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Predominance of cyclooxygenase 1 over cyclooxygenase 2 in the generation of proinflammatory prostaglandins in autoantibody-driven KBxN serumtransfer arthritis.

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Vol. 58, No. 5, May 2008, pp 1354–1365
DOI 10.1002/art.23453
© 2008, American College of Rheumatology
Predominance of Cyclooxygenase 1 Over Cyclooxygenase 2 in
the Generation of Proinflammatory Prostaglandins in
Autoantibody-Driven K/BxN Serum–Transfer Arthritis
Mei Chen,1 Eric Boilard,1 Peter A. Nigrovic,1 Patsy Clark,2 Daigen Xu,2 Garret A. FitzGerald,3
Laurent P. Audoly,2 and David M. Lee1
COX-2ⴚ/ⴚ mice as well as isoform-specific inhibitors.
The relative importance of PGE2 and PGI2 (prostacyclin) was determined using mice deficient in microsomal
PGE synthase 1 (mPGES-1) and in the receptors for
Results. High levels of PGE2 and 6-keto-PGF1␣
(a stable metabolite of PGI2) were detected in arthritic
joint tissues, correlating strongly with the intensity of
synovitis. Pharmacologic inhibition of PG synthesis
prevented arthritis and ameliorated active disease.
While both COX isoforms were found in inflamed joint
tissues, only COX-1 contributed substantially to clinical
disease; COX-1ⴚ/ⴚ mice were fully resistant to disease,
whereas COX-2ⴚ/ⴚ mice remained susceptible. These
findings were confirmed by isoform-specific pharmacologic inhibition. Mice lacking mPGES-1 (and therefore
PGE2) developed arthritis normally, whereas mice incapable of responding to PGI2 exhibited a significantly
attenuated arthritis course, confirming a role of PGI2 in
this arthritis model.
Conclusion. These findings challenge previous
paradigms of distinct “housekeeping” versus inflammatory functions of the COX isoforms and highlight the
potential pathogenic contribution of prostanoids synthesized via COX-1, in particular PGI2, to inflammatory
Objective. Prostaglandins (PGs) are found in
high levels in the synovial fluid of patients with rheumatoid arthritis, and nonsteroidal blockade of these
bioactive lipids plays a role in patient care. The aim of
this study was to explore the relative contribution of
cyclooxygenase (COX) isoforms and PG species in the
autoantibody-driven K/BxN serum–transfer arthritis.
Methods. The prostanoid content of arthritic
ankles was assessed in ankle homogenates, and the
importance of this pathway was confirmed with pharmacologic blockade. The presence of COX isoforms
was assessed by Western blotting and their functional
contribution was compared using COX-1ⴚ/ⴚ and
Drs. Chen and Boilard’s work was supported by grants from
the Arthritis Foundation. Dr. FitzGerald’s work was supported by a
grant from the National Heart, Blood, and Lung Institute, NIH
(P01-HL-62250). Dr. Lee’s work was supported by grants from the
Arthritis Foundation, the NIH (P01-AI-065858-01), and the Cogan
Family Foundation.
Mei Chen, MD, PhD, Eric Boilard, PhD, Peter A. Nigrovic,
MD, David M. Lee, MD, PhD: Brigham and Women’s Hospital,
Harvard Medical School, Boston, Massachusetts; 2Patsy Clark, MSc,
Daigen Xu, PhD, Laurent P. Audoly, PhD: Merck Frosst Centre for
Therapeutic Research, Kirkland, Quebec, Canada; 3Garret A.
FitzGerald, MD: University of Pennsylvania School of Medicine,
Dr. FitzGerald has received honoraria or fees for consulting
or speaking (less than $10,000 each) from Merck, Novartis, Daiichi,
and NicOx. Dr. Audoly owns stock or stock options in Merck &
Company, Inc. Dr. Lee has received honoraria or fees for consulting or
speaking (less than $10,000 each) from Resolvyx, UCB Pharma, and
Religen; owns stock or stock options in Synovex; and has received
research support from MedImmune, Biogen, and Genentech.
Address correspondence and reprint requests to David M.
Lee, MD, PhD, Department of Medicine, Division of Rheumatology,
Immunology and Allergy, Brigham and Women’s Hospital, Harvard
Medical School, Boston, MA 02115. E-mail: dlee@rics.bwh.
Submitted for publication July 17, 2007; accepted in revised
form February 1, 2008.
Prostaglandins (PGs) are lipid mediators that, in
addition to their role in numerous physiologic activities,
contribute to the pathogenesis of pathologic inflammation. They are generated by conversion of arachidonic
acid by the cyclooxygenase (COX) enzymes to PGH2,
which is further catalyzed by distinct synthases to 5
major bioactive prostaglandins (PGE2, PGI2, PGF2␣,
PGD2, and thromboxane A2) (1). Two isoforms of COX,
designated COX-1 and COX-2, exist outside the brain
(2,3). Classically, COX-1 is constitutively expressed in
most tissues, whereas COX-2 is induced by a range of
mitogenic and inflammatory stimuli. These expression
patterns gave rise to the hypothesis that COX-1 provides
“housekeeping” synthetic activity, while prostaglandin
synthesis in inflammatory conditions is largely attributable to COX-2. However, in vivo studies have called into
question this paradigm of a clear division of labor
between COX-1 and COX-2, with synthetic contributions from COX-2 to healthy gastric and renal physiology and contributions from COX-1 to inflammatory
states (4–10). Thus, there is an increasing appreciation
that pathways of prostanoid generation in health and
disease are not readily described by simple paradigms.
In rheumatoid arthritis (RA) and other inflammatory joint diseases, high concentrations of prostaglandin species have been detected in synovial fluid (11,12).
To assess the potential pathogenic contribution of these
mediators, investigators have turned to mouse models of
arthritis, in particular, collagen-induced arthritis (CIA).
In this model, immunization with type II collagen in
Freund’s complete adjuvant elicits a chronic inflammatory arthritis. This model exhibits a significant reliance
on the COX-2 isoform for the development of anticollagen antibodies and clinical synovitis (13,14). Dissection
of the role of individual prostanoid species in mouse
CIA has established a requirement for PGE2, whose
synthesis is reliant on microsomal PGE synthase 1
(mPGES-1) (15), and for PGI2 (prostacyclin) via its
receptor (the IP receptor) (16). Further analysis of the
mechanisms of the PGE2 contribution has focused on
the function of receptors for PGE2, revealing a dual
contribution by the EP2 and EP4 receptors in CIA (16).
A variation of the mouse model of CIA, using pathogenic anticollagen antibodies and lipopolysaccharide
(LPS) (collagen antibody–induced arthritis [CAIA]), has
also demonstrated significant contributions from both
PGE2 (via its receptor EP4) and prostacyclin to disease
Given the dynamic regulation of COX isoform
expression, we elected to reevaluate the pathogenic role
of COX enzymes and downstream mediators in a model
system that requires neither adjuvant nor LPS. In the
K/BxN serum–transfer model of arthritis, administration
of serum from arthritic K/BxN mice induces inflammatory arthritis in most recipient strains, which is mediated
by IgG autoantibodies; coadministration of additional
agents is not required (19–22). In the present study, we
examined the contribution of prostaglandins to both the
induction and the perpetuation of arthritis in this model.
We found a significant elevation of prostanoids in the
joint that mirrored disease progression. Genetic and
pharmacologic studies demonstrated a prominent contribution of prostanoids to disease initiation and to
propagation of chronic inflammation. Interestingly, we
showed a particular requirement for the COX-1 isoform,
whereas the COX-2 isoform was apparently dispensable.
Finally, we found that disease progressed in the absence
of PGE2 and that there was a partial dependence on
PGI2 acting via the IP receptor. These findings provide
a further counterexample to the paradigm that inflammatory prostaglandin production is dependent on
COX-2 and underscore the rationale for continued
examination of prostanoid pathways in human arthritis.
Mice. We used mice ages 6–10 weeks for these studies.
C57BL/6 mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). Male COX-1⫺/⫺, COX-2⫺/⫺, and wild-type
(WT) mice (C57BL/6 ⫻ 129/Ola founders [7,23] intercrossed
for ⬎30 generations) were purchased from Taconic (Germantown, NY). Microsomal PGE synthase 1–null mice (N5 backcross onto the C57BL/6 background) (15), PGE2 receptor
EP1– and PGI receptor IP–null mice (N ⬎10 on the C57BL/6
background) were bred locally (24,25). K/BxN mice were
maintained as described elsewhere (20). All procedures were
approved by the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute (Boston, MA).
Drugs. The following drugs were used. Sulindac sulfone ([Z]-5-fluoro-2-methyl-1-[p-(methylsulfonyl)benzylidene]indene-3-acetic acid) was obtained from Sigma (St. Louis,
MO). SC-560 (5-[4-chlorophenyl]-1-[4-methoxyphenyl]-3[trifluoromethyl]-1H-pyrazole) was purchased from Cayman
Chemical (Ann Arbor, MI). MF-tricyclic (MFT; 3-[3,4-difluorophenyl]-4-[4-(methylsulfonyl)phenyl]-2-[5H]-furanone) was
provided by Merck Frosst Centre for Therapeutic Research
(Kirkland, Quebec, Canada) (26). These drugs were suspended
and diluted in 1% methylcellulose. Medications were administered orally via gavage once daily. The doses used, 10 mg/kg
for sulindac, 10 mg/kg for SC-560, and 3 mg/kg for MFT, were
chosen based on previously defined pharmacokinetic profiles
of these drugs in mice (26–29). A vehicle control (1% methylcellulose) was administered orally in the same volume and
frequency to control mice.
The biochemical activity of COX inhibitors was assessed in ex vivo analyses of whole blood using enzyme-linked
immunosorbent assay measurement of thromboxane B2 (a
surrogate for COX-1 activity) or LPS-stimulated PGE2 (for
COX-2 activity) production according to the manufacturer’s
instructions (Cayman Chemical) (30–34).
Serum transfer protocol and arthritis scoring. Arthritogenic K/BxN serum was transferred to recipient mice to
induce arthritis, as described previously (20). Briefly, 150 ␮l of
serum was administered intraperitoneally on experimental
days 0 and 2. Clinical indices were recorded at 24–48-hour
intervals. Ankle thickness was measured at the malleoli with
the ankle in a fully flexed position, using spring-loaded dial
calipers (Long Island Indicator Service, Hauppauge, NY). The
clinical index of arthritis was graded on a scale of 0–12 as
described previously (35).
Measurements of prostanoids in the inflamed joints by
liquid chromatography mass spectrometry (LC-MS). At selected times after injection with K/BxN serum, ankle tissues
were harvested, weighed, and frozen in liquid nitrogen (18).
Frozen joint tissues were pulverized using a mortar and pestle
to obtain a fine powder. This powder was homogenized
(Polytron PRO200 homogenizer; PRO Scientific, Oxford, CT)
at 4°C in phosphate buffered saline (PBS) supplemented with
10 ␮M indomethacin and 1⫻ Complete Protease Inhibitor
mixture (Roche Applied Science, Laval, Quebec, Canada).
The homogenates were subsequently sonicated on ice for 10–
30 seconds (Cole-Parmer Ultrasonic homogenizer, 50% output; Cole-Parmer, Montreal, Quebec, Canada) and centrifuged at 1,000g for 10 minutes at 4°C. Supernatants were isolated and stored at ⫺80°C until further analyses were performed.
The measurement of PGE2 and 6-keto-PGF1␣ (stable
breakdown product of PGI2) by LC-MS was performed as
described previously (36). Briefly, samples (100 ␮l) were
protein-precipitated by the addition of 150 ␮l of acetonitrile
containing 2 ng/ml of deuterated prostanoids that served as
internal standards for quantification. Samples were mixed
thoroughly by pipetting, centrifuged at 1,200g for 10 minutes
at 4°C, and supernatants were transferred to a new 96-well
plate for analysis by LC-MS. The detection limit for LC-MS is
0.002 ng/mg of protein for PGE2 and 6-keto-PGF1␣. The concentrations for PGE2 and 6-keto-PGF1␣ were normalized
relative to tissue wet weight.
Isolation of protein and Western blot analysis. Ankles
were harvested at the indicated times, placed in 1.5 ml of
ice-cold lysis buffer (10 mM Tris, pH 7.4, 140 mM NaCl, 10 mM
EDTA, 1% Triton X-100, 0.05% sodium dodecyl sulfate [SDS]
and freshly added 5 mM phenylmethylsulfonyl fluoride, as well
as a protease inhibitor cocktail [catalog no. P8340; Sigma]), cut
into small pieces with a scalpel, and homogenized using a
Brinkmann homogenizer (Brinkmann, Westbury, NY). Lysates
were subsequently clarified by centrifugation (9,500g for 10
minutes at 4°C), and the protein concentration in the lysates
was determined using Bradford reagent (Sigma). Typically,
lysates contained 2–7 mg/ml of protein. Proteins (80 ␮g) boiled
in Laemmli sample buffer (37) were separated on 10% SDS–
polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 30 minutes
at room temperature with 5% milk proteins in Tris buffered
saline–Tween (TBST; 190 mM NaCl, 0.05% Tween 20, 25 mM
Tris, pH 7.6), washed in TBST, and incubated with anti–
COX-1 (1:400 dilution), anti–COX-2 (1:400 dilution) (both
from Cayman Chemical), or anti-GAPDH (1:5,000 dilution)
(Abcam, Cambridge, MA) for 18 hours at 4°C.
Membranes were washed 6 times for 5 minutes each in
TBST and incubated for 1 hour with horseradish peroxidase–
conjugated donkey anti-rabbit or donkey anti-mouse IgG
(1:15,000 dilution; Jackson ImmunoResearch, West Grove,
PA). After 6 washes with TBST, the membranes were developed using Western Lightning chemiluminescence reagent
(PerkinElmer, Boston, MA). For protein band densitometry, a
MultiImage Light Cabinet (Alpha Innotech, San Leandro,
CA) was used to capture images, and spot densitometry was
performed using ChemiImager 4400 software (Alpha Innotech).
Histologic examination. For histomorphometric analysis, ankle tissues were fixed for 24 hours in 4% paraformaldehyde in PBS and decalcified for 48–72 hours with modified
Kristensen’s solution (38). Tissues were then dehydrated,
embedded in paraffin, sectioned at 5 ␮m thickness and stained
with hematoxylin and eosin. Histologic scoring of inflammation, cartilage erosion, and bone erosion was performed as
described previously (39,40). Briefly, inflammation was scored
on a scale of 0–5, where 0 ⫽ normal, 1 ⫽ minimal infiltration
of inflammatory cells, 2 ⫽ mild infiltration, 3 ⫽ moderate
infiltration, 4 ⫽ marked infiltration, and 5 ⫽ severe infiltration. Bone erosion was scored on a scale of 0–5, where 0 ⫽
normal, 1 ⫽ small areas of resorption, 2 ⫽ more numerous
areas of resorption, 3 ⫽ obvious resorption of trabecular and
cortical bone, 4 ⫽ full-thickness defects in the cortical bone
and marked trabecular bone loss, and 5 ⫽ full-thickness
defects in the cortical bone and marked trabecular bone loss,
with distortion of the profile of the remaining cortical surface.
Cartilage erosion was scored on a scale of 0–5, where 0 ⫽ no
cartilage injury, 1 ⫽ synovial adherence to margins of cartilage
in fewer than 3 sites, 2 ⫽ synovial adherence to margins of
cartilage in 3 or more sites, 3 ⫽ synovial adherence to cartilage
not limited to margins, but no full-thickness injury (damage
does not extend beyond the tidemark), 4 ⫽ full-thickness
injury in fewer than 3 sites, and 5 ⫽ full-thickness injury in 3 or
more sites.
Statistical analysis. Results are presented as the
mean ⫾ SEM. The statistical significance for comparisons
between groups was determined using Student’s unpaired
2-tailed t-test or two-way analysis of variance, followed by
Bonferroni correction using the Prism software package (version 4.00; GraphPad Software, San Diego, CA). P values less
than 0.05 were considered significant.
PGs in joint inflammation induced by K/BxN
serum transfer. To understand the potential contribution of prostanoids to K/BxN arthritis, we started by
assaying specific PGs in arthritic, chronically inflamed
joint tissue from K/BxN mice. As shown in Figure 1, high
levels of PGE2 and the PGI2 metabolite 6-keto-PGF1␣
were evident in joint tissues from K/BxN mice with
chronic arthritis. We then proceeded to examine the
kinetics of prostanoid elevations after arthritis induction via K/BxN serum transfer. There was a close
temporal association between the development of joint
inflammation (Figure 1C) and the elevation of tissue
Figure 1. Prostaglandin levels in joint tissues from mice with K/BxN serum–transfer arthritis.
Concentrations of A, prostaglandin E2 (PGE2) and B, 6-keto-PGF1␣ in chronically inflamed joint
tissues from 10-week-old K/BxN mice or in joint tissues from wild-type (WT) mice were measured
by liquid chromatography mass spectrometry on days 0, 4, and 8 after administration of
arthritogenic K/BxN serum (n ⫽ 13 mice per group and per time point). Differences in PGE2 levels
were statistically significant at P ⬍ 0.01 for WT mice on day 8 versus day 0 and for K/BxN mice
versus WT mice on day 0; differences in 6-keto-PGF1␣ levels were statistically significant at P ⬍
0.001 for WT mice on day 4 and on day 8 versus day 0 and for K/BxN mice versus WT mice on day
0. C, Clinical index of arthritis after administration of arthritogenic K/BxN serum to WT mice (n ⫽
13 per time point). Differences were statistically significant at P ⬍ 0.001 on day 4 and on day 8
versus day 0. Values are the mean and SEM of pooled data from 3 individual experiments. P values
were determined by Student’s unpaired 2-tailed t-test.
PGE2 (Figure 1A) and 6-keto-PGF1␣ (Figure 1B) levels.
Thus, the kinetics of prostanoid generation are consistent with the participation of these molecules in joint
Amelioration of K/BxN serum–transfer arthritis
by pharmacologic inhibition of prostaglandin synthesis.
Having demonstrated the presence of prostanoids concurrent with joint inflammation, we next examined the
functional contribution of this pathway to disease induction and perpetuation. To this end, we administered oral doses of sulindac, a potent inhibitor of both
COX isoforms (27,41), or vehicle control to WT mice
and induced arthritis via passive transfer of K/BxN
As shown in Figure 2, initiation of sulindac prior
to arthritis induction substantially prevented joint inflammation in this model (Figure 2A). Furthermore,
administration of sulindac to mice with established
joint inflammation rapidly reduced clinical signs of
arthritis (Figure 2B). These clinical changes corresponded to a clear decrease in inflammation as well as
joint injury, as assessed by histologic scoring (Figures 2C
and D). As anticipated, PGE2 (Figure 2E) and 6keto-PGF1␣ (Figure 2F) production was inhibited in
joint tissues from the mice administered sulindac. Thus,
prostanoids contribute to both the initiation and the
perpetuation of joint inflammation in this model.
Role of COX-1, but not COX-2, as an obligate
participant in arthritis. Western blot analysis of joint
lysates demonstrated increased levels of COX-1 and
COX-2 after disease induction (by day 4); these increased levels were maintained during the course of the
disease (Figures 3A and B). The specificity of the
COX-1 staining was confirmed by the disappearance of
the 70-kd COX-1 band in immunoblots of ankle lysates
prepared from COX-1⫺/⫺ mice and following preincubation of the primary antibody with a COX-1–blocking
peptide (murine amino acids 274–288; Cayman Chemical) (data not shown).
We next used a genetic approach to examine the
functional contribution of the COX isoforms. We found
that COX-1⫺/⫺ mice were remarkably resistant to the
development of K/BxN serum–induced inflammatory
arthritis, whereas COX-2⫺/⫺ mice showed no diminu-
Figure 2. Prevention and treatment of K/BxN serum–induced arthritis by inhibition of cyclooxygenase.
A and B, C57BL/6J mice (n ⫽ 15 per group) were given oral doses of sulindac or vehicle control (1%
methylcellulose) beginning on day –2 before administration of arthritogenic K/BxN serum (A) or day 6
after administration of arthritogenic K/BxN serum (B). Differences were statistically significant at P ⬍
0.001 for pretreated mice versus vehicle-treated controls and at P ⬍ 0.01 for treated mice versus
vehicle-treated controls. C and D, Histomorphometric quantification of inflammation, bone erosion,
and cartilage erosion was performed on day 14 after K/BxN serum transfer in pretreated mice (C) and
treated mice (D). Differences were statistically significant at P ⬍ 0.001 for pretreated mice and treated
mice versus vehicle-treated controls. E and F, Levels of prostaglandin E2 (PGE2) (E) and 6-keto-PGF1␣
(F) were measured in joint tissues from pretreated and treated mice. Differences in PGE2 and
6-keto-PGF1␣ levels were statistically significant at P ⬍ 0.001 for pretreated mice and for treated mice
versus vehicle-treated controls. Values are the mean ⫾ SEM of pooled data from 3 experiments. P
values in A and B were determined by two-way analysis of variance; those in C–F were determined by
Student’s unpaired 2-tailed t-test.
tion in disease activity (Figures 3C and D). We measured tissue levels of PGE2 (Figure 3E) and 6-ketoPGF1␣ (Figure 3F) to confirm that COX-1⫺/⫺ mice
(with intact COX-2) lacked elevations in tissue prostanoids, and we found levels consistent with those in
healthy joint tissues.
Histomorphometric examination of inflammation, bone erosion, and cartilage erosion in joint tissues from these mice confirmed the clinical findings
(Figures 3G–L). Whereas WT and COX-2⫺/⫺ mice
demonstrated synovial hyperplasia, leukocytic infiltration, and the presence of synovial erosion into bone
and cartilage (Figures 3G, J, and K), COX-1⫺/⫺ joint
tissues retained a normal appearance, with little evi-
dence of these inflammatory changes (Figure 3H).
These data implicate COX-1 in the development of
arthritis after K/BxN serum transfer, whereas COX-2
appears to be dispensable.
Effects of COX-1 and COX-2 inhibitors on
K/BxN serum–induced arthritis. Given the divergence
of our findings with those documented in other arthritis
models, we used COX isoform–specific pharmacologic
inhibition to confirm the substantial COX-1 contribution to arthritis induction as well as to examine the role
of these isoforms in the perpetuation of K/BxN serum–
transfer arthritis. Indeed, we found that administration
of SC-560, a highly selective oral inhibitor of COX-1
(28), both prevented the development of disease when
Figure 3. Expression and function of cyclooxygenase (COX) isoforms in K/BxN serum–transfer
arthritis. A and B, Levels of COX-1 and COX-2 protein in mouse ankle joints. C57BL/6J mice (n ⫽
5 mice per time point) were administered arthritogenic K/BxN serum, and ankle tissues were
harvested at the indicated times. Western blots of COX-1, COX-2, and GAPDH protein in ankle
lysates (A) (representative of 5 independent experiments) and densitometric quantification of the
ratio of each COX protein to GAPDH (B) were performed. Expression levels were normalized
relative to day 0, which was arbitrarily assigned a ratio of 1. Levels of both COX isoforms increased
after K/BxN serum transfer. C and D, Clinical index of arthritis in COX-1⫺/⫺ (C) and COX-2⫺/⫺
(D) mice and their wild-type (WT) controls (n ⫽ 10–12 mice per group). Differences were
significant at P ⬍ 0.001 for COX-1⫺/⫺ mice versus WT mice. E and F, Production of prostaglandin
E2 (PGE2) (E) and 6-keto-PGF1␣ (F) in joint tissues from COX-1⫺/⫺ mice 2 weeks after K/BxN
serum transfer. Differences in PGE2 and 6-keto-PGF1␣ levels were statistically significant at P ⬍
0.001 for COX-1⫺/⫺ mice versus WT mice. G, H, J, and K, Histologic features of arthritis on day
14 after K/BxN serum transfer in COX-1 WT (G), COX-1⫺/⫺ (H), COX-2 WT (J), and COX-2⫺/⫺
(K) mice. Ca ⫽ cartilage; Bn ⫽ Bone; S ⫽ synovium. Bar ⫽ 100 ␮m. I and L, Histomorphometric
quantification of arthritis on day 14 after K/BxN serum transfer in COX-1⫺/⫺ (I) and COX-2⫺/⫺
(L) mice and their WT controls. Differences were significant at P ⬍ 0.001 for COX-1⫺/⫺ mice
versus WT mice. Values in B–F, I, and L are the mean ⫾ SEM of pooled data from 3 experiments.
P values in C and D were determined by two-way analysis of variance; those in E, F, I, and L were
determined by Student’s unpaired 2-tailed t-test.
Figure 4. Effects of selective pharmacologic inhibition of cyclooxygenase (COX) on K/BxN
serum–induced arthritis. A–D, C57BL/6J mice (n ⫽ 15 per group) were given oral doses of the
COX-1 inhibitor SC-560 (10 mg/kg) (A and B) or the COX-2 inhibitor MF-tricyclic (MFT; 3 mg/kg)
(C and D) beginning on day –2 before administration of arthritogenic K/BxN serum (A and C) or
day 6 after administration of arthritogenic K/BxN serum (B and D), and the clinical index of
arthritis was monitored. Differences were statistically significant at P ⬍ 0.001 for SC-560–
pretreated mice versus vehicle-treated controls and at P ⬍ 0.01 for SC-560–treated mice versus
vehicle-treated controls. Values are the mean ⫾ SEM. E and F, Ex vivo effect of a single 10-mg/kg
oral dose of SC-560 or a single 3-mg/kg oral dose of MFT on COX-1 (E) and COX-2 (F) activity
in whole blood, as determined by measuring serum levels of thromboxane B2 (TXB2) (E) in
coagulated blood and plasma levels of prostaglandin E2 (PGE2) (F) in lipopolysaccharide-treated
blood from C57BL/6J mice (n ⫽ 10 per group) at 2 hours after oral dosing. Values are the mean
and SEM of pooled data from 2 experiments. Differences in TXB2 levels were statistically
significant at P ⬍ 0.001 for SC-560–treated mice versus vehicle-treated controls; differences in
PGE2 levels were statistically significant at P ⬍ 0.001 for MFT-treated mice versus vehicle-treated
controls. P values in A–D were determined by two-way analysis of variance; those in E and F were
determined by Student’s unpaired 2-tailed t-test.
administered prior to arthritogenic serum and rapidly
diminished the clinical signs of arthritis when used to
treat established disease (Figures 4A and B). In contrast,
administration of the selective COX-2 inhibitor MFT
(26,42) resulted in no discernible decrease in arthritis
activity when used either in the prevention or the
treatment of disease at doses that fully inhibited LPSinduced PGE2 in peripheral blood leukocytes (Figures
4C, D, and F).
Substantial contribution of PGI2, but not PGE2,
to K/BxN serum–induced arthritis. Since our studies
demonstrated increasing levels of PGE2 and 6-ketoPGF1␣ in arthritic joint tissues and the lack of PGE2 and
6-keto-PGF1␣ production in COX-1⫺/⫺ mice, we explored the contribution of the prostanoid species PGE2
and PGI2 in K/BxN serum–induced arthritis. We used
mPGES-1–deficient mice (15) to assess the involvement of PGE2. The mPGES-1⫺/⫺ mice developed arthri-
Figure 5. Dispensability of prostaglandin E2 (PGE2) in K/BxN serum–induced arthritis. A, Clinical
index of arthritis in mPGES-1⫺/⫺ and wild-type (WT) control mice after administration of
arthritogenic K/BxN serum (n ⫽ 15 mice per group). B and C, Concentration of PGE2 (B) and
6-keto-PGF1␣ (C) in arthritic joint tissues from mPGES-1⫺/⫺ and WT control mice (n ⫽ 10 per
group). Differences in PGE2 levels were significant at P ⬍ 0.001 and differences in 6-keto-PGF1␣
levels were significant at P ⬍ 0.05 for mPGES-1⫺/⫺ mice versus WT mice. D and E, Histologic
features of ankle tissues from WT (D) and mPGES-1⫺/⫺ (E) mice on day 14 after K/BxN serum
transfer. Ca ⫽ cartilage; Bn ⫽ bone; S ⫽ synovium. Bar ⫽ 100 ␮m. F, Histomorphometric
quantification of arthritis on day 14 after K/BxN serum transfer in mPGES-1⫺/⫺ and WT control
mice. Values in A–C and F are the mean ⫾ SEM of pooled data from 3 experiments. P values were
determined by Student’s unpaired 2-tailed t-test.
tis that was clinically and histologically indistinguishable
from that in WT controls (Figures 5A, D, E, and F).
Since previous studies demonstrated the presence of at
least 3 distinct PGE2 synthases (43,44), we quantified
joint tissue levels of PGE2 and 6-keto-PGF1␣ in
mPGES-1⫺/⫺ mice and confirmed a lack of significant
PGE2 production, while the 6-keto-PGF1␣ levels were
intact (Figures 5B and C). Thus, while mPGES-1 remains the primary source of synovial PGE2 production
in our experimental system, PGE2 itself appears to be
dispensable for the initiation and perpetuation of arthritis in the K/BxN model.
Having found no discernible contribution from
PGE2, we examined the role of PGI2 by using mice
deficient in the PGI2 receptor IP. In these experiments,
we found a significant, albeit partial, decrease in clinical
arthritis in IP⫺/⫺ mice, to 31% of the level in WT mice
(Figure 6A). Histomorphometric analyses again confirmed the clinical findings, with reductions in mean
scores for inflammation, bone erosion, and cartilage
erosion of 59%, 61%, and 77%, respectively, in IP⫺/⫺
mice (Figure 6B). Since high concentrations of PGI2
may activate the EP1 receptor (45), we assessed a
potential in vivo contribution from PGI2 via EP1 by
examining arthritis in EP1-deficient mice. As shown in
Figures 6C and D, we find no amelioration of clinical or
histologic arthritis activity in this strain. These data
indicate that a substantial proportion of the prostanoid
contribution to joint inflammation in K/BxN serum–
transfer arthritis can be accounted for by an interaction
of PGI2 and its receptor IP.
Our studies revealed a striking contribution of
prostaglandins to autoantibody-driven joint inflammation in the K/BxN serum–transfer model. Perhaps most
surprising was the apparent reliance on the COX-1
Figure 6. Contribution of prostaglandin I2 (PGI2) to K/BxN serum–induced arthritis. A, Clinical
index of arthritis in IP⫺/⫺ and wild-type (WT) mice after administration of arthritogenic K/BxN
serum. B, Histomorphometric quantification of arthritis on day 14 after K/BxN serum transfer in
IP⫺/⫺ and WT mice (n ⫽ 15 per group). Differences were significant at P ⬍ 0.001 for IP⫺/⫺ mice
versus WT mice. C, Clinical index of arthritis in EP1⫺/⫺ and WT mice after establishment of
arthritis. D, Histomorphometric quantification of arthritis on day 14 after K/BxN serum transfer in
EP1⫺/⫺ and WT mice (n ⫽ 12–15 per group). Values are the mean ⫾ SEM of pooled data from
3 experiments. P values in A and C were determined by two-way analysis of variance; those in B and
D were determined by Student’s unpaired 2-tailed t-test.
isoform and the dispensability of COX-2 for both the
initiation and the perpetuation of arthritis. This conclusion is based on our findings from experiments in
genetically deficient animals as well as through isoformspecific pharmacologic inhibition in normal mice. While
our results do not exclude a contribution from COX-2,
we showed that COX-1 is an essential and sufficient
source of arthritogenic prostaglandins in both normal
and inflamed murine joints. Our findings thus provide a
counterpoint to the paradigm that COX-1 contributes
only to “housekeeping” prostaglandin synthesis, while
prostaglandins generated under inflammatory conditions reflect the activity of the highly inducible COX-2
Furthermore, we found that a significant proportion of the arthritogenic activity of prostaglandins in
K/BxN serum–transfer arthritis may be attributed to the
action of PGI2 (prostacyclin) via its receptor, the IP
receptor. Potent proinflammatory activities of prostacyclin have only recently been reported in other models of
arthritis (16,18), and its role has been explored in a
limited number of other disease states (46). The demonstrated functional contribution in K/BxN arthritis
provides added rationale for further examination of the
predominant synthetic sources of PGI2 as well as the
proinflammatory effector functions elicited in target
cells in the joint. Furthermore, our observations raise
the possibility that suppression of COX-1–derived PGI2
may contribute to antiinflammatory efficacy independently of COX-2–derived PGI2, whose suppression has
been associated with cardiovascular hazard in human
trials (47,48). Indeed, sparing COX-2–dependent prostacyclin production may confer cardiovascular protection to arthritis patients, a population with demonstrated
increased cardiovascular risk.
These results add yet another pathway to a
surprisingly large number of effector mechanisms required for the full expression of arthritis in the K/BxN
serum–transfer model. Thus far, lack of intact Fc receptor signaling (Fc␥RIII), complement anaphylatoxins (via
C5a receptor [CD88]), neutrophils, mast cells, natural
killer T cells, and the mediators interleukin-1 (IL-1; via
IL-1 receptor type I), leukotriene B4 (LTB4), and prostaglandins all confer dramatic resistance to arthritis
mediated by passive transfer of arthritogenic autoantibodies (35,39,49–54). A major unresolved question that
arises from these observations is whether these pathways
operate sequentially or in parallel in a codominant
manner. Also unclear are the interactions between these
pathways at the level of cellular effector responses.
Further insight into these regulatory events will clarify
whether autoantibody-driven arthritis proceeds as a linear series of events or whether a network of parallel
inflammatory pathways conspire to propagate disease.
In either case, further understanding will inform our
views regarding pathogenic processes in human inflammatory arthritis.
The dispensability of PGE2 and its synthetic
enzyme in K/BxN arthritis was unexpected, given the
substantial levels of this species in arthritic joint tissues
and the previous studies documenting PGE2-stimulated
effector functions on cellular populations represented
within the arthritic joint (55–57). As with COX isoform
dependence (13,14), these results differ from those of
studies using the CIA or CAIA models (15,17,18) and
represent a rare divergence in effector mechanisms
between these arthritis models (58). Anti-GPI and anticollagen models share a common requirement for cytokines (IL-1 and tumor necrosis factor), IgG Fc receptors,
complement, and eicosanoids. The eicosanoid requirement in both models includes intact cytosolic phospholipase A2 (59,60), the capacity to synthesize LTB4
(49,50), and an important, albeit partial, reliance on
PGI2 (61,62).
While the mechanistic basis for this divergence
remains undefined, 2 possibilities deserve mention. The
first is the potential contribution of LPS or adjuvant
used in CIA and CAIA. These agents are known to
stimulate the production of prostaglandins and induce
the expression of COX-2 (15,63–65). Thus, it is possible
that these stimuli contribute to the dominance of COX-2
in those experimental systems. Indeed, we find that the
administration of LPS enables the initiation of K/BxN
arthritis in COX-1⫺/⫺ animals, though whether this
effect operates via COX-2 is unknown (Chen M, Lee D:
unpublished observations).
A second consideration is the impact of COX
isoform deficiency on T cell and B cell function. Modulation of adaptive immune responses by prostanoids has
been demonstrated in multiple experimental systems
(66–68), and COX-2–null mice with CIA had markedly
decreased levels of anticollagen antibodies (14). Thus,
whether COX-2 interruption impedes the development
of a lymphocyte-dependent anticollagen response, interferes with synovial inflammatory networks, or both,
remains unclarified. K/BxN serum transfer proceeds in
the absence of lymphocytes (20), thereby affording a
focus on effector-phase arthritis mechanisms that should
not be impacted by a potential COX-2 contribution to
lymphocyte function. These issues notwithstanding, the
findings in these arthritis models demonstrate that inflammatory arthritis can proceed via disparate pathways
of prostaglandin synthesis and that multiple prostanoid
species may contribute to the final common pathway of
Finally, our findings provide a rationale to reexamine the contribution of COX-1 and specific downstream prostaglandins to inflammatory arthritis in humans. While nonsteroidal antiinflammatory drugs
(NSAIDs) have been used frequently for amelioration of
symptoms, these agents have not been demonstrated to
have disease-modifying activity. However, the extent to
which prostaglandin synthesis within the RA joint is
blocked by these agents has never, to our knowledge,
been studied. Therefore, the magnitude of the pathogenic contribution of prostaglandins to human RA remains an open question. This consideration is particularly relevant for the COX-2–selective NSAIDs, where
clinical development was focused on gastrointestinal
safety and equivalence of pain relief. Indeed, both COX
isozymes are expressed in the rheumatoid synovium
(69,70). Our data suggest that COX-2–selective agents,
even if tolerated at higher dosages because of reduced
gastrointestinal toxicity, could miss important prostanoid pathways in the inflamed joint. The recent demonstration of cardiovascular toxicity from treatment with
COX-2–selective NSAIDs has introduced a further limitation on the use of these drugs (71). Thus, the identification of predominant roles for COX-1 and discrete
prostaglandin species by our group and others suggests
that antagonism of specific prostaglandin species may be
an effective therapeutic strategy if it can be achieved
without the limiting toxicity that has thus far plagued the
use of such inhibitors clinically.
We are grateful to Dr. B. H. Koller (University of
North Carolina, Chapel Hill, NC) for providing the mPGES1–null mice and the EP1-deficient mice (derived and maintained with support from NIH grant HL-068141 to Dr. Koller).
We also acknowledge the expert histotechnical assistance of
Teresa Bowman.
Dr. Lee 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
Study design. Chen, Audoly, Lee.
Acquisition of data. Chen, Boilard, Clark, Xu, Lee.
Analysis and interpretation of data. Chen, Boilard, Nigrovic, Lee.
Manuscript preparation. Chen, Boilard, Nigrovic, FitzGerald, Audoly,
Statistical analysis. Chen, Lee.
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