expressed in rheumatoid arthritis and augmentprostaglandin production in cultured synovial cells Seiko Masuda1, Makoto Murakami1, Kazuo Komiyama2, Motoko Ishihara3, Yukio Ishikawa3, Tosh
Trang 1expressed in rheumatoid arthritis and augment
prostaglandin production in cultured synovial cells
Seiko Masuda1, Makoto Murakami1, Kazuo Komiyama2, Motoko Ishihara3, Yukio Ishikawa3,
Toshiharu Ishii3and Ichiro Kudo1
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 A2 (sPLA2) is a group of
disulfide-rich, low molecular mass, lipolytic enzymes
with a His-Asp catalytic dyad [1,2] To date, 10 sPLA2
enzymes (IB, IIA, IIC, IID, IIE, IIF, III, V, X and
XIIA) have been identified in mammals Of these
enzymes, sPLA2s 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 sPLA2s 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 A2;
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: mako@pharm.showa-u.ac.jp
(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 A2 (sPLA2-IIA) is known to
be abundantly present in the joints of patients with rheumatoid arthritis (RA), expression of other sPLA2s in this disease has remained unknown
In this study, we examined the expression and localization of six sPLA2s (groups IIA, IID, IIE, IIF, V and X) in human RA Immunohistochemis-try of RA sections revealed that sPLA2-IIA was generally located in syn-ovial lining and sublining cells and cartilage chondrocytes, sPLA2-IID in lymph follicles and capillary endothelium, sPLA2-IIE in vascular smooth muscle cells, and sPLA2-V in interstitial fibroblasts Expression levels of these group II subfamily sPLA2s appeared to be higher in severe RA than
in inactive RA sPLA2-X was detected in synovial lining cells and intersti-tial fibers in both active and inactive RA sections Expression of sPLA2 -IIF was partially positive, yet its correlation with disease states was unclear Expression of sPLA2 transcripts was also evident in cultured nor-mal human synoviocytes, in which sPLA2-IIA and -V were induced by interleukin-1 and sPLA2-X was expressed constitutively Adenovirus-mediated expression of sPLA2s in cultured synoviocytes resulted in increased prostaglandin E2 production at low ngÆmL)1 concentrations Thus, multiple sPLA2s 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; sPLA2, secretory phospholipase A2; TNFa, tumor necrosis factor a; VSMC, vascular smooth muscle cells.
Trang 2injury, and cancer Although sPLA2s 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
sPLA2 activity [41], and enzyme purification and
molecular cloning studies have ascribed this activity
to sPLA2-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 sPLA2s
has raised a fundamental question of whether only
sPLA2-IIA is induced or other sPLA2s are also
pre-sent in inflamed tissues The remarkable
species-associated difference in the tissue distribution of
indi-vidual sPLA2s [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 sPLA2s in human pathology,
because individual sPLA2s 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 sPLA2s can release
cellular AA via at least three distinct mechanisms, the
occurrence of which appears to be cell type or stimulus
specific First, sPLA2s release AA intracellularly prior
to secretion [43] Second, after secretion into the
extra-cellular space, sPLA2s with high interfacial binding
capacity to phosphatidylcholine (e.g sPLA2-V and -X)
act on the phosphatidylcholine-rich outer plasma
mem-brane [20,21,25–29,32,33] Third, sPLA2s with affinity
for heparanoids (e.g sPLA2-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,
sPLA2s act as ligands for a transmembrane protein
called M-type sPLA2 receptor, which in turn activates
group IVA cytosolic PLA2a (cPLA2a) to initiate AA
metabolism [44]
In this study, we performed immunohistochemistry
with antibodies specific for each sPLA2 to evaluate the
expression and localization of six sPLA2s (IIA, IID,
IIE, IIF, V and X) in human joints affected by RA
We further examined the possible contribution of these
sPLA2s to AA metabolism in cultured normal human
synovial cells Our results indicate that these sPLA2s
are diversely expressed in RA tissues and are able to
augment prostaglandin E2 (PGE2) production in syn-ovial cells
Results
Detection of various sPLA2s in RA tissues
by RT-PCR
It is well established that human joints affected by
RA contain large amounts of sPLA2-IIA, and its expression levels are correlated with disease severity [41,42] In order to assess whether these tissues also express other sPLA2 enzymes, we initially performed RT-PCR with primers specific for individual sPLA2s (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 sPLA2-IIA and COX-2 (see below), which has been shown to correlate with the disease states [41,42,45] As expected, the sPLA2-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 sPLA2-IIA, diverse expression of other sPLA2s was also found in these samples Thus, sPLA2-IID and -IIE were detec-ted only in sample b, and sPLA2-V and -X were detected in both samples almost equally (Fig 1A) Expression of sPLA2-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) sPLA2-IB was not detected at all (Fig 1A)
Immunoblotting of the same RA samples with anti-bodies specific for individual sPLA2s yielded similar results (Fig 1B) Thus, 14–18 kDa immunoreactive bands for sPLA2-IIA, -V and -X were detected in both samples a and b, and those of sPLA2-IID and -IIE were detectable only in sample b (Fig 1B) sPLA2-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 cPLA2a, 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)
Trang 3Immunohistochemistry of RA tissues
Given these observations, we aimed to determine the
cellular localization of these sPLA2s in synovial tissues
of RA patients by immunohistochemistry A previous
immunohistochemical study showed that, in RA
tis-sues, sPLA2-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
sPLA2-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 sPLA2-IIA, whereas staining
of the infiltrating fibroblasts was weak (Fig 2D)
Vascular smooth muscle cells (VSMC) also provided
positive staining for sPLA2-IIA (Fig 2E) These
distri-butions of sPLA2-IIA in RA tissues are largely in
agreement with a previous study [46]
Staining of PLA2-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 sPLA2-IID
Prom-inent sPLA2-IID staining was evident in the lymphoid
aggregates and capillary endothelial cells in three
dis-tinct active RA sections (Fig 3C–E) Staining of
sPLA2-IID in synovial lining cells was also evident (Fig 3D,E), even though weak and less frequent than that of sPLA2-IIA (Fig 2B,C), -V, and -X (see below) Staining of cartilage chondrocytes was very weak (Fig 3E), compared with that of sPLA2-IIA (Fig 2A) Thus, sPLA2-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 sPLA2-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, sPLA2 -IIE is induced rather specifically in VSMC in synovial tissues with active RA
In inactive RA sections, sPLA2-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 sPLA2-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 sPLA2-IIF (Fig 3Dc) These results, together with the results of RT-PCR and western blot (Fig 1), implies that the expression of sPLA2-IIF in
RA is rather lower than that of other sPLA2s 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 sPLA2-V in inactive RA sections was either undetectable (Fig 5A) or very weak (Fig 5B)
Fig 1 Expression of sPLA2s and other PGE2-biosynthetic enzymes in human joints affected by RA Expression of sPLA2s 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 PGE2synthesis in the two RA samples was assessed by immunoblotting.
Trang 4In the latter case, weak staining was locally detected in
the interstitium (Fig 5B) Intense sPLA2-V
immuno-reactivity was observed in wide areas of three active
RA sections In all cases, sPLA2-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 sPLA2-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 sPLA2-V, whereas fibroblasts infiltrating into the cartilage tissues were intensely stained (Fig 5Ec), thus exhibiting a reciprocal pattern compared with sPLA2-IIA (Fig 2d)
sPLA2-X immunoreactivity was evident in two inac-tive (Fig 6A,B) and three acinac-tive (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, sPLA2-X stain-ing was evident in the extracellular matrix fibers (Fig 6Cc) and neuronal fibers (Fig 6Cd)
Expression of endogenous sPLA2s in cultured human synovial cells
We next used RT-PCR to examine the expression of these sPLA2s in cultured normal human synovial cells (a mixed population of synovial lining cells and inter-stitial fibroblasts) Although sPLA2-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 sPLA2s were also weakly induced by tumor necrosis factor (TNF)a, whereas the effect of interferon (IFN)-c was minimal sPLA2-X transcript was weakly but constitutively expressed in synoviocytes with no appreciable induc-tion by cytokines (Fig 7A) In contrast, sPLA2-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 sPLA2-IIA, -V and -X were located, whereas sPLA2-IID and -IIE were barely detected, in
Fig 2 Immunohistochemical localization of sPLA2-IIA in human joints affected by RA Staining of sPLA2-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), sPLA2-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 infiltratStain-ing into the cartilage was faint (D, red arrows).
Trang 5synovial lining cells and interstitial fibroblastic cells
(Figs 2–6) However, expression levels of endogenous
sPLA2-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
sPLA2-IIA and data not shown for sPLA2-V and -X),
suggesting that some additional factors, which may
exist in synovial tissue microenvironments, are further
required for optimal sPLA2 induction in normal
syn-ovial cells Indeed, synsyn-ovial fibroblasts from RA
patients express sPLA2-IIA protein in primary culture
[47]
As assessed by immunoblotting, cPLA2a, 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 E2 (PGE2) generation over 24 h, whereas TNFa and IFN-c exhibited poor PGE2-biosynthetic effects (Fig 7D) Time course experiments showed that the accu-mulation of PGE2 in the medium of IL-1b-stimulated
E
Fig 3 Immunohistochemical localization of sPLA2-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, sPLA2-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).
Trang 6cells reached a plateau peak over 12–24 h (Fig 7E).
IL-1b-stimulated PGE2 generation was suppressed by
the cPLA2 inhibitor methyl arachidonoyl
fluorophos-phate by > 80%, suggesting the contribution of
cPLA2a to this biosynthetic response Indeed, cPLA2a
dependence of PGE2 production in IL-1b-stimulated synovial cells was reported previously [48]
PGE2production by sPLA2s in cultured human synovial cells
To examine the effect of individual sPLA2s on PGE2 production by cultured synoviocytes, these cells were infected with adenoviruses harboring cDNAs for sPLA2-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 sPLA2 -IID and -IIF, which were not intrinsically expressed in this cell type (Figs 3 and 4), and with cPLA2a, which was used as a positive control for increased PGE2 pro-duction, using the same strategy After 36 h of adeno-virus infection, the expression of each sPLA2 and cPLA2a 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 sPLA2s Significant portions of sPLA2-IIA, -IID and -V ( 55, 30 and 45%) were detected in the membrane-bound fractions, whereas sPLA2-IIF and -X was predominantly distributed in the supernatants (Fig 8B) These distribution patterns (supernatant vs cell-surface bound) of individual sPLA2s are consistent with those in several reports using other cell types [17–21] The concentrations of individual sPLA2s 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 sPLA2 standards (which were measured after dilution
in culture medium) (Fig 8B) Because the concentra-tions of sPLA2-IIA often reach the order of lgÆmL)1
in synovial fluids of RA patients [41,42], the levels of sPLA2s 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 sPLA2-IIA protein band was detected in cells infected with sPLA2-IIA-bearing adenovirus (Fig 8C, upper) Similar immunoblot results were obtained in cells infected with adenovirus for sPLA2-V and -X (data not shown) In the case of sPLA2-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 sPLA2s form a homodimer or undergo some post-translational modification (such as glycosy-lation) in cultured synovial cells, a possibility that is under investigation
Fig 4 Immunohistochemical localizations of sPLA 2 -IIE (A,B) and
-IIF (C,D) in human joints affected by RA Although sPLA2-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 sPLA2-IIE (Bd) Staining of
sPLA2-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 sPLA2-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 sPLA2-IIF (Dc).
Trang 7As shown in Fig 8A, IL-1b-stimulated production
of PGE2 was markedly augmented in cells transfected
with these sPLA2s and cPLA2a over that in control
cells in a manner dependent upon adenovirus doses
(i.e PLA2 expression levels) There was no increase in
PGE2 production in cells infected with adenoviruses
for the catalytically inactive sPLA2-IIA and -X
mutants (G30S; a mutation in the Ca2+-binding loop
[20] (Fig 8D), implying that the enzymatic activity is
essential for augmented PGE2 generation by sPLA2s
To assess the intracellular localization of these
sPLA2s in synovial cells, we performed
immunocyto-staining of cells that had been infected with
adeno-viruses for sPLA2s for 36 h and then incubated for an
additional 12 h with or without IL-1b Signals for
sPLA2-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 sPLA2-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 sPLA2-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 sPLA2-IIA, whereas the Golgi staining was unaffec-ted (Fig 9A, lower) These cytoplasmic punctate sig-nals for sPLA2-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 sPLA2-IID and -V (data not shown) These results suggest that the punctate signals for sPLA2-IIA observed in IL-1b-stimulated synovial cells
A
B
D
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 sPLA2-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).
Trang 8represent 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 sPLA2-X, an
HSPG-nonbinding enzyme [20,21], even after IL-1b
stimulation (Fig 9C, middle) Weak and diffused
stain-ing of sPLA2-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 PGE2 synthesis downstream of PLA2 [18–20], was located predominantly in the perinuclear membrane of IL-1b-stimulated cells (Fig 9F)
A
B
E
Fig 6 Immunohistochemical localizations of sPLA2-X in human joints affected by RA Staining of sPLA2-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).
Trang 9Because of the heparin-sensitive caveolae localization
of a small fraction of sPLA2-IIA (Fig 9A), we
antici-pated that this pool of the enzyme might contribute to
the promotion of PGE2 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 PGE2 generation by sPLA2-IIA (Fig 8E) or by
other sPLA2s (data not shown) This indicates that,
even though HSPG-dependent shuttling of sPLA2-IIA
(and other HSPG-binding sPLA2s) into caveolae
vesi-cles could occur in IL-1b-stimulated synovial cells, this event is not associated with increased PGE2 synthesis
by this enzyme under current experimental conditions
Discussion
Eicosanoids, especially PGE2, are critical mediators of
RA [49–51] Administration of PGE2 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 PGE2, inclu-ding cPLA2a [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 sPLA2-IIA as previously reported [41,42,46], various sPLA2s exist in human synovial tissues affected by
RA sPLA2-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 PLA2s in the PGE2 -biosy-nthetic pathway, are also expressed in synovial lining cells in the RA joints Distribution of sPLA2-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 sPLA2-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 sPLA2-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 sPLA2s and RA pathology, our results have opened new insights into the expression of mul-tiple sPLA2s in human inflammatory tissues
In cultured normal human synovial cells, expression
of sPLA2-IIA and -V is cytokine-dependent, whereas that of sPLA2-X is rather constitutive (Fig 7A) Simi-larly, more sPLA2-IIA, -IID, -IIE and -V are detected immunohistochemically in active RA than inactive RA tissues (Figs 2–5), while sPLA2-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 sPLA2s and sPLA2-X are distinct Importantly, even the induction
of individual group II subfamily sPLA2s requires dis-tinct cytokines in different cell types [57,58], implying
A
B
C
Fig 7 Expression of sPLA2s and other PGE2-biosynthetic enzymes
in human cultured synovial cells (A) Expression of endogenous
sPLA2s 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 PGE2production 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.
Trang 10the existence of cell type-specific transcriptional
machinery for each enzyme As shown in our series of
studies [14,23], expression of sPLA2-X in many types
of cells and tissues appears to be relatively constitutive,
even though elevated expression can occur in
associ-ation with cell differentiassoci-ation and maturassoci-ation [59]
Because sPLA2-X, but not group II subfamily sPLA2s,
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 sPLA2s, producing PGE2 in response to all sPLA2s when expressed at low ngÆmL)1concentrations (Fig 8) This sPLA2 sensitivity is remarkable because 100–1000 ngÆmL)1 or even more sPLA2s are generally required for triggering eicosanoid biosynthesis when
A
B
C
D
E
Fig 8 Adenovirus-mediated transfer of PLA2s into human cultured synovial cells (A) PGE2generation 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) sPLA2activities 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 sPLA2s (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 sPLA2s (C) Western blotting of synovial cells infected with adenovirus for sPLA2-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 sPLA2 In the case of sPLA2-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 sPLA2s was verified by northern blotting (inset) (E) Synovial cells infected with adenovirus for sPLA2-IIA or LacZ were preincu-bated with 500 lgÆmL)1heparin or 0.5 unitÆmL)1heparinase 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).