SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Antibodies directed to citrulline-containing proteins are highly specific for rheumatoid arthritis (RA) and can be detected in up to 80% of patients with RA. Citrulline is a nonstandard amino acid that can be incorporated into proteins only by posttranslational modification of arginine by peptidylarginine deiminase (PAD) enzymes. The objective of this study was to investigate the presence of anticitrulline antibodies, PAD enzymes, and citrullinated antigens in mouse models of both acute and chronic destructive arthritis: streptococcal cell wall (SCW)–induced arthritis and collagen-induced arthritis (CIA), respectively.

Methods

Synovial tissue biopsy specimens were obtained from naive mice, mice with CIA, and mice with SCW-induced arthritis. The expression of messenger RNA (mRNA) for PAD enzymes was analyzed by reverse transcriptase–polymerase chain reaction; the presence of PAD proteins and their products (citrullinated proteins) was analyzed by Western blotting and by immunolocalization. The presence of anticitrullinated protein antibodies was investigated by an anti–cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) and an ELISA using in vitro citrullinated fibrinogen.

Results

In both mouse models, PAD type 2 (PAD2) mRNA was present in the synovium but was not translated into PAD2 protein. In contrast, PAD4 mRNA, although absent from healthy synovium, was readily transcribed and translated by polymorphonuclear neutrophils infiltrating the synovial tissue during inflammation. As a consequence, several synovial proteins were subjected to citrullination. One of these proteins was identified as fibrin, which has been reported to be citrullinated also in synovium of patients with RA. Although generation of citrullinated antigens during synovial inflammation in the mice was eminent, no anti-CCP antibodies could be detected.

Conclusion

Citrullination of synovial antigens is an active process during joint inflammation in both mice and humans, but the induction of autoantibodies directed to these proteins is a more specific phenomenon, detectable only in human RA patients.

Rheumatoid arthritis (RA) is the most prevalent systemic rheumatic disorder, affecting ∼1% of the world population. It is characterized by chronic inflammation of the joints, eventually causing irreversible joint damage that can lead to severe disability. The inflamed joints are infiltrated by large numbers of activated mononuclear cells that contribute to the destruction of articular cartilage.

One of the major features of RA is that many circulating autoantibodies directed to self antigens, including the well-known rheumatoid factor (RF) antibodies, can be found in patient sera (for review, see ref.1). The most specific autoantibody system for RA is the family of autoantibodies directed to citrulline-containing proteins (for review, see ref.2), including antiperinuclear factor (APF) (3), antikeratin antibody (4), antifilaggrin autoantibodies (5), anti-Sa (6), and anti–cyclic citrullinated peptide (anti-CCP) (5, 7). The essential part of the antigenic determinant recognized by these antibodies is the unusual amino acid citrulline (7, 8). Citrulline can be generated by posttranslational modification of arginine residues (guanido group[RIGHTWARDS ARROW] ureido group) (Figure 1). This modification is catalyzed by peptidylarginine deiminase (PAD) enzymes, of which 4 mammalian isotypes have been described (9). Antibodies to citrullinated proteins can be detected in up to 80% of patients with RA, with >98% specificity (2, 10).

thumbnail image

Figure 1. Enzymatic conversion of peptidylarginine to peptidylcitrulline by peptidylarginine deiminase in the presence of Ca2+.

Download figure to PowerPoint

Many murine models of arthritis exist, and 2 such models were studied here. Type II collagen (CII)–induced arthritis (CIA) is a widely used model of arthritis, based on autoreactivity against cartilage CII. Immunization of susceptible strains of mice with foreign CII consequently leads to cross-reactivity of the T cell response and an antibody-mediated immune reaction to homologous CII. After a delayed onset (∼4 weeks), arthritis progresses to a chronic stage, characterized by severe erosions of cartilage and bone (11, 12). Streptococcal cell wall (SCW)–induced arthritis in mice can be produced by intraarticular injection of bacterial fragments. An acute, macrophage-initiated inflammation is induced, with severe joint swelling accompanied by the release of chemokines, leading to rapid infiltration of predominantly polymorphonuclear neutrophils (PMNs) and macrophages into the joint. This acute local inflammation does not lead to a chronic or polyarticular arthritis; uninjected joints remain unaffected (13, 14).

In animal models, RF has been reported in MRL/lpr mice (15) and was recently observed in interleukin-1 receptor antagonist–deficient (16) and HLA–DQ8–transgenic mice with CIA (17). There are no reports on the presence of antibodies to citrullinated proteins or citrullinated synovial antigens in mouse models. This is the first report on the synovial expression of PAD enzymes and the presence of citrullinated proteins in CIA and SCW-induced arthritis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Animals.

For CIA, male DBA/1 mice were obtained from Bomholtgård (Ry, Denmark). For SCW-induced arthritis, male C57BL6 mice were obtained from Charles River Deutschland (Sulzfeld, Germany). The mice were housed in filter-top cages, and water and food were provided ad libitum. The mice were 10–12 weeks old at the time of study.

Initiation of CIA.

Mice received 100 μg of bovine CII in Freund's complete adjuvant enriched with Mycobacterium tuberculosis H37Ra (4 mg/ml), by injection into the base of the tail. Bovine collagen was isolated as described elsewhere (11). On day 21, the mice received intraperitoneal booster injections with 100 μg of collagen diluted in phosphate buffered saline (PBS). Mice were killed, in groups of at least 5 animals, on days 14, 21, 28, 35, and 42 postimmunization.

SCW preparation, and initiation of SCW-induced arthritis.

Streptococcus pyogenes T12 cells were cultured overnight in Todd-Hewitt broth. Cell walls were prepared as described previously (18). The resulting 10,000g supernatant was used for arthritis induction. Unilateral arthritis was induced by intraarticular injection of 25 μg SCW in 5 μl PBS into the right knee joint of naive mice. As a control, PBS was injected into the left knee joint of each mouse. Mice were killed, in groups of at least 5 animals, after 1 or 2 days.

Assessment of arthritis.

Mice were carefully examined, and scores for disease activity were assigned, as previously described (19, 20). The clinical severity of arthritis was graded on a scale of 0–2 for each paw, according to changes in redness and swelling (0 = no changes, 0.5 = significant, 1.0 = moderate, 1.5 = marked, 2.0 = maximal swelling and redness followed by ankylosis). After removal of the skin around the knee joint, inflammation of the knee joint was rated according to the same scoring system. The total arthritis score was expressed as the mean ± SD score for all paws and knee joints.

Collection of mouse tissue samples.

Following orbita punction to collect blood for serum analysis, mice were killed by cervical dislocation. The patella, with adjacent synovium, was immediately dissected. Two biopsy samples (3-mm diameter, 1 from the lateral side and 1 from the medial side) were punched out using a biopsy punch (Steifel, Watersbach, Germany). The synovium samples were immediately frozen in liquid nitrogen and stored at −80°C. For immunohistochemical analysis, total knee joints were fixed in 4% formaldehyde for 4 days. After decalcification in 5% formic acid, the specimens were processed for paraffin embedding.

Measurement of antibodies to citrullinated proteins.

The presence of antibodies to citrullinated proteins in mouse serum and synovial fluid was investigated by 2 enzyme-linked immunosorbent assays (ELISAs): the Rapscan RA second generation kit (CCP-2 test; Euro-Diagnostica, Arnhem, The Netherlands), and an ELISA with citrullinated fibrinogen. The CCP-2 test was performed according to the manufacturer's instructions. For the ELISA with citrullinated fibrinogen, 1 mg of human fibrinogen (Sigma, St. Louis, MO) was citrullinated in vitro with 2 units rabbit PAD type 2 (PAD2) (Sigma) in 0.1M Tris HCl (pH 7.6), 10 mM CaCl2, and 5 mM dithioerythritol (DTE) for 2 hours at 37°C. Progression of the citrullination was followed by immunoblotting with Senshu antibodies (described below). The ELISA was performed as described previously (21). Wells were coated with citrullinated fibrinogen (0.5 mg/well); wells coated with unmodified fibrinogen were used as a negative control. Sera obtained from RA patients (positive controls) were tested in a 200-fold dilution, mouse serum was tested in a 10-fold dilution, and mouse synovial fluid was tested in a 5-fold dilution.

Antibodies.

Rabbit antibodies directed against chemically modified citrulline (Senshu antibodies) have been described previously (22, 23). Monoclonal antibodies NIMP-R14 and F4/80 were obtained from Serotec (Kidlington, UK). Antibodies against mouse fibrin(ogen) were obtained from Dako (Glostrup, Denmark). Isotype-specific antibodies directed against PAD2 or PAD4 were produced by immunization of rabbits with PAD isotype–specific peptides (for PAD2, amino acids 3–8 and 516–531; for PAD4, amino acids 210–225 and 517–531) (Eurogentec, Seraing, Belgium).

To affinity purify the anti-PAD antisera, 1 mg of each of the 2 peptides was conjugated to 8 mg bovine serum albumin (BSA) in the presence of 0.05% glutaraldehyde. After 1 hour at room temperature, the reaction was stopped by adding 0.1 volume of 1M glycine NaOH (pH 7.2). The mixture was dialyzed against coupling buffer (0.1M NaHCO3 [pH 8.3], 500 mM NaCl) and then coupled to 0.5 gm CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) according to the manufacturer's instructions. The column was equilibrated in washing buffer (1× PBS [pH 7.4], 350 mM NaCl, 0.05% Nonidet P40 [NP40]) before serum was added. Unbound antibodies were removed by extensive washing. Anti-PAD antibodies were eluted with elution buffer (100 mM glycine HCl [pH 2.5], 500 mM NaCl, 0.05% NP40). Collected fractions were neutralized with 1M Tris (pH 10).

Preparation of synovial tissue protein extracts.

Frozen synovial biopsy specimens (10 specimens; 2 each from 5 mice per time point) were ground to a fine powder using the Micro-Dismembrator II (Braun, Melsungen, Germany). Proteins were extracted by resuspending the powder in lysis buffer (50 mM Tris HCl [pH 7.4], 100 mM KCl, 20 mM EGTA, 1 mM DTE, 1% NP40, 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) followed by sonification using a microtip probe (Branson Ultrasonics, Danbury, CT). Insoluble material was removed from the aqueous extract by centrifugation (12,000g for 15 minutes) and extracted with guanidine buffer (6M guanidine HCl, 20 mM EGTA, 10 mM DTE, 0.02% NaN3, 1 mM PMSF). After precipitation with 4 volumes of acetone, the guanidine-extracted proteins were redissolved in sodium dodecyl sulfate (SDS) sample buffer (250 mM Tris [pH 6.8], 2% SDS, 5% β-mercaptoethanol).

Western blotting.

Proteins were separated on 10% or 13% SDS–polyacrylamide gels and transferred onto nitrocellulose membranes by electroblotting. Blots used to detect citrullinated proteins were chemically modified prior to immunostaining, as described by Senshu et al (22). Blots were blocked in blotting buffer (PBS containing 4% nonfat dried milk and 0.1% NP40) for 2 hours at room temperature and incubated for 1–3 hours with the antibody of interest diluted in blotting buffer. After washing with blotting buffer, bound antibodies were detected by incubation with horseradish peroxidase (HRP)–conjugated swine anti-rabbit IgG antibodies (Dako), followed by chemiluminescence.

RNA isolation and reverse transcriptase–polymerase chain reaction (RT-PCR).

Total RNA was isolated from the ground tissue powder with 1 ml TRIzol reagent (Gibco BRL, Breda, The Netherlands) according to the manufacturer's instructions. Synthesis of complementary DNA (cDNA) from 1 μg of RNA was performed using the Reverse Transcription System (Promega, Leiden, The Netherlands) with 500 ng of random primers, according to the manufacturer's instructions. The reaction mixture was incubated for 10 minutes at room temperature, followed by 90 minutes at 42°C. After incubation, cDNA samples were diluted with water to 100 μl. For RT-PCR, 10 μl cDNA was added to 40 μl of mastermix containing 75 mM Tris HCl [pH 8.8], 20 mM (NH4)2SO4, 0.01% (volume/volume) Tween 20, 2 mM MgCl2, 0.2 mM dNTPs, 0.5 μM forward and reverse primers, and 1 unit Red Hot DNA polymerase (ABgene, Leusden, The Netherlands). PCR was performed using a T3 thermocycler (Biometra, Göttingen, Germany). Conditions were as follows: 2 minutes at 94°C; 25–35 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 15 seconds at 72°C; and 2 minutes at 72°C. Fifteen microliters of PCR product was analyzed on a 1.5% agarose gel using the Gel Doc system (Bio-Rad, Hercules, CA). The following primer pairs were used (the size of the amplicon is shown in parentheses): for β-actin (239 bp), 5′-ATTCCATCATGAAGTGTGACG-3′ and 5′-CGTACTCCTGCTTGCTGATCC-3′; for mammalian PAD1 (mPAD1) (460 bp), 5′-TGCTAACCATTTGAAG-3′ and 5′-CTTGTCATTGCGGCCGTGG-3′; for mPAD2 (173 bp), 5′-CATGTCTCAGATGATCCT-3′ and 5′-GCTGGTAGAGCTTCTGCC-3′; for mPAD3 (315 bp), 5′-CTGTGCGGACCGGCAGG-3′ and 5′-CACACTTATAGGCCTCACAG-3′; and for mPAD4 (373 bp), 5′-ATGGACTTTGAGGATGAC-3′ and 5′-TGTCTTGGAACACACGGG-3′. Cloned cDNA served as a positive control.

Immunolocalization.

For the immunolocalization of PAD enzymes, deparaffinized sections (7 μm) were treated for 15 minutes with 3% H2O2 in methanol, washed with PBS, and incubated for 60 minutes at room temperature with the purified rabbit anti-PAD2 or anti-PAD4 primary antibody. After washing, the sections were incubated for 30 minutes at room temperature with biotinylated swine anti-rabbit secondary antibody (E0431; Dako) diluted 1:200 in PBS–1% BSA. After another wash, the samples were incubated for 30 minutes at room temperature with 4 μg/ml HRP-conjugated streptavidin (P0397; Dako). The sections were developed for 10 minutes at room temperature with 0.5 mg/ml diaminobenzidine in 50 mM Tris (pH 7.6) and 0.02% H2O2 and counterstained with either hematoxylin or methylene green (to facilitate the detection of nuclear staining of PAD4). The sections were embedded in Permount (Fisher Scientific, Fair Lawn, NJ) and evaluated using a Leica (Rijswijk, The Netherlands) DMR microscope. Purified preimmune IgG from the same rabbits was used as a negative control. PMNs and mononuclear cells were detected by staining with the monoclonal antibodies NIMP-R14 and F4/80, respectively, as described previously (24, 25).

Citrullinated proteins were detected as described by Asaga and Senshu (26). Prior to incubation with the primary antibody, sections were treated for 3 hours at 37°C in modification solution consisting of 2 parts solution A (0.025% [weight/volume] FeCl3, 4.6M H2SO4, 3.0M H3PO4), 1 part solution B (1% diacetyl monoxime, 0.5% antipyrine, 1M acetic acid), and 1 part H2O. Control sections were incubated in a mixture of 2 parts solution A and 2 parts H2O. After extensive washing with PBS, slides were subsequently incubated with 3% H2O2 in methanol (15 minutes), with 5% normal swine serum (X0901; Dako) and 1% BSA in PBS (30 minutes). Samples were incubated overnight at room temperature with 0.125 μg/ml anti–modified citrulline (Senshu antibodies) in PBS–1% BSA. After washing, the sections were incubated for 30 minutes at room temperature with biotinylated swine anti-rabbit secondary antibody (E0431; Dako) diluted 1:200 in PBS–1% BSA. Finally, the slides were incubated for 30 minutes with Vectastain ABC reagent (Vector, Burlingame, CA) and subsequently developed as described above.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Absence of antibodies to citrullinated proteins in serum and synovial fluid of mice with experimental arthritis and control mice.

Autoantibodies to citrullinated proteins are very specific markers for RA and can be detected by the CCP-2 test in up to 80% of all patients with RA (10, 27). The antibodies constitute a higher proportion of IgG in synovial fluid compared with serum (ref.28, and Vossenaar E, et al: unpublished observations), which is indicative of local antibody production in the inflamed synovium. This production requires the local presence of citrullinated antigens. One such antigen, citrullinated fibrin, is specifically present in actively inflamed synovial tissue of patients with RA (29). It has been suggested that other proteins might also be citrullinated in the inflamed synovium (6, 30).

Because there have been no reports on the presence of antibodies to citrullinated proteins in animal models of arthritis, we investigated whether these antibodies can be detected in the serum and synovial fluid of mice with CIA and mice with SCW-induced arthritis, using 2 methods: the CCP-2 test and an in-house ELISA with in vitro citrullinated fibrinogen. All serum samples (n = 50) and synovial fluid samples (n = 10) from mice with CIA or SCW-induced arthritis, as well as all samples from control animals (n = 20), tested negative, suggesting that antibodies to citrullinated proteins are not produced during the course of disease in these models of experimental arthritis. Serum samples obtained from CIA mice 70 days postimmunization (n = 5) also tested negative.

Expression of PAD enzymes in synovial tissue.

Although anti-CCP antibodies could not be detected in the examined mouse models, it has been shown that PAD enzymes, which catalyze the formation of peptidylcitrulline (Figure 1), are expressed in leukocytes (31, 32). The joints of the arthritis-affected mice are infiltrated by large numbers of these cells. Therefore, we investigated the expression of PAD enzymes in inflamed mouse synovium.

First, the expression of messenger RNA (mRNA) for PAD enzymes was investigated. The 4 mammalian PAD enzymes are highly conserved. To distinguish between each type of PAD mRNA, isotype-specific PCR primer sets were developed. Total RNA was isolated from synovial biopsy specimens obtained from mice with CIA and mice with SCW-induced arthritis, and each isotype of PAD was amplified by RT-PCR using the isotype-specific primers. Mouse PAD1 is normally expressed only in epidermis and the uterus (9, 33), while mPAD3 expression is restricted to hair follicles (34). It is therefore not surprising that mRNA coding for mPAD1 and mPAD3 were undetectable in the mouse synovium (Figure 2).

thumbnail image

Figure 2. Expression of mRNA for peptidylarginine deiminase (PAD) enzymes in mouse synovial tissue. Total RNA, isolated from synovial biopsy specimens, was analyzed by reverse transcriptase–polymerase chain reaction with isotype-specific primers. Mouse PAD type 1 (mPAD1) mRNA and mPAD3 mRNA (panels 1 and 3, respectively) were undetectable. Expression of mPAD2 mRNA was observed in comparable amounts in both naive and arthritic mice (with collagen-induced arthritis [CIA] or streptococcal cell wall [SCW]–induced arthritis) (panel 2), whereas mPAD4 expression (panel 4) was detected only in arthritis-affected mice (for lane 2, average inflammation score = 1.4; for lane 3, average inflammation score = 1.8; for lane 5, average inflammation score = 1.5; for lane 6, average inflammation score = 1.7). β-actin (panel β) was used as a control for RNA input. Cloned cDNA served as a positive control (lane 8). As a negative control, reverse transcription was performed without template RNA (lane 7).

Download figure to PowerPoint

Mouse PAD2 is expressed in a broad range of tissues, including skeletal muscle, uterus, brain, salivary glands, and pancreas (33, 35). As shown in Figure 2, mPAD2 mRNA was readily expressed in mouse synovial tissue, and its expression level remained essentially unchanged during the inflammation process. PAD4 is also expressed in a wide variety of tissues, including spleen, pancreas, ovary, and granulocytes (9, 36, 37). In uninflamed mouse synovium, however, PAD4 was undetectable (Figure 2, lanes 1 and 4). Interestingly, expression of mRNA for mPAD4 was observed in the inflamed mouse synovium (Figure 2, lanes 2, 3, 5, and 6), and mRNA expression levels appeared to correlate with the degree of inflammation (results not shown). These results indicate that normally only the mRNA encoding for mPAD2 is present in mouse synovium. After induction of synovial inflammation, however, large numbers of activated leukocytes enter the joint, and mPAD4 mRNA expression can be detected.

Second, the protein expression of the PAD enzymes was investigated. Because no mRNA for mPAD1 or mPAD3 could be detected, we focused on PAD2 and PAD4. Protein extracts were prepared from biopsy specimens of synovial tissue and analyzed by immunoblotting. Furthermore, sections of synovial tissue were analyzed immunohistochemically. Using the PAD2 isotype–specific antibodies, mPAD2 protein could be detected on blots from neither naive nor inflamed mouse synovium (Figure 3A). Also, synovial tissue sections from mice with CIA (Figure 4A), mice with SCW-induced arthritis, and control mice (results not shown) were negative for mPAD2 protein. As expected, abundant mPAD2 staining was observed in the muscle tissue adjacent to the synovium (35, 38) (Figure 4B). From these experiments, we conclude that although the mRNA encoding mPAD2 is present, it is not translated into protein in either control mice or mice with CIA or SCW-induced arthritis.

thumbnail image

Figure 3. Immunoblots showing that mouse peptidylarginine deiminase type 4 (PAD4) protein is detected only in tissue extracts from inflamed synovium. Protein extracts from mouse synovial tissue were analyzed by immunoblotting using isotype-specific anti-PAD antisera. A, Immunoblot stained for PAD2. No PAD2 protein could be observed in either naive mice, mice with collagen-induced arthritis (CIA), or mice with streptococcal cell wall (SCW)–induced arthritis. Purified recombinant PAD2 (recPAD2) served as a positive control, and purified recombinant PAD4 served as a negative control. B, Identical immunoblot stained for PAD4. PAD4 protein expression could be observed only in inflamed synovial tissue (CIA and SCW) and was undetectable in naive mice. Purified recombinant PAD4 served as a positive control, and purified recombinant PAD2 served as a negative control.

Download figure to PowerPoint

thumbnail image

Figure 4. Immunolocalization showing that mouse peptidylarginine deiminase type 2 (PAD2) protein is absent from synovial tissue. Sections of mouse synovial tissue were stained for PAD2. No PAD2 protein could be detected in synovium from mice with collagen-induced arthritis (A), naive mice, or mice with streptococcal cell wall–induced arthritis (results not shown). Sections of mouse skeletal muscle (B) served as a positive control. Purified IgG from preimmune serum (insets) was used as a negative control. Sections were counterstained with hematoxylin to reveal tissue morphology. Bars = 100 μm.

Download figure to PowerPoint

The findings regarding mPAD4 were completely different. Using the PAD4 isotype–specific antibodies, mPAD4 protein could be detected in inflamed synovial tissue (from both mice with CIA and mice with SCW-induced arthritis) but not in synovial tissue from naive mice (Figure 3B). The amount of mPAD4 protein appeared to correlate with the degree of inflammation (results not shown). To identify which cells were expressing PAD4, we proceeded with immunolocalizations on synovial tissue sections. In sections obtained from naive control mice, no mPAD4 staining could be seen (Figure 5A), while in inflamed synovium, staining of the leukocyte infiltrate was observed (Figures 5B and E). Serial sections stained for monocyte/macrophages (F4/80) (Figures 5C and F) and PMNs (NIMP/R14) (Figures 5D and G) revealed a clear colocalization of mPAD4 expression with PMNs but not with monocyte/macrophages. These results indicate that during inflammation, mPAD4 protein is expressed by PMNs infiltrating the mouse synovium.

thumbnail image

Figure 5. Immunolocalization of mouse peptidylarginine deiminase type 4 (mPAD4) in naive and inflamed synovium. No mPAD4 staining was observed in synovium from naive mice (A). In contrast, PAD4 could be detected in the leukocyte infiltrate of mice with collagen-induced arthritis (B; day 42, inflammation score 2) and mice with streptococcal cell wall–induced arthritis (E; day 2, inflammation score 2). Serial sections stained for monocyte/macrophages (F4/80) (C and F) and polymorphonuclear neutrophils (PMNs; NIMP/R14) (D and G) indicate a colocalization of mPAD4 expression with PMNs but not with monocyte/macrophages. In B and E, the sites of strongest PAD4 expression are shown in the white elipses. Purified IgG from preimmune serum (insets) was used as a negative control. Sections were counterstained with hematoxylin (A) or methylene green (B–G) to reveal tissue morphology. Bars = 100 μm.

Download figure to PowerPoint

Presence of citrullinated proteins in inflamed synovial tissue.

The presence of PAD enzymes does not necessarily mean that citrullinated proteins—the products of these enzymes—are also present, because calcium and possibly other factors are needed for activation of the enzymes. For detection of citrullinated proteins in protein extracts or tissue sections, Senshu et al developed a method in which the citrulline side chain is specifically modified into an artificial amino acid side chain that is so bulky that the influence of flanking amino acids for epitope recognition becomes negligible (22, 23, 26). Noncitrullinated proteins cannot be modified by the chemical treatment and thus are not recognized by the specific antibodies. This method has been used successfully for the specific detection of citrullinated proteins in various studies (22, 23, 26, 29, 39–44). We used these Senshu antibodies to investigate the presence of citrullinated proteins in mouse synovium.

Staining of tissue sections revealed that citrullinated proteins could be detected in the synovial leukocyte infiltrate of both mice with CIA and mice with SCW-induced arthritis (Figures 6B and C). Synovial specimens obtained from naive control mice were completely negative (Figure 6A). In the inflamed synovium, oxygen metabolism is in disequilibrium, which leads to sites with oxygen excess (and subsequent generation of reactive oxygen species) and, in contrast, to sites of hypoxia, which can cause synovial tissue microinfarctions. At these sites, plaques of extravascular fibrin can be observed. Masson-Bessière et al have reported that in RA, some of this fibrin is citrullinated (29). To investigate whether the citrullinated protein mass observed in the sections of inflamed mouse synovial tissue could contain fibrin, sequential sections were stained for fibrin(ogen). No extravascular fibrin(ogen) could be detected in healthy control synovium (Figure 6D). In the arthritis-affected synovium, however, large plaques of extravascular fibrin could be observed, which showed—at least partly—colocalization with the citrullinated proteins (Figures 6E and F). These results strongly suggest that extravascular fibrin and probably other proteins are citrullinated during inflammation of mouse synovium.

thumbnail image

Figure 6. Immunolocalization of citrullinated proteins in naive and inflamed mouse synovium. A, No citrullinated protein could be detected in synovial tissue from naive mice. In the synovial tissue of arthritis-affected mice, however, citrullinated protein could be detected in the leukocyte-infiltrated areas of B, mice with collagen-induced arthritis (CIA; day 42, inflammation score 2) and C, mice with streptococcal cell wall (SCW)–induced arthritis (day 2, inflammation score 2). Serial sections stained for fibrin(ogen) revealed a colocalization of fibrin(ogen) and citrullinated proteins in E, mice with CIA and F, mice with SCW-induced arthritis. D, No fibrin(ogen) could be detected in synovial tissue from naive mice; only staining in blood vessels and bone marrow could be observed. Citrullinated proteins were detected with Senshu antibodies against chemically modified peptidylcitrulline. A–C, Unmodified sections were used as a negative control for citrulline staining (insets). D–F, For fibrin(ogen) staining, purified IgG from normal rabbit serum was used as a negative control (insets). Sections were counterstained with hematoxylin to reveal tissue morphology. Bars = 100 μm.

Download figure to PowerPoint

For biochemical analysis of citrullinated proteins, biopsy specimens of synovial tissue were ground to a fine powder in liquid nitrogen, and extracts from this powder were analyzed by immunoblotting. As expected, results for naive mice were negative, while the extracts obtained from arthritis-affected mice decorated several citrullinated proteins (Figure 7A). Several citrullinated protein bands could be detected in the aqueous and guanidine extracts (compare lanes 4 and 5 with lanes 7 and 8). Some bands were also decorated by antifibrin(ogen) antibodies of an identical immunoblot (indicated by arrows). From these experiments we conclude that citrullination of synovial fibrinolytic fragments and also of other synovial proteins is induced during the inflammatory processes in CIA and SCW-induced arthritis models.

thumbnail image

Figure 7. Immunoblots of synovial tissue extracts, revealing the presence of citrullinated proteins in inflamed synovium. A, No citrullinated proteins are detectable in extracts from naive mice (lanes 3 and 6). In arthritis-affected synovial tissue (collagen-induced arthritis [CIA] and streptococcal cell wall [SCW]–induced arthritis), multiple bands of citrullinated proteins are stained (lanes 4, 5, 7, and 8). Citrullinated proteins were detected with Senshu antibodies against chemically modified peptidylcitrulline. Arrows indicate citrullinated fibrinolytic fragments that react with antifibrin(ogen) antibodies. In vitro citrullinated human fibrinogen (cit-fib; 25 ng) served as a positive control. Noncitrullinated human fibrinogen (fib; 25 ng) was included as a specificity control. The fibrinogen subunits (Aα, Bβ, and γ) are indicated. The γ subunit cannot be citrullinated because it contains no arginines. B, Identical immunoblot stained for fibrin(ogen). The amount of fibrin(ogen) present is strongly elevated in inflamed synovial tissue, due to the occurrence of plaques of extravascular fibrin. Several fibrinolytic fragments are present in the extracts of inflamed synovial tissue. Arrows indicate citrullinated fibrinolytic fragments that are recognized by Senshu antibodies. A number of these bands can be recognized in the citrulline immunoblot (indicated by arrows). Human fibrinogen (citrullinated and noncitrullinated, both 25 ng) was included as a positive control.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

In mouse models of both acute and chronic arthritis, PAD2 mRNA is present in the synovium but is not translated into PAD2 protein. In contrast, PAD4 mRNA, although absent from naive synovium, is readily transcribed and translated in PMNs infiltrating the synovial tissue during inflammation. As a consequence, several synovial proteins are subjected to citrullination. One of these proteins was identified as fibrin, which has been reported to be citrullinated also in the synovium of patients with RA. Although in mice the generation of citrullinated antigens during synovial inflammation was evident, no anticitrullinated protein antibodies could be detected.

Mouse PAD2 mRNA expression was substantial in both naive and arthritis-affected mice. The primers used to amplify mPAD2 are selected on separate exons, so there is no risk of possible artifacts caused by the presence of genomic DNA in the RNA isolates. Nevertheless, mPAD2 protein could not be detected in the synovial tissue, by either Western blotting or immunohistochemical analysis. In contrast, mPAD2 protein was abundantly present in the muscle tissue surrounding the synovium. These observations strongly indicate that the synthesis of PAD2 protein in synovial cells is strictly regulated at the translational level. We assume that additional, as yet unknown, factors are needed for the translation of mPAD2 mRNA. The exact nature of such factors is not clear at present, but the results presented here indicate that mPAD2 protein is not involved in the citrullination of synovial proteins in these mouse models of arthritis. Mouse PAD4 is expressed by PMNs infiltrating the synovium during inflammation. In the synovium of naive mice, no mPAD4 expression could be detected, whereas in the inflamed synovium mPAD4 was found at both the RNA and the protein levels. The amount of mPAD4 present in the synovium was consistent with the degree of inflammation: weak expression in mildly inflamed synovium and stronger expression in severely inflamed synovium. The immunolocalization showed a clear overlap of mPAD4 expression with PMNs, but not with monocytes or macrophages.

Expression of the human homolog of rodent PAD4 in human PMNs has been reported (37, 39). This human homolog was previously known as PAD5. The human enzyme shows slightly different reaction kinetics toward artificial substrates (40). This is why the human enzyme was first believed to be a novel PAD, hence its name PAD5. Recent data (for review, see ref.45), however, all indicate that human PAD5 corresponds to the rodent PAD4 (37, 39). Sequence data show that human PAD4/5 is more strongly conserved to rodent PAD4 (71% identical amino acids) than to other PAD isotypes (∼50% identical amino acids). Its position within the PAD gene cluster (all PADs are encoded on human chromosome 1p36, mouse chromosome 4E1, and rat chromosome 5q36) is conserved. Furthermore, PAD4/5 contains a conserved nuclear localization signal motif, which has been shown to be required and sufficient for nuclear localization, whereas all other PAD isotypes are located in the cytosol (39). Based on these data, our suggestion to change the name of human PAD5 to PAD4 was recently approved by the Human Genome Organisation Gene Nomenclature Committee (45, 46).

PAD4 expression has been observed in human and mouse peripheral blood granulocytes (refs.37 and47, and Vossenaar E, et al: unpublished observations) and in HL60-derived granulocyte-like cells (40). In both mouse models, infiltration of PMNs into the synovium is a crucial step in the onset of joint inflammation. These results strongly suggest that the infiltrating PMNs are responsible for the synovial expression of mPAD4 protein.

For the activation of mPAD4 protein, Ca2+ (and possibly other factors) is needed. PAD enzymes are present in the cytosol or nucleoplasm (39, 48), where the Ca2+ concentration usually is very low (∼10−7M) and strictly regulated. The threshold Ca2+ concentration for PAD activation is approximately 10−5M (ref.49 and Nijenhuis S, et al: unpublished observations); the cytosolic Ca2+ concentration is thus too low for activation of PAD. Several studies have shown that PAD enzymes can be activated by stimulation of PAD-containing cells with a calcium ionophore (39, 44, 47, 50). Also in human buccal mucosa cells (which contain the APF antigen filaggrin) (3), PAD enzymes are activated only very late in the terminal differentiation of the cells (41, 51). By that time, the integrity of the plasma membrane of the cells is compromised, causing influx of Ca2+ from the extracellular space and subsequent activation of PAD. In the inflamed synovium, many cells undergo apoptosis or necrosis. Especially PMNs have a very short lifespan, estimated to be only a couple of days. When the membrane integrity is lost during the death of these cells, Ca2+ can freely enter the cell and activate PAD enzymes that are already present. An alternative possibility is that PAD enzymes could leak out of the dying cells, become activated (the extracellular Ca2+ concentration is ∼10−3M), and induce the citrullination of extracellular proteins such as fibrin. Citrullinated proteins could be detected in the leukocyte infiltrate in the inflamed synovium (Figure 6). This area contains large numbers of mPAD4-positive PMNs, which corroborates the idea that PAD4 is responsible for the citrullination of synovial proteins during inflammation.

In sections of inflamed synovial tissue, large deposits of extravascular fibrin could be seen (Figures 6E and F). These fiber-like structures can be visualized with antibodies against fibrin(ogen) but are known to contain other proteins as well (such as fibronectin and collagen) (52, 53). These fiber-like structures are also intensively decorated with Senshu antibodies, indicating that they contain citrullinated proteins. Western blots confirm that fibrin is one of these citrullinated proteins (Figure 7), although other citrullinated proteins appear to be present as well. The identity of these proteins is the subject of our further investigations.

Our finding that extravascular fibrin is citrullinated during joint inflammation in mouse models of arthritis is very interesting, because the presence of citrullinated fibrin has also been reported in human RA (29). It therefore appears that the generation of citrullinated fibrin in both mice and humans is the result of synovial inflammation and is not a particularly human RA–specific phenomenon.

Although citrullinated proteins were present during synovial inflammation in mice, no antibodies directed to them could be detected, either in the serum or in the synovial fluid. In fact, in none of the many animal models of arthritis tested (including rodent, canine, and primate models) does anti-CCP appear to be positive (van Boekel M: personal communication). There are 2 likely explanations for this phenomenon.

First, there is a huge difference in the duration of the human disease as compared with the mouse models. In the SCW model, joint inflammation is induced within 24 hours, while in the CIA model onset of inflammation occurs within a few weeks. The development of RA takes many years, possibly more than a decade. Two independent studies have recently shown that former blood donors in whom RA later developed became RF positive and anti-CCP positive years before appearance of the first clinical symptoms of RA (54, 55). Anti-CCP production and, consequently, citrullination are clearly very early processes in the development of RA. Nevertheless, it could take many months or even years before anti-CCP autoimmunity develops. The duration of disease in the animal models (a maximum of 10 weeks in the CIA model) may well be too short for the evolution of anti-CCP antibodies.

Second, the role of genetic factors (e.g., HLA alleles as a risk factor for RA) has been known for more than 25 years. The HLA–DR4 (HLA–DRB1*0401 and *0404) phenotypes are especially important in this respect (56). Recent molecular modeling data indicate that peptides containing citrulline, but not arginine, can be bound by *0401 major histocompatibility complex molecules (57). This citrulline-specific interaction might be the basis of a citrulline-specific immune response. T cell proliferation assays with HLA–DRB1*0401–transgenic mice showed that stimulation with citrullinated peptides, not arginine peptides, induced proliferation and activation of T cells (58). These data suggest that the specific structure of HLA–DR4 molecules plays an important role in the induction of autoimmunity to citrullinated proteins. The mice used in the CIA model (DBA/1) and the SCW-induced arthritis model (C57BL6) do not have the arthritis-prone alleles. Their respective HLA haplotypes, H-2q and H-2b, are not associated with a risk of developing RA.

Taken together, these factors are compatible with the danger model hypothesis (59): an initial small inflammation caused by an external pathogen can cause the death of cells that cause or participate in inflammation, and consequently induce the citrullination of synovial proteins. In a certain genetic environment, this may lead to presentation of citrullinated peptides by antigen-presenting cells and consequently to activation of T cells. If the right conditions are present, this initial immune response can snowball into a systemic disease that is manifested many years later.

Citrullination of synovial proteins is not a process specific to RA. In the models of both acute and chronic arthritis, several synovial proteins, including fibrin, are subjected to citrullination during inflammation. Although these mouse models can be very useful in understanding the arthritis process, they do not exactly mimic RA (especially not the SCW model, which is a model of acute arthritis). The humoral response that is so characteristic of RA is absent in these mice. We conclude that the generation of citrullinated antigens is an inflammation-associated process, but that the antibody response to citrullinated proteins is highly specific to RA and possibly is involved in the perpetuation of the human disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Birgitte Oppers and Dr. Erik Lubberts (Nijmegen, The Netherlands) for useful discussions and excellent technical assistance, and Ben de Jong and Kalok Cheung for their valuable help in the ELISA experiments.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  • 1
    Mageed RA. The RF antigen. In: van VenrooijWJ, MainiRN, editors. Manual of biological markers of disease. Dordrecht (The Netherlands): Kluwer Academic Publishers; 1996. Section B1.1. p. 127.
  • 2
    Van Boekel MA, Vossenaar ER, van den Hoogen FH, van Venrooij WJ. Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value. Arthritis Res 2002; 4: 8793.
  • 3
    Hoet RM, Boerbooms AM, Arends M, Ruiter DJ, van Venrooij WJ. Antiperinuclear factor, a marker autoantibody for rheumatoid arthritis: colocalisation of the perinuclear factor and profilaggrin. Ann Rheum Dis 1991; 50: 6118.
  • 4
    Simon M, Girbal E, Sebbag M, Gomes Daudrix V, Vincent C, Salama G, et al. The cytokeratin filament-aggregating protein filaggrin is the target of the so-called “antikeratin antibodies,” autoantibodies specific for rheumatoid arthritis. J Clin Invest 1993; 92: 138793.
  • 5
    Girbal-Neuhauser E, Durieux JJ, Arnaud M, Dalbon P, Sebbag M, Vincent C, et al. The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated on various sites of (pro)filaggrin by deimination of arginine residues. J Immunol 1999; 162: 58594.
  • 6
    Menard HA, Lapointe E, Rochdi MD, Zhou ZJ. Insights into rheumatoid arthritis derived from the Sa immune system. Arthritis Res 2000; 2: 42932.
  • 7
    Schellekens GA, Visser H, de Jong BA, van den Hoogen FH, Hazes JM, Breedveld FC, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 2000; 43: 15563.
  • 8
    Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest 1998; 101: 27381.
  • 9
    Rus'd AA, Ikejiri Y, Ono H, Yonekawa T, Shiraiwa M, Kawada A, et al. Molecular cloning of cDNAs of mouse peptidylarginine deiminase type I, type III and type IV, and the expression pattern of type I in mouse. Eur J Biochem 1999; 259: 6609.
  • 10
    Van Venrooij WJ, Hazes JM, Visser H. Anticitrullinated protein/peptide antibody and its role in the diagnosis and prognosis of early rheumatoid arthritis. Neth J Med 2002; 60: 3838.
  • 11
    Van den Berg WB, Joosten LA. Murine collagen-induced arthritis. In: MorganDW, MarschallLA, editors. In vivo models of inflammation. Basel: Birkhauser Verlag; 1999. p. 5175.
  • 12
    Luross JA, Williams NA. The genetic and immunopathological processes underlying collagen-induced arthritis. Immunology 2001; 103: 40716.
  • 13
    Carlson RP, Jacobson PB. Comparison of adjuvant and streptococcal cell wall-induced arthritis in the rat. In: MorganDW, MarschallLA, editors. In vivo models of inflammation. Basel: Birkhauser Verlag; 1999. p. 150.
  • 14
    Schimmer RC, Schrier DJ, Flory CM, Dykens J, Tung DK, Jacobson PB, et al. Streptococcal cell wall-induced arthritis: requirements for neutrophils, P-selectin, intercellular adhesion molecule-1, and macrophage-inflammatory protein-2. J Immunol 1997; 159: 41038.
  • 15
    Jonsson R, Pitts A, Mestecky J, Koopman W. Local IgA and IgM rheumatoid factor production in autoimmune MRL/lpr mice. Autoimmunity 1991; 10: 714.
  • 16
    Horai R, Saijo S, Tanioka H, Nakae S, Sudo K, Okahara A, et al. Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. J Exp Med 2000; 191: 31320.
  • 17
    Taneja V, Taneja N, Paisansinsup T, Behrens M, Griffiths M, Luthra H, et al. CD4 and CD8 T cells in susceptibility/protection to collagen-induced arthritis in HLA-DQ8-transgenic mice: implications for rheumatoid arthritis. J Immunol 2002; 168: 586775.
  • 18
    Van den Broek MF, van den Berg WB, van de Putte LB, Severijnen AJ. Streptococcal cell wall-induced arthritis and flare-up reaction in mice induced by homologous or heterologous cell walls. Am J Pathol 1988; 133: 13949.
  • 19
    Joosten LA, Helsen MM, van de Loo FA, van den Berg WB. Anticytokine treatment of established type II collagen–induced arthritis in DBA/1 mice: a comparative study using anti-TNFα, anti-IL-1α/β, and IL-1Ra. Arthritis Rheum 1996; 39: 797809.
  • 20
    Van den Berg WB, Joosten LA, Helsen M, van de Loo FA. Amelioration of established murine collagen-induced arthritis with anti-IL-1 treatment. Clin Exp Immunol 1994; 95: 23743.
  • 21
    Verheijen R, de Jong BA, Oberye EH, van Venrooij WJ. Molecular cloning of a major CENP-B epitope and its use for the detection of anticentromere autoantibodies. Mol Biol Rep 1992; 16: 4959.
  • 22
    Senshu T, Sato T, Inoue T, Akiyama K, Asaga H. Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane. Anal Biochem 1992; 203: 94100.
  • 23
    Senshu T, Akiyama K, Kan S, Asaga H, Ishigami A, Manabe M. Detection of deiminated proteins in rat skin: probing with a monospecific antibody after modification of citrulline residues. J Invest Dermatol 1995; 105: 1639.
  • 24
    Van Lent PL, Holthuysen AE, van den Bersselaar LA, van Rooijen N, Joosten LA, van de Loo FA, et al. Phagocytic lining cells determine local expression of inflammation in type II collagen–induced arthritis. Arthritis Rheum 1996; 39: 154555.
  • 25
    Austyn JM, Gordon S. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur J Immunol 1981; 11: 80515.
  • 26
    Asaga H, Senshu T. Combined biochemical and immunocytochemical analyses of postmortem protein deimination in the rat spinal cord. Cell Biol Int 1993; 17: 52532.
  • 27
    Vasishta A. Diagnosing early-onset rheumatoid arthritis: the role of anti-CCP antibodies. Am Clin Lab 2002; 21: 346.
  • 28
    Smeets TJ, Vossenaar ER, Kraan MC, van Mansum WAM, Raats JM, van Venrooij WJ, et al. Expression of citrulline-containing antigens in RA synovium [abstract]. Arthritis Res 2002; 4 Suppl 3: 4.
  • 29
    Masson-Bessière C, Sebbag M, Girbal-Neuhauser E, Nogueira L, Vincent C, Senshu T, et al. The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- and beta-chains of fibrin. J Immunol 2001; 166: 417784.
  • 30
    Masson-Bessière C, Sebbag M, Durieux JJ, Nogueira L, Vincent C, Girbal-Neuhauser E, et al. In the rheumatoid pannus, anti-filaggrin autoantibodies are produced by local plasma cells and constitute a higher proportion of IgG than in synovial fluid and serum. Clin Exp Immunol 2000; 119: 54452.
  • 31
    Vossenaar ER, Radstake TR, van der Heijden A, Barrera P, van Venrooij WJ. Anti-citrulline antibodies and citrullinating enzymes in RA [abstract]. Arthritis Rheum 2002; 46 Suppl 9: S502.
  • 32
    Nagata S, Senshu T. Peptidylarginine deiminase in rat and mouse hemopoietic cells. Experientia 1990; 46: 724.
  • 33
    Terakawa H, Takahara H, Sugawara K. Three types of mouse peptidylarginine deiminase: characterization and tissue distribution. J Biochem (Tokyo) 1991; 110: 6616.
  • 34
    Rogers G, Winter B, McLaughlan C, Powell B, Nesci A. Hair follicle peptidylarginine deiminase. Exp Dermatol 1999; 8: 3623.
  • 35
    Watanabe K, Senshu T. Isolation and characterization of cDNA clones encoding rat skeletal muscle peptidylarginine deiminase. J Biol Chem 1989; 264: 1525560.
  • 36
    Yamakoshi A, Ono H, Nishijyo T, Shiraiwa M, Takahara H. Cloning of cDNA encoding a novel isoform (type IV) of peptidylarginine deiminase from rat epidermis. Biochim Biophys Acta 1998; 1386: 22732.
  • 37
    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: 4651.
  • 38
    Sugawara K, Oikawa Y, Ouchi T. Identification and properties of peptidylarginine deiminase from rabbit skeletal muscle. J Biochem (Tokyo) 1982; 91: 106571.
  • 39
    Nakashima K, Hagiwara T, Yamada M. Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes. J Biol Chem 2002; 277: 495628.
  • 40
    Nakashima K, Hagiwara T, Ishigami A, Nagata S, Asaga H, Kuramoto M, et al. Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1α,25-dihydroxyvitamin D(3). J Biol Chem 1999; 274: 2778692.
  • 41
    Senshu T, Kan S, Ogawa H, Manabe M, Asaga H. Preferential deimination of keratin K1 and filaggrin during the terminal differentiation of human epidermis. Biochem Biophys Res Commun 1996; 225: 7129.
  • 42
    Senshu T, Akiyama K, Nomura K. Identification of citrulline residues in the V subdomains of keratin K1 derived from the cornified layer of newborn mouse epidermis. Exp Dermatol 1999; 8: 392401.
  • 43
    Akiyama K, Senshu T. Dynamic aspects of protein deimination in developing mouse epidermis. Exp Dermatol 1999; 8: 17786.
  • 44
    Asaga H, Yamada M, Senshu T. Selective deimination of vimentin in calcium ionophore-induced apoptosis of mouse peritoneal macrophages. Biochem Biophys Res Commun 1998; 243: 6416.
  • 45
    Vossenaar ER, Zendman AJW, van Venrooij WJ, Pruijn G. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays 2003. In press.
  • 46
    Povey S, Lovering R, Bruford E, Wright M, Lush M, Wain H. The HUGO Gene Nomenclature Committee (HGNC). Hum Genet 2001; 109: 67880.
  • 47
    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: 97983.
  • 48
    Takahara H, Tsuchida M, Kusubata M, Akutsu K, Tagami S, Sugawara K. Peptidylarginine deiminase of the mouse: distribution, properties, and immunocytochemical localization. J Biol Chem 1989; 264: 133618.
  • 49
    Takahara H, Okamoto H, Sugawara K. Calcium-dependent properties of peptidylarginine deiminase from rabbit skeletal muscle. Agric Biol Chem 1986; 50: 2899904.
  • 50
    Mizoguchi M, Manabe M, Kawamura Y, Kondo Y, Ishidoh K, Kominami, et al. Deimination of 70-kD nuclear protein during epidermal apoptotic events in vitro. J Histochem Cytochem 1998; 46: 13039.
  • 51
    Ishida-Yamamoto A, Senshu T, Eady RA, Takahashi H, Shimizu H, Akiyama M, et al. Sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and keratin 1 deimination. J Invest Dermatol 2002; 118: 2827.
  • 52
    Weinberg JB, Pippen AM, Greenberg CS. Extravascular fibrin formation and dissolution in synovial tissue of patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum 1991; 34: 9961005.
  • 53
    Cheung HS, Ryan LM, Kozin F, McCarty DJ. Synovial origins of rice bodies in joint fluid. Arthritis Rheum 1980; 23: 726.
  • 54
    Rantapää-Dahlqvist S, de Jong BAW, Berglin E, Hallmans G, Wadell G, Stenlund H, et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum. In press.
  • 55
    Nielen MMJ, van Schaardenburg D, van de Stadt RJ, van der Horst-Bruinsma IE, de Koning MHM, Habibuw M, et al. Autoantibodies in serum of blood donors precede symptoms of rheumatoid arthritis (RA) by 1 to 6 years [abstract]. Arthritis Rheum 2002; 46 Suppl 9: S370.
  • 56
    Silman AJ, Pearson JE. Epidemiology and genetics of rheumatoid arthritis. Arthritis Res 2002; 4 Suppl 3: S26572.
  • 57
    Hill JA, Jevnikar AM, Bell DA, Cairns E. The role of HLA–DRB1*0401 MHC class II in autoimune responses to citrullinated antigens [abstract]. Arthritis Rheum 2002; 46 Suppl 9: S298.
  • 58
    Hill JA, Jevnikar AM, Bell DA, Cairns E. Citrullinated vimentin and fibrinogen peptides activate CD4+ T cells in HLA–DRB1*0401 transgenic mice [abstract]. Arthritis Rheum 2002; 46 Suppl 9: S299.
  • 59
    Matzinger P. The danger model: a renewed sense of self. Science 2002; 296: 3015.