T cell regulation of collagen-induced arthritis in mice. II. Immunomodulation of arthritis by cytotoxic T cell hybridomas specific for type II collagen

Injection of native type II collagen (CII) to susceptible strains of mice (H-2^q) induces a rheumatoid arthritis-like disease. To study the role of CD8⁺ T cells in the collagen-induced arthritis (CIA),we generated CII-specific T cell hybridomas by fusion of cells from arthritic C3H.Q mice and an AKR thymoma. Two hybrid clones (P3G8 and P2D9) were selected for their ability to lyse syngeneic CH-pulsed macrophages and recognize different antigenic epitopes in association with K^q molecules. When these T cell clones were irradiated and inoculated into (C3H.Q × AKR)F₁ mice 21 days prior to priming with native CII/ complete Freund's adjuvant, the incidence and the duration of CIA were significantly reduced in comparison to groups receiving saline or control T cell hybridoma. Furthermore, both anti-CII T cell hybridomas were able to attenuate CIA in highly susceptible inbred strains of mice and this suppression was antigen and disease specific. The protective activity seems to require intact cells as neither membrane fractions nor cytosolic preparations of the hybridoma T cells retained the vaccinating activity. Most importantly, one of the hybrid clones (P3G8) had a therapeutic effect on CIA since its administration to arthritic DBA/1 mice on day 30 after priming down-regulated the ongoing disease. Taken together, these findings suggest that anti-CII cytotoxic T cell clones can vaccinate against CIA and even reverse the disease.     

Characterization of monoclonal antibody specific for human alpha interferon

A mouse hybridoma cell line has been isolated, which secretes a monoclonal antibody specific of human leukocyte (alpha) interferon. The antibody secreted by the hybridoma belongs to the IgG1 class. It neutralizes biological activities (cellular and antiviral) of alpha interferon. Mass production of the antibody in ascitic fluid has been obtained. A convenient method of purification of the IgG from the ascitic fluid on DEAE-Trisacryl M is described.     

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.     

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.     

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.     

HLA-B27 transgenic mice are susceptible to collagen-induced arthritis: type II collagen as a potential target in human disease

HLA-B27 is highly linked with a group of human diseases called spondyloarthropathies (SpA). Many of these disorders begin after an infection with an enterobacteria. The symptoms seen in patients with spondyloarthropathies are inflammatory pain in the spine and asymmetrical arthritis of lower limbs. Additional symptoms related to SpA include inflammation in the eyes, bowel, and skin. The autoantigen(s) in SpA are not known. Proteins such as collagen and proteoglycans have been thought to be potent autoantigens in arthritidis including B27-associated human diseases. Type II collagen is a common denominator among eyes and joints, affected tissues in B27-linked diseases. Moreover, a few reports indicated CII specific T cells and antibodies in patients with spondyloarthropathies. We and others have previously described development of spontaneous arthritis and nail disease in HLA-B27 transgenic animals. To determine whether CII may be a target antigen in the B27-linked diseases, B27 + m beta 2 m% (HLA-B27) transgenic mice lacking mouse beta 2m with and without human beta 2m) mice were immunized with type II collagen inside the barrier facility. Male HLA-B27 transgenic mice developed collagen-induced arthritis compared to transgene negative littermates or female counterparts. There was no difference in the incidence of arthritis in HLA-B27 transgenic mice with and without human beta 2m. Our data suggest that beta 2m free heavy chain of HLA-B27 may present soluble antigens such as type II collagen to trigger specific T cells contributing in the development of arthritis. Our data also suggest that CII may be a potential target antigen in the cartilage during the disease process.     

Suppression of collagen-induced arthritis by oral or nasal administration of type II collagen.

We directly compared the effects of oral and nasal administration of collagen type II (CII) on disease progression, cytokine production and T cell responses in DBA/1 mice. Lymphocytes were assayed for proliferation and cytokine production and cell lines established. T cells from fed or nasally treated groups proliferated significantly less and produced markedly less IFN-gamma than the non-fed immunized group 10 days after immunization and prior to onset of arthritis. T cell lines established from fed or nasally treated mice showed a pattern of cytokine production involving IL-4, IL-10 and TGF-beta, whereas T cell lines from the control group produced more IFN-gamma and IL-2. Suppression of clinical measures of arthritis was equivalent in the nasal and orally treated groups. Animals were then tested for IFN-gamma production 70 days after a booster immunization at a time when disease was apparent. Mucosally treated animals secreted less IFN-gamma as compared to controls, even at this late time point. Suppression of collagen induced arthritis (CIA) by nasal treatment of mice with CII was associated with diminished levels of TNF-alpha and IL-6 mRNA expression in the joints of tolerized mice, two cytokines known to be involved in the inflammatory and pathological process of CIA. These results demonstrate the induction of antigen specific Th2 and TGF-beta secreting regulatory cells following both oral and nasal treatment, which is associated with suppression of local inflammation in the joints and decreased Th1 type responses in the periphery throughout the course of the illness.     

Exposure of female mice to type II collagen reduces susceptibility to collagen-induced arthritis in offspring

The effect of exposure of female DBA/1 mice to collagen II (CII) prior to breeding on the susceptibility of their offspring to CII-induced arthritis (CIA) was investigated. It was found that female offspring, born within 3 months after exposure of the mothers to CII, had a significantly reduced incidence of CIA, following immunization with CII. Just prior to this immunization, no anti-CII could be detected in the offspring. Offspring born more than 3 months after exposure of the mothers to CII showed no differences in susceptibility to induction of CIA, if optimal conditions for induction were used. However, when suboptimal conditions for induction of CIA were used, offspring of females that had been exposed to CII developed less severe arthritis and had a delayed onset of arthritis as compared with controls. It is concluded that exposure of female mice to CII prior to mating results in changes in the immune response to CII in the offspring, leading to a subtle decrease in susceptibility to CIA.     

Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen-induced arthritis

Immunization of mice with type II collagen (CII) leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis. T cell recognition of CII is believed to be a critical step in CIA development. We have analyzed the T cell determinants on CII and the TCR used for their recognition, using twenty-nine T cell hybridomas derived from C3H.Q and DBA/1 mice immunized with rat CII. All hybridomas were specific for the CII(256 – 270) segment. However, posttranslational modifications (hydroxylation and variable O-linked glycosylation) of the lysine at position 264 generated five T cell determinants that were specifically recognized by different T cell hybridoma subsets. TCR sequencing indicated that each of the five T cell epitopes selected its own TCR repertoire. The physiological relevance of this observation was shown by in vivo antibody-driven depletion of TCR Vα2-positive T cells, which resulted in an inhibition of the T cell proliferative response in vitro towards the non-modified CII(256 – 270), but not towards the glycosylated epitope. Most hybridomas (20/29) specifically recognized CII(256 – 270) glycosylated with a monosaccharide (β-D -galactopyranose). We conclude that this glycopeptide is immunodominant in CIA and that posttranslational modifications of CII create new T cell determinants that generate a diverse TCR repertoire.     

Characterization of a murine monoclonal antibody specific for human early lymphohemopoietic cells

This paper summarizes the results of the characterization of A10, a murine monoclonal antibody which recognizes an epitope not restricted to cells of a definite lineage, but which seems to be specific for an early differentiation antigen present on precursors of circulating T and B cells. The structure recognized by the A10 monoclonal antibody, although strikingly structurally similar to the HLA-A,B,C complex, is immunologically different both from histocompatibility antigens and from beta 2 microglobulin. Furthermore, the distribution of the A10 antigen is analyzed in different cell and tissue samples, both in health and disease conditions.     

抗VEGF中和抗体对Ⅱ型胶原诱导的关节炎形成的影响

目的：了解抗血管内皮生长因子（VEGF）中和抗体对Ⅱ型胶原（CoⅡ）诱导的关节炎（CIA）形成的影响。方法：于8～10wk龄的DBA／1J小鼠尾根部皮内注射鸡CoⅡ进行被动免疫，建立小鼠CIA模型，以CIA发生率、关节炎指数及关节组织的病理变化为指标，评价抗VEGF抗体对CIA形成的影响。结果：与对照组相比较．在CIA形成的早期，注射抗VEGF中和抗体能有效地抑制关节炎的产生及减轻其严重程度（P〈0．05）；而在CIA已形成后再注射抗VEGF中和抗体对关节炎的严重程度则无明显影响（P〉0．05）。结论：抗VEGF中和抗体明显抑制滑膜细胞增殖、血管新生及血管炎，因此有望成为类风湿性关节炎（RA）治疗的新型制剂。     

Glycosylation of type II collagen is of major importance for T cell tolerance and pathology in collagen-induced arthritis

Type II collagen (CII) is a candidate cartilage-specific autoantigen, which can become post-translationally modified by hydroxylation and glycosylation. T cell recognition of CII is essential for the development of murine collagen-induced arthritis (CIA) and also occurs in rheumatoid arthritis (RA). The common denominator of murine CIA and human RA is the presentation of an immunodominant CII-derived glycosylated peptide on murine Aq and human DR4 molecules, respectively. To investigate the importance of T cell recognition of glycosylated CII in CIA development after immunization with heterologous CII, we treated neonatal mice with different heterologous CII-peptides (non-modified, hydroxylated and galactosylated). Treatment with the galactosylated peptide (galactose at position 264) was superior in protecting mice from CIA. Protection was accompanied by a reduced antibody response to CII and by an impaired T cell response to the glycopeptide. To investigate the importance of glycopeptide recognition in an autologous CIA model, we treated MMC-transgenic mice, which express the heterologous CII epitope with a glutamic acid in position 266 in cartilage, with CII-peptides. Again, a strong vaccination potential of the glycopeptide was seen. Hence CII-glycopeptides may be the optimal choice of vaccination target in RA, since humans share the same epitope as the MMC mouse.     

Low affinity antibodies against collagen type II are associated with pathology in collagen-induced arthritis in mice

The relationship between the functional affinity of antibodies against type II collagen (CII) and the development of arthritis was studied in mice with collagen-induced arthritis. The responses of DBA/1 strain mice were compared with those of mice selectively bred to produce antibodies of high functional affinity (HA mice) and low functional affinity (LA mice). HA and LA mice did not develop arthritis in response to immunization with CII whereas 86% of DBA/1 mice did, with 33% showing severe and 53% mild disease. Anti-CII antibodies of the highest titre, the lowest functional affinity, and the greatest affinity heterogeneity were associated with the development of the severest arthritis in DBA/1 mice: even in DBA/1 mice with moderate or no disease the amount of antibody and heterogeneity were higher and functional affinity lower than in either HA or LA mice. Antibodies of the G1, 2a, 2b and 3 subclasses were produced in all mice, and none of these alone accounted for the overall difference in IgG antibody titres or affinity in the groups of mice. Antibodies of the IgG2a subclass showed the closest association with the development of arthritis in the different groups. It is concluded that anti-CII antibodies of low functional affinity, and presumably also of the IgG2a subclass, influence the disease process in collagen arthritis.     

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.     

Gastric administration of type II collagen delays the onset and severity of collagen-induced arthritis in rats.

Rats were per-gastrically dosed by gavage with collagen type II (CII) in solution and were shown, as a result, to be subsequently resistant to the induction of disease by CII administered parenterally in Freund's incomplete adjuvant. Disease onset was delayed and the severity was reduced. There was no concomitant reduction in the systemic antibody response to CII. Gavage with CII did not in itself induce systemic anti-CII antibodies and did not cause symptoms of arthritis. The results indicate that materials specifically cross-reactive with autoantigens can be absorbed through the gut and can modify susceptibility to autoimmune disease.     

Differential activities of immunogenic collagen type II peptides in the induction of nasal tolerance to collagen-induced arthritis

Nasal tolerance has recently been used to modulate immune responses in animal models of autoimmunity. We have compared immunogenic collagen type II (CII) peptides for induction of nasal tolerance in DBA/1 mice to collagen-induced arthritis (CIA). Three synthetic peptides corresponding to T cell-stimulating sequences of alpha1(II)-CB11, 260-270, 245-270 and 259-273, one peptide analog 245-270 (A260B261N263) and one myelin basic protein (MBP) peptide 89-101 were administered intranasally to DBA/1 mice respectively (total 300 microg peptide/mouse on three consecutive days) 10 days prior to CII immunization. Forty percent of CII245-270 (P<0.05) and 20% CII260-270 (P>0.05) treated mice did not develop arthritis whilst all of the mice treated with CII245-270 (A260B261N263) or CII259-273 developed arthritis compared to those in control groups (PBS- and MBP89-101-treated). The mice in either the CII245-270- or CII260-270-treated group which developed arthritis had a significantly delayed onset and their disease was less severe both clinically and histologically. All mice in both CII245-270- and CII260-270-treated groups had a reduced serum level of anti-CII antibody (P<0.01), with a marked reduction of IgG2a. Drain lymph node (LN) cells taken 7 days after CII immunization from these mice showed a significant reduction of interferon (IFN)-gammaP<0.01) production uponin vitro stimulation with CII. These results indicate that intranasal administration of synthetic CII peptides can control CIA, which is achieved by down-regulating the Th1 CII-induced responses. In addition, they stress that a fine 'tuning' of the peptide able to induce 'tolerance' is required to achieve the optimal effect.     

Efficient promotion of collagen antibody induced arthritis (CAIA) using four monoclonal antibodies specific for the major epitopes recognized in both collagen induced arthritis and rheumatoid arthritis

An antibody response to defined epitopes located on the triple helical portion of type II collagen (CII) is associated with the development of collagen-induced arthritis (CIA) and rheumatoid arthritis (RA). Monoclonal antibodies to epitopes associated with arthritis, but not antibodies specific for epitopes not associated with arthritis, induce arthritis in mice, the so-called collagen antibody induced arthritis (CAIA) model. We have selected monoclonal IgG antibodies specific for four well-defined major epitopes on triple helical CII, the C1, J1, D3 and U1 epitopes. These antibodies bind the epitopes specifically as determined using recombinant or synthetic triple helical epitopes. They are encoded from somatically mutated V genes. They all bind cartilage in vivo in normal mice. All of the antibodies induce mild arthritis after injection intravenously and if injected as a cocktail they induce severe clinical arthritis. Intravenous injection of a total of 4 mg antibodies (0.5 mg antibodies per clone) induced arthritis in several different mouse strains without any secondary immune stimulus and intraperitoneal injection of LPS 7 days later dramatically raised the severity. Thus, this method is recommended as a new protocol for the induction of CAIA.