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Induction of Anti-RA33 Hnrnp Autoantibodies and Transient

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Induction of Anti-RA33 Hnrnp Autoantibodies and Transient Induction of anti-RA33 hnRNP autoantibodies and transient spread to U1-A snRNP complex of spliceosome by idiotypic manipulation with anti-RA33 antibody preparation in mice G. Steiner1,2, O. Shovman3, K. Skriner1,2, B. Gilburd4, P. Langevitz5, M. Miholits1,2, R. Hoet6, Y. Levy 3, G. Za n d m a n - G o dd a r d3, E. Hoefle r 7,J.S . Smolen1, 7 , Y. Shoenfel d 3, 4 , 5 1Division of Rheumatology, Department of Internal Medicine III and 2Institute of Biochemistry, University of Vienna, Austria; 3Department of Medicine B, 4Center for Autoimmune Diseases, and 5Division of Rheumatology, Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine Tel-Aviv University, Tel-Aviv; 6Department of Biochemistry, University of Nijmegen, The Netherlands; 7Second Department of Medicine, Lainz Hospital, Vienna, Austria. Abstract Objective Anti-RA33 antibodies occur in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD) and target the A2/B1 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex 4 which forms part of the spliceosome. The aim of the present study was to evaluate the immune response and pathological features induced in mice immunized with anti-RA33 antibodies or patient-derived recombinant single-chain variable fragments (scFv) of anti-RA33 antibodies. Methods In the first set of the experiment, two strains of mice (C57BL/6J and BALB/c) were immunized with IgG preparations obtained from two patients with RA and one normal donor. One of the patients had high titer anti-RA33 antibodies; the other one showed weak borderline reactivity. In the second set of the experiment three groups of C57BL/6J mice were immunized, respectively, with affinity-purified (AP) anti-RA33 antibodies, scFv of anti-RA33 antibodies and normal human IgG. The immunological response induced in immunized mice was studied by immunoblotting and line immunoassay (LIA). The presence of arthritis, serositis or myositis was assessed six-months following initial immunization. Results While anti-RA33 antibodies developed in only two of the mice immunized with different human IgG fractions, anti- RA33 antibodies were clearly detected in 7 sera of 13 mice immunized with AP anti-RA33 antibodies three months after the boost immunization and, moreover, also in 2 sera of 13 mice immunized with scFv of anti-RA33 antibodies. In contrast, mice immunized with normal human IgG did not develop anti-RA33 antibodies. Interestingly, transient autoantibody production against another nuclear autoantigen, U1 snRNP, was observed in 3 C57BL/6J mice immu- nized with scFv and in 1 mouse immunized with AP autoantibodies. However, these immunological responses were not associated with pathological findings. Conclusions Active immunization of naive mice with AP anti-RA33 antibodies and scFv of anti-RA33 antibodies resulted on the one hand in the production of murine anti-RA33 antibodies and led, on the other hand, to transient “autoantibody spread” to snRNP component of the spliceosome and other nuclear autoantigens. This “autoantibody spread” probably reflected disregulation of the idiotypic anti-idiotypic cascade. Key words Affinity purified anti-RA33 antibodies, recombinant single-chain variable fragments of anti-RA33 antibodies, rheumatoid arthritis, systemic lupus erythematosus, mixed connective tissue disease, hnRNP complex. Clinical and Experimental Rheumatology 2002; 20: 517-524. Anti-RA33 autoantibodies in mice / G. Steiner et al. #G. Steiner, MD; #O. Shovman, MD; Introduction N-terminus, and hnRNP B2 which is K. Skriner, MD; B. Gilburd, MD; Most connective tissue diseases are assumed to be another altern at ive ly P. Langevitz, MD; M. Miholits, MD; characterized by the presence of auto- spliced variant of hnRNP-A2/B1 (13, R. Hoet, MD; Y. Levy, MD; E. Hoefler, antibodies to nucleic acid binding pro- 14). Accordingly, the RA33 autoanti- MD; J.S. Smolen, MD; Y. Shoenfeld,MD. # These two authors contributed equally teins. The targets of these autoantibod- gen is actually composed of thre e to the work. ies are, in general, protein components hnRNP pro t e i n s : A 2 , B l , B 2 , wh i ch This study was supported by a grant from of multimolecular ri b o nu cl e o p ro t e i n m ay be designated the “RA33 com- the Austrian National Bank and by the ICP (RNP) structures (1,2). These proteins p l ex”. Subsequently, anti-RA33 anti- Programme of the Austrian Federal Min- f re q u e n t ly play an important role in bodies were also detected in sera from istry for Education, Science and Culture. various vital cellular functions such as patients with SLE and MCTD (14, 15). Please address correspondence to: D NA rep l i c ation and tra n s c ri p t i o n , Similar observations were made with Dr. Yehuda Shoenfeld, Department of RNA processing, or messenger RNA the closely related hnRNP Al protein Medicine ‘B’, Sheba Medical Center, (mRNA) translation (3). For example, (15, 16). Sackler Faculty of Medicine, Tel-Aviv p roteins of the small nu clear RNP The most interesting fact is that this University, Tel-Hashomer 52621, Israel. (snRNP) complexe s , wh i ch fo rm the s u b fa m i ly of hnRNP proteins rep re- E-mail: [email protected] major constituent of the spliceosome, sents the only known spliceosome as- Received on October 8, 2001; accepted are characteristically targeted by auto- s o c i ated autoantigen wh i ch is re c og- in revised form on April 17, 2002. antibodies from patients with mixe d nized by patients with RA, emphasiz- © Copyright CLINICAL AND connective tissue disease (MCTD) and ing the immu n o l ogical re l at i o n s h i p EXPERIMENTAL RHEUMATOLOGY 2002. systemic lupus ery t h e m atosus (SLE): between RA, SLE and MCTD (14, 16). thus, Ul snRNP specific antigens are H oweve r, in SLE and MCTD anti- re c og n i zed by virt u a l ly all pat i e n t s RA33 antibodies usually occur togeth- with MCTD and by 20-50% of patients er with antibodies to other components with SLE, whereas the Sm antigen (i. e. of the spliceosome - Ul snRNP or Sm the common snRNP proteins) is a very proteins, respectively (14, 16, 17). A si- s p e c i fic autoantigen in patients with milar association has been recently ob- SLE (4, 5). Other major components of served in lupus prone MRL/lpr mice, the spliceosome are heteroge n e o u s wh e reas in NZB/NZW mice neither nuclear RNP (hnRNP) particles which anti-RA33 nor anti-snRNP antibodies are much less well characterized than could be detected. (18). Remarkably, in snRNP (3). These hnRNP are compos- MRL/lpr mice antibodies to short pep- ed of newly transcribed pre-mRNA and tide epitopes of hnRNP-A2/Bl and Sm- ~30 different proteins (6). Six of these - D protein developed very early and pre- Al, A2, Bl, B2, Cl, and C2 - have been ceded antibodies to the whole proteins termed hnRNP “core” proteins. They and, even more importantly to dsDNA are structurally related and have molec- by several weeks. Therefore, in SLE ular weights (MW) of 34-43 kd (7). (and MCTD) anti-hnRNP and anti- In contrast to snRNPs, proteins of the snRNP (i.e. anti-spliceosomal) reactiv- hnRNP complex have long been ne- ities appear to be early and closely link- glected as potential autoantigens in ed immune responses (14, 17) wh i ch c o n n e c t ive tissue diseases (8, 9). In might represent some of the first auto- 1989 a new IgG autoantibody was de- immune events, at least in the particular tected in sera from patients with rheu- disease model of SLE. On the other matoid arthritis (RA), which was di- hand, the more restrictive response to rected against a nuclear protein with the spliceosome in RA patients may MW of 33kD, therefore this antibody reflect a different type of autoimmune was designated as anti-RA33 (10). Sev- response indicative of a probable insult eral experimental studies demonstrated of anti-RA33 in the pat h ogenesis of the RA33 antigen to be indistinguish- RA. Our objective in the present study able from the hnRNP-A2 pro t e i n , was to eva l u ate the effi c a cy of anti- thereby identifying a hnRNP protein as RA33 antibodies to induce immune re- an important autoantigen in rheumatic sponses and, p o s s i bly, p at h o l ogi c a l diseases (11, 12). A dditional studies symptoms in naive mice. showed that anti-RA33 antibodies rec- ognized also hnRNP Bl, an alternative- Materials and methods ly spliced va riant diffe ring from A 2 Materials only by a 12 amino acid insertion at the Sera were obtained from two patients 518 Anti-RA33 autoantibodies in mice / G. Steiner et al. with RA; one of them had high titer i m mu n i zed intra d e rm a l ly in the hind data obtained by immunoblotting the anti-RA33 antibodies, the other one foot pads with different IgG prepara- recently developed line immunoassay showed borderline reactivity, and from tions 10 µg/mouse) diluted in Freund’s ( I n n oge n e t i c s , G h e n t , B e l gium) wa s one additional healthy donor. None of complete adjuvant. Group 1 was immu- used which employs a panel of immo- the sera exhibited any reactivity to the nized with normal human IgG, group 2 bilized (mostly recombinant) antigens U1-A snRNP antigen.
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