Citation for published version (APA): Cantaert, T. (2008). How is autoimmunity against citrullinated proteins regulated?

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1 UvA-DARE (Digital Academic Repository) How is autoimmunity against citrullinated proteins regulated? Cantaert, T. Link to publication Citation for published version (APA): Cantaert, T. (2008). How is autoimmunity against citrullinated proteins regulated? General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 22 Nov 2018

2 ACPA levels are associated with synovial intracellular citrullinated proteins CHAPTER 4 colocalizingwith synovial intracellular citrullinated proteins peptidyl arginine aredeiminase pathophysiologically relevant antigenic of determinants rheumatoid arthritis- specific humoral autoimmunity81.

3 CHAPTER 4 L. De Rycke*, A. P. Nicholas*, T. Cantaert, E. Kruithof, J. D. Echols, B. Vandekerckhove, E. M. Veys, F. De Keyser, D. Baeten. * Both authors contributed equally to this study. Arthritis and Rheumatism, 2005, 52(8):

4 ACPA levels are associated with synovial intracellular citrullinated proteins Abstract Objectives The present study addresses the ongoing debate of the specificity of synovial citrullinated proteins for rheumatoid arthritis (RA) and analyses their pathophysiological relevance in the induction or perpetuation of the RA-specific anti-citrullinated protein antibodies (ACPA). Patients and methods Synovium of 19 RA and 19 non-ra controls was immunostained for the presence of citrullinated proteins with a mouse monoclonal antibody (F95), for the citrullinating enzyme peptidyl arginine deiminase type 2 (PAD2), and for the free citrullineproducing enzyme inducible nitric oxide synthetase (inos). Extending the RA cohort to 61 patients, the anti-citrulline stainings in synovium were related to serum and synovial fluid ACPA levels as measured by ELISA. Results F95 staining indicated the presence of synovial intracellular citrullinated proteins in 53% of RA versus 5% of control samples, whereas extracellular staining was not RAspecific. Immunoblotting and inhibition experiments confirmed that the antibody recognized citrulinated proteins but not free citrulline. Accordingly, inos was equally found in RA and control synovium and in intracellular citrullinated positive and negative samples. In contrast, intracellular citrullinated proteins colocalized with PAD2, which was found in 59% of RA versus 17% of control samples. Independent of local disease activity, the presence of the RA-specific synovial intracellular citrullinated proteins was associated with significantly higher systemic and local ACPA levels and with local ACPA production in the joint. Conclusion These data confirm the presence of RA-specific intracellular citrullinated proteins in synovium. The link with PAD2 and local and systemic ACPA levels emphasizes their pathophysiological relevance for RA-specific humoral autoimmunity. 83.

5 CHAPTER 4 Introduction Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA)(1-6). The occurence of ACPA early in the disease course and even before onset of clinical symptoms (7,8) as well as the correlation with disease severity and prognosis (9,10) suggested that ACPA are not only valuable clinical biomarkers but are also pathophysiologically involved in the disease. However, epithelial (pro)filaggrin, which is the first identified antigenic target of ACPA (11-13), is not expressed in the joint and RA does not affect filaggrin containing epithelial tissues such as skin and buccal mucosa. Based on crucial observations that ACPA can be produced locally in the joint (14) and that posttranslational modification of arginine into citrulline residues in the context of specific amino acid sequences is essential for recognition by ACPA (15-17), the assumption was made that citrullinated filaggrin could be a cross-reacting in vitro antigen rather than a genuine in vivo antigenic target of ACPA and, subsequently, that distinct citrullinated proteins present in the inflamed synovium may be involved in the induction or perpetuation of the RA-specific humoral autoimmune responses. Firstly, Serre and coworkers demonstrated elegantly the presence of several deiminated proteins in RA synovium, of which the deiminated forms of the α- and β-chains of fibrin appeared to be major antigenic targets of ACPA (18). Moreover, in vitro deiminated fibrinogen is a highly specific and sensitive substrate for the detection of serum ACPA (19). However, the mere presence of deiminated fibrin in synovium does not fully explain the RA-specific induction of ACPA since citrullinated fibrin was equally detected in inflamed synovium in non-ra patients and in animal arthritis, where they do not induce ACPA (20-22). Secondly, Menard and coworkers described a 50 kda antigen called Sa in extracts from human placenta, spleen, and RA synovium (23). Anti-Sa antibodies are specific for RA and recognize deiminated vimentin, which was previously shown to be present in apoptotic macrophages in vitro (24,25). Thirdly, we demonstrated the presence of intracellular citrullinated proteins which colocalized with ACPA-reactivity in RA synovium (26). Although the biochemical nature of these intracellular citrullinated proteins remains ill-defined, the major interest in comparison with both previously mentioned potential antigenic targets is that they are specifically present in RA synovium (20,26,27). Since the latest observation might indicate a pathophysiological role in the induction of ACPA in vivo, the present study aimed to confirm the RA-specificity of synovial intracellular citrullinated proteins, with special reference to the presence of the citrullinating enzyme peptidyl arginine deiminase (PAD) (28,29). Considering the presence of several deiminated proteins in inflamed synovium, we further aimed to provide direct evidence that these RA-specific synovial intracellular citrullinated 84.

6 ACPA levels are associated with synovial intracellular citrullinated proteins proteins are genuine antigenic targets of the humoral autoimmune process by analyzing their influence on local and systemic ACPA levels. Material and methods Patients and samples For the analysis of intracellular citrullinated proteins, PAD2, and inos the study included 19 RA patients fulfilling the ACR criteria (30). All patients had active disease as characterized by a swollen joint count (SJC) of 7,5±6,2 (mean ± standard deviation) with at least one swollen knee joint for needle arthroscopy, a serum C- reactive protein (CRP) level of 78±71 mg/l, and an erythrocyte sedimentation rate (ESR) of 55±28 mm/h. The control group (n=19) consisted of 8 patients with spondyloarthropathy fulfilling the ESSG criteria (31), 6 patients with osteoarthritis, 4 patients with juvenile chronic arthritis, and 1 patient with villonodular synovitis. As for the RA group, all patients had active joint disease with at least one swollen knee joint for needle arthroscopy. For the analysis of intracellular citrullinated proteins in relation with ACPA titers, we extended the RA cohort with an additional 42 RA patients fulfilling the ACR criteria (30). Similarly to the first cohort, all patients had at least one swollen knee joint for needle arthroscopy and had active disease with a SJC of 6,6±4,9, a serum CRP of 48±37 mg/l, and an ESR of 46±23 mm/hour. All patients signed a written informed consent before inclusion in the study, which was approved by the Ethics Committee of the Ghent University Hospital. From all patients, synovial tissue samples were obtained from clinically involved knee joints by needle arthroscopy as described previously (32). Paired synovial fluid (SF) and serum samples were collected. Antibodies Synovial intracellular citrullinated proteins were detected using a well described and validated mouse IgM monoclonal antibody (mab), called F95, which was raised against a deca-citrullinated peptide linked to the carrier protein, keyhole limpet hemocyanin (33-35). For the detection of the citrullinating PAD enzymes we focused on PAD type 2 (PAD2), based on the presence of both PAD2 and PAD4 but not PAD1 and PAD3 subtypes in synovial extracts (36), the expression of PAD4 in the nucleus rather than the cytoplasma (37-39), and the absence of correlation between PAD4 85.

7 CHAPTER 4 polymorphisms and intracellular citrullinated proteins (unpublished data). A single chain protein against human PAD2, similar to a light chain of an IgG antibody, was developed by phage display technology. It was directed against the PAD2 antigen VEKNNPKKASWTWGPEGQGA, which is 100% homologous for human, rat, and mouse PAD2 and has less than 50% homology with peptides from other PAD subtypes. Since this single chain protein against human PAD2 contained the antigenic marker FLAG, rabbit anti-flag IgG were loaded with the FAB ends against PAD2 and used for immunohistochemical detection of PAD2 in synovium. Blot experiments using recombinant PAD confirmed that only PAD2 and not PAD4 was recognized (40). Additionally, the following commercially available antibodies were used: rabbit anti-l-citrulline polyclonal antibody (pab) (Biogenesis, Poole, UK)(26) and mouse anti-inducible nitric oxide synthase (inos) mab (R&D Systems, Minneapolis, MN, USA). Immunohistochemistry Frozen sections of synovial tissue were stained by incubation with the primary antibody followed by sequentially a biotinylated second antibody, a streptavidineperoxidase complex, and amino-ethyl carbazole as substrate (LSAB+ kit, Dako, Glostrup, Denmark), as extensively described before (26,27). For F95, the same protocol was used as extensively described previously for rat and human brain tissue (34,35). For all stainings, isotype and concentration matched irrelevant antibodies were used as negative control. For tissue blocking experiments, F95 was incubated with 0,9 µm decacitrullinated peptide or free citrulline for 24 hours and subsequently used for immunohistochemistry. Global synovial inflammation was assessed by the degree of inflammatory infiltration and synovial vascularity and the number of CD3+ T lymphocytes, CD20+ B lymphocytes, and plasma cells, as described previously (26,27). The stained sections were blinded for diagnosis and were evaluated by two independent observers on a 0 to 3 semiquantitative scale, using the mean score of the two observers in case of discrepancy (26,27). Immunoblotting Dot blots containing 25 µg of either decacitrullinated peptide, free citrulline, or mouse brain extracts were dried on PVDF membrane (Perkin Elmer, Boston, MA, USA), which was then cut lentghwise to transect each blot. The top half of the 86.

8 ACPA levels are associated with synovial intracellular citrullinated proteins membrane was blocked with 3% BSA for 2 hours, washed, and incubated with F95 (1/50) for 1 hour. After washing, binding of F95 was detected with anti-mouse IgM- HRP (Jackson Immunoresearch, West Grove, PA, USA) using the Western Lighting Chemiluminescence Kit (Perkin Elmer). The bottom half of the membrane was used as positive control to detect either free amino acids (free citrulline) by staining with 0,25% Ninhydrin in 10% acetone and 10% acetic acid, or peptide bounds (decacitrullinated peptide and brain extract) by staining with Biuret reagent (Sigma, St Louis, MO, USA) and Folin (Sigma). Double immunofluorescence Double immunofluorescence experiments were performed with the previously mentioned antibodies detected by goat anti-rabbit immunoglobulins-tritc (Dako), goat anti-mouse immunoglobulins-fitc (Dako), or goat anti-rabbit immunoglobulins- Alexa Fluor 555 (Molecular Probes Inc., Eugene, USA) as appropriate. ACPA measurement ACPA levels in serum and SF were detected by the commercially available anti-ccp2 ELISA containing synthetic cyclic citrullinated peptides as substrate (Immunoscan RA, mark 2, Eurodiagnostica, Arnhem, The Netherlands). To compare ACPA between serum and SF, the measured titers were corrected for total IgG as measured by the Human IgG ELISA Quantitation Kit (ImTec Diagnostics NV, Antwerp, Belgium). Both ELISAs were performed according to the manufacturers instructions. Statistics Comparisons between 2 groups were performed with the Mann-Whitney U test. Chi-square test was used for comparison of proportions. Correlations were assessed by the Spearman s rho correlation coefficient. For ACPA levels, the median values are given. P<0,05 was considered as statistically significant. 87.

9 CHAPTER 4 Results Detection of RA-specific intracellular citrullinated proteins in the inflamed synovium Investigating the presence of intracellular citrullinated proteins in RA and control synovium, the mouse anti-citrulline mab F95 stained single mononuclear cells in RA synovium (Fig1a). Controle staining with an irrelevant concentration and isotype matched primary antibody was completely negative (Fig 1b). Intracellular staining was observed in 10 out of 19 RA samples and in only one of the control samples (p=0,004)(fig 1c). Confirming these findings, F95 staining correlated significantly with the results obtained with the rabbit anti-citrulline pab, which we previously used to indicate the RA-specificity of intracellular citrullinated proteins in synovium (r=0,943 p<0,001)(fig 1d)(26). Of interest, F95 also gave an extracellular staining of extra-synovial deposits infiltrated by high numbers of polymorphonuclear cells, the surface of the lining layer, and occasionally the deeper synovium (Fig 1e and f); in sharp contrast with the intracellular staining, however, the extracellular staining was not RA-specific since it was also seen in 9 out of the 19 controls samples. To exclude that the staining observed with F95 is due to the detection of free citrulline, additional immunoblotting experiments indicated that F95 recognized decacitrullinated peptide and mouse brain extracts but not free citrulline (Fig 1g). Moreover, decacitrullinated peptide but not free citrulline could inhibit the F95 staining in RA synovium (Fig 1h and i). RA-specificity of intracellular citrullinated proteins is related to PAD2 Exploring potential mechanisms related to the RA-specificity of synovial intracellular citrullinated proteins, immunohistochemical stainings demonstrated the pronounced presence of the citrullinating enzyme PAD2 in RA synovium. PAD2 showed a predominant cellular expression pattern both in synovium and in extra-synovial deposits, with occasionally associated extra-cellular staining (Fig2ac). PAD2 expression was significantly higher in RA (median 1, range 0-3) than in control synovial tissue (median 0, range 0-2)(p=0,041), with PAD2 expression in 59% of the RA samples versus 17% of the controls (p=0,026). In RA synovium, expression of intracellular citrullinated proteins was higher in the PAD2 positive samples (median 2, range 0-3) than in the PAD2 negative samples (median 0, range 0-1)(p=0,054). Furthermore, double immunofluorescence demonstrated that intracellular citrullinated proteins always colocalized with PAD2 staining in RA 88.

10 ACPA levels are associated with synovial intracellular citrullinated proteins Figure 1: Rheumatoid arthritis (RA)-specific staining of intracellular citrullinated proteins in synovial biopsy samples. Frozen synovial tissue sections stained with a monoclonal mouse anti-citrulline antibody (F95) showed positively staining cells in RA synovium (panel A), whereas staining with the negative controle antibody was negative (panel B) and no cellular staining was seen in all but one control samples (panel C). Analysis of 19 RA synovia showed that intracellular staining (scored on a semiquantitative 0-3 scale) by the monoclonal mouse anti-citrulline antibody (F95) correlated well with staining by the previously used polyclonal rabbit anti-citrulline antibody (panel D). In contrast with the RA-specific intracellular staining, extracellular staining by F95 was observed on the synovial surface (panel E) and occasionally the deeper synovium (panel F) in both RA and non-ra samples. In order to exclude that this staining was due to the detection of free citrulline rather than citrullinated proteins, deca-citrullinated peptide (panel G4), free citrulline (panel G5), and mouse brain extract (panel G6) were blotted on a membrane; F95 recognized deca-citrullinated peptide (panel G1) and mouse brain extract (panel G3) but not free citrulline (panel G2). Accordingly, the staining of intracellular citrullinated proteins in RA synovium by F95 could not be inhibited by free citrulline (panel H) but was completely abolished by preincubation with deca-citrullinated peptide (panel I). synovium whereas not all PAD2 positive cells stained for intracellular citrullinated proteins (Fig2 d-f), which is in agreement with the notion that not the mere presence but rather the activation of PAD2 leads to deimination. In sharp contrast, cellular expression and occasional extracellular staining of the free citrulline producing enzyme inos was found in the lining layer, the sublining layer, and the vascular endothelium of both RA (71% inos positive) and non-ra (95% inos positive) synovia (Fig2 g-i). There was no difference in inos expression levels between RA (median 1.5, range 0-3) and controls (median 2, range 0-3) and, within the RA group, there was no significant association with synovial intracellular citrullinated proteins. Synovial intracellular citrullinated proteins determine systemic ACPA levels Based on the RA-specificity, the pathophysiological link with distinct synovial PAD2 expression, and the previously demonstrated colocalization with ACPA-reactivity in RA synovium (26), we next assessed if the synovial intracellular citrullinated proteins were pathophysiologically relevant antigenic determinants of ACPA or rather crossreactive substrates such as assumed for epithelial filaggrin. ACPA serum levels were significantly higher in patients with (n=27/61) versus without (n=34/61) intracellular citrullinated proteins in synovium (865 U/ml versus 324 U/ml; p=0,017)(fig3). Of interest, the previously described link between ACPA and HLA-DR shared epitope 89.

11 CHAPTER 4 Figure 2: Synovial intracellular citrullinated proteins colocalize with peptidyl arginine deiminase type 2 (PAD2). The citrullinating enzyme PAD2 is expressed with a cellular pattern in both the synovial lining and sublining layer of rheumatoid arthritis (RA) synovium (panel A). The cellular staining can be associated with extracellular presence of PAD2 (panel B). Both the expression of PAD2 and the number of PAD2 positive samples is higher in RA than in controls, but strong cellular PAD2 expression can occasionally be observed in other types of inflammatory arthritis such as spondyloarthropathy (panel C). PAD2 expression (in green, panel D) was found in all synovial intracellular citrullinated protein-positive cells (in red, panel E), although not all PAD2-positive cells colocalized with intracellular citrullinated proteins (merged picture, panel F). In sharp contrast with PAD2, cellular staining in the synovial lining and sublining layer for the free citrulline producing enzyme inos was equally found in intracellular citrullinated protein positive RA synovium (panel G), in intracellular citrullinated protein negative RA synovium (panel H), and in non-ra control synovium (panel I). (6,10,41-44) was clearly more pronounced in the synovial intracellular citrullinated protein positive patients (1105 U/ml in shared epitope positive patients versus 167 U/ml in shared epitope negative patients; p=0,050) than in the synovial intracellular citrullinated protein negative patients (446 U/ml in shared epitope positive patients versus 110 U/ml in shared epitope negative patients; p=ns)(fig3). Since both ACPA and HLA-DR shared epitope are associated with disease severity (6,9,44,45), we assessed if the presence of intracellular citrullinated proteins in RA synovium was not merely the consequence of higher local inflammation. Parameters of synovial inflammation (infiltration, vascularity, CD3+ T cells, CD20+ B cells, and plasma cells) did not correlate with serum ACPA levels or shared epitope status. In contrast, vascularity (p=0,026) and CD20+ B lymphocytes (p=0,025) were even higher in shared epitope negative patients. Accordingly, there were no significant differences in these parameters of synovial inflammation between intracellular citrullinated protein positive versus negative synovium, with exception of vascularity which was higher in the former group (p=0,012). Finally, analysis of synovial biopsies obtained from an additional cohort of RA patients without knee synovitis demonstrated intracellular citrullinated protein in 5 out of 16 cases (data not shown). Taken together, these data indicated that the presence of synovial intracellular citrullinated proteins is associated with ACPA levels independently of local inflammation. 90.

12 ACPA levels are associated with synovial intracellular citrullinated proteins Figure 3: Serum anti-citrullinated protein antibodies (ACPA) levels (median and interquartile range) as determined by anti-ccp2 ELISA (U/ml) were significantly higher in rheumatoid arthritis (RA) patients with synovial intracellular citrullinated proteins than in RA patients without synovial intracellular citrullinated proteins (*p=0,017)(panel A). The previously demonstrated link between serum ACPA levels and HLA-DR shared epitope was more pronounced in intracellular citrullinated positive patients (*p=0,050 for shared epitope positive versus negative)(panel B) than in the intracellular citrullinated protein negative group (no significant difference between shared epitope positive and negative)(panel C). Synovial intracellular citrullinated proteins determine local ACPA levels Considering the previous demonstration of local production of ACPA in the synovial membrane (14) and the indicated link between serum ACPA levels and synovial intracellular citrullinated proteins, we next demonstrated that the difference in ACPA levels between the intracellular citrullinated protein positive and negative group was even more pronounced in SF (858 U/ml versus 127 U/ml, p=0,013) than in serum. After correction of the ACPA levels for total IgG in serum and SF, respectively, the resulting relative amount of IgG directed against citrullinated proteins was higher in SF than in serum for the intracellular citrullinated protein positive group (median ratio=1,49) but lower in SF than in serum for the intracellular citrullinated protein negative group (median ratio=0,91)(comparison between both groups: p=0,024). The increase of local ACPA levels specifically in those joints characterized by the presence of synovial intracellular citrullinated proteins suggested local ACPA production rather than trapping of systemically produced ACPA since inhibition experiments indicated that free ACPA but not ACPA complexed with citrullinated proteins such as citrullinated fibrinogen were measured by the anti-ccp2 ELISA (data not shown). 91.

13 CHAPTER 4 Discussion Considering the high specificity of ACPA and their occurence before the clinical onset of RA (3-8), identification of the molecular and cellular players involved in the citrullinated protein/acpa conflict is of major interest for unravelling the pathogenesis of RA. Previous data indicated that not all in vivo or in vitro substrates reactive with ACPA are pathophysiologically relevant, as illustrated by deiminated filaggrin, and that the relevant antigenic target(s) are probably present in the inflamed joint (14,46). Therefore, the present study addressed the pathophysiological relevance of synovial targets of ACPA by analyzing directly human synovial tissue and paired serum and SF samples. Extending our earlier report (26), staining with the monoclonal F95 anti-citrulline antibody indicated the presence of RA-specific intracellular citrullinated proteins in synovium. Whereas the results of our first study could have been biased by the detection of free citrulline or by the non-specific binding of the anti-l-citrulline antibody to plasma cells due to its polyclonal rabbit origin (47), the F95 antibody is of mouse origin and does not recognize free citrulline. Taken together with previous experiments demonstrating the specificity of F95 for citrullinated but not native proteins (33 and unpublished data), these data confirm that the RA-specific targets recognized by F95 in synovium are intracellular citrullinated proteins. We additionally demonstrated that the RA-specific intracellular staining with the F95 antibody correlates strongly with the data obtained with the polyclonal rabbit anticitrulline antibody. In contrast with the RA-specificity of the intracellular staining, F95 stained also extracellular citrullinated proteins which are not RA-specific. These observations are in line with a recent report using different phage display antibodies and purfied human ACPA (48) as well as with our findings on deiminated fibrin using an antibody recognizing chemically modified citrulline epitopes independently of the amino acid context (20). The detection of different synovial deiminated proteins, some of which are RA-specific, by distinct antibodies is not contradictory but rather indicates that not only citrulline but also neighbouring amino acids determine the specific epitopes recognized by the different anti-citrulline antibodies, mimicking the in vivo situation with ACPA (16). Of interest, the extra- versus intracellular localization of deiminated antigens may be of crucial importance since it was demonstrated that the abundant extracellular presence usually leads to B cell tolerance whereas intracellular localization as such may be sufficient to convert a tolerogenic self-antigen into a potent autoimmunogen (49). Ongoing biochemical studies are investigating if the RA-specific intracellular citrulinated proteins are distinct intracellular molecules or rather epitopes processed from deiminated extracellular proteins such as fibrin. 92.

14 ACPA levels are associated with synovial intracellular citrullinated proteins Considering the presence of several deiminated proteins in RA synovium (18,20,24,26), the repeated finding that only the intracellular citrullinated proteins described in the present study are highly specific for RA synovium (20,26,27) was further investigated. In sharp contrast with the free citrulline-producing enzyme inos, we demonstrated that both the number of samples containing PAD2 and the degree of PAD2 expression was significantly higher in RA than in control synovium and that intracellular citrullinated proteins colocalized with the citrullinating enzyme PAD2. However, it should be taken into account that not the mere presence but also the activation of PAD2 is pathophysiologically relevant, as illustrated by the fact that not all PAD2-positive cells contained citrullinated proteins in RA synovium and that PAD2 was also found in some control samples. Ongoing investigation is assessing if this is related to programmed cell death, as demonstrated in vitro (25,28,29), or to other mechanisms leading to the increase of the intracellular calcium levels needed for the activation of the citrullinating PAD enzymes in human RA synovium. Of interest, we demonstrated previously the presence of not only PAD2 but also PAD4 in human synovial tissue and both subtypes were also found in synovial fluid mononuclear cells (29,36). However, the association between functional haplotypes of PADI4, the gene encoding for PAD4, and RA remains controversial (50-53). Furthermore, in contrast with PAD2, the PAD4 protein was extensively distributed throughout RA synovium, did not colocalize selectively with intracellular citrullinated proteins, and was also found in osteoarthritic synovium (54). It would be most interesting to assess if PAD2 and PAD4 have different cellular origins, synovial localization, and substrate specificities in the joint, and if this relates to the difference between the RAspecificity of intracellular citrullinated proteins and the more ubiquitous presence of other deiminated proteins in synovium. Beside the RA-specificity and the link with the distinct presence of PAD2 in RA synovium, the higher ACPA titers in synovial intracellular citrullinated proteinpositive versus -negative patients is a third major argument provided by this study for the pathophysiological importance of these distinct proteins. The relevance of this finding is emphasized by several facts. Firstly, previous studies failed to demonstrate a link between ACPA and non RA-specific citrullinated proteins, including fibrin (20,48). Secondly, we indicated that the presence of intracellular citrullinated proteins is independent of the degree of synovial inflammation and that the demonstrated link with ACPA is thus not merely a secondary phenomenon due to higher ACPA levels leading to enhanced inflammation. Thirdly, the link between ACPA and intracellular citrullinated proteins was related to the presence of the HLA-DR shared epitope, corroborating with increasing evidence of an interaction between the HLA-DR genetic background and anti-citrullinated protein reactivity (41-44). Finally, the relationship between intracellular citrullinated proteins and ACPA was stronger locally in SF than systemically in serum, with higher SF than 93.

15 CHAPTER 4 serum ACPA levels in intracellular citrullinated protein-positive but not -negative joints. Since this can not be fully explained by immobilization of ACPA by complex formation with citrullinated targets in the joints, these data are in line with the previous demonstration of local ACPA production (14,48) and additionally suggest that this could be driven by specific synovial deiminated proteins. In conclusion, the present study indicates the RA-specific presence in synovium of intracellular citrullinated proteins, which is, at least partially, related to the distinct presence of the citrullinating PAD2 enzyme. Additionally, the association of synovial intracellular citrullinated proteins with systemic and local ACPA levels provides direct evidence for the pathophysiological relevance of these proteins as RA-specific antigenic targets of the humoral autoimmune process in vivo. 94.

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19 CHAPTER 4 citrullinated epitopes: its characterization and some applications to immunohistochemistry in human brain. Glia 2002;37: Nicholas AP, King JL, Sambandam T, Echols JD, Gupta KB, McInnes C, Whitaker JN. Immunohistochemical localization of citrullinated proteins in adult rat brain. J Comp Neurol 2003;459: Nicholas AP, Sambandam T, Echols JD, Tourtellotte WW. Increased citrullinated glial firbillary acidic protein in secondary progressive multiple sclerosis. J Comp Neurol 2004;473: Chapuy-Regaud S, Sebbag M, Nachat R, Baeten D, Foulquier V, Simon M, et al. Peptidylarginine deiminase isoforms expressed in the synovial membrane of rheumatoid arthritis patients. Arthritis Res Ther 2003;5(Suppl 1): Asaga H, Nakashima K, Senshu T, Ishigami A, Yamada M. Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils. J Leukoc Biol 2001;70: Nakashima K, Hagiwara T, Yamada M. Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes. J Biol Chem 2002;277: Hagiwara T, Nakashima K, Hirano H, Senshu T, Yamada M. Deimination of arginine residues in nucleophosmin/b23 and histones in HL-60 granulocytes. Biochem Biophys Res Commun 2002;290: Sambandam T, Belousova M, Accaviti-Loper MA, Blanquicett C, Guercello V, Raijmakers R, Nicholas AP. Increased peptidylarginine deiminase type II in hypoxic astrocytes. Biochem Biophys Res Commun 2004;325: Hill JA, Southwood S, Sette A, Jevnikar AM, Bell DA, Cairns E. Cutting edge: the conversion of arginine to citrulline allows for high affinity peptide interaction with the rheumatoid arthritis associated HLA-DRB1*0401 MHC class II molecule. J Immunol 2003;171: Zeng X, Ai M, Tian X, Gan X, Shi Y, Song Q, Tang F. Diagnostic value of anticyclic citrullinated peptide antibody in patients with rheumatoid arthritis. J Rheumatol 2003;30: Goldbach-Mansky R, Lee J, McCoy A, Hoxworth J, Yarboro C, Smolen JS, et al. Rheumatoid arthritis associated autoantibodies in patients with synovitis of recent onset. Arthritis Res 2000;2: van Gaalen FA, van Aken J, Huizinga TWJ, Schreuder GMT, Breedveld FC, Zanelli E, et al. Association between HLA class II genes and autoantibodies to cyclic citrullinated peptides (CCPs) influences the severity of rheumatoid arthritis. Arthritis Rheum 2004;50; Vallbracht I, Rieber J, Oppermann M, Förger F, Siebert U, Helmke K. Diagnostic and clinical value of anti-cyclic citrullinated peptide antibodies compared with rheumatoid factor isotypes in rheumatoid arthritis. Ann 98.

20 ACPA levels are associated with synovial intracellular citrullinated proteins Rheum Dis 2004;63: Reparon-Schuijt CC, van Esch WJ, van Kooten C, Schellekens GA, de Jong BA, van Venrooij WJ, et al. Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in rheumatoid arthritis. Arthritis Rheum 2001;44: Smeets TJ, Vossenaar ER, van Venrooij WJ, Tak PP. Is expression of intracellular citrullinated proteins in synovial tissue specific for rheumatoid arthritis? Comment on the article by Baeten et al. Arthritis Rheum 2002;46: Vossenaar ER, Smeets TJ, Kraan MC, Raats JM, van Venrooij WJ, Tak PP. The presence of citrullinated proteins is not specific for rheumatoid synovial tissue. Arthritis Rheum 2004;50: Ferry H, Jones M, Vaux DJ, Roberts ISD, Cornall RJ. The cellular location of self-antigen determines the positive and negative selection of autoreactive B cells. J Exp Med 2003;198: Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, Suzuki M, et al. Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nature Genetics 2003;34: Barton A, Bowes J, Eyre S, Spreckley K, Hinks A, John S, Worthington J. A functional haplotype of the PADI4 gene associated with rheumatoid arthritis in a Japanese population is not associated in a United Kingdom population. Arthritis Rheum 2004;50: Caponi L, Petit-Teixeira E, Sebbag M, Bongiorni F, Moscato S, Pratesi F, et al. A family-based study shows no association between rheumatoid arthritis and the PADI4 gene in a French caucasian population. Ann Rheum Dis (Epub ahead of print). 53. Cantaert T, Coucke P, De Rycke L, Veys EM, De Keyser F, Baeten D. Rheumatoid arthritis specific synovial intracellular citrullinated proteins and anti-citrullinated protein antibodies are not associated with functional haplotypes of PADI4. Ann Rheum Dis 2005 (Epub ahead of publication). 54. Chang X, Yamada R, Suzuki A, Sawada T, Yoshino S, Tokuhiro S, Yamamoto K. Localization of peptidylarginine deiminase 4 (PADI4) and citrullinated protein in synovial tissue of rheumatoid arthritis. Rheumatology 2004;