Impaired activation-induced cell death promotes spontaneous arthritis in antigen cartilage proteoglycanspecific T cell receptortransgenic mice.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 62, No. 10, October 2010, pp 2984–2994 DOI 10.1002/art.27614 © 2010, American College of Rheumatology Impaired Activation-Induced Cell Death Promotes Spontaneous Arthritis in Antigen (Cartilage Proteoglycan)–Specific T Cell Receptor–Transgenic Mice Ferenc Boldizsar,1 Katalin Kis-Toth,2 Oktavia Tarjanyi,1 Katalin Olasz,1 Akos Hegyi,2 Katalin Mikecz,2 and Tibor T. Glant2 Objective. To investigate whether genetic preponderance of a T cell receptor (TCR) recognizing an arthritogenic peptide of human cartilage proteoglycan (PG) is sufficient for development of arthritis. Methods. We performed a longitudinal study using BALB/c mice expressing a TCR that recognizes the arthritogenic ATEGRVRVNSAYQDK peptide of human cartilage PG. PG-specific TCR–transgenic (PG-TCR– Tg) mice were inspected weekly for peripheral arthritis until 12 months of age. Peripheral joints were examined histologically, and T cell responses, T cell activation markers, serum cytokines, and autoantibodies were measured. Apoptosis and signaling studies were performed in vitro on T cells from aged PG-TCR–Tg mice. Results. Spontaneous arthritis developed as early as 5–6 months of age, and the incidence increased to 40–50% by 12 months of age. Progressive inflammation began with cartilage and bone erosions in the interphalangeal joints, and later expanded to the proximal joints of the front and hind paws. Spontaneous arthritis was associated with a high proportion of activated CD4ⴙ T cells, enhanced interferon-␥ and interleukin-17 (IL-17) production, and elevated levels of serum autoantibodies. PG-TCR–Tg mice lacking IL-4 developed arthritis earlier and at a higher incidence than IL-4–sufficient mice. Antigen-specific activation–induced cell death was diminished in vitro in CD4ⴙ T cells of PG-TCR–Tg mice with spontaneous arthritis, especially in those lacking IL-4. Conclusion. The presence of CD4ⴙ T cells expressing a TCR specific for an arthritogenic PG epitope is sufficient to trigger spontaneous autoimmune inflammation in the joints of BALB/c mice. IL-4 appears to be a negative regulator of this disease, through attenuation of activation-induced cell death. Extracellular matrix components of avascular hyaline cartilage contain “tissue-restricted” antigens, such as those encrypted in a tertiary complex (e.g., the G1 domain of proteoglycan [PG] aggrecan) or hidden in the triple helix of type II collagen. Although some of these cartilage macromolecules are involved in central tolerance (1), they are considered potential autoantigens in rheumatoid arthritis (RA) (2–5). Epitope mapping studies in PG-induced arthritis (PGIA) have identified a dominant arthritogenic epitope within the G1 domain, 5/4E8 (ATEGRVRVNSAYQDK [core peptide is underlined]) (6–8). Importantly, this peptide sequence is partially or completely incorporated in peptides that have been shown to stimulate T cells from patients with RA (9–11). Of note, a synthetic peptide containing the citrullinated 5/4E8 epitope (citrullinated at the T cell receptor [TCR]–binding arginine  [boldface in sequence shown above]) induced positive T cell responses in ⬃60% of human patients with RA (11). The T cell hybridoma specific for this 5/4E8 peptide has been used to generate TCR-transgenic (referred to below as “PG-TCR–Tg”) mice (13). When compared with wild-type BALB/c mice, PG-TCR–Tg mice on the BALB/c background developed exacerbated Drs. Mikecz and Glant’s work was supported by the Grainger Foundation, Forest Park, Illinois. Dr. Glant received additional support from the NIH (grant AR-040310) and holds the J. O. Galante Endowed Chair at Rush University Medical Center. 1 Ferenc Boldizsar, MD, PhD, Oktavia Tarjanyi, MD, Katalin Olasz, MSc: Rush University Medical Center, Chicago, Illinois, and University of Pecs, Pecs, Hungary; 2Katalin Kis-Toth, PhD (current address: Beth Israel Hospital and Harvard University Medical School, Boston, Massachusetts), Akos Hegyi, MS, Katalin Mikecz, MD, PhD, Tibor T. Glant, MD, PhD: Rush University Medical Center, Chicago, Illinois. Address correspondence and reprint requests to Tibor T. Glant, MD, PhD, Section of Molecular Medicine, Rush University Medical Center, Cohn Research Building, Room 708, 1735 West Harrison Street, Chicago, IL 60612. E-mail: firstname.lastname@example.org. Submitted for publication March 31, 2010; accepted in revised form June 10, 2010. 2984 IMPAIRED AICD AND SPONTANEOUS ARTHRITIS arthritis upon PG immunization (14). Because T cell autoreactivity plays a central role in the etiology and pathologic mechanisms of RA and of disease in corresponding mouse models (15–17), (auto)antigen-specific TCR signaling is of special interest. Therefore, the PG-TCR–Tg BALB/c mouse is a useful model for studying T cell activation by self peptides as well as the link between autoreactivity and arthritis development. Depending on the threshold of stimulation, TCR signaling might result in either activation (proliferation and differentiation) or apoptosis (18), both of which are regulated by costimulatory molecules and cytokine receptor signaling pathways (19–21). TCR signal–induced apoptosis, also called activation-induced cell death (AICD), is a key mechanism in deleting activated T cell clones to down-regulate superfluous immune responses (22). Thus, defective AICD may underlie the sustained T cell activation that is usually associated with autoimmune disease (23). Interleukin-4 (IL-4) is an antiinflammatory cytokine that controls several target genes through the activation of STAT-6. Deficiency of either IL-4 or STAT-6 on the BALB/c background has been shown to increase the severity of PGIA (24). The regulatory function of IL-4 in AICD has also been demonstrated (25). Therefore, autoepitope-specific (PG-TCR–Tg) CD4⫹ T cells in combination with IL-4 deficiency, especially on an arthritis-prone genetic background (BALB/c), may mediate an accelerated autoimmune response. Spontaneous arthritis has been reported in a number of genetically modified/altered mouse strains. For example, the K/BxN mouse was generated by intercrossing KRN TCR-Tg mice, specific for bovine pancreas ribonuclease, with the NOD strain (26). In the context of NOD class II major histocompatibility complex (MHC) (H2g7), the KRN transgenic TCR recognizes an epitope in glucose-6-phosphate isomerase, which is the actual autoantigen in the K/BxN spontaneous arthritis model (27,28). SKG mice develop arthritis due to a spontaneous mutation in the SH2 domain of Zap70 (17). Altered thymic selection in these SKG mice leads to the survival of otherwise negatively selected T cell clones, which then spontaneously differentiate into Th17 cells in the periphery and attack the joints. IL-1 receptor antagonist protein (IRAP)–knockout mice, in contrast, develop spontaneous arthritis due to increased production of proinflammatory cytokines (IL-1␤, IL-6, IL-17, and tumor necrosis factor ␣) and autoantibodies, because a negative regulator of IL-1 signaling is absent (29,30). Importantly, spontaneous arthritis develops in 2985 SKG and IRAP-deficient mice only on the BALB/c genetic background (17,29,30). Herein we report that PG-TCR–Tg mice on the BALB/c background develop spontaneous arthritis at an advanced age. Inflammation of the interphalangeal joints is observed in association with cartilage and bone erosions after 6 months of age. Inflammation expands slowly but steadily, involving the metacarpal/metatarsal and then the wrist/ankle joints. The morphologic alterations are associated with increasing activation of CD4⫹ T cells and production of increasing amounts of anti-PG autoantibodies in PG-TCR–Tg mice. The lack of the antiinflammatory cytokine IL-4 results in a further increase in the severity of inflammation and an earlier disease onset. Based on these observations, we conclude that the dominant presence of an arthritogenic epitope– specific TCR is sufficient to trigger and maintain spontaneous autoimmune inflammation in the joints of aging mice on an appropriate (BALB/c) genetic background. MATERIALS AND METHODS Chemicals. All chemicals were purchased from Sigma or Fisher Scientific, unless indicated otherwise. Mouse recombinant cytokines and enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems or BD Biosciences. Phosphate buffered saline (PBS [pH 7.4]) was used for washing and short-term storage of cells until use. Cell surface labeling with monoclonal antibodies (all from BD Biosciences) was carried out in flow cytometry wash buffer (PBS containing 0.1% NaN3 and 0.1% bovine serum albumin). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum under standard tissue culture conditions. Mice and clinical assessment of arthritis. All animal procedures were conducted under a protocol approved by the Institutional Animal Care and Use Committee of Rush University Medical Center. IL-4–deficient (IL-4⫺/⫺) mice (The Jackson Laboratory) and PG-TCR–Tg mice, in which ⬎95% of the CD4⫹ T cells express PG (5/4E8 epitope)–specific V␤4/V␣1.1 TCR (13), both on the BALB/c background, were intercrossed. Previously, exacerbated arthritis was demonstrated in both IL-4⫺/⫺ and PG-TCR–Tg mice upon PG immunization (14,24). All experiments were performed with homozygous female PG-TCR–Tg/IL-4⫹/⫹ and PG-TCR–Tg/ IL-4⫺/⫺ mice. Mice were monitored for clinical signs of arthritis once per week from 1 month of age until the end of the experiment. Redness and swelling of the toes were considered the first signs of inflammation. Animals were killed at different time points up to 12 months of age to assess disease progression histologically. T cell separation, activation of transgenic T cells with peptide-presenting irradiated A20 cells, and detection of TCR signaling. T cells from the spleens of PG-TCR–Tg mice were purified using the EasySep magnetic T cell enrichment kit (Stem Cell Technologies). T cells were seeded on irradiated 2986 A20 antigen-presenting cells (American Type Culture Collection), which can present the 5/4E8 peptide (31). A20 cells (1 ⫻ 105 cells/well) were plated in 48-well plates, precultured for 12 hours with or without synthetic 5/4E8 peptide (50 g/ml), and then washed with serum-free DMEM. For the signaling studies, 3 ⫻ 105 purified T cells were spun onto the A20 cell layer by brief centrifugation (900g for 5 minutes), and then T cells were harvested after 1, 2, 3, and 5 hours of coculture. Phosphorylation of Zap70 and ERK-1/2 was detected by phospho-flow technique according to the instructions of the manufacturer (BD Biosciences) (32). Cells were labeled with peridinin chlorophyll A protein–Cy5.5– conjugated anti-CD4 and phospho-specific antibodies (either phycoerythrin [PE]–conjugated anti-mouse Zap70 [clone 17A/ P-Zap70] recognizing pY319 or PE-conjugated mouse anti– ERK-1/2 [clone 20A] recognizing pT203/pY205 in ERK-1 and pT183/pY185 in ERK-2) (both from BD Biosciences). Apoptosis detection using annexin V/propidium iodide (PI) staining. Annexin V/PI staining was used to distinguish between early and late apoptotic cells (33). Live cells are negative for both annexin V and PI, early apoptotic cells are positive for annexin V and negative for PI, and late apoptotic cells are positive for both annexin V and PI. Labeling was done according to the instructions of the manufacturer (BD Biosciences), and cells were immediately analyzed by flow cytometry. Monoclonal antibodies, fluorescent cell surface labeling, and flow cytometry. Fluorochrome-labeled rat anti-mouse antibodies specific for TCR V␤4, CD3, CD4, CD5, CD8, CD19, CD23, CD25, CD43, CD44, CD62L, CD69, B220, IgD, and IgM, as well as fluorescein isothiocyanate–conjugated annexin V and PI, were purchased from BD Biosciences. We used a multicolor labeling technique for simultaneous detection of multiple cell surface molecules on cells harvested from the spleens of mice, as previously described (34). Samples were measured using a FACS Canto II flow cytometer, and data were analyzed with FACS Diva software (BD Flow Cytometry Systems). Cell populations were defined by surface marker expression as previously described (34). Specific cell populations were expressed as a percentage of total cells unless otherwise stated. We used fluorescence histogram plots to compare mean fluorescence intensities of different samples and to calculate the proportions of positively stained cells. Measurement of PG-specific antibodies and T cell responses. Serum samples and spleen cells were harvested from mice at the end of the experiment. Mouse PG-specific IgG1 or IgG2a (auto)antibodies in serum were measured by ELISA as previously described (35). Antigen-specific T cell responses were measured in quadruplicate samples of spleen cells (3 ⫻ 105 cells/well in 200 l in 96-well plates) cultured in the absence or presence of 25 g 5/4E8 peptide/ml. T cell proliferation was assessed by 3H-thymidine incorporation on day 3 of culture (14,36). Spontaneous and antigen-specific production of IL-4, IL-6, IL-17, tumor necrosis factor ␣, and interferon-␥ (IFN␥) in spleen cell culture supernatants (1.8 ⫻ 106 cells/well in 600 l in 48-well plates) harvested on day 4 was measured by capture ELISA, and the results were expressed as ng of cytokine secreted by 1 ⫻ 106 cells (14). Statistical analysis. Descriptive statistics were used to determine group means and SEM. The significance of differences between groups was tested by Student’s t-test (for 2 groups) or analysis of variance (for ⱖ3 groups). P values less than or equal to 0.05 were considered significant. BOLDIZSAR ET AL RESULTS Spontaneous development of arthritis in PGTCR–Tg mice at an advanced age. Mice transgenic for the 5/4E8 sequence–specific PG TCR on the BALB/c background were developed to study the mechanisms of PGIA and the role of antigen-specific TCR signaling and AICD in the clinical phenotype of arthritis (13,14). During the process of backcrossing onto the BALB/c background, we occasionally noted mild interphalangeal joint swelling in aging naive PG-TCR–Tg mice. This observation compelled us to perform a longitudinal study of homozygous PG-TCR–Tg mice to monitor for spontaneous development of arthritis. PG-TCR–Tg mice developed inflammation in the interphalangeal joints beginning at age 5–6 months (Figure 1A). The incidence increased gradually from 10–20% at 6 months of age to ⬃40% at 12 months of age (Figure 1A). Inflammation typically started in the distal interphalangeal joints, first in the hind paws, developing ⬃2 weeks later in the front paws as well (Figures 2A–C). Additional (proximal) interphalangeal joints became inflamed, followed by metacarpophalangeal, metatarsophalangeal, carpometacarpal, and tarsometatarsal joints as the mice aged (Figure 2C). Although stiffness of affected digits was characteristic, cartilaginous or bony ankylosis did not occur, but cartilage and bone were eroded, especially in tarsal and carpal joints at an advanced age. Repeated inflammatory episodes in joints led to the thickening of digits, nails were lost, and finally a “drumstick finger” deformity developed (Figure 2B). Ultimately, inflammation expanded to the proximal joints (wrist and ankle) in older animals, which was more pronounced in animals with the earliest onset of arthritis. Whereas approximately half of the PG-TCR–Tg mice were clinically healthy at the age of 12 months, histologic analysis frequently revealed proliferation of synovial lining cells and mild cartilage and bone erosions, even in symptom-free animals after 5–6 months of age. Although not all asymptomatic animals were examined histologically, by 12 months of age all animals tested were positive for synovial inflammation, and this finding persisted through 15 months of age (most recent information; results not shown). Therefore, we expect that with sufficient time all PG-TCR–Tg mice would develop arthritis, although their life expectancy is ⬃25– 30% shorter than their wild-type littermates. Such changes were never seen in age-matched nontransgenic BALB/c mice (results not shown). Immunologic characterization of mice with spontaneous arthritis. To gain insight into the potential mechanisms involved in the spontaneous development IMPAIRED AICD AND SPONTANEOUS ARTHRITIS 2987 Figure 1. Timeline of the development of spontaneous arthritis in the 5/4E8 peptide (ATEGRVRVNSAYQDK)–specific proteoglycan T cell receptor–transgenic (PG-TCR–Tg) mice. Swelling and mild redness of the distal interphalangeal joints were considered the first signs of inflammation. A, Spontaneous arthritis in 28 female interleukin-4 (IL-4)–sufficient PG-TCR–Tg BALB/c mice. B, Spontaneous arthritis in 20 female IL-4–deficient PG-TCR–Tg BALB/c mice. of arthritis in PG-TCR–Tg mice, groups of mice were killed at different ages. Sera and spleens were harvested, and T and B cell responses and serum cytokines were assayed. Because spontaneous arthritis was usually observed in mice older than 6 months and immunosenescence appears to play a role in PGIA (37), we compared the T and B cell responses of old (12 months of age, arthritic or as yet nonarthritic) and young (1.5 months of age) PG-TCR–Tg mice (Tables 1 and 2). These ⬃1.5month-old mice were chosen as “young” controls because BALB/c mice at this age have been found to be resistant to PGIA (37). The age-related expansion of CD69 high or CD25 high (activated) or CD44 high (activated/memory) transgenic CD4⫹ T cells, with a marked decrease of the CD62Lhigh (naive) population (Table 1), appeared to create an optimal milieu for the development of autoimmunity. There was no significant difference in 5/4E8 epitope peptide–induced proliferation of spleen cells in young and old (arthritic or still symptom-free) mice (Table 2). This was more or less the same when a number of in vitro stimulation–induced cytokines were measured in supernatants of spleen cell cultures; the exception was peptide stimulation–induced IFN␥ production, which was significantly higher in aged (12month-old) mice than in young mice (Table 2). Thus IFN␥, a Th1 proinflammatory cytokine, may play a role in the development of spontaneous arthritis, similar to that reported in PGIA (38). Higher incidence of spontaneous arthritis and higher frequency of autoreactive CD4ⴙ T cells in PGTCR–Tg/IL-4ⴚ/ⴚ mice than in PG-TCR–Tg/IL-4ⴙ/ⴙ mice. Earlier studies from our laboratory showed that IL-4 regulates arthritis severity in a STAT-6–dependent manner (24). Therefore, we intercrossed PG-TCR–Tg mice with IL-4–knockout mice (both on a BALB/c background) to determine whether IL-4 has a regulatory role in spontaneous arthritis. As shown in Figure 1, earlier onset of spontaneous arthritis was observed in IL-4–deficient PGTCR–Tg mice as compared with IL-4–sufficient PGTCR–Tg mice (PG-TCR–Tg/IL-4⫹/⫹). Interphalangeal joint inflammation developed in ⬃10% of PG-TCR–Tg/ IL-4⫺/⫺ mice at 4 months of age, and this increased gradually to ⬃60% by 12 months of age (Figure 1B). This difference in onset time and incidence indicated that IL-4 was involved in the regulation of spontaneous arthritis. However, the macroscopic abnormalities and histopathologic features were similar in PG-TCR–Tg/IL-4⫹/⫹ and PG-TCR–Tg/IL-4⫺/⫺ mice (Figure 2). Spleen cells harvested from arthritic PGTCR–Tg mice (either IL-4–deficient or IL-4–sufficient) produced high concentrations of IFN␥ upon 5/4E8 peptide stimulation in vitro (Table 2). Proinflammatory IL-1␤, IL-6, and IL-17 were found in the sera of arthritic PG-TCR–Tg/IL-4⫺/⫺ mice, but, except for IL-1␤ in aged nonarthritic mice, these cytokines were not detected in the nonarthritic old or young control groups (Table 2). The discrepancy between the serum and in vitro– produced cytokines reflects the difference between the cytokine levels measured in the circulation versus a more selective group of cells examined in in vitro tests (mainly T lymphocytes in the spleen in response to antigen stimulation). Similar to findings in the PG-TCR–Tg/IL4⫹/⫹ BALB/c mice, anti-mouse PG autoantibodies (only the IgG2 isotype) were detected in the sera of arthritic PG-TCR–Tg/IL-4⫺/⫺ mice (Table 2). 2988 BOLDIZSAR ET AL Figure 2. Representative macroscopic and histologic images of the front and hind paws. A–C, Macroscopic findings in the hind paws and in the front paws (insets) in a wild-type (healthy 9-month-old BALB/c) mouse (A) and a PG-TCR–Tg/IL-4⫹/⫹ mouse (B) and PG-TCR–Tg/IL4⫺/⫺ mouse (C) with spontaneous arthritis. Corresponding histologic sections (at low and high magnifications) of digits are depicted in A1 and A2, B1 and B2, and C1 and C2 (boxed areas in A1, B1, and C1 are shown at higher magnification in A2, B2, and C2). Thickening of the distal interphalangeal joints and phalanges and loss of nails were the earliest macroscopic abnormalities, which were followed by progression of inflammation to the proximal interphalangeal, metatarsophalangeal, and tarsometatarsal or carpometacarpal joints. C3 is a low-magnification montage picture of a hind paw (ankle area) from a 1-year-old PG-TCR–Tg/IL-4⫺/⫺ BALB/c mouse (⬃6 months after arthritis onset). Insets C31–C33 are higher-magnification views of the boxed areas in C3. Extensive cartilage and bone erosions of affected joints are the prominent histopathologic abnormalities (C31 and C33; contours of multinuclear osteoclasts are indicated by white outlines and arrowheads in C33). Predominantly mononuclear cells infiltrate the synovium and the joint cavities (C32). Dotted ovals in insets in A–C indicate the digits used for the histologic analyses shown in A1–C1 and A21–C2; dotted oval in C indicates the ankle used for the histologic analysis shown in C3. (Hematoxylin and eosin stained; original magnification ⫻ 4 in A1, B1, C1, and C3, ⫻ 10 in A2, B2, and C2, and ⫻ 40 in C31, C32, and C33.) See Figure 1 for definitions. IMPAIRED AICD AND SPONTANEOUS ARTHRITIS 2989 Table 1. Cellular composition of the spleen in PG-TCR⫺Tg/IL-4⫹/⫹ or PG-TCR⫺Tg/IL-4⫺/⫺ spontaneously arthritic and healthy control mice, as assessed by flow cytometry* PG-TCR⫺Tg/IL-4⫹/⫹ Arthritic PG-TCR⫺Tg/IL-4⫺/⫺ Nonarthritic Arthritic Nonarthritic Age 12 months (n ⫽ 6) Age 12 months (n ⫽ 3) Age 1.5 months (n ⫽ 4) Age 12 months (n ⫽ 19) Age 12 months (n ⫽ 3) Age 1.5 months (n ⫽ 4) 23.1 ⫾ 1.2 2.8 ⫾ 0.1 96.1 ⫾ 0.1 3.2 ⫾ 0.1 7.4 ⫾ 1.5 61.3 ⫾ 1.5 15.9 ⫾ 1.5 37.3 ⫾ 0.8 5.5 ⫾ 0.2§ 27.2 ⫾ 1.4 2.7 ⫾ 0.4 95.2 ⫾ 1.0 4.0 ⫾ 0.5# 6.8 ⫾ 1.6# 50.3 ⫾ 2.0# 19.5 ⫾ 2.2# 36.3 ⫾ 1.0 8.1 ⫾ 0.3 28.7 ⫾ 1.5 0.5 ⫾ 0.01 98.7 ⫾ 0.1 1.0 ⫾ 0.1 3.4 ⫾ 0.2 90.6 ⫾ 1.0 5.5 ⫾ 0.3 39.2 ⫾ 0.5 6.6 ⫾ 0.4 19.0 ⫾ 0.9 5.2 ⫾ 0.4§ 92.0 ⫾ 0.8 9.3 ⫾ 1.2§ 10.2 ⫾ 0.9 50.1 ⫾ 1.7 31.4 ⫾ 1.4§ 34.1 ⫾ 1.1 4.5 ⫾ 0.3§ 17.8 ⫾ 1.6 2.8 ⫾ 0.5 93.6 ⫾ 1.8 2.7 ⫾ 0.6 6.8 ⫾ 1.9# 57.5 ⫾ 5.5# 12.4 ⫾ 1.0 33.3 ⫾ 4.7 8.2 ⫾ 0.6 25.1 ⫾ 0.3 0.4 ⫾ 0.1 98.8 ⫾ 0.1 1.1 ⫾ 0.1 3.4 ⫾ 0.2 91.5 ⫾ 0.1 5.8 ⫾ 0.3 41.2 ⫾ 0.9 8.6 ⫾ 0.4 T cell (V␤4⫹CD3⫹)† CD8⫹ T cell‡ CD4⫹ T cell‡ CD69highCD4⫹ T cell¶ CD25highCD4⫹ T cell¶ CD62LhighCD4⫹ T cell¶ CD44highCD4⫹ T cell¶ B cell (B220⫹)† B1 and marginal zone and transitional B cells† * Values are the mean ⫾ SEM. PG-TCR⫺Tg ⫽ proteoglyan-specific T cell receptor⫺transgenic; IL-4 ⫽ interleukin-4. † Percent of total cells. ‡ Percent of CD3⫹ T cells. § P ⬍ 0.05 versus age-matched nonarthritic mice. ¶ Percent of CD4⫹ T cells. # P ⬍ 0.05 versus 1.5-month-old nonarthritic mice. Impaired antigen-specific AICD may promote the development of spontaneous arthritis in PGTCR–Tg BALB/c mice. Strong TCR signals lead to activation of T cells, followed by AICD. Perturbed AICD is assumed to underlie autoimmune processes through accumulation of activated (and potentially selfreactive) T cells (23). Because aged PG-TCR–Tg mice developed arthritis spontaneously and arthritis was associated with the accumulation of activated self-reactive T cells (Tables 1 and 2), we next decided to characterize Table 2. Immunologic parameters and biomarkers in the PG-TCR⫺Tg/IL-4⫹/⫹ and PG-TCR⫺Tg/IL-4⫺/⫺ BALB/c mice at 6 weeks and 12 months of age* PG-TCR⫺Tg/IL-4⫹/⫹ Arthritic Proliferation, ⌬cpm ⫻104 In vitro 5/4E8 peptide⫺induced spleen cell cytokine production, ng/106 cells IL-4 IL-6 IL-17 IFN␥ TNF␣ Serum cytokines, pg/ml IL-1␤ IL-4 IL-6 IL-17 IFN␥ TNF␣ Serum autoantibodies, g/ml IgG1 IgG2a PG-TCR⫺Tg/IL-4⫺/⫺ Nonarthritic Arthritic Nonarthritic Age 12 months (n ⫽ 6) Age 12 months (n ⫽ 3) Age 1.5 months (n ⫽ 4) Age 12 months (n ⫽ 19) Age 12 months (n ⫽ 3) Age 1.5 months (n ⫽ 4) 8.5 ⫾ 0.4 7.5 ⫾ 0.3 7.1 ⫾ 0.1 7.2 ⫾ 0.4 6.3 ⫾ 1.2 7.5 ⫾ 0.7 ND† 0.22 ⫾ 0.09 0.51 ⫾ 0.38 6.76 ⫾ 2.71‡ 0.15 ⫾ 0.04 ND† 0.10 ⫾ 0.01 0.40 ⫾ 0.10 4.59 ⫾ 0.27 0.15 ⫾ 0.03 ND 0.70 ⫾ 0.03 1.01 ⫾ 0.14 ND 0.39 ⫾ 0.08 ND 0.19 ⫾ 0.02 0.89 ⫾ 0.12 6.16 ⫾ 0.46‡ 0.13 ⫾ 0.01 ND 0.10 ⫾ 0.03 0.63 ⫾ 0.24 4.94 ⫾ 0.53 0.14 ⫾ 0.02 ND 0.66 ⫾ 0.04 1.19 ⫾ 0.12 0.89 ⫾ 0.63 0.47 ⫾ 0.04 3.3 ⫾ 8.9 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 6.3 ⫾ 1.7 ND 29.7 ⫾ 2.1 78.2 ⫾ 7.1 ND ND 2.3 ⫾ 1.4 ND ND ND ND ND ND ND ND ND ND ND ND 29.5 ⫾ 16.2 ND ND ND ND ND 33.6 ⫾ 13.8 ND ND ND ND * Values are the mean ⫾ SEM. ND ⫽ not detectable; IFN␥ ⫽ interferon-␥; TNF␣ ⫽ tumor necrosis factor ␣ (see Table 1 for other definitions). † Although sera were negative for IL-4 at age 12 months, T cells isolated from younger (age 6⫺7 months) PG-TCR⫺Tg mice secreted significant amounts of IL-4 in vitro in response to human PG or recombinant human G1 (antigen) stimulation (data not shown). ‡ P ⬍ 0.05 versus 1.5-month-old nonarthritic mice. 2990 Figure 3. Antigen-specific activation-induced apoptosis in purified CD4⫹ splenic T cells from PG-TCR–Tg mice. In vitro 5/4E8 peptide stimulation–induced apoptosis was compared in CD4⫹ T cells from IL-4–deficient and IL-4–sufficient PG-TCR–Tg BALB/c mice. A, Percent of live (annexin V [AnnV] negative/propidium iodide [PI] negative], early apoptotic (annexin V positive/PI negative), and late apoptotic (annexin V positive/PI positive) cells. Values are the mean ⫾ SEM from 3 mice in each group on days 2 and 3. ⴱ ⫽ P ⬍ 0.05. B and C, Representative flow cytometric contour plots showing the distribution of PG-TCR–Tg/IL-4⫹/⫹ (B) and PG-TCR–Tg/IL-4⫺/⫺ (C) mouse T cells according to their annexin V and PI staining after 3-day culture in the presence of TCR-specific 5/4E8 peptide presented by semiconfluent irradiated A20 cells. Numbers in the quadrants are the percent of total cells. See Figure 1 for other definitions. the antigen (5/4E8 epitope)–specific TCR signal– induced apoptosis in PG-TCR–Tg mice. A regulatory role for IL-4 in AICD has been demonstrated in mice with IL-4 and/or STAT-6 deficiency (25); therefore, the use of PG-TCR–Tg/IL-4⫺/⫺ mice appeared to be appropriate to study the regulatory role of IL-4 on TCR signaling and apoptosis. Because only subtle differences were found between IL-4–deficient and IL-4–sufficient PG-TCR–Tg BOLDIZSAR ET AL mice in onset and incidence of spontaneous arthritis (Figure 1) but there were more pronounced differences in the percentage of activated CD4⫹ T cells (Table 1), we hypothesized that IL-4 was involved in the regulation of AICD. Therefore, we compared the 5/4E8 peptide– induced apoptosis of T cells from PG-TCR–Tg/IL-4⫹/⫹ and PG-TCR–Tg/IL-4⫺/⫺ mice (Figure 3). Approximately 60–70% of the CD4⫹ cells from PG-TCR–Tg mice (either IL-4–deficient or IL-4–sufficient) were positive for annexin V after 2 days, when cultured in the presence of 5/4E8 synthetic peptide presented by A20 cells (Figure 3A). The percentage of early apoptotic cells was still ⬎50% in PG-TCR–Tg/IL-4⫹/⫹ mouse T cell cultures on day 3, whereas it was reduced to 30–40% in PG-TCR–Tg/IL-4⫺/⫺ mouse T cell cultures (Figures 3B and C). At both time points, there were significantly more live cells (especially evident on day 3) and fewer late apoptotic cells in the absence of IL-4 (Figure 3). To determine whether the IL-4–dependent differences in apoptosis could be explained by alteration of the TCR signaling threshold, we performed intracellular staining with phospho-specific antibodies against Zap70 and ERK-1/2, 2 key members of the TCR signaling cascade (39) (Figure 4). The phosphorylation of Zap70 and ERK-1/2 in spleen CD4⫹ T cells reached a peak at 2 hours of in vitro stimulation with 5/4E8 peptide– coated A20 cells (Figure 4). In the absence of IL-4, the amplitude of Zap70 phosphorylation was considerably lower (Figures 4A and C), while the ERK-1/2 phosphorylation was only subtly lower in IL-4–deficient versus IL-4–sufficient PG-TCR–Tg mice (Figures 4B and D). DISCUSSION Alterations in T cell activation and apoptosis have been shown to contribute to the development of autoimmune diseases (23). PG-TCR–Tg mice, which possess CD4⫹ T cells specific for the 5/4E8 peptide sequence, a dominant arthritogenic epitope in the G1 domain of the PG-aggrecan molecule (14), are a useful model for studying the potential role of antigen-specific AICD in PGIA. Our findings in PG-TCR–Tg mice deficient in IL-4 confirmed that IL-4 contributes to the regulation of AICD in PGIA (25). Nonetheless, both IL-4⫹/⫹ and IL-4⫺/⫺ PG-TCR–Tg mice developed spontaneous arthritis at an advanced age (beginning at age 4–6 months), with the earliest signs of inflammation being localized to the interphalangeal joints. In PG-TCR–Tg mice, almost all T cells recognize the dominant arthritogenic epitope GRVRVNSAY (14). Cross-reactivity with the homologous mouse se- IMPAIRED AICD AND SPONTANEOUS ARTHRITIS 2991 Figure 4. Phosphorylation changes upon TCR stimulation in PG-TCR–Tg mouse T cells. In vitro 5/4E8 peptide–induced signaling was compared by flow cytometry using purified T cells (⬎95% CD4⫹V␤4⫹) harvested from the spleens of aged (ⱖ9 months old) PG-TCR–Tg/IL4⫹/⫹ or PG-TCR–Tg/IL-4⫺/⫺ mice. A and B, Mean fluorescence intensity (MFI) in 4 representative samples, measured in fluorescence channel 2 (FL2) after cells were labeled with phycoerythrin (PE)–conjugated anti–phospho-Zap70 antibodies (A) or PE-conjugated anti–phospho–ERK-1/2 antibodies (B). C, Representative FL2 histogram plots, showing Zap70 phosphorylation (C) and ERK-1/2 phosphorylation (D) measured at different time points in the CD4⫹ T cells of PG-TCR–Tg/IL-4⫹/⫹ and PG-TCR–Tg/IL-4⫺/⫺ mice. Numbers in the panels are the MFI value; vertical lines show the MFI value of the control sample. See Figure 1 for other definitions. quence GQVRVNSIY has been confirmed (6,13). It is of special importance that T cell responses to the human 5/4E8 epitope in its native form (10) or citrullinated form (11) were frequently detected in RA patients. PG-TCR–Tg BALB/c mice were shown to be highly susceptible to PGIA, with very early onset and high severity of the disease (14). However, no previous study has determined whether the presence of antigen-specific TCR–transgenic T cells is sufficient to induce arthritis without injection of exogenous antigen, either in our model or in type II collagen–specific TCR–Tg mice (40). In the present investigation we confirmed that arthritis indeed develops spontaneously in PG-TCR–Tg mice at an advanced age, and disease develops earlier in the absence of IL-4. The clinical symptoms and the early histopathologic abnormalities, however, are markedly different from those observed in “classic” PGIA or collagen-induced arthritis (4,5). In PG-TCR–Tg mice, the disease begins with mild lesions of the digits, with gradual development (over a period of months) of more severe deformities, loss of nails, and thickening of the toes, but the whole paw is affected in only a minority of animals. In contrast, PGIA usually starts 10–15 days after the second intraperitoneal injection of PG with adjuvant, and the complete clinical picture (redness and swelling of entire paws, and early joint deformities and ankylosis) develops rapidly after the third immunization (36). The dominant proximal joint (digit) involvement, the late onset, and the histopathologic features of affected small joints observed in this study are similar to findings in a previous study of HLA–DR4–transgenic 2992 mice with spontaneous arthritis (35). Replacement of the I-Ad molecule (class II MHC in BALB/c mice) with human HLA–DR4 on a BALB/c background led to the spontaneous development of arthritis, which resembled psoriatic arthritis (35). The association of HLA–DR4 with RA was first described ⬎20 years ago (41), and this MHC molecule can likely initiate activation of T cells through presentation of potentially arthritogenic peptide fragments, leading to autoimmunity in susceptible individuals (7). The DR4 molecule has been shown to present 20 peptide fragments (epitopes) of the human cartilage PG (aggrecan) molecule, including the 5/4E8 epitope (7). Based on the similarity of the spontaneous arthritis in PG-TCR–Tg mice described here and that observed earlier in HLA–DR4–Tg mice (35), we hypothesized that a common immunologic mechanism operates in both cases. In addition to the same BALB/c genetic background, age seems to be a factor. Age-related cartilage degeneration could be a common triggering event. Over time, small amounts of arthritogenic cartilage components (including PG fragments) are released from the joints, leading to activation of T and B cells. On an appropriate genetic background, this stimulation could lead to a breach of tolerance, resulting in an autoimmune response that culminates in arthritis development. While 100% incidence was observed in DR4-Tg mice (35), only 40–60% of PG-TCR–Tg mice developed spontaneous arthritis, depending on the presence or absence of IL-4. In DR4-Tg mice, the DR4 molecule can present a broad spectrum of epitopes to a diverse T cell repertoire, which could lead to a “polyclonal” activation of T cells (7). In PG-TCR–Tg mice, in contrast, the majority of T cells are specific for 5/4E8, the dominant arthritogenic peptide epitope. Therefore, only one of the potentially arthritogenic epitopes, presented by the native I-Ad, is recognized, which leads to a limited “monoclonal” activation of T cells, resulting in lower disease incidence in these mice. Another possible explanation for the lower incidence of spontaneous arthritis in PG-TCR–Tg mice could be that the 5/4E8 homologous sequence in the mouse PG molecule has lower affinity for the TCR of the Tg mice than the human peptide (8). In either case, arthritogenic T cells may be continuously activated by PG fragments released from cartilage catabolism in aging animals, gradually paving the way to autoimmune joint inflammation. Studying T cell apoptosis is of special interest in elucidating the pathogenesis of autoimmune arthritis (23,42). According to our present results, T cell apoptosis might play a role in the pathogenesis of the sponta- BOLDIZSAR ET AL neous arthritis in PG-TCR–Tg mice. IL-4 is an antiinflammatory cytokine that contributes to the regulation of PGIA (24). We found a slightly higher incidence of the spontaneous disease and a more pronounced accumulation of activated T cells in PG-TCR–Tg mice deficient in IL-4. The lack of IL-4 resulted in decreased in vitro TCR signaling and impaired apoptosis in CD4⫹ T cells. This is consistent with results from a previous study showing that IL-4 potentiates T cell apoptosis (25). We propose that in PG-TCR–Tg mice, repeated endogenous antigen exposure leads to T cell activation, which, in the absence of IL-4, is followed by reduced apoptosis. Thus, accumulation of activated T cells in the absence of IL-4 may ultimately lead to higher disease incidence. The threshold of TCR signaling is one of the key regulators, or critical “checkpoints,” of AICD. Zap70 and ERK-1/2 have been shown to be involved in regulating T cell apoptosis (43,44). In the present study, decreased phosphorylation of Zap70 (at Y319) and ERK-1/2 (at T203/Y205 in ERK-1 and T183/Y185 in ERK-2) was detected upon TCR stimulation in IL-4– deficient PG-TCR–Tg mouse CD4⫹ T cells when compared with PG-TCR–Tg/IL-4⫹/⫹ mouse CD4⫹ T cells. A point mutation study has shown that loss of the Y319 activator phosphorylation site in Zap70 abrogates phospholipase C␥ and LAT phosphorylation, while SH2 domain–containing protein of 76 kd and ERK signaling remain unaffected (45). The fact that TCR-induced Y319 phosphorylation of Zap70 in PG-TCR–Tg CD4⫹ T cells is affected by the absence of IL-4 raised the possibility of crosstalk between the TCR and IL-4 signaling pathways. Modification of IL-4 signaling by the Ras–MAPK pathway after TCR engagement has been reported (46). However, this is the first report of a study in which attenuation of TCR signaling by IL-4 deficiency has been demonstrated in spontaneous self-reactive transgenic CD4⫹ T cells. IL4–induced phosphorylation of p56 Lck and p59 Fyn was shown in CD3-activated killer cells (47). Decreased phosphorylation of Zap70 in the absence of IL-4, described here, might thus be attributed to lower activity of Lck and Fyn, which are important upstream regulators of Zap70 (39). The ERK pathway is hyperresponsive in T cells from RA patients, and this is not limited to activated T effector cells, but involves all naive and central memory CD4⫹ and CD8⫹ T cells (48). This observation is of interest because, in an earlier study (49), it was proposed that phospho-ERK, which is central to TCR threshold tuning, makes the decision between responding to exogenous high-affinity antigens (such as human PG in the case of the present study) and maintaining low response IMPAIRED AICD AND SPONTANEOUS ARTHRITIS or tolerance to low-affinity self peptides (such as mouse PG in the present study). Activation of the ERK pathway in RA patients or in PG-TCR–Tg BALB/c mice could shift this delicate balance. Higher responsiveness of the ERK pathway is found not only in patients with established RA but also in SKG mice before they develop arthritis, and it may be a critical step toward the breach of tolerance by allowing for expansion and differentiation of autoreactive T cells (49). In conclusion, aging PG-TCR–Tg mice develop spontaneous arthritis, which could be triggered by sustained low-threshold T cell activation by self cartilage components coupled with impaired AICD. In addition, IL-4 was confirmed to be a regulator of antigen-specific AICD through Zap70 and ERK-1/2, two key signaling components of TCR activation. PG-TCR–Tg mice provide a useful model for studying antigen (PG)–specific signaling pathways and the role of the threshold of T cell activation and T cell apoptosis in the pathogenesis of arthritis. 2993 5. 6. 7. 8. 9. 10. 11. ACKNOWLEDGMENTS The authors thank Beata Tryniszewska, BS, for assistance with animal breeding and Dr. T. Kobezda for collecting human cartilage. We appreciate our earlier coauthors, Dr. Suzanne E. Berlo, Dr. Chris P. Broeren (who passed away in 2003), and Prof. Willem van Eden, Utrecht, The Netherlands, who were technically involved in, or intellectually contributed to, the development of the transgenic mice. 12. 13. 14. 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