The effects of interleukin-18 on rat articular chondrocytes: a study of mRNA expression and protein synthesis of proinflammatory substances.

Interleukin (IL)-18 is a potent stimulator of immunity and augments the severity of type II collagen-induced arthritis (CIA) in rats and mice by enhancing T helper 1 (Th1) cell activation, which increases the production of proinflammatory cytokines and arthritogenic antibodies. In this study, we show that recombinant IL-18 (rIL-18) also has a direct effect on normal rat chondrocytes maintained in vitro inducing them to produce proinflammatory factors including IL-6, regulated upon activation normal T cell expressed and secreted (RANTES), prostaglandin E(2) (PGE(2)) and prostaglandin F(2alpha) (PGF(2alpha)) in a dose- and time-dependent manner. The production of matrix metalloproteinase (MMP)-13, nitric oxide (NO), tumour necrosis factor (TNF)-alpha and IL-1beta were also enhanced, although less intensely. Neutralizing polyclonal anti-rIL-18 antibodies effectively blocked the production of IL-6, PGE(2) and RANTES, as well as mRNA expression for the same products in addition to IL-18 and TNF-alpha. In contrast, neutralizing antibodies to IL-1beta, TNF-alpha and IL-6 were ineffective in suppressing any of these products. Together, these findings suggest that IL-18 may play an important, possibly direct, role in mediating cartilage injury, which might not be amenable to treatment with currently utilized anti-cytokine agents. These findings suggest further that IL-18 antagonists might prove beneficial as anti-inflammatory and chondroprotective agents in the treatment of arthritis, and that the development of such agents for human use is worth consideration.

The roles of interleukin-18 in collagen-induced arthritis in the BB rat.

Interleukin (IL)-18 is a member of the IL-1 cytokine family. Its expression is increased in rheumatoid arthritis synovium, and its proinflammatory effects have been demonstrated in experimental models of murine arthritis. Here, we investigate the actions of varying doses of recombinant rat IL-18 (rIL-18) on the course of type II collagen-induced arthritis (CIA) in BB rats, including clinical and immune events, plus splenic cytokine production. Small doses of rIL-18 (10 and 50 microg/rat) administered intraperitoneally (i.p.) increased arthritis incidence and severity (P < 0.01) when a low-potency CII preparation was used for immunization. IgG1 and IgG2a anti-CII antibody levels were significantly greater in rats given 10 and 50 microg rIL-18 doses than controls. rIL-18 significantly increased levels of proinflammatory cytokines [interferon (IFN)-gamma, IL-2, tumour necrosis factor (TNF)-alpha and IL-6] produced by splenocyte cultures. Larger doses of rIL-18 (300 microg/rat) suppressed arthritis and immunity. To ascertain whether the pro-arthritic effects of IL-18 could be attenuated, rats were treated with neutralizing rabbit anti-rIL-18 IgG before immunization with a high-potency CII preparation. When given serially for 3 weeks, the incidence and severity of CIA, in addition to anti-CII IgG2a and splenic IL-6 and IFN-gamma production, were all significantly reduced. Similar results were noted when antibody was given twice, just before arthritis onset. These results demonstrate that IL-18 plays an important proinflammatory role in the pathogenesis of CIA which is achieved, in part, by an immunostimulatory action. Neutralizing endogenous IL-18 with antibodies attenuated CIA, CII immunity and cytokine responses. These studies support the use of IL-18 antagonists as treatments for inflammatory arthritis.

Immunogenicity of recombinant type IX collagen in murine collagen-induced arthritis.

OBJECTIVE: Past attempts to isolate type IX collagen (CIX) from cartilage using limited proteolysis yielded partially degraded material. Recent application of recombinant technology, however, has allowed the preparation of intact native CIX. We used the murine collagen-induced arthritis model to characterize the immunologic properties of recombinant human CIX (rCIX) produced using a baculovirus expression system. METHODS: A panel of B10 congenic mice was immunized with rCIX emulsified with Freund's complete adjuvant (CFA). The ability of the rCIX to induce tolerance and suppress arthritis was determined by administration intravenously or orally before challenge with CII/CFA. RESULTS: None of the mice immunized with rCIX developed overt arthritis, although 2 of 5 HLA-DR1 transgenic mice developed limited digital erythema and swelling. Recombinant CIX administered by either route effectively induced suppression of arthritis, although the suppression was less pronounced than that induced with CII. Immune responses to CIX and CII were specific, suggesting that bystander suppression, rather than cross-reactivity with CII, was instrumental in suppressing arthritis. CONCLUSION: These data show that CIX down-regulates arthritis in mice while having no associated risk of inducing arthritis.

Lack of efficacy of oral bovine type II collagen added to existing therapy in rheumatoid arthritis.

OBJECTIVE: To investigate the efficacy of oral type II collagen (CII) in the treatment of rheumatoid arthritis (RA), when added to existing therapy. METHODS: Patients with active RA (n = 190) were randomized into a 6-month, double-blind, placebo-controlled trial. Patients continued to take their current arthritis medications. Patients received either placebo or bovine CII, 0.1 mg/day for 1 month, then 0.5 mg/day for 5 months. RESULTS: There were no significant differences between the baseline characteristics of either group. The primary response parameter was the American College of Rheumatology (ACR) preliminary definition of improvement in RA (ACR 20). There was no statistically significant difference in the ACR 20 after 6 months (20.0% of placebo patients; 16.84% of bovine CII patients). There were significant differences in several clinical variables after treatment, all favoring the placebo group. CONCLUSION: Oral solubilized bovine CII, added to existing therapy, did not improve disease activity in patients with RA.

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 < or = 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 microg 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.

Evidence for preferential T cell receptor V beta gene usage and T cell clonal expansion in the synovium of BB rats with early-onset collagen-induced arthritis.

Type II collagen (CII) is a potent arthritogen in the BB rat. To determine whether a restricted group of T cells is involved in the pathogenesis of collagen-induced arthritis, lymphocytes from synovium, peripheral blood, and lymph nodes of arthritic rats were studied for T cell receptor (TCR) V beta gene usage using polymerase chain reaction (PCR). Oligoclonal TCR V beta usage was found only in synovium recovered day 2 post-arthritis onset, but not day 7; lymph node and peripheral blood T cells showed diverse TCR usage at both times. To determine whether T cell local clonal expansion occurred in synovium at day 2 of arthritis, cDNA for four TCR beta families was sequenced through VDJ regions. Strong selective expansion of TCR V beta 8.2, 4, and 17 was noted. Importantly, the dominant clonotype of V beta 8.2 was identical to that of a lymph node-derived T hybridoma specific for the immunodominant epitope in CII(181-210). Cells from synovium (day 2 postonset) analyzed by flow cytometry also showed V beta 8.2+ cell enrichment. These observations, plus finding that T cells from inflamed synovium respond to CII(181-201) in vitro, suggest the local recruitment and clonal expansion of some T cells families, possibly driven by autologous CII released during cartilage degradation.

The cartilage collagens: a review of their structure, organization, and role in the pathogenesis of experimental arthritis in animals and in human rheumatic disease.

This contribution reviews the structure and organization of collagen molecules found in cartilage and the roles that they may play in rheumatic diseases. Cartilage is unique in its physical properties and molecular composition, and contains sufficient amounts of types II, IX, X, and XI collagen to deem these molecules as "cartilage-specific." The vitreous body of the eye, a "cartilage-like" tissue is also rich in the same collagens but is type X deficient. Types VI and XII collagen are present in cartilage as well as noncartilaginous tissues. Types II, IX, and XI collagen are organized into matrix fibrils, where type II constitutes the bulk of the fibril, type XI regulates fibril size, and type IX facilitates fibril interaction with proteoglycan macromolecules. Genetic defects in these collagens can produce mild to severe developmental abnormalities, including spondyloepiphyseal dysplasia often accompanied by an accelerated form of osteoarthritis. Sensitization with collagen can produce experimental rheumatic diseases. Type II collagen induces an erosive polyarthritis in certain strains of rats, mice, and higher primates which can resemble rheumatoid arthritis and relapsing polychondritis. Type XI collagen is arthritogenic in rats but not mice; type IX induces autoimmunity in both species but not arthritis. Arthritis is initiated by complement fixing antibodies that bind to type II collagen in autologous cartilage, and the production of these antibodies is MHC restricted and T cell dependent. It is unclear whether T cells alone can induce arthritis, although they probably help sustain it. Mapping and characterizing the of T cell epitopes on type II collagen has resulted in the synthesis of small homolog and substituted peptides of type II collagen which suppress arthritis in an antigen-specific manner by a variety of routes, including mucosal. Moreover, collagen-induced arthritis has proven a valuable model to study the contribution of cytokines and other biological agents in the pathogenesis of joint injury and how they might be used to develop new therapies. Collagen autoimmunity has been implicated in the pathogenesis rheumatoid arthritis and polychondritis. Circulating antibodies to type II collagen are found in both diseases. Antibodies to types IX and XI collagen are also present in rheumatoid sera but are less prevalent. Rheumatoid cartilage and synovium contain antibodies to type II collagen at a prevalence far greater than serum, suggesting an intra-articular antigen-driven immune process. Although effective in animal models, attempts to treat rheumatoid arthritis with orally administered type II collagen have proven elusive. Different approaches using newer formulations and selected or modified oligopeptides remain to be tested and could prove effective in the treatment of the human rheumatic diseases.

Characterization of recombinant type II collagen: arthritogenicity and tolerogenicity in DBA/1 mice.

Recombinant human type II collagen (rhCII) was produced using both the HT1080 mammalian cell expression system (rhCIIht) and a baculovirus expression system (rhCIIbac). The biosynthesis of CII requires extensive post-translational modifications, such as the hydroxylation of prolyl and lysyl residues and glycosylation of hydroxylysyl residues. Amino acid analyses indicated that the rhCIIbac was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived hCII, while rhCIIht was hyperhydroxylated and hyperglycosylated at lysyl residues. When the murine collagen-induced arthritis (CIA) model was used to investigate the immunological properties of the two forms of recombinant CII, each induced a high incidence of arthritis following immunization of susceptible mice when emulsified with complete Freund's adjuvant (CFA). However, the severity of the arthritis, as assessed by the number of affected limbs, was significantly higher in mice immunized with rhCIIht than in mice immunized with rhCIIbac. These data indicate that the degree of hydroxylysine glycosylation may play a role in the induction of the arthritogenic response to CII. Each of the recombinant collagens was comparable to tissue-derived hCII in their ability to induce tolerance and suppress arthritis when given as intravenous or oral tolerogens. Taken together, our data suggest that recombinant CII can be prepared in adequate amounts for therapeutic uses and that the material is immunologically comparable to tissue-derived hCII when used to induce tolerance.

Collagen-induced arthritis, an animal model of autoimmunity.

Collagen induced arthritis (CIA) is an autoimmune model that in many ways resembles rheumatoid arthritis (RA). Immunization of genetically susceptible strains of rodents and primates with type II collagen (CII) leads to the development of a severe polyarticular arthritis that is mediated by an autoimmune response. Like RA, synovitis and erosions of cartilage and bone are hallmarks of CIA, and susceptibility to both RA and CIA is linked to the expression of specific MHC class II molecules. Although not identical to RA, CIA clearly establishes the biological plausibility that an autoimmune reaction to a cartilage component can lead to a chronic, destructive, polyarthritis. Although it is induced in susceptible animals by immunization with heterologous CII, it is the autoreactive component of the immune response that leads to disease. A wealth of evidence indicates that synovitis is initiated by the production of pathogenic autoreactive antibodies capable of fixing and activating complement. The elucidation of the specific amino acid sequences of collagen that are recognized by the MHC molecules has enabled at least two approaches to specific immunotherapy to be considered. Firstly, small synthetic peptides representing dominant epitopes have been used as effectively as the original antigen as a tolerogen. The rather fastidious physicochemical properties of collagen that make it difficult for its routine use in therapy are thereby circumvented by the use of oligopeptides. Secondly, analysis of the specific amino acid side chains that are involved in MHC contact and TCR recognition enables analog peptides to be devised which can specifically and exquisitely inhibit the response to CII, preventing the onset of arthritis. Further investigations involving this model may contribute to the development of specific immunotherapies in the human disorder.

Induction by chronic autoimmune arthritis in DBA/1 mice by oral administration of type II collagen and Escherichia coli lipopolysaccharide.

We have developed a new model of autoimmune arthritis in DBA/1 mice by feeding chick type II collagen (CII) for 2-3 week intervals over a 15 week period. Clinically evident arthritis occurred in 8/10 mice receiving native CII (nCII; 100 micrograms/mouse) alone at 9-13 weeks. Arthritis was aggravated by the further ingestion of CII, while remission occurred after withdrawal of the CII. Heat-denatured CII (dCII; 200 micrograms/mouse) was also arthritogenic if co-administered with ovoinhibitor (OVI; 2 mg/mouse), a proteinase inhibitor. Co-oral administration of lipopolysaccharide (LPS; 10 micrograms/mouse) with CII enhanced the antibody production and T-cell responses to CII, and induced a more chronic arthritis that progressed spontaneously without further administration of CII or LPS. Long-term oral administration of LPS alone also induced a mild arthritis characterized by destruction of bone rather than cartilage. These observations suggest that abnormal gastrointestinal absorption of dietary mimic antigens and intestinal bacterial toxins can potentially disrupt self-tolerance mechanisms, thereby precipitating or exacerbating autoimmune disease in genetically susceptible individuals.

The mechanism of autoantibody formation to cartilage in rheumatoid arthritis: possible cross-reaction of antibodies to dietary collagens with autologous type II collagen.

In order to study the mechanism of autoantibody formation to type II collagen in rheumatoid arthritis (RA), IgG and IgA antibodies in sera from 259 RA patients and 285 non-RA controls were evaluated for their specificity as to collagen type (I and II) and species (chick, bovine, and porcine) using an improved enzyme-linked immunosorbent assay. IgG and IgA anti-type II collagen antibodies were commonly found in both RA (IgG, 41%; and IgA, 45%) and non-RA (IgG, 36%; and IgA, 31%) sera. Both IgG and IgA collagen antibodies were highly reactive with one or more heterologous type II or type I collagen; however, approximately 35% of IgG and 50% of IgA antibody-positive sera from both RA patients and non-RA controls cross-reacted with human type II collagen (HII) to some degree. However, no antibodies specific to HII were observed in either RA or control sera. In individual patient sera, IgG and IgA antibodies had identical collagen-type and species specificities. Importantly, IgG anti-HII antibodies purified from RA sera by affinity chromatography reacted equally with human, chick and bovine type II collagens, suggesting reactivity with conserved epitopes shared by all three species. In contrast, purified IgG anti-HII antibodies from non-RA control sera commonly lacked reactivity with one or the other of the heterologous type II collagens, suggesting reactivity limited to epitopes shared by HII and only one of the heterologous type II collagens. These data suggest that dietary collagens could elicit circulating IgG and IgA anti-collagen antibodies that cross-react with autologous type II collagen. Also the epitope specificity of IgG autoantibodies may be relevant to the pathogenesis of RA.

Variable-region gene family usage for type II collagen autoantibodies in arthritis-susceptible DBA/1 mice.

Collagen-induced arthritis is mediated by autoantibodies to type II collagen (CII). This experimental model has proven useful in determining the molecular and cellular mechanisms responsible for autoimmune arthritis. We have shown that polyarthritis can be transferred to normal mice by administering combinations of three or four complement-fixing monoclonal antibodies (mAbs) which recognize cross-reactive epitopes on the alpha 1(II)-CB11 region of chick and mouse CII. Currently, the light- and heavy-chain variable-region structures on a panel of alpha 1 (II)-CB11-specific mAbs that cross-react with chick and mouse CII, or react solely with chick CII, have been analyzed. The results indicate biased usage of VK19 and VK21 families of light-chain variable-region genes but random VH gene usage. Interestingly, two mAbs derived from different mice recognized identical epitopes on mouse CII and had nearly identical light- and heavy-chain variable-region structure including junctionally derived sequence.

Collagen-induced arthritis in mice: synergistic effect of E. coli lipopolysaccharide bypasses epitope specificity in the induction of arthritis with monoclonal antibodies to type II collagen.

DBA/1 mice develop a chronic peripheral arthritis after immunization with type II collagen termed collagen-induced arthritis. We have localized the main arthritogenic determinants of CB11, a CNBr-generated arthritogenic fragment of chick type II collagen (CII), using 3 smaller peptide fragments of CB11 generated by endoproteinase LysC, LysC1 (CII 124-290), LysC2 (CII 291-374) and LysC3 (CII 375-402) and a panel of monoclonal antibodies (mAb) specific to CB11. MAb specific to the arthritogenic region of CB11 were also used to study the synergistic effect of E. coli lipopolysaccharide (LPS) on antibody-mediated arthritis in naive DBA/1 mice. LysC2 contained a minimum essential arthritogenic fragment of type II collagen: LysC2 induced arthritis by active immunization, also, a combination of four mAb specific to LysC2 passively transferred arthritis to naive mice. A single i.p. injection of LPS (50 micrograms/mouse) reduced the threshold values of the arthritogenic dose of mAb from 1 mg to 50 micrograms/clone per mouse, and decreased the number of mAb required for inducing arthritis from 4 to 2 clones. These observations suggest that LysC2, an 84 amino acid residue fragment, contains the main arthritogenic determinants within chick CB11. Importantly, LPS, a strong inducer of pro-inflammatory cytokines, negates the required multiple epitope specificity of autoantibodies in the passive transfer model and acts synergistically in the induction of arthritis by autoantibody.

Type XI and II collagen-induced arthritis in rats: characterization of inbred strains of rats for arthritis-susceptibility and immune-responsiveness to type XI and II collagen.

To determine the relationship between susceptibility to bovine type XI and II (BXI and BII) collagen-induced arthritis, we immunized 14 inbred and one outbred strains of rats with BXI and BII. Susceptibility to BXI-arthritis corresponded largely with susceptibility, or resistance, to BII-arthritis. LEW, BB, WF, DA, and WKY were readily susceptible to BXI- and BII-arthritis. Likewise, BII-resistant F344 and BN rats were BXI-resistant. Some strains responded differently to BXI and BII. BUF and COP, which are moderately susceptible to BII, were BXI-resistant, whereas the BII-resistant rats, DA.1N and WF.1N, were partially susceptible to BXI. (F344 x BN) F1 hybrids responded to both collagens suggesting gene complementation. Arthritis occurred in all strains producing the highest titer antisera (LEW, WF and BB). Antibody responses to BXI and BII were generally commensurate within individual strains. DA were susceptible to arthritis but produced low levels of antibody comparable to BN rats which were arthritis-resistant. BXI and BII-susceptibility was variable in rats producing intermediate antibody responses. Antibodies to RXI were detected in all BXI-immunized rats, whereas antibodies to RV and RII were uniformly weaker. DTH to RXI and RII was strong in both groups of rats, correlating poorly with arthritis and antibody responses. These studies show that phenotypic susceptibility to BXI- and BII-arthritis are largely concordant among inbred rat strains but clear differences exist in certain strains; multiple genes are likely involved.

Type XI collagen-induced arthritis in the Lewis rat. Characterization of cellular and humoral immune responses to native types XI, V, and II collagen and constituent alpha-chains.

Autoimmune arthritis can be induced in rats by immunization with cartilage-specific types II and XI collagen. Type XI collagen is composed of alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains, in which alpha 3(XI) is essentially identical to alpha 1(II) of type II, and alpha 1(XI) and alpha 2(XI) are similar to alpha 1(V) and alpha 2(V) of type V collagen. To characterize the immune response to type XI collagen, Lewis rats were injected with bovine type XI collagen, and the cellular and humoral responses were compared with those of rats injected with types V and II collagen. Arthritis, IgG deposits in cartilage, and joint destruction were seen in rats immunized with types XI and II collagen. Type XI elicited strong cellular responses to rat types XI, V, and II; conversely, types II and V collagen elicited strong responses to rat type XI. Antitype XI Abs reacted with rat type XI, moderately with rat type V, but poorly with rat type II. Direct and inhibition ELISA showed that cross-reactions between types XI and V collagen resulted from recognition of determinants shared by their respective alpha 2(XI) and alpha 1(V) chains. Abs eluted from joints of rats immunized with type XI collagen, however, reacted only with native rat type XI collagen. These data demonstrate that type XI collagen induces diverse populations of Abs differing in collagen-type specificity, and suggest that only those Abs to native rat type XI collagen are central to the pathogenesis of type XI collagen-induced arthritis.

Collagen-induced arthritis in rats. Examination of the epitope specificities of circulating and cartilage-bound antibodies produced by outbred and inbred rats using cyanogen bromide-derived peptides purified from heterologous and homologous type II collagens.

To determine the number and location of antibody binding epitopes on type II collagen, outbred and inbred rats were immunized with chick, bovine, human, and rat type II collagen (CII, BII, HII, and RII); all sera were assayed for reaction with a panel of CB peptides purified and renatured from the immunizing collagen and from RII. Antibody reaction patterns (profiles) varied among individual outbred rats but were essentially constant over time and changed little after boosting. The strongest antibody reactions were to CB11, CB9-7, and CB12 followed by CB8, CB10, and CB6. Antibody profiles varied depending on the species of collagen used for immunization and the strain of rat immunized. Except for CB10, where antibodies were largely specific for heterologous collagens, antibodies reactive with all other CB peptides cross-reacted strongly with renatured rat CB peptides. Sera from inbred BB rats immunized with BII, CII, or HII reacted best with CB11, unlike antisera to RII that reacted strongly with CB9-7. Inbred LEW, COP, WKY, F344, and BUF rats immunized with BII reacted strongest with CB9-7 and variably with CB11 and CB12. BBxLEW F1 hybrid rats reacted almost equally with CB11 and CB9-7 producing an antibody profile intermediate to those elicited in the parent strains. Finally, antibodies reactive with rat CB11, CB9-7, and CB12 could be eluted from normal rat cartilage incubated in anti-BII serum; antibody eluate profiles generally paralleled the profile produced by the sera applied to cartilage. Taken together, these findings indicate that multiple antibody-reactive epitopes on type II collagen may be instrumental in the initiation of collagen-induced arthritis in rats, particularly shared or cross-reactive epitopes located within CB11, CB9-7, CB12, and CB8.

Acceleration of onset of collagen-induced arthritis by intra-articular injection of tumour necrosis factor or transforming growth factor-beta.

We examined whether tumour necrosis factor (TNF) or transforming growth factor-beta 1 (TGF-beta 1) could alter the course of collagen-induced arthritis (CIA). Injection of 100 ng TNF or 500 ng TGF-beta 1 into ankle joints of normal rats induced a very limited inflammatory response, observable only upon histological analysis. However, when injected into ankle joints of rats 9 days after immunization with bovine type II collagen (CII), identical doses of TNF or TGF-beta 1 induced a sustained, clinically obvious inflammation and oedema that began within 8 h on average, as compared to 90 h in CII-immunized control rats given no injections or intra-articular injections of buffer. The incidence of arthritis at 2 weeks post-immunization was 100% for TNF-injected hindpaws, compared with 55% for the control groups, a statistically significant difference. In rats passively immunized with a subarthritic dose of affinity purified antibody to rat-CII, intra-articular injection of 100 ng TNF or 500 ng of TGF-beta 1 also induced intense, though transient arthritis. The rapid proinflammatory effects in CIA described in this study and the synergy demonstrated between anti-CII IgG and either cytokine, suggest that these cytokines can participate locally in the pathogenesis of arthritis.

Immunogenetics of collagen-induced arthritis in rats. Both MHC and non-MHC gene products determine the epitope specificity of immune response to bovine and chick type II collagens.

Seven inbred, RT1-congenic rat strains were immunized with native bovine (BII), porcine (PII), or chick (CII) type II collagen and observed for onset, incidence, and severity of arthritis. Clinical results were compared with IgG reactive with native rat type II collagen (RII) and the purified, renatured cyanogen-bromide peptides of BII, CII, or RII. Immunodominant responses to CB11, CB9,7, and CB12 of RII were identified. Secondary responses to CB8 and CB10 also occurred. Reproducible patterns of peptide reactivity were defined in each strain and reflected both RT1 and non-RT1 genotypes plus the species of immunizing collagen. BN non-RT1 gene products moderated clinical arthritis but increased the levels of reactivity to CB11 in three strains carrying RT1l,n,av1 haplotypes. WF (RT1u) rats were susceptible to collagen-induced arthritis (CIA) and developed very high levels of autoantibodies with dominant responses to rat CB11 after CII injections and to rat CB11 and CB9,7 after BII injections. DA (RT1av1) rats developed the most severe arthritis but had only moderate (total) levels of anti-RII IgG: a broad response to CB11, CB10, and CB9,7 after CII injections but predominantly to CB12 and CB9,7 after BII injections. Three RT1n strains--DA.1N(BN), WF.1N(MAXX), and BN--were resistant to BII-induced CIA but developed mild arthritis after immunization with CII. After BII: BN IgG reacted with CB9-7, CB11, and CB12; DA.1N and WF.1N IgG reacted with CB9,7 and CB12. After CII: BN IgG reacted broadly with CB11, CB9-7, CB12, and CB8; WF.1N IgG reacted to CB9-7, CB11, CB8, and CB12; DA.1N IgG reacted with CB8, CB11, and CB9-7. Thus, selective induction of CIA in BN, WF.1N, and DA.1N rats by CII correlated with serum IgG reactivity to rat CB11, but overall strain results identified no single cyanogen-bromide peptide as expressing the sole "arthritogenic" epitope in CIA.

Induction of arthritis with monoclonal antibodies to collagen.

mAb were developed from DBA/1 mice immunized with chick type II collagen. A total of 69 IgG antibodies was isolated and characterized. The majority (36%) reacted with a CNBr-derived peptide CB11 previously identified as containing a major immunogenic and arthritogenic epitope(s). Seven of the antibodies reactive with CB11 crossreacted strongly with mouse type II collagen. These were administered to DBA/1 mice in an attempt to induce arthritis. Individual antibodies were able to induce mild lesions consisting of minimal synovial proliferation but not overt arthritis. However, a combination of antibodies induced severe arthritis with marked destruction of articular cartilage. The minimal effective combination consisted of three antibodies. Arthritis developed within 48 to 72 h after injection of the antibodies and persisted for the duration of the observation period of 3 wk. Antibody levels were measured at intervals and persisted for the 3 wk observation period although at diminishing levels. Competitive binding assays demonstrated that each of the effective antibodies bound independently suggesting that some spatial or quantitative relationship was important possibly related to their ability to activate complement.

Immunity to type XI collagen in mice. Evidence that the alpha 3(XI) chain of type XI collagen and the alpha 1(II) chain of type II collagen share arthritogenic determinants and induce arthritis in DBA/1 mice.

To determine whether native bovine type XI collagen (BXI) is arthritogenic, five strains of inbred mice were immunized with BXI/CFA. Arthritis was not observed in any of these strains, though it was prevalent in DBA/1 and B10.RIII controls immunized with bovine type II collagen (BII). Antisera from BXI-immunized mice reacted with mouse type XI collagen (MsXI), weakly with the alpha-chains of BXI, and minimally with mouse type II collagen (MsII). However, antisera to BII reacted with MsII and MsXI, indicating antibodies to conformation-independent epitopes shared by alpha 1(II) and alpha 3(XI). Mice immunized with BXI containing a small amount of BII developed arthritis much like those immunized with BII; sera from these mice reacted with MsXI and MsII. Delayed-type hypersensitivity responses differed from IgG responses, i.e., BXI elicited responses to alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II); BII, to alpha 3(XI) and alpha 1(II) exclusively. To determine whether alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II) are arthritogenic, DBA/1J mice were immunized with each alpha-chain. Arthritis was seen in mice injected with alpha 3(XI) or alpha 1(II). Sera to both alpha-chains reacted similarly with MsII and peptide fragment alpha 1(II)-CB11. Epitope mapping using polyclonal and mAb to type II collagen revealed that all polyclonal and 11 of 14 mAb reacted with alpha 3(XI) and alpha 1(II), whereas three mAb reacted only with alpha 1(II). In conclusion, BXI is immunogenic but not arthritogenic in five strains of mice, whereas alpha 3(XI) and alpha 1(II) are arthritogenic and immunogenic in DBA/1 mice and share greater than or equal to 11 epitopes recognized by autoantibody.