Aberrant Expression of the Autoantigen Heterogeneous Nuclear Ribonucleoprotein-A2 (RA33) and Spontaneous Formation of Rheumatoid This information is current as Arthritis-Associated Anti-RA33 of September 27, 2021. in TNF- α Transgenic Mice Silvia Hayer, Makiyeh Tohidast-Akrad, Silva Haralambous, Beatrice Jahn-Schmid, Karl Skriner, Sylvie Trembleau,

Hélène Dumortier, Serafin Pinol-Roma, Kurt Redlich, Georg Downloaded from Schett, Sylviane Muller, George Kollias, Josef Smolen and Günter Steiner J Immunol 2005; 175:8327-8336; ; doi: 10.4049/jimmunol.175.12.8327

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Aberrant Expression of the Autoantigen Heterogeneous Nuclear Ribonucleoprotein-A2 (RA33) and Spontaneous Formation of -Associated Anti-RA33 Autoantibodies in TNF-␣ Transgenic Mice1

Silvia Hayer,* Makiyeh Tohidast-Akrad,† Silva Haralambous,‡ Beatrice Jahn-Schmid,§ Karl Skriner,2*¶ Sylvie Trembleau,ʈ He´le`ne Dumortier,# Serafin Pinol-Roma,** Kurt Redlich,* Georg Schett,* Sylviane Muller,# George Kollias,†† Josef Smolen,*†ʈ and Gu¨nter Steiner3*†¶ʈ

Human TNF-␣ transgenic (hTNFtg) mice develop erosive arthritis closely resembling rheumatoid arthritis (RA). To investigate

mechanisms leading to pathological autoimmune reactions in RA, we examined hTNFtg animals for the presence of RA-associated Downloaded from autoantibodies including Abs to citrullinated epitopes (anti-cyclic citrullinated peptide), heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (anti-RA33), and heat shock proteins (hsp) (anti-hsp). Although IgM anti-hsp Abs were detected in 40% of hTNFtg and control mice, IgG anti-hsp Abs were rarely seen, and anti-cyclic citrullinated peptide Abs were not seen at all. In contrast, >50% of hTNFtg mice showed IgG anti-RA33 autoantibodies, which became detectable shortly after the onset of arthritis. These Abs were predominantly directed to a short epitope, which was identical with an epitope previously described in MRL/lpr mice.

Incidence of anti-RA33 was significantly decreased in mice treated with the osteoclast inhibitor osteoprotegerin and also in http://www.jimmunol.org/ c-fos-deficient mice lacking osteoclasts. Pronounced expression of hnRNP-A2 and a smaller splice variant was seen in joints of hTNFtg mice, whereas expression was low in control animals. Although the closely related hnRNP-A1 was also overexpressed, autoantibodies to this protein were infrequently detected. Because expression of hnRNP-A2 in thymus, spleen, brain, and lung was similar in hTNFtg and control mice, aberrant expression appeared to be restricted to the inflamed joint. Finally, immunization of hTNFtg mice with recombinant hnRNP-A2 or a peptide harboring the major B cell epitope aggravated arthritis. These findings suggest that overproduction of TNF-␣ leads to aberrant expression of hnRNP-A2 in the rheumatoid joint and subsequently to autoimmune reactions, which may enhance the inflammatory and destructive process. The Journal of Immunology, 2005, 175: 8327–8336. by guest on September 27, 2021

he pathogenesis of rheumatoid arthritis (RA)4 is still un- class II genes, the frequent presence of autoantibodies, and the resolved. Although effects mediated by proinflammatory efficacy of T cell-directed therapies suggest involvement of T cytokines are pivotal in the development of this chronic the adaptive immune response, primarily in the initial phases of the destructive disorder (1), the strong association of RA with MHC disease but also in its subsequent course (2–4). With respect to autoimmune responses, apart from rheumatoid factor (RF), i.e., to IgG, autoantibodies to a variety of Ags have been *Department of Rheumatology, Internal Medicine III, Medical University of Vienna, Vienna, Austria; †Ludwig Boltzmann Institute for Rheumatology and Balneology, Vi- described in recent years (5). In particular, the discovery of auto- enna, Austria; ‡Laboratory of Molecular Genetics, Hellenic Pasteur Institute, Athens, antibodies to citrullinated proteins such as fibrin or vimentin in Greece; §Institute of Pathophysiology, Medical University of Vienna, Vienna, Austria; ʈ patients with RA was one of the most important finding in rheu- ¶Institute of Medical Biochemistry, Medical University of Vienna, Vienna, Austria; Cen- ter of Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; #In- matology research, pointing to the potential importance of post- stitute of Molecular and Cellular Biology, Centre National de la Recherche Scientifique, translational modifications in the generation of autoepitopes (6, 7). Strasbourg, France; **Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, NY 10029; and ††Institute of Immunology, Biomedical Sciences Another well-characterized autoantigen of interest is the heteroge- Research Center “Alexander Fleming,” Vari, Greece neous nuclear ribonucleoprotein (hnRNP)-A2, also known as the Received for publication May 5, 2005. Accepted for publication October 3, 2005. RA33 Ag. Autoantibodies to this protein (anti-RA33) are detect- The costs of publication of this article were defrayed in part by the payment of page able in one third of RA patients and show similar specificity for charges. This article must therefore be hereby marked advertisement in accordance RA as RF (8, 9). Importantly, pronounced Th1-like reactivity to with 18 U.S.C. Section 1734 solely to indicate this fact. hnRNP-A2 has recently been described to occur in ϳ50% of RA 1 This work was supported by the Center of Molecular Medicine of the Austrian Academy of Sciences, Vienna. patients but not in disease controls, suggesting possible pathogenic 2 Current address: Department of Rheumatology and Clinical Immunology, Charite´ involvement of this autoreactivity (10). University Medicine, Humboldt University and Free University of Berlin, Berlin, Autoantibodies may be already present very early or even years Germany. before the onset of disease, as recently demonstrated for RF and 3 Address correspondence and reprint requests to Dr. Gu¨nter Steiner, Department of anti-cyclic citrullinated peptide (CCP) Abs (11, 12). This indicates Rheumatology, Internal Medicine III, Medical University of Vienna, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria. E-mail address: [email protected] a role of such autoimmune response in the pathogenesis of RA, but 4 Abbreviations used in this paper: RA, rheumatoid arthritis; RF, rheumatoid factor; a definitive proof for this is still lacking. In contrast, there is abun- hnRNP, heterogeneous nuclear ribonucleoprotein; CCP, cyclic citrullinated peptide; dant evidence for the role of cytokines in RA synovitis and the hsp, heat shock protein; hTNF, human TNF; tg, transgenic; OPG, osteoprotegerin; ␣ TRAP, tartrate resistant acid phosphatase; RRM, RNA recognition motif; snRNP, ensuing joint destruction. In particular, TNF- , IL-1, and IL-6 ap- small nuclear RNP; wt, wild type; SLE, systemic erythematosus. pear to constitute the most important proinflammatory mediators

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 8328 DEVELOPMENT OF AUTOANTIBODIES IN TNF␣ TRANSGENIC MICE of the RA process. The role of TNF-␣ is undisputed, given its Histological analysis abundant presence in the joint and the successful therapeutic in- Serial paraffin sections (1–2 ␮m) of hind and front paws and knees were tervention with biological agents inhibiting TNF-␣ (1). Another stained with H&E for histological analyses of arthritis. Areas of inflam- important piece of evidence for the pivotal role of TNF-␣ in RA mation and bone erosion were counted in mm2. To investigate expression pathogenesis stems from the observation that mice carrying human of hnRNP-A2, paraffin sections were boiled in 0.01 M Na-citrate buffer (pH TNF-␣ as transgene develop a severe erosive inflammatory poly- 6) in a microwave oven for 2 min at 780 W and for 10 min at 180 W. After cooling to room temperature, sections were stained with the 10D1 mAb arthritis demonstrating typical features of human RA, such as sy- (0.7 mg/ml) directed to hnRNP-A2 diluted 1/100–1/200 (10, 21). For de- novial hypercellularity, inflammatory infiltrates, pannus formation, tection of osteoclasts, sections were stained for tartrate-resistant acid phos- cartilage destruction, bone erosions, and, finally, crippling of paws phatase (TRAP) using the Leukocyte Acid Phosphatase staining kit (Sig- (13). Other experimental models in which arthritis is triggered by ma-Aldrich) as described previously (16). defined Ags, such as type II collagen, and in which proinflamma- tory cytokines including TNF-␣ and IL-1 also play a major role, Recombinant hnRNP-A2 and synthetic peptides provide evidence for the importance of (auto)immune mecha- For cellular stimulation and immunization experiments, the B1 splice vari- nisms. Most noteworthy, autoimmunity is clearly the trigger in the ant of hnRNP-A2 (hnRNP-A2/B1) was used, which differs from KRN ϫ NOD model in which an autoreactive transgenic TCR hnRNP-A2 by a 12-aa insertion close to the N terminus. For stimulation assays, His-tagged hnRNP-A2/B1 was used (10); for immunizations, drives formation of autoantibodies to the enzyme glucose-6-phos- highly purified untagged recombinant protein (manufactured by BioMay) phate isomerase, leading to a severe and destructive arthritis was used. For characterization of Ab binding regions, three overlapping (14, 15). recombinant fragments were used that contained one or both RNA recog-

ϩ Downloaded from Despite these compelling data, the initial processes leading to nition motifs (RRM): RRM1 (aa 1–89), RRM2 (aa 80–182), and RRM1 2 (aa 1–182) (22). For fine mapping of epitopes, a series of 13 overlapping loss of tolerance against “self” proteins in autoimmune diseases synthetic peptides covering the N-terminal portion of hnRNP-A2 (aa are still in the dark. Thus, it is not clear which events lead to 1–206) was used as described previously (23). Two selected peptides activation of autoreactive T cells and the formation of autoanti- (p50-70 and p140-160) were used for immunization of hTNFtg and bodies and why only a limited number of Ags appear to be targeted control mice. in RA. In previous studies (16, 17), we have extensively used human TNF-␣ transgenic (hTNFtg) mice to investigate molecular Detection of autoantibodies by Western blotting http://www.jimmunol.org/ and cellular mechanisms of tissue destruction. In the course of Autoreactivities against nuclear proteins were analyzed by immunoblot- these studies, we became interested to know whether TNF-␣-me- ting, using HeLa nuclear extracts as described previously (8). Nitrocellu- diated inflammation could lead to loss of tolerance against self lose membranes were cut into strips and blocked with blocking buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, 3% nonfat components. Therefore, we investigated sera of hTNFtg mice for dried milk) for1hatroom temperature. Strips were incubated for 45 min the presence of autoantibodies typically found in RA including at room temperature with serum diluted 1/50 in blocking buffer. Human Abs to citrullinated epitopes, hnRNP-A2, and heat shock proteins autoimmune sera served as positive controls for detection of autoantibodies (hsp). The data obtained reveal the frequent presence of IgG anti- to hnRNPs, Sm, U1, small nuclear RNP (snRNP), Ro, La, topoisomerase I, and ribosomal P proteins. After washing, strips were incubated for 30 RA33 autoantibodies in the sera of arthritic animals, whereas IgG min with alkaline phosphatase-conjugated anti-mouse IgG or anti-mouse by guest on September 27, 2021 Abs to citrullinated epitopes were not detectable, and IgG anti-hsp IgM secondary Ab (Accurate Chemical & Scientific) diluted 1/1000 in Abs were rarely seen. Furthermore, hnRNP-A2 was highly over- blocking buffer. To quantitate the Ab response, selected sera showing mod- expressed in the joint of hTNFtg mice, and immunization of these erate to high reactivity were serially diluted (up to 1/800) and probed with hnRNP-A2 partially purified from HeLa cells, as described previously (8). mice with the recombinant protein aggravated disease, suggesting For detection of autoantibodies to stress proteins, 10 ␮g each of recom- that TNF-␣-driven autoimmune reactions to hnRNP-A2 may con- binant mouse hsp60, mycobacterial hsp65, rat hsp70, and hamster hsp78 tribute to the inflammatory and destructive processes in the ar- (BiP) obtained from StressGen Biotechnologies were separated by SDS- thritic joint. PAGE on preparative minigels (Bio-Rad) and transferred to nitrocellulose membranes; transfer efficiency was controlled by staining the membranes with Ponceau S. A mAb to hsp60 (clone LK1; StressGen Biotechnologies) served as positive control. Materials and Methods Animals Detection of autoantibodies by ELISA Tg197 hTNFtg mice carrying a 3ЈUTR-modified human TNF (hTNF)-␣ Mouse sera were analyzed for the presence of autoantibodies to citrulli- gene construct were used throughout this study (18). These mice develop nated epitopes by a commercial ELISA (Axis Shields Diagnostics) in a severe erosive inflammatory arthritis starting within 4–6 wk of age. In which a CCP is used as Ag. The assay was used according to the manu- addition, sera from Tg5453 hTNFtg mice expressing only membrane- facturer’s instructions, except that HRP-conjugated anti-mouse IgG or IgM bound hTNF-␣ (19) and sera from the previously described Tg197 ϫ (Accurate Chemical & Scientific) diluted 1/2,000 was used as secondary c-fosϪ/Ϫ mice (16) that lack a functional c-fos gene were analyzed. Control Ab; anti-CCP-positive human sera served as positive controls. Occurrence strains included non-tg (transgenic) CBA ϫ C57BL/6 littermates, BALB/c, of Abs in mice immunized with recombinant hnRNP-A2 was monitored by C57BL/6, and DBA/1 mice obtained from Harlan-Winckelmann. All ani- ELISA, using 2 ␮g of Ag per well and HRP-conjugated anti-mouse IgG mal procedures were approved by the local ethics committee. Treatment of (Accurate Chemical & Scientific) diluted 1/2,000 as secondary Abs. Re- Tg197 mice with anti-TNF-␣ mAb (Infliximab; Centocor) and osteopro- activities to peptides were measured by ELISA as described previously tegerin (OPG; Amgen) has been described in detail previously (20). (23). Briefly, microtiter plates (Falcon) were coated overnight at 37°C with 2 ␮M of each peptide diluted in 0.05 M carbonate buffer (pH 9.6) and blocked by adding PBS containing 0.05% Tween 20 and 0.5% BSA. As a Clinical assessment control, mouse sera were also tested in a noncoated well incubated with coating buffer. Serum dilution was 1/1,000, and HRP-conjugated goat anti- Clinical evaluation was performed in a blinded manner between 4 and 9 wk mouse IgG (Jackson ImmunoResearch Laboratories) diluted 1/20,000 was of age, as described previously (16). Briefly, joint swelling was examined used as secondary Ab. using a clinical score graded from 0 to 3 (0, no swelling; 1, mild swelling; 2, moderate swelling; 3, severe swelling of toes and ankle). In addition, Indirect immunofluorescence grip strength of each paw was analyzed on a wire 3 mm in diameter, using a score from 0 to Ϫ4 (0, normal grip; Ϫ1, mildly reduced grip strength; For detection of antinuclear IgG Ab, a commercial HEp-2 test kit (Hema- Ϫ2, moderately reduced grip strength; Ϫ3, severely reduced grip strength; gen Diagnostics) was used. Sera were initially diluted 1/25 in PBS and Ϫ4, no grip strength at all). incubated for1hatroom temperature in a humid chamber. After washing, The Journal of Immunology 8329 slides were incubated with FITC-conjugated rabbit anti-mouse IgG (Da- automatic cell-harvesting device (Packard Instrument), and DNA-incorpo- koCytomation) diluted 1/40 for 30 min and subsequently analyzed by flu- rated radioactivity was measured using a Matrix 9600 direct beta counter orescence microscopy. Sera were obtained from hTNFtg mice either pos- (Packard Instrument). For control stimulations, cells were incubated with 5 itive (n ϭ 6) or negative (n ϭ 2) for anti-RA33 Ab, non-tg littermates (n ϭ ␮g/ml Con A (Sigma-Aldrich). 2), BALB/c mice immunized with hnRNP-A2 (n ϭ 2), and naive BALB/c mice (n ϭ 2). Statistical analysis Ϯ Analysis of protein expression in joints and organs by Western Histological data are given as mean SEM, and group mean values were compared by unpaired two-tailed Student’s t test (see Fig. 5). Differences blotting between groups in the prevalence of anti-RA33 Ab were calculated using After removal of skin and muscles, synovial tissue from knee joints and Fisher’s exact test (see Table II). tarsal areas of hind and front paws were mechanically homogenized in Results Schreiber buffer (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1.5 mM MgCl2,1 mM DTT, 1 mM EDTA, 0.1 mM EGTA, 20% glycerol) using an Ultra- Spontaneous autoimmune response to hnRNP-A2 in hTNFtg Thurrax homogenizer (24). Mouse organs were homogenized in the same mice buffer. The tissue extracts were then centrifuged for 15 min at 14,000 rpm. Joint (300 ␮g) and organ extracts (200 ␮g) were separated by SDS-PAGE To elucidate pathways leading to loss of tolerance and formation on minigels (joint extracts) or large gels (organ extracts) and transferred of autoantibodies in RA, we were interested whether immune re- onto nitrocellulose membranes. Membranes were blocked for1hatroom activities against autoantigens commonly targeted by patients with temperature and subsequently incubated for1hatroom temperature under RA occur in hTNFtg mice, an animal model of inflammatory ero- constant shaking with mAb directed to hnRNP-A2 (10D1, diluted 1/500 in ␣ blocking buffer), hnRNP-A1 (4B10 (Ref. 25), diluted 1/500), the A protein sive arthritis that is entirely driven by TNF- (13). Therefore, we of the U1 snRNP (9A9 (Ref. 26), diluted 1/100), or a rabbit anti-actin Ab investigated sera from two hTNFtg strains for the presence of IgG Downloaded from (diluted 1/250; Sigma-Aldrich). After washing, the membrane was incu- and IgM autoantibodies to citrullinated epitopes (anti-CCP), bated for 45 min at room temperature with a HRP-conjugated secondary hnRNP-A2 (anti-RA33), and four different stress proteins includ- Ab (DakoCytomation) diluted 1/2000 in blocking solution. After washing, immunostained proteins were visualized using the ECL detection kit (ECL ing murine hsp60, mycobacterial hsp65, rat hsp70, and hamster Western Blotting Detection Reagents; Amersham Biosciences). hsp78 (BiP). Strains under investigation were strain Tg197 ex- pressing hTNF-␣ in both soluble and membrane-bound form and Immunization experiments strain Tg5453 expressing only the transmembrane form (18, 19). http://www.jimmunol.org/ Tg197 mice were immunized s.c. with recombinant hnRNP-A2/B1 or syn- Using the commercially available anti-CCP ELISA, neither IgG thetic peptides emulsified in CFA (Sigma-Aldrich) at 4 wk of age and nor IgM Abs to citrullinated epitopes were found (Table I). In boosted 3 wk later with the same amount of Ag in IFA (Sigma-Aldrich). contrast, IgM Abs to stress proteins were detected by immuno- One group of hTNFtg mice was immunized with 100 ␮g of recombinant hnRNP-A2/B1 protein, one with 30 ␮g of peptide p50-70, and one with 30 blotting in 40% of hTNFtg and control sera and were predomi- ␮g of peptide p140-160. Controls included mice treated with PBS or ad- nately directed to hsp70 and hsp78 (data not shown). IgG Abs, juvant and untreated mice. Each group consisted of five animals. Disease however, were observed in only 7% of hTNFtg sera and not at all activity was assessed weekly by evaluating paw swelling and grip strength, in control sera (Table I). as described previously (16). Nine-week-old mice were sacrificed by In contrast to citrullinated Ags and hsps, pronounced IgG cervical dislocation, and blood was taken by heart puncture. In addition, immunizations were performed in mouse strains with different MHC- anti-RA33 reactivities were detected by immunoblotting in sera of by guest on September 27, 2021 backgrounds BALB/c (H-2d), CBA (H-2k), C57BL/6 (H-2b), and in arthri- hTNFtg mice. Interestingly, the autoantibody response was more tis-prone DBA/1 mice, which were additionally immunized with chicken prevalent and more pronounced in the transmembrane strain (Ta- type II collagen. ble I). Anti-RA33 Abs first became detectable at the age of 8 wk Cellular assays shortly after the onset of arthritis. Titer and incidence of Abs increased until the age of 14–16 wk when animals were sacrificed Lymph node and spleen cells were isolated by standard procedures and (Fig. 1, A and B). Thus, at this late stage, 50–70% of the mice were resuspended at a concentration of 5 ϫ 106 cells/ml in culture medium, and 100 ␮l of this suspension were added to microtiter wells (Costar) contain- positive for IgG anti-RA33 Abs with titers ranging between 1/100 ing 100 ␮l of medium, with different concentrations of protein or peptides and Ͼ1/800, whereas IgM Abs were generally low-titered (1/50– as described previously (23). Each concentration was tested in triplicate, 1/100) and less frequently seen. Interestingly, IgG anti-RA33 Abs and tests were repeated at least three times in independent experiments. were only weakly reactive with the recombinant protein, whereas After 24 h, 50 ␮l of supernatant were taken to analyze for the production of IFN-␥, IL-2, or IL-4 by ELISA (BD Pharmingen). After 54 h, the cul- IgM Abs were similarly reactive with the natural and recombinant tures were pulsed for 18 h with 1 ␮Ci/well [3H]thymidine (Amersham Ag (data not shown). Anti-RA33-positive sera produced a large Biosciences). The cells were subsequently harvested on filters using an speckled nuclear pattern on Hep-2 cells (Fig. 1C) that is known as

Table I. IgG autoantibodies to hnRNP-A2 (anti-RA33), citrullinated epitopes (anti-CCP), and hsp (anti-hsp) in hTNFtg micea

Age Mouse Strain No. (weeks) Anti-RA33 Anti-CCP Anti-hsp

Tg197 64 8–14 25 (39%) 0/20 ND Tg5453 91 8–16 55 (60%) 0/40 3/40 (7%) C57BL6 ϫ CBA 20 8–14 0 0/20 0/15 DBA/1-CIA 10 14 0 ND ND DBA/1-control 10 14 0 ND ND

a Sera from strain Tg197 (expressing both soluble and membrane-bound hTNF-␣) and strain Tg5453 (expressing only membrane-bound hTNF-␣), non-tg littermates (C57BL6 ϫ CBA), and from DBA/1 mice immunized with collagen II (DBA/ 1-CIA) were investigated by Western blotting for the presence of anti-RA33 autoantibodies. In addition, anti-CCP Abs were measured by ELISA in 80 hTNFtg and control mice, and Abs to hsps including murine hsp60, mycobaterial hsp65, rat hsp70, and hamster hsp78 were determined by Western blotting in 55 animals. Anti-RA33 Abs were more prevalent and generally higher titered in strain Tg5453 (1/100–Ͼ1/800 in older animals), whereas titers of Tg197 mice did not exceed 1/400. IgG anti-hsp Abs were reactive with hsp70 and hsp65, IgM Abs (which were detected in 40% of hTNFtg and wt mice) reacted predominantly with hsp70 and hsp78. Data are given as numbers and percentages (in brackets) of positive sera. 8330 DEVELOPMENT OF AUTOANTIBODIES IN TNF␣ TRANSGENIC MICE

recognized by ϳ15% of the sera, most of which contained also pronounced reactivities to hnRNP-A2. Of note, nuclear Ags com- monly targeted by patients with other systemic autoimmune dis- eases were not recognized by these sera, including Sm and U1 snRNP Ags, Ro, La or topoisomerase I. No significant difference in disease onset and severity was seen between mice with and without autoantibodies. Because in previous investigations RF had also not been detectable (18, 19), anti-RA33 appeared to be the only dominant RA-associated autoreactivity in hTNFtg animals.

Epitope recognition To characterize the autoimmune response to hnRNP-A2 in more detail, epitope mapping studies were performed using recombinant fragments encompassing either both RRMs (aa 1–182), or RRM1 (aa 1–89), or RRM2 (aa 80–182), as well as 13 overlapping pep- tides covering the N-terminal portion of the protein (aa 1–206). All sera reacted with the fragment containing both RRMs in a similar manner as with the complete protein, indicating that the C-terminal portion (which could not be expressed separately due to its high Downloaded from glycine content) did not contain a major epitope (data not shown). Subsequent peptide mapping studies led to identification of a dom- inant epitope between aa 50 and 70, which was recognized by the sera of all 11 mice tested (aged between 8 and 16 wk) but not by sera from five control animals (Fig. 2). Interestingly, in previous http://www.jimmunol.org/

FIGURE 1. Autoantibodies to hnRNP-A2 in sera of hTNFtg mice. A, Sera from wt control and hTNFtg mice were analyzed for the presence of IgG autoantibodies against nuclear proteins (from HeLa cells) by immu-

noblotting. Control mice showed no reactivities (lanes 1–5), whereas by guest on September 27, 2021 hTNFtg mice developed Abs against hnRNP-A2 in the course of disease: no reactivities can be seen in sera from mice 4 wk old (lanes 6–10), whereas they are visible in three sera of mice aged 8 wk (lanes 11–15) and very pronounced in older animals suffering from severe arthritis (lanes 16–20). Sera stain the characteristic hnRNP-A2/B1/B2 triplett and addi- tionally a 40-kDa protein presumably corresponding to hnRNP-A3. This pattern is similar to that produced by the human autoimmune serum shown in the last lane. In addition, some sera (week 8, 1st serum; week 14, 2nd and 3rd serum) stain the characteristic hnRNP-A1 band migrating below the hnRNP-A2 band. B, Sequential sera of three hTNFtg mice analyzed at week 5, 7, 9, 12, and 14. All mice became reactive with hnRNP-A2, and mouse 1 also reacted with hnRNP-A1. A human serum from a patient with RA is shown at right. C, Detection of antinuclear Abs by indirect immu- nofluorescence. Sera from a hTNFtg mouse positive for anti-RA33, a hTNFtg mouse negative for anti-RA33, a BALB/c mouse immunized with hnRNP-A2, and a naive BALB/c mouse were analyzed using Hep-2 cells as substrate. A speckled nuclear staining pattern is produced by the serum of the positive hTNFtg mouse and by the serum of the immunized BALB/c mouse; titers of hTNFtg sera ranged from 1/25 to 1/400.

FIGURE 2. Epitope mapping of hnRNP-A2. A, Schematic representa- nuclear matrix pattern and typically produced by Abs to hnRNP tion of hnRNP-A2. The protein shows a modular structure consisting of proteins (27). A similar pattern was obtained with sera from two conserved RRMs and a glycine-rich auxiliary domain. The two most BALB/c mice immunized with hnRNP-A2 (Fig. 1C). Mouse sera conserved parts of RRM1 and RRM2 are indicated by dark gray bars. The recognized protein bands of 36, 38, and 39 kDa corresponding to 13 peptides overlapping by 5–10 aas used for epitope mapping are shown. hnRNP-A2, and its alternatively spliced variants hnRNP-B1 and The two peptides used for immunization of hTNFtg mice are in black. Numbers indicate the N- and C-terminal amino acids of each peptide. B, -B2, and a 40-kDa protein that comigrated with the closely related ELISA reactivities to p50-70 and p140-160 in Tg197 (1–11) and age- hnRNP-A3 (28). Thus, hTNFtg mice showed a similar reactivity matched non-tg control mice (12–16). Values are given in arbitrary units, pattern as patients with RA (8). As can be seen in Fig. 1, apart from with 1000 U corresponding to an OD value of 1.0. All Tg197 mice clearly this set of proteins only a few, if any, other (nuclear) proteins were showed reactivity to p50-70 but did not react with p140-160, which con- recognized. The double band migrating below the hnRNP-A2 band tains the same conserved motif as p50-70. None of the control mice showed corresponded to the closely related hnRNP-A1 (29) and was reactivity to any of the 13 peptides tested. The Journal of Immunology 8331 studies, the same sequence had been found to harbor a major specific T cell reactivity could be detected in these primary culture epitope recognized by lupus-prone MRL/lpr mice (23). Two sera assays, in contrast to immunized control animals in which both T were additionally reactive with peptide p90-116 located in the N- and B cell responses were readily detectable (data not shown). terminal part of RRM2. Of note, neither of these two peptides was recognized by non-tg control mice immunized with recombinant hnRNP-A2 is overexpressed in the joints of hTNFtg mice hnRNP-A2, confirming previous observations (23). Investigations previously performed in human synovial tissue had revealed hnRNP-A2 to be highly overexpressed in synovial tissue Therapeutic interventions reduce autoimmune reactivity against of RA patients (10). To study expression in mouse joints, tissue hnRNP-A2 sections of hTNFtg and wt control mice were analyzed by immu- To elucidate the effects of anti-inflammatory and antidestructive nohistochemistry. Although in the joints of control animals ex- therapies on the spontaneous autoimmune response to hnRNP-A2, pression of hnRNP-A2 was hardly detectable, massive expression mouse sera collected during previous therapeutic trials were as- was seen in the inflamed joints of hTNFtg mice (Fig. 3, A and B). sessed for the presence of anti-RA33 autoantibodies. Sera were The protein was highly expressed in synovial macrophages and from mice treated with either an anti-TNF-␣ mAb (Infliximab) or fibroblasts, particularly at sites where synovial tissue invades the OPG, the natural inhibitor of osteoclast differentiation and prolif- bone and also in chondrocytes of articular cartilage. Pronounced eration. As reported previously, pannus formation and bone de- expression was also seen in multinucleated TRAP-positive oste- struction were largely reduced by these treatments, whereas in- oclasts close to the cartilage-pannus junction (Fig. 3, C and D). flammation was inhibited only by the anti-TNF-␣ mAb but not by To further investigate the aberrant expression of hnRNP-A2 in OPG (20). However, both therapies affected autoantibody forma- the joints of hTNFtg mice, protein extracts were prepared from the Downloaded from tion: whereas autoantibodies were observed in Ͼ50% of untreated knees and hind paws of hTNFtg and wt mice and analyzed by mice, their incidence was significantly lower in both therapy immunoblotting. This analysis confirmed the immunohistochem- groups (Table II). To get further insight into the underlying mech- istry data, because the 10D1 mAb stained several protein bands in anism, hTNFtg mice lacking a functional c-fos gene were exam- extracts from hTNFtg mice, whereas no staining was seen in ex- ined for the presence of anti-RA33 autoantibodies. Osteoclast dif- tracts from wt animals (Fig. 4A). However, the expression pattern ferentiation is impaired in c-fos-deficient mice, and consequently was different from that obtained with a HeLa nuclear extract: http://www.jimmunol.org/ c-fos-deficient hTNFtg mice do not develop bone erosions, whereas in HeLa cells four bands were stained (hnRNP-A2, its whereas joint inflammation is not reduced (16). The incidence of splice variants hnRNP-B1/B2, and the 40-kDa protein), neither anti-RA33 autoantibodies was indeed decreased in c-fos-deficient hnRNP-B2 nor the 40-kDa band were detected in the joint extract. Tg197 mice, confirming the results obtained with OPG-treated an- In contrast, and in contrast to HeLa cells, a smaller variant with an imals. Thus, these data suggested that osteoclasts may be involved estimated molecular mass of 32-kDa appeared to be almost as in the generation of the autoimmune response to hnRNP-A2. strongly expressed as hnRNP-A2. This protein presumably repre- sented a recently described hnRNP-B1 splice variant lacking exon Cellular responses 9 (31). To study spontaneous T cell responses to hnRNP-A2, Tg197, and To control for specificity of expression, we additionally inves- by guest on September 27, 2021 non-tg controls were sacrificed at different stages of the disease (8 tigated expression of two structurally and/or functionally related wk, 13 wk, and 15 wk). Non-tg mice immunized with hnRNP- nuclear proteins: hnRNP-A1, which shows 70% sequence identity A2/B1 served as positive controls. Spleen cells and peripheral with hnRNP-A2 (29); and the A protein of the U1 snRNP (U1-A), lymph node cells were stimulated with 5 ␮g/ml recombinant Ag or which, except for the presence of two RRMs, is structurally unre- purified protein derivative as control, and proliferation as well as lated to hnRNP-A2 but colocalizes with hnRNP proteins in spli- production of IFN-␥ was determined. As compared with cells de- ceosomal complexes (32). This analysis revealed hnRNP-A1 to be rived from wild-type (wt) animals, the mitogen (Con A)-induced overexpressed in the joint in a similar manner as hnRNP-A2, response of Tg197 cells was reduced by ϳ20%. However, neither whereas expression of U1-A was clearly observed in joints of both hnRNP-A2/B1 nor purified protein derivative elicited proliferative wt and hTNFtg mice and appeared to be only slightly increased responses, and T cell cytokines such as IFN-␥ or IL-4 were not hTNFtg animals (Fig. 4B). detected in culture supernatants. Because chronic exposure to Finally, we analyzed expression of hnRNP-A2 (and cross-react- TNF-␣ renders T cells anergic (30), assays were performed also in ing proteins) in mouse organs. Protein extracts from thymus, the presence of neutralizing anti-TNF-␣ mAb, which, however, spleen, liver, kidney, brain, lung, heart, and skeletal muscle were had no or only insignificant effects on proliferation. Thus, no Ag- separated by SDS-PAGE and analyzed by immunoblotting (Fig.

Table II. Anti-RA33 autoantibodies in hTNFtg mice treated with anti-TNF mAb, OPG, or lacking a functional c-fos genea

Anti-RA33 Positive

Animals nn% p valueb

Anti-TNF mAb treated 20 3 15 0.001 OPG treated 8 1 12 0.05 c-fos deficient 13 2 15 0.035 Controls for anti-TNF trial 20 14 70 Controls for OPG and c-fos trial 24 13 54

a Mice were treated with either an anti-TNF-␣ mAb (Infliximab) or recombinant OPG, and anti-RA33 autoantibodies were measured by Western blotting at the end of the trial; in addition, autoantibodies were determined in c-fos-deficient Tg197 mice (16). A significant reduction of anti-RA33 autoreactivity was observed in all three groups. b Statistical significance vs untreated control group determined by Fisher’s exact test. 8332 DEVELOPMENT OF AUTOANTIBODIES IN TNF␣ TRANSGENIC MICE Downloaded from

FIGURE 3. Immunohistochemical analysis of hnRNP-A2 expression in the joints of wt and hTNFtg mice. A, Joint of a wt mouse showing a monolayered synovial membrane surrounding the joint and articular carti- lages covering healthy bones. Expression of hnRNP-A2 is virtually unde- tectable. B, Joint of a hTNFtg mouse characterized by a highly inflamma- tory multilayered pannus invading and eroding subchondral bone at sites of http://www.jimmunol.org/ bone-cartilage junctions (arrow). Massive overexpression of hnRNP-A2 (brown staining) can be seen in inflammatory pannus tissue and also in chondrocytes of articular cartilage. C and D, Serial sections stained for hnRNP-A2/B1 (brown color; C) and TRAP (purple color; D). Pronounced expression of hnRNP-A2/B1 can be seen at the site where the pannus invades into subchondral bone. The majority of stained cells appear to be synovial fibroblasts and macrophages, but staining can also be observed in some large multinucleated cells showing an osteoclast-like phenotype as FIGURE 4. Immunoblot analysis of hnRNP-A2 expression in joint and revealed by positive TRAP staining (arrow). Original magnification is organs of wt and hTNFtg mice. A, Expression of hnRNP-A2 in the joint.

ϫ100 (A and B) and ϫ400 (C and D). Protein extracts were prepared from knees and hind paws of wt and by guest on September 27, 2021 hTNFtg mice and separated by SDS-PAGE on minigels (300 ␮g/lane). For comparison, a HeLa nuclear extract is shown on the left. No expression can be seen in extracts from wt mice, whereas the mAb 10D1 stains three distinct bands in extracts from hTNFtg animals corresponding to hnRNP- 4C). The most pronounced expression of hnRNP-A2 was detected A2, hnRNP-B1, and the 32-kDa splice variant of hnRNP-B1. B, Compar- in the thymus and spleen. In these extracts, the mAb recognized ison of expression of hnRNP-A2 (1st panel), hnRNP-A1 (2nd panel), and the characteristic triplett (hnRNP-A2/B1/B2) plus a 40-kDa pro- the U1A protein of the U1 snRNP (3rd panel). Joint extracts from three tein that appeared to be identical with the 40-kDa protein observed hTNFtg and three wt mice were probed with mAb to these proteins. Ex- in HeLa extracts. Remarkably, however, the 32-kDa variant was pression of actin served as loading control (4th panel). Both hnRNP-A2/B1 not detected. Strong expression of hnRNP-A2 was further detected and hnRNP-A1 (and the A1b splice variant) are overexpressed in joints of hTNFtg mice, whereas expression of the U1A protein appears to be only in lung and brain, whereas no expression was seen in heart and slightly increased in hTNFtg mice. C, Expression of hnRNP-A2 in organs. skeletal muscle. In lung the 32-kDa hnRNP-B1 variant was ex- Organ extracts were prepared from spleen, thymus, lung, kidney, liver, pressed, whereas in brain a 31-kDa protein was detected, which heart, skeletal muscle, and brain, and separated by SDS-PAGE on large was most likely identical with a recently described hnRNP-A2 gels (200 ␮g/lane). In spleen, lung, thymus, and brain, pronounced staining splice variant lacking exon 9 (31). In brain a 41-kDa band was seen of bands corresponding to hnRNP-A2/B1/B2 can be seen. In addition, a additionally, which appeared to be selectively expressed in kidney 40-kDa band is strongly stained in thymus and brain and weakly in spleen and liver, whereas expression of hnRNP-A2/B1/B2 was undetect- and lung. In brain a 41-kDa band is detected, which is the only band visible able in these two organs. Interestingly, in these analyses no dif- in liver and kidney extracts. The 31-kDa band visible in brain and the ferences were observed between organs from wt and hTNFtg mice 32-kDa band visible in lung extracts represent the smaller splice variants of (data not shown). Thus, overexpression of hnRNP-A2 appeared to hnRNP-A2 and hnRNP-B1, respectively. exclusively occur in the joint of hTNFtg mice and, furthermore, resulted in a unique expression pattern (hnRNP-A2/B1 and the of the Ab response to other nuclear proteins was also not observed 32-kDa variant) that was not seen in any of the organs analyzed (data not shown). (Table III). Because we assumed that the failure to induce arthritis was due to the low expression of hnRNP-A2 in the joints of healthy mice, Immunization with hnRNP-A2 enhances arthritis in hTNFtg mice we next investigated whether immunization with hnRNP-A2/B1 To investigate whether immunization could induce disease in would influence the progression of arthritis in hTNFtg mice. An- non-tg mice, arthritis-prone DBA/1 mice were immunized with imals (five mice per group) were immunized s.c. at the age of 4 wk, hnRNP-A2/B1 or peptide p50-70 or collagen II as positive control. i.e., before onset of clinical disease, with either the recombinant Although a strong Ab response was evoked, no signs of arthritis protein, with peptide p50-70 or with peptide p140-160. As de- emerged within the observation period (up to 6 mo), and spreading scribed above, p50-70 contains a major B cell epitope, whereas The Journal of Immunology 8333

Table III. Expression profiles of hnRNP-A2 and related proteins reactive with mAb 10D1 in mouse organsa

Molecular Protein Joint Joint Mass kDa hnRNP Spleen Thymus Brain Lung Liver Kidney (normal) hTNFtg

41 A3b ϪϪϩϩϪϩϩϩϪ Ϫ 40 A3b ϩ/Ϫϩϩϩϩ/ϪϪ Ϫ Ϫ Ϫ 39 B2 ϩϩϩ ϩϩϩ ϩ ϩϩ Ϫ Ϫ Ϫ Ϫ 38 B1 ϩϩϩ ϩϩϩ ϩ ϩϩ Ϫ Ϫ Ϫ ϩ 36 A2 ϩϩϩ ϩϩϩ ϩϩ ϩϩ Ϫ Ϫ Ϫ ϩϩ 32 B1bc ϪϪϩ/ϪϩϩϪ Ϫ Ϫ ϩϩ 31 A2bc Ϫ Ϫ ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ

a Note the unique expression pattern (A2/B1/B1b) that was exclusively observed in the joints of hTNFtg mice and the absence of these proteins in the joints of normal control animals. b Assumed to be identical with hnRNP-A3 isoforms based on migration in SDS-PAGE (28). c Smaller splice variants of hnRNP-A2/B1 lacking exon 9 (31). p140-160, despite its structural similarity with p50-70, is not tar- Ag or p50-70, whereas animals immunized with p140-160 did not geted by autoantibodies. Control animals received adjuvant only. differ from controls that had received CFA only (Fig. 5C). In line Blood was taken after 9 wk when the animals were sacrificed. As with the increased inflammatory response, areas of erosions were can be seen in Fig. 5A, a pronounced Ab response against twice as large as those of controls (Fig. 5D). Thus, immunization Downloaded from hnRNP-A2 was observed in 4 of 5 hTNFtg mice immunized with with the full-length protein or the major B cell epitope aggravated the full-length Ag: sera recognized the characteristic set of protein disease, suggesting potential involvement of the anti-RA33 auto- bands between 36 and 40 kDa, and similar responses were seen in immune response in TNF-driven erosive arthritis. wt mice (data not shown). In contrast, none of the hTNFtg mice immunized with p50-70 reacted with any of these bands while Discussion showing pronounced reactivity in the peptide-specific ELISA (data A remarkable feature of systemic autoimmune diseases is the pro- http://www.jimmunol.org/ not shown). Remarkably, 2 of 5 hTNFtg mice immunized with duction of autoantibodies directed to ubiquitously expressed pro- p140-160 and one mouse immunized with CFA developed a rel- teins. Most of the major autoantigens are part of large nuclear atively strong reactivity to hnRNP-A2, which was much more pro- complexes composed of DNA or RNA and multiple proteins such nounced than the reactivities seen in untreated mice. as nucleosomes, spliceosomal complexes, or ribosomes. Although To study the effects of immunization on the development of autoantibodies against these structures may be generated in a arthritis, clinical assessment of grip strength and paw swelling was rather disease-specific manner, the pathogenetic role of most of performed weekly (Fig. 5B). To evaluate histological effects, the these Ags has remained elusive (33–35). RA differs from other area of inflammatory synovial tissue and the area of bone erosions systemic autoimmune diseases both clinically and serologically. by guest on September 27, 2021 was assessed in the joints and tarsal area of the hind paws. Al- Thus, RA is characterized by the occurrence of high-titered RF and though clinical evaluation did not show significant differences autoantibodies against citrulline-containing proteins such as fibrin among the four groups, histological analyses, performed blinded or vimentin, whereas nuclear Ags are rarely targeted (5, 7, 36, 37). for treatment groups, revealed a significantly higher ( p Ͻ 0.05) An exception is hnRNP-A2, which seems to form a relatively dis- degree of inflammation in mice immunized with either full-length ease-specific target for autoantibodies and autoreactive Th1-like

FIGURE 5. Immunization of hTNFtg mice with hnRNP-A2/B1 and synthetic peptides. Animals were immunized s.c. at the age of 4 wk with either full-length recombinant hnRNP-A2/B1 or the two synthetic pep- tides p50-70 and p140-160 (see also Fig. 2); controls received CFA only. Animals were sacrificed at the age of 9 wk. A, Detection of Ab to HeLa nuclear proteins in sera of immunized wt and hTNFtg mice by immuno- blotting. No reactivities can be seen in sera of peptide- immunized wt mice and also not in sera of hTNFtg mice immunized with p50-70, whereas pronounced reactivi- ties are visible in sera of hTNFtg mice immunized with the complete Ag. Reactivities are visible in 2 of 5 sera from mice immunized with p140-160 and also in two sera from the CFA control group. Weak reactivity can be seen also in one serum of the untreated control group. B, Clinical evaluation of grip strength. No significant differences were seen among the four groups. However, the most pronounced decrease in grip strength was ob- served in mice immunized with the full-length Ag (red graph). C and D, Histological analysis of inflammation and bone erosion. The size of areas of inflammatory and eroded tissue was determined in a semiquantitative man- ner. As compared with CFA controls, inflammation (C) and bone erosion (D) was increased in mice immunized with hnRNP-A2/B1 or p50-70 (p Ͻ 0.05, Student’s t test). 8334 DEVELOPMENT OF AUTOANTIBODIES IN TNF␣ TRANSGENIC MICE cells (8–10). Despite this finding, and although RF and anti-CCP tion of autoimmune reactions, this is certainly not sufficient, and Abs are significantly associated with a more severe disease course, other factors including posttranslational modifications, expression it is still unclear which role these autoimmune responses play in of unusual splice variants, and, last but not least, immunogenetic the pathogenesis of erosive arthritis, which is a unique feature of factors may substantially contribute. The fact that posttranslational RA that is not seen in other rheumatic disorders. modifications of hnRNP-A2 (e.g., phosphorylation or methylation) In contrast to systemic lupus erythematosus (SLE), no sponta- may play an important role in the generation of the autoantibody neous animal model of RA exists, and, therefore, several inducible response is strongly suggested by the weaker reactivity of IgG or tg models are being used that cover only certain aspects of the anti-RA33 Ab with the recombinant protein. Thus, autoantibodies disease. Thus, hTNFtg mice are an excellent model to study cel- may be primarily directed to modified epitopes, or (and more lular and molecular mechanisms triggered by TNF-␣, i.e., by a likely) the modifications may induce structural changes that might state of chronic inflammation, whereas they are less suitable to reveal cryptic epitopes such as p50-70. HnRNP-A2 is known to study the initial events that lead to TNF-␣ overproduction (13). In become reversibly phosphorylated and methylated in vivo, and contrast, because autoimmune reactions may arise as a conse- methylation has been recently shown to be essential for nuclear quence of chronic inflammatory processes, we considered this localization of this protein (51). model quite useful for investigating this aspect. Thus, it is now Interestingly, hTNFtg animals developed neither RF nor anti- widely assumed that chronic inflammation may lead to aberrant CCP Abs and, apart from a few exceptions, no IgG Ab to stress expression of self proteins thereby creating neoepitopes, which proteins, although IgM anti-hsp reactivities were clearly seen in may become targets of autoreactive T and B cells. Potential mech- ϳ40% of hTNFtg and control mice. Stress proteins are of partic- anisms include overexpression over longer periods leading to an- ular interest because autoimmunity to them has been suggested to Downloaded from tigenic overload, aberrant localization, overexpression of minor play a (possibly beneficial) role in the pathogenesis of RA (52–54), splice variants, or posttranslational modifications (7, 38–42). In and overexpression of hsp60 and other stress proteins has been this respect TNF may play a dual role, being able to both promote observed in the joints of mice with adjuvant arthritis as well as in and abrogate pathological autoimmune reactions (43). patients with RA (52, 55–57). In contrast, anti-hsp autoimmunity The data presented in this study further confirm that such mech- does not seem to be specific for any (autoimmune) disease and

anisms exist, and that they can lead to the induction of an auto- may commonly occur during infections and even in healthy indi- http://www.jimmunol.org/ immune response that is characteristically observed in patients viduals (58–60). Because it is plausible to assume that hsps were with RA. Thus, abundant overexpression of hnRNP-A2 was de- also overexpressed in the inflamed joint of hTNFtg mice, it was tected in the joints of hTNFtg mice, and the animals developed somewhat unexpected that the immune system of the animals ap- autoantibodies to this protein, which increased in titer as disease peared to be tolerant to these proteins, despite the presence of progressed. In contrast, therapies reducing joint destruction, and preformed IgM autoreactivities. This finding indicates that hTNFtg particularly bone erosion, also significantly reduced the autoanti- mice were unable to mount a potent T cell response to stress pro- body response, even if inflammation (and synovial overexpression teins, which might be predominantly of an anti-inflammatory na- of hnRNP-A2) persisted. This was most impressively seen in c-fos- ture as described for patients with juvenile idiopathic arthritis deficient hTNFtg mice, which developed no erosions at all, al- (53, 61). by guest on September 27, 2021 though the inflammatory state of their joints was as severe as in So far, anti-CCP Abs have been exclusively found in humans hTNFtg animals (16). and not in animal models of arthritis and other inflammatory dis- Apart from hnRNP-A2 and its major splice variant hnRNP-B1, eases, although synovial expression of citrullinated proteins was a 32-kDa protein was expressed that presumably corresponded to observed in various arthritis models (62, 63). In patients with RA, a recently described smaller splice variant reported to be expressed anti-CCP Abs seem to be closely linked to the presence of the in testis, brain, and skin (31). Remarkably, in our analyses this shared epitope, a pentameric sequence found in RA-associated variant was undetectable in lymphoid organs. This suggests that HLA-DR alleles (64, 65). Thus, it may be possible that citrulli- aberrant expression of the smaller variant in the inflamed joint nated epitopes cannot be efficiently presented by murine MHC might form the molecular basis for pathological autoimmune re- class II molecules. However, it should be taken into consideration actions to an Ag that, due to its high expression in lymphoid or- that the anti-CCP assay was developed for detection of human gans, is normally tolerized by the immune system. An analogous autoantibodies, and, therefore, it is possible that mice may generate observation was recently made in rats susceptible to experimental an autoimmune response to a citrullinated epitope that does not autoimmune encephalitis, where T cell autoimmunity was directed cross-react with the cyclic peptide used in the anti-CCP assay. to a brain-specific splice variant of proteolipid protein that was not However, sera of hTNFtg mice were also not reactive with citrul- expressed in the thymus (44). In this context, the identification of linated (human) filaggrin and fibrinogen (our unpublished obser- a major epitope in RRM1 (p50-70) was of particular interest be- vation), supporting the assumption that these mice do not mount an cause the same epitope had previously been found to be targeted autoimmune response to citrullinated epitopes. Although these by MRL/lpr mice, one of the most widely used models of SLE findings cannot be completely extrapolated to the human disease, (23). In contrast to other lupus models, MRL/lpr mice may de- they may allow the hypothesis that anti-CCP Ab as well as RF are velop erosive arthritis and RF and suffer from a SLE/RA overlap not induced by an inflammatory process, which is bolstered by the disease with SLE symptoms predominating (45). Interestingly, the very early appearance of these Abs often years before clinical epitope was not recognized upon immunization with the full- symptoms manifest (11, 12). Nevertheless, further studies using length Ag and thus may represent a cryptic epitope that under murine proteins are required to definitely answer this question. normal conditions is not exposed to the immune system (46–50). Taken together, these observations suggest a cascade of events Remarkably, Ab to hnRNP-A1, which shows ϳ70% identity leading to the generation of pathological autoimmune reactions with hnRNP-A2 (29, 32), were rarely detectable, although this against hnRNP-A2 in the inflamed joint: 1) TNF-triggered inflam- protein was as highly expressed in the joint as hnRNP-A2. Of note, mation induces aberrant expression of a protein that under normal hnRNP-A1 is also infrequently targeted in human RA and if so conditions is not or only weakly expressed in the joint; 2) aberrant mostly by cross-reacting anti-hnRNP-A2 Ab (8). Thus, even if expression leads to aberrant presentation of the Ag by Ag-present- aberrant expression of a self protein may be necessary for induc- ing cells and subsequently to the activation of autoreactive T cells The Journal of Immunology 8335 and the generation of autoantibodies; 3) this process is enhanced 4. Smolen, J. S., S. Hayer, G. Schett, K. Redlich, M. Aringer, G. Kollias, E. Wagner, by tissue destruction leading to the release of large amounts of and G. Steiner. 2004. Autoimmunity and rheumatoid arthritis. Autoimmun. Rev. 3(Suppl. 1): S23. autoantigens; 4) autoantibodies and autoreactive T cells may fur- 5. Steiner, G., and J. Smolen. 2002. Autoantibodies in rheumatoid arthritis and their ther enhance the inflammatory and destructive processes by form- clinical significance. Arthritis Res. 4(Suppl 2): S1–S5. 6. Doyle, H. A., and M. J. Mamula. 2001. Post-translational protein modifications ing immune complexes, activation of the complement cascade, and in antigen recognition and autoimmunity. Trends Immunol. 22: 443–449. recruitment of macrophages and other inflammatory cells, estab- 7. Vossenaar, E. R., and W. J. van Venrooij. 2004. Citrullinated proteins: sparks that lishing a vicious circle in which inflammation and autoimmunity may ignite the fire in rheumatoid arthritis. Arthritis Res. Ther. 6: 107–111. 8. Hassfeld, W., G. Steiner, A. Studnicka Benke, K. Skriner, W. Graninger, mutually enhance each other until the target tissue is destroyed. I. Fischer, and J. S. Smolen. 1995. Autoimmune response to the spliceosome: an Thus, even if the autoimmune response to hnRNP-A2 arises immunologic link between rheumatoid arthritis, mixed connective tissue disease, secondarily to tissue destruction, it may nevertheless contribute to and systemic lupus erythematosus. Arthritis Rheum. 38: 777–785. 9. Steiner, G., K. Skriner, and J. S. Smolen. 1996. Autoantibodies to the A/B pro- the pathophysiology of erosive arthritis. The arthritis-enhancing teins of the heterogeneous nuclear ribonucleoprotein complex: novel tools for the effect of immunization with either the complete protein or the pep- diagnosis of rheumatic diseases. Int. Arch. Allergy Immunol. 111: 314–319. tide containing the major B cell epitope supports such assumption. 10. Fritsch, R., D. Eselbock, K. Skriner, B. Jahn-Schmid, C. Scheinecker, B. Bohle, M. Tohidast-Akrad, S. Hayer, J. Neumuller, S. Pinol-Roma, et al. 2002. Char- Nevertheless, our data do not exclude a primary role for this au- acterization of autoreactive T cells to the autoantigens heterogeneous nuclear toimmune reaction in human disease, at least in those patients who ribonucleoprotein A2 (RA33) and filaggrin in patients with rheumatoid arthritis. J. Immunol. 169: 1068–1076. show autoantibodies to hnRNP-A2 very early in the course of their 11. Rantapaa-Dahlqvist, S., B. A. de Jong, E. Berglin, G. Hallmans, G. Wadell, disease (66, 67). H. Stenlund, U. Sundin, and W. J. van Venrooij. 2003. Antibodies against cyclic So far, there is only limited knowledge available on the func- citrullinated peptide and IgA rheumatoid factor predict the development of rheu- matoid arthritis. Arthritis Rheum. 48: 2741–2749. Downloaded from tions of hnRNP-A2 and its variants. Functions ascribed to hnRNP- 12. Nielen, M. M. J., D. van Schaardenburg, H. W. R. Reesink, R. J. van de Stadt, A2/B1 include roles in regulation of alternative splicing, mRNA I. E. van der Horst-Bruinsma, M. H. de Koning, M. R. Habibuw, transport, and translation (68–70), whereas nothing is known J. P. Vandenbroucke, and B. A. Dijkmans. 2004. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood about the smaller variants that show a more restricted expression donors. Arthritis Rheum. 50: 380–386. pattern. They appear to be developmentally regulated because they 13. Kollias, G., E. Douni, G. Kassiotis, and D. Kontoyiannis. 1999. On the role of are mainly expressed in young animals (71). Therefore, their pres- tumor necrosis factor and receptors in models of multiorgan failure, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. Immunol. Rev. 169: ence in inflamed tissue of older mice and patients with RA (our 175–194. http://www.jimmunol.org/ unpublished observation) is intriguing, pointing to an unusual role 14. Kyburz, D., and M. Corr. 2003. The KRN mouse model of inflammatory arthritis. Springer Semin. Immunopathol. 25: 79–90. in cells exposed to proinflammatory and stressful conditions. Of 15. Monach, P. A., C. Benoist, and D. Mathis. 2004. The role of antibodies in mouse note, several studies from other investigators have reported over- models of rheumatoid arthritis, and relevance to human disease. Adv. Immunol. expression and cytoplasmic accumulation of hnRNP-A2 in various 82: 217–248. 16. Redlich, K., S. Hayer, R. Ricci, J. P. David, M. Tohidast Akrad, G. Kollias, kinds of cancers, suggesting a functional role in the altered cellular G. Steiner, J. S. Smolen, E. F. Wagner, and G. Schett. 2002. Osteoclasts are metabolism of transformed cells (72–74). essential for TNF-␣-mediated joint destruction. J. Clin. Invest. 110: 1419–1427. In summary, the data obtained in the course of our investigations 17. Zwerina, J., S. Hayer, M. Tohidast Akrad, H. Bergmeister, K. Redlich, U. Feige, C. Dunstan, G. Kollias, G. Steiner, J. Smolen, and G. Schett. 2004. Single and are in line with the hypothesis of the “altered” self, suggesting that combined inhibition of tumor necrosis factor, interleukin-1, and RANKL path- altered expression of an autoantigen in the course of an inflam- ways in tumor necrosis factor-induced arthritis: effects on synovial inflammation, by guest on September 27, 2021 matory process may lead to altered processing, altered Ag presen- bone erosion, and cartilage destruction. Arthritis Rheum. 50: 277–290. 18. Keffer, J., L. Probert, H. Cazlaris, S. Georgopoulos, E. Kaslaris, D. Kioussis, and tation, and, finally, to activation of autoreactive T and B cells. G. Kollias. 1991. 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Arthritis Rheum. 46: 785–792. merous potential candidate autoantigens immunogenic in patients 21. Marcu, A., B. Bassit, R. Perez, and S. Pinol-Roma. 2001. Heterogeneous nuclear ribonucleoprotein complexes from Xenopus laevis oocytes and somatic cells. Int. with autoimmune diseases and the respective animal models. J. Dev. Biol. 45: 743–752. 22. Skriner, K., W. H. Sommergruber, V. Tremmel, I. Fischer, A. Barta, J. S. Smolen, and G. Steiner. 1997. Anti-A2/RA33 autoantibodies are directed to the RNA Acknowledgments binding region of the A2 protein of the heterogeneous nuclear ribonucleoprotein We thank Elisabeth Ho¨fler for expert technical assistance with autoanti- complex: differential epitope recognition in rheumatoid arthritis, systemic lupus body detection, Jean-Paul Briand (Institut de Biologie Moleculaire et Cel- erythematosus, and mixed connective tissue disease. J. Clin. Invest. 100: 127–135. lulaire, Centre National de la Recherche Scientifique, Strasbourg, France) 23. 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