Collagen-induced arthritis--use of the direct and indirect haemolytic plaque-assay to study the humoral response to collagen

Using both the direct and indirect haemolytic plaque assay, lymphocytes secreting antibodies to native type II collagen could be detected in the lymph nodes, spleen and blood of collagen-immunised rats. Sprague-Dawley, but not Alderley Park, strain rats showed clear differences in the number of plaque-forming cells in the lymph nodes between arthritic and non-arthritic rats. Differences also occurred between these strains in their response to the production of indirect plaques. The presence of native or denatured type II collage, but not type I collagen, was necessary to increase or maintain, in vitro, the lymphocytes producing anti-collagen antibody and the increase could be inhibited by serum from immunised rats or by colchicine.     

Native type II collagen-induced arthritis in the rat. I. Incidence and humoral response to collagen.

An acute inflammatory arthritis has been induced in 76% of rats injected intradermally with native bovine type II collagen emulsified in Freund's complete (CFA) or incomplete (ICFA) adjuvant. The arthritis became chronic in 14 out of 31 rats, and ear and tail lesions were noted in some rats. No arthritis was induced by native type I collagen, denatured type II collagen, rabbit IgG, or buffer alone injected intradermally with adjuvant. Using a solid-phase radioimmunoassay for serum antibodies we have shown that IgM and IgG levels to native bovine type II collagen were significantly higher in arthritic than nonarthritic rats. Antiglobulin antibody levels were not raised in arthritic rats. Equivalent antibody levels to native type II collgen were obtained whether this antigen was emulsified in ICFA or CFA. However, native type I collagen produced high antibody levels only when emulsified in CFA, indicating a difference in the immunogenicity of these collagens. These studies suggest that native type II collagen possesses arthritogenic properties in the rat and that humoral immunity may play a role in the induction of this arthritis.     

Arthritogenicity of minor cartilage collagens (types IX and XI) in mice

Native type II collagen, the major cartilage collagen, is immunogenic and arthritogenic in rodents. To investigate whether minor cartilage collagens are arthritogenic, we immunized DBA/1 mice with the pepsin-soluble fractions of type IX or type XI collagen emulsified in Freund's complete adjuvant. Both collagens were arthritogenic in DBA/1 mice after only 1 injection. However, the incidence of the polyarthritis was lower and the severity was lesser than with that induced by bovine type II collagen, even when a booster injection was administered. All mice developed a humoral response to the immunizing antigen, without any relationship to the arthritic status. Interestingly, competition experiments showed that antibodies raised against type XI collagen also bound with high avidity to type II collagen. In contrast, sera from type IX collagen-immunized mice did not react with either type II or type XI collagen. We conclude that types IX and XI minor cartilage collagens are both arthritogenic and immunogenic in DBA/1 mice. Whether the recognition of epitopes common to different collagens is relevant to the articular pathology remains to be elucidated.     

Collagen-induced arthritis: antibody production by lymphocytes in vitro

The in vitro production of anti-bovine type II collagen antibodies by lymphocytes from rats immunised with native bovine type II collagen and adjuvant was measured using a solid-phase enzyme-linked immunoassay. Antibodies to native bovine type II collagen could be detected in culture supernatants from the lymphocytes of rats only if they had been immunised with native bovine type II collagen, but not if immunised with native type I collagen, with keyhole limpet haemocyanin, with buffer or if un-immunised. The antibodies produced also bound to native rat, human and chick type II collagens, to native bovine 1 alpha 2 alpha 3 alpha collagen but not to native type I collagen. The amount of antibody in the cultures was altered by the presence of serum from type II collagen immunised rats or by the presence of either cyclohexamide, colchicine, Concanavalin A, catalase or lipopolysaccharide. Pre-treatment of the lymphocytes with mitomycin-C reduced the amount of anti-collagen antibody. This system can be used to investigate mechanisms controlling anti-collagen antibody production.     

Native type II collagen-induced arthritis in the rat. III. Relationship between the cellular immune response to native type II collagen and arthritis.

The relationship between cell mediated immunity to collagen and arthritis was studied with lymphocytes from arthritic and nonarthritic rats after immunisation with native bovine type II collagen. With the in-vivo radiometric ear assay arthritic rats gave a significantly higher response to native type II collagen than did nonarthritic rats. However, there was an overlap of values, and some arthritic rats gave no response to collagen even on the day of onset of arthritis. There was no difference in the response of lymphocytes from arthritic and nonarthritic rats with in-vitro transformation to native type II collagen, responses being found in both groups. All rats which developed arthritis had serum antibodies to native type II collagen, but not all responded to the tests for cell mediated immunity. These findings suggest that antibodies to collagen are more associated with the development of arthritis than is cell mediated immunity to collagen.     

Collagen-induced and adjuvant-induced arthritis in rats. Post-immunization treatment with collagen to suppress or abrogate the arthritic response

Immunization of Lewis rats with native type II collagen results in an inflammatory arthritis and increased humoral and cellular immune responses to type II collagen. The exposure of rats to native type II collagen at day 7 or 10 after immunization suppressed the incidence of arthritis and anticollagen antibody levels, although the cellular response was not affected. The exposure to denatured type II collagen offered partial protection, while type I collagen had no significant effect. Rats immunized with Mycobacterium tuberculosis also showed reduced arthritic response when subsequently treated with type II collagen. The common modalities between the 2 models and the possible role of type II collagen in the interference with the inflammatory arthritic events are discussed.     

Collagen-induced and adjuvant-induced arthritis in rats

Immunization of Lewis rats with native type II collagen results in an inflammatory arthritis and increased humoral and cellular immune responses to type II collagen. The exposure of rats to native type II collagen at day 7 or 10 after immunization suppressed the incidence of arthritis and anticollagen antibody levels, although the cellular response was not affected. The exposure to denatured type II collagen offered partial protection, while type I collagen had no significant effect. Rats immunized with Mycobacterium tuberculosis also showed reduced arthritic response when subsequently treated with type II collagen. The common modalities between the 2 models and the possible role of type II collagen in the interference with the inflammatory arthritic events are discussed.     

Diversity of the immune response to bovine type II and XI collagens in rats.

Immunisation of rats with native type II or type XI collagen produced an inflammatory arthritis in certain strains of rats. Antibodies to the native and denatured whole molecules, to the individual alpha chains and to the cyanogen bromide derived peptides of these chains were studied. Inter- and intra-strain variation in the specificity of the antibodies produced was seen and there were also changes with time, especially with the rats immunised with type XI collagen. Both collagen type-specific and cross-reacting antibodies were produced following immunisation with either type II or type XI collagen. Although no specific pattern of antibodies was unique to the presence or absence of arthritis in all rats, antibodies that bound to the CB-11 peptide or antibodies that bound to the CB-9,7 peptide of type II collagen occurred at the time of onset of arthritis in type II or type XI immunised, arthritic rats. Antibodies to these peptides occur in many patients with rheumatoid arthritis (RA) who also have antibodies to type II collagen. Therefore these findings suggest that epitopes on these peptides may be important in the continued production of antibodies to these collagens in patients with RA and that these antibodies may indeed be pathogenic.     

Involvement of endogenous tumor necrosis factor alpha and transforming growth factor beta during induction of collagen type II arthritis in mice.

Both tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) are found in synovial fluid from arthritic joints of humans and of rodents with experimental arthritis. The role of endogenously produced TGF-beta and TNF in the pathogenesis of collagen type II-induced arthritis (CIA) in DBA/1 mice was examined by determining the effect of neutralizing monoclonal antibodies to these factors on the course of the disease. Endogenously produced as well as systemically administered TGF-beta 1 and TNF-alpha had opposite effects, since TGF-beta 1 and anti-TNF protected against CIA, whereas anti-TGF-beta and TNF-alpha increased CIA incidence and/or severity. Intraperitoneally injected TGF-beta 1 at a dose of 2 micrograms per day for 14 days significantly ameliorated arthritis, even when started at the time of arthritis development, although it did not reverse established disease. The resistance to CIA induction caused by a prior intravenous injection of collagen type II was not significantly influenced by the simultaneous injection of TGF-beta 1, TNF-alpha, or interleukin 1 alpha. It is concluded that the endogenous production of TNF and TGF-beta is important in determining the course of CIA.     

Type II collagen induced arthritis: comparison of histological changes in arthritis-susceptible and arthritis-resistant rats.

Wistar rats (type II collagen-induced arthritis-susceptible) and PVG.RT1u rats (arthritis-resistant) were immunised with type II collagen and histological changes in the synovial tissues of the hind feet and in the popliteal lymph nodes draining these feet were examined and compared with unimmunised rats. There were no apparent differences in the joints of unimmunised rats of each strain, but the popliteal lymph nodes of the Wistar rats appeared more "activated", suggesting possible differences between strains in the continuous, low-grade release of antigens from the joints. Whilst the joints of arthritic rats showed the most marked histological changes, the joints of immunised PVG.RT1u rats and of non-arthritic Wistar rats showed some minor changes in the synovia and loss of cartilage with a concomitant increase in the size of the popliteal lymph nodes. The number of mast cells per unit area increased in the lymph nodes of immunised PVG.RT1u rats in proportion to the size increase, but decreased in those draining arthritic Wistar rat feet. The number of mast cells per unit area of synovium decreased most in arthritic feet and the staining pattern of the mast cells altered in the most severely arthritic feet, suggesting a change in mast cell population. These differences in mast cells may be directly related to whether or not clinical arthritis develops in a particular joint. Further study of mast cell sub-populations is warranted.     

Antibodies to type II and XI collagens: evidence for the formation of antigen specific as well as cross reacting antibodies in patients with rheumatoid arthritis.

Antigen specific and cross reacting antibodies to native and denatured types II and XI collagen were detected in the sera of rats immunised with either of these antigens. The antibodies from rats immunised with type XI collagen initially showed the strongest binding to the alpha 2(XI) chain of type XI collagen but later binding to the alpha 3(XI) chain was seen. Sera from patients with rheumatoid arthritis had antibodies that bound to both type II and XI collagens. Immunoblotting studies showed that most patients had antibodies which bound to the alpha 1(II) chain of type II collagen and to the alpha 3(XI) chain of type XI collagen. Some patients also had antibodies which bound to the alpha 1(XI) and to the alpha 2(XI) chains of type XI collagen. Thus antibodies to unique as well as to common epitopes on each of the two types of collagen molecule occur in some patients with rheumatoid arthritis.     

Collagen-induced arthritis in rats

An oil emulsion of purified type I1 collagen from bovine articular cartilage when injected intradermally into rats induced an inflammatory polyarthritis in 4 of 12 animals. When similarly injected, collagen purified from bovine vitreous induced arthritis in 6 of 12 animals. Studies of humoral and cell-mediated immunity to both collagen preparations demonstrated complete crossreactivity. It is concluded that vitreous collagen shares the arthritogenic property of cartilage-derived type I1 collagen and that collagen from the two sources is indistinguishable in arthritogenic and immunologic properties. An oil emulsion of purified type II collagen from bovine articular cartilage when injected intradermally into rats induced an inflammatory polyarthritis in 4 of 12 animals. When similarly injected, collagen purified from bovine vitreous induced arthritis in 6 of 12 animals. Studies of humoral and cell-mediated immunity to both collagen preparations demonstrated complete cross-reactivity. It is concluded that vitreous collagen shares the arthritogenic property of cartilage-derived type II collagen and that collagen from the two sources is indistinguishable in arthritogenic and immunologic properties.     

Type ii collagen—induced arthritis in mice

The passive transfer of high levels of anti-type II collagen antibody from mice with type II collagen-induced arthritis may transfer arthritis to syngeneic recipient animals. However, the transfer of either arthritogenic or sub-arthritogenic doses of polyclonal anti-type II collagen antibody, or non-arthritogenic monoclonal anti-type II collagen antibody may protect mice against the subsequent induction of arthritis by type II collagen. A protective effect was also observed in mice given free native type II collagen intravenously before the administration of collagen in an arthritogenic protocol.     

Homologous type II collagen-induced arthritis in rats. Characterization of the disease and demonstration of clinically distinct forms of arthritis in two strains of rats after immunization with the same collagen preparation

Arthritis was induced in Lewis and DA rat strains after immunization with native homologous type II collagen (CII). Differences were noted in the clinical signs of arthritis in the 2 rat strains, which were immunized with the same arthritogenic preparation of CII. DA rats showed disease onset characterized by symmetric involvement of the interphalangeal joints of the hind feet. Lewis rats showed disease onset characterized by involvement of only the ankle or knee joints. Moreover, the arthritis tended to be chronic (i.e., persistent and variable redness and swelling seen in interphalangeal joints) in DA rats, but not in Lewis rats. Analysis of the delayed-type hypersensitivity reaction to CII and anti-CII autoantibody production demonstrated the presence of T cell, as well as B cell, reactivity to rat CII in both strains of rat. A spontaneous T cell reactivity to CII, as measured by rat CII-induced ear swelling, was observed in nonimmunized DA rats, but not in nonimmunized Lewis rats. The demonstration that clinical features of arthritis induced with identical arthritogenic stimuli are different depending on the genetic background of the affected animal may be relevant in understanding the heterogeneity of the clinical features of human inflammatory joint disease.     

Homologous type ii collagen—induced arthritis in rats

Arthritis was induced in Lewis and DA rat strains after immunization with native homologous type II collagen (CII). Differences were noted in the clinical signs of arthritis in the 2 rat strains, which were immunized with the same arthritogenic preparation of CII. DA rats showed disease onset characterized by symmetric involvement of the interphalangeal joints of the hind feet. Lewis rats showed disease onset characterized by involvement of only the ankle or knee joints. Moreover, the arthritis tended to be chronic (i.e., persistent and variable redness and swelling seen in interphalangeal joints) in DA rats, but not in Lewis rats. Analysis of the delayed-type hypersensitivity reaction to CII and anti-CII autoantibody production demonstrated the presence of T cell, as well as B cell, reactivity to rat CII in both strains of rat. A spontaneous T cell reactivity to CII, as measured by rat CII-induced ear swelling, was observed in nonimmunized DA rats, but not in nonimmunized Lewis rats. The demonstration that clinical features of arthritis induced with identical arthritogenic stimuli are different depending on the genetic background of the affected animal may be relevant in understanding the heterogeneity of the clinical features of human inflammatory joint disease.     

Type II collagen-induced arthritis in mice. II. Passive transfer and suppression by intravenous injection of anti-type II collagen antibody or free native type II collagen

The passive transfer of high levels of anti-type II collagen antibody from mice with type II collagen-induced arthritis may transfer arthritis to syngeneic recipient animals. However, the transfer of either arthritogenic or sub-arthritogenic doses of polyclonal anti-type II collagen antibody, or non-arthritogenic monoclonal anti-type II collagen antibody may protect mice against the subsequent induction of arthritis by type II collagen. A protective effect was also observed in mice given free native type II collagen intravenously before the administration of collagen in an arthritogenic protocol.     

SEGREGATION ANALYSIS OF THE STRUCTURAL GENES OF THE MAJOR FIBRILLAR COLLAGENS PROVIDES FURTHER EVIDENCE OF MOLECULAR HETEROGENEITY IN TYPE II EHLERS--DANLOS SYNDROME

Type II Ehlers—Danlos syndrome (EDS is one of a groupof disorders characterized by striking abnormalities of thesoft connective tissues. The major fibrillar collagens (typesI and III) found in these tissues have important stress—bearingfunctions and abnormal collagen could therefore account forthe clinical features of this condition. We have used a numberof restriction site dimorphisms, tightly linked to the structuralgenes of type I collagen (COL1A1 COL1A2) and type III collagen(COL3A1), to investigate the segregation of corresponding allelesin three pedigrees in which type II EDS was clearly inheritedas a dominant trait. Discordant segregation of all three collagengenes was seen in a large pedigree that included 17 affectedindividuals with the typical phenotype of type II EDS. Thus,mutations in neither type I nor type III collagen genes wereresponsible for the disease in this family. In a second smallpedigree discordant segregation of the disease with both typeI collagen loci was observed while the concordant segregationseen at COL3A1 could easily have arisen by chance (P =0.5).The third pedigree was uninformative at all three collagen locibecause of inability to discriminate between the parental alleles.These results suggest that there may be molecular heterogeneityof type II EDS since abnormalities of type I collagen have beendescribed in other individuals phenotypically similar to thosein our study. KEY WORDS: COL1A1, COL1A2, COL3A1, Connective tissue ^*Current address: Biotechnology Department, ICI Pharmaceuticals,Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG.     

Intron-mediated recombination may cause a deletion in an alpha 1 type I collagen chain in a lethal form of osteogenesis imperfecta.

To understand the nature of the mutation in type I collagen genes in cells from an infant with the perinatal lethal form of osteogenesis imperfecta (type II), we cloned and sequenced almost 2 kilobases of a normal alpha 1(I) collagen gene and the corresponding region of a mutant alpha 1(I) gene from cell strain CRL 1262. The mutant gene had undergone recombination between two non-homologous introns, which resulted in the loss of three exons coding for 84 amino acids in the triple-helical domain. The deletion predicted the loss of amino acid residues surrounding and including the methionine at the junction between the CNBr peptides alpha 1(I) CB8 and alpha 1(I) CB3, a result confirmed by analysis of the cleavage peptides from the product of the mutant gene. Although large deletions from collagen genes are uncommon causes of the osteogenesis imperfecta type II phenotype, analysis of the de novo change in gene structure in this cell strain suggests that similar rearrangements may have occurred during the evolution of the large collagen genes.     

The role of cartilage minor collagens in inducing arthritis

Arthritogenic properties of native types IX, XI, and II collagen were investigated in female Wistar rats. Immunization with native type-XI or -II collagen led to the arthritic reaction in 60% of investigated rats. After boosting, a distinct increase in anticollagen antibodies was observed followed by temperature rise (swelling and redness of affected joints). At the end of the experiment (after 7 weeks) subchondral bone destruction was detectable upon x-ray examination. Histological observation of the affected knee and ankle joints showed progressive destruction of the articular cartilage and subchondral bone accompanied by proliferative synovitis with extensive formation of fibrous tissue found in joints of rats immunized with native type-XI collagen. Immune response to collagen types (determined by ELISA) reached its peak between the 14th and the 21st day. From the acute stage to the chronic stage a decrease of serum anticollagen antibodies was observed to remain constant. Rats immunized with native type IX and denatured type XI collagen did not develop arthritis.     

Enhancement (by native type II collagen) of the humoral immune response to native type I collagen in the rat

Both native type II collagen and rabbit IgG were good immunogens in the rat when emulsified in incomplete Freund's adjuvant but native type I collagen and denatured type II collagen were not. Both native type II collagen and rabbit IgG had an enhancing effect upon the production of antibodies to native type I collagen when rats were immunised with a mixture of native type I collagen and either of these immunogens but denatured type II collagen did not have this effect. These immunogens, however, did not enhance the production of antibodies to ovalbumin when emulsified with it. Although both native type II collagen and rabbit IgG showed this enhancing effect with native type I collagen, only rats immunised with native type II collagen or mixtures containing native type II collagen developed arthritis. Thus, a specific immune response to collagen rather than this enhancing effect appears to be necessary for the induction of arthritis.