ORIGINAL ARTICLE

Hypoxia-induced endogenous prostaglandin E2 negativelyregulates hypoxia-enhanced aberrant overgrowth of rheumatoidsynovial tissue

Hirofumi Mitomi • Hidehiro Yamada • Hiroshi Ito • Toshiko Nozaki Shibata •

Yoshioki Yamasaki • So Nomoto • Atsushi Kusaba • Hiroki Yamashita •

Shoichi Ozaki

Received: 3 August 2012 / Accepted: 26 October 2012 / Published online: 25 November 2012

� Japan College of Rheumatology 2012

Abstract

Objective During isometric exercise, the synovial joint

tissue is prone to hypoxia, which is further enhanced in the

presence of synovial inflammation. Hypoxia is also known to

induce inflammatory cascades, suggesting that periodic

hypoxia perpetuates synovitis in rheumatoid arthritis. We

previously established an ex vivo cellular model of rheu-

matoid arthritis using the synovial tissue-derived inflam-

matory cells, which reproduced aberrant synovial

overgrowth and pannus-like tissue development in vitro.

Using this model, we investigated the regulatory mechanism

of synovial cells against hypoxia in rheumatoid arthritis.

Methods Inflammatory cells that infiltrated synovial tis-

sue from patients with rheumatoid arthritis were collected

without enzyme digestion, and designated as synovial tis-

sue-derived inflammatory cells. Under normoxia or peri-

odic hypoxia twice a week, their single-cell suspension was

cultured in medium alone to observe an aberrant over-

growth of inflammatory tissue in vitro. Cytokines produced

in the culture supernatants were measured by enzyme-

linked immunosorbent assay kits.

Results Primary culture of the synovial tissue-derived

inflammatory cells under periodic hypoxia resulted in the

attenuation of the spontaneous growth of inflammatory tis-

sue in vitro compared to the culture under normoxia.

Endogenous prostaglandin E2 (PGE2) production was

enhanced under periodic hypoxia. When endogenous PGE2

was blocked by indomethacin, the aberrant tissue over-

growth was more enhanced under hypoxia than normoxia.

Indomethacin also enhanced the production of tumor

necrosis factor-a (TNF-a), macrophage colony-stimulating

factor (M-CSF), and matrix metalloproteinase-9 (MMP-9)

under periodic hypoxia compared to normoxia. The EP4-

specific antagonist reproduced the effect of indomethacin.

Exogenous PGE1 and EP4-specific agonist effectively

inhibited the aberrant overgrowth and the production of the

inflammatory mediators under periodic hypoxia as well as

normoxia.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10165-012-0794-7) contains supplementarymaterial, which is available to authorized users.

H. Mitomi � H. Yamada (&) � H. Ito � T. N. Shibata �Y. Yamasaki � S. Ozaki

Division of Rheumatology and Allergology, Department of

Internal Medicine, St. Marianna University School of Medicine,

2-16-1 Sugao, Miyamae-ku, Kawasaki 216-8511, Japan

e-mail: guriko@marianna-u.ac.jp

H. Mitomi

e-mail: mitomihirofumi@marianna-u.ac.jp

H. Ito

e-mail: h4ito@marianna-u.ac.jp

T. N. Shibata

e-mail: no-yes@marianna-u.ac.jp

Y. Yamasaki

e-mail: yams@marianna-u.ac.jp

S. Ozaki

e-mail: ozk@marianna-u.ac.jp

S. Nomoto

Department of Orthopedic Surgery and Rheumatology,

Saiseikai Yokohamashi Tobu Hospital, 3-6-1, Shimosueyoshi,

Tsurumi-ku, Yokohama 230-8765, Japan

e-mail: so.nomoto@nifty.com

A. Kusaba � H. Yamashita

Institute of Rheumatology, Ebina General Hospital, 1320,

Kawaraguchi, Ebina 243-0433, Japan

e-mail: weardebris@aol.com

H. Yamashita

e-mail: izy00277@nifty.com

123

Mod Rheumatol (2013) 23:1069–1075

DOI 10.1007/s10165-012-0794-7

Conclusions The enhancing effect of periodic hypoxia on

the aberrant overgrowth of rheumatoid synovial tissue was

effectively down-regulated by the simultaneously induced

endogenous PGE2.

Keywords Rheumatoid arthritis � Hypoxia � Synovial

overgrowth � PGE2 � NSAIDs

List of Abbreviations

RA Rheumatoid arthritis

IL Interleukin

PG Prostaglandin

TNF Tumor necrosis factor

M-CSF Macrophage colony-stimulating factor

MIP Macrophage inflammatory protein

VEGF Vascularendothelial growth factor

HIF-1a Hypoxia-inducible factor-1aMMP Matrix metalloproteinase

IFN Interferon

COX Cyclooxygenase

normo Normoxia

hypo Periodic hypoxia

Introduction

Inflammation in general is fundamentally a protective

response against cellular and tissue injury caused by diverse

pathological stimuli, and it is closely intertwined with the

process of repair. In some circumstances, inflammation and

tissue repair are not successfully completed and inflamma-

tion perpetuates chronically. Rheumatoid arthritis is char-

acterized by chronic inflammation of the synovial

membrane. A critical event during the chronic inflammation

is the transformation of the synovial membrane into an

aggressive granulation tissue (so-called pannus), resulting in

cartilage and bone destruction [1, 2]. The joint synovial

tissue is prone to hypoxia during exercise as well as ische-

mia–reperfusion injury, which is further enhanced in the

presence of synovial inflammation [3]. It is reported that

inflammatory effusions had lower oxygen tensions than non-

inflammatory effusions and the more severe histological

grading of synovial changes correlated with lower oxygen

tension [3]. It is also reported that low tissue oxygen pressure

levels in the inflamed synovial joint tissue was significantly

associated with the degree of synovitis and the level of TNF-

a, interleukin (IL)-1b, interferon (IFN)-c and the chemokine

macrophage inflammatory protein (MIP) 3a [4].

Cytokine networks and cell–cell interaction, as well as

other inflammatory mediators, such as prostanoids, con-

tribute to the development of pannus tissue. This complex

system of rheumatoid synovitis includes both positive and

negative feedback regulation of the inflammatory response.

Therefore, a human cell model that represents this complex

will be useful to study the role of hypoxia in the patho-

genesis of rheumatoid arthritis. We previously established

an ex vivo cellular model using the synovial tissue-derived

inflammatory cells, which reproduced pannus-like tissue

growth in vitro [5]. Using this model, we demonstrated that

periodic hypoxia enhanced aberrant synovial overgrowth,

which was negatively regulated by the simultaneously

induced PGE2 in the hypoxic condition.

Materials and methods

Reagents

PGE1 was purchased from Sigma-Aldrich (St. Louis, MO).

Indomethacin was from Wako (Osaka, Japan). The EP4-

specific agonist (ONO-AE1-329 and ONO-AE1-437) and

EP4-specific antagonist (ONO-AE3-208) were provided

from Ono Pharmaceuticals co (Osaka, Japan). In mem-

branes of Chinese hamster ovary cells expressing the

respective mouse EP receptors, the above EP agonist had

the following Ki values in 3H-PGE2 binding assays: ONO-

AE1-329, 0.0097 lM at the EP4 receptor; and ONO-AE1-

437, 0.0007 lM at the EP4 receptor [6, 7, 8]. The 50 %

maximum response concentration values were as follows:

ONO-AE3-329, 0.0031 lM at the EP4 receptor ([10 lM at

the EP1 receptor, 1.2 lM at the EP2 receptor, and 5.8 lM at

the EP3 receptor); ONO-AE1-437, 0.0007 lM at the EP4

receptor ([10 lM at the EP1 receptor, 0.62 lM at the EP2

receptor, and 0.056 lM at the EP3 receptor). The Ki values

of EP4 receptor antagonist ONO-AE3-208 were 0.0013 lM

for the EP4 receptor, 0.03 lM for the EP3 receptor, and

[10 lM for the mouse EP1, EP2, DP receptors [9], and it

antagonized both an increase of intracellular cAMP induced

by 100 nM PGE2 with a 50 % inhibition concentration

(IC50) value of 1.7 nM and an increase of cytosolic calcium

induced by 10 nM PGE2 with an IC50 value of 120 nM.

Synovial tissue specimens

Synovial tissue specimens were obtained from patients who

fulfilled the 1987 revised classification criteria of the

American College of Rheumatology who underwent knee

joint replacement. In compliance with institutional policies,

informed consent was obtained from all patients. The ethics

committee of each institution approved this study.

In vitro reconstruction of inflammatory tissue

by synovial tissue-derived inflammatory cells

Synovial tissue-derived inflammatory cells were prepared as

previously described [5]. In brief, synovial tissue specimen

1070 Mod Rheumatol (2013) 23:1069–1075

123

were cut into small pieces and cultured in 100-mm dishes

containing RPMI 1640 (Asahi Technoglass, Chiba, Japan)

with 10 % fetal calf serum (FCS) and 100 units/ml of peni-

cillin G sodium and 100 lg/ml streptomycin sulfate (Gibco

BRL, Grand Island, NY). After 1–3 days’ incubation, tissue

was removed and single cells were collected by vigorous

pipetting. Cell suspensions were washed once and viable cells

were collected onto Lymphocyte Separation Solution (Nac-

alai Tesque, Kyoto, Japan). Single suspension of synovial

tissue-derived inflammatory cells were seeded at a density of

5 9 105/well in 48-well culture plates and cultured in Dul-

becco’s modified Eagle’s medium (DMEM; Gibco BRL)

containing 10 % FCS, 100 units/ml of penicillin G sodium

and 100 lg/ml streptomycin sulfate. The culture was

observed for morphologic changes under an inverted phase-

contrast microscope twice a week for 4 weeks. When cul-

tured in DMEM/10 % FCS in the absence or presence of

EP4-specific antagonist (100 ng/ml) or indomethacin (1 lM)

under hypoxic condition or normoxic condition, synovial

tissue-derived inflammatory cells started to aggregate,

forming foci within a few days. Further culturing resulted in a

3-dimensional (3-D) growth, which ultimately produced

macroscopic tissue 2 mm in size within 4 weeks. Morpho-

logic changes were semiquantitatively scored on a scale of

0–4, according to the degree of tissue development, where

0 = no cellular foci or aggregations, 1 = formation of cel-

lular foci or aggregations, 2 = further growth of cellular

aggregations, 3 = further 3-D growth with a multilayered

structure, and 4 = development of macroscopic tissue. A

cumulative tissue growth score was calculated by the total

sum of the tissue growth scores obtained twice a week for

4 weeks of culture. Half of the supernatants were collected

twice a week and replaced with fresh medium or the addition

of a dose of EP4-specific antagonist or indomethacin.

Supernatants were frozen at -80 �C until assayed.

Hypoxic culture conditions

In each experiment, synovial tissue-derived inflammatory

cells from one donor were seeded in two 48 well culture

plates. One plate was incubated under a normoxic condi-

tion in a 5 % CO2 incubator (Sanyo Co., Osaka, Japan) as a

control, another plate was exposed to a 1 % O2 condition

for 24 h twice a week in a hypoxic chamber, PERSONAL

CO2 MULTI GAS INCUBATOR (ASTEC Co., Fukuoka,

Japan) which create an atmosphere composed of 1 % O2,

5 % CO2 and 94 % N2. Joint movements in daily life

frequently result in the hypoxia-reperfusion of synovial

tissue especially in patients with rheumatoid arthritis. In

order to mimic this condition, we cultured the synovial

cells under periodic hypoxia. Another reason was the fact

that continuous hypoxia during 4 weeks culture resulted in

the loss of cell viability in our pilot experiments.

Cytokine assay

Supernatants obtained from each course of cell cultures

were stored at -80 �C until assayed, and levels of vascular

endothelial growth factor (VEGF), PGE2, TNF-a, M-CSF

(all from R&D Systems, Minneapolis, MN, USA), and

matrix metalloproteinase 9 (MMP-9; Amersham Biosci-

ences, Buckinghamshire UK) released into the culture

supernatants were measured using enzyme-linked immu-

nosorbent assay kits according to the manufacturers’

recommendations.

Result

Periodic hypoxia induces VEGF release

It is well known that hypoxia increases VEGF production

by synovial cells [10, 11, 12]. To confirm our culture

system for hypoxia, we measured VEGF release from

synovial tissue-derived inflammatory cells. As shown in

Fig. 1, VEGF release was significantly enhanced by our

hypoxic condition.

Effect of periodic hypoxia on the aberrant synovial

overgrowth

Hypoxia is well known to up-regulate inflammatory

response. We have reported that synovial tissue-derived

inflammatory cells showed aberrant synovial overgrowth

and spontaneous development of pannus-like tissue in vitro

Fig. 1 Hypoxic condition induces VEGF release. Synovial tissue-

derived inflammatory cells were exposed to either 1 % oxygen for

24 h twice a week (periodic hypoxia: hypo) or 21 % oxygen

(normoxia: normo) for 4 weeks. VEGF was measured by an

enzyme-linked immunosorbent assay of cell culture supernatants

(n = 33). Data was analysed versus normoxia by the Wilcoxon

signed rank test: *P \ 0.0001

Mod Rheumatol (2013) 23:1069–1075 1071

123

[5]. Therefore, we examined whether aberrant synovial

overgrowth was enhanced by periodic hypoxia. Paired

experiments using the same synovial tissue-derived

inflammatory cells, however, they showed that the aberrant

synovial overgrowth was attenuated under periodic

hypoxia compared with normoxia (Fig. 2).

Effect of periodic hypoxia on the PGE2 release

by rheumatoid synovial tissues-derived inflammatory

cells

It is reported that the hypoxic condition induces cycloox-

ygenase-2 (COX-2) expression by rheumatoid synovial

fibroblast [13] as well as PGE2 release from cancer cells

[14, 15]. We previously showed that endogenous PGE2

produced in rheumatoid synovium negatively regulated

aberrant synovial overgrowth through the EP4 receptor

[16]. Therefore, we hypothesized that attenuation of the

aberrant synovial overgrowth under periodic hypoxia could

be due to enhanced release of PGE2. As shown in Fig. 3,

PGE2 release was significantly enhanced under periodic

hypoxia.

Effect of indomethacin and EP4-specific antagonist

on the aberrant synovial overgrowth under periodic

hypoxia

To further confirm the hypothesis, we studied the effect of

indomethacin, an inhibitor of endogenous prostanoids

production, on the aberrant synovial overgrowth under

periodic hypoxia. As shown in Fig. 4a, indomethacin

enhanced the aberrant synovial overgrowth by the synovial

tissue-derived inflammatory cells. This effect was signifi-

cantly more enhanced under periodic hypoxia. The aberrant

synovial overgrowth was also enhanced by the addition of

the EP4-specific antagonist, this enhancement was signifi-

cantly higher under periodic hypoxia than normoxia.

Both the exogenous PGE1 and an EP4-specific agonist

significantly inhibited the aberrant synovial overgrowth in

the presence of indomethacin under periodic hypoxia as

well as normoxia. There was no significant difference

between normoxia and periodic hypoxia (Fig. 4b).

Effect of periodic hypoxia on the production

of cytokines and growth factor in the supernatants

of synovial tissue-derived inflammatory cells

Excessive and prolonged release of cytokines, such as

M-CSF, TNF-a MMP-9, and IL-6 seems to play a role in

the pathogenesis of inflammatory bone and joint destruc-

tion in RA. We have also shown that a large amount of

these proteins was produced in the supernatants of the

synovial tissue-derived inflammatory cells [5]. Both indo-

methacin and EP4-specific antagonist significantly

enhanced spontaneous production of M-CSF, TNF-a and

MMP-9 by the synovial tissue-derived inflammatory cells.

Fig. 2 Effect of periodic hypoxia on the in vitro tissue growth.

Synovial tissue-derived inflammatory cells were incubated in medium

alone (n = 7) under either 1 % oxygen for 24 h twice a week

(periodic hypoxia: hypo) or 21 % oxygen (normoxia: normo) for

4 weeks, and the development of in vitro tissue growth was observed

and scored as described in ‘‘Materials and methods’’. Cumulative

tissue growth scores observed in 1 % oxygen (hypoxia) and 21 %

oxygen (normoxia). Date was analysed versus normoxia by Wicoxon

signed rank test: *P \ 0.05

Fig. 3 Effect of periodic hypoxia on the PGE2 release by rheumatoid

synovial tissues. Synovial tissue-derived inflammatory cells were

incubated in medium alone (n = 13). The level of PGE2 in culture

supernatants was measured by enzyme-linked immunosorbent assay.

Date was analysed versus normoxia by Wilcoxon signed rank test:

*P \ 0.01

1072 Mod Rheumatol (2013) 23:1069–1075

123

This effect was significantly more enhanced under periodic

hypoxia than normoxia. On the other hand, there was no

significant difference of the effect of indomethacin or EP4-

antagonist on the spontaneous production of IL-6 between

normoxia and periodic hypoxia (Table 1).

Discussion

Rheumatoid arthritis is characterized by chronic inflam-

mation of the synovial membrane, which results in the

development of an invasive pannus tissue and the

Fig. 4 Effect of indomethacin (Ind) and EP4-specific antagonist

(EP4-ant) on the in vitro tissue growth. Synovial tissue-derived

inflammatory cells were cultured with indomethacin (1 lM), EP4-

specific antagonist (100 nM) for 4 weeks under either 1 % oxygen for

24 h twice a week (periodic hypoxia: hypo) or 21 % oxygen

(normoxia: normo), and the development of in vitro tissue growth

was observed and scored as described in ‘‘Materials and methods’’.

Cumulative tissue growth scores observed with indomethacin, EP4-

specific antagonist were compared to that observed with medium

alone. The relative value represents the percent of the cumulative

tissue growth score observed with indomethacin and EP4-specific

antagonist compared to that observed with medium alone. *P \ 0.05,

by Wilcoxon signed rank test. Both the exogenous PGE1 and an EP4-

specific agonist significantly inhibited the in vitro tissue growth in the

presence of indomethacin. There was no significant difference

between normoxia and periodic hypoxia

Table 1 Effect of periodic hypoxia on the production of cytokines

and growth factor. Tumor necrosis factor-a (TNF-a), macrophage

colony-stimulated factor (M-CSF), matrix metalloproteinase-9

(MMP-9), interleukin-6 (IL-6) synthesis in response to normoxic

and hypoxic synovial tissue-derived inflammatory cells supernatants

Each cytokine and growth factor was measured by enzyme-linked immunosorbent assay (n = 12)

The relative value represents the TNF-a, M-CSF, MMP-9 observed with indomethacin and EP4-specific antagonist compared to that observed

with medium alone

* P \ 0.05, by Wilcoxon signed rank test

** P \ 0.05, by Wilcoxon signed rank test, versus medium� No significance, by Wilcoxon signed rank test, versus medium

Mod Rheumatol (2013) 23:1069–1075 1073

123

subsequent destruction of cartilage and bone. The hyper-

plasia of the synovial lining layer results in microenvi-

ronmental alteration causing hypoxia and hypoperfusion.

The microenvironment of the inflamed joint is character-

ized by a low partial pressure of oxygen. Low oxygen

tension measurements were first recorded in the synovial

fluid of patients with rheumatoid arthritis [17], and sub-

sequent studies demonstrated decreased oxygen tension

and glucose levels alongside raised carbon dioxide, lactate

and acetate levels, consistent with anaerobic metabolism

[18, 19].

It is suggested that hypoxia plays a pivotal role in the

development and persistence of synovial inflammation and

subsequent joint destruction in both the adjuvant-induced

arthritis model [20] and rheumatoid synovial cells through

the induction of hypoxia-inducible factor-1a (HIF-1a) and

inflammatory cytokines [21–24]. However, little is known

about the net effects of hypoxia, cytokine and cell–cell

interaction that might contribute to the development of

pannus tissue. Therefore, a human cell model that repre-

sents this complex system of the aberrant synovial over-

growth will be useful to study the role of hypoxia in the

pathogenesis of rheumatoid arthritis. We have reported that

synovial tissue-derived inflammatory cells showed spon-

taneous development of the aberrant synovial overgrowth

and pannus-like tissue in vitro [5].

The objective of our study was, therefore, to examine the

potential in vivo consequences of periodic hypoxia in

rheumatoid arthritis in terms of synovial proliferation and

pannus growth, by periodically exposing synovial tissue-

derived inflammatory cells to 1 % oxygen (hypoxia). Using

a human cellular model of pannus, we have demonstrated

that periodic hypoxia induced negative feedback regulation

by PGE2 production, as well as proinflammatory cascades

including TNF-a, M-CSF and MMP-9. The positive effect

of hypoxia on aberrant synovial overgrowth by the synovial

tissue-derived inflammatory cells was effectively down-

regulated by the simultaneously induced endogenous PGE2.

Inhibition of either endogenous prostanoids by indo-

methacin or EP4 receptor by the EP4-specific antagonist

resulted in the enhancement of aberrant synovial over-

growth. Therefore, the net effects of periodic hypoxia may

depend upon the balance between the positive and negative

regulatory responses.

There is a controversy in the effects of hypoxia on PGE2

production. Demasi et al. [13] reported that hypoxia

enhanced IL-1b-stimulated COX-2 expression and PGE2

production by fibroblast-like synoviocytes. Choi et al. [25]

reported that hypoxia decreased IL-1b-stimulated PGE2

production by fibroblast-like synoviocytes even though it

increased expression of COX-2. The major differences

between their and our results may be the fact that we

measured spontaneous production of PGE2 during the

mixed cell–cell interaction. Choi et al. [25] also reported

that hypoxia did not affect IL-1b-stimulated IL-6 and IL-8

production by fibroblast-like synoviocytes. This is consis-

tent with our finding that periodic hypoxia did not show

any effect on the production of IL-6. On the other hand,

M-CSF production was significantly enhanced by periodic

hypoxia in the present study. Our preliminary experiments

indicated that M-CSF production was best correlated with

an in vitro tissue growth score among other cytokines such

as TNF-a, IL-6 and IL-8. Furthermore, it was reported that

the M-CSF level correlated with disease activity in rheu-

matoid arthritis [26].

The recent study clearly demonstrated that COX-2

specific (as well as the bispecific) NSAIDs, by reducing the

levels of PGE2, were able to increase expression of the key

proinflammatory cytokine TNF-a in rheumatoid synovium

[27]. We also showed that endogenous PGE2 inhibition by

NSAIDs enhanced aberrant synovial overgrowth and

osteoclastic activity [16].

Proinflammatory cytokines, including TNF-a, IL-1, and

IL-6 play a critical role in the progression of synovitis and

joint destruction mainly through activation of NF-jB [28–

30], while they directly induce COX-2 and PGE2 expres-

sion [29]. PGE2 up-regulates COX-2 expression via EP2/

EP4 receptors and the cyclic adenosine monophosphate-

dependent signaling pathway [31], which in turn modulates

the production of the proinflammatory molecules. The link

between proinflammatory molecules and PGE2 could have

considerable importance in the regulation of inflammatory

cells of rheumatoid arthritis. The paracrine and autocrine

feedback mechanisms via COX-2, PGE2, EP2 and EP4

could help to avoid the potential pathological damage

caused by the excessive production of inflammatory

mediators in response to various biological stimuli such as

periodic hypoxia in rheumatoid arthritis.

Acknowledgments The authors are grateful to Dr. Kuniomi

Yamasaki for continuing encouragement and his financial support for

our work, and we also thank Kiyomi Matsuo for the excellent tech-

nical assistance.

Conflict of interest Hidehiro Yamada received research funds from

Ono Pharmaceuticals Co. All other authors have declared no conflicts

of interest.

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