activity was evaluated using gelatinase zymography. Oestrogen treatment delayed the time-point of disease onset and reduced the incidence and degree of manifest immunoarthritis significantly, assessed by macroscopic evaluation of hind paw inflammation and paw volume. Measures of serum or tissue levels of CTX-II showed significantly reduced type II collagen degradation elicited by oestrogen treatment. In alignment, a decreased activity of MMP-2 and MMP-9 was found in the paw protein extracts. We have demonstrated that the anti-inflammatory effect of ET is linked to chondroprotective effects in an animal model of systemic immunoarthritis. As ET has positive rather than negative effects on bone health in contrast to prednisolone, these observations may be important for potential combination therapy.