Carbamylation-Dependent Activation of T Cells: A Novel Mechanism in the Pathogenesis of Autoimmune Arthritis

This information is current as Piotr Mydel, Zeneng Wang, Mikael Brisslert, Annelie of September 28, 2021. Hellvard, Leif E. Dahlberg, Stanley L. Hazen and Maria Bokarewa J Immunol published online 19 May 2010 http://www.jimmunol.org/content/early/2010/05/19/jimmun

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 19, 2010, doi:10.4049/jimmunol.1000075 The Journal of Immunology

Carbamylation-Dependent Activation of T Cells: A Novel Mechanism in the Pathogenesis of Autoimmune Arthritis

Piotr Mydel,* Zeneng Wang,† Mikael Brisslert,* Annelie Hellvard,* Leif E. Dahlberg,‡ Stanley L. Hazen,x and Maria Bokarewa*

The posttranslational modification of proteins has the potential to generate neoepitopes that may subsequently trigger immune responses. The carbamylation of residues to form homocitrulline may be a key mechanism triggering inflammatory responses. We evaluated the role of carbamylation in triggering immune responses and report a new role for this process in the induction of arthritis. Immunization of mice with homocitrulline-containing peptides induced chemotaxis, T cell activation, and Ab production. The mice also developed erosive arthritis following intra-articular injection of peptides derived from homo- and citrulline. Adoptive transfer of T and B cells from homocitrulline-immunized mice into normal recipients induced

arthritis, whereas systemic injection of homocitrulline-specific Abs or intra-articular injection of homocitrulline-Ab/citrulline- Downloaded from peptide mixture did not. Thus, the T cell response to homocitrulline-derived peptides, as well as the subsequent production of anti- homocitrulline Abs, is critical for the induction of autoimmune reactions against citrulline-derived peptides and provides a novel mechanism for the pathogenesis of arthritis. The Journal of Immunology, 2010, 184: 000–000.

ollowing synthesis, proteins undergo the process of post- peptides and the development of RA (8, 9). Despite the immense

translational modification (PTM), thus extending their value of these anti-Cit Abs as an early and specific predictive http://www.jimmunol.org/ F range of function through the modification of amino acids marker for RA (anti-citrullinated Abs are detectable before the with various functional groups (1, 2). These modifications have onset on clinical disease) (10, 11), the specific role played by a critical influence on protein structure and biological function, in the development of arthritis remains elusive. In- especially in the context of aging, physiological stress, and in- creased levels of Cit-modified residues have been found in myelin flammation. The role of PTM in the generation of neoepitopes on basic protein, which is now used as an Ag for the induction of self proteins that are subsequently responsible for the pathogenesis experimental autoimmune encephalomyelitis (12); however, sim- of autoimmune diseases, such as multiple sclerosis, diabetes ilar attempts using anti-Cit Abs to trigger experimental arthritis mellitus, systemic lupus erythematosus, and rheumatoid arthritis have yielded only modest results. Therefore, the existence of an- (RA), has only recently been recognized (3–6). other triggering factor has been postulated (13, 14). Factors that by guest on September 28, 2021 Citrullination, the posttranslational conversion of (Arg) have been identified as predisposing to the production of anti-Cit residues to citrulline (Cit) residues by peptidylarginine deiminase Abs include HLA-DR alleles and smoking (15–17). enzymes, has been extensively studied in relation to autoimmune Carbamylation (homocitrullination) is a PTM that has been arthritis (Fig. 1A) (7). The potentially harmful effects of these Cit- studied for many years in the context of uremia (18–20). It involves 2 modified proteins have attracted a lot of attention recently because the nonenzymatic reaction of -derived cyanate with free NH2 of the close association between the production of Abs to Cit- groups on lysine (Lys) residues to yield homocitrulline (Hcit) (Fig. 1B). This process can be mediated in vivo by myeloperoxi- dase (MPO), the enzyme responsible for the inflammation-driven *Department of Rheumatology and Inflammation Research, Sahlgrenska University 2 † carbamylation of proteins via the MPO/H O /SCN system. Thus, Hospital, University of Go¨teborg, Go¨teborg; Department of Cell Biology and 2 2 ‡Department of Orthopedics, University Hospital of Malmo¨, University of Lund, MPO has recently attracted attention as a potential trigger factor for x Malmo¨, Sweden; and Department of Cardiovascular Medicine, Cleveland Clinic, atherogenesis and inflammation (21). Hcit residues affect the Cleveland, OH 44195 charge distribution within a peptide in a way that may result in im- Received for publication January 12, 2010. Accepted for publication April 14, 2010. pairment or even loss of function. Loss of enzymatic function upon This work was supported by grants from the Medical Society of Go¨teborg, the carbamylation has been reported for matrix metalloproteinase- Swedish Association against Rheumatism, the King Gustaf V:s Foundation, the Swedish Medical Research Council, the Nanna Swartz Foundation, the AME Wolff 2, tissue inhibitor of metalloproteinase-2, and insulin (22–24). 2 Foundation, the Rune and Ulla Amlo¨vs Trust, the Swedish Foundation for Strategic Plasma SCN levels are known to be significantly higher in smok- Research, the Pharmacist Hedberg’s Foundation, the University of Go¨teborg, the ers, leading to increased carbamylation of proteins (21, 25); fur- Tho¨len and Kristlers Foundation, Bo¨rje Dahlin’s Foundation, the National Inflam- mation Network, the Lundberg Foundation, and the regional agreement on medical thermore, carbamylation has emerged as a potential pathogenic training and clinical research between the Western Go¨taland county council and the factor in renal insufficiency (26), cardiovascular disease (21, 27), University of Go¨teborg. and cataracts (28). Extracellular matrix proteins, such as collagen Address correspondence and reprint requests to Dr. Piotr Mydel, Department of and fibrinogen, are thought to accumulate structural damage eli- Rheumatology and Inflammation Research, University of Go¨teborg, Guldhedsgatan 10, S41346 Go¨teborg, Sweden. E-mail address: [email protected] cited by PTMs because of their long half-life and low turnover The online version of this article contains supplemental material. rates. Notably, it was shown that collagen is easily carbamylated Abbreviations used in this paper: aCCP, Ab to cyclic citrullinated peptides; Arg, in vivo and that such modification may be directly linked to gran- arginine; Cit, citrulline; Hcit, homocitrulline; KCNO, potassium cyanate; Lys, lysine; ulocyte activation and protease release (29, 30). mALB, mouse albumin; MPO, myeloperoxidase; n.a., not available; PTM, posttrans- In this study, we show that carbamylation provides the missing lational modification; RA, rheumatoid arthritis. link between Cit-peptides and the development of erosive arthritis. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 Using experimental models and patient material, we show that the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000075 2 CARBAMYLATION AND AUTOIMMUNE ARTHRITIS presence of carbamylated Lys residues triggers primary immune proved 98% of cells to be CD3+. A total of 2 3 106 of CD3+ cells were + injected i.v. into naive BALB/c mice. Controls received the same amount of responses, including the proliferation and chemotaxis of CD4 + T cells and the production of IL-10 and the proinflammatory CD3 cells isolated from unmanipulated BALB/c mice. Following 14 d of CD3+ cell transfer, mice received an intra-articular injection of Cit-peptide cytokines IFN-g and IL-17. Our results provide a novel mecha- (sequence E, 1 mg/knee). The injected knees were histologically evaluated nism for the pathogenesis of arthritis involving activation of 7 d later. Adoptive transfer of B cells was done as described above. B cells T cells in combination with a powerful Ab response, which leads were purified by negative selection using the Mouse B Cell Enrichment Kit to the local recognition of Cit-peptides in the joints and the sub- (StemCell Technologies, Vancouver, British Columbia, Canada). The purity of the B cell population (CD19+) was confirmed by flow cytometry. sequent development of erosive arthritis. Ab transfer Materials and Methods Total IgG was purified from serum obtained on immunization day 28 using Patients with RA and control individuals protein A affinity column (Amersham, Uppsala, Sweden). Following di- alysis against PBS, the reactivity of IgG with Hcit-peptide (Table I, se- Plasma and synovial fluid were collected from 72 consecutive RA patients quence B) was proved by ELISA. Purified IgG (Hcit-Ab) was injected i.p. (age range, 34–82 y; 17 males and 55 females; disease duration, 1–36 y) into naive recipients (6.7 mg/kg, 0.2 mg/mouse) in the volume of 0.2 ml admitted to Rheumatology Clinics at Sahlgrenska University Hospital for PBS, 24 h prior to intra-articular challenge. Hcit-Ab was incubated with acute joint effusion. Clinical and demographic characteristics of the patients deiminated (Cit) peptide (sequence E) in equal proportions for 30 min at are presented in Supplemental Table I. Plasma of 41 healthy individuals, room temperature to allow complex formation. The Hcit-Ab/Cit-peptide matched to RA patients by age and gender, and synovial fluid of patients (age mixture (total protein content 1 mg/knee) was instilled in the knee joints of n range, 28–82 y, 17 males and 23 females) with knee trauma ( = 24) and healthy mice. Control mice received intra-articular injection of Cit-peptide osteoarthritis (n = 16) were used as control. Collected samples were in complex with monoclonal Cit-Ab (ACC3), kindly provided by Prof. Downloaded from 3 g 2 centrifuged at 800 for 15 min, aliquoted, and stored frozen at 70˚C Rikard Holmdahl (Karolinska Institute, Stockholm, Sweden) (32). until use. The study was approved by the Ethical Committee of the Uni- versity of Go¨teborg. Informed consent was obtained from all patients. Ero- sive RA was defined by the presence of bone erosions on recent posterior– Cell stimulation and proliferation assay anterior radiographs of hands and feet. The presence of Ab to cyclic Spleens were excised on immunization day 21, and a single-cell suspension citrullinated peptides (aCCPs) were measured by ELISA (Immunoscan 3 6 . was prepared as described (33). The cultures (1 10 cells/ml) were CCPlus, Euro-Diagnostica, Malmo¨, Sweden), and levels 50 U/ml were maintained in 96-well plates (Nunc, Roskilde, Denmark) at 37˚C in 5% considered positive. http://www.jimmunol.org/ CO2 and 95% humidity and stimulated with study peptides and anti-CD3 Abs (BD Pharmingen, San Diego, CA). The supernatants were collected Peptides after 24 and 48 h. Syntheticpeptidescontainingfilaggrin-derivedsequencesweresynthesizedby Proimmune (Oxford, U.K.) (Table I, sequences A and D). The modifications Mass spectrometry of proteins recovered from and introduced in the peptides included carbamylation/homocitrullination of synovial fluid Lys (Hcit, sequences B and C), and deimination/citrullination of Arg (Cit, sequence E). Sequences C, D, and E have a cyclic structure due to the dis- Stable isotope-dilution liquid chromatography mass spectrometry analysis ulfide bond between the cysteine residues. Lyophilized peptides were dis- was performed on Arg and Lys and their derivatives, Cit and Hcit, in proteins solved in 10% acetic acid to a final concentration 4 mg/ml, aliquoted, and kept recovered from blood and synovial fluid, as described (21). In short, [13C6] frozen at 220˚C. Arg, [13C6, 15N2] Lys, and [13C6, 15N] Hcit were added before hydrolysis by guest on September 28, 2021 and used as internal standards to quantify Arg, Lys, Cit, and Hcit. Proteins Carbamylation of BSA and mouse albumin in vitro were hydrolyzed with 6 N HCl at 110˚C under vacuum. After acid hydro- lysis and clean-up with a mini solid-phase DSC-SCX extraction column BSA (20 mg/ml) and mouse albumin (2 mg/ml) (Sigma-Aldrich, St. Louis, (Discovery DSC-SCX SPE tubes; 1 ml; Sigma-Aldrich), analytes were re- MO) were carbamylated by incubation with 0.1 M potassium cyanate solved on a Phenyl column (4.6 3 250 mm, 5 mm Rexchrom Phenyl, Regis (KCNO) in 0.15 M phosphate buffer (pH 7.4) at 37˚C for 24 h. KCNO Technologies, Morton Grove, IL) using a gradient generated between aque- was removed by excessive dialysis at 4˚C against ultrapure water for 48 h ous ammonium formate versus methanol/0.1% formic acid/5 mM ammo- (31). A control protein sample treated with 0.1 M KCl instead of KCNO was nium formate mobile phases. Amino acids were analyzed on an API 365 also prepared using the procedure described above. The number of Hcit triple-quadrupole mass spectrometer with Ionics EP 10+ upgrade (Concord, residues generated during the carbamylation of BSA was measured by Ontario, Canada) interfaced to a Cohesive Technologies Aria LX Series nano-liquid chromatography electrospray ionization mass spectrometry HPLC multiplexing system using electrospray ionization on positive-ion (linear ion trap-Fourier transform ion cyclotron resonance mass spectrom- mode with multiple reaction monitoring of parent and characteristic daugh- eter), which showed that 78% of the total number of Lys were carbamylated. ter ions specific for each analyte monitored. The results for amino acids are reported in absolute concentrations. Animals, immunization procedure, and experimental arthritis In vitro migration assay For in vivo experiments, NMRI, BALB/c, and C57bl/6 mice were purchased from B & K International (Sollentuna, Sweden). An Asn breeding pair was The migratory capacity of splenocytes was tested using the Transwell provided by Dr. Mariam Gregorian (Cancer Institute, Copenhagen, Denmark) system with a pore size of 3 mm (AH Diagnostics, Stockholm Sweden), as and were bred in the animal facility of the University of Go¨teborg. Eight to 10 previously described (34). In short, splenocytes (5 3 105/well) were placed mice were kept per cage under standard environmental conditions and had in the upper chamber and migrated for 12 h toward CXCL13 (100 ng/ml) free access to standard laboratory chow and drinking water. Ethical permis- or 0.1% BSA-PBS dilution buffer as negative control added to the lower sion was obtained from the Animal Research Ethics Committee of Go¨teborg chamber. To exclude chemokinesis, 100 ng/ml CXCL13 was included in a University. The peptides were resuspended in carbonate/bicarbonate buffer set of wells in the lower and upper chambers. Migrated cells were collected (pH 9.6) to a final concentration 0.75 mg/ml and emulsified in an equal from the lower chamber and subjected to phenotype analyses by flow volume of CFA (Sigma-Aldrich). On days 0 and 14, mice were immunized cytometry, as described below. with 75 mg peptide provided intradermally. On day 21, mice were challenged with an intra-articular injection of Cit-peptide (1 mg/knee). Mice were sacri- Flow cytometry ficed on immunization day 28, and the lower paws were removed for histo- logic assessment. Mice were bled on days 0, 7, 21, and 28 for assessment of Migrated cells were stained with allophycocyanin-conjugated monoclonal Ab production. rat anti-mouse CD3 Abs, PE-conjugated monoclonal rat anti-mouse CD19 Abs, PerCP-conjugated monoclonal rat anti-mouse CD4 Abs, and Pacific Adoptive transfer of T cells Blue-conjugated monoclonal rat anti-mouse CD8 Abs (all Abs were from BD Biosciences, Erembodegem, Belgium) for 30 min on ice in FACS buffer CD3+ cells were isolated from spleens of BALB/c mice on day 21 following (PBS, 1% FCS, and 0.02% azide [pH 7.2]). Cells were washed, collected in immunization with Hcit peptide (sequence B) using the Dynal Mouse T cell the cytometer (FACSCanto II, BD Biosciences), and analyzed with FACS- Negative Isolation Kit (Invitrogen, Stockholm, Sweden). Flow cytometry Diva software using FlowJo software (Tree Star, Ashland, OR). Gating of The Journal of Immunology 3

FIGURE 1. Schematic illustration of PTMs of Arg and Lys. A, Deimination (citrullination) is an enzy- matic modification characterized by cleavage of NH3 groups from Arg residues mediated by peptidylarginine deiminases. B, Carbamylation (homocitrullination) is 2 characterized by the addition of a CONH2 group to Lys residues. the cells was based on the isotype control values, as well as fluorochrome Results minus one settings when needed. Immunization with carbamylated peptides predisposes to the Immunohistochemistry and histopathology development of erosive arthritis Lower paws of sacrificed mice were removed, fixed, decalcified in Parengy’s NMRI mice were immunized on days 0 and 14 with synthetic buffer, embedded in paraffin, and cut into 4-mm thin sections. Tissue peptide sequences derived from filaggrin, an epidermal skin protein sections were stained with H&E. For immunohistochemical evaluation, (Table I). On day 21, the knee joints were injected with carba- paws were decalcified using 0.5 M EDTA (pH 7.1). Tissue sections were deparaffinized and stained using rat anti-mouse CD3 Abs (10 mg/ml, mylated/Hcit-peptide (sequence B) or deiminated/Cit-peptide

MCA1477, Serotec, Oxford, U.K.), followed by biotinylated goat anti-rat (sequence E). Examination of knee joints injected with peptides Downloaded from Abs (2.5 mg/ml, Vector Laboratories, Burlingame, CA). Color reaction was identical to the one of immunization showed that Hcit-immunized completed with the Vectastain Elite ABC kit (Vector Laboratories) and 3- mice had clear signs of arthritis (arthritis index, 1.05 6 0.19) and amino-9-ethyl-carbazole containing H2O2. Sections were counterstained cartilage damage. In contrast, the Cit-immunized mice injected with hematoxylin. Rat serum constituted negative control. The sections were evaluated with respect to synovitis and erosion of bone/cartilage by intra-articularly with Cit-peptide developed arthritis only occa- a blinded examiner. Synovitis was defined as a membrane thickness of more sionally (arthritis index, 0.56 6 0.11) (Fig. 2A,2B). Interestingly, than two cell layers (35) and scored as follows: 1, mild; 2, moderate; and 3, the Hcit-immunized mice injected with Cit-peptide developed the http://www.jimmunol.org/ severe synovitis and joint damage (36). One half point was added to the most pronounced arthritis (arthritis index, 1.98) (Fig. 2A,2C1), score if erosions were present. Final arthritis index in the group was calcu- lated as a sum of all scores divided by the number of joints. with a prevalence of 92%. The severity of arthritis in the latter case was 2.21-fold greater than that seen in nonimmunized or Cit- Detection of peptide-specific Abs immunized mice given subsequent intra-articular injections of Cit- The 96-well plates (Maxisorp, Nunc) were coated with Cit-peptides (se- peptide (Fig. 2C2). We found these results to be consistent over five quence E, 0.5 mg/ml) or Hcit-peptides (sequence B, 0.1 mg/ml) in carbon- independent experiments using NMRI, BALB/c, and A/Sn mice ate-bicarbonate buffer (pH 9.6) and incubated overnight at 4˚C. Following strains (n = 126). The severity of the arthritis seen in the Hcit- blocking with 2% skim milk/PBS and washing with 0.05% Tween/PBS, immunized mice varied among the strains, being greatest in the samples (diluted 1/50 and 1/100 in blocking solution) were applied and d incubated for 6 h at room temperature. Rabbit anti-human IgG/HRP BALB/c (H2-A haplotype) and outbred NMRI mice (arthritis by guest on September 28, 2021 (100 ng/ml, Dako A/S, Glostrup, Denmark) was used as detection Ab. index, 2.23 6 0.49 and 2.06 6 0.21, respectively). A/Sn mice To measure peptide-specific Abs in mice, serum plates were coated with developed less severe arthritis (arthritis index, 1.09 6 0.27), peptides A–E at 0.1 mg/ml. Serum samples were diluted 1:200 in 1% BSA whereas the arthritis index for the C57BL/6 (H2-Ab haplotype) and incubated overnight at 4˚C. Rabbit anti-mouse IgG/HRP 10 ng/ml in 6 blocking solution were used as detection Abs. Secretion of Ig by spleno- mice was significantly lower (0.45 0.28), about the same as for cytes from mice that underwent adoptive transfer of T and B cells was nonimmunized controls. Taken together, these findings suggest that detected using ELISPOT, as described (37). the development of arthritis in this particular model is dependent upon the MHC-H2 haplotype. Measurements of cytokine levels Intra-articular injection of Hcit into nonimmunized or Cit- IL-2, -6, -10, and -17A and INF-g in supernatants were quantified using immunized mice (Fig. 1) led to the development of synovitis Quantikine ELISAs (R&D Systems, Minneapolis, MN). (arthritis index, 1.33 6 0.2 and 0.77 6 0.17, respectively) (Fig. Statistical analysis 2B,2C3). This was less severe than the arthritis that we saw pre- viously, with the development of erosions in only a few cases Continuous parameters were expressed as mean 6 SEM. Comparisons between the matched blood and synovial fluid samples were analyzed by compared with the mice immunized with Hcit and intra- the Kruskal–Wallis test, followed by Dunn post hoc comparisons. The articularly injected with Cit-peptide (p , 0.0001) (Fig. 2C1). Kruskal–Wallis test was appropriate because of the comparison of three No significant difference was observed in the frequency or severity samples of data that were not distributed normally. (Figs. 2, 3, 5). Differ- of arthritis between the Cit-immunized and nonimmunized mice ences among the groups shown in Fig. 4 were calculated using the Mann– Whitney U test, with the Dunn post hoc adjustment. All statistical analyses that were intra-articularly injected with Cit-peptide (Fig. 2C2, were performed using GraphPad Prism, version 5.02 for Mac (GraphPad, 2C4, respectively), nor did we see any evidence of cartilage de- San Diego, CA), and p values , 0.05 were considered significant. struction.

Table I. Filaggrin-derived peptides predispose to Cit-induced arthritis

Peptide Sequence Sequence Name Arthritis Index Erosions (%) A SHQEST--K------GKSKGKSGKSGS Lys lineal 0.22 6 0.09 25 BHQCHQESTK-Hcit-GKSKGKCGKSGS Carbamylated, cyclic (Hcit) 1.98 6 0.27* 69* C SHQESTKHcit------GKSKGKSGKSGS Carbamylated, lineal 0.97 6 0.23** 37.5** DHQCHQEST--K------GKSKGKCGKSGS Lys cyclic 0.39 6 0.12 8 EHQCHQESTR-Cit--GRSRGRCGRSGS Deiminated, cyclic (Cit) 0.28 6 0.15 18 Mice were immunized with peptides A–E (75 mg/mouse on days 0 and 14); all mice received Cit-peptide intra-articularly (1 mg/knee). pp , 0.005; ppp , 0.05; compared with nonimmunized mice injected with Cit-peptide intra-articularly (arthritis index 0.33 6 0.13; n = 18). 4 CARBAMYLATION AND AUTOIMMUNE ARTHRITIS

animals received intra-articular injections of Cit-peptide (se- quence E) (1 mg/knee). Histological evaluation of the injected joints showed significant levels of synovitis and cartilage erosion only in mice immunized with the cyclic or linear Hcit-peptides (sequences B and C, respectively) (Table I), although the histo- logical changes were less severe in the mice immunized with linear Hcit compared with those immunized with cyclic Hcit. The arthritis index of mice immunized with the Lys-containing peptides (sequences A and D) or with the Cit-containing peptide (sequence E) was significantly lower than for mice immunized with either cyclic or linear Hcit (Table I). Hcit-peptides induce Hcit-specific chemotaxis and T cell activation in immunized mice Immunohistological staining of the Cit-injected joints from Hcit- immunized mice showed that substantial numbers of CD3+ cells had infiltrated the synovia (Fig. 2C5,2C6), but none were found in the synovia of Cit-immunized mice. This interesting observation

prompted us to look at the effects of Hcit-immunization on the Downloaded from activity and function of T cells. We examined the ability of splenocytes harvested from Hcit- and Cit-immunized mice to migrate in response to a chemotactic agent (CXCL13) in vitro. Flow cytometric analysis of the transmigrated cells showed a preponderance of CD4+ cells compared with CD8+ + + or CD19 cells (p , 0.001). This increased migration of CD4 http://www.jimmunol.org/ cells occurred in cultures derived from Hcit-immunized mice, but not in those from Cit-immunized mice or nonimmunized controls (Fig. 3A). Interestingly, the addition of Hcit- or Cit-peptides (500 ng/ml) to the lower chamber resulted in no difference in spleno- cyte migration between immunized and nonimmunized mice. We next examined whether splenocytes isolated from Hcit- and Cit-immunized mice were able to proliferate and produce cytokines in response to CD3 stimulation. Lymphocytes isolated from Hcit- immunized mice had a significantly greater proliferative capacity by guest on September 28, 2021 compared with those from Cit-immunized mice or nonimmunized controls (p , 0.0001) (Fig. 3B). The culture supernatants from CD3-stimulated cells isolated from Hcit-immunized mice also con- tained significantly greater concentrations of IL-10 and -17 and FIGURE 2. Immunization with carbamylated peptide predisposes to the g development of erosive arthritis. Mice were immunized with Hcit-peptide IFN- compared with those of Cit-immunized mice or nonimmu- (Hcit) and Cit-peptide (Cit) (75 mg/mouse) on days 0 and 14. Intra- nized controls (Fig. 3C). However, IL-6 levels were similar in all articular injections of Cit-peptide (A) and Hcit-peptide (B)(1mg/knee) culture supernatants. were given on day 21. Histological evaluation of injected knees was un- The adoptive transfer of T and B lymphocytes from dertaken on day 28; data are presented as arthritis index. Values from three independent experiments using NMRI mice were pooled (n = 60). The Hcit-immunized mice induces specific Ab response and arthritis index for Hcit-immunized mice was 2.21-fold higher compared arthritis in unmanipulated recipients with Cit-immunized and nonimmunized mice (p , 0.001). Values repre- To evaluate the role of T cells in Cit-induced arthritis, CD3+ T cells 6 sent the mean SEM. Horizontal lines indicate the medians. C, Repre- were isolated by negative selection from the spleens of sentative histological changes seen in the injected joints of mice BALB/c Hcit-immunized mice and injected i.v. into unmanipulated immunized with Hcit (1) and Cit (2) followed by intra-articular injection BALB/c recipients (5 3 106 cells/mouse, n = 5). Control of Cit-peptide, and nonimmunized controls (3, 4). Arthritis induction was + performed in five independent experiments, giving a 92% incidence of BALB/c mice received the same number of CD3 T cells isolated arthritis in three wild-type strains (NMRI, BALB/c, and Asn) (n = 126). from unmanipulated BALB/c mice (n = 5). Two weeks later, all Immunological staining of Hcit-immunized and Cit-immunized mice was mice were challenged with an intra-articular injection of Cit- done using rat anti-mouse CD3 Ab. Large numbers of CD3+ cells (brown peptide (1 mg/knee). Histological examination of the injected joints staining) were observed in the synovia of Hcit-immunized mice (5) com- of mice that received T cells from Hcit-immunized mice showed pared with Cit-immunized mice (6). Original magnification 3500. Tissue evidence of severe arthritis, with cartilage destruction (erosion) and sections were stained with H&E (C1–C4). Immunostaining for CD3 was bone loss (Fig. 4A1). Mice that received T cells from unmanipu- developed with 3-amino-9-ethylcarbazole. Hematoxylin was used as coun- lated mice showed no morphological signs of arthritis (Fig. 4A2). terstain (C5, C6). Also, we found that splenocytes isolated from the Hcit-immunized T cell recipients had significantly greater proliferation rates and To confirm that the injected peptide must contain carbamylated produced more IFN-g in response to CD3 stimulation than did residues to induce arthritis, we immunized mice with peptides control mice that received naive T cells (Fig. 4B). Transfer of containing unmodified Lys residues (Table I, sequences A and D), CD19+ B cells from Hcit-immunized animals triggered arthritis Cit-containing peptide (sequence E), or with peptides containing in a manner similar to T cell transfer, with an arthritis index of cyclic or linear Hcit-peptides (sequences B and C). On day 21, all 1.41 6 0.23. Interestingly, arthritis triggered by adoptive transfer The Journal of Immunology 5

FIGURE 3. Immunization with carbamylated peptide (Hcit) increases cell migration, T cell proliferation, and cytokine production upon CD3-mediated

activation. A, Splenocytes from Hcit-immunized (n = 5), Cit-immunized (n = 5), and nonimmunized mice (n = 4) were allowed to migrate along a CXCL13 Downloaded from gradient (1 mg/ml) for 12 h. Flow cytometry analysis of the transmigrated cells showed more CD4+ T cells (1) in cultures from Hcit-immunized mice compared with Cit-immunized mice (p , 0.001) and nonimmunized controls. The transmigrated CD8+ (2) and CD19+ (3) populations were similar in Hcit- and Cit-immunized mice. Values represent mean 6 SEM. Horizontal lines indicate the median. B, Thymidine [3H] proliferation assay following stimulation of spleen cells (1 3 106 cells/ml) from Hcit-immunized mice (n = 8), Cit-immunized mice (n = 8), and nonimmunized controls (n = 6) with anti-CD3 Abs (1 mg/ml) shows a 2.2-fold increase in the proliferation rate of Hcit-immunized mice compared with Cit-immunized mice (p , 0.0001). Splenocytes from Cit-immunized mice and nonimmunized controls show no differences in their proliferation rates. Nonstimulated cells were used as a negative control.

Results represent pooled values from three independent experiments. Values are presented as mean 6 SEM. Horizontal lines indicate the median. http://www.jimmunol.org/ C, Cytokine production was measured in the supernatants of anti-CD3–stimulated splenocyte cultures. Cells from Hcit-immunized mice produced higher levels of IL-10 (1), IFN-g (2), and IL-17 (3) than Cit-immunized mice or nonimmunized controls (p , 0.01). of B cells was only occasionally destructive for joint cartilage and Immunization with carbamylated peptide induces efficient Ab bone. Adoptive transfer of T and B cells also triggered peptide- production specific Ab production in the recipients. In both cases, we observed When we analyzed blood samples taken from mice on day 0 and on significantly more specific Abs to Hcit-peptide in our ELISPOT days 7, 21, and 28 postimmunization, we found that the anti–

assay compared with control mice that received unmanipulated Hcit-Ab titers rapidly increased after the second round of immu- by guest on September 28, 2021 lymphocytes (Fig. 4C). nization and that the Ab titer remained high throughout the course

FIGURE 4. T and B lymphocytes of mice immunized with carbamylated peptide (Hcit) predispose to Cit-induced arthritis. CD3+ and CD19+ cells were isolated by negative selection from the spleens of Hcit-immunized BALB/c mice and injected i.v into naive BALB/c recipients (5 3 106 cells/mouse, n = 10). Control BALB/c mice received the same number of CD3+ or CD19+ cells isolated from naive BALB/c mice (n = 10). On day 12 following lymphocyte transfer, all mice received intra-articular injections of Cit-peptide (1 mg/knee). A, Histological evaluation of injected knee joints 7 d later showed development of severe arthritis in 100% of the recipients of CD3+ cells from Hcit-immunized mice (arthritis index, 2.19 6 0.54) (1, 3), whereas only two recipients of naive CD3+ cells showed evidence of arthritis (arthritis index, 0.4 6 0.41; p = 0.003) (2, 3). Knees were embedded in paraffin, sectioned, and stained with H&E. Original magnification 3500. B, Recipients of Hcit-immunized CD3+ cells also showed a significant increase in the T cell proliferation rate (p , 0.0001) (1) and produced more IFN-g compared with controls (2). C, Recipients of Hcit-immunized CD3+ or CD19+ cells showed significantly higher levels of Hcit-specific Abs, as assessed by ELISPOT. Values represent mean 6 SEM. Horizontal lines indicate the median. 6 CARBAMYLATION AND AUTOIMMUNE ARTHRITIS of the experiment. In contrast, mice immunized with the Cit- displayed signs of severe arthritis, with a prevalence of 91% (ar- peptide produced low levels of Abs. Specificity studies showed thritis index, 1.45 6 0.18) compared with control animals immu- that the IgG Abs produced by the Hcit-immunized (cyclic) mice nized with unmodified albumin (arthritis index, 0.26 6 0.17; p = bound exclusively to the Hcit-peptides and not to Cit-peptide (E). 0.0024). Immunization with Hcit-BSA showed a similarly high We decided to analyze the specificity of these anti-Hcit Abs fur- prevalence of erosive arthritis (87%; arthritis index, 1.56 6 ther, using plates coated with peptides A–E. The anti-Hcit Abs 0.23) compared with controls (arthritis index, 0.5 6 0.24; p = cross-reacted to some extent with peptides C and D (Table II). 0.04). We also detected high levels of Abs specific for the Hcit- Despite the fact that mice immunized with peptides A and D protein following immunization with Hcit-mAlb or Hcit-BSA. produced high levels of Abs that cross-reacted with the Hcit- These results clearly show that carbamylated proteins can trigger peptide, they showed no morphological signs of arthritis following processes that lead to the development of arthritis in vivo. Taken intra-articular injections of the Cit-peptide (Table I). Immuniza- together, our findings strongly suggest that this mechanism is not tion with Cit-peptide resulted in low Ab titers that had almost no peptide specific and that the immune responses that result in joint cross-reactivity with any of the peptides studied (Table II). destruction are activated by carbamylated residues within proteins. Systemic injection of anti-Hcit Abs or intra-articular injection of Quantitative analysis of carbamylated and deiminated residues the Hcit-Ab/Cit-peptide mixture does not induce susceptibility to in the blood and synovial fluid from RA patients arthritis We then looked at the levels of carbamylated Lys (Hcit) and dei- We used two approaches to elucidate the role of Hcit-Abs in the de- minated Arg (Cit) residues in the proteins recovered from the blood

velopment of arthritis. First, purified IgG from Hcit-immunized and synovial fluid of RA patients (n = 72), patients with knee trauma Downloaded from mice was injected i.p. into unmanipulated NMRI mice (0.2 mg/ (n = 24), and patients with osteoarthritis (n = 16), using mass mouse, n = 5), followed by an intra-articular injection of Cit- spectrometry, and compared them with the levels of unmodified peptide (1 mg/knee) 1 d later. Control mice (n = 6) were treated in Lys and Arg residues. The RA patients were further stratified the same way but received IgG from nonimmunized mice. When we according to radiological findings (erosive RA versus nonerosive examined the injected joints histologically, we saw no signs of ar- RA) and the presence of anti-Cit Abs (aCCPs). We found that the

thritis in the test or control mice (data not shown). In the second circulating levels of Cit- and Hcit-peptides were greater in patients http://www.jimmunol.org/ approach, anti–Hcit-Ab/Cit-peptide complexes were formed ex vivo with erosive RA than in those with nonerosive RA or controls and then injected directly into the knee joints of naive mice (total (p = 0.001; Fig. 5A,5B). In contrast, the levels of unmodified protein content 1 mg/knee, n = 8). Control mice (n = 8) received Arg and Lys residues in the blood and synovial fluid were not intra-articular injections of Cit-peptide complexed with the mono- significantly different (data not shown). Furthermore, the levels clonal anti–Cit-Ab, ACC3 (32). Histological evaluation of the of Hcit were greatest in patients with erosive RA plus positive injected joints showed mild synovitis in two of eight joints injected anti–Cit-Ab titers (p = 0.023; Fig. 5B). The levels of Cit were high with the Hcit-Ab/Cit-peptide complex (arthritis index, 0.25 6 0.12), in all patients with erosive RA compared with the nonerosive group whereas no signs of arthritis were seen in the joints injected with (p = 0.0008), and they were not influenced by whether the patient + 2 ACC3/Cit-peptide complex. Because of the low affinity of Hcit-Abs was anti–Cit-Ab or anti–Cit-Ab (Fig. 5A). The levels of Hcit- by guest on September 28, 2021 to Cit-peptide, only a restricted complex formation is expected. Abs were significantly greater in patients with erosive RA than in However, the experiments showed that the simultaneous presence those with nonerosive RA or in controls (Fig. 5C). of Hcit-Abs and Cit-peptide, as well as fully formed complexes of Cit-peptide with its mAb (ACC3), in the joint cavity was not suffi- Discussion cient to induce arthritis. In this study, we showed that the carbamylation of Lys residues may be the missing link in the chain of pathogenesis of autoimmune Carbamylated proteins have arthritogenic properties arthritis. Indeed, immunization of mice with carbamylated peptides To try to establish the role of carbamylation in triggering auto- (Hcit-immunization) induced T and B cell activation, followed by immune arthritis and to exclude the possibility of specific reactions high levels of proliferation and cytokine and autoantibody produc- against the synthetic peptides themselves, we immunized mice with tion. This clearly suggests that Hcit-modification changes immuno- exogenous (BSA) and endogenous (mouse albumin [mAlb]) pro- logically inert peptides into autoantigens. More importantly, Hcit- teins carbamylated using potassium cyanate (KNOC). Forty-seven immunized mice became susceptible to arthritis induced by the NMRI mice were immunized on days 0 and 14 with carbamylated intra-articular injection of Cit-peptides. BSA (Hcit-BSA) or mAlb (Hcit-mAlb), followed by intra-articular We found that splenocytes isolated from Hcit-immunized mice injection of Cit-peptide on day 28. Histological examination of the showed a .2-fold increase in proliferation in response to CD3 stim- injected joints showed that mice immunized with Hcit-mAlb ulation compared with those from nonimmunized or Cit-immunized

Table II. Ab production following immunization with fillagrin-derived peptides and their cross-reactivity in solid phase

Immunization

Plate Coating Lys Lineal (A) Hcit, Cyclic (B) Hcit, Lineal (C) Lys Cyclic (D) Cit, Cyclic (E) A 0.514 0.075 0.112 0.617 0.086 B 0.187 0.522 0.386 0.048 0.039 C 0.094 0.149 0.471 0.057 0.054 D 0.755 0.145 0.231 0.574 0.074 E 0.096 0.041 n.a. 0.037 0.228 Bold font indicates level of specific Ab. n.a., not available. The Journal of Immunology 7 Downloaded from FIGURE 5. Quantitative evaluation of carbamylated (Hcit) and citrullinated (Cit) peptides in proteins recovered from blood and synovial fluid of patients with RA. A, Quantitative evaluation of Hcit and Cit proteins recovered from blood and synovial fluid was done by mass spectrophotometry and expressed as absolute concentrations (mmol). Hcit Ab levels are presented as OD at 450 nm with 1:100 serum dilution. Levels of Hcit-Abs in serum and synovial fluid were measured using ELISA. Patient material was stratified according to radiological findings into erosive, nonerosive, and control groups. The erosive group has higher levels of Hcit-Ab compared with controls (p = 0.019) and the nonerosive group (p = 0.024). B, Levels of Hcit-peptides measured by mass spectrometry in proteins recovered from blood are increased in erosive and aCCP+ RA patients compared with erosive and aCCP2 patients and the control group (p , 0.05). C, Cit-peptide levels in blood of RA patients with erosive changes are higher compared with the control group but not within the http://www.jimmunol.org/ nonerosive group. D, Hcit-Abs in synovial fluid of erosive RA patients are higher that in patients with nonerosive RA (p , 0.05). E, Absolute levels of Hcit- peptides are identical among the groups. F, Cit-peptide levels in synovial fluid are higher than in control group, regardless of aCCP status of RA patients. Blood and synovial fluid samples were collected from RA patients (n = 72) and controls with knee trauma (n = 41) and osteoarthritis (n = 40). Values are presented as mean 6 SEM. Horizontal lines indicate the median. mice. Additionally, IL-10 and -17 and IFN-g levels were significantly the Cit-injected joints of Hcit-immunized mice showed high numbers higher in the culture supernatants of splenocytes isolated from Hcit- of CD3+ cells. In comparison, we found no T cell infiltration of the immunized mice. Phenotypic analysis of spleen cells from Hcit- synovia following Cit-immunization. To further confirm the role of by guest on September 28, 2021 immunized mice migrating along a CXCL13 gradient showed a high T cells in the development of disease, we performed the adoptive proportion of CD4+ cells (Th lymphocytes)comparedwiththose from transfer of T cells from Hcit-immunized mice into unmanipulated Cit-immunized and nonimmunized mice, whereas the numbers BALB/c mice. Our results consistently showed that the arthritis index of transmigrated CD8+ (cytotoxic T lymphocytes) and CD19+ cells of the Cit-injected knees was significantly higher in recipients of Hcit- (B cells) were similar. IL-10 and IFN-g are cytokines produced immunized CD3+ cells compared with recipients of unstimulated by CD4+ T cells and are known to play a pivotal role in Ag pre- CD3+ cells. In line with the histological findings, recipients of the sentation (38–40). Hcit-immunized CD3+ cells exhibited greater T cell proliferation rates We went on to investigate the pathogenic significance of Hcit- and IFN-g production, mimicking the characteristics found in mice immunization in a murine model of experimental arthritis. The directly immunized with Hcit-containing peptides. results of these experiments showed that the injection of Hcit- Hcit-immunization led to relatively strong Ab responses, peptide into nonimmunized mice gave rise to moderate arthritis; whereas immunization with the Cit-peptide did not. Evaluation this was not the case for Cit-peptide. Surprisingly, intra-articular of Hcit-Ab specificity showed binding to the linear Hcit-peptide, injection of Cit-modified peptides into Hcit-immunized mice as well as the linear and cyclic forms of the Lys-containing pep- resulted in the greatest arthritis index and severe erosive arthritis. tides. However, despite high Ab titers, immunization with the un- Immunization with Hcit-peptides, followed by intra-articular injec- modified Lys-containing peptides did not result in arthritis. With tion of Cit-peptides, triggered a severe erosive arthritis in .90% of the help of Ag-specific T cells, B cells can produce isotype- mice. In contrast, Cit-immunization followed by the intra-articular switched Abs with high specificity to Ags (41–43). The adoptive- injection of Cit-peptide had no arthritogenic effects. This predis- transfer experiments showed that the immunization of mice gen- position to Cit-induced arthritis was independent of the structure erated T cells with the ability to stimulate B cells for production of (linear or cyclic) of the Hcit peptide and supports the role of Hcit-specific Abs. Low cross-reactivity between the Abs induced carbamylation in the induction of this disease. by immunization with Hcit or Cit and the monoclonal anti-Cit Our model of arthritis shows a clear MHC class II dependency, Ab (ACC3) was detected. Taken together, these results show being most severe in BALB/c mice (H2-Ad haplotype), whereas the importance of carbamylation in the initiation of autoim- C57BL/6 mice (H2-Ab haplotype) are resistant. This strong mune responses. association of Hcit-triggered arthritis with MHC class II empha- To rule out the possibility that immune complexes may be re- sizes the clinical relevance of our model to human RA, because sponsible for the development of arthritis in our model, we injected RA susceptibility in humans is known to be strongly associated Hcit-Ab/Cit-modified peptide immune complexes into the joints of with HLA-DRB1 (14). naive mice. Our results suggested that immune complex formation WehavealsoprovidedevidenceforthepivotalroleofHcit-sensitized between Hcit-Abs/Cit-peptide is not the major mechanism respon- T cells in the etiology of Cit-induced arthritis. Immunostaining of sible for arthritis in Cit-injected joints. Similarly, the intraperitoneal 8 CARBAMYLATION AND AUTOIMMUNE ARTHRITIS transfer of Hcit-specific Ig prior to intra-articular injection of the Cit- 2. Mann, E., M. J. McDermott, J. Goldman, R. Chiesa, and A. Spector. 1991. peptide was not sufficient to trigger arthritis. Phosphorylation of alpha-crystallin B in Alexander’s disease brain. FEBS Lett. 294: 133–136. We have also provided evidence supporting the importance of 3. Doyle, H. A., and M. J. Mamula. 2005. Posttranslational modifications of self- Cit-peptides, rather than anti-Cit Abs, in the pathogenesis of arthri- antigens. Ann. N. Y. Acad. Sci. 1050: 1–9. 4. Utz, P. J., M. Hottelet, P. H. Schur, and P. Anderson. 1997. Proteins phosphorylated tis. Cit-peptides are highly arthritogenic in animals that have been during stress-induced apoptosis are common targets for autoantibody production in exposed to peptide/proteins containing carbamylated Lys residues patients with systemic lupus erythematosus. J. Exp. Med. 185: 843–854. (Hcit-peptide). In agreement with previous reports (44, 45), we 5. Zamvil, S. S., D. J. Mitchell, A. C. Moore, K. Kitamura, L. Steinman, and J. B. Rothbard. 1986. T-cell epitope of the autoantigen myelin basic protein that showed that injection of Cit-peptides into the joints of naive mice induces encephalomyelitis. Nature 324: 258–260. failed to induce arthritis and that immunization with Cit-peptide 6. Masson-Bessie`re, C., M. Sebbag, E. Girbal-Neuhauser, L. Nogueira, C. Vincent, elicited only a modest Ab response. To exclude any potential bias T. Senshu, and G. Serre. 2001. The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- related to the synthetic origin of the peptides used in the study, we and beta-chains of fibrin. J. Immunol. 166: 4177–4184. conducted experiments using carbamylated endogenous mAlb 7. Klareskog, L., J. Ro¨nnelid, K. Lundberg, L. Padyukov, and L. Alfredsson. 2008. (Hcit-mAlb) and exogenous BSA (Hcit-BSA) as immunizing Immunity to citrullinated proteins in rheumatoid arthritis. Annu. Rev. Immunol. 26: 651–675. agents. Immunization with Hcit-mAlb and Hcit-BSA resulted in 8. Nishimura, K., D. Sugiyama, Y. Kogata, G. Tsuji, T. Nakazawa, S. Kawano, levels of T cell activation similar to those seen with the Hcit- K. Saigo, A. Morinobu, M. Koshiba, K. M. Kuntz, et al. 2007. Meta-analysis: peptide and predisposed the animals to Cit-induced arthritis. Thus, diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis. Ann. Intern. Med. 146: 797–808. taken together, our experimental data support the role of carbamy- 9. Avouac, J., L. Gossec, and M. Dougados. 2006. Diagnostic and predictive value lated Lys residues within peptides as the trigger for a chain re- of anti-cyclic citrullinated protein antibodies in rheumatoid arthritis: a systematic action that results in an increased sensitivity to Cit-peptide at the literature review. Ann. Rheum. Dis. 65: 845–851. Downloaded from 10. Burkhardt, H., B. Sehnert, R. Bockermann, A. Engstro¨m, J. R. Kalden, and site of its accumulation. To test our hypothesis, this sequence of R. Holmdahl. 2005. Humoral immune response to citrullinated collagen type II events was challenged in a human setting. Quantitative evaluation determinants in early rheumatoid arthritis. Eur. J. Immunol. 35: 1643–1652. of carbamylated and citrullinated residues in blood and synovial 11. Schellekens, G. A., H. Visser, B. A. de Jong, F. H. van den Hoogen, J. M. Hazes, F. C. Breedveld, and W. J. van Venrooij. 2000. The diagnostic properties of fluid-recovered proteins, as well as levels of specific IgG against rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Ar- these PTMs, was done in patients with RA. Increased levels of thritis Rheum. 43: 155–163. 12. Cao, L., D. Sun, and J. N. Whitaker. 1998. Citrullinated myelin basic protein Hcit-peptides and Cit-peptides characterized patients with RA and http://www.jimmunol.org/ induces experimental autoimmune encephalomyelitis in Lewis rats through were associated with an erosive course of arthritis. Although lev- a diverse T cell repertoire. J. Neuroimmunol. 88: 21–29. els of Cit-peptides were high in the circulation of RA patients, 13. Cantaert, T., L. De Rycke, T. Bongartz, E. L. Matteson, P. P. Tak, A. P. Nicholas, they also accumulated locally in synovial fluid. Importantly, high and D. Baeten. 2006. Citrullinated proteins in rheumatoid arthritis: crucial...but not sufficient! Arthritis Rheum. 54: 3381–3389. levels of Cit-peptides were found, irrespectively of the Cit-Ab 14. Klareskog, L., A. I. Catrina, and S. Paget. 2009. Rheumatoid arthritis. Lancet status of the patients, whereas Hcit-Abs were increased in erosive 373: 659–672. 15. Hill, J. A., D. A. Bell, W. Brintnell, D. Yue, B. Wehrli, A. M. Jevnikar, RA. It is important to mention that Arg and Lys levels showed no D. M. Lee, W. Hueber, W. H. Robinson, and E. Cairns. 2008. Arthritis induced discrepancy between the groups of RA patients and controls, as by posttranslationally modified (citrullinated) fibrinogen in DR4-IE transgenic measured by mass spectrometry. This distribution pattern of Hcit- mice. J. Exp. Med. 205: 967–979.

16. Klareskog, L., P. Stolt, K. Lundberg, H. Ka¨llberg, C. Bengtsson, J. Grunewald, by guest on September 28, 2021 and Cit-peptides provides the necessary support to our experimen- J. Ro¨nnelid, H. E. Harris, A. K. Ulfgren, S. Rantapa¨a¨-Dahlqvist, et al. 2006. A tal data. Smoking has been repeatedly shown as an unfavorable new model for an etiology of rheumatoid arthritis: smoking may trigger prognostic factor in RA (46). Interestingly, pathogenic links of HLA-DR (shared epitope)-restricted immune reactions to autoantigens modified by citrullination. Arthritis Rheum. 54: 38–46. smoking go to the enhanced MPO-dependent carbamylation (21) 17. Hill, J. A., S. Southwood, A. Sette, A. M. Jevnikar, D. A. Bell, and E. Cairns. and to peptidylarginine-deiminase–dependent citrullination of 2003. Cutting edge: the conversion of arginine to citrulline allows for a high- proteins (47). Thus, smoking might provide an environmental affinity peptide interaction with the rheumatoid arthritis-associated HLA- DRB1p0401 MHC class II molecule. J. Immunol. 171: 538–541. basis for the presence of PTMs and contribute to arthritis. 18. Erill, S., R. Calvo, and R. Carlos. 1980. Plasma protein carbamylation and de- To summarize, we have clearly shown that carbamylated proteins creased acidic drug protein binding in uremia. Clin. Pharmacol. Ther. 27: 612– are able to trigger primary immune responses, inducing the che- 618. + 19. Flu¨ckiger, R., W. Harmon, W. Meier, S. Loo, and K. H. Gabbay. 1981. Hemo- motaxis and proliferation of CD4 T cells and the subsequent pro- globin carbamylation in uremia. N. Engl. J. Med. 304: 823–827. duction of IFN-g and IL-10 and -17 production. This activation of 20. Van Lente, F., A. McHugh, and C. E. Pippenger. 1986. Carbamylation of apo- T cells, combined with a strong Ab response, leads to the local aspartate aminotransferase: a possible mechanism for enzyme inactivation in uremic patients. Clin. Chem. 32: 2107–2108. recognition of citrullinated peptides within the joints and, ultimately, 21. Wang, Z., S. J. Nicholls, E. R. Rodriguez, O. Kummu, S. Ho¨rkko¨, J. Barnard, to the development of erosive arthritis. In this study, we compared W. F. Reynolds, E. J. Topol, J. A. DiDonato, and S. L. Hazen. 2007. Protein the effects of two PTMs, carbamylation/homocitrullination and dei- carbamylation links inflammation, smoking, uremia and atherogenesis. Nat. Med. 13: 1176–1184. mination/citrullination, and observed their distinct effects with re- 22. Kraus, L. M., L. Gaber, C. R. Handorf, H. P. Marti, and A. P. Kraus, Jr. 2001. spect to T cell activation and the induction of cytokine and Ab Carbamylation of glomerular and tubular proteins in patients with failure: production. Despite their functional distinctions, the peptides pro- a potential mechanism of ongoing renal damage. Swiss Med. Wkly. 131: 139–4. 23. Higashi, S., and K. Miyazaki. 1999. Reactive site-modified tissue inhibitor of duced by these PTMs complement each other. The immune- metalloproteinases-2 inhibits the cell-mediated activation of progelatinase A. J. activating effects of carbamylation enhance the arthritogenic prop- Biol. Chem. 274: 10497–10504. 24. Oimomi, M., H. Hatanaka, Y. Yoshimura, K. Yokono, S. Baba, and Y. Taketomi. erties of the Cit-peptides; therefore, for the first time, we have been 1987. Carbamylation of insulin and its biological activity. Nephron 46: 63–66. able to show that the complementary effects of these two posttransla- 25. Sirpal, S. 2009. Myeloperoxidase-mediated lipoprotein carbamylation as a mech- tionally modified peptides provide a novel mechanism for the path- anistic pathway for atherosclerotic vascular disease. Clin. Sci. (Lond.) 116: 681– 695. ogenesis of autoimmune arthritis. 26. Balion, C. M., T. F. Draisey, and R. J. Thibert. 1998. Carbamylated hemoglobin and carbamylated plasma protein in hemodialyzed patients. Kidney Int. 53: 488– Disclosures 495. The authors have no financial conflicts of interest. 27. Asci, G., A. Basci, S. V. Shah, A. Basnakian, H. Toz, M. Ozkahya, S. Duman, and E. Ok. 2008. Carbamylated low-density lipoprotein induces proliferation and increases adhesion molecule expression of human coronary artery smooth mus- cle cells. Nephrology (Carlton) 13: 480–486. References 28. Beswick, H. T., and J. J. Harding. 1984. Conformational changes induced in 1. Anderton, S. M. 2004. Post-translational modifications of self antigens: impli- bovine lens alpha-crystallin by carbamylation. Relevance to cataract. Biochem. J. cations for autoimmunity. Curr. Opin. Immunol. 16: 753–758. 223: 221–227. The Journal of Immunology 9

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