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.     

Regulation of cellular and humoral immune responses to collagen type I or collagen type II.

We have investigated the characteristics of antigen-specific reductions in murine immune responses to rat collagen type I (R-CI), chick collagen type II (C-CII) or bovine collagen type II (B-CII). Intravenous pretreatment with the appropriate soluble collagen or collagen-coupled spleen cells led to the development of antigen-specific reduced immune responses, the former treatment being more effective than the latter. In the case of CII, pretreatment with R-CI or non-related antigens was ineffective. However, pretreatment with denatured bovine-CII, native bovine-CII or chick-CII led to immune hyporesponsiveness for either the homologous or heterologous CII molecule. A delayed development of the diminished immune responses was observed for the cell-mediated immune response (CMI), as measured by in vivo delayed-type hypersensitivity (DTH), in that no reduction was evident at Day 7 but a significantly decreased response was observed at Day 14. Collagen-specific IgG and IgM antibody responses were consistently reduced by the pretreatment and remained reduced during the study period. The antigen-specific hyporesponsive state was not sensitive to cyclophosphamide treatment and was not transferable with hyporesponsive spleen cells. Additionally, we have induced unresponsiveness to CII by treating mice with an antibody directed to T helper cells (GK1.5). This treatment led to profound reductions in CII CMI responses as well as CII antibody levels. However, this unresponsive state is not permanent and not transferable with spleen cells from treated mice. These two types of procedures, soluble B-CII i.v. or GK1.5 treatment, not only resulted in CII hyporesponsive states, but also produced delayed onset and decreased incidence of arthritis in the appropriate strains.     

Identification of antibody epitopes in the CB-11 peptide of bovine type II collagen recognized by sera from arthritis-susceptible and -resistant rhesus monkeys.

Sera from eight rhesus monkeys that had been immunized with native bovine type II collagen were tested for antibodies to cyanogen bromide peptides (CB peptides) of type II collagen by Western blotting. The monkeys produced IgG antibodies to a number of different CB peptides, with five out of eight animals producing antibodies to the CB-11 peptide (four arthritic, one non-arthritic). Antibody epitopes on the CB-11 peptide of bovine type II collagen recognized by these sera were investigated by epitope mapping. Peptides (8-mers overlapping by seven amino acids) representing the CB-11 region were synthesised and the sera screened for binding to these peptides to determine areas of high IgG antibody binding to this region of type II collagen. The profiles obtained were not identical, though there were some epitopes that were commonly recognized. Antibodies to one epitope, also present in human type II collagen, were found only in the sera of two animals with the severest arthritis. The technique of epitope mapping has successfully identified a number of epitopes within the CB-11 peptide of type II collagen recognized by antibodies from bovine type II collagen-immunized monkeys. Studies on the relevance of responses to the identified epitopes can now be undertaken.     

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.     

Growth of Leishmania amastigotes in macrophages from normal and immune mice

Summary An in vitro system of prolonged culture of Leishmania tropica amastigotes in mouse macrophages is presented. The division rate of parasites was monitored by microscopic observations and by ³H-thymidine incorporation. The dynamics of macrophage infection and parasite division are influenced by the initial rate of promastigotes per cells in culture. Parasites multiply, gradually infect and finally destroy all available macrophages from normal mice releasing large numbers of viable amastigotes. Macrophages from immune donors were inferior in their ability to support parasite multiplication and did not survive long periods in culture.     

Effective induction of type 1 helper IgG2a and cytotoxic T-cell responses in mice following immunization with human papillomavirus type 16 E2 in MF59

Human papillomavirus (HPV) 16 E2-specific cell-mediated immunity to the early viral antigen E2 is associated with regression of natural infection in patients with cervical dysplasia. Vaccination strategies that activate this type of immune response may have application in the immunotherapeutic treatment of pre-existing HPV infections. The objective of this study was to test if cell-mediated immunity to E2 could be activated when delivered with the already licensed adjuvant MF 59.We found that immunization of mice with E2 in MF 59 stimulated T-cell responses when administered either intraperitoneally (IP) or subcutaneously (SC), and that the response was polarized to a Th-1 type IgG2a response in the IP immunized mice. The magnitude of the lymphoproliferative response was augmented by reducing the time interval between the primary and secondary immunizations from 12 to 4 wk. Stronger responses to the C-terminal third of E2 were detected, suggesting that one or more immunodominant epitopes were localized to this region. Significantly, immunization with E2 in MF 59 IP was sufficient to stimulate an E2-specific cytotoxic T-cell response.This immunization regimen activates the components of a cell-mediated immunity that are predicted to be efficacious in clearance of pre-existing infection, and supports its testing in a papillomavirus challenge model, as the next step in the progression toward its development as an immunotherapeutic vaccine for use in humans.     

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

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

Resistance to collagen-induced arthritis in rats and rhesus monkeys after immunization with attenuated type II collagen

Immunization of susceptible rodent or primate species with type II collagen (b-CII) from bovine origin induces type II collagen-induced arthritis (CIA). The disease is characterized as a systemic polyarthritis associated with humoral and cellular autoimmunity to CII and shares similarity with human arthritic diseases. The objective of this study was to develop a procedure for induction of resistance to CIA in animals, which possess a certain major histocompatibility complex phenotype that makes them prone to develop CIA (susceptible). It is shown that by immunization with an attenuated form of CII, in which arthritogenic epitopes have been destroyed by heat denaturation, disease resistance is induced in a susceptible inbred rat strain (RT-1^U) and in an outbred population of susceptible rhesus monkeys (lacking the Mamu-A26 allele). In both species the disease resistance is connected with modulation of anti-CII autoantibodies of the IgM isotype. This protocol may provide a basis for effective and safe methods to induce protection to autoimmune arthritis in those subjects that are genetically prone to develop such a disease.     

Humoral immune response to citrullinated collagen type II determinants in early rheumatoid arthritis

Collagen type II (CII) is a relevant joint-specific autoantigen in the pathogenesis of rheumatoid arthritis (RA). Whereas the reasons for the breakage of self tolerance to this major cartilage component are still enigmatic, T cell responses to glycosylated CII determinants in RA patients indicate that post-translational modifications play a role. Since the conversion of arginine into citrulline by peptidylarginine deiminases (PAD) in some non-joint-specific antigens such as filaggrin or fibrin has been shown to give rise to RA-specific humoral immune responses, we investigated whether PAD modification of cartilage-specific CII might affect its recognition by circulating autoantibodies in early RA. In vitro treatment with purified PAD led to arginine deimination of native CII or of synthetic CII peptides as evidenced by amino acid analysis. The citrullination resulted in modified recognition of the immunodominant CII epitope C1(III) (amino acid residues 359-369) by murine and human antibodies. In a cohort of early RA patients (n=286), IgG antibodies directed toward a synthetic citrullinated C1(III) peptide (citC1(III)-P) were detectable with a prevalence of 40.4%. The partial autoantibody cross-reactivity between citC1(III)-P and citrullinated peptides mimicking epitopes of the cytoskeletal autoantigen filaggrin suggests that autoimmunity to cartilage-specific modified self might be a critical intermediate bridging recognition of PAD-modified extra-articular autoantigens with the disruption of tolerance to native cartilage constituents.     

The immune response of guinea-pigs to type II collagen: poor cross-reactivity with homologous type II collagen accounts for resistance to collagen-induced arthritis.

In order to determine the susceptibility of guinea-pigs to collagen-induced arthritis (CIA), Hartley and Strain 13 guinea-pigs were immunized with heterologous or homologous type II collagen. None of the animals developed CIA. Because immunity to type II collagen plays a critical role in CIA, we characterized the guinea-pig's immune response to determine the basis for this resistance. Guinea-pigs develop cellular and humoral reactivity to heterologous type II collagen similar to that of CIA-susceptible rats. The reactions distinguish type I from type II collagen but not among several heterologous type II collagens. The cell-mediated immune (CMI) response was specific for determinants on the primary amino acid structure of collagen, whether native or denatured collagen was used for immunization; however, the humoral response was specific for the form of the molecule used for immunization. Guinea-pigs differ from CIA-susceptible rats in that immunization with homologous or heterologous type II collagen fails to induce significant cross-reactive immunity with the homologous antigen. A transient arthritis could be induced in animals immunized with heterologous type II collagen by injecting them intra-articularly with heterologous but not with homologous type II collagen. Our results show that the disparity between immunity to type II collagen and the susceptibility to develop CIA in guinea-pigs is due to their poor cross-reactive immune response to autologous type II collagen.     

Identification of an immunodominant B-cell epitope in bovine type II collagen and the production of antibodies to type II collagen by immunization with a synthetic peptide representing this epitope.

Using epitope scanning of 272 short, synthetic peptides representing the amino acid sequence of the CB-11 peptide of type II collagen, we have shown that five strains of rat, immunized with type II collagen, produce antibodies to a region 37-45 amino acids from the amino end of CB-11 peptide. Antibodies to this region always gave the highest binding values suggesting that it is an immunodominant region. Wistar rats immunized with a synthetic peptide representing this region, coupled to keyhole limpet haemocyanin, produced antibodies to this peptide which could still be detected at 1:4000 to 1:8000 dilution but none developed clinical arthritis. All sera also showed binding of antibodies to denatured bovine type II collagen but not to native type II collagen, keyhole limpet haemocyanin or to bovine serum albumin by ELISA. Sera from peptide-immunized rats were examined for antibody binding to the 272 short peptides of the CB-11 peptide and to the synthetic peptides representing shortened forms of the immunodominant region and forms of it with substituted amino acids. These results showed that the antibodies in the peptide-immunized rats were not identical to those produced to that peptide by rats immunized with type II collagen but may represent subpopulations of them. These findings suggest caution in interpreting the role of antibodies to individual peptides in arthritis induction without knowledge of their fine specificity.     

Retarded skeletal development in transgenic mice with a type II collagen mutation.

Transgenic mice harboring specific mutations provide a tool for systematic studies on the effects of dominant mutations on embryonic development and growth. In the present study, several different techniques were employed to follow the development of growth abnormalities and other phenotypic consequences in the mouse litters from matings of heterozygous Del1 mice carrying six copies of a mouse type II collagen transgene with a small engineered deletion mutation. Skeletal staining of complete litters with alcian blue/alizarin red S revealed that the onset of chondrogenesis and subsequent endochondral ossification were delayed in heterozygous and homozygous Del1 embryos. The lengths, widths, and shapes of long bones were all affected. Histological and in situ hybridization analyses revealed several types of abnormalities in the epiphyseal growth plates of homozygous Del1 embryos including disorganization of growth plate architecture, abnormal appositional growth activity, an increase in the number of hypertrophic chondrocytes, a deficiency in the formation of metaphyseal cancellous bone, and development of necrotic areas in the epiphyseal heads. Many of these findings parallel those seen in human chondrodysplasias. The basic information obtained on the effects of a specific deletion mutation in the type II collagen gene on the development and growth of the transgenic embryos provides a background for testing the efficacy of novel therapeutic strategies for prevention of such growth abnormalities.     

Secondary immune amplification following live poliovirus immunization in humans

Eight subjects inoculated orally with live attenuated poliovirus were investigated to study the effects of live virus infection on human T-cell responses. Proliferation to poliovirus and unrelated recall antigens were measured serially over a 3-week period. Five of eight subjects inoculated demonstrated a clear anamnestic response to poliovirus, but three did not. Only the five subjects demonstrating an anamnestic response to poliovirus were found to have augmented secondary immune responses to two unrelated recall antigens (tetanus toxoid and reovirus) and in the autologous mixed lymphocyte response (AMLR). No consistent changes were found in circulating T-cell surface activation antigens whether or not the subjects responded to poliovirus. These studies suggest that an asymptomatic poliovirus infection associated with immunization in humans can induce nonspecific secondary immune amplification as measured by in vitro T-cell proliferative response. This amplification pathway is a potential mechanism for immune responses against antigens other than those of the infecting virus.     

Uptake of intravenously administered soluble immune complexes by type I alveolar cells and macrophages in mice

Mice (C57BL/6, n = 5) were given a single intravenous injection of soluble bovine serum albumin (BSA)-rabbit anti-BSA immune-complexes (IC) prepared at five-fold antigen excess. The mice were sacrificed 15 and 30 min, and 1, 2, 4 and 12 h after injection. Granular immunofluorescence for BSA and rabbit IgG, in a pattern consistent with IC aggregation, was observed in the alveolar walls and in the cytoplasm of alveolar cells. Moderate immunofluorescence was observed 15 min after IC injection and maximal immunofluorescence was observed at 30-60 min which decreased rapidly 2 h after IC administration. Only a trace amount of immunofluorescence was observed at 4 h and none was seen at 12 h. Light microscopic immunoperoxidase staining showed localization of IC in the interstitium of the alveolar septa and in type I alveolar cells and alveolar macrophages. By immunoelectronmicroscopy, the uptake of IC in the cytoplasm of type I alveolar cells and alveolar macrophages was confirmed.     

IgG responses to intranasal immunization with cholera-toxin-immobilized polymeric nanospheres in mice

IgG responses to antigen-nanosphere hybrids were studied in mice. Cholera toxin (CT) was covalently immobilized onto the surface of polymeric nanospheres (NS) with a nanophase-separated structure consisting of a polystyrene core and a poly(methacrylic acid) graft corona. Reaction conditions favoring the dehydroxide condensation reaction of the amino group of the CT with the carboxyl group of NS effectively immobilized CT onto their surface. When CT-immobilized nanospheres (CT-NS) were suspended in aqueous solution and administrated to mice either intranasally or intramuscularly, serum IgG titers elevated with increasing time and reached a maximum level at 8 weeks after immunization. On the other hand, intranasal administration of CT alone induced an even higher serum IgG titer than that of CT-NS at 4 weeks. However, the titer gradually decreased thereafter. Thus, polymeric NS may be an effective substrate to covalently immobilize antigen on their surface, steadily inducing a high level of IgG production in response to the intranasal administration.     

IgG responses to intranasal immunization with cholera-toxin-immobilized polymeric nanospheres in mice

IgG responses to antigen-nanosphere hybrids were studied in mice. Cholera toxin (CT) was covalently immobilized onto the surface of polymeric nanospheres (NS) with a nanophase-separated structure consisting of a polystyrene core and a poly(methacrylic acid) graft corona. Reaction conditions favoring the dehydroxide condensation reaction of the amino group of the CT with the carboxyl group of NS effectively immobilized CT onto their surface. When CT-immobilized nanospheres (CT-NS) were suspended in aqueous solution and administrated to mice either intranasally or intramuscularly, serum IgG titers elevated with increasing time and reached a maximum level at 8 weeks after immunization. On the other hand, intranasal administration of CT alone induced an even higher serum IgG titer than that of CT-NS at 4 weeks. However, the titer gradually decreased thereafter. Thus, polymeric NS may be an effective substrate to covalently immobilize antigen on their surface, steadily inducing a high level of IgG production in response to the intranasal administration.     

Absence of immunity to type II collagen and proteoglycan in osteoarthritic C57BL mice

We measured immunity to type II collagen and proteoglycans in osteoarthritic C57BL mice to determine whether it is related to osteoarthritis pathogenesis. Histological examination revealed articular cartilage lesions in all mice and synovitis in only a few mice. Immunological responses to type II collagen were found in collagen arthritic mice, but not in C57BL mice. Furthermore, immunological responses to proteoglycans were observed in proteoglycan-immunized mice, but not in C57BL mice. Therefore, articular cartilage degeneration may not result in autoimmunity to type II collagen and proteoglycans in osteoarthritis of C57BL mice, and immune responses to these components may not be a primary etiology of osteoarthritis in this model.     

Common epitopes in Clq and collagen type II

An epitope common for collagen type II and Clq was demonstrated by specific binding of a monoclonal anti-collagen type II antibody, MAb B1, to purified Clq. This was further substantiated by the affinity shown between F(ab')2 fragments of anti-Clq antibodies and rat chondrosarcoma collagen type II. The interaction between MAb B1 and Clq was demonstrated in hemolytic assays, in an enzyme-linked biotin-avidin assay and by the binding of Clq to MAb B1 immobilized on Sepharose 4B beads. MAb B1 recognized only purified Clq and not the macromolecular Cl complex, indicating that the epitope for MAb B1 was situated in the collagen-like region in Clq, where Clq and Cls are anchored. The binding of the purified collagen-like fragment of Clq to radiolabelled MAb B1 confirmed these findings. The affinity between MAb B1 and Clq was significantly increased if Clq was first reacted with heat aggregated IgG, indicating a demasking of the reactive epitope on binding to the aggregated IgG. The present findings raise the question of the pathogenetic significance of the presence of anti-collagen type II antibodies and free Clq, both of which are frequently seen in high amounts in rheumatoid arthritis.     

Patterns of autoreactivity to collagen type II in autoimmune MRL/l mice.

The kinetics and mechanisms for secretion of antibodies against native and denatured collagen type II have been studied in spontaneously arthritic MRL/l mice. Circulating antibodies were quantified by an ELISA assay and frequencies of specific antibody secreting spleen cells by an ELISPOT assay. The degree of humoral immunity to collagen type II increased at late stages of the disease (6 months of age) whereas severe synovitis was seen earlier (5 months of age). Both the appearance of anti-collagen II producing cells and development of synovitis was preceded by and not correlated with a general state of polyclonal B cell activation. In MRL/l mice, collagen II specific antibodies appeared spontaneously and titres were largely unaffected by collagen II immunization. The levels of circulating anti-collagen II antibodies in MRL/l mice were lower, and the antibodies displayed lower avidities and different specificities as compared with the antibodies generated in collagen II high responder DBA/l mice after immunization with collagen II. It is suggested that the antibody response in MRL/l mice against collagen type II does not need MHC-restricted T cell help and that induction of antibody production to collagen II in MRL/l mice is triggered by joint cartilage destruction and subsequent collagen II release.