Immunology 1991 72 486-490

ADONIS

001928059100082M

Down-regulation of collagen arthritis after in vivo treatment with a syngeneic monoclonal anti-idiotypic antibody to a cross-reactive idiotope on collagen II auto-antibodies C. NORDLING, R. HOLMDAHL & L. KLARESKOG Department of Medical and Physiological Chemistry, Center, Uppsala University, Sweden

Biomedical

Acceptedfor publication 1I December 1990

SUMMARY Monoclonal anti-idiotypic antibodies previously shown to react with a cross-reactive idiotope of anti-collagen II auto-antibodies were used for in vivo treatment of DBA/1 mice receiving immunization with arthritogenic native rat collagen type II. Injection of 100 pg of the anti-idiotypic antibody 3 weeks before the collagen immunization resulted in a significant suppression of collagen arthritis, compared with mice treated with a monoclonal control antibody. The treatment with antiidiotypic antibody 3 weeks before collagen immunization could also cause a marked down-regulation of the total serum levels of anti-collagen II antibodies. When the anti-idiotypic antibodies were administered near the time for induction of arthritis (2 days after collagen immunization) a significant effect was seen on the collagen arthritis, but not on the levels of anti-collagen antibody. As collageninduced arthritis is a disease where both T- and B-cell mediated immunity are believed to play critical roles, the present effects of the in vivo anti-idiotype treatment on arthritis development could provide an interesting system for the study of idiotype regulation on both B- and T-cell arthritis-associated autoimmunity.

murine CII-induced arthritis constitutes a well-defined model for experimental autoimmunity where both B- and T-cell mediated immune reactions appear to contribute to development ofdisease. Thus, the disease can be induced both with antiCII antibodies and with CII-reactive T cells'0-'2 and the cellular and humoral CII response seem to act in synergy.'3 During our efforts to characterize the regulation of autoanti-CII immunity and arthritis development in the mouse using the DBA/I (H-2q) mouse as the model system, we have produced a panel of CII-reactive monoclonal antibodies. We have shown that passive transfer of CII-reactive monoclonal antibodies can induce synovitis.'4 We have also described a monoclonal anti-idiotypic antibody that not only recognizes a cross-reactive idiotope present on auto-anti-CII antibodies, but also recognizes a determinant present on isolated Fc fragments of normal autologous IgG, but not on the intact IgG.'5 Considering that this auto-anti-idiotypic antibody may be representative for antibodies with a potential to regulate autoimmunity to CII as well as development of CII arthritis in vivo, we carried out experiments where mice were treated with the anti-idiotypic antibody before and after CII immunization, and investigated the mice for development of arthritis and antiCII antibody response. The results show that the anti-idiotypic antibody treatment suppressed development of CII-induced arthritis and could down-regulate the total levels of the anti-CII antibody response.

INTRODUCTION Immune reactions in general and autoimmune reactions in particular are subject to a number of regulating events affecting both cellular and humoral immunity. Determinants on lymphocyte receptor molecules, idiotopes, have been suggested to be a part of this regulation of the immune system. The regulation could involve both unique private idiotopes and, perhaps more likely, the widely distributed cross-reactive idiotopes (CRI). Idiotope-specific anti-idiotypic antibodies are supposed to be acting together with the idiotopes in this network regulation.' This kind of network regulation has been proposed to operate in autoimmune situations2 and a regulatory effect of anti-idiotypic antibodies on autoantibody production has been shown in experimental autoimmune diseases.3 Autoimmunity to cartilage-derived collagen type II (CII) has been demonstrated to occur spontaneously in rheumatoid arthritis4-6 and induction of an anti-CII immunity is in some rats,7 mice8 and monkeys9 associated with the development of experimental arthritis. Whereas the relationship between disease and CII auto-immunity still remains unclear in man, the Abbreviations: CII, collagen type II; CRI, cross-reactive idiotope; ELISA, enzyme-linked immunosorbent assay, Ig, immunoglobulin. Correspondence: Dr C. Nordling, Dept. of Medical and Physiological Chemistry, Box 575, 75123 Uppsala, Sweden.

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Down-regulation of collagen arthritis MATERIALS AND METHODS Animals DBA/I mice, originally obtained from Jackson Laboratories (Bar Harbour, ME), were kept and bred at the Biomedical Center, Uppsala, Sweden. In the present experiments we used male mice at an age of 4-8 weeks.

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Monoclonal antibodies The DBA/l-derived hybridomas producing the IgGI monoclonal anti-idiotypic antibody denoted ClC3'6 and the control IgGI monoclonal antibody denoted El 1 (specific for human parathyroid cells)'7 were grown in vitro. The anti-idiotypic antibody CIC3 was purified by ammonium sulphate precipitation, followed by DEAE ion-exchange chromatography and Sephadex G200 gel chromatography.'8 The purification method was found to be critical for both antibody-affinity, detected in ELISA, and for effect on arthritis development. As previously shown,'5 the anti-idiotypic antibody ClC3 recognizes a crossreactive idiotope present on four out of five monoclonal IgG anti-collagen antibodies that all recognize different epitopes on the CII molecule, but is unreactive with Fab fragments of normal syngeneic IgG. Cl C3 is a not binding-site related antiidiotype. The control antibody E 1 was purified with protein ASepharose (Pharmacia, Uppsala, Sweden) affinity chromatography in a way that did not affect the binding characteristics. Fragmentation and characterization of antibody fragments Antibodies were fragmented with papain and characterized for purity from uncleaved material as described before.'5

Enzyme-linked immunoassays ELISA methods were used to analyse the serum levels of antibodies to native rat CII and the specificity of the purified anti-idiotypic antibodies. Microelisaplates (Dynatech, Plochingen, Germany) were coated over night at 40 with 50 pl of phosphate-buffered saline containing 10 pg/ml of native rat CII or of Fab fragments from the respective antibodies. Subsequently the sera or purified antibody fractions were added diluted in Tris-buffered saline (0- 13 M NaCl, 10 mm Tris HCI pH 7.4) and incubated over night at 4°. The binding of anti-CII antibodies or purified antibodies was detected with alkaline phosphatase conjugated to sheep anti-mouse IgG Fc antibodies. The amount of bound enzyme was quantified with p-nitrophenol as the substrate and measured at 405 nm in a Titertek multiscan spectrophotometer. All washings were made in Trisbuffered saline. Anti-CII antibodies were quantified by relating absorbance values to a standard curve made with purified antiCIT antibodies in a way slightly modified from the method described by Holmdahl et al.'9 Immunization, antibody treatment and scoring for arthritis The mice were immunized subcutaneously with 50 ,g of native CII that was dissolved in 0-1 M HAc and emulsified in Freund's complete adjuvant. The CII was purified from a rat chondrosardescribed earlier. '4 The C1C3 and El antibodies, respectively, were injected intraperitoneally 3 weeks before or 2 days after the CII immunization. The mice were bled from the tail on Day 28 after CII immunization and sera from individual mice were frozen and kept at 70° until they were simultaneously tested in

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Figure 1. ELISA test showing binding of purified anti-idiotypic antibody C1C3 to Fab fragments of anti-CI monoclonal antibody C2 (-) and to Fab fragments of normal, pooled syngeneic IgG (*).

ELISA for anti-CII activity. Clinical scoring for arthritis was carried out using a system where macroscopic inspection yields a score of 0-3 on each foot (1 point = swelling and erythema in a single joint; 2 points = swelling and erythema in more than one joint; 3 points = severe swelling of the entire paw) and where the summary score for the two forefeet and two hindfeet in arthritic animals is used as the indicator for arthritis development. Statistics Continuous variables were analysed by Student's t-test. Dichotomous variables were analysed by their proportionate group frequencies (chi-square test) and the significance of arthritic scores was analysed by the Mann-Whitney U-test.

RESULTS

Specificity of monoclonal anti-idiotypic antibody The anti-idiotypic antibody C I C3 has previously been shown to react with a CRI present on syngeneic anti-CIT antibodies. 15 The anti-idiotypic antibody was grown and purified as described in the Materials and Methods and was analysed in ELISA. From Fig. l it can be seen that the antibody binds to the Fab fragments ofmonoclonal anti-CIT antibody C2, which carries the CRI, and that it lacks affinity for Fab fragments of normal pooled syngeneic IgG. The control antibody E 11 does not react with anti-CIT or anti-anti-CII antibodies (data not shown). Effects of treatment with anti-idiotypic antibodies on the development of collagen arthritis Mice were treated with anti-idiotypic antibody 2 days after the immunization with CII, and in a control group mice were similarly treated with irrelevant antibodies (El 1). The mice were followed for arthritis development using macroscopic scoring for arthritis. The mice receiving the control antibody developed a severe polyarthritis, i.e. they closely followed the clinical course that has previously been noted for male DBA/l mice receiving CII immunization. As also can be seen from Fig. 2,

C. Nordling, R. Holmdahl & L. Klareskog

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suppression of the CII arthritis could be seen in the mice receiving the anti-idiotypic antibody. Among the mice that developed arthritis, there was no significant difference in severity of disease between the two groups. In another experiment mice were given the treatment with anti-idiotypic and control antibodies, respectively, 3 weeks before CII immunization. As can be seen from Fig. 3 this scheme of treatment significantly affected the severity of arthritis in the mice that were treated with anti-idiotypic antibody compared with the mice given the control antibody, although the incidence was not affected.

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Days after immunization

Figure 2. Development of CII arthritis in DBA/1 mice after treatment with the anti-idiotypic anti-anti-CII antibody CIC3 (-) and the irrelevant antibody E lI (0) 2 days after CII immunization. The severity of arthritis is shown as incidence of disease (22 mice were included in each experimental group). Levels of significance: X= P< 0 05.

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Down-regulation of collagen arthritis after in vivo treatment with a syngeneic monoclonal anti-idiotypic antibody to a cross-reactive idiotope on collagen II auto-antibodies.

Monoclonal anti-idiotypic antibodies previously shown to react with a cross-reactive idiotope of anti-collagen II auto-antibodies were used for in viv...
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