Cytosolic Phospholipase A2 Expression Research Articles

STAT-3-dependent Cytosolic Phospholipase A2 Expression Is Required for Thrombin-induced Vascular Smooth Muscle Cell Motility

Vascular smooth muscle cell (VSMC) migration from media to intima and its multiplication in intima is a contributing factor in the pathogenesis of atherosclerosis and restenosis after angioplasty. Previously, we have demonstrated that STAT-3-dependent cytosolic phospholipase A(2) (cPLA(2)) expression is needed for VSMC motility induced by platelet-derived growth factor-BB, a receptor tyrosine kinase agonist (Neeli et al. (2005) J. Biol. Chem. 279, 46122-46128). In order to learn more about the STAT-3-cPLA(2) axis in motogenic signaling, here we have studied its role in VSMC motility in response to a G protein-coupled receptor (GPCR) agonist, thrombin. Thrombin induced VSMC motility in a dose-dependent manner with a maximum effect at 0.5 units/ml. Thrombin activated STAT-3 as measured by its tyrosine phosphorylation and translocation from the cytoplasm to the nucleus. Forced expression of a dominant negative mutant of STAT-3 reduced thrombin-induced STAT-3 tyrosine phosphorylation and its translocation from the cytoplasm to the nucleus. Thrombin stimulated STAT-3-DNA binding and reporter gene activities in VSMC, and these responses were blocked by FS3DM, a dominant negative mutant of STAT-3. FS3DM also attenuated thrombin-induced VSMC motility. Thrombin induced the expression of cPLA(2) in a time- and STAT-3-dependent manner. In addition, pharmacological inhibition of cPLA(2) blocked thrombin-induced VSMC motility. Furthermore, exogenous addition of arachidonic acid rescued thrombin-induced VSMC motility from inhibition by blockade of STAT-3 activation. Forced expression of cPLA(2) also surpassed the inhibitory effect of dominant negative STAT-3 on thrombin-induced VSMC motility. Together, these results show that thrombin-induced VSMC motility requires STAT-3-dependent induction of expression of cPLA(2).

Celecoxib inhibits angiogenesis by inducing endothelial cell apoptosis in human pancreatic tumor xenografts

Previous studies suggest that antagonists of cyclooxygenases 1 and 2 (COX-1, -2) inhibit angiogenesis in tumor xenografts, but the molecular mechanisms involved remain unclear. Here we characterized the effects of non-selective (indomethacin) and selective (NS398, celecoxib) cyclooxygenase inhibitors on parameters of angiogenesis in human pancreatic adenocarcinoma cells. COX-1 expression was constitutive in 9/9 pancreatic cancer cell lines, whereas COX-2 and cytosolic phospholipase A2 (cPLA2 ) expression were observed in 4/9 cell lines (BxPC3, Capan2, Cfpac1, and L3.6pl). Production of the COX product, prostaglandin E2, correlated with expression of cPLA2 and COX-2 and was blocked by non-steroidal anti-inflammatory drugs (NSAIDs, indomethacin or NS398). In contrast to the findings of others, neither indomethacin nor NS398 affected tumor cell proliferation or secretion of angiogenic factors (VEGF, bFGF, IL-8) at concentrations that produced maximal inhibition of PGE2 production, and higher concentrations increased angiogenic factor production. We also studied the effects of celecoxib in orthotopic L3.6pl xenografts. Immunofluorescence analyses revealed high-level expression of COX-2 in endothelial cells in L3.6pl xenografts that increased following therapy with celecoxib, whereas the tumor cells expressed uniformly low levels of COX- 2. Celecoxib did not decrease tumor-associated VEGF levels in orthotopic human L3.6pl xenografts, but the drug did decrease tumor microvessel density (MVD) and increase apoptosis in tumor-associated endothelial cells in a dose-dependent fashion. Together, our results demonstrate that the anti-angiogeneic effects of NSAIDs in human pancreatic cancer cells are exerted via direct effects on endothelial cells.

Arachidonic Acid Rescues Defects on PMA-Elicited NADPH Oxidase Activity in Rac2-Deficient Neutrophils.

Rac2 is a hematopoietic-specific Rho-GTPase implicated as an important constituent of the NADPH oxidase complex. We previously showed that Rac2 plays a stimulus-specific role in regulating NADPH oxidase activation and other functional responses in neutrophils [Kim and Dinauer, JI 166, 2001]. Here we investigate the effect of arachidonic acid (AA) on Rac2-regulated NADPH oxidase activity. Superoxide production in rac2-/- neutrophils was significantly lower (~4-fold) than that of wild-type when stimulated with PMA or AA alone. However, exogenously added AA (10 μM) fully restored the defect in PMA-elicited NADPH oxidase activity in rac2−/ − neutrophils, while having no effect on FMLP-elicited superoxide production. Impaired PMA- or AA-induced F-actin polymerization in rac2−/ − neutrophils was also not restored by co-stimulation with PMA and AA. Taken together, these observations suggest that there are agonist- and pathway-specific differences in the underlying basis of functional defects in rac2−/ − neutrophils. To further investigate possible mechanisms of AA-mediated rescue of PMA-stimulated NADPH oxidase activation in rac2−/ − neutrophils, we measured protein expression and activity of cytosolic phospholipase A2 (cPLA2) and protein kinase C (PKC). The expression of cPLA2 and PMA-stimulated release of AA was similar between wild-type and rac2−/ − neutrophils, suggesting that defects in AA production by PMA-stimulated rac2−/ − neutrophils do not account for the effect of exogenous AA on oxidase activity. The neutrophil expression of PKC isoforms (α, β, δ, ζ) was also similar between genotypes. The cytosolic p47phox and p67phox components of NADPH oxidase were translocated to the plasma membrane upon stimulation with PMA in both genotypes, and no additional translocation in either wild-type or rac2−/ − neutrophils was detected upon co-stimulation with AA. The level of activated Rac1-GTP was similar between genotypes following stimulation, and was not increased by co-stimulation with PMA and AA. These studies indicate that the addition of exogenous AA reconstitutes PMA-elicited superoxide production in rac2−/ − neutrophils independent of the effects on translocation of p47phox and p67phox and activation of Rac1 GTPase. We hypothesize that the effect of AA is exerted through conformational changes of the assembled NADPH oxidase.

Labour is associated with increased expression of type-IIA secretory phospholipase A2 but not type-IV cytosolic phospholipase A2 in human myometrium.

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the type-IV cytosolic phospholipase A2 (cPLA2-IV) and the type IIA secretory phospholipase A2 (sPLA2-IIA) in myometrium in association with labour onset at term and preterm deliveries. These enzymes are important for the release of the prostaglandin precursor, arachidonic acid, from phospholipid membrane stores. RT-PCR was used to determine differences in gene expression between non-labour and labour groups. Expression of sPLA2-IIA in human myometrium was significantly increased with pregnancy, and with labour, both at term and preterm. Expression of cPLA2-IV in myometrium was not significantly altered with respect to pregnancy or labour. Immunohistochemical analysis demonstrated differences in the spatial localization of cPLA2-IV and sPLA2-IIA protein in upper and lower segment myometrium. cPLA2-IV was predominantly in vascular endothelial cells, while sPLA2-IIA was observed in vascular, endothelial and smooth muscle cells. In addition, sPLA2-IIA showed a distinct nuclear or perinuclear localization in myometrial smooth muscle cells of the lower segment. We postulate that the increased expression of sPLA2-IIA rather than cPLA2-IV in the myometrium may play a role in the onset and/or maintenance of human parturition.

Tumor necrosis factor-alpha stimulates gastric epithelial cell proliferation.

TNF-alpha is a cytokine produced during gastric mucosal injury. We examined whether TNF-alpha could promote mucosal repair by stimulation of epithelial cell proliferation and explored further the underlying mechanisms in a rat gastric mucosal epithelial cell line (RGM-1). TNF-alpha treatment (1-10 ng/ml) for 12 or 24 h significantly increased cell proliferation but did not induce apoptosis in RGM-1 cells. TNF-alpha treatment significantly increased cytosolic phospholipase A(2) and cyclooxygenase-2 (COX-2) protein expression and PGE(2) level but did not affect the protein levels of EGF, basic fibroblast growth factor, and COX-1 in RGM-1 cells. The mRNA of TNF receptor (TNF-R) 2 but not of TNF-R1 was also increased. Dexamethasone dose dependently inhibited the stimulatory effect of TNF-alpha on cell proliferation, which was associated with a significant decrease in cellular COX-2 expression and PGE(2) level. A selective COX-2 inhibitor 3-(3-fluorophenyl)-4-[4-(methylsulfonyl)phenyl]-5,5-dimethyl-(5)H-furan-2-one (DFU) by itself had no effect on basal cell proliferation but significantly reduced the stimulatory effect of TNF-alpha on RMG-1 cells. Combination of dexamethasone and DFU did not produce an additive effect. PGE(2) significantly reversed the depressive action of dexamethasone on cell proliferation. These results suggest that TNF-alpha plays a regulatory role in epithelial cell repair in the gastric mucosa via the TNF-alpha receptor and activation of the arachidonic acid/PG pathway.

Additive blockade of beta 2-integrin adhesion of eosinophils by salmeterol and fluticasone propionate.

Migration of human eosinophils is regulated by integrin expression, conformational change, and activation of cytosolic phospholipase A2 (cPLA2). Corticosteroids have been shown to inhibit cPLA2 hydrolysis in human eosinophils. The objective of this study was to determine the mechanisms of fluticasone propionate (FP) alone or in combination with salmeterol (SM) in blocking adhesion mediated by beta 2-integrin in human eosinophils. Human eosinophils were isolated by negative magnetic selection. beta 2-integrin-mediated eosinophil adhesion was measured by residual eosinophil peroxidase activity. Eosinophils were pretreated for 12 h to 24 h with FP and with or without SM for 30 min. Both SM alone and FP alone inhibited eosinophil adhesion in concentration- and time-dependent manner. SM alone modestly (approximately 30%) inhibited interleukin (IL)-5-induced eosinophil adhesion. Blockade of IL-5-induced eosinophil adhesion caused by 10(-7) M FP at 24 h was augmented by 10(-7) M SM from 41.5% to 72.5%. Similar blockade was also observed for eotaxin-induced eosinophil adhesion. Neither SM, FP, nor FP + SM blocked either: 1) upregulation of CD11b surface expression; or 2) phosphorylation of cPLA2. Blockade of beta 2-integrin-mediated eosinophil adhesion by fluticasone propionate is augmented by salmeterol. Decreased adhesion results from augmented blockade of nuclear translocation of cytosolic phospholipase A2 caused by addition of salmeterol to fluticasone.

Site-specific augmentation of amnion cyclooxygenase-2 and decidua vera phospholipase-A2 expression in labor: possible contribution of mechanical stretch and interleukin-1 to amnion prostaglandin synthesis.

To investigate a possible site-specific augmentation of prostaglandin (PG) synthesis in the fetal membranes during labor. We used reverse transcriptase-polymerase chain reaction or Western blot analysis to evaluate the expression of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-1, -2 (COX-1, -2), in both the upper and lower parts of the amnion, chorion laeve, and decidua vera tissues from term pregnant women before (n = 8) and after labor (n = 24). Prostaglandin E2 (PGE2) secretion from amnion-derived WISH cells was assessed using enzyme-linked immunosorbent assay after stimulation by cyclic mechanical stretching and interleukin-1 (IL-1). The expression of cPLA2 and COX-1 and COX-2 mRNAs was detected in all samples examined. Western blot analysis revealed that COX-2 expression in the upper part of the amnion, chorion laeve, and decidua vera tissues after labor was 4.7-, 4.9-, and 3.7-fold higher than that before labor, respectively (P < .05 for all). The cPLA2 protein expression in the upper part of the amnion and chorion laeve tissues after labor was 14.0- and 8.8-fold higher than that before labor, respectively (P < .05 for both). Moreover, in specimens obtained after labor, the amnion COX-2 expression and the decidua vera cPLA2 expression in the lower part of the fetal membrane was 1.9- and 2.6-fold higher than the respective levels in the upper part (P < .05 for both). In an in vitro study, cyclic mechanical stretching significantly enhanced IL-1-augmented PGE2 secretion from WISH cells. In the lower part of the amnion and decidua vera tissues, adjacent to the dilating cervical canal, PG synthesis was upregulated site specifically after labor. Such enhancement of amnion PG synthesis might be regulated at least partly by IL-1 and cyclic distension.

Regulation of prostaglandin biosynthesis in dispersed choriodecidual cells in culture

We have evaluated the prostaglandin (PG) production and PG biosynthetic gene expression in a choriodecidual dispersed cell culture system. Cells dispersed from human choriodecidual membranes by dispase and trypsin digestion were evaluated after 1, 3, 5 and 7 days of culture for basal and tumour necrosis factor α (F- α) stimulated PGE2 production. The highest rates of production (P<0.05) were obtained with cells treated after 3 days of culture, (3.7±1)×102 pg PGE2 per 16 h per μg total cellular protein (mean±SEM), which was 3.9 times basal rate after 3 days culture. In choriodecidual cells treated after 3 days in culture, expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) mRNA was similarly responsive to TNF- α (3.9 times basal within 3 h of 30 ng/ml TNF- α) while there was little effect on PGHS-1 or cytosolic phospholipase A2 expression. Hence, the dispersed choriodecidual cell culture system described retains TNF- α responsive PG biosynthetic capacity which is at least in part upregulated via increased expression of PGHS-2 mRNA.

In vivo and in vitro studies of cytosolic phospholipase A2 expression in Helicobacter pylori infection.

Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10 H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2 activity in the gastric mucosae of patients with H. pylori infection and no change in epithelial cell lines exposed to H. pylori.

Increased expression of cytosolic phospholipase A2, 5-lipoxygenase and 5-lipoxygenase-activating protein in rat peritoneal macrophages during ovalbumin-induced sensitization.

Macrophages are involved in immediate hypersensitivity reactions by their ability to release leukotrienes involved in the symptomatology of allergy. To date it is unknown whether this ability to secrete leukotrienes has been favoured by modifications, occurring during the sensitization phase, of the enzymes involved in leukotriene metabolism. We used ovalbumin-sensitized rats to study the expression of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in peritoneal macrophages during active sensitization. We compared basal and challenged (PMA, A23187 and allergen) arachidonic acid (AA) metabolism of macrophages from control (cPM) and sensitized (sPM) rats. Then we tested, in cultured cPM, whether IL-4, the predominant cytokine of sensitization process, could reproduce the enzymatic modifications occurring in macrophages during sensitization. cPLA2, 5-LO and FLAP expression was assessed by Western blotting. The arachidonic acid (AA) metabolism study was performed after incorporation of tritiated AA in macrophages and analysis of secreted tritiated eicosanoids. Ovalbumin-sensitization of rats increased cPLA2, 5-LO and FLAP expression in peritoneal macrophages. These increased expressions were not paralleled by modifications of basal and PMA- or A23187-stimulated AA metabolism of sPM. However, when macrophages encountered the specific allergen for a second time, sPM secreted higher levels of leukotrienes than cPM. IL-4 induced FLAP expression in cPM but had no effect on cPLA2 and 5-LO expression. Active sensitization of rats induces an increase, in peritoneal macrophages, of the enzymes involved in leukotriene metabolism. The increased leukotriene secretion of sPM in response to ovalbumin challenge may be favoured by this increased expression of cPLA2, 5-LO and FLAP that, however, is not able to lead to modifications of macrophage AA metabolism in any circumstance. Our results also suggest that IL-4 is not the major element originating the enzymatic modification induced by sensitization in peritoneal macrophages.

Secretory Phospholipase A2 Mediates Cooperative Prostaglandin Generation by Growth Factor and Cytokine Independently of Preceding Cytosolic Phospholipase A2 Expression in Rat Gastric Epithelial Cells

Transforming growth factor (TGF)-alpha and interleukin (IL)-1beta are responsible for the healing of gastric lesions through, in part, prostaglandin (PG) generation. We examined the contribution of cytosolic and secretory phospholipase A(2)s (cPLA(2) and sPLA(2)) to the PG generation by rat gastric epithelial cells in response to both stimuli. Stimulation with TGF-alpha for 24 h increased cPLA(2) and cyclooxygenase (COX)-2 markedly, PGE(2) slightly, and type IIA sPLA(2) and COX-1 not at all, whereas IL-1beta increased sPLA(2) only. Both stimuli synergistically increased PGE(2), sPLA(2), and the two COXs but not cPLA(2). The onset of the PGE(2) generation paralleled the sPLA(2) release but was apparently preceded by increases in cPLA(2) and the two COXs. The increase in PGE(2) was impaired by inhibitors for sPLA(2) and COX-2 but not COX-1. cPLA(2) inhibitors suppressed PGE(2) generation by TGF-alpha alone but not augmentation of PGE(2) generation or sPLA(2) release by IL-1beta in combination with TGF-alpha. Furthermore, despite an increase in cPLA(2) including its phosphorylated form (phosphoserine), -induced arachidonic acid liberation was impaired in the TGF-alpha/IL-1beta-stimulated cells, in which p11, a putative cPLA(2) inhibitory molecule, was also increased and co-immunoprecipitated with cPLA(2). These results suggest that synergistic stimulation of sPLA(2) and COX-2 expression by TGF-alpha and IL-1beta results in an increase in PGE(2). Presumably, the preceding cPLA(2) expression is not involved in the PGE(2) generation, because of impairment of its hydrolytic activity in the stimulated cells.

Feedback Control of the Arachidonate Cascade in Rheumatoid Synoviocytes by 15-deoxy-Δ12,14-Prostaglandin J2

Rheumatoid arthritis (RA) is a chronic polyarticular joint disease associated with massive synovial proliferation, inflammation, and angiogenesis. PPAR-γ ligands, both 15-deoxy-Δ12,14-prostaglandin J2 (15d- PGJ2) and troglitazone (TRO), can inhibit the growth of RA synoviocytes in vitro, and suppress the chronic inflammation of adjuvant-induced arthritis in rats, but the potency of 15d-PGJ2 is higher than TRO. Prostaglandin (PG) E2 plays important roles in joint erosion and synovial inflammation. In the present study, 15d-PGJ2, but not TRO and other prostanoids, suppressed interleukin (IL)-1β-induced PGE2 synthesis in rheumatoid synovial fibroblasts (RSFs) through the inhibition of cyclooxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) expression. Furthermore, the inhibition was not affected by pretreatment with anti-PPAR-γ antibody. It means that this anti-inflammatory effect of 15d-PGJ2 for PG synthesis may be independent of PPAR-γ and 15d-PGJ2 is a key regulator of negative feedback of the arachidonate cascade on the COX pathway. These findings provide new insight into the feedback mechanism of the arachidonate cascade.

Cytosolic phospholipase A2and 15-hydroxyprostaglandin dehydrogenase mRNA expression in murine uterine and gestational tissues during late pregnancy

We evaluated the changes in mRNA expression of cytosolic phospholipase A2(cPLA2) and 15-hydroxyprostaglandin dehydrogenase (PGDH) in intrauterine and gestational tissues during mid-late murine pregnancy. Tissues (decidual caps, fetal membranes, and placentae, uterus, and cervix) were collected from pregnant mice at days 12, 14, 16, 18, and 19 (am and pm) of gestation. Total RNA was isolated and evaluated for cPLA2and PGDH expression by northern blot analysis normalized to GAPDH expression. Expression of mRNA for cPLA2increased in the placentae and decidual caps on day 18 and 19 pm, respectively. There was also increased expression for PGDH mRNA in the placenta and fetal membranes at the later stages of pregnancy. The tissue specific differences in expression of cPLA2and PGDH suggest that changes in enzymatic regulation of PG production and degradation may be crucial for the initiation of labour.

Sustained activity of luteal cytosolic phospholipase A2 during luteolysis in pseudopregnant rats: its possible implication in tissue involution.

We investigated the expression and activity of cytosolic phospholipase A2 (cPLA2) in the corpus luteum during spontaneous and induced luteolysis in pseudopregnant rats. In both models, luteal PLA2 activity rose in association with functional regression and persisted during the following structural regression. Tissue concentration of prostaglandin F2alpha with a luteolytic potency showed a similar fluctuation. The enzyme activity was almost completely suppressed by a cPLA2-specific inhibitor. Expression of cPLA2, analyzed by immunohistochemistry, became enhanced during luteolysis with preferential localization to phagocytotic and fibrotic replacement sites. Taken together with our previous finding, the data indicate a persistent elevation in luteal cPLA2 expression and activity that may affect tissue involution in vivo.

B cell receptor-stimulated mitochondrial phospholipase A2 activation and resultant disruption of mitochondrial membrane potential correlate with the induction of apoptosis in WEHI-231 B cells.

Cross-linking of the Ag receptors on the immature B cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. We now show that although commitment to such B cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A(2) activation, disruption of mitochondrial function, and ATP depletion, it is executed independently of caspase activation. First, we demonstrate a pivotal role for mitochondrial function in determining B cell fate by showing up-regulation of cytosolic phospholipase A(2) expression, induction of mitochondrial phospholipase A(2) activity, arachidonic acid-mediated collapse of mitochondrial transmembrane inner potential (Delta psi(m)), and depletion of cellular ATP under conditions of apoptotic, but not proliferative, signaling via the BCR. Importantly, disruption of Delta psi(m), ATP depletion, and apoptosis can be prevented by rescue signals via CD40 or by Delta psi(m) stabilizers such as antimycin or oligomycin. Second, we show that commitment and postmitochondrial execution of BCR-mediated apoptosis are not dependent on caspase activation by demonstrating that such apoptotic signaling does not induce release of cytochrome c from the mitochondria or activation of effector caspases, as evidenced by poly(ADP-ribose) polymerase or Bcl-x(L) cleavage. Indeed, apoptotic signaling via the BCR in WEHI-231 B cells does not stimulate the activation of caspase-3 and, consistent with this, BCR-mediated disruption of Delta psi(m) and commitment to apoptosis take place in the presence of caspase inhibitors. In contrast, BCR signaling induces the postmitochondrial activation of cathepsin B, and resultant apoptosis is blocked by the cathepsin B inhibitor, (23,35)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester (EST) suggesting a key role for this executioner protease in Ag receptor-driven apoptosis of WEHI-231 immature B cells.

Intraarticular Osteoid Osteoma Associated with Synovitis: A Possible Role of Cyclooxygenase-2 Expression by Osteoblasts in the Nidus

To clarify the condition of development of synovitis associated with intraarticular osteoid osteoma (OO), expressions of cyclooxygenase-2 (COX-2) protein and its messenger ribonucleic acid were investigated both in the nidus and the synovial tissue using immunohistochemical and reverse transcription-polymerase chain reaction analyses. Diffuse and strong COX-2 immunoreactivity was found in osteoblast-like tumor cells in the nidus of all six cases of OO (three of six cases were intraarticular OO associated with synovitis) and one case of osteoblastoma associated with synovitis. Expression of COX-2 messenger ribonucleic acid was demonstrated in one case of OO associated with synovitis, and was higher in the nidus than that in the inflamed synovial tissue. However, there were no significant difference between the nidus and synovium in the expression of cytosolic phospholipase A2, one of the enzymes involved in arachidonic acid metabolism, and inflammatory cytokines such as interleukin-1β and tumor necrosis factor alpha. Finally, as there was only one case in which the examinations of gene expression were performed, no definitive overall conclusions could be reached; rather it is suggested that COX-2 expressed primarily by osteoblasts in the nidus of intraarticular OO may play a role in activating the pathway of arachidonic acid metabolism, resulting in synovitis of the involved joint.

New induction of leukotriene A4 hydrolase by interleukin-4 and interleukin-13 in human polymorphonuclear leukocytes

Interleukin (IL)-4, IL-10, and IL-13, Th2 cell–derived cytokines, play major roles in the pathophysiology of allergic diseases. These cytokines up-regulate or down-regulate the production of arachidonic acid metabolites. In this study, we have investigated the effect of IL-4, IL-10, IL-13, and other cytokines on A23187-stimulated synthesis of leukotriene (LT) B4 in human polymorphonuclear leukocytes (PMNs). Production of LTB4 was measured by specific radioimmunoassay and high performance liquid chromatography. Messenger RNA (mRNA) expression of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and LTA4 hydrolase, which were involved in the synthesis of LTB4, was determined by reverse transcription–polymerase chain reaction and Northern blot analysis. Protein synthesis of their enzymes was determined by Western blot analysis. IL-4 and IL-13 enhanced A23187-stimulated LTB4 synthesis and increased mRNA expression and protein synthesis of LTA4hydrolase, but not those of cPLA2 or 5-LO. These results indicate that IL-4 and IL-13 transcriptionally or post-transcriptionally up-regulate the synthesis of LTB4, a potent chemotactic factor to PMNs, at the enzyme level of LTA4 hydrolase, and this up-regulation mechanism may participate in the development of allergic inflammation.

New induction of leukotriene A4 hydrolase by interleukin-4 and interleukin-13 in human polymorphonuclear leukocytes

AbstractInterleukin (IL)-4, IL-10, and IL-13, Th2 cell–derived cytokines, play major roles in the pathophysiology of allergic diseases. These cytokines up-regulate or down-regulate the production of arachidonic acid metabolites. In this study, we have investigated the effect of IL-4, IL-10, IL-13, and other cytokines on A23187-stimulated synthesis of leukotriene (LT) B4 in human polymorphonuclear leukocytes (PMNs). Production of LTB4 was measured by specific radioimmunoassay and high performance liquid chromatography. Messenger RNA (mRNA) expression of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and LTA4 hydrolase, which were involved in the synthesis of LTB4, was determined by reverse transcription–polymerase chain reaction and Northern blot analysis. Protein synthesis of their enzymes was determined by Western blot analysis. IL-4 and IL-13 enhanced A23187-stimulated LTB4 synthesis and increased mRNA expression and protein synthesis of LTA4hydrolase, but not those of cPLA2 or 5-LO. These results indicate that IL-4 and IL-13 transcriptionally or post-transcriptionally up-regulate the synthesis of LTB4, a potent chemotactic factor to PMNs, at the enzyme level of LTA4 hydrolase, and this up-regulation mechanism may participate in the development of allergic inflammation.

Elevated cytosolic phospholipase A2 expression and activity in human neutrophils during sepsis

Sepsis is defined as the systemic inflammatory response to infection. Phospholipase A(2) (PLA(2)) plays an important role in inflammation processes by initiating the production of inflammatory mediators. The role of cytosolic PLA (cPLA(2)) has not yet been identified in inflammatory and infectious disease clinical settings. The aim of the present research was to determine whether cPLA(2) activity has a role during sepsis. Since neutrophil activation has been documented during sepsis, these cells were chosen as a model to evaluate the function of cPLA(2) in this clinical setting. cPLA(2 )was studied at 3 levels: activity, protein expression, and messenger RNA (mRNA). Neutrophils from 32 septic patients with and without bacteremia were examined. cPLA(2) activity was measured using labeled phosphatidyl choline vesicles as a substrate, and total PLA(2) was determined by the release of labeled arachidonic acid from prelabeled cells. A significant increase in cPLA(2) activity, protein expression, and total PLA(2) activity in neutrophils was detected during sepsis. mRNA levels, detected by reverse transcriptase-polymerase chain reaction, were significantly higher during sepsis, indicating that the increase in the amount of cPLA(2) is regulated on the mRNA level. The significant elevation of cPLA(2) activity and expression in neutrophils during sepsis suggests that this enzyme plays a major role in neutrophil function in this clinical setting. (Blood. 2000;95:660-665)

Granulocyte-Macrophage Colony-Stimulating Factor Upregulates Reduced 5-Lipoxygenase Metabolism in Peripheral Blood Monocytes and Neutrophils in Acquired Immunodeficiency Syndrome

AbstractLeukotrienes (LT) are mediators derived from the 5-lipoxygenase (5-LO) pathway, which play a role in host defense, and are synthesized by both monocytes (peripheral blood monocyte [PBM]) and neutrophils (PMN). Because 5-LO metabolism is reduced in alveolar macrophages and PMN from acquired immunodeficiency syndrome (AIDS) subjects, we investigated the synthesis of LT by PBM and PMN from these subjects. There was a reduction (74.2% ± 8.8% of control) in LT synthesis in PBM from human immunodeficiency virus (HIV)-infected compared with normal subjects. Expression of 5-LO (51.2% ± 8.8% of control), and 5-LO activating protein (FLAP) (48.5% ± 8.0% of control) was reduced in parallel. We hypothesized that this reduction in LT synthetic capacity in PBM and PMN was due to reduced cytokine production by CD4 T cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We treated 10 AIDS subjects with GM-CSF for 5 days. PBM 5-LO metabolism ex vivo was selectively increased after GM-CSF therapy and was associated with increased 5-LO and FLAP expression. PMN leukotriene B4(LTB4) synthesis was also augmented and associated with increased 5-LO, FLAP, and cytosolic phospholipase A2 expression. In conclusion, as previously demonstrated for PMN, PBM from AIDS subjects also demonstrate reduced 5-LO metabolism. GM-CSF therapy reversed this defect in both PBM and PMN. In view of the role of LT in antimicrobial function, cytokine administration in AIDS may play a role as adjunct therapy for infections.