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Impaired activation-induced cell death promotes spontaneous arthritis in antigen cartilage proteoglycanspecific T cell receptortransgenic mice.

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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 [12] [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: tglant@rush.edu.
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.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Glant had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Boldizsar, Kis-Toth, Tarjanyi, Olasz,
Hegyi, Mikecz, Glant.
Acquisition of data. Boldizsar, Kis-Toth, Tarjanyi, Olasz, Hegyi,
Mikecz, Glant.
Analysis and interpretation of data. Boldizsar, Olasz, Mikecz, Glant.
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