Various secretory phospholipase A2 enzymes are expressed in rheumatoid arthritis and augment prostaglandin production in cultured synovial cells Seiko Masuda1, Makoto Murakami1, Kazuo Komiyama2, Motoko Ishihara3, Yukio Ishikawa3, Toshiharu Ishii3 and 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
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
in cultured synoviocytes
sPLA2s
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- its correlation with disease states was IIF was partially positive, yet unclear. Expression of sPLA2 transcripts was also evident in cultured nor- in which sPLA2-IIA and -V were induced by mal human synoviocytes, interleukin-1 and sPLA2-X was expressed constitutively. Adenovirus- resulted in mediated expression of low ngÆmL)1 concentrations. increased prostaglandin E2 production at 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.
low molecular mass,
to have structural characteristics and are thought diverged from a common ancestor gene by successive gene duplication events. The expression of individual sPLA2s is tissue specific and often stimulus inducible leading to the proposal that they may play [3–15], tissue tissue-specific functions during inflammation,
is a group of Secretory phospholipase A2 (sPLA2) disulfide-rich, 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 these XIIA) have been identified in mammals. Of enzymes, sPLA2s in the I ⁄ II ⁄ V ⁄ X branch share many
Abbreviations AA, arachidonic acid; COX, cyclooxygenase; cPGES, cytosolic prostaglandin E synthase; cPLA2, cytosolic PLA2; ER, endoplasmic reticulum; HSPG, heparan sulfate proteoglycan; IFN-c, interferon-c; IL-1b, interleukin-1b; mPGES, membrane-bound prostaglandin E synthase; NaCl ⁄ Pi, phosphate-buffered saline; PtdCho, phosphatidylcholine; PG, prostaglandin; RA, rheumatoid arthritis; sPLA2, secretory phospholipase A2; TNFa, tumor necrosis factor a; VSMC, vascular smooth muscle cells.
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
655
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
augment prostaglandin E2 (PGE2) production in syn- ovial cells.
Results
is well established that
injury, 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
Detection of various sPLA2s in RA tissues by RT-PCR
functions of individual
sPLA2s sPLA2s display distinct
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- in inflamed tissues. The remarkable species- sent 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 in human pathology, the enzy- because matic activities toward phospholipids in mammalian cellular membranes [16–33], lung surfactant [34], bac- [35,36], and lipoprotein particles terial membranes [37,38].
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 the sPLA2-IIA tran- states [41,42,45]. As expected, 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- sPLA2-IB was not detected at all ting (Fig. 1A). (Fig. 1A).
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
Immunoblotting of the same RA samples with anti- bodies specific for individual sPLA2s yielded similar (Fig. 1B). Thus, 14–18 kDa immunoreactive results 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).
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
656
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
A
B
C
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 PGE2 synthesis in the two RA samples was assessed by immunoblotting.
Immunohistochemistry of RA tissues
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.
Given these observations, we aimed to determine the cellular localization of these sPLA2s in synovial tissues of RA patients by immunohistochemistry. A previous in RA tis- immunohistochemical study showed that, 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- lining cells in the section sely expressed in synovial 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].
sPLA2-IIF (Fig. 3Dc). These
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 results, stained for 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).
Staining of PLA2-IID was weak in a section of inac- tive RA tissues, in which scattered staining was located lymph aggregates (lymph follicles) in the subintimal (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- (Fig. 3C–E). Staining of tinct active RA sections
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
657
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
Immunohistochemical
localization of sPLA2-IIA in human Fig. 2. 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 lining cells (B and C dark arrowheads). Aggregates of in synovial 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).
tissues were negative for
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- sPLA2-V, whereas lage fibroblasts infiltrating into the cartilage tissues were intensely stained (Fig. 5Ec), thus exhibiting a reciprocal pattern compared with sPLA2-IIA (Fig. 2d).
chondrocytes
in all
sPLA2-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 samples (Fig. 6). In the subintimal interstitium, sPLA2-X stain- in the extracellular matrix fibers ing was evident (Fig. 6Cc) and neuronal fibers (Fig. 6Cd).
Expression of endogenous sPLA2s in cultured human synovial cells
In 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
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
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
658
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
E
A
C
B
D
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 sPLA2-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).
synovial fibroblasts
Indeed,
synovial 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. 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
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
659
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
A
C
dependence of PGE2 production in IL-1b-stimulated synovial cells was reported previously [48].
PGE2 production by sPLA2s in cultured human synovial cells
B
D
cell-surface bound) of
equivalent
Immunohistochemical
least
localizations of sPLA2-IIE (A,B) and Fig. 4. -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 In an inactive RA section, a few intimal cells (D) RA tissues. 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).
To examine the effect of individual sPLA2s on PGE2 production by cultured synoviocytes, these cells were for infected with adenoviruses harboring cDNAs 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- the expression of each sPLA2 and virus infection, 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 ((cid:1) 55, (cid:1) 30 and (cid:1) 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. 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 to adenoviruses were 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 two orders of magnitude lower than those in the pathologic range. On immuno- blotting, a 14 kDa sPLA2-IIA protein band was infected with sPLA2-IIA-bearing detected in cells immunoblot (Fig. 8C, upper). Similar adenovirus 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.
cells 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
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
660
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
C
E
A
B
D
Fig. 5. Immunohistochemical localizations of sPLA2-V in human joints affected by RA. (A,B) In two inactive RA sections, sPLA2-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).
the
catalytically inactive
the intracellular
localization of
To assess
that
As 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 sPLA2-IIA and -X for 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. 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 the punctate signals for sPLA2-IIA observed in IL-1b-stimulated synovial cells
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
661
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
A
C
D
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).
represent a pool of this enzyme sorted into caveolae- in an HSPG-dependent manner, derived vesicles 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).
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
662
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
A
B
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
C
D
E
sublining lesions
(Fig. 5)
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 PGE2-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) PGE2 production by synovial cells treated for 24 h with or without cyto- kines. (E) Time course of PGE2 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.
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 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 in the extracellular matrix is HSPG binding [20,21], 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.
the group II
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 subfamily sPLA2s and regulation of 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
Because 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 the cells with [19–21,28,31]. However, treatment of 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-
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
663
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
A
C
D
B
E
Fig. 8. Adenovirus-mediated transfer of PLA2s into human cultured synovial cells. (A) PGE2 generation by synovial cells infected with the indicated doses of adenoviruses for PLA2s or control (LacZ) for 36 h, followed by stimulation with IL-1b for 12 h. Expression of each PLA2 was verified by northern blotting (upper). (B) sPLA2 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 sPLA2s (multiplicity of infection [MOI] ¼ 10). Val- ues indicate the amounts of sPLA2s 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 sPLA2-IIA and -X for 36 h, and then stimulated for 12 h with IL-1b to assess PGE2 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)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 PGE2 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).
cell
type-specific
existence of
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)1 concentrations (Fig. 8). This sPLA2 sensitivity is remarkable because 100–1000 ngÆmL)1 or even more sPLA2s are generally required for triggering eicosanoid biosynthesis when
the 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 differentiation and maturation [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-
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
664
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
A
C
Immunocytostaining of sPLA2s adenovirally expressed in Fig. 9. cultured synovial cells. Synovial cells infected with adenovirus for sPLA2-IIA (A), -IID (B) or -X (C) were immunostained with respect- ive antibodies in combination with FITC-conjugated secondary anti- body. sPLA2-IIA (A) and -IID (B) were mainly localized in the perinuclear Golgi apparatus (red arrows), and sPLA2-X resides in the Golgi and ER (C). In IL-1b-stimulated cells, punctate signals for sPLA2-IIA (A, middle), but not sPLA2-X (C, middle), appeared in the cytoplasm, which was abrogated by treatment with 500 lgÆmL)1 heparin (A, lower). (D) Double immunostaining of sPLA2-IIA and the Golgi marker GM130. Signals for sPLA2-IIA (green) and GM130 (red) were largely overlapped (yellow). (E) Double immunostaining of sPLA2-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 sPLA2-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.
B
F
D
E
treatment of
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 sPLA2s (e.g. sPLA2-IIA and -IID) might be because the HSPG-shuttling pathway, which confers cellular sensitivity to HSPG-binding sPLA2s [17–21,25–31], is operative in synovial cells, whereas the ability of sPLA2-X and -IIF to increase PGE2 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 sPLA2s 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 PGE2 generation in synovial cells is unli- kely in our case because, even though a small fraction of HSPG-binding sPLA2s are located in caveolae-rich vesicles, cells with exogenous these heparin or heparinase, which perturbs the HSPG- dependent pathway [19–21,28,31], did not affect PGE2 production by these (Fig. 8E), and the sPLA2s amounts of sPLA2s 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.
the possibility that sPLA2s could act intracellularly without requirement for prior secretion should be taken into account, as recently proposed [43]. This model can explain why exogenously added sPLA2s is orders of magnitude less efficient at phospholipid hydrolysis than those produced within the cell; the concentration of sPLA2s (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
Considering that the cellular sensitivities to exogen- ously added vs. endogenously produced sPLA2s differ considerably [17–28,32,33] and that the majority of sPLA2s resides in the Golgi (and ER) in these cells,
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
665
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
synovial cells
to sPLA2s;
It has previously been shown that,
sPLA2s or overexpression of
niscent of the fact that sPLA2-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, sPLA2-IID expression is dramatically altered in mice deficient in lymphotoxin a [68], a cytokine that plays a crucial role in lymph node development [69]. sPLA2-IIE is predominantly distri- buted in the arterial VSMC of active RA tissues (Fig. 4B). In our preliminary study, sPLA2-IIE immu- noreactivity is also distributed rather specifically in VSMC of other organs, such as heart, mammary tract and male reproductive gland, gastrointestinal organs (S Masuda, M Murakami, Y Ishikawa, T Ishii and I Kudo, unpublished observations). Thus, sPLA2- IIE might play specific roles in the regulation of VSMC functions.
Experimental procedures
Materials
scenario, the AA released at the Golgi membrane by the de novo synthesized sPLA2s is supplied to the peri- nuclear COX-2 in synovial cells. It has been reported that cPLA2a primarily targets to the Golgi membrane location may allow efficient [62]. Thus, this spatial functional coupling between PLA2 and COX enzymes. In addition, there might be alternative mechanism for for high sensitivity of 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 sPLA2s. in addition to increasing AA release, high concentrations of exogen- sPLA2s often ous augment COX-2 induction, which contributes to amplification of PGE2 production, in several cell types, including primary rheumatoid synovial fibroblasts [19,20,47]. However, adenoviral expression of sPLA2s in this study did not significantly affect the inducible expression of COX-2 and mPGES-1 over control cells (data not shown), probably because sPLA2 expression was adjusted to low ngÆmL)1 levels or because COX-2 induction by sPLA2s is a cell type-specific event. Inter- estingly, high concentrations of exogenous sPLA2-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 sPLA2-IIA.
Nevertheless, given that
Northern blotting
the levels of sPLA2-IIA often reach the order of lgÆmL)1 in RA tissues [41,42], it is likely that the multiple sPLA2s expressed in RA tissues can contribute to arthritic inflammation by aug- menting PGE2 production. In support of this, injection of sPLA2-IIA into rat adjuvant arthritis tissues leads to exacerbation of edema [63], and exogenous addition of sPLA2-IIA to rheumatoid synoviocytes results in increased generation of PGE2, an effect reversed by an sPLA2 inhibitor [47,64]. Moreover, sPLA2-V-deficient mice exhibit reduced inflammatory response, which is accompanied by reduction of eicosanoids [65]. Because in collagen-induced arthritis occurs only mildly cPLA2a knockout mice [50], cPLA2a and sPLA2s may cooperate in the process of this disease, thereby contri- buting to amplification of the inflammatory cascades. functional cross-talk between cPLA2a and Indeed, sPLA2s has been observed in various cell types [24,29,30,66]. Also, sPLA2s may release AA and lyso- phospholipids from microvesicles shed from activated cells, which are enriched in RA fluid [67].
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 sPLA2s were des- cribed previously [70,71]. The specificity of these anti-sPLA2 to individual sPLA2s was verified by immunoblotting with various sPLA2-transfected cells [70,71]. Goat anti-human COX-1 and anti-human COX-2, rabbit anti-human group IVA cPLA2a, 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). for sPLA2s and cPLA2a have been described cDNAs 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).
In active RA tissues, sPLA2-IID is mainly located in the lymph follicles (Fig. 3C–E). This location is remi-
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
666
Equal amounts ((cid:1) 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-
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
Immunohistochemistry
RT-PCR
lon-N membranes (Millipore, Bedford, MA, USA). The resulting blots were then probed with their respective cDNA probes that had been labeled with [32P]dCTP (Amersham Bioscience, UK) by random priming (Takara Biomedicals, Ohtsu, Japan). Hybridization and subsequent membrane washing were carried out as described previously [17–21].
formation of
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, lymph follicles, and infiltration of lymphocytes and plasma cells were obvious, whereas these features were poorly observed in ‘inactive RA’ cases.
Expression of PLA2s by the adenovirus system
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) H2O2, washed three times with NaCl ⁄ Pi for 5 min each, incubated with 5% (v ⁄ v) skim milk for 30 min, washed three times with NaCl ⁄ Pi ⁄ Tween for 5 min each, and incubated for 2 h with anti-human sPLA2 (1 : 200–500 dilutions) in NaCl ⁄ Pi. 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.
SDS ⁄ PAGE immunoblotting
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 sPLA2. For amplifi- cation of sPLA2-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 sPLA2-IB, -IIA, -IID, -IIE, -V, and -X were 94 (cid:1)C for 30 s and then 30–33 cycles of amplification at 94 (cid:1)C for 5 s and 68 (cid:1)C for 4 min, using the Advantage cDNA polymerase mix (Clon- tech, Palo Alto, CA, USA) [23,24]. The PCR conditions for sPLA2-IIF were 94 (cid:1)C for 30 s and then 35 cycles of ampli- fication at 94 (cid:1)C for 30 s, 58 (cid:1)C for 30 s, and 72 (cid:1)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 sPLA2 cDNAs as probe, as required for the experiments. RT-PCR for mPGES-1 was performed as described previously [45].
tissue homogenates phosphate-buffered
cells competent (Invitrogen),
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
667
Adenovirus bearing each PLA2 cDNA was prepared with a ViraPower Adenovirus Expression System (Invitrogen) according to the manufacturers instructions. Briefly, the full-length cDNAs for sPLA2s and cPLA2a, 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 the transformed Top10 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 Lysates from 105 cultured cells or 20 lg protein equivalents of saline in (NaCl ⁄ Pi) were subjected to SDS ⁄ PAGE using 7.5% (for cPLA2a and COXs), 12.5% (for PGESs), and 15% (for sPLA2s) 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 ⁄ Pi containing 0.05% Tween-20 (NaCl ⁄ Pi ⁄ Tween), the membranes were probed with the respective antibodies for 2 h. Dilutions of the anti- bodies in NaCl ⁄ Pi ⁄ Tween were as follows: cPLA2a, COX- 2, cPGES, and mPGES-2, 1 : 5000; COX-1, 1 : 10 000; and sPLA2s and mPGES-1, 1 : 2000. After three washes with NaCl ⁄ Pi–Tween, the membranes were incubated with horse- radish peroxidase-conjugated anti-goat or anti-rabbit IgG (1 : 5000 dilution in NaCl ⁄ Pi ⁄ Tween) for 2 h, washed six times, and were visualized using the ECL western blot sys- tem (NENTM Life Science Products, Boston, MA, USA), as described previously [17–21].
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
adenovirus-containing resulting
Cell culture experiments using adenovirus
(Invitrogen) was 4 (cid:1)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, supernatants were the 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 trans- fected into 293 A cells to produce LacZ-bearing adenovirus.
PLA2s for 2 days and then stimulated with IL-1b before fixation, as required for the experiments. After three washes with NaCl ⁄ Pi, 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 ⁄ Pi for 1 h, with anti-sPLA2 and anti-COX-2 1 h in NaCl ⁄ Pi con- taining 1% albumin (1 : 200–500 dilutions), and then with FITC-conjugated anti-(rabbit IgG) for 1 h in NaCl ⁄ Pi con- taining 1% albumin (1 : 200 dilution), with three washes with NaCl ⁄ Pi at each interval. For control staining, sPLA2- expressing cells were treated with normal rabbit IgG or cells infected with lacZ-adenoviruses were treated with anti- sPLA2, with which fluorescent signals were negligible (data not shown). For double immunostaining, cells stained with anti-sPLA2 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 ⁄ Pi, the fluorescent signal was visualized with a laser scanning confocal microscope (IX70; Olympus, Tokyo, Japan), as described previously [19,21].
Acknowledgements
recombinant proteins and antibodies
Measurement of sPLA2 activity
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, for sPLA2s. This work was supported by grants-in aid for scientific research from the Ministry of Education, Sci- ence, Culture, Sports and Technology of Japan.
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 PLA2s 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 PGE2 measurement using a PGE2 enzyme (Cayman Chemicals, Ann Arbor, MI, immunoassay kit USA) or for PLA2 enzyme assay, and cells were subjected to northern and western blotting to assess the expression of individual PLA2s or other related enzymes. Replicate adenovirus-infected cells were incubated for 30 min with medium containing 1 m NaCl, and PLA2 activities solubi- lized into the supernatants were measured [17–21].
References
1 Murakami M & Kudo I (2001) Diversity and regulatory functions of mammalian secretory phospholipase A2s. Adv Immunol 77, 163–194. 2 Kudo I & Murakami M (2002) Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat 68–69, 3–58. 3 Nakano T, Ohara O, Teraoka H & Arita H (1990)
Glucocorticoids suppress group II phospholipase A2 pro- duction by blocking mRNA synthesis and post-transcrip- tional expression. J Biol Chem 265, 12745–12748.
Confocal laser microscopy
sPLA2 activity was assayed by measuring the amounts of radiolabeled linoleic acid released from the substrate 1-palmitoyl-2-[14C]linoleoyl-phosphatidylethanolamine (Amer- sham Bioscience). The substrate in ethanol was dried under a stream of N2 and dispersed in water by sonication. Each reaction mixture (total volume of 250 lL) consisted of the required sample, 100 mm appropriate amounts of Tris ⁄ HCl (pH 7.4), 4 mm CaCl2 and 10 lm substrate. After [14C]linoleic acid was incubation for 20 min at 37 (cid:1)C, 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 sPLA2s released from the cells into the culture super- natants, the activities of pure recombinant sPLA2s (provi- ded by MH Gelb, University of Washington) diluted with synovial cell culture medium were measured. 4 Chen J, Engle SJ, Seilhamer JJ & Tischfield JA (1994) Cloning and recombinant expression of a novel human low molecular weight Ca2+-dependent phospholipase A2. J Biol Chem 269, 2365–2368.
5 Chen J, Engle SJ, Seilhamer JJ & Tischfield JA (1994) Cloning and characterization of novel rat and mouse low molecular weight Ca2+-dependent phospholipase A2s containing 16 cysteines. J Biol Chem 269, 23018– 23024.
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
668
6 Cupillard L, Koumanov K, Mattei MG, Lazdunski M & Lambeau G (1997) Cloning, chromosomal mapping, 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 ⁄ Pi. Cells were infected with adenoviruses bearing
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
and expression of a novel human secretory phospholi- pase A2. J Biol Chem 272, 15745–15752. secretory phospholipase A2s are functionally redundant and act in concert with cytosolic phospholipase A2. J Biol Chem 273, 14411–14423.
7 Ishizaki J, Suzuki N, Higashino K, Yokota Y, Ono T, Kawamoto K, Fujii N, Arita H & Hanasaki K (1999) Cloning and characterization of novel mouse and human secretory phospholipase A2s. J Biol Chem 274, 24973–24979. 18 Murakami M, Kambe T, Shimbara S & Kudo I (1999) Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prosta- noid biosynthetic pathways. J Biol Chem 274, 3103– 3115. 19 Murakami M, Kambe T, Shimbara S, Yamamoto S,
8 Suzuki N, Ishizaki J, Yokota Y, Higashino K, Ono T, Ikeda M, Fujii N, Kawamoto K & Hanasaki K (2000) Structures, enzymatic properties, and expression of novel human and mouse secretory phospholipase A2s. J Biol Chem 275, 5785–5793.
Kuwata H & Kudo I (1999) Functional association of type IIA secretory phospholipase A2 with the glycosyl phosphatidylinositol-anchored heparan sulfate proteo- glycan in the cyclooxygenase-2-mediated delayed prostanoid biosynthetic pathway. J Biol Chem 274, 29927–29936. 20 Murakami M, Kambe T, Shimbara S, Higashino K, 9 Valentin E, Ghomashchi F, Gelb MH, Lazdunski M & Lambeau G (1999) On the diversity of secreted phos- pholipases A2. Cloning, tissue distribution, and func- tional expression of two novel mouse group II enzymes. J Biol Chem 274, 31195–31202. 10 Valentin E, Ghomashchi F, Gelb MH, Lazdunski M &
Lambeau G (2000) Novel human secreted phospholipase A2 with homology to the group III bee venom enzyme. J Biol Chem 275, 7492–7496.
Hanasaki K, Arita H, Horiguchi M, Arita M, Arai H, Inoue K et al. (1999) Different functional aspects of the group II subfamily (types IIA and V) and type X secre- tory phospholipase A2s in regulating arachidonic acid release and prostaglandin generation: implication of cyclooxygenase-2 induction and phospholipid scrambla- se-mediated cellular membrane perturbation. J Biol Chem 274, 31435–31444. 11 Gelb MH, Valentin E, Ghomashchi F, Lazdunski M & Lambeau G (2000) Cloning and recombinant expression of a structurally novel human secreted phospholipase A2. J Biol Chem 275, 39823–39826.
12 Ho IC, Arm JP, Bingham CO 3rd, Choi A, Austen KF & Glimcher L (2001) A novel group of phospholipase A2s preferentially expressed in type 2 helper T cells. J Biol Chem 276, 18321–18326.
21 Murakami M, Koduri RS, Enomoto A, Shimbara S, Seki M, Yoshihara K, Singer A, Valentin E, Ghoma- shchi F, Lambeau G et al. (2001) Distinct arachidonate- releasing functions of mammalian secreted phospholipase A2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms. J Biol Chem 271, 30041–30051. 22 Murakami M, Yoshihara K, Shimbara S, Lambeau G, 13 Sawada H, Murakami M, Enomoto A, Shimbara S & Kudo I (1999) Regulation of type V phospholipase A2 expression and function by proinflammatory stimuli. Eur J Biochem 263, 826–835.
Gelb MH, Singer AG, Sawada M, Inagaki N, Nagai H, Ishihara M et al. (2002) Cellular arachidonate-releasing function and inflammation-associated expression of group IIF secretory phospholipase A2. J Biol Chem 277, 19145–19155. 23 Murakami M, Masuda S, Shimbara S, Bezzine S, 14 Murakami M, Yoshihara K, Shimbara S, Sawada M, Inagaki N, Nagai H, Naito M, Tsuruo T, Moon TC, Chang HW et al. (2002) Group IID heparin-binding secretory phospholipase A2 is expressed in human colon carcinoma cells and human mast cells and upregulated in mouse inflammatory tissues. Eur J Biochem 269, 2698–2707. 15 Murakami M, Yoshihara K, Shimbara S, Lambeau G,
Ladzunski M, Lambeau G, Gelb MH, Matsukura S, Kokubu F, Adachi M et al. (2003) Cellular arachido- nate-releasing function of novel classes of secretory phospholipase A2s (group III and XII). J Biol Chem 278, 10657–10667. 24 Hamaguchi K, Kuwata H, Yoshihara K, Masuda S, Singer A, Gelb MH, Sawada M, Inagaki N, Nagai H & Kudo I (2002) Arachidonate release and eicosanoid gen- eration by group IIE phospholipase A2. Biochem Bio- phys Res Commun 292, 689–696. 16 Pfeilschifter J, Schalkwijk C, Briner VA & van den
Shimbara S, Oh-ishi S, Murakami M & Kudo I (2003) Induction of distinct sets of secretory phospholipase A2 in rodents during inflammation. Biochim Biophys Acta 1635, 37–47. 25 Kim KP, Rafter JD, Bittova L, Han SK, Snitko Y, Bosch H (1993) Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. Its contribu- tion to arachidonic acid release and prostaglandin synthesis by cultured rat glomerular cells. J Clin Invest 92, 2516–2523.
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
669
17 Murakami M, Shimbara S, Kambe T, Kuwata H, Win- stead MV, Tischfield JA & Kudo I (1998) The functions of five distinct mammalian phospholipase A2s in regu- lating arachidonic acid release: type IIA and type V Munoz NM, Leff AR & Cho W (2001) Mechanism of human group V phospholipase A2 (PLA2)-induced leu- kotriene biosynthesis in human neutrophils. A potential role of heparan sulfate binding in PLA2 internalization and degradation. J Biol Chem 276, 11126–11134.
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
37 Sartipy P, Johansen B, Gasvik K & Hurt-Camejo E 26 Han SK, Kim KP, Koduri R, Bittova L, Munoz NM,
(2000) Molecular basis for the association of group IIA phospholipase A2 and decorin in human atherosclerotic lesions. Circ Res 86, 707–714. Leff AR, Wilton DC, Gelb MH & Cho W (1999) Roles of Trp31 in high membrane binding and proinflamma- tory activity of human group V phospholipase A2. J Biol Chem 274, 11881–11888.
38 Hurt-Camejo E, Camejo G, Peilot H, Oorni K & Kova- nen P (2001) Phospholipase A2 in vascular disease. Circ Res 89, 298–304.
27 Kim YJ, Kim KP, Rhee HJ, Das S, Rafter JD, Oh YS & Cho W (2002) Group V phospholipase A2 induces leukotriene biosynthesis in human neutrophils through the activation of group IVA phospholipase A2. J Biol Chem 277, 9358–9365.
39 MacPhee M, Chepenik KP, Liddell RA, Nelson KK, Siracusa LD & Buchberg AM (1995) The secretory phospholipase A2 gene is a candidate for the Mom1 locus, a major modifier of ApcMin-induced intestinal neoplasia. Cell 81, 957–966. 40 Enomoto A, Murakami M, Valentin E, Lambeau G,
28 Munoz NM, Kim YJ, Meliton AY, Kim KP, Han SK, Boetticher E, Oı´ Leary E, Myou S, Zhu X, Bonventre JV et al. (2003) Human group V phospholipase A2 induces group IVA phospholipase A2-independent cysteinyl leukotriene synthesis in human eosinophils. J Biol Chem 278, 38813–38820. 29 Balsinde J, Balboa MA & Dennis EA (1998) Functional Gelb MH & Kudo I (2000) Redundant and segregated functions of granule-associated heparin-binding group II subfamily of secretory phospholipases A2 in the regula- tion of degranulation and prostaglandin D2 synthesis in mast cells. J Immunol 165, 4007–4014.
coupling between secretory phospholipase A2 and cyclooxygenase-2 and its regulation by cytosolic group IV phospholipase A2. Proc Natl Acad Sci USA 95, 7951–7956. 41 Pruzanski W & Vadas P (1991) Phospholipase A2: a mediator between proximal and distal effectors of inflammation. Immunol Today 12, 143–146. 42 Kramer RM, Hession C, Johansen B, Hayes G,
McGray P, Chow EP, Tizard R & Pepinsky RB (1989) Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem 264, 5768–5775. 43 Mounier CM, Ghomashchi F, Lindsay MR, James S, 30 Shinohara H, Balboa MA, Johnson CA, Balsinde J & Dennis EA (1999) Regulation of delayed prostaglandin production in activated P388D1 macrophages by group IV cytosolic and group V secretory phospholipase A2s. J Biol Chem 274, 12263–12268.
31 Balboa MA, Shirai Y, Gaietta G, Ellisman MH, Balsin- de J & Dennis EA (2003) Localization of group V phos- pholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells. J Biol Chem 278, 48059– 48065. Singer AG, Parton RG & Gelb MH (2004) Arachidonic acid release from mammalian cells transfected with human groups IIA and X secreted phospholipase A2 occurs predominantly during the secretory process and with the involvement of cytosolic phospholipase A2a. J Biol Chem 279, 25024–25038. 44 Lambeau G & Lazdunski M (1999) Receptors for a
growing family of secreted phospholipases A2. Trends Pharmacol Sci 20, 162–170. 45 Murakami M, Nakashima K, Kamei D, Masuda S, 32 Hanasaki K, Ono T, Saiga A, Morioka Y, Ikeda M, Kawamoto K, Higashino K, Nakano K, Yamada K, Ishizaki J et al. (1999) Purified group X secretory phospholipase A2 induced prominent release of arachi- donic acid from human myeloid leukemia cells. J Biol Chem 274, 34203–34211. 33 Bezzine S, Koduri RS, Valentin E, Murakami M,
Ishikawa Y, Ishii T, Ohmiya Y, Watanabe K & Kudo I (2003) Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2. J Biol Chem 278, 37937– 37947. 46 Jamal OS, Conaghan PG, Cunningham AM, Brooks
Kudo I, Ghomashchi F, Sadilek M, Lambeau G & Gelb MH (2000) Exogenously added human group X secreted phospholipase A2 but not the group IB, IIA, and V enzymes efficiently release arachidonic acid from adherent mammalian cells. J Biol Chem 275, 3179– 3191. 34 Touqi L & Arbibe L (1999) A role for phospholipase
A2 in ARDS pathogenesis. Mol Med Today 5, 244–249. 35 Koduri RS, Gronroos JO, Laine VJ, Le Calvez C, Lam- beau G, Nevalainen TJ & Gelb MH (2002) Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A2. J Biol Chem 277, 5849– 5857. PM, Munro VF & Scott KF (1998) Increased expression of human type IIa secretory phospholipase A2 antigen in arthritic synovium. Ann Rheum Dis 57, 550–558. 47 Bidgood MJ, Jamal OS, Cunningham AM, Brooks PM & Scott KF (2000) Type IIA secretory phospholipase A2 up-regulates cyclooxygenase-2 and amplifies cytokine-mediated prostaglandin production in human rheumatoid synoviocytes. J Immunol 165, 2790–2797. 48 Hulkower KI, Wertheimer SJ, Levin W, Coffey JW,
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
670
36 Gronroos JO, Laine VJ, Janssen MJ, Egmond MR & Nevalainen TJ (2001) Bactericidal properties of group IIA and group V phospholipases A2. J Immunol 166, 4029–4034. Anderson CM, Chen T, DeWitt DL, Crowl RM, Hope WC & Morgan DW (1994) Interleukin-1b induces cyto- solic phospholipase A2 and prostaglandin H synthase in rheumatoid synovial fibroblasts. Evidence for their roles
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
60 Nabi IR & Le PU (2003) Caveolae ⁄ raft-dependent endocytosis. J Cell Biol 161, 673–677. in the production of prostaglandin E2. Arthritis Rheum 37, 653–661. 61 van Deurs B, Roepstorff K, Hommelgaard AM &
Sandvig K (2003) Caveolae: anchored, multifunctional platforms in the lipid ocean. Trends Cell Biol 13, 92–100. 49 Portanova JP, Zhang Y, Anderson GD, Hauser SD, Masferrer JL, Seibert K, Gregory SA & Isakson PC (1996) Selective neutralization of prostaglandin E2 blocks inflammation, hyperalgesia, and interleukin 6 production in vivo. J Exp Med 184, 883–891.
62 Evans JH & Leslie CC (2004) The cytosolic phospho- lipase A2 catalytic domain modulates association and residence time at Golgi membranes. J Biol Chem 279, 6005–6016. 63 Murakami M, Kudo I, Nakamura H, Yokoyama Y,
50 Mehindate K, al-Daccak R, Dayer JM, Kennedy BP, Kris C, Borgeat P, Poubelle PE & Mourad W (1995) Superantigen-induced collagenase gene expression in human IFN-c-treated fibroblast-like synoviocytes involves prostaglandin E2. Evidence for a role of cyclo- oxygenase-2 and cytosolic phospholipase A2. J Immunol 155, 3570–3577. Mori H & Inoue K (1990) Exacerbation of rat adjuvant arthritis by intradermal injection of purified mammalian 14-kDa group II phospholipase A2. FEBS Lett 268, 113–116.
51 Vane JP & Botting RM (1995) New insights into the mode of action of anti-inflammatory drugs. Inflamm Res 44, 1–10. 52 Hegen M, Sun L, Uozumi N, Kume K, Goad ME,
64 Triggiani M, Granata F, Oriente A, Gentile M, Petrar- oli A, Balestrieri B & Marone G (2002) Secretory phos- pholipases A2 induce cytokine release from blood and synovial fluid monocytes. Eur J Immunol 32, 67–76. 65 Satake Y, Diaz BL, Balestrieri B, Lam BK, Kanaoka
Nickerson-Nutter CL, Shimizu T & Clark JD (2003) Cytosolic phospholipase A2a-deficient mice are resistant to collagen-induced arthritis. J Exp Med 197, 1297– 1302.
Y, Grusby MJ & Arm JP (2004) Role of group V phos- pholipase A2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene dis- ruption. J Biol Chem 279, 16488–16494. 66 Han WK, Sapirstein A, Hung CC, Alessandrini A &
53 Myers LK, Kang AH, Postlethwaite AE, Rosloniec EF, Morham SG, Shlopov BV, Goorha S & Ballou LR (2000) The genetic ablation of cyclooxygenase 2 pre- vents the development of autoimmune arthritis. Arthritis Rheum 43, 2687–2693. 54 Trebino CE, Stock JL, Gibbons CP, Naiman BM,
Bonventre JV (2003) Cross-talk between cytosolic phos- pholipase A2a (cPLA2a) and secretory phospholipase A2 (sPLA2) in hydrogen peroxide-induced arachidonic acid release in murine mesangial cells: sPLA2 regulates cPLA2a activity that is responsible for arachidonic acid release. J Biol Chem 278, 24153–24163.
Wachtmann TS, Umland JP, Pandher K, Lapointe JM, Saha S, Roach ML et al. (2003) Impaired inflammatory and pain responses in mice lacking an inducible prosta- glandin E synthase. Proc Natl Acad Sci USA 100, 9044– 9049.
55 McCoy JM, Wicks JR & Audoly LP (2002) The role of prostaglandin E2 receptors in the pathogenesis of rheu- matoid arthritis. J Clin Invest 110, 651–658. 67 Fourcade O, Simon MF, Viode C, Rugani N, Leballe F, Ragab A, Fournie B, Sarda L & Chap H (1995) Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesi- cles shed from activated cells. Cell 80, 919–927.
68 Shakhov AN, Rubtsov AV, Lyakhov IG, Tumanov AV & Nedospasov SA (2000) SPLASH (PLA2IID), a novel member of phospholipase A2 family, is associated with lymphotoxin deficiency. Genes Immun 1, 191–199. 69 Tumanov AV, Grivennikov SI, Shakhov AN, Rybtsov 56 Siegle I, Klein T, Backman JT, Saal JG, Nusing RM & Fritz P (1998) Expression of cyclooxygenase 1 and cyclooxygenase 2 in human synovial tissue: differential elevation of cyclooxygenase 2 in inflammatory joint dis- eases. Arthritis Rheum 41, 122–129. 57 Thomas G, Bertrand F & Saunier B (2000) The differ-
ential regulation of group IIA and group V low molecu- lar weight phospholipases A2 in cultured rat astrocytes. J Biol Chem 275, 10876–10886. SA, Koroleva EP, Takeda J, Nedospasov SA & Kuprash DV (2003) Dissecting the role of lymphotoxin in lymphoid organs by conditional targeting. Immunol Rev 195, 106–116. 58 van der Helm HA, Buijtenhuijs P & van den Bosch H 70 Degousee N, Ghomashchi F, Stefanski E, Singer A,
(2001) Group IIA and group V secretory phospholipase A2: quantitative analysis of expression and secretion and determination of the localization and routing in rat mesangial cells. Biochim Biophys Acta 1530, 86–96. 59 Gurrieri S, Furstenberger G, Schadow A, Haas U, Smart BP, Borregaard N, Reithmeier R, Lindsay TF, Lichtenberger C, Reinisch W et al. (2002) Groups IV, V, and X phospholipases A2s in human neutrophils: role in eicosanoid production and gram-negative bac- terial phospholipid hydrolysis. J Biol Chem 277, 5061– 5073.
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
671
Singer AG, Ghomashchi F, Pfeilschifter J, Lambeau G, Gelb MH & Kaszkin M (2003) Differentiation-depe- ndent regulation of secreted phospholipases A2 in mur- ine epidermis. J Invest Dermatol 121, 156–164. 71 Ito M, Ishikawa Y, Kiguchi H, Komiyama K, Murakami M, Kudo I, Akasaka Y & Ishii T (2004) Intrahepatic distribution of type V secretory phospholipase A2
S. Masuda et al.
sPLA2s in human rheumatoid arthritis
expression under hepatocyte injuries by liver diseases. J Gastroenterol Hepatol 19, 1140–1149.
73 Kamei D, Murakami M, Nakatani Y, Ishikawa Y, Ishii T & Kudo I (2003) Potential role of microsomal prosta- glandin E synthase-1 in tumorigenesis. J Biol Chem 278, 19396–19405.
FEBS Journal 272 (2005) 655–672 ª 2005 FEBS
672
74 Clegg DO & Ward JR (1987) Diagnostic criteria in rheumatoid arthritis. Scand J Rheumatol Suppl 65, 3–11. 72 Tanioka T, Nakatani Y, Semmyo N, Murakami M & Kudo I (2000) Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J Biol Chem 275, 32775–32782.
