Tài liệu Báo cáo khoa học: Various secretory phospholipase A2 enzymes are expressed in rheumatoid arthritis and augment prostaglandin production in cultured synovial cells docx

Various secretory phospholipase A
2
enzymes are
expressed in rheumatoid arthritis and augment
prostaglandin production in cultured synovial cells
Seiko Masuda
1
, Makoto Murakami
1
, Kazuo Komiyama
2
, Motoko Ishihara
3
, Yukio Ishikawa
3
,
Toshiharu Ishii
3
and Ichiro Kudo
1
1 Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan
2 Department of Pathology, Division of Immunology and Patho-Biology at Dental Research Center, Nihon University School of Dentistry,
Tokyo, Japan
3 Department of Pathology, Toho University School of Medicine, Tokyo, Japan
Secretory phospholipase A
2
(sPLA
2
) is a group of
disulfide-rich, low molecular mass, lipolytic enzymes
with a His-Asp catalytic dyad [1,2]. To date, 10 sPLA

2
enzymes (IB, IIA, IIC, IID, IIE, IIF, III, V, X and
XIIA) have been identified in mammals. Of these
enzymes, sPLA
2
s in the I ⁄ II ⁄ V ⁄ X branch share many
structural characteristics and are thought to have
diverged from a common ancestor gene by successive
gene duplication events. The expression of individual
sPLA
2
s is tissue specific and often stimulus inducible
[3–15], leading to the proposal that they may play
tissue-specific functions during inflammation, tissue
Keywords
immunohistochemistry; phospholipase A
2
;
prostaglandin; rheumatoid arthritis; synovial
cell
Correspondence
M. Murakami, Department of Health
Chemistry, School of Pharmaceutical
Sciences, Showa University, 1-5-8
Hatanodai, Shinagawa-ku, Tokyo 142-8555,
Japan
Fax: +81 3 37848245
Tel: +81 3 37848197
E-mail:
(Received 8 May 2004, revised 26 October

2004, accepted 17 November 2004)
doi:10.1111/j.1742-4658.2004.04489.x
Although group IIA secretory phospholipase A
2
(sPLA
2
-IIA) is known to
be abundantly present in the joints of patients with rheumatoid arthritis
(RA), expression of other sPLA
2
s in this disease has remained unknown.
In this study, we examined the expression and localization of six sPLA
2
s
(groups IIA, IID, IIE, IIF, V and X) in human RA. Immunohistochemis-
try of RA sections revealed that sPLA
2
-IIA was generally located in syn-
ovial lining and sublining cells and cartilage chondrocytes, sPLA
2
-IID in
lymph follicles and capillary endothelium, sPLA
2
-IIE in vascular smooth
muscle cells, and sPLA
2
-V in interstitial fibroblasts. Expression levels of
these group II subfamily sPLA
2
s appeared to be higher in severe RA than

in inactive RA. sPLA
2
-X was detected in synovial lining cells and intersti-
tial fibers in both active and inactive RA sections. Expression of sPLA
2
-
IIF was partially positive, yet its correlation with disease states was
unclear. Expression of sPLA
2
transcripts was also evident in cultured nor-
mal human synoviocytes, in which sPLA
2
-IIA and -V were induced by
interleukin-1 and sPLA
2
-X was expressed constitutively. Adenovirus-
mediated expression of sPLA
2
s in cultured synoviocytes resulted in
increased prostaglandin E
2
production at low ngÆmL
)1
concentrations.
Thus, multiple sPLA
2
s are expressed in human RA, in which they may play
a role in the augmentation of arachidonate metabolism or exhibit other cell
type-specific functions.
Abbreviations

AA, arachidonic acid; COX, cyclooxygenase; cPGES, cytosolic prostaglandin E synthase; cPLA
2
, cytosolic PLA
2
; ER, endoplasmic reticulum;
HSPG, heparan sulfate proteoglycan; IFN-c, interferon-c;IL-1b, interleukin-1b; mPGES, membrane-bound prostaglandin E synthase; NaCl ⁄ P
i
,
phosphate-buffered saline; PtdCho, phosphatidylcholine; PG, prostaglandin; RA, rheumatoid arthritis; sPLA
2
, secretory phospholipase A
2
;
TNFa, tumor necrosis factor a; VSMC, vascular smooth muscle cells.
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS 655
injury, and cancer. Although sPLA
2
s have been impli-
cated in various biological events, including arachid-
onic acid (AA) metabolism [16–33] and others [34–40],
their precise in vivo functions are still a subject of
debate.
It is well established that synovial fluid from
patients with rheumatoid arthritis (RA) contains high
sPLA
2
activity [41], and enzyme purification and
molecular cloning studies have ascribed this activity
to sPLA
2

-IIA [42]. Elevated levels of this enzyme
have also been observed in the plasma of patients
with various types of inflammatory disease (e.g. sep-
sis, Crohn’s disease and acute pancreatitis) [1,2,41].
However, subsequent identification of novel sPLA
2
s
has raised a fundamental question of whether only
sPLA
2
-IIA is induced or other sPLA
2
s are also pre-
sent in inflamed tissues. The remarkable species-
associated difference in the tissue distribution of indi-
vidual sPLA
2
s [3–15] underlines the need to investi-
gate the expression of each enzyme in human tissues.
This issue is of particular importance to understand
the functions of sPLA
2
s in human pathology,
because individual sPLA
2
s display distinct enzy-
matic activities toward phospholipids in mammalian
cellular membranes [16–33], lung surfactant [34], bac-
terial membranes [35,36], and lipoprotein particles
[37,38].

Current evidence suggests that sPLA
2
s can release
cellular AA via at least three distinct mechanisms, the
occurrence of which appears to be cell type or stimulus
specific. First, sPLA
2
s release AA intracellularly prior
to secretion [43]. Second, after secretion into the extra-
cellular space, sPLA
2
s with high interfacial binding
capacity to phosphatidylcholine (e.g. sPLA
2
-V and -X)
act on the phosphatidylcholine-rich outer plasma mem-
brane [20,21,25–29,32,33]. Third, sPLA
2
s with affinity
for heparanoids (e.g. sPLA
2
-IIA, -IID and -V) often
bind to cell surface heparan sulfate proteoglycans
(HSPGs; e.g. glypican), internalized through caveo-
lae ⁄ raft-dependent endocytosis, and then exert their
function [17–19,21,28,31]. As an additional mechanism,
sPLA
2
s act as ligands for a transmembrane protein
called M-type sPLA

2
receptor, which in turn activates
group IVA cytosolic PLA
2
a (cPLA
2
a) to initiate AA
metabolism [44].
In this study, we performed immunohistochemistry
with antibodies specific for each sPLA
2
to evaluate the
expression and localization of six sPLA
2
s (IIA, IID,
IIE, IIF, V and X) in human joints affected by RA.
We further examined the possible contribution of these
sPLA
2
s to AA metabolism in cultured normal human
synovial cells. Our results indicate that these sPLA
2
s
are diversely expressed in RA tissues and are able to
augment prostaglandin E
2
(PGE
2
) production in syn-
ovial cells.

Results
Detection of various sPLA
2
s in RA tissues
by RT-PCR
It is well established that human joints affected by
RA contain large amounts of sPLA
2
-IIA, and its
expression levels are correlated with disease severity
[41,42]. In order to assess whether these tissues also
express other sPLA
2
enzymes, we initially performed
RT-PCR with primers specific for individual sPLA
2
s
(IB, IID, IIE, IIF, V and X, as well as IIA as a pos-
itive control), followed by high-sensitivity Southern
blotting, on RNA samples obtained from synovial tis-
sues of two patients with distinct pathologic states,
which relied on historical determination on the basis
of the morphology of the sections as well as on the
expression of sPLA
2
-IIA and COX-2 (see below),
which has been shown to correlate with the disease
states [41,42,45]. As expected, the sPLA
2
-IIA tran-

script was detected intensely in both samples with
more expression in severe RA (sample b) than in mild
RA (sample a) (Fig. 1A). In addition to sPLA
2
-IIA,
diverse expression of other sPLA
2
s was also found in
these samples. Thus, sPLA
2
-IID and -IIE were detec-
ted only in sample b, and sPLA
2
-V and -X were
detected in both samples almost equally (Fig. 1A).
Expression of sPLA
2
-IIF was low, but a trace level of
its expression was detected in sample a when RT-
PCR was followed by high-sensitivity Southern blot-
ting (Fig. 1A). sPLA
2
-IB was not detected at all
(Fig. 1A).
Immunoblotting of the same RA samples with anti-
bodies specific for individual sPLA
2
s yielded similar
results (Fig. 1B). Thus, 14–18 kDa immunoreactive
bands for sPLA

2
-IIA, -V and -X were detected in both
samples a and b, and those of sPLA
2
-IID and -IIE
were detectable only in sample b (Fig. 1B). sPLA
2
-IIF
protein was undetectable by immunoblotting, probably
because of its low expression level. In agreement with
the fact that the arthritic symptoms were more severe
in the patient from which sample b was derived than
in the patient providing sample a, expression of
cPLA
2
a, COX-2 and membrane-bound prostaglan-
din E synthase (mPGES)-1, which are elevated in
severe RA [45], was higher in sample b than in sample
a, whereas expression of COX-1, mPGES-2 and cyto-
solic prostaglandin E synthase (cPGES), which are
constitutively expressed in many cell types [45], was
similar between both samples (Fig. 1C).
sPLA2s in human rheumatoid arthritis S. Masuda et al.
656 FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
Immunohistochemistry of RA tissues
Given these observations, we aimed to determine the
cellular localization of these sPLA
2
s in synovial tissues
of RA patients by immunohistochemistry. A previous

immunohistochemical study showed that, in RA tis-
sues, sPLA
2
-IIA is distributed in various cells, such as
synovial lining and sublining cells and vascular cells,
as well as in extracellular matrix fibers [46]. In our
study, synovial membranes from a patient with inac-
tive RA (i.e. inflammatory symptoms were temporarily
ceased after therapy) showed only weak staining for
sPLA
2
-IIA (Fig. 2A), whereas the enzyme was inten-
sely expressed in synovial lining cells in the section
from a patient with active RA (Fig. 2B,C). Staining of
the synovial sublining area was also significant, and
there was scattered expression in mononuclear cells
(Fig. 2C). Cartilage chondrocytes in active RA tissues
were intensely positive for sPLA
2
-IIA, whereas staining
of the infiltrating fibroblasts was weak (Fig. 2D).
Vascular smooth muscle cells (VSMC) also provided
positive staining for sPLA
2
-IIA (Fig. 2E). These distri-
butions of sPLA
2
-IIA in RA tissues are largely in
agreement with a previous study [46].
Staining of PLA

2
-IID was weak in a section of inac-
tive RA tissues, in which scattered staining was located
in the subintimal lymph aggregates (lymph follicles)
(Fig. 3A). In another inactive RA section, the lymph
aggregates (Fig. 3Ba) and microvascular endothelium
(Fig. 3Bb) were weakly stained for sPLA
2
-IID. Prom-
inent sPLA
2
-IID staining was evident in the lymphoid
aggregates and capillary endothelial cells in three dis-
tinct active RA sections (Fig. 3C–E). Staining of
sPLA
2
-IID in synovial lining cells was also evident
(Fig. 3D,E), even though weak and less frequent than
that of sPLA
2
-IIA (Fig. 2B,C), -V, and -X (see below).
Staining of cartilage chondrocytes was very weak
(Fig. 3E), compared with that of sPLA
2
-IIA (Fig. 2A).
Thus, sPLA
2
-IID appears to be preferentially induced
in the lymph follicular cells and capillary endothelial
cells in synovial tissues with active RA.

Although no staining of sPLA
2
-IIE was observed in
two inactive RA sections (Fig. 4A), it was intense in
VSMC in three distinct active RA sections (Fig. 4Ba–
c). In contrast, staining of synovial lining cells and
sublining interstitum (Fig. 4Ba–c), as well as cartilage
chondrocytes (Fig. 4Bd), was negligible. Thus, sPLA
2
-
IIE is induced rather specifically in VSMC in synovial
tissues with active RA.
In inactive RA sections, sPLA
2
-IIF showed sporadic
and weak staining in individual cells (Fig. 4Ca,b), and
a few interstitial cells provided intense staining in one
sample (Fig. 4Cb). In two active RA sections, scattered
staining of sPLA
2
-IIF was detected in the subintima,
in which only a limited population of plasma cells
showed immunoreactivity (Fig. 4Ca,b), consistent with
a previous report [22]. Cartilage condrocytes were not
stained for sPLA
2
-IIF (Fig. 3Dc). These results,
together with the results of RT-PCR and western blot
(Fig. 1), implies that the expression of sPLA
2

-IIF in
RA is rather lower than that of other sPLA
2
s and does
not show any obvious difference in staining between
tissues derived from the two patients with active and
those with inactive RA.
Expression of sPLA
2
-V in inactive RA sections was
either undetectable (Fig. 5A) or very weak (Fig. 5B).
ABC
Fig. 1. Expression of sPLA
2
s and other PGE
2
-biosynthetic enzymes in human joints affected by RA. Expression of sPLA
2
s in mild (a) and
severe (b) RA joint tissues was assessed by RT-PCR (A) and immunoblotting (B). (A) Amplified fragments were visualized by ethidium bro-
mide in agarose gels (left), followed by Southern blotting (right). PCR cycle numbers are indicated. (C) Expression of other enzymes involved
in PGE
2
synthesis in the two RA samples was assessed by immunoblotting.
S. Masuda et al. sPLA2s in human rheumatoid arthritis
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS 657
In the latter case, weak staining was locally detected in
the interstitium (Fig. 5B). Intense sPLA
2
-V immuno-

reactivity was observed in wide areas of three active
RA sections. In all cases, sPLA
2
-V staining was evi-
dent in synovial lining cells and especiallly in sublining
granulation tissue, which was composed of massive
fibrosis with extracellular matrix fibers (Fig. 5C–E). The
vascular walls, including VSMC and endothelial cells,
provided no detectable signals for sPLA
2
-V (Fig. 5C,D).
Scattered staining was also observed in the lymph aggre-
gates (Fig. 5Eb). Staining of chondrocytes in the carti-
lage tissues were negative for sPLA
2
-V, whereas
fibroblasts infiltrating into the cartilage tissues were
intensely stained (Fig. 5Ec), thus exhibiting a reciprocal
pattern compared with sPLA
2
-IIA (Fig. 2d).
sPLA
2
-X immunoreactivity was evident in two inac-
tive (Fig. 6A,B) and three active (Fig. 6C–E) RA sam-
ples. Although the staining intensities of individual
samples were variable, the enzyme was consistently
localized in the synovial lining layers and the intersti-
tium that precludes the lymphoid aggregates, vascular
walls, and cartilage chondrocytes in all samples

(Fig. 6). In the subintimal interstitium, sPLA
2
-X stain-
ing was evident in the extracellular matrix fibers
(Fig. 6Cc) and neuronal fibers (Fig. 6Cd).
Expression of endogenous sPLA
2
s in cultured
human synovial cells
We next used RT-PCR to examine the expression of
these sPLA
2
s in cultured normal human synovial cells
(a mixed population of synovial lining cells and inter-
stitial fibroblasts). Although sPLA
2
-IIA and -V tran-
scripts were barely detectable in unstimulated cells,
they were markedly induced in cells stimulated with
interleukin (IL)-1b (Fig. 7). These two sPLA
2
s were
also weakly induced by tumor necrosis factor (TNF)a,
whereas the effect of interferon (IFN)-c was minimal.
sPLA
2
-X transcript was weakly but constitutively
expressed in synoviocytes with no appreciable induc-
tion by cytokines (Fig. 7A). In contrast, sPLA
2

-IID
and -IIE were undetectable in these cells even after sti-
mulation with cytokines (Fig. 7A) and five more cycles
of PCR amplification (data not shown). These results
are in good agreement with the immunohistochemical
study, in which sPLA
2
-IIA, -V and -X were located,
whereas sPLA
2
-IID and -IIE were barely detected, in
Fig. 2. Immunohistochemical localization of sPLA
2
-IIA in human
joints affected by RA. Staining of sPLA
2
-IIA in an inactive RA tissue
was rare, and only a few synovial lining regions (dark arrowheads)
showed weak immunoreactivity (A). In an active RA tissue (B–E),
sPLA
2
-IIA was found virtually in all areas of RA tissues, in particular
in synovial lining cells (B and C dark arrowheads). Aggregates of
mononuclear cells (C, blue arrows) and fibroblasts (C, red arrows)
in the sublining region, cartilage chondrocytes (D, yellow arrow-
heads), and VSMC (E, green arrows) were positively stained. Stain-
ing of fibroblast-like cells infiltrating into the cartilage was faint (D,
red arrows).
sPLA2s in human rheumatoid arthritis S. Masuda et al.
658 FEBS Journal 272 (2005) 655–672 ª 2005 FEBS

synovial lining cells and interstitial fibroblastic cells
(Figs 2–6). However, expression levels of endogenous
sPLA
2
-IIA, -V and -X proteins in cultured synovial
cells were below the detection limit of immunoblotting
even 24 h after stimulation with IL-1b (see Fig. 8C for
sPLA
2
-IIA and data not shown for sPLA
2
-V and -X),
suggesting that some additional factors, which may
exist in synovial tissue microenvironments, are further
required for optimal sPLA
2
induction in normal syn-
ovial cells. Indeed, synovial fibroblasts from RA
patients express sPLA
2
-IIA protein in primary culture
[47].
As assessed by immunoblotting, cPLA
2
a, COX-1,
cPGES and mPGES-2 were uniformly expressed in
synovial cells before and after cytokine stimulation
(Fig. 7B). COX-2 was undetectable in unstimulated
cells and was markedly induced in cells stimulated with
IL-1b, but not with TNFa or IFN-c (Fig. 7B). Induc-

tion of COX-2 was already evident at 6 h, reaching a
plateau by 24 h (Fig. 7C). Although expression of
mPGES-1 protein was below the detection limit by
immunoblotting (data not shown), its expression was
detectable by RT-PCR, where it was weakly expressed
in unstimulated cells and induced by all three cyto-
kines, with IL-1b and TNFa exhibiting more potent
effect than IFN-c (Fig. 7B). In the case of IL-1b sti-
mulation, increased expression of COX-2 and mPGES-
1 was observed over 6–24 h (Fig. 7C). Consistent with
the elevated expression of COX-2 and mPGES-1, sti-
mulation of these cells with IL-1b resulted in marked
prostaglandin E
2
(PGE
2
) generation over 24 h, whereas
TNFa and IFN-c exhibited poor PGE
2
-biosynthetic effects
(Fig. 7D). Time course experiments showed that the accu-
mulation of PGE
2
in the medium of IL-1b-s timulat ed
AC
BD
E
Fig. 3. Immunohistochemical localization of sPLA
2
-IID in human joints affected by RA. (A,B) Staining of two inactive RA tissues. In both sam-

ples, weak and scattered staining of sPLA
2
-IID was seen in the lymph follicles (red arrows). Some microvascular endothelial cells (light blue
arrowheads) were also weakly positive (Bb). (C–E) Staining of three active RA tissues. In all sections, sPLA
2
-IID was intensely stained in the
lymph follicles and microvascular endothelium. Synovial lining cells (dark arrowheads) were also partially stained (Da,b, Ea,b). Cartilage
condrocytes showed weak staining (Ec).
S. Masuda et al. sPLA2s in human rheumatoid arthritis
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS 659
cells reached a plateau peak over 12–24 h (Fig. 7E).
IL-1b-stimulated PGE
2
generation was suppressed by
the cPLA
2
inhibitor methyl arachidonoyl fluorophos-
phate by > 80%, suggesting the contribution of
cPLA
2
a to this biosynthetic response. Indeed, cPLA
2
a
dependence of PGE
2
production in IL-1 b-stimulated
synovial cells was reported previously [48].
PGE
2
production by sPLA

2
s in cultured human
synovial cells
To examine the effect of individual sPLA
2
s on PGE
2
production by cultured synoviocytes, these cells were
infected with adenoviruses harboring cDNAs for
sPLA
2
-IIA, -V and -X, which were detected in synovial
cells both in RA tissues (Figs 2,5 and 6) and in culture
(Fig. 7A). We also transfected these cells with sPLA
2
-
IID and -IIF, which were not intrinsically expressed in
this cell type (Figs 3 and 4), and with cPLA
2
a, which
was used as a positive control for increased PGE
2
pro-
duction, using the same strategy. After 36 h of adeno-
virus infection, the expression of each sPLA
2
and
cPLA
2
a in the transfectants was verified by northern

blotting (Fig. 8A, upper). Figure 8B represents the
enzyme activities in the supernatants and cell-surface-
associated (1 m NaCl-solubilized) fractions of synovio-
cytes transfected with sPLA
2
s. Significant portions of
sPLA
2
-IIA, -IID and -V ( 55,  30 and  45%) were
detected in the membrane-bound fractions, whereas
sPLA
2
-IIF and -X was predominantly distributed in
the supernatants (Fig. 8B). These distribution patterns
(supernatant vs. cell-surface bound) of individual
sPLA
2
s are consistent with those in several reports
using other cell types [17–21]. The concentrations of
individual sPLA
2
s produced by cells infected with a
high dose of adenoviruses were equivalent to
4–6 ngÆmL
)1
, as estimated from their enzymatic activit-
ies in comparison with those of pure recombinant
sPLA
2
standards (which were measured after dilution

in culture medium) (Fig. 8B). Because the concentra-
tions of sPLA
2
-IIA often reach the order of lgÆmL
)1
in synovial fluids of RA patients [41,42], the levels of
sPLA
2
s overexpressed in cultured synovial cells in this
experiment were at least two orders of magnitude
lower than those in the pathologic range. On immuno-
blotting, a 14 kDa sPLA
2
-IIA protein band was
detected in cells infected with sPLA
2
-IIA-bearing
adenovirus (Fig. 8C, upper). Similar immunoblot
results were obtained in cells infected with adenovirus
for sPLA
2
-V and -X (data not shown). In the case of
sPLA
2
-IID (Fig. 8C, lower) and -IIF (data not shown),
a larger band (26–30 kDa) was also detected in cells
infected with their adenoviruses. Although the entity
of this larger band is unknown at present, we speculate
that these two sPLA
2

s form a homodimer or undergo
some post-translational modification (such as glycosy-
lation) in cultured synovial cells, a possibility that is
under investigation.
AC
BD
Fig. 4. Immunohistochemical localizations of sPLA
2
-IIE (A,B) and
-IIF (C,D) in human joints affected by RA. Although sPLA
2
-IIE was
undetectable in two inactive RA tissues (Aa,b), it was detected in
VSMC (green arrows) in three active RA tissues (Ba–c). Cartilage
chondrocytes were not stained for sPLA
2
-IIE (Bd). Staining of
sPLA
2
-IIF was weak and scattered in both inactive (C) and active
(D) RA tissues. In an inactive RA section, a few intimal cells
showed immunoreactivity (Cb). In two active RA sections, scat-
tered staining of sPLA
2
-IIF was detected in the subintima, in which
it was expressed only in a few plasma cells (red arrowheads)
(Da,b). Cartilage chondrocytes were not stained for sPLA
2
-IIF (Dc).
sPLA2s in human rheumatoid arthritis S. Masuda et al.

660 FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
As shown in Fig. 8A, IL-1b-stimulated production
of PGE
2
was markedly augmented in cells transfected
with these sPLA
2
s and cPLA
2
a over that in control
cells in a manner dependent upon adenovirus doses
(i.e. PLA
2
expression levels). There was no increase in
PGE
2
production in cells infected with adenoviruses
for the catalytically inactive sPLA
2
-IIA and -X
mutants (G30S; a mutation in the Ca
2+
-binding loop
[20] (Fig. 8D), implying that the enzymatic activity is
essential for augmented PGE
2
generation by sPLA
2
s.
To assess the intracellular localization of these

sPLA
2
s in synovial cells, we performed immunocyto-
staining of cells that had been infected with adeno-
viruses for sPLA
2
s for 36 h and then incubated for an
additional 12 h with or without IL-1b. Signals for
sPLA
2
-IIA (Fig. 9A), -IID (Fig. 9B), and -V (data not
shown) were mainly localized near the nucleus, being
largely colocalized with the Golgi marker GM130
(Fig. 9D). Signals for sPLA
2
-X (Fig. 9C) and -IIF (data
not shown) were also located in the Golgi, but showed
more disperse distribution with reticular pattern, indica-
ting that a large portion of these enzymes also resides in
the endoplasmic reticulum (ER). We also noted that
IL-1b stimulation resulted in the appearance of punctate
signals for sPLA
2
-IIA in the cytoplasm, even though the
Golgi staining was still predominant (Fig. 9A, middle).
Treatment of IL-1b-stimulated cells with cell-imperme-
able heparin abrogated the cytoplasmic punctate signals
for sPLA
2
-IIA, whereas the Golgi staining was unaffec-

ted (Fig. 9A, lower). These cytoplasmic punctate sig-
nals for sPLA
2
-IIA in IL-1b-stimulated cells were
largely colocalized with caveolin (Fig. 9E), a marker for
caveolae-derived vesicles. Similar staining was observed
in cells expressing sPLA
2
-IID and -V (data not shown).
These results suggest that the punctate signals for
sPLA
2
-IIA observed in IL-1b-stimulated synovial cells
A
B
D
CE
Fig. 5. Immunohistochemical localizations of sPLA
2
-V in human joints affected by RA. (A,B) In two inactive RA sections, sPLA
2
-V immuno-
reactivity was very low, with only moderate staining in the interstitium (purple arrows). (C–E) Staining of sPLA
2
-V in three active RA tissues.
In all cases, intense staining of the granulation tissue in the sublining interstitium was evident. Staining of the granulation tissue and lymph
aggregates (red arrow) is magnified (Eb). Synovial lining cells (dark arrowheads) were also positive. In contrast, the vascular walls (green
arrows) were largely negative (C, D), as magnified (Cb). Cartilage chondrocytes (yellow arrowheads) (Ec) were negatively stained, whereas
fibroblasts infiltrating into the cartilage were intensely positive (Ec).
S. Masuda et al. sPLA2s in human rheumatoid arthritis

FEBS Journal 272 (2005) 655–672 ª 2005 FEBS 661
represent a pool of this enzyme sorted into caveolae-
derived vesicles in an HSPG-dependent manner,
whereas the Golgi localization represents the de novo
synthesized pool of the enzyme entering into the secre-
tory pathway. The cytoplasmic punctate signals were
barely detectable in cells expressing sPLA
2
-X, an
HSPG-nonbinding enzyme [20,21], even after IL-1b
stimulation (Fig. 9C, middle). Weak and diffused stain-
ing of sPLA
2
-X in the cytoplasm is likely to reflect its
localization in the ER (Fig. 9C) because of its secreted
property and because of its failure to colocalize with
caveolin (data not shown). Endogenous COX-2, which
is an absolute requirement for cytokine-stimulated
PGE
2
synthesis downstream of PLA
2
[18–20], was
located predominantly in the perinuclear membrane of
IL-1b-stimulated cells (Fig. 9F).
A
B
CD
E
Fig. 6. Immunohistochemical localizations of sPLA

2
-X in human joints affected by RA. Staining of sPLA
2
-X in two inactive (A,B) and three
active (C–E) RA tissues revealed its expression in synovial lining cells (dark arrowheads) as well as in the interstitium (purple arrows).
Lymph aggregates (red arrows) and vascular walls (green arrows) were negative. Neural fibers of the synovial sublining region showed
intense staining (Cd, orange arrowheads). Although cartilage chondrocytes were negative, fibroblasts infiltrating into the cartilage were
intensely stained (Eb).
sPLA2s in human rheumatoid arthritis S. Masuda et al.
662 FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
Because of the heparin-sensitive caveolae localization
of a small fraction of sPLA
2
-IIA (Fig. 9A), we antici-
pated that this pool of the enzyme might contribute to
the promotion of PGE
2
production via the HSPG-
dependent pathway, as reported in several other cells
[19–21,28,31]. However, treatment of the cells with
heparin or heparinase, which perturbs the HSPG-
dependent pathway [19–21,28,31], did not significantly
alter PGE
2
generation by sPLA
2
-IIA (Fig. 8E) or by
other sPLA
2
s (data not shown). This indicates that,

even though HSPG-dependent shuttling of sPLA
2
-IIA
(and other HSPG-binding sPLA
2
s) into caveolae vesi-
cles could occur in IL-1b-stimulated synovial cells, this
event is not associated with increased PGE
2
synthesis
by this enzyme under current experimental conditions.
Discussion
Eicosanoids, especially PGE
2
, are critical mediators of
RA [49–51]. Administration of PGE
2
into the hind
paws of rats with adjuvant arthritis (a rat model of
RA) exacerbates edema [49], and gene targeting of
enzymes involved in the biosynthesis of PGE
2
, inclu-
ding cPLA
2
a [52], COX-2 [53] and mPGES-1 [54], as
well as of the PGE receptor EP4 [55], leads to marked
amelioration of collagen-induced arthritis (a mouse
model of RA). We now show that, in addition to
sPLA

2
-IIA as previously reported [41,42,46], various
sPLA
2
s exist in human synovial tissues affected by
RA. sPLA
2
-IIA (Fig. 2), -V (Fig. 5), and -X (Fig. 6)
are expressed in synovial lining and sublining cells, an
observation further supported by in vitro synovial cell
culture (Fig. 7A). COX-2 [56] and mPGES-1 [45],
which lie downstream of PLA
2
s in the PGE
2
-biosy-
nthetic pathway, are also expressed in synovial lining
cells in the RA joints. Distribution of sPLA
2
-V in the
synovial sublining lesions (Fig. 5) is noteworthy
because this enzyme shows fibroblastic location in sev-
eral other tissues (S Masuda, M Murakami, M Mitsui-
shi, K Komiyama, Y Ishikawa, T Ishii and I Kudo,
unpublished observation). The presence of sPLA
2
-IIA
and -V in the extracellular matrix fibers is compatible
with their association with negatively charged sulfated
sugar chains of proteoglycans [17–19], whereas the

location of sPLA
2
-X, which does not show appreciable
HSPG binding [20,21], in the extracellular matrix is
suggestive of its interaction with unknown matrix com-
ponents. Although examination of more samples,
including those from normal subjects, is needed to
clarify the precise relationship between the expression
of individual sPLA
2
s and RA pathology, our results
have opened new insights into the expression of mul-
tiple sPLA
2
s in human inflammatory tissues.
In cultured normal human synovial cells, expression
of sPLA
2
-IIA and -V is cytokine-dependent, whereas
that of sPLA
2
-X is rather constitutive (Fig. 7A). Simi-
larly, more sPLA
2
-IIA, -IID, -IIE and -V are detected
immunohistochemically in active RA than inactive RA
tissues (Figs 2–5), while sPLA
2
-X is diversely expressed
in both inactive and active RA tissues (Fig. 6). These

results indicate that the mechanisms of transcriptional
regulation of the group II subfamily sPLA
2
s and
sPLA
2
-X are distinct. Importantly, even the induction
of individual group II subfamily sPLA
2
s requires dis-
tinct cytokines in different cell types [57,58], implying
A
DE
B
C
Fig. 7. Expression of sPLA
2
s and other PGE
2
-biosynthetic enzymes
in human cultured synovial cells. (A) Expression of endogenous
sPLA
2
s in synovial cells before and after stimulation with or without
IL-1b, TNFa and IFN-c for 24 h, as assessed by RT-PCR (30 cycles).
(B) Expression of other PGE
2
-biosynthetic enzymes in synovial cells
with or without cytokine stimulation for 24 h, as assessed by
immunoblotting. Expression of mPGES-1 was evaluated by RT-

PCR. (C) Time course of the induction of COX-2 (immunoblotting)
and mPGES-1 (RT-PCR) in IL-1b-stimulated synovial cells. (D) PGE
2
production by synovial cells treated for 24 h with or without cyto-
kines. (E) Time course of PGE
2
production by synovial cells treated
with or without IL-1b for the indicated periods. In (D) and (E), val-
ues are mean ± SE of three experiments.
S. Masuda et al. sPLA2s in human rheumatoid arthritis
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS 663
the existence of cell type-specific transcriptional
machinery for each enzyme. As shown in our series of
studies [14,23], expression of sPLA
2
-X in many types
of cells and tissues appears to be relatively constitutive,
even though elevated expression can occur in associ-
ation with cell differentiation and maturation [59].
Because sPLA
2
-X, but not group II subfamily sPLA
2
s,
has an N-terminal propeptide that is removed by pro-
teolysis to produce an active enzyme [32], up-regula-
tion of this enzyme might generally be controlled by
this post-translational processing rather than by gene
induction.
Cytokine-stimulated synovial cells are highly sus-

ceptible to sPLA
2
s, producing PGE
2
in response to all
sPLA
2
s when expressed at low ngÆmL
)1
concentrations
(Fig. 8). This sPLA
2
sensitivity is remarkable because
100–1000 ngÆmL
)1
or even more sPLA
2
s are generally
required for triggering eicosanoid biosynthesis when
A
B
C
D
E
Fig. 8. Adenovirus-mediated transfer of PLA
2
s into human cultured synovial cells. (A) PGE
2
generation by synovial cells infected with the
indicated doses of adenoviruses for PLA

2
s or control (LacZ) for 36 h, followed by stimulation with IL-1b for 12 h. Expression of each PLA
2
was verified by northern blotting (upper). (B) sPLA
2
activities in the supernatant (S, shaded bars) and membrane-associated (1 M NaCl-solubi-
lized) (M, closed bars) fractions of synovial cells after infection with adenoviruses bearing sPLA
2
s (multiplicity of infection [MOI] ¼ 10). Val-
ues indicate the amounts of sPLA
2
s released into the medium, as estimated from the enzymatic activities of the respective standard
recombinant sPLA
2
s. (C) Western blotting of synovial cells infected with adenovirus for sPLA
2
-IIA (upper), -IID (lower), or control (LacZ) for
36 h, followed by stimulation with IL-1b for 12 h. Arrow indicates a specific band for each sPLA
2
. In the case of sPLA
2
-IID (lower), another
high molecular mass band was detected in the transfectants (shaded arrow). (D) Synovial cells were infected with adenovirus for wild-type
(WT) or catalytically inactive mutants (Mut) for sPLA
2
-IIA and -X for 36 h, and then stimulated for 12 h with IL-1b to assess PGE
2
generation.
Expression of sPLA
2

s was verified by northern blotting (inset). (E) Synovial cells infected with adenovirus for sPLA
2
-IIA or LacZ were preincu-
bated with 500 lgÆmL
)1
heparin or 0.5 unitÆmL
)1
heparinase for 2 h and then stimulated for 12 h with IL-1b in the continued presence of
heparin or heparinase to assess PGE
2
generation. In (A,B,D,E), values are mean ± SE of three independent experiments. Position of 18S
ribosomal RNA in northern blotting is indicated in (A) and (D).
sPLA2s in human rheumatoid arthritis S. Masuda et al.
664 FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
they are added exogenously to various cells, including
rheumatoid synovial fibroblasts [25–28,32,33,47]. We
initially thought that the high sensitivity of synovial
cells to HSPG-binding sPLA
2
s (e.g. sPLA
2
-IIA and
-IID) might be because the HSPG-shuttling pathway,
which confers cellular sensitivity to HSPG-binding
sPLA
2
s [17–21,25–31], is operative in synovial cells,
whereas the ability of sPLA
2
-X and -IIF to increase

PGE
2
production in synovial cells depends on the
external plasma membrane pathway, as observed in
several other cell types [20,22,32,33]. Indeed, there was
accumulation of a small pool of the HSPG-binding
sPLA
2
s into caveolin-rich vesicles, a process that was
sensitive to heparin, in cytokine-stimulated synovial
cells (Fig. 9), consistent with a recent proposal that
caveolae-mediated endocytosis often occurs after cell
activation [60,61]. However, the contribution of these
pathways to PGE
2
generation in synovial cells is unli-
kely in our case because, even though a small fraction
of HSPG-binding sPLA
2
s are located in caveolae-rich
vesicles, treatment of these cells with exogenous
heparin or heparinase, which perturbs the HSPG-
dependent pathway [19–21,28,31], did not affect PGE
2
production by these sPLA
2
s (Fig. 8E), and the
amounts of sPLA
2
s released into the culture medium

(an order of low ngÆmL
)1
) seem to be insufficient to
promote both the HSPG-dependent and the external
plasma membrane pathways.
Considering that the cellular sensitivities to exogen-
ously added vs. endogenously produced sPLA
2
s differ
considerably [17–28,32,33] and that the majority of
sPLA
2
s resides in the Golgi (and ER) in these cells,
the possibility that sPLA
2
s could act intracellularly
without requirement for prior secretion should be
taken into account, as recently proposed [43]. This
model can explain why exogenously added sPLA
2
sis
orders of magnitude less efficient at phospholipid
hydrolysis than those produced within the cell; the
concentration of sPLA
2
s (and even of the phospho-
lipids substrates) within the secretory compartments
may be orders of magnitude higher than those secreted
and dispersed into the extracellular medium. In this
Fig. 9. Immunocytostaining of sPLA

2
s adenovirally expressed in
cultured synovial cells. Synovial cells infected with adenovirus for
sPLA
2
-IIA (A), -IID (B) or -X (C) were immunostained with respect-
ive antibodies in combination with FITC-conjugated secondary anti-
body. sPLA
2
-IIA (A) and -IID (B) were mainly localized in the
perinuclear Golgi apparatus (red arrows), and sPLA
2
-X resides in
the Golgi and ER (C). In IL-1b-stimulated cells, punctate signals for
sPLA
2
-IIA (A, middle), but not sPLA
2
-X (C, middle), appeared in the
cytoplasm, which was abrogated by treatment with 500 lgÆ mL
)1
heparin (A, lower). (D) Double immunostaining of sPLA
2
-IIA and the
Golgi marker GM130. Signals for sPLA
2
-IIA (green) and GM130
(red) were largely overlapped (yellow). (E) Double immunostaining
of sPLA
2

-IIA (green) and caveolin (red) in IL-1b-stimulated cells.
Caveloin was located in the caveolae-derived vesicles and Golgi, as
has been reported previously [71,72]. Many, if not all, cytoplasmic
vesicles as well as Golgi showed colocalization of sPLA
2
-IIA and
caveolin. (F) Localization of endogenous COX-2 in the perinuclear
membrane of IL-1b-stimulated cells. Weak COX-2 signal around the
perinuclear membrane may represent the ER. Cells infected with
LacZ adenovirus showed no obvious staining for all antibodies used
(not shown). Representative results of three independent experi-
ments are shown.
A
B
D
E
F
C
S. Masuda et al. sPLA2s in human rheumatoid arthritis
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS 665
scenario, the AA released at the Golgi membrane by
the de novo synthesized sPLA
2
s is supplied to the peri-
nuclear COX-2 in synovial cells. It has been reported
that cPLA
2
a primarily targets to the Golgi membrane
[62]. Thus, this spatial location may allow efficient
functional coupling between PLA

2
and COX enzymes.
In addition, there might be alternative mechanism for
high sensitivity of synovial cells to sPLA
2
s; for
instance, unique membranous features (such as phos-
pholipid composition and asymmetry, curvature, ruf-
fling, oxidation, and putative accessory molecules)
might allow synovial cells to be susceptible to sPLA
2
s.
It has previously been shown that, in addition to
increasing AA release, high concentrations of exogen-
ous sPLA
2
s or overexpression of sPLA
2
s often
augment COX-2 induction, which contributes to
amplification of PGE
2
production, in several cell types,
including primary rheumatoid synovial fibroblasts
[19,20,47]. However, adenoviral expression of sPLA
2
s
in this study did not significantly affect the inducible
expression of COX-2 and mPGES-1 over control cells
(data not shown), probably because sPLA

2
expression
was adjusted to low ngÆmL
)1
levels or because COX-2
induction by sPLA
2
s is a cell type-specific event. Inter-
estingly, high concentrations of exogenous sPLA
2
-IIA
are capable of inducing COX-2 expression in synovial
fibroblasts obtained from RA patients [47], suggesting
that certain microenvironmental rheumatoid factor(s)
may allow synovial cells to express more COX-2 in
response to sPLA
2
-IIA.
Nevertheless, given that the levels of sPLA
2
-IIA
often reach the order of lgÆmL
)1
in RA tissues [41,42],
it is likely that the multiple sPLA
2
s expressed in RA
tissues can contribute to arthritic inflammation by aug-
menting PGE
2

production. In support of this, injection
of sPLA
2
-IIA into rat adjuvant arthritis tissues leads
to exacerbation of edema [63], and exogenous addition
of sPLA
2
-IIA to rheumatoid synoviocytes results in
increased generation of PGE
2
, an effect reversed by an
sPLA
2
inhibitor [47,64]. Moreover, sPLA
2
-V-deficient
mice exhibit reduced inflammatory response, which is
accompanied by reduction of eicosanoids [65]. Because
collagen-induced arthritis occurs only mildly in
cPLA
2
a knockout mice [50], cPLA
2
a and sPLA
2
s may
cooperate in the process of this disease, thereby contri-
buting to amplification of the inflammatory cascades.
Indeed, functional cross-talk between cPLA
2

a and
sPLA
2
s has been observed in various cell types
[24,29,30,66]. Also, sPLA
2
s may release AA and lyso-
phospholipids from microvesicles shed from activated
cells, which are enriched in RA fluid [67].
In active RA tissues, sPLA
2
-IID is mainly located in
the lymph follicles (Fig. 3C–E). This location is remi-
niscent of the fact that sPLA
2
-IID is expressed in the
spleen and lymph nodes (second lymphoid organs)
[7,9,68], allowing us to speculate that this enzyme may
have a unique regulatory role for lymphoid cells and
tissues. Related to this view, sPLA
2
-IID expression is
dramatically altered in mice deficient in lymphotoxin a
[68], a cytokine that plays a crucial role in lymph node
development [69]. sPLA
2
-IIE is predominantly distri-
buted in the arterial VSMC of active RA tissues
(Fig. 4B). In our preliminary study, sPLA
2

-IIE immu-
noreactivity is also distributed rather specifically in
VSMC of other organs, such as heart, mammary
gland, gastrointestinal tract and male reproductive
organs (S Masuda, M Murakami, Y Ishikawa, T Ishii
and I Kudo, unpublished observations). Thus, sPLA
2
-
IIE might play specific roles in the regulation of
VSMC functions.
Experimental procedures
Materials
Normal human synovial cells and culture medium (CS-C
Complete Medium kit 4Z0-500) were obtained from Cell
Systems (Kirkland, WA, USA). The cells were maintained
on collagen-coated six-well plates (Iwaki Glass Co., Tokyo,
Japan) and were used within three passages after thawing.
Rabbit antisera for individual human sPLA
2
s were des-
cribed previously [70,71]. The specificity of these anti-sPLA
2
to individual sPLA
2
s was verified by immunoblotting with
various sPLA
2
-transfected cells [70,71]. Goat anti-human
COX-1 and anti-human COX-2, rabbit anti-human group
IVA cPLA

2
a, and goat anti-human caveolin-2 (sc-1858)
were purchased from Santa Cruz (Santa Cruz, CA, USA).
Rabbit antibodies against cPGES and mPGES-1 and -2
have been described previously [45,72,73]. Goat anti-human
GM130 in the Organelle Sampler Kit was obtained from
Transduction Laboratories (Newington, NH, USA).
cDNAs for sPLA
2
s and cPLA
2
a have been described
previously [17–21]. Human IL-1b, TNFa and IFN-c were
purchased from Genzyme (Boston, MA, USA). Fluorescein
isothiocyanate-, Cy3-, and horseradish peroxidase-conju-
gated anti-IgG were purchased from Zymed (South San
Francisco, CA, USA). Heparin and heparinase (Flavobacte-
rium heparinum) were obtained from Sigma (St. Louis, MO,
USA). Primers for RT-PCR were from Greiner Japan
(Tokyo, Japan).
Northern blotting
Equal amounts ( 5 lg) of total RNA obtained from cells
by use of TRIzol reagent (Invitrogen, San Diego, CA, USA)
were applied to separate lanes of 1.2% (w ⁄ v) formaldehyde–
agarose gels, electrophoresed, and transferred to Immobi-
sPLA2s in human rheumatoid arthritis S. Masuda et al.
666 FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
lon-N membranes (Millipore, Bedford, MA, USA). The
resulting blots were then probed with their respective cDNA
probes that had been labeled with [

32
P]dCTP (Amersham
Bioscience, UK) by random priming (Takara Biomedicals,
Ohtsu, Japan). Hybridization and subsequent membrane
washing were carried out as described previously [17–21].
RT-PCR
Synthesis of cDNAs was performed with 0.5 lg of total
RNA from human cell lines or tissues and AMV reverse
transcriptase, according to the manufacturerı
´
s instructions
supplied with the RNA PCR kit (Takara Biomedicals).
Subsequent amplification of the cDNA fragments was per-
formed using 0.5 lL of the reverse-transcribed mixture as a
template with specific primers for each sPLA
2
. For amplifi-
cation of sPLA
2
-IB, -IIA, -IID, -IIE, -IIF, -V, and -X
cDNAs, we used a set of 23-bp oligonucleotide primers cor-
responding to the 5¢- and 3¢-nucleotide sequences of their
open reading frames. The PCR conditions for sPLA
2
-IB,
-IIA, -IID, -IIE, -V, and -X were 94 °C for 30 s and then
30–33 cycles of amplification at 94 °C for 5 s and 68 °C for
4 min, using the Advantage cDNA polymerase mix (Clon-
tech, Palo Alto, CA, USA) [23,24]. The PCR conditions for
sPLA

2
-IIF were 94 °C for 30 s and then 35 cycles of ampli-
fication at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for
30 s, using ExTaq polymerase (Takara Biomedicals) [23,24].
The PCR products were analyzed by 1% agarose gel elec-
trophoresis with ethidium bromide. The gels were further
subjected to Southern blot hybridization using sPLA
2
cDNAs as probe, as required for the experiments. RT-PCR
for mPGES-1 was performed as described previously [45].
SDS ⁄ PAGE immunoblotting
Lysates from 10
5
cultured cells or 20 lg protein equivalents
of tissue homogenates in phosphate-buffered saline
(NaCl ⁄ P
i
) were subjected to SDS ⁄ PAGE using 7.5% (for
cPLA
2
a and COXs), 12.5% (for PGESs), and 15% (for
sPLA
2
s) gels under reducing conditions. The separated pro-
teins were electroblotted onto nitrocellulose membranes
(Schleicher and Schuell, Keene, Germany) using a semidry
blotter (MilliBlot-SDE system; Millipore). After blocking
with 3% (w ⁄ v) skim-milk in NaCl ⁄ P
i
containing 0.05%

Tween-20 (NaCl ⁄ P
i
⁄ Tween), the membranes were probed
with the respective antibodies for 2 h. Dilutions of the anti-
bodies in NaCl ⁄ P
i
⁄ Tween were as follows: cPLA
2
a, COX-
2, cPGES, and mPGES-2, 1 : 5000; COX-1, 1 : 10 000; and
sPLA
2
s and mPGES-1, 1 : 2000. After three washes with
NaCl ⁄ P
i
–Tween, the membranes were incubated with horse-
radish peroxidase-conjugated anti-goat or anti-rabbit IgG
(1 : 5000 dilution in NaCl ⁄ P
i
⁄ Tween) for 2 h, washed six
times, and were visualized using the ECL western blot sys-
tem (NEN
TM
Life Science Products, Boston, MA, USA), as
described previously [17–21].
Immunohistochemistry
Synovial tissue sections were obtained from RA patients (all
female, 68–74 years old) undergoing surgery at Toho Uni-
versity Ohmori Hospital following approval from the ethical
committee of the Faculty and informed consent from the

patients. All patients were RA factor-seropositive cases
according to the RA criteria [74] and were treated temporar-
ily with steroid and nonsteroidal anti-inflammatory drugs
for similar periods (> 10 years) and in similar ways. RA
states were evaluated basically on the morphology of the tis-
sue sections; in the ‘active RA’ cases, outgrowth of synovial
cells, formation of lymph follicles, and infiltration of
lymphocytes and plasma cells were obvious, whereas these
features were poorly observed in ‘inactive RA’ cases.
Immunohistochemistry was performed as described previ-
ously [48,49]. Briefly, the tissue sections (4 lm thick) were
incubated with Target Retrieval Solution (DAKO, Carpin-
tenia, CA, USA) as required, incubated for 10 min with
3% (v ⁄ v) H
2
O
2
, washed three times with NaCl ⁄ P
i
for 5 min
each, incubated with 5% (v ⁄ v) skim milk for 30 min,
washed three times with NaCl ⁄ P
i
⁄ Tween for 5 min each,
and incubated for 2 h with anti-human sPLA
2
(1 : 200–500
dilutions) in NaCl ⁄ P
i
. The sections were treated with a

CSA system staining kit (DAKO) with diaminobenzidine
substrate. The cell type was identified from conventional
hematoxylin and eosin staining of serial sections adjacent
to the specimen used for immunohistochemistry.
Expression of PLA
2
s by the adenovirus system
Adenovirus bearing each PLA
2
cDNA was prepared with
a ViraPower Adenovirus Expression System (Invitrogen)
according to the manufacturers instructions. Briefly, the
full-length cDNAs for sPLA
2
s and cPLA
2
a, amplified by
PCR with Pyrobest proofreading polymerase (Takara Bio-
medicals), were subcloned into the pENTER ⁄ D-TOPO
vector with a pENTER Directional TOPO Cloning kit
(Invitrogen). After purification of the plasmids from the
transformed Top10 competent cells (Invitrogen), the
sequences of the cDNA inserts were verified with a Taq
cycle sequencing kit (Takara Biomedicals) and an auto-
fluorometric DNA sequencer (310 Genetic Analyzer;
Applied Biosystems, Foster City, CA, USA). The cDNA
inserts were then transferred to the pAd ⁄ CMV ⁄ V5-DEST
vector (Invitrogen) by means of the Gateway system using
LR clonase (Invitrogen). After purification from the trans-
formed Top10 cells, the plasmids were linearized by diges-

tion with PacI (New England BioLabs, Bervery, MA, USA)
and transfected into subconfluent 293A cells (Invitrogen)
with Lipofectamine 2000 (Invitrogen) in Opti-MEM med-
ium (Invitrogen). After 1–2 weeks of culture in RPMI-1640
containing 10% fetal bovine serum until most cells had floa-
ted, the culture medium and cells were harvested together,
freeze-thawed twice, and centrifuged at 2 000 g for 5 min at
S. Masuda et al. sPLA2s in human rheumatoid arthritis
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS 667
4 °C to obtain the adenovirus-enriched supernatants. Aliqu-
ots of the supernatants were added to fresh 293A cells, and
the culture was continued for appropriate periods in order
to amplify adenoviruses. After 2–4 cycles of amplification,
the resulting adenovirus-containing supernatants were
used as virus stocks. Viral titers were determined by the
plaque-forming assay with 293A cells. As a control, the
pAd ⁄ CMV ⁄ V5-GW lacZ vector (Invitrogen) was trans-
fected into 293 A cells to produce LacZ-bearing adenovirus.
Cell culture experiments using adenovirus
Human synovial cells were seeded into 24-well plates and
cultured to near confluency. After replacing with fresh cul-
ture medium, aliquots of adenoviruses for individual PLA
2
s
and controls were added to each well, and the culture was
continued for 36 h. After replacing with fresh culture med-
ium, the cells were incubated with or without 1 ngÆmL
)1
IL-1b, 100 UÆmL
)1

TNFa or 10 ngÆmL
)1
IFN-c in 250 lL
of culture medium per well for 12 h. The supernatants were
then taken for PGE
2
measurement using a PGE
2
enzyme
immunoassay kit (Cayman Chemicals, Ann Arbor, MI,
USA) or for PLA
2
enzyme assay, and cells were subjected
to northern and western blotting to assess the expression of
individual PLA
2
s or other related enzymes. Replicate
adenovirus-infected cells were incubated for 30 min with
medium containing 1 m NaCl, and PLA
2
activities solubi-
lized into the supernatants were measured [17–21].
Measurement of sPLA
2
activity
sPLA
2
activity was assayed by measuring the amounts
of radiolabeled linoleic acid released from the substrate
1-palmitoyl-2-[

14
C]linoleoyl-phosphatidylethanolamine (Amer-
sham Bioscience). The substrate in ethanol was dried under
a stream of N
2
and dispersed in water by sonication. Each
reaction mixture (total volume of 250 l L) consisted of
appropriate amounts of the required sample, 100 mm
Tris ⁄ HCl (pH 7.4), 4 mm CaCl
2
and 10 lm substrate. After
incubation for 20 min at 37 °C, [
14
C]linoleic acid was
extracted by Dole’s method, and the radioactivity was
quantified by liquid scintillation counting, as described pre-
viously [17–21]. For rough quantification of individual
sPLA
2
s released from the cells into the culture super-
natants, the activities of pure recombinant sPLA
2
s (provi-
ded by MH Gelb, University of Washington) diluted with
synovial cell culture medium were measured.
Confocal laser microscopy
Cells grown in glass-bottomed dishes (Matsunami Glass,
Tokyo, Japan) precoated with 10 lgÆmL
)1
fibronectin (Sig-

ma) were fixed with 3% (v ⁄ v) paraformaldehyde for 30 min
in NaCl ⁄ P
i
. Cells were infected with adenoviruses bearing
PLA
2
s for 2 days and then stimulated with IL-1b before
fixation, as required for the experiments. After three washes
with NaCl ⁄ P
i
, the fixed cells were sequentially treated with
1% (w ⁄ v) bovine serum albumin (for blocking) containing
0.1% (w ⁄ v) saponin (for permeabilization) in NaCl ⁄ P
i
for
1 h, with anti-sPLA
2
and anti-COX-2 1 h in NaCl ⁄ P
i
con-
taining 1% albumin (1 : 200–500 dilutions), and then with
FITC-conjugated anti-(rabbit IgG) for 1 h in NaCl ⁄ P
i
con-
taining 1% albumin (1 : 200 dilution), with three washes
with NaCl ⁄ P
i
at each interval. For control staining, sPLA
2
-

expressing cells were treated with normal rabbit IgG or
cells infected with lacZ-adenoviruses were treated with anti-
sPLA
2
, with which fluorescent signals were negligible (data
not shown). For double immunostaining, cells stained with
anti-sPLA
2
were incubated with goat anti-GM130 (1 : 250
dilution) or anti-caveolin-2 (1 : 100 dilution) for 2 h, fol-
lowed by incubation with Cy3-conjugated anti-(goat IgG)
(1 : 100 dilution) for 2 h. After six washes with NaCl ⁄ P
i
,
the fluorescent signal was visualized with a laser scanning
confocal microscope (IX70; Olympus, Tokyo, Japan), as
described previously [19,21].
Acknowledgements
We would like to thank Drs M H Gelb (University of
Washington, Seattle, WA) and G Lambeau (CNRS-
UPR 411, Sophia Antipolis, France) for providing us
cDNAs, recombinant proteins and antibodies for
sPLA
2
s. This work was supported by grants-in aid for
scientific research from the Ministry of Education, Sci-
ence, Culture, Sports and Technology of Japan.
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