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of June 17, 2017.
Modulation of Orphan Nuclear Receptor
NURR1 Expression by Methotrexate in
Human Inflammatory Joint Disease Involves
Adenosine A2A Receptor-Mediated
Responses
Jennifer A. Ralph, Alice N. McEvoy, David Kane, Barry
Bresnihan, Oliver FitzGerald and Evelyn P. Murphy
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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
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 175:555-565; ;
doi: 10.4049/jimmunol.175.1.555
http://www.jimmunol.org/content/175/1/555
The Journal of Immunology
Modulation of Orphan Nuclear Receptor NURR1 Expression
by Methotrexate in Human Inflammatory Joint Disease
Involves Adenosine A2A Receptor-Mediated Responses1
Jennifer A. Ralph,* Alice N. McEvoy,* David Kane,‡ Barry Bresnihan,† Oliver FitzGerald,† and
Evelyn P. Murphy2*
M
ethotrexate (MTX)3 was developed as a chemotherapeutic agent in the treatment of childhood acute lymphocytic leukemia in the late 1940s (1). The antiproliferative properties of high-dose MTX are due to inhibition of
dihydrofolate reductase and other folate-dependent enzymes,
which inhibit both purine and pyrimidine nucleotide biosynthesis
(2–5). At high levels, MTX inhibits cells in the S phase of cell cycle
and slows the entry of cells from the G1 phase into S phase (6, 7).
Since 1985, low-dose intermittent MTX has been demonstrated
to be an effective therapy for rheumatoid arthritis (RA) and is the
most commonly used disease-modifying antirheumatic drug in the
*Department of Veterinary Biochemistry and Physiology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, and †Department of Rheumatology, St. Vincent’s University Hospital, Elm Park, Dublin,
Ireland; and ‡Department of Rheumatology, Freeman Hospital, Newcastle upon Tyne,
United Kingdom
Received for publication December 10, 2004. Accepted for publication April
13, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
E.P.M. was supported by a grant from the Health Research Board of Ireland.
2
Address correspondence and reprint requests to Dr. Evelyn P. Murphy, Department
of Veterinary Biochemistry and Physiology, University College Dublin, Belfield,
Dublin 4, Ireland. E-mail address: [email protected]
3
Abbreviations used in this paper: MTX, methotrexate; RA, rheumatoid arthritis;
PsA, psoriatic arthritis; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; PGE2, prostaglandin E2; ST, synovial tissue; LL, lining layer;
RI, Ritchie articular index; ESR, erythrocyte sedimentation rate; CRP, C-reactive
protein; DAS, Disease Activity Score; NECA, 5⬘-N-ethylcarboxamideadenosine;
DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; DMPX, 3,7-dimethyl-1-propargylxanthine; ZM-241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a[]1,3,5]triazin-5ylamino]ethyl)phenol; MRS 1220, N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene; hpf, high-power field; SL, sublining; 6-MP, 6-mercaptopurine.
Copyright © 2005 by The American Association of Immunologists, Inc.
treatment of this disease (4, 8). Currently, its use is increasing in
other immune-mediated pathologies including psoriatic arthritis
(PsA), psoriasis, Crohn’s disease, and graft-vs-host disease (9 –
13). In human inflammatory joint disease, MTX acts by reducing
the levels of soluble mediators of inflammation such as metalloproteases and cytokines (14 –16). MTX also significantly reduces
cellular infiltration through down-regulation of cell surface adhesion molecules (17, 18). Several lines of evidence now indicate
that the immunosuppressive and anti-inflammatory actions of lowdose MTX are not due to inhibition of folate metabolism but are
mediated by its capacity to increase extracellular adenosine levels
(16, 19 –22). Adenosine, acting at one or more of its membrane
receptors, is responsible for the anti-inflammatory actions of MTX
in animal models of both acute and chronic inflammation (20, 21,
23). Despite a growing body of evidence to indicate that the mechanisms of MTX action are closely related to enhanced release of
adenosine, the molecular targets modulated by MTX or adenosine
action in vivo remain to be elucidated.
NURR1, together with NOR-1 and NUR77, constitute the
NURR subfamily of orphan receptors within the steroid/thyroid
receptor superfamily (24, 25). Unlike most nuclear receptors, the
NURR subfamily are products of immediate early genes, the expression of which can be induced in response to a variety of extracellular stimuli (26 –32). All three proteins bind to the same
cis-acting consensus sequence (NBRE) to regulate target gene expression, and can function as constitutively active transcription
factors (33–35). These genes are involved in neuroendocrine regulation, neural differentiation, liver regeneration, cell apoptosis,
and mitogenic stimuli in different cell types (28, 30, 31, 36 – 40).
NURR1 is involved in certain diseases involving dopamine transmission including Parkinson’s disease (41). The NOR-1 gene was
0022-1767/05/$02.00
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Modulation by proinflammatory mediators indicate that NURR1 induction represents a point of convergence of distinct signaling
pathways, suggesting an important common role for this transcription factor in mediating multiple inflammatory signals. The
present study identifies NURR1 as a molecular target of methotrexate (MTX) action in human inflammatory joint disease and
examines the mechanism through which MTX modulates NURR1 expression. MTX significantly suppresses expression of NURR1
in vivo in patients with active psoriatic arthritis (n ⴝ 10; p < 0.002) who were prescribed low-dose MTX for management of
peripheral arthritis. Importantly, reduction in NURR1 levels correlate (n ⴝ 10; r ⴝ 0.57; p ⴝ 0.009) with changes in disease
activity score (both clinical and laboratory parameters). MTX selectively modulates NURR1 levels induced by inflammatory
stimuli and growth factors in resident cell populations of synovial tissue. In primary human synoviocytes and microvascular
endothelial cells, we observe dose-dependent differential effects of MTX on steady-state and inducible NURR1 levels. Our data
confirms that adenosine, and its stable analog 5ⴕ-N-ethylcarboxamideadenosine, can mimic the differential effects of MTX on
NURR1 transcription. In addition, we verify that the inhibitory effect of low-dose MTX on NURR1 activation is mediated through
the adenosine receptor A2. More specifically, our data distinguishes the selective involvement of the A2A receptor subtype in these
responses. In summary, these findings establish the nuclear orphan receptor NURR1 as a molecular target of MTX action in
human inflammatory joint disease and demonstrate that the immunomodulatory actions of MTX on NURR1 expression are
mediated through adenosine release. The Journal of Immunology, 2005, 175: 555–565.
556
NURR1 MODULATION BY MTX IN HUMAN INFLAMMATORY JOINT DISEASE
Materials and Methods
Patient details and tissue collection
All ST biopsies were obtained from patients with inflammatory arthritis
and active knee synovitis who were undergoing knee arthroscopy. PsA was
defined according to established criteria (53). All patients attended the
Early Arthritis Clinic at St. Vincent’s University Hospital (Dublin, Ireland), and ethical permission was obtained from the Ethics Committee in
accordance with the Declaration of Helsinki principles. MTX was com-
menced at a dose of 7.5 mg/wk P.O. and could be increased by the supervising rheumatologist to a maximum of 15 mg/wk, depending on the degree of clinical response and/or side effects. Folic acid supplementation
was also prescribed to all patients (54). Patients were continued on stable
nonsteroidal anti-inflammatory drugs. Intra-articular steroids were contraindicated for the duration of the study.
Biopsies were taken before MTX treatment and between six and 12 mo
during MTX treatment, either when a clinical response had been obtained
or a maximal dose of 15 mg of MTX had been prescribed. At the time of
the first biopsy, all patients (n ⫽ 10) had active knee synovitis (median, 18
mo; range, 0.25–168 mo). Patients received a weekly dose of MTX (median, 13.75 mg/wk) and were not receiving any other disease-modifying
agents, including prednisolone. Patients were assessed on the day of each
arthroscopy. Assessments included the Ritchie articular index (RI) (55), the
European League Against Rheumatism swollen joint count (maximum 44
joints), the erythrocyte sedimentation rate (ESR) (measured by standard
Westergren technique), the C-reactive protein (CRP) level (measured by
standard nephelometry), and the 3-variable Disease Activity Score
(DAS) (56).
Arthroscopic synovial biopsy of the knee was performed on patients
under local anesthesia using a 2.7-mm Storz arthroscope and 1.5-mm
grasping forceps. Biopsy samples were obtained from all compartments of
the knee joint, embedded in TissueTek OCT compound (Sakura Finetek),
snap frozen, and stored in liquid nitrogen until used. Cryostat sections (7
␮M) were mounted on 3-aminopropyltriethoxysilane-coated glass slides,
air dried overnight, wrapped in foil, and stored at ⫺80°C until immunohistochemical analysis was performed.
Cell culture
ST obtained from the knee by arthroscopy was treated for 4 h with 1 mg/ml
collagenase (type I; Worthington Biochemical) in RPMI 1640 at 37°C in
5% CO2. Dissociated cells were plated in RPMI 1640 supplemented with
10% FCS (Invitrogen Life Technologies), penicillin (100 U/ml), and streptomycin (100 U/ml) (52). To eliminate nonadherent mononuclear cells
from synovial cell populations, the plated cells were cultured for at least
24 h. The cells were then washed extensively with PBS. Primary PsA
synoviocytes were used between the third and seventh passage for subsequent experiments (32, 52). Primary PsA synoviocyte cells were found to
be morphogically homogenous fibroblast-like cells and did not react with
specific Abs to the macrophage/monocyte Ag CD14. The immortalized
normal human K4 IM synoviocyte cell line was grown as described previously (39, 57, 58). Primary human microvascular endothelial cells (BioWhittaker) were grown using EGM-MV BulletKit (BioWhittaker) (58). All
cells were grown in serum-free medium for 24 h before stimulation. MTX,
dexamethasone, adenosine, 5⬘-N-ethylcarboxamideadenosine (NECA), alloxazine, VEGF, bFGF, PGE2, 8-cyclopentyl-1,3-dipropylxanthine
(DPCPX), 3,7-dimethyl-1-propargylxanthine (DMPX; Sigma-Aldrich), recombinant human TNF-␣, IL-1␤ (Calbiochem), 4-(2-[7-amino-2-(2furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol
(ZM-241385), and N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene (MRS 1220; Tocris Cookson) were resuspended according to the manufacturer’s guidelines and included at concentrations as
indicated. The concentration of MTX and duration of exposure used had no
effect on cell viability (data not shown) (22, 59).
Immunohistochemistry
ST immunohistochemistry was conducted on matched tissue sections from
biopsies taken before and during MTX treatment (n ⫽ 10). The primary Ab
(1:100) used for NURR1 (Santa Cruz Biotechnology) is a rabbit polyclonal
Ab mapping to the amino terminus of human and rat NURR1 (32, 39).
Tissue sections were incubated in diluted normal goat serum (Vector Laboratories) for 1 h. The primary Ab for NURR1 was diluted (1/100) in 10%
normal human serum and incubated on the tissue sections for 1 h. Following the addition of biotinylated anti-rabbit secondary Ab (1:500) (Vector
Laboratories), sections were incubated with the avidin-biotin-peroxidase
complex (VECTASTAIN Elite ABC Kit; Vector Laboratories). For negative controls, isotype-matched nonimmune IgG were included, and the primary Ab was preabsorbed with its specific synthetic peptide (blocking
peptide; Santa Cruz Biotechnology) (32, 39).
Immunocytochemistry on primary PsA synoviocytes was conducted using identical procedures as that used for ST immunohistochemistry. Primary cells were grown on 8-chamber culture slides (BD Biosciences) and
maintained in serum-free RPMI 1640 for 24 h before treatment.
Microscopic analysis
Only ST sections in which the LL was identifiable were included in the
analysis. Two people, who were blinded to the identities of the sections at
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identified originally through its involvement in the chromosomal
rearrangement associated with extraskeletal myxoid chondrosarcoma (42). Recent studies identify the NURR subfamily as effector
proteins of cytokine action (32, 39, 43, 44). Data from our laboratory and others (39, 45– 47) propose that the NURR subfamily
could serve as novel targets in the treatment of inflammatory arthritis, atherosclerosis, and lung cancer.
Inflammation and hyperplasia of the synovium are hallmarks of
RA and PsA (48, 49). The normal synovium is a delicate tissue
lining the joint capsule: however, in arthritis, the synovium transforms into an aggressive, tumor-like structure called pannus (48,
49). Angiogenesis, the formation of new blood vessels, is one of
the earliest histopathological findings in disease pathogenesis and
is required for pannus development (49, 50). The endothelium lining the blood vessels is recognized as playing an active and pivotal
role in the recruitment of leukocytes to the inflamed synovium (50,
51). Macrophage-derived cytokines are potent mitogens for proliferating synoviocytes (fibroblast-like) in the vicinity of the affected cartilage that produce matrix-degrading molecules (48, 49).
Synoviocytes and macrophages within the synovium orchestrate a
self-perpetuating inflammatory response via the autocrine actions
of cytokines (e.g., TNF-␣ and IL-1␤), growth factors (e.g., basic
fibroblast growth factor (bFGF) and vascular endothelial growth
factor; VEGF), and prostaglandins (e.g., prostaglandin E2; PGE2)
(48, 49, 52). It is the persistent invasive and destructive growth of
synovial tissue (ST) that ultimately leads to joint erosion (48, 49).
Markedly enhanced expression of NURR1 is observed in ST of
patients with RA and PsA compared with normal subjects (32).
Enhanced NURR1 expression in the synovial lining layer (LL),
subsynovial synoviocytes, and vascular endothelium suggests that
NURR1 is expressed primarily in cells believed to be at the leading
edge of invasive pannus (32). Modulation by proinflammatory mediators in primary and normal synoviocytes suggests that PGE2,
TNF-␣, and IL-1␤ markedly enhance NURR1 mRNA and protein
levels in contrast to other family members, NUR77 and NOR-1
(39). NURR1 induction represents a point of convergence of
distinct signaling pathways, suggesting an important common
role for this transcription factor in mediating multiple inflammatory signals (39).
In this study, we report that MTX significantly suppresses
NURR1 expression in vivo in patients with PsA (n ⫽ 10) who
were prescribed low-dose MTX for management of peripheral arthritis. Importantly, reduction in NURR1 levels correlate with
changes in both clinical and laboratory parameters. We investigated whether MTX modulates endogenous NURR1 levels induced by inflammatory stimuli and growth factors in resident cell
populations of ST. In primary synoviocytes and microvascular endothelial cells, we observe dose-dependent differential effects of
MTX on NURR1 levels. At pharmacologically relevant doses,
MTX differentially modulates induced NURR1 mRNA and protein
levels. Our data confirms that adenosine can mimic the differential
effects of MTX on steady-state and inducible NURR1 expression.
Finally, we confirm that the inhibitory effects of low-dose MTX on
NURR1 activation are mediated through the adenosine receptor
type A2A.
The Journal of Immunology
the time of scoring, evaluated all tissue sections randomly. The results of
the analysis were scored on a 0 –5 scale: 0, ⬍1% positive cells; 1, 1–10%;
2, 11–25%; 3, 26 –50%; 4, 51–75%; and 5, 76 –100%. Scoring was performed in randomly selected high-power fields (hpf) at ⫻400 magnification; a minimum of 17 hpf from three separate sections and the mean score
were calculated. For each hpf, a separate score for percentage positive cells
was made for LL, sublining (SL) layer, and blood vessels. Percentage
degree of localization of NURR1 (cytoplasmic and nuclear) was also evaluated (data not shown). These techniques have been validated previously
and reported (60 – 62).
Northern blot analysis
Western blot analysis
Cell cultures grown in 25-cm2 tissue culture flasks were maintained in
serum-free RPMI 1640 for 24 h before treatment. Cells were harvested
after specific treatments, and nuclear protein extracts were prepared as
described previously (31, 58). SDS-PAGE was performed under reducing
conditions on 10% polyacrylamide gels using concentrations of nuclear
lysates as indicated. The resolved proteins were transferred onto nitrocellulose sheets, which were immunoblotted overnight at 4°C with antiNURR1 mouse mAb (1:1000) (BD Biosciences). Secondary Abs to mouse
IgG, conjugated to HRP, were used for NURR1 detection and detected by
SuperSignal West Dura Extended Duration Substrate (Pierce) according to
the manufacturer’s instructions.
Synthesis of cDNA and PCR
Complementary DNA was prepared by reverse transcription of 1 ␮g of
each total RNA sample from cultured synoviocytes as described previously
(32). For PCR, the sense (5⬘-CGA CAT TTC TGC CTT CTC C-3⬘) and
antisense (5⬘-GGT AAA GTG TCC AGG AAA AG-3⬘) primer pair was
used to amplify human NURR1, yielding a 277-bp product. The sense
(5⬘-CCA CCC ATG GCA AAT TCC ATG GCA-3⬘) and antisense (5⬘TCT AGA CGG CAG GTC AAG TCC ACC-3⬘) primer pair was used to
amplify GAPDH, yielding a 465-bp product. Both primer pairs flanked
intron sequences. The conditions for amplification were as follows: an
initial denaturation step at 97°C for 2 min, followed by denaturation at
97°C for 30 s, primer annealing at 55°C for 30 s, and extension at 72°C for
30 s. PCR was performed in 50-␮l volumes, and each PCR sample underwent a 30-cycle amplification, which ensured that the reactions had not
reached the plateau phase of amplification. PCR products were electrophoresed on a 1.5% agarose gel (ImageMaster VDS; Amersham Biosciences).
were treated with a mean MTX dose of 13.75 mg/wk at the time
of follow-up biopsy. During treatment, there was a significant improvement in clinical measures of disease activity including RI
( p ⫽ 0.014) and swollen joint count ( p ⫽ 0.018), with improvements in laboratory measures of ESR and CRP also observed (Table I). All patients experienced a significant reduction ( p ⫽ 0.009)
in their DAS, which is a composite of both clinical and laboratory
measurements (Table I).
At baseline arthroscopy, all patient ST (n ⫽ 10) demonstrated
macroscopic features of synovial inflammation. Microscopic analysis confirmed inflammatory cell infiltrate, increased LL, thickness, and extensive SL vascularization (Fig. 1, A–F) (61, 62). Consistent with previous reports on the immunomodulatory effects of
MTX in inflammatory joint disease (18, 63), all patient tissue demonstrated significant reduction in LL thickness and inflammatory
cell infiltrate (Fig. 1, G–L).
Before treatment, NURR1 immunostaining was extensive in all
samples (n ⫽ 10), with abundant staining observed in synoviocyte
and macrophage cell populations of the synovial LL and SL regions (Fig. 1, A–F). Synovial vascular endothelium was also a site
of NURR1 expression in all inflamed ST studied. This pattern of
NURR1 staining is consistent with patterns of NURR1 expression
in RA ST (32, 39, 64). During MTX treatment, cells positive for
NURR1 immunostaining in the LL and SL regions (Fig. 1, G–L)
decreased significantly (Table I). Reduction in NURR1 levels correlated significantly with changes in DAS (n ⫽ 10; r ⫽ 0.57; p ⫽
0.009). Vascular endothelial expression of NURR1 was significantly reduced but not eliminated during MTX treatment. Importantly, most NURR1 immunostaining that remained during treatment was predominately cytoplasmic (Fig. 1, G–L) compared with
dominant nuclear localization in inflamed ST (Fig. 1, A–F). The
specificity of NURR1 staining was verified by loss of staining
when NURR1 Ab was preincubated with an excess of Ag (blocking peptide) and tissue incubation with isotype-matched nonimmune IgG (data not shown) (32, 39, 64).
MTX selectively inhibits inducible NURR1 mRNA levels in
primary human synoviocyte and microvascular endothelial cells
Proinflammatory mediators (PGE2, IL-1␤, and TNF-␣) and growth
factors (bFGF and VEGF) associated with joint inflammation (48,
49, 58) modulate NURR1 mRNA expression in normal human
synoviocytes, primary RA and PsA synoviocytes, and vascular endothelium (32, 39, 64). Characteristic of immediate early response
Table I. Clinical responses and changes in NURR1 expression prior to
and during MTX treatmenta
Statistical analysis
Analysis of the data was performed using nonparametric analysis in InStat
software (GraphPad). Mann-Whitney U test was performed to compare
groups. Spearman rank correlation coefficient was used to test for correlation of variables.
Results
MTX suppresses NURR1 expression in vivo
Previous findings from our laboratory demonstrated that inflamed
RA and PsA ST produces markedly increased levels of NURR1
compared with normal ST (32, 39). To determine the effects of
MTX treatment on NURR1 expression in vivo, ST was obtained
before and during treatment from 10 patients with active disease
who were beginning MTX therapy. All patients presented with
active synovitis of one or more knees with a median of 7.5 swollen
joints at presentation. The median duration of disease at the point
of entry to the study was 18 mo (range, 0.25–168 mo). Patients
Before MTX
Clinical features
RI
Swollen joint
count
ESR, mm/hr
CRP mg/l
DAS (3variable)b
Synovial membrane
analysis of
NURR1
LL
SL layer
During MTX
p
10.0 ⫾ 2.5 (9.0)
9.6 ⫾ 3.0 (7.5)
3.0 ⫾ 1.2 (1.5)
2.7 ⫾ 0.8 (1.5)
0.014
0.018
29.1 ⫾ 12.3 (19)
46.5 ⫾ 23.5 (15.5)
3.3 ⫾ 0.48 (2.9)
9.5 ⫾ 3.6 (4)
7.7 ⫾ 3.8 (1.5)
1.6 ⫾ 0.3 (1.4)
0.103
0.268
0.009
4.6 ⫾ 0.2 (5.0)
4.3 ⫾ 0.2 (4.5)
2.1 ⫾ 0.4 (1.7)
2.0 ⫾ 0.3 (2.0)
0.002
0.001
a
Values are the mean ⫾ SEM (median). ST findings were scored semiquantitatively on a scale of 0 –5, where 0 ⫽ ⬍1% positive cells, 1 ⫽ 1–10%, 2 ⫽ 11–25%,
3 ⫽ 26 –50%, 4 ⫽ 51–75%, and 5 ⫽ 76 –100%. p values were determined by MannWhitney U test.
b
DAS, a composite score of ESR, swollen joint count, and RI.
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Cell cultures grown in 25-cm2 tissue culture flasks were maintained in
serum-free RPMI 1640 for 24 h before treatment. Total RNA was isolated
using RNeasy (Qiagen) after specific treatments as indicated. RNA was
quantitated by UV absorption, and 10 ␮g total RNA was electrophoresed
on a standard Northern gel and transferred to nylon membrane (Bio-Rad).
NURR1 cDNA, spanning the amino-terminal region to avoid cross-hybridization, and GAPDH cDNA were radiolabeled to a high specific activity
using [␣-32P]dCTP and a random primer labeling system (Promega) (31).
Blots were exposed to film at ⫺80°C using intensifying screens, and autoradiographic intensity was quantitated using an imaging densitometer.
Densitometric values provided are representative of at least three separate
experiments and are expressed as fold induction over basal levels relative
to GAPDH expression ⫾ SEM.
557
558
NURR1 MODULATION BY MTX IN HUMAN INFLAMMATORY JOINT DISEASE
genes, the induction of NURR1 by such mediators exhibits a rapid
turnover, with maximal NURR1 mRNA levels peaking at 1 h (32,
39). Consistently, PGE2 has the most potent effect in stimulating
NURR1 mRNA levels in these cells (32, 39). To further characterize the ability of MTX to modulate NURR1 expression, we
examined the effect of MTX on proinflammatory mediator- and
growth factor-induced mRNA levels using primary human synoviocytes and primary human microvascular endothelial cells. Importantly, induction of NURR1 mRNA levels by all mediators
tested peaked at 1–2 h and returned to basal levels within 3– 4 h of
treatment (n ⫽ 4). PGE2 (35.9 ⫾ 5.5-fold), TNF-␣ (14.5 ⫾ 3.3fold), IL-1␤ (10.76 ⫾ 1.4-fold), bFGF (5.7 ⫾ 0.8-fold), and VEGF
(16.5 ⫾ 2.5-fold) robustly up-regulate NURR1 mRNA (Fig. 2G).
Treatment of inflammatory joint disease with low-dose MTX
can result in serum concentrations of up to 50 ␮g/ml (110 ␮M)
(59, 65). At this concentration, MTX has been shown previously to
have no toxic effect on fibroblast or endothelial cells (22). Therefore, primary human synoviocytes (n ⫽ 3) and microvascular endothelial cells (n ⫽ 3) were preincubated for 2 h (22) with 50 ␮M
or 100 ␮M MTX (59) and treated with PGE2 (10⫺6 M), TNF-␣ (10
ng/ml), IL-1␤ (10 ng/ml), bFGF (10 nM), or VEGF (10 nM) for
1 h. Following treatment, total RNA was prepared and assayed for
NURR1 mRNA levels by Northern blot analysis (n ⫽ 3) (Fig. 2).
As shown in Fig. 2, A and B, MTX inhibited PGE2-induced
NURR1 mRNA levels in both cell types. Interestingly, the most
potent effect of MTX was mediated at 50 ␮M. In the presence of
50 ␮M MTX, PGE2-induced NURR1 mRNA levels were reduced
significantly from 35.9 ⫾ 5.5-fold to 14.8 ⫾ 0.6-fold induction.
MTX at higher concentrations (100 ␮M) had no significant effect
on PGE2-induced NURR1 mRNA levels. In the presence of 100
␮M MTX, PGE2-induced NURR1 mRNA levels were reduced
from 35.9 ⫾ 5.5-fold to 28.4 ⫾ 6.8-fold induction. The extent of
50 ␮M MTX inhibition on NURR1 mRNA levels was comparable
to the anti-inflammatory actions of dexamethasone (32). In the
presence of dexamethasone (10⫺8 M), PGE2-induced NURR1
mRNA levels were reduced from 35.9 ⫾ 5.5-fold to 17.7 ⫾ 2.5fold induction. Modest effects of MTX (50 ␮M) on IL-1␤- and
TNF-␣-induced NURR1 mRNA levels in primary endothelial cells
and synoviocytes were observed (Fig. 2, C, D, and G). In contrast,
MTX, at the concentrations tested, had no inhibitory effect on
bFGF and VEGF-dependent NURR1 expression (Fig. 2, E–G).
MTX modulates endogenous and PGE2-induced NURR1
expression in a dose-dependent manner
To determine the effects of MTX alone on endogenous NURR1
transcription, human synoviocyte cells (Fig. 3A) and endothelial
cells (data not shown) were treated with increasing concentrations
of MTX (n ⫽ 3). Low doses of MTX had no effect on endogenous
NURR1 mRNA turnover; however, at 100 ␮M MTX, levels of
NURR1 mRNA begin to accumulate, and, at 200 ␮M MTX, modulation of NURR1 transcription was comparable to levels reached
by PGE2 stimulation (Fig. 3A). To further analyze the differential
inhibitory effects of MTX on PGE2-dependent NURR1 expression,
synoviocytes and endothelial cells were preincubated with increasing concentration of MTX (5–200 ␮M) for 2 h and stimulated with
PGE2 (10⫺6 M) for 1 h (Fig. 3, B and C). In both cell types, MTX
inhibited PGE2-induced NURR1 mRNA levels in a dose-dependent manner, with maximal inhibition occurring at the lower concentration of ⬃10 ␮M and maintained at 50 ␮M levels (Fig. 3, B
and C). Consistent with initial observations (Fig. 2), concentrations
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FIGURE 1. MTX suppresses NURR1 expression in vivo. Immunohistochemical staining for NURR1 in ST sections obtained from patients with PsA.
Representative ST sections from patients with PsA (#1, #2, and #3) were stained with NURR1-specific antiserum before commencing MTX treatment (A–F)
and during treatment (G–L). Positive immunoreactivity appears as brown/black. Nuclei are counterstained with hematoxylin (blue/purple). Before treatment,
specific nuclear NURR1 staining was extensive throughout the lining (LL) and SL layers and vascular endothelium (3). During treatment, cells positive
for NURR1 decreased significantly within all regions of the synovium. Vascular endothelial NURR1 expression was reduced but not eliminated. The
majority of NURR1 immunostaining that remained during MTX treatment is predominantly cytoplasmic compared with the predominant nuclear localization before treatment. Original magnification, ⫻100 in A–C and G–I; and ⫻400 in D–F and J–L.
The Journal of Immunology
559
of 100 ␮M MTX failed to significantly modulate induced NURR1
levels. Interestingly, higher MTX concentrations (200 ␮M) augmented the effects of PGE2 on NURR1 transcription, which is
consistent with the stimulatory effects of 100 –200 ␮M MTX on
endogenous NURR1 gene transcription (Fig. 3A).
In human synoviocytes, basal levels of NURR1 protein are induced, and maximum levels were observed within 1–2 h following
PGE2 stimulation (data not shown). Immunohistochemical studies
using isolated primary PsA synoviocytes confirm that MTX can
potently inhibit PGE2-induced NURR1 protein levels (Fig. 4).
NURR1 is primarily localized to the nucleus in PGE2-stimulated
synoviocytes. Following incubation with MTX (n ⫽ 3), PGE2induced levels are inhibited, and, comparable to its cellular localization during MTX treatment in vivo, NURR1 is primarily confined to the cytoplasm (Fig. 4A).
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FIGURE 2. MTX selectively inhibits NURR1 mRNA levels in primary synoviocyte and microvascular
endothelial cells. Representative
Northern blot analysis of NURR1
mRNA transcription in primary PsA
synoviocytes and microvascular endothelial cells. A, Primary synoviocytes and endothelial cells (B–F)
were left untreated or preincubated
for 2 h with MTX 50 ␮M, 100 ␮M,
or dexamethasone (Dex) 10⫺8 M.
Following preincubation, cells were
left untreated or stimulated for 1 h
with PGE2 (10⫺6 M), TNF-␣ (10 ng/
ml), IL-1␤ (10 ng/ml), bFGF (10
nM), or VEGF (10 nM). Following
treatments, total RNA was isolated
and assayed for NURR1 and GAPDH
mRNA levels as described in Materials and Methods. G, Densitometric
analysis representing NURR1 mRNA
levels from four separate experiments. Values are expressed as fold
induction over basal levels relative to
GAPDH expression ⫾ SEM.
Cells were pretreated with increasing concentrations of MTX
(10 –200 ␮M), exposed to PGE2 (10⫺6 M) for 1 h, and then examined for nuclear NURR1 protein levels by Western blot analysis
(n ⫽ 3) (Fig. 4B). Consistent with our mRNA analysis, inhibition
of NURR1 protein levels was observed following incubation with
10 ␮M MTX; inhibited levels were maintained with 25–50 ␮M
MTX and began to increase, reaching PGE2-dependent levels at
100 –200 ␮M MTX (Fig. 4, B and C).
MTX suppresses PGE2-induced NURR1 expression through
adenosine release
Recent reports provide convincing evidence that the nucleoside
adenosine mediates the anti-inflammatory and immunoregulatory
effects of MTX in in vivo models of acute and chronic inflammation (19 –23). To determine whether adenosine alone can modulate
560
NURR1 MODULATION BY MTX IN HUMAN INFLAMMATORY JOINT DISEASE
steady-state NURR1 mRNA levels, synoviocyte cells (Fig. 5A)
were treated with increasing concentrations of adenosine (0.1–500
␮M). Our results indicate that following incubation with 0.1–10
␮M adenosine, steady-state levels of endogenous NURR1 mRNA
remain unchanged. At 100 ␮M adenosine, levels of NURR1
mRNA begin to increase, and, at 500 ␮M adenosine, modulation
of NURR1 transcription is comparable to levels reached by PGE2
stimulation (Fig. 5A). Thus, confirming that adenosine can mimic
the effects of MTX on endogenous NURR1 transcription.
To determine whether adenosine modulates PGE2-mediated
NURR1 transcription, human synoviocytes were pretreated with
different amounts of adenosine (0.5–500 ␮M) and then examined
for effects on PGE2-induced NURR1 transcription. Our results indicate that preincubation with adenosine for 2 h abrogates PGE2-
FIGURE 4. Effect of MTX on PGE2-dependent
NURR1 protein levels. A, Immunohistochemical staining of primary PsA synoviocytes with anti-NURR1 immune serum. Synoviocytes were left untreated (NT) or
preincubated with MTX (50 ␮M) for 2 h. Following
preincubation, cells were left untreated (NT) or treated
for 1 h with PGE2 (10⫺6 M). B, Representative Western
blot analysis of nuclear extracts of primary synoviocytes left untreated (NT) or preincubated for 2 h with
MTX (5–200 ␮M) then treated with PGE2 10⫺6 M for
1 h. Nuclear lysates (15 ␮g) were isolated and loaded
onto a 10% SDS-polyacrylamide gel, electrophoresed
and subsequently transferred onto nitrocellulose. Immunoblots were probed with an Ab specific for
NURR1. (MW, m.w. markers; ⫹, positive control cell
lysate). C, Densitometric analysis representing trends
of relative amounts of PGE2-dependent NURR1 levels
following MTX treatment in synoviocytes from three
separate Western blot experiments, expressed as mean
values ⫾ SEM.
FIGURE 5. Adenosine can mimic the effects of MTX on endogenous
and PGE2-dependent NURR1 transcription. Total RNA was isolated from
synoviocytes that were left untreated or treated with PGE2 (10⫺6 M) for 1 h
or adenosine (0.1–500 ␮M) for 2 h (A), left untreated or preincubated with
adenosine (0.1–500 ␮M) for 2 h and then treated with PGE2 (10⫺6 M) for
1 h (B), and left untreated or preincubated with extended does of adenosine
(10 –1500 ␮M) for 2 h and then treated with PGE2 (10⫺6 M) for 1 h (C).
Following treatments, total RNA was assayed by Northern blot analysis for
NURR1 and GAPDH mRNA levels.
induced NURR1 mRNA levels in a dose-dependent manner at all
concentrations tested (Fig. 5B). The stimulatory effects of higher
concentrations of adenosine on steady-state NURR1 mRNA levels
were not detected in the presence of PGE2 (10⫺6 M) (Fig. 5B).
Therefore, we decided to extend the dose range of adenosine from
10 ␮M to 1500 ␮M and examine the effects of higher adenosine
levels on PGE2-induced NURR1 levels (Fig. 5C). In the presence
of PGE2, the inhibitory effect of adenosine on PGE2-induced
NURR1 transcription was maintained over an extended concentration range (Fig. 5C).
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FIGURE 3. MTX modulates endogenous and PGE2-dependent NURR1
expression in a dose-dependent manner. A, Northern blot analysis of
NURR1 mRNA levels in synoviocytes left untreated or treated with PGE2
(10⫺6 M) or MTX (5–200 ␮M) for 1 h. Synoviocytes (B) and microvascular endothelial cells (C) that were left untreated or preincubated with
increasing concentrations of MTX (5–200 ␮M) for 2 h. Following MTX
preincubation, cells were left untreated or treated with PGE2 (10⫺6 M) for
1 h. NURR1 and GAPDH mRNA levels were measured using 10 ␮g total
RNA as described in Materials and Methods.
The Journal of Immunology
FIGURE 6. The inhibitory effects of MTX on PGE2-dependent NURR1
mRNA levels are mediated through the type 2 adenosine receptor. The
effects of selective adenosine receptor type 1 and type 2 antagonists on the
modulation of low-dose MTX on PGE2-dependent NURR1 mRNA levels
were analyzed by Northern blot analysis. Total RNA was isolated from
primary synoviocytes that were left untreated or preincubated for 2 h with
10 ␮M MTX alone, or in combination with increasing concentrations of A1
receptor antagonist (DPCPX) or A2 receptor antagonist (DMPX) (10 or 20
␮M). Following preincubation, cells were treated with PGE2 (10⫺6 M) for
1 h, and total RNA was isolated and assayed for NURR1 and GAPDH
mRNA levels.
FIGURE 7. NECA signaling through the A2A receptor is a potent modulator of endogenous and PGE2-dependent NURR1 transcription. The effects of NECA and NECA signaling via the type 2A receptor on endogenous and PGE2-dependent NURR1 mRNA levels were analyzed by
Northern blot analysis. Total RNA was isolated from primary synoviocytes
that were (A) left untreated or treated with PGE2 (10⫺6 M) for 1 h or NECA
(0.01–10 ␮M) for 2 h, (B) left untreated or preincubated with NECA
(0.01–10 ␮M) for 2 h and then treated with PGE2 (10⫺6 M) for 1 h, and (C)
left untreated or preincubated for 2 h with 1 ␮M NECA alone, or in combination with ZM-241385 (0.1 or 1 ␮M). Following preincubation, cells
were treated with PGE2 (10⫺6 M) for 1 h, and total RNA was isolated and
assayed for NURR1 and GAPDH mRNA levels.
7C, ZM-241385 reversed the inhibitory effects of NECA (1 ␮M),
confirming that adenosine effects on PGE2-induced NURR1
mRNA levels can be mediated through this receptor subtype.
Finally, the involvement of A2B and A3 receptors on NURR1
gene expression following stimulation by high doses of adenosine
was examined. The specific effects of antagonizing the A2B and
A3 receptors were analyzed using the selective A2B receptor antagonist alloxazine and the A3 receptor antagonist MRS 1220.
NECA at high concentrations (ⱖ10 ␮M) induces NURR1 gene
transcription in a dose-dependent manner (Fig. 8). The presence of
alloxazine or MRS 1220 with high concentrations of NECA had no
significant effect on NECA-induced NURR1 gene transcription
(Fig. 8). These data suggest that the A2B and A3 receptors are not
involved in the modulation of NURR1 expression by high-dose
FIGURE 8. The stimulatory effects of high-dose adenosine on NURR1
mRNA levels are not mediated through type A2B or A3 adenosine receptors. Representative RT-PCR products generated following amplification
of mRNA from primary synoviocytes with NURR1- and GAPDH-specific
primers. Primary synoviocytes were either left untreated or incubated for
2 h with NECA 10 ␮M or 20 ␮M in the presence or absence of alloxazine
(10 ␮M) or MRS 1220 (1 ␮M). The m.w. markers are shown on the left.
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Adenosine, acting at one or more of its receptors (A1, A2A,
A2B, and A3), can exert a number of actions on a variety of cell
types relevant to the anti-inflammatory actions of MTX (4, 66 –
68). In support of this mechanism of adenosine action, in vivo
studies suggest that adenosine receptor antagonists can reverse or
prevent the anti-inflammatory effects of MTX (21). To further investigate whether the inhibitory effects of MTX on NURR1 activation is mediated through adenosine, we examined the effect of
type A1 and type A2 adenosine receptor antagonists. As shown in
Fig. 6, MTX suppressed PGE2-induced NURR1 mRNA, and the
selective A2 receptor antagonist (DMPX) reversed this effect in a
concentration-dependent manner. In contrast to the potent effects
of DMPX, the selective A1 receptor antagonist (DPCPX) had no
effect at any of the concentrations tested (Fig. 6).
Adenosine bioavailability for signaling at its receptor sites is
determined by production, release, and cellular uptake. Because
adenosine is metabolized efficiently, the “effective” concentration
necessary for adenosine receptor-mediated modulation of NURR1
gene transcription may be significantly lower than that observed in
our experiments (Fig. 5). Therefore, a stable adenosine analog,
NECA, was used to further examine adenosine effects on NURR1
gene transcription in primary human synoviocytes. Our results indicate that following incubation with NECA alone (0.01–1 ␮M),
endogenous NURR1 mRNA levels remain unchanged (Fig. 7A).
At the higher concentration of NECA (10 ␮M), steady-state
NURR1 gene transcription begins to increase (Fig. 7A), a trend
similar to that observed with higher adenosine concentrations (100
␮M) (Fig. 5A). NECA potently abrogates PGE2-induced NURR1
transcription in a concentration-dependant manner (0.01–1 ␮M)
(Fig. 7B). The concentrations of adenosine (0.5–100 ␮M) eliciting
similar responses as NECA (0.01–1 ␮M) are ⬃50- to 100-fold
higher. Therefore, the effective concentrations of NECA required
to inhibit PGE2-induced NURR1 transcription are analogous to
appreciably higher concentrations of adenosine (Fig. 5B).
Having established that the effects of low-dose MTX on NURR1
gene expression are mediated through the adenosine receptor A2
(Fig. 6), we sought to determine the selective involvement of A2
receptor isoforms in modulating adenosine responses in our system. The adenosine receptor subtype, A2A, is a critical player in a
physiological negative feedback mechanism involved in the limitation and termination of inflammatory responses (67, 68). Thus,
the consequences of A2A receptor inhibition were tested using the
selective A2A receptor antagonist, ZM-241385. As shown in Fig.
561
562
NURR1 MODULATION BY MTX IN HUMAN INFLAMMATORY JOINT DISEASE
adenosine and lends further support to our conclusion that modulation of NURR1 expression by MTX involves adenosine A2A
receptor-mediated responses.
Discussion
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Members of the NURR subfamily of nuclear orphan receptors are
expressed aberrantly in human lung cancer cells, atherosclerotic
lesions, and inflamed ST compared with normal tissue (32, 39,
45– 47). Prolonged or inappropriate inflammatory responses contribute to the pathogenesis of these diseases (69, 70). Consistent
with our earlier findings that transcription of the NURR1 gene is
activated by inflammatory mediators associated with inflamed ST,
recent reports also identify NURR receptors as effector molecules
of cytokine signaling (32, 39, 43, 44). In this present study, we
describe our observation that NURR1 is a molecular target of
MTX action in vivo in human inflammatory joint disease. Furthermore, our data establishes that the immunomodulatory effect of
MTX on endogenous and cytokine-induced NURR1 expression
can be mimicked by adenosine and mediated through adenosine
receptor-mediated responses.
In the five decades since Farber et al. (1–5, 8, 66) first described
clinical remissions in children with acute leukemia after treatment
with the folate antagonist aminopterin, antifolate drugs dominated
by MTX have been used to treat millions of patients with malignant and immune-mediated diseases. It is estimated that MTX is
now prescribed to at least 500,000 patients with RA and other
forms of inflammatory arthritis (71). As demonstrated by several
studies, the potent anti-inflammatory efficacy of low-dose intermittent MTX cannot be due entirely to folate antagonism and diminished purine synthesis. The effect of MTX treatment on lymphocyte and monocyte proliferation and apoptosis in patients is
transient and is reversed by administration of folic acid (8, 66, 71).
Significantly, folic acid or folinic acid supplementation (1–5 mg/
day) during MTX treatment of patients with inflammatory joint
disease moderates MTX toxicity with no consequences on the antiinflammatory efficacy of the drug (54, 72). MTX reduces ST inflammation by decreasing infiltration of lymphocytes and monocytes, possibly through down-regulation of cytokine levels
including IL-1␤ and TNF-␣ and consequently a reduction in cytokine-induced expression of adhesion molecules (18, 63, 66). The
molecular mechanisms by which MTX decreases cytokine levels
in inflamed ST remain to be elucidated (4, 66).
Enhanced NURR1 expression in the synovial LL, subsynovial
synoviocytes, and vascular endothelial cells of inflamed ST confirms that NURR1 is expressed primarily in cells believed to be at
the leading edge of invasive tumor-like synovium (pannus) into
adjacent cartilage and bone (32, 39, 49). During MTX treatment,
NURR1 expression is significantly reduced in cells of the lining
( p ⫽ 0.002) and SL regions ( p ⫽ 0.001). The significant changes
in NURR1 levels correlate with clinical response and improvements in patients’ DAS (r ⫽ 0.57; p ⫽ 0.009). The principal site
of NURR1 expression during treatment is limited to the vascular
endothelium; importantly, this pattern of expression is identical
with NURR1 cellular localization in normal ST (32, 39). We have
demonstrated previously that NURR1 is primarily confined to the
nucleus of cells in inflamed tissue compared with a predominant
cytoplasmic localization in normal tissue (32, 39). Comparison of
NURR1 cellular localization in ST before and during MTX treatment reveals a distinct adjustment from nuclear to cytoplasmic
staining in response to MTX. Taken together, these data suggest
that MTX may modulate the transcriptional regulatory capacity of
NURR1 in vivo.
Inflamed ST produces large quantities of proinflammatory mediators and growth factors. PGE2, IL-1␤, and TNF-␣ occupy a
prominent place in the hierarchy of proinflammatory mediators
associated with inflammatory, immune, and arthritic diseases (48,
49). Peptide growth factors, including bFGF and VEGF, enhance
synoviocyte and endothelial cell migration and proliferation and
promote extracellular matrix protein turnover, processes which
play an important role in synovitis (49). The pathological importance of NURR1 was verified by establishing the ability of these
proinflammatory agonists, produced locally by inflamed synovium, to stimulate NURR1 transcription in synoviocytes (32, 39).
Moreover, the NURR1 promoter responds to these same immunological stimuli in a manner similar to the response of endogenous
NURR1 expression (39). Consistently, PGE2 has the most potent
effect in stimulating NURR1 mRNA in these cells (39). In this
study, we have established that low-dose MTX selectively inhibits
NURR1 mRNA levels induced by PGE2 and other inflammatory
agents. The potency of low-dose MTX inhibition is comparable to
dexamethasone, a powerful anti-inflammatory agonist previously
shown to inhibit cytokine and PGE2-induced NURR1 expression
(32). Importantly, with increasing MTX concentrations, we observe a molecular switch of MTX signaling. MTX, at concentrations ⬎50 ␮M, fails to inhibit cytokine-induced NURR1 gene transcription and has significant stimulatory effects on endogenous
NURR1 mRNA and protein levels. The immunomodulatory actions of MTX are not confined to specific cell types because analogous responses were observed in primary synoviocyte and microvascular endothelial cells.
In RA, activated synoviocytes demonstrate increased proliferative and invasive activity, and they release proteolytic enzymes,
cytokines, and prostanoids leading to cartilage erosion (49). MTX
suppresses synoviocyte invasion and ameliorates cartilage destruction in the SCID mouse model for human RA (73). Cytokinestimulated release of resorptive agents, such as matrix metalloproteinases, occurs in association with a change from a fibroblast-like
to a stellate morphology (74). This study and our previous analysis
demonstrate that cytokine and PGE2 induction of NURR1 also
occurs in parallel with a transformation of synoviocytes from a
fibroblast-like to a stellate morphology (32, 39). In primary synoviocytes, MTX can amend this induced stellate morphology and
reduce cytokine-induced NURR1 protein levels. The predominant
cytoplasmic localization of NURR1 initiated by MTX in vitro is
comparable to NURR1 cellular location observed during MTX
treatment in vivo. These data identify NURR1 as a molecular target of MTX signaling and verify that MTX effects on NURR1
levels observed in vivo are direct and not solely due to diminished
infiltration of lymphocytes and monocytes and a reduction in cytokine levels.
The observation that the in vitro action of adenosine and its
stable analog, NECA, mimic the effects of low-dose MTX on
NURR1 in vivo supports several reports that the anti-inflammatory
effects of MTX are due in large part to its capacity to enhance
extracellular adenosine at sites of inflammation. Adenosine regulates inflammation via interaction with one or more of its four
known receptors (A1, A2A, A2B, and A3) (67). Stimulation of A2
and A3 type adenosine receptors has been shown to alter the cytokine milieu by decreasing inflammatory cytokine secretion by
macrophage cells in vitro (59, 75–77). In animal models of acute
and chronic inflammation, nonselective adenosine receptor antagonists can reverse the anti-inflammatory effects of MTX treatment
(17, 20, 21). A recent report using the murine air-pouch model of
acute inflammation confirms that MTX fails to suppress inflammation in the air pouches of A2A and A3 receptor-deficient mice
(23). Our demonstration of A2A receptor involvement in mediating MTX effects is consistent with the role of the A2A receptor as
a regulator of inflammatory events. A2A receptor signaling is
The Journal of Immunology
Acknowledgments
We thank Dr. Eithne Murphy and the late Dr. Leanne Stafford for synovial
biopsy collection and Martina Gogarty for tissue sectioning.
Disclosures
The authors have no financial conflict of interest.
References
1. Farber, S., L. K. Diamond, R. D. Mercer, R. F. Sylvester, and J. A. Wolff. 1948.
Temporary remissions in acute leukemia in children produced by folic antagonist
4-amethopteroylglutamic acid (aminopterin). N. Engl. J. Med. 238: 787–793.
2. Purcell, W. T., and D. S. Ettinger. 2003. Novel antifolate drugs. Curr. Oncol.
Rep. 5: 114 –125.
3. Zhao, R., and I. D. Goldman. 2003. Resistance to antifolates. Oncogene 22:
7431–7457.
4. Kremer, J. M. 2004. Toward a better understanding of methotrexate. Arthritis
Rheum. 50: 1370 –1382.
5. Cronstein, B. N. 1996. Molecular therapeutics: methotrexate and its mechanism
of action. Arthritis Rheum. 39: 1951–1960.
6. Budzik, G. P., L. M. Colletti, and C. R. Faltynek. 2000. Effects of methotrexate
on nucleotide pools in normal human T cells and the CEM T cell line. Life Sci.
66: 2297–2307.
7. Linke, S. P., K. C. Clarkin, A. Di Leonardo, A. Tsou, and G. M. Wahl. 1996. A
reversible, p53-dependent G0-G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage. Genes Dev. 10: 934 –947.
8. Weinblatt, M. E., A. L. Maier, P. A. Fraser, and J. S. Coblyn. 1998. Longterm
prospective study of methotrexate in rheumatoid arthritis: conclusion after 132
months of therapy. J. Rheumatol. 25: 238 –242.
9. Abu-Shakra, M., D. D. Gladman, J. C. Thorne, J. Long, J. Gough, and
V. T. Farewell. 1995. Longterm methotrexate therapy in psoriatic arthritis: clinical and radiological outcome. J. Rheumatol. 22: 241–245.
10. Cutolo, M., B. Seriolo, C. Pizzorni, C. Craviotto, and A. Sulli. 2002. Methotrexate in psoriatic arthritis. Clin. Exp. Rheumatol. 20: S76 –S80.
11. Bright, R. D. 1999. Methotrexate in the treatment of psoriasis. Cutis 64: 332–334.
12. Feagan, B. G., R. N. Fedorak, E. J. Irvine, G. Wild, L. Sutherland,
A. H. Steinhart, G. R. Greenberg, J. Koval, C. J. Wong, M. Hopkins, et al. 2000.
A comparison of methotrexate with placebo for the maintenance of remission in
Crohn’s disease: North American Crohn’s Study Group Investigators. N. Engl.
J. Med. 342: 1627–1632.
13. Nash, R. A., L. A. Pineiro, R. Storb, H. J. Deeg, W. E. Fitzsimmons, T. Furlong,
J. A. Hansen, T. Gooley, R. M. Maher, P. Martin, et al. 1996. FK506 in combination with methotrexate for the prevention of graft-versus-host disease after
marrow transplantation from matched unrelated donors. Blood 88: 3634 –3641.
14. Barrera, P., A. M. Boerbooms, P. N. Demacker, L. B. van de Putte, H. Gallati,
and J. W. van der Meer. 1994. Circulating concentrations and production of
cytokines and soluble receptors in rheumatoid arthritis patients: effects of a single
dose methotrexate. Br. J. Rheumatol. 33: 1017–1024.
15. Seitz, M., M. Zwicker, and P. Loetscher. 1998. Effects of methotrexate on differentiation of monocytes and production of cytokine inhibitors by monocytes.
Arthritis Rheum. 41: 2032–2038.
16. Boyle, D. L., F. G. Sajjadi, and G. S. Firestein. 1996. Inhibition of synoviocyte
collagenase gene expression by adenosine receptor stimulation. Arthritis Rheum.
39: 923–930.
17. Asako, H., R. E. Wolf, and D. N. Granger. 1993. Leukocyte adherence in rat
mesenteric venules: effects of adenosine and methotrexate. Gastroenterology
104: 31–37.
18. Dolhain, R. J., P. P. Tak, B. A. Dijkmans, P. De Kuiper, F. C. Breedveld, and
A. M. Miltenburg. 1998. Methotrexate reduces inflammatory cell numbers, expression of monokines and of adhesion molecules in synovial tissue of patients
with rheumatoid arthritis. Br. J. Rheumatol. 37: 502–508.
19. Cronstein, B. N., M. A. Eberle, H. E. Gruber, and R. I. Levin. 1991. Methotrexate
inhibits neutrophil function by stimulating adenosine release from connective
tissue cells. Proc. Natl. Acad. Sci. USA 88: 2441–2445.
20. Cronstein, B. N., D. Naime, and E. Ostad. 1993. The anti-inflammatory mechanism of methotrexate: increased adenosine release at inflamed sites diminishes
leukocyte accumulation in an in vivo model of inflammation. J. Clin. Invest. 92:
2675–2682.
21. Montesinos, M. C., J. S. Yap, A. Desai, I. Posadas, C. T. McCrary, and
B. N. Cronstein. 2000. Reversal of the antiinflammatory effects of methotrexate
by the nonselective adenosine receptor antagonists theophylline and caffeine:
evidence that the antiinflammatory effects of methotrexate are mediated via multiple adenosine receptors in rat adjuvant arthritis. Arthritis Rheum. 43: 656 – 663.
22. Majumdar, S., and B. B. Aggarwal. 2001. Methotrexate suppresses NF-␬B activation through inhibition of I␬B␣ phosphorylation and degradation. J. Immunol.
167: 2911–2920.
23. Montesinos, M. C., A. Desai, D. Delano, J. F. Chen, J. S. Fink, M. A. Jacobson,
and B. N. Cronstein. 2003. Adenosine A2A or A3 receptors are required for
inhibition of inflammation by methotrexate and its analog MX-68. Arthritis
Rheum. 48: 240 –247.
24. Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono,
B. Blumberg, P. Kastner, M. Mark, P. Chambon, and R. M. Evans. 1995. The
nuclear receptor superfamily: the second decade. Cell 83: 835– 839.
25. Giguere, V. 1999. Orphan nuclear receptors: from gene to function. Endocr. Rev.
20: 689 –725.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
thought to comprise an endogenous immunosuppressive loop capable of distinguishing beneficial and harmful inflammatory responses, whereby accumulated extracellular adenosine signals
through cAMP-elevating A2A receptors in a delayed negative
feedback manner (67, 68). Ohta and Sitkovsky (68) have demonstrated the involvement of the A2A receptor in the down-regulation of inflammation and inhibition of tissue damage in vivo. In
this study, subthreshold doses of an inflammatory stimulus that
caused minimal tissue damage in wild-type mice were sufficient to
induce extensive tissue damage, more prolonged and higher levels
of proinflammatory cytokines, and death of animals deficient in the
A2A receptor.
Adenosine exerts autocrine and paracrine responses when released from inflamed tissue (67, 78). Adenosine A2 receptor-mediated inhibition of inflammation is dominant when cells are exposed to adenosine concentrations at ⱖ0.5 ␮M (79). In both
synoviocyte and endothelial cells, adenosine inhibition of PGE2stimulated NURR1 expression was dose dependent and maximal at
concentrations ⬎0.5 ␮M and sustained, in the presence of PGE2,
over a wide concentration range. A novel function of PGE2 reveals
that this prostanoid may exert its effect by coupling the chemokine
CCR7 receptor to signal transduction modules (80). In the presence of PGE2, stimulation with CCR7 ligands led to enhanced
phosphorylation of protein kinases and subsequent downstream
effects. Thus, it is possible that PGE2 may influence adenosine
receptor-mediated signaling in a similar manner. Both PGE2 and
adenosine have been shown to signal, at least in part, via cAMP/
protein kinase A-dependent and -independent pathways. However, our preliminary analysis reveals no alterations in protein
kinase A-dependent CREB phosphorylation in cells exposed to
adenosine in the presence of PGE2 (J.A. Ralph, unpublished
observations). This data may be consistent with the observation
that engagement of multiple adenosine receptors, capable of
stimulating and inhibiting intracellular cAMP levels, contribute
to discoordinate gene expression in the amelioration of inflammatory disease (21, 23, 81– 83).
In the absence of PGE2, and similar to the effects of MTX on
steady-state NURR1 mRNA turnover, adenosine with increasing
concentration displays stimulatory effects on endogenous NURR1
mRNA levels. Recent findings indicate this nuclear orphan receptor may play a role in mediating the intracellular effects of the
purine antimetabolite 6-mecaptopurine (6-MP) (84). Ordentlich et
al. (84) showed that 6-MP, only at concentrations ⱖ50 ␮M, is a
potent regulator of NURR1 transcriptional activity. Significantly,
the effect of 6-MP on NURR1 transcriptional activity is fully inhibited in the presence of 15 ␮M adenosine. Taken together, these
data suggest that the cellular content of purine nucleotides may
modulate both the expression and transcriptional activity of this
nuclear orphan receptor.
In summary, these findings provide new insights into signaling
pathways through which MTX can mediate its effect in human
inflammatory arthritis. The identification of NURR1 as a target of
MTX and adenosine action may facilitate the development of new
and more effective therapies for the treatment of inflammatory disease. The clinical potential of nuclear receptors as drug targets has
been proven already in the case of steroid receptors, in particular
those for estrogen, androgens, and glucocorticoids (24, 25). The
possibility that the activity of nuclear receptors might be regulated
by the direct action of natural and synthetic compounds make orphan receptors excellent targets for drug discovery even in the
absence of known natural ligands. In the case of NURR1, the further characterization of molecular signaling cascades both regulating and modulated by NURR1 may provide new approaches for
intervention using this transcription factor as a molecular target.
563
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NURR1 MODULATION BY MTX IN HUMAN INFLAMMATORY JOINT DISEASE
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS
Lett. 372: 83– 87.
Veale, D., S. Rogers, and O. Fitzgerald. 1994. Classification of clinical subsets in
psoriatic arthritis. Br. J. Rheumatol. 33: 133–138.
Whittle, S. L., and R. A. Hughes. 2004. Folate supplementation and methotrexate
treatment in rheumatoid arthritis: a review. Rheumatology 43: 267–271.
Ritchie, D. M., J. A. Boyle, J. M. McInnes, M. K. Jasani, T. G. Dalakos,
P. Grieveson, and W. W. Buchanan. 1968. Clinical studies with an articular index
for the assessment of joint tenderness in patients with rheumatoid arthritis.
QJ Med. 37: 393– 406.
van der Heijde, D. M., M. A. van’t Hof, P. L. van Riel, L. A. Theunisse,
E. W. Lubberts, M. A. van Leeuwen, M. H. van Rijswijk, and
L. B. van de Putte. 1990. Judging disease activity in clinical practice in
rheumatoid arthritis: first step in the development of a disease activity score.
Ann. Rheum. Dis. 49: 916 –920.
Haas, C., W. K. Aicher, A. Dinkel, H. H. Peter, and H. Eibel. 1997. Characterization of SV40T antigen immortalized human synovial fibroblasts: maintained
expression patterns of EGR-1, HLA-DR and some surface receptors. Rheumatol.
Int. 16: 241–247.
McEvoy, A. N., B. Bresnihan, O. FitzGerald, and E. P. Murphy. 2004. Cyclooxygenase 2-derived prostaglandin E2 production by corticotropin-releasing hormone contributes to the activated cAMP response element binding
protein content in rheumatoid arthritis synovial tissue. Arthritis Rheum. 50:
1132–1145.
Cutolo, M., A. Bisso, A. Sulli, L. Felli, M. Briata, C. Pizzorni, and
B. Villaggio. 2000. Antiproliferative and antiinflammatory effects of methotrexate on cultured differentiating myeloid monocytic cells (THP-1) but not
on synovial macrophages from patients with rheumatoid arthritis. J. Rheumatol. 27: 2551–2557.
Youssef, P. P., M. Kraan, F. Breedveld, B. Bresnihan, N. Cassidy, G. Cunnane,
P. Emery, O. Fitzgerald, D. Kane, S. Lindblad, et al. 1998. Quantitative microscopic analysis of inflammation in rheumatoid arthritis synovial membrane samples selected at arthroscopy compared with samples obtained blindly by needle
biopsy. Arthritis Rheum. 41: 663– 669.
Youssef, P. P., T. J. Smeets, B. Bresnihan, G. Cunnane, O. Fitzgerald,
F. Breedveld, and P. P. Tak. 1998. Microscopic measurement of cellular infiltration in the rheumatoid arthritis synovial membrane: a comparison of semiquantitative and quantitative analysis. Br. J. Rheumatol. 37: 1003–1007.
Kane, D., J. Roth, M. Frosch, T. Vogl, B. Bresnihan, and O. FitzGerald. 2003.
Increased perivascular synovial membrane expression of myeloid-related proteins
in psoriatic arthritis. Arthritis Rheum. 48: 1676 –1685.
Balsa, A., C. Gamallo, E. Martin-Mola, and J. Gijon-Banos. 1993. Histologic
changes in rheumatoid synovitis induced by naproxen and methotrexate. J. Rheumatol. 20: 1472–1477.
McEvoy, A., B. Bresnihan, O. FitzGerald, and E. P. Murphy. 2002. Corticotropin-releasing hormone signaling in synovial tissue vascular endothelium is mediated through the cAMP/CREB pathway. Ann. NY Acad. Sci. 966: 119 –130.
Cutolo, M., A. Sulli, C. Craviotto, L. Felli, C. Pizzorni, B. Seriolo, and
B. Villaggio. 2002. Antiproliferative-antiinflammatory effects of methotrexate
and sex hormones on cultured differentiating myeloid monocytic cells (THP-1).
Ann. NY Acad. Sci. 966: 232–237.
Chan, E. S., and B. N. Cronstein. 2002. Molecular action of methotrexate in
inflammatory diseases. Arthritis Res. 4: 266 –273.
Gomez, G., and M. V. Sitkovsky. 2003. Targeting G protein-coupled A2a adenosine receptors to engineer inflammation in vivo. Int. J. Biochem. Cell Biol. 35:
410 – 414.
Ohta, A., and M. V. Sitkovsky. 2001. Role of G protein-coupled adenosine receptors in down-regulation of inflammation and protection from tissue damage.
Nature 414: 916 –920.
Shacter, E., and S. A. Weitzman. 2002. Chronic inflammation and cancer. Oncology 16: 217–226.
McPherson, J. A., K. G. Barringhaus, G. G. Bishop, J. M. Sanders, J. M. Rieger,
S. E. Hesselbacher, L. W. Gimple, E. R. Powers, T. Macdonald, G. Sullivan, et
al. 2001. Adenosine A(2A) receptor stimulation reduces inflammation and neointimal growth in a murine carotid ligation model. Arterioscler. Thromb. Vasc.
Biol. 21: 791–796.
Furst, D. E. 1997. The rational use of methotrexate in rheumatoid arthritis and
other rheumatic diseases. Br. J. Rheumatol. 36: 1196 –1204.
Ortiz, Z., B. Shea, M. Suarez Almazor, D. Moher, G. Wells, and P. Tugwell.
2000. Folic acid and folinic acid for reducing side effects in patients receiving
methotrexate for rheumatoid arthritis. Cochrane Database Syst. Rev.
CD000951.
Fiehn, C., E. Neumann, A. Wunder, S. Krienke, S. Gay, and U. Muller-Ladner.
2004. Methotrexate (MTX) and albumin coupled with MTX (MTX-HSA) suppress synovial fibroblast invasion and cartilage degradation in vivo. Ann. Rheum.
Dis. 63: 884 – 886.
Werb, Z., R. M. Hembry, G. Murphy, and J. Aggeler. 1986. Commitment to
expression of the metalloendopeptidases, collagenase, and stromelysin: relationship of inducing events to changes in cytoskeletal architecture. J. Biol. Chem.
102: 697–702.
Hasko, G., C. Szabo, Z. H. Nemeth, V. Kvetan, S. M. Pastores, and E. S. Vizi.
1996. Adenosine receptor agonists differentially regulate IL-10, TNF-␣, and
nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice.
J. Immunol. 157: 4634 – 4640.
Sajjadi, F. G., K. Takabayashi, A. C. Foster, R. C. Domingo, and G. S. Firestein.
1996. Inhibition of TNF-␣ expression by adenosine: role of A3 adenosine receptors. J. Immunol. 156: 3435–3442.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
26. Hazel, T. G., D. Nathans, and L. F. Lau. 1988. A gene inducible by serum growth
factors encodes a member of the steroid and thyroid hormone receptor superfamily. Proc. Natl. Acad. Sci. USA 85: 8444 – 8448.
27. Law, S. W., O. M. Conneely, F. J. DeMayo, and B. W. O’Malley. 1992. Identification of a new brain-specific transcription factor, NURR1. Mol. Endocrinol.
6: 2129 –2135.
28. Scearce, L. M., T. M. Laz, T. G. Hazel, L. F. Lau, and R. Taub. 1993. RNR-1, a
nuclear receptor in the NGFI-B/Nur77 family that is rapidly induced in degenerating liver. J. Biol. Chem. 286: 8855– 8861.
29. Mages, H. W., O. Rilke, R. Bravo, G. Senger, and R. A. Kroczek. 1994. NOT, a
human immediate-early response gene closely related to the steroid/thyroid hormone receptor NAK1/TR3. Mol. Endocrinol. 8: 1583–1591.
30. Tetradis, S., O. Bezouglaia, and A. Tsingotjidou. 2000. Parathyroid hormone
induces expression of the nuclear orphan receptor NURR1 in bone cells. Endocrinology 142: 663– 670.
31. Murphy, E. P., and O. M. Conneely. 1997. Neuroendocrine regulation of the
hypothalamic pituitary adrenal axis by the nurr1/nur77 subfamily of nuclear receptors. Mol. Endocrinol. 11: 39 – 47.
32. Murphy, E. P., A. McEvoy, O. M. Conneely, B. Bresnihan, and O. FitzGerald.
2001. Involvement of the nuclear orphan receptor NURR1 in the regulation of
corticotropin-releasing hormone expression and actions in human inflammatory
arthritis. Arthritis Rheum. 44: 782–793.
33. Wilson, T. E., T. J. Fahrner, M. Johnston, and J. Milbrandt. 1991. Identification
of the DNA binding site for NGFI-␤ by genetic selection in yeast. Science 252:
1296 –1300.
34. Ohkura, N., M. Hijikuro, A. Yamamoto, and K. Miki. 1994. Molecular cloning
of a novel thyroid/steroid receptor superfamily gene from cultured rat neuronal
cells. Biochem. Biophys. Res. Commun. 205: 1959 –1965.
35. Murphy, E. P., A. D. W. Dobson, C. H. Keller, and O. M. Conneely. 1996.
Differential regulation of transcription by the NURR1/NUR77 subfamily of nuclear transcription. Gene Expression 5: 169 –179.
36. Zetterstrom, R. H., L. Solomin, L. Jansson, B. J. Hoffer, L. Olson, and
T. Perlmann. 1997. Dopamine neuron agenesis in Nurr1-deficient mice. Science
276: 248 –250.
37. Wagner, J., P. Akerud, D. S. Castro, P. C. Holm, J. M. Canals, E. Y. Snyder,
T. Perlmann, and E. Arenas. 1999. Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes. Nat. Biotechnol. 17: 653– 659.
38. Woronicz, J. D., B. Calnan, V. Ngo, and A. Winoto. 1994. Requirement for the
orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas. Nature 367:
277–281.
39. McEvoy, A. N., E. A. Murphy, T. Ponnio, O. M. Conneely, B. Bresnihan,
O. FitzGerald, and E. P. Murphy. 2002. Activation of nuclear orphan receptor
NURR1 transcription by NF-␬B and cyclic adenosine 5⬘-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue. J. Immunol. 168: 2979 –2987.
40. Liu, D., H. Jia, D. I. Holmes, A. Stannard, and I. Zachary. 2003. Vascular endothelial growth factor-regulated gene expression in endothelial cells: KDR-mediated induction of Egr3 and the related nuclear receptors Nur77, Nurr1, and
Nor1. Arterioscler. Thromb. Vasc. Biol. 23: 2002–2007.
41. Le, W. D., P. Xu, J. Jankovic, H. Jiang, S. H. Appel, R. G. Smith, and
D. K. Vassilatis. 2003. Mutations in NR4A2 associated with familial Parkinson
disease. Nat. Genet. 33: 85– 89.
42. Brody, R. I., T. Ueda, A. Hamelin, S. C. Jhanwar, J. A. Bridge, J. H. Healey,
A. G. Huvos, W. L. Gerald, and M. Ladanyi. 1997. Molecular analysis of the
fusion of EWS to an orphan nuclear receptor gene in extraskeletal myxoid chondrosarcoma. Am. J. Pathol. 150: 1049 –1058.
43. Suzuki, S., N. Suzuki, C. Mirtsos, T. Horacek, E. Lye, S. K. Noh, A. Ho,
D. Bouchard, T. W. Mak, and W. C. Yeh. 2003. Nur77 as a survival factor in
tumor necrosis factor signaling. Proc. Natl. Acad. Sci. USA 100: 8276 – 8280.
44. Hong, C. Y., J. H. Park, R. S. Ahn, S. Y. Im, H. S. Choi, J. Soh, S. H. Mellon,
and K. Lee. 2004. Molecular mechanism of suppression of testicular steroidogenesis by proinflammatory cytokine tumor necrosis factor ␣. Mol. Cell. Biol. 24:
2593–2604.
45. Arkenbout, E. K., V. de Waard, M. van Bragt, T. A. van Achterberg,
J. M. Grimbergen, B. Pichon, H. Pannekoek, and C. J. de Vries. 2002. Protective
function of transcription factor TR3 orphan receptor in atherogenesis: decreased
lesion formation in carotid artery ligation model in TR3 transgenic mice. Circulation 106: 1530 –1535.
46. Martinez-Gonzalez, J., J. Rius, A. Castello, C. Cases-Langhoff, and L. Badimon.
2003. Neuron-derived orphan receptor-1 (NOR-1) modulates vascular smooth
muscle cell proliferation. Circ. Res. 92: 96 –103.
47. Kolluri, S. K., N. Bruey-Sedano, X. Cao, B. Lin, F. Lin, Y. H. Han,
M. I. Dawson, and X. K. Zhang. 2003. Mitogenic effect of orphan receptor TR3
and its regulation by MEKK1 in lung cancer cells. Mol. Cell. Biol. 23:
8651– 8667.
48. Choy, E. H., and G. S. Panayi. 2001. Cytokine pathways and joint inflammation
in rheumatoid arthritis. N. Engl. J. Med. 344: 907–916.
49. Tak, P., and B. Bresnihan. 2000. The pathogenesis and prevention of joint damage in rheumatoid arthritis. Arthritis Rheum. 43: 2619 –2633.
50. Koch, A. E. 1998. Review: angiogenesis: implications for rheumatoid arthritis.
Arthritis Rheum. 41: 951–962.
51. Middleton, J., L. Americh, R. Gayon, D. Julien, L. Aguilar, F. Amalric, and
J. P. Girard. 2004. Endothelial cell phenotypes in the rheumatoid synovium:
activated, angiogenic, apoptotic and leaky. Arthritis Res. Ther. 6: 60 –72.
52. Ben-Av, P., L. J. Crofford, R. L. Wilder, and T. Hla. 1995. Induction of vascular
endothelial growth factor expression in synovial fibroblasts by prostaglandin E
The Journal of Immunology
77. Link, A. A., T. Kino, J. A. Worth, J. L. McGuire, M. L. Crane, G. P. Chrousos,
R. L. Wilder, and I. J. Elenkov. 2000. Ligand-activation of the adenosine A2a
receptors inhibits IL-12 production by human monocytes. J. Immunol. 164:
436 – 442.
78. Cronstein, B. N. 1994. Adenosine, an endogenous anti-inflammatory agent.
J. Appl. Physiol. 76: 5–13.
79. Merrill, J. T., C. Shen, D. Schreibman, D. Coffey, O. Zakharenko, R. Fisher,
R. G. Lahita, J. Salmon, and B. N. Cronstein. 1997. Adenosine A1 receptor
promotion of multinucleated giant cell formation by human monocytes: a mechanism for methotrexate-induced nodulosis in rheumatoid arthritis. Arthritis
Rheum. 40: 1308 –1315.
80. Scandella, E., Y. Men, D. F. Legler, S. Gillessen, L. Prikler, B. Ludewig, and
M. Groettrup. 2004. CCL19/CCL21-triggered signal transduction and migration
of dendritic cells requires prostaglandin E2. Blood 103: 1595–1601.
565
81. Schrier, D. J., M. E. Lesch, C. D. Wright, and R. B. Gilbertsen. 1990. The
antiinflammatory effects of adenosine receptor agonists on the carrageenan-induced pleural inflammatory response in rats. J. Immunol. 145: 1874 –1879.
82. Lesch, M. E., M. A. Ferin, C. D. Wright, and D. J. Schrier. 1991. The effects of
(R)-N-(1-methyl-2-phenylethyl) adenosine (L-PIA), a standard A1-selective
adenosine agonist on rat acute models of inflammation and neutrophil function.
Agents Actions 34: 25–27.
83. Salvatore, C. A., S. L. Tilley, A. M. Latour, D. S. Fletcher, B. H. Koller, and
M. A. Jacobson. 2000. Disruption of the A(3) adenosine receptor gene in mice
and its effect on stimulated inflammatory cells. J. Biol. Chem. 275: 4429 – 4434.
84. Ordentlich, P., Y. Yan, S. Zhou, and R. A. Heyman. 2003. Identification of the
antineoplastic agent 6-mercaptopurine as an activator of the orphan nuclear hormone receptor Nurr1. J. Biol. Chem. 278: 24791–24799.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017