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

Various secretory phospholipase 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 Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan Department of Pathology, Division of Immunology and Patho-Biology at Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan 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 37848245 Tel: +81 37848197 E-mail: mako@pharm.showa-u.ac.jp (Received 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 Immunohistochemistry of RA sections revealed that sPLA2-IIA was generally located in synovial 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 interstitial fibers in both active and inactive RA sections Expression of sPLA2IIF was partially positive, yet its correlation with disease states was unclear Expression of sPLA2 transcripts was also evident in cultured normal human synoviocytes, in which sPLA2-IIA and -V were induced by interleukin-1 and sPLA2-X was expressed constitutively Adenovirusmediated expression of sPLA2s in cultured synoviocytes resulted in increased prostaglandin E2 production at low ngỈmL)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 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 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 sPLA2s in human rheumatoid arthritis injury, and cancer Although sPLA2s have been implicated in various biological events, including arachidonic 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 sepsis, 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 present in inflamed tissues The remarkable speciesassociated difference in the tissue distribution of individual sPLA2s [3–15] underlines the need to investigate 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 enzymatic activities toward phospholipids in mammalian cellular membranes [16–33], lung surfactant [34], bacterial 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 extracellular space, sPLA2s with high interfacial binding capacity to phosphatidylcholine (e.g sPLA2-V and -X) act on the phosphatidylcholine-rich outer plasma membrane [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 caveolae ⁄ 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 656 S Masuda et al augment prostaglandin E2 (PGE2) production in synovial 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 positive control), followed by high-sensitivity Southern blotting, on RNA samples obtained from synovial tissues 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 transcript 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 detected 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 RTPCR was followed by high-sensitivity Southern blotting (Fig 1A) sPLA2-IB was not detected at all (Fig 1A) Immunoblotting of the same RA samples with antibodies 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 prostaglandin 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 cytosolic 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 S Masuda et al sPLA2s in human rheumatoid arthritis A B C Fig 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 bromide 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 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 tissues, 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 inactive RA (i.e inflammatory symptoms were temporarily ceased after therapy) showed only weak staining for sPLA2-IIA (Fig 2A), whereas the enzyme was intensely 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 distributions 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 inactive 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 Prominent sPLA2-IID staining was evident in the lymphoid aggregates and capillary endothelial cells in three distinct active RA sections (Fig 3C–E) Staining of FEBS Journal 272 (2005) 655–672 ª 2005 FEBS 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, sPLA2IIE 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) 657 sPLA2s in human rheumatoid arthritis S Masuda et al Fig 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 arrowheads), and VSMC (E, green arrows) were positively stained Staining of fibroblast-like cells infiltrating into the cartilage was faint (D, red arrows) 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 aggregates (Fig 5Eb) Staining of chondrocytes in the cartilage 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 inactive (Fig 6A,B) and three active (Fig 6C–E) RA samples Although the staining intensities of individual samples were variable, the enzyme was consistently localized in the synovial lining layers and the interstitium that precludes the lymphoid aggregates, vascular walls, and cartilage chondrocytes in all samples (Fig 6) In the subintimal interstitium, sPLA2-X staining was evident in the extracellular matrix fibers (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 immunoreactivity was observed in wide areas of three active RA sections In all cases, sPLA2-V staining was evident in synovial lining cells and especiallly in sublining granulation tissue, which was composed of massive 658 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 interstitial fibroblasts) Although sPLA2-IIA and -V transcripts 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 induction by cytokines (Fig 7A) In contrast, sPLA2-IID and -IIE were undetectable in these cells even after stimulation 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 S Masuda et al sPLA2s in human rheumatoid arthritis A C B E D Fig Immunohistochemical localization of sPLA2-IID in human joints affected by RA (A,B) Staining of two inactive RA tissues In both samples, 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 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 synovial cells Indeed, synovial 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 FEBS Journal 272 (2005) 655–672 ª 2005 FEBS IL-1b, but not with TNFa or IFN-c (Fig 7B) Induction of COX-2 was already evident at 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 cytokines, with IL-1b and TNFa exhibiting more potent effect than IFN-c (Fig 7B) In the case of IL-1b stimulation, increased expression of COX-2 and mPGES1 was observed over 6–24 h (Fig 7C) Consistent with the elevated expression of COX-2 and mPGES-1, stimulation 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 accumulation of PGE2 in the medium of IL-1b-stimulated 659 sPLA2s in human rheumatoid arthritis A C S Masuda et al 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 Fig Immunohistochemical localizations of sPLA2-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, scattered 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) cells reached a plateau peak over 12–24 h (Fig 7E) IL-1b-stimulated PGE2 generation was suppressed by the cPLA2 inhibitor methyl arachidonoyl fluorophosphate by > 80%, suggesting the contribution of cPLA2a to this biosynthetic response Indeed, cPLA2a 660 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 sPLA2IID and -IIF, which were not intrinsically expressed in this cell type (Figs and 4), and with cPLA2a, which was used as a positive control for increased PGE2 production, using the same strategy After 36 h of adenovirus 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-surfaceassociated (1 m NaCl-solubilized) fractions of synoviocytes 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 ngỈmL)1, as estimated from their enzymatic activities in comparison with those of pure recombinant sPLA2 standards (which were measured after dilution in culture medium) (Fig 8B) Because the concentrations of sPLA2-IIA often reach the order of lgỈmL)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 immunoblotting, 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 glycosylation) in cultured synovial cells, a possibility that is under investigation FEBS Journal 272 (2005) 655–672 ª 2005 FEBS S Masuda et al A sPLA2s in human rheumatoid arthritis C E B D Fig Immunohistochemical localizations of sPLA2-V in human joints affected by RA (A,B) In two inactive RA sections, sPLA2-V immunoreactivity 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) 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 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 immunocytostaining of cells that had been infected with adenoviruses 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 FEBS Journal 272 (2005) 655–672 ª 2005 FEBS (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, indicating 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-impermeable heparin abrogated the cytoplasmic punctate signals for sPLA2-IIA, whereas the Golgi staining was unaffected (Fig 9A, lower) These cytoplasmic punctate signals 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 661 sPLA2s in human rheumatoid arthritis A S Masuda et al C B D E Fig 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 caveolaederived vesicles in an HSPG-dependent manner, whereas the Golgi localization represents the de novo synthesized pool of the enzyme entering into the secretory 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 stain662 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 S Masuda et al A sPLA2s in human rheumatoid arthritis 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 B Discussion C D E Fig 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 RTPCR (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 cytokines (E) Time course of PGE2 production by synovial cells treated with or without IL-1b for the indicated periods In (D) and (E), values are mean ± SE of three experiments Because of the heparin-sensitive caveolae localization of a small fraction of sPLA2-IIA (Fig 9A), we anticipated that this pool of the enzyme might contribute to the promotion of PGE2 production via the HSPGdependent pathway, as reported in several other cells [19–21,28,31] However, treatment of the cells with heparin or heparinase, which perturbs the HSPGdependent 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 vesiFEBS Journal 272 (2005) 655–672 ª 2005 FEBS 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, including 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-biosynthetic 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 several other tissues (S Masuda, M Murakami, M Mitsuishi, 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 components 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 multiple 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) Similarly, 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 distinct cytokines in different cell types [57,58], implying 663 sPLA2s in human rheumatoid arthritis A S Masuda et al C D B E Fig 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-solubilized) (M, closed bars) fractions of synovial cells after infection with adenoviruses bearing sPLA2s (multiplicity of infection [MOI] ¼ 10) Values 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 preincubated with 500 lgỈmL)1 heparin or 0.5 unitỈmL)1 heparinase for 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) the 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 association with cell differentiation and maturation [59] Because sPLA2-X, but not group II subfamily sPLA2s, has an N-terminal propeptide that is removed by proteolysis to produce an active enzyme [32], up-regula664 tion of this enzyme might generally be controlled by this post-translational processing rather than by gene induction Cytokine-stimulated synovial cells are highly susceptible to sPLA2s, producing PGE2 in response to all sPLA2s when expressed at low ngỈmL)1 concentrations (Fig 8) This sPLA2 sensitivity is remarkable because 100–1000 ngỈmL)1 or even more sPLA2s are generally required for triggering eicosanoid biosynthesis when FEBS Journal 272 (2005) 655–672 ª 2005 FEBS S Masuda et al Fig Immunocytostaining of sPLA2s adenovirally expressed in cultured synovial cells Synovial cells infected with adenovirus for sPLA2-IIA (A), -IID (B) or -X (C) were immunostained with respective antibodies in combination with FITC-conjugated secondary antibody 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 lgỈmL)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 experiments are shown sPLA2s in human rheumatoid arthritis A B 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 unlikely in our case because, even though a small fraction of HSPG-binding sPLA2s are located in caveolae-rich vesicles, treatment of these cells with exogenous heparin or heparinase, which perturbs the HSPGdependent pathway [19–21,28,31], did not affect PGE2 production by these sPLA2s (Fig 8E), and the amounts of sPLA2s released into the culture medium (an order of low ngỈmL)1) seem to be insufficient to promote both the HSPG-dependent and the external plasma membrane pathways Considering that the cellular sensitivities to exogenously 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 C F D E 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 phospholipids substrates) within the secretory compartments may be orders of magnitude higher than those secreted and dispersed into the extracellular medium In this 665 sPLA2s in human rheumatoid arthritis scenario, the AA released at the Golgi membrane by the de novo synthesized sPLA2s is supplied to the perinuclear COX-2 in synovial cells It has been reported that cPLA2a primarily targets to the Golgi membrane [62] Thus, this spatial location may allow efficient functional coupling between PLA2 and COX enzymes In addition, there might be alternative mechanism for high sensitivity of synovial cells to sPLA2s; for instance, unique membranous features (such as phospholipid composition and asymmetry, curvature, ruffling, oxidation, and putative accessory molecules) might allow synovial cells to be susceptible to sPLA2s It has previously been shown that, in addition to increasing AA release, high concentrations of exogenous sPLA2s or overexpression of sPLA2s often 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 ngỈmL)1 levels or because COX-2 induction by sPLA2s is a cell type-specific event Interestingly, 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 the levels of sPLA2-IIA often reach the order of lgỈmL)1 in RA tissues [41,42], it is likely that the multiple sPLA2s expressed in RA tissues can contribute to arthritic inflammation by augmenting 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 collagen-induced arthritis occurs only mildly in cPLA2a knockout mice [50], cPLA2a and sPLA2s may cooperate in the process of this disease, thereby contributing to amplification of the inflammatory cascades Indeed, functional cross-talk between cPLA2a and sPLA2s has been observed in various cell types [24,29,30,66] Also, sPLA2s may release AA and lysophospholipids from microvesicles shed from activated cells, which are enriched in RA fluid [67] In active RA tissues, sPLA2-IID is mainly located in the lymph follicles (Fig 3C–E) This location is remi666 S Masuda et al 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 distributed in the arterial VSMC of active RA tissues (Fig 4B) In our preliminary study, sPLA2-IIE immunoreactivity is also distributed rather specifically in VSMC of other organs, such as heart, mammary gland, gastrointestinal tract and male reproductive organs (S Masuda, M Murakami, Y Ishikawa, T Ishii and I Kudo, unpublished observations) Thus, sPLA2IIE might play specific roles in the regulation of VSMC functions Experimental procedures Materials Normal human synovial cells and culture medium (CS-C Complete Medium kit 4Z0-500) were obtained from Cell Systems (Kirkland, WA, USA) The cells were maintained on collagen-coated six-well plates (Iwaki Glass Co., Tokyo, Japan) and were used within three passages after thawing Rabbit antisera for individual human sPLA2s were described 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) cDNAs for sPLA2s and cPLA2a have been described previously [17–21] Human IL-1b, TNFa and IFN-c were purchased from Genzyme (Boston, MA, USA) Fluorescein isothiocyanate-, Cy3-, and horseradish peroxidase-conjugated anti-IgG were purchased from Zymed (South San Francisco, CA, USA) Heparin and heparinase (Flavobacterium heparinum) were obtained from Sigma (St Louis, MO, USA) Primers for RT-PCR were from Greiner Japan (Tokyo, Japan) Northern blotting Equal amounts ( 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- FEBS Journal 272 (2005) 655–672 ª 2005 FEBS S Masuda et al 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] RT-PCR Synthesis of cDNAs was performed with 0.5 lg of total RNA from human cell lines or tissues and AMV reverse transcriptase, according to the manufacturerı´ s instructions supplied with the RNA PCR kit (Takara Biomedicals) Subsequent amplification of the cDNA fragments was performed using 0.5 lL of the reverse-transcribed mixture as a template with specific primers for each sPLA2 For amplification of sPLA2-IB, -IIA, -IID, -IIE, -IIF, -V, and -X cDNAs, we used a set of 23-bp oligonucleotide primers corresponding 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 °C for 30 s and then 30–33 cycles of amplification at 94 °C for s and 68 °C for min, using the Advantage cDNA polymerase mix (Clontech, Palo Alto, CA, USA) [23,24] The PCR conditions for sPLA2-IIF were 94 °C for 30 s and then 35 cycles of amplification at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, using ExTaq polymerase (Takara Biomedicals) [23,24] The PCR products were analyzed by 1% agarose gel electrophoresis 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] SDS ⁄ PAGE immunoblotting Lysates from 105 cultured cells or 20 lg protein equivalents of tissue homogenates in phosphate-buffered saline (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 proteins 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 h Dilutions of the antibodies in NaCl ⁄ Pi ⁄ Tween were as follows: cPLA2a, COX2, cPGES, and mPGES-2, : 5000; COX-1, : 10 000; and sPLA2s and mPGES-1, : 2000 After three washes with NaCl ⁄ Pi–Tween, the membranes were incubated with horseradish peroxidase-conjugated anti-goat or anti-rabbit IgG (1 : 5000 dilution in NaCl ⁄ Pi ⁄ Tween) for h, washed six times, and were visualized using the ECL western blot system (NENTM Life Science Products, Boston, MA, USA), as described previously [17–21] FEBS Journal 272 (2005) 655–672 ª 2005 FEBS sPLA2s in human rheumatoid arthritis Immunohistochemistry Synovial tissue sections were obtained from RA patients (all female, 68–74 years old) undergoing surgery at Toho University 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 temporarily 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 tissue sections; in the ‘active RA’ cases, outgrowth of synovial cells, formation of lymph follicles, and infiltration of lymphocytes and plasma cells were obvious, whereas these features were poorly observed in ‘inactive RA’ cases Immunohistochemistry was performed as described previously [48,49] Briefly, the tissue sections (4 lm thick) were incubated with Target Retrieval Solution (DAKO, Carpintenia, CA, USA) as required, incubated for 10 with 3% (v ⁄ v) H2O2, washed three times with NaCl ⁄ Pi for each, incubated with 5% (v ⁄ v) skim milk for 30 min, washed three times with NaCl ⁄ Pi ⁄ Tween for each, and incubated for 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 Expression of PLA2s by the adenovirus system 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 Biomedicals), were subcloned into the pENTER ⁄ D-TOPO vector with a pENTER Directional TOPO Cloning kit (Invitrogen) After purification of the plasmids from the transformed Top10 competent cells (Invitrogen), the sequences of the cDNA inserts were verified with a Taq cycle sequencing kit (Takara Biomedicals) and an autofluorometric 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 transformed Top10 cells, the plasmids were linearized by digestion with PacI (New England BioLabs, Bervery, MA, USA) and transfected into subconfluent 293A cells (Invitrogen) with Lipofectamine 2000 (Invitrogen) in Opti-MEM medium (Invitrogen) After 1–2 weeks of culture in RPMI-1640 containing 10% fetal bovine serum until most cells had floated, the culture medium and cells were harvested together, freeze-thawed twice, and centrifuged at 000 g for at 667 sPLA2s in human rheumatoid arthritis °C to obtain the adenovirus-enriched supernatants Aliquots of the supernatants were added to fresh 293A cells, and the culture was continued for appropriate periods in order to amplify adenoviruses After 2–4 cycles of amplification, the resulting adenovirus-containing supernatants were used as virus stocks Viral titers were determined by the plaque-forming assay with 293A cells As a control, the pAd ⁄ CMV ⁄ V5-GW lacZ vector (Invitrogen) was transfected into 293 A cells to produce LacZ-bearing adenovirus Cell culture experiments using adenovirus Human synovial cells were seeded into 24-well plates and cultured to near confluency After replacing with fresh culture 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 medium, the cells were incubated with or without ngỈmL)1 IL-1b, 100 mL)1 TNFa or 10 ngỈmL)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 immunoassay kit (Cayman Chemicals, Ann Arbor, MI, 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 with medium containing m NaCl, and PLA2 activities solubilized into the supernatants were measured [17–21] Measurement of sPLA2 activity sPLA2 activity was assayed by measuring the amounts of radiolabeled linoleic acid released from the substrate 1-palmitoyl-2-[14C]linoleoyl-phosphatidylethanolamine (Amersham 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 appropriate amounts of the required sample, 100 mm Tris ⁄ HCl (pH 7.4), mm CaCl2 and 10 lm substrate After incubation for 20 at 37 °C, [14C]linoleic acid was extracted by Dole’s method, and the radioactivity was quantified by liquid scintillation counting, as described previously [17–21] For rough quantification of individual sPLA2s released from the cells into the culture supernatants, the activities of pure recombinant sPLA2s (provided by MH Gelb, University of Washington) diluted with synovial cell culture medium were measured Confocal laser microscopy Cells grown in glass-bottomed dishes (Matsunami Glass, Tokyo, Japan) precoated with 10 lgỈmL)1 fibronectin (Sigma) were fixed with 3% (v ⁄ v) paraformaldehyde for 30 in NaCl ⁄ Pi Cells were infected with adenoviruses bearing 668 S Masuda et al PLA2s for 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 h, with anti-sPLA2 and anti-COX-2 h in NaCl ⁄ Pi containing 1% albumin (1 : 200–500 dilutions), and then with FITC-conjugated anti-(rabbit IgG) for h in NaCl ⁄ Pi containing 1% albumin (1 : 200 dilution), with three washes with NaCl ⁄ Pi at each interval For control staining, sPLA2expressing cells were treated with normal rabbit IgG or cells infected with lacZ-adenoviruses were treated with antisPLA2, with which fluorescent signals were negligible (data not shown) For double immunostaining, cells stained with anti-sPLA2 were incubated 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655–672 ª 2005 FEBS ... human synovial cells Our results indicate that these sPLA2s are diversely expressed in RA tissues and are able to 656 S Masuda et al augment prostaglandin E2 (PGE2) production in synovial cells. .. staining for sPLA2-IIA (Fig 2A), whereas the enzyme was intensely expressed in synovial lining cells in the section from a patient with active RA (Fig 2B,C) Staining of the synovial sublining area... Thus, sPLA2IIE 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