p38 MAPK regulates group IIa phospholipase A2 expression in interleukin-1beta -stimulated rat neonatal cardiomyocytes.

Group IIa phospholipase A(2) (GIIa PLA(2)) is released by some cells in response to interleukin-1beta. The purpose of this study was to determine whether interleukin-1beta would stimulate the synthesis and release of GIIa PLA(2) from cardiomyocytes, and to define the role of p38 MAPK and cytosolic PLA(2) in the regulation of this process. Whereas GIIa PLA(2) mRNA was not identified in untreated cells, exposure to interleukin-1beta resulted in the sustained expression of GIIa PLA(2) mRNA. Interleukin-1beta also stimulated a progressive increase in cellular and extracellular GIIa PLA(2) protein levels and increased extracellular PLA(2) activity 70-fold. In addition, interleukin-1beta stimulated the p38 MAPK-dependent activation of the downstream MAPK-activated protein kinase, MAPKAP-K2. Treatment with the p38 MAPK inhibitor, SB202190, decreased interleukin-1beta stimulated MAPKAP-K2 activity, GIIa PLA(2) mRNA expression, GIIa PLA(2) protein synthesis, and the release of extracellular PLA(2) activity. Infection with an adenovirus encoding a constitutively active form of MKK6, MKK6(Glu), which selectively phosphorylates p38 MAPK, induced cellular GIIa PLA(2) protein synthesis and the release of GIIa PLA(2) and increased extracellular PLA(2) activity 3-fold. In contrast, infection with an adenovirus encoding a phosphorylation-resistant MKK6, MKK6(A), did not result in GIIa PLA(2) protein synthesis or release by unstimulated cardiomyocytes. In addition, infection with an adenovirus encoding MKK6(A) abrogated GIIa PLA(2) protein synthesis and release by interleukin-1beta-stimulated cells. These results provide direct evidence that p38 MAPK activation was necessary for interleukin-1beta-induced synthesis and release of GIIa PLA(2) by cardiomyocytes.

Mitogenic effect of lipoproteins on human vascular smooth muscle cells: the impact of hydrolysis by gr II A phospholipase A(2).

Multifactorial interaction among lipoproteins, vascular wall cells, and inflammatory mediators has been recognized as the basis of atherogenesis. In the arterial wall high-density lipoprotein (HDL) and human secretory phospholipase A(2) (sPLA(2)) colocalize with vascular smooth muscle cells and concentrate in the atherosclerotic lesions. It has been shown that gr IIA sPLA(2) hydrolyzes lipoproteins, altering their structure and releasing active agents such as lyso-phosphatidylcholine (PtdCho) and free fatty acids. We investigated the impact of normal HDL(3) (NHDL(3)), acute phase HDL(3) (APHDL(3)), and low-density lipoprotein (LDL), both unhydrolyzed and sPLA(2)-hydrolyzed, and some products of hydrolysis, such as lyso-PtdCho, oleic and linoleic acid, on [(3)H] thymidine incorporation by DNA of cultured human vascular smooth muscle cells (VSMC). NHDL(3) markedly enhanced mitogenic activity of VSMC in a dose- and time-dependent manner. Doubling of thymidine incorporation was usually achieved by 40 microg/ml of NHDL(3) after 4 hours of incubation. APHDL(3) had invariably a stronger inducing effect on the mitogenic activity than NHDL(3); 40 microg/ml more than tripled [(3)H] thymidine incorporation after 4 hours of incubation. NHDL(3) preincubated with human apo serum amyloid A apolipoprotein-induced higher mitogenic activity in VSMC than NHDL(3) alone. Hydrolysis of NHDL(3), APHDL(3), or LDL by gr IIA sPLA(2) markedly enhanced mitogenic activity of VSMC as compared with unhydrolyzed lipoproteins. sPLA(2) concentrations that can be found in atherosclerotic vascular walls markedly enhanced lipoprotein-induced mitogenic activity of VSMC. sPLA(2) per se did not affect thymidine incorporation and VSMC did not release sPLA(2) into the medium. There was no evidence for hydrolysis of the wall of VSMC by gr IIA sPLA(2). The presence of the products of hydrolysis of lipoproteins such as oleic and linoleic acids and lyso-PtdCho or their combinations with NHDL(3) explains in part markedly enhanced mitogenic activity of VSMC. It is conceivable that sPLA(2,) which is known to colocalize with lipoproteins in the vascular wall in the domain of VSMC, is capable of induction of the mitogenic activity in these cells in vivo and should be considered as a proatherogenic enzyme.

Comparative analysis of lipid composition of normal and acute-phase high density lipoproteins.

In the acute-phase response and in diseases with prolonged acute phases, normal HDL (NHDL) is converted into acute-phase HDL (APHDL) and becomes proinflammatory and unable to protect LDL against oxidative modification. Earlier work had demonstrated that these changes are associated with alterations in apolipoprotein composition and enzymatic activity of APHDL, but the effect of the acute-phase condition on the lipid composition of APHDL had remained obscure. The present study shows marked quantitative differences in lipid composition between NHDL and APHDL. Specifically, APHDL contained 25% less total lipid per milligram of protein. Up to 50% of cholesteryl ester in the lipid core of APHDL was replaced by triacylglycerol; however, the total phospholipid/total neutral lipid ratios were the same as in NHDL, both lipoproteins giving similar calculated lipid core radii. Furthermore, the phosphatidylcholine/sphingomyelin ratio in APHDL was nearly double that in NHDL, indicating a relative loss of sphingomyelin. A decrease was also seen in diacyl and alkenylacyl glycerophosphatidylethanolamine as well as in phosphatidylinositol of APHDL when compared with NHDL. APHDL contained proportionally more saturated and less polyunsaturated and isoprostane-containing species of phosphatidylcholine, as well as more saturated than unsaturated cholesteryl esters. APHDL also contained significantly more free fatty acids, lysophosphatidylcholine, and free cholesterol. These changes in the lipid composition of HDL are consistent with the alterations in the apoprotein composition and enzymatic activity of APHDL and indicate proinflammatory and proatherogenic roles for APHDL.

Three Gaucher-disease-producing mutations in a patient with Gaucher disease: mechanism and diagnostic implications.

As Gaucher disease is an autosomal recessive disorder, most patients are either homozygotes or compound heterozygotes for glucocerebrosidase mutations. We have encountered a patient with three mutations, two c.1226A-->G (1226G, N370S) and one c.1448 T-->C (1448C, L444P). This was shown to be due to a gene conversion event in which the sequence of the glucocerebrosidase pseudogene that includes the 1448C mutation had been imposed on a glucocerebrosidase gene that already had the 1226G mutation. The patient had relatively mild disease which had been discovered after an attack of infectious mononucleosis, a relationship that has been observed previously. If it had not been recognized that this patient had the 1226G/1226G,1448C genotype, prenatal testing might have falsely identified a 1226G,1448C/wt (wild type) fetus as having Gaucher disease.

Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL.

Group IIA secretory phospholipase A2 is an acute phase enzyme, co-expressed with serum amyloid A protein. Both are present in atherosclerotic lesions. We report that human normal and acute phase high density lipoproteins and low density lipoprotein are effective substrates for human group IIA phospholipase A2. The enzyme hydrolyzed choline and ethanolamine glycerophospholipids at the sn -2 position resulting in an accumulation of the corresponding lysophospholipids, including the unhydrolyzed alkyl and alkenyl ether derivatives. The hydrolysis of acute phase high density lipoprotein was 2- to 3-fold more rapid and intensive than of normal high density lipoprotein. The hydrolysis of lipoproteins was noted at enzyme concentration as low as 0.05 microgram/mg protein, which was within the range observed in the circulation in acute and chronic inflammatory diseases. The enzyme hydrolyzed the different molecular species of the residual glycerophospholipids in proportion to their mass, showing no preference for the release of arachidonic acid. Group IIA phospholipase A2 preferentially attacked the hydroxy and hydroperoxy linoleates and possibly other oxygenated fatty acids, which were released from the glycerophospholipids at early times of incubation. There was no effect on the content or molecular species composition of the sphingomyelins or neutral lipids of the lipoproteins. In conclusion, human plasma lipoproteins are the first reported natural biological substrates for human group IIA phospholipase A2. The enhanced hydrolysis of acute phase high density lipoproteins is probably due to its association with serum amyloid A protein, which enhances the activity of the enzyme and may promote its penetration to the lipid monolayer. As sPLA2-induced hydrolysis of the lipoproteins leads to accumulation of lysophosphatidylcholine and potentially toxic oxygenated fatty acids, overexpression of this enzyme may be proatherogenic.

Chemically modified non-antimicrobial tetracyclines inhibit activity of phospholipases A2.

OBJECTIVE: Tetracyclines have been recognized as useful agents for therapy of inflammatory arthritides. However, prolonged use of tetracyclines is limited by their detrimental antimicrobial properties. Recently, a group of chemically modified tetracyclines (CMT) devoid of antimicrobial properties has been synthesized. Some CMT were found to inhibit various matrix metalloproteinases (MMP). We reported previously that antimicrobial tetracyclines inhibit the activity of proinflammatory secretory group II phospholipase A2 (sPLA2). The objective of this study was to detect whether non-antimicrobial CMT also inhibit sPLA2 and other phospholipases A2. METHODS: Ten synthetic CMT were tested for inhibition of sPLA2 human and porcine PLA2, and Naja naja PLA2. PLA2 activity was assessed by radiolabeled Escherichia coli assay using standard and high calcium concentrations. RESULTS: Six of 10 CMT inhibited sPLA2 activity at concentrations close to or lower than 50 microg/ml. All 6 CMT had identical C1-3 and C10-12a positions in the 4-ringed nucleus of the tetracycline molecule. Calcium concentrations up to 20 mM did not eliminate the inhibitory activity of CMT. Inhibition of other PLA2 was induced by some CMT, all but one (CMT-9) belonging to the group of strong inhibitors of sPLA2. Thus, inhibition of PLA2 different from sPLA2 does not necessarily require identical C1-3/C10-12a residues. CONCLUSION: Since CMT, which inhibit proinflammatory sPLA2, are also inhibitors of some MMP, they may be useful for therapy of inflammatory diseases in which both MMP and sPLA2 are overexpressed.

Regulation of the cellular expression of secretory and cytosolic phospholipases A2, and cyclooxygenase-2 by peptide growth factors.

Secretory group II (sPLA2) and cytosolic (cPLA2) phospholipases A2 and cyclooxygenase-2 (Cox-2) play a pivotal role in release of proinflammatory eicosanoids. Excessive activity of sPLA2 per se can also propagate inflammation. Endogenous control of the above enzymes has not been completely elucidated. We investigated the combined impact of promoting cytokines and inhibitory peptide growth factors on the expression of mRNA of the above enzymes, on protein content and extracellular release of sPLA2 and on PGE2 production in osteoblasts (FRCO). The synthesis and release of sPLA2 were enhanced by about 20-fold by 0.5 ng/ml IL-1beta or by 50 ng/ml of TNFalpha. Coaddition of both cytokines resulted in synergistic 150-fold increase in the release of sPLA2 implying the existence of two paths of induction. IL-1beta and TNFalpha markedly enhanced the transcription of sPLA2 mRNA. Kinetic study showed that IL-1/TNF initiated sPLA2 release after 12 h, reaching maximum at 48 h. IL-1alpha was a weak stimulator of sPLA2 release, whereas IL-6, IL-8, IGF, IFN-gamma, growth hormone, insulin and GM-CSF were not stimulatory. Peptide growth hormones TGFbeta, PDGF-BB, EGF and bFGF markedly inhibited the extracellular release of sPLA2. TGFbeta and PDGF-BB significantly reduced the level of sPLA2 mRNA, thus acting upon transcription whereas EGF and bFGF were not inhibitory, acting rather upon the translational or posttranslational steps. IL-1/TNF and growth factors had no significant effect on cPLA2 mRNA expression. Cox-2 mRNA expression was markedly enhanced by IL-1/TNF and suppressed by all growth factors tested. Cytokines enhanced the extracellular release of PGE2 and further enhancement was induced by growth factors with the exception of TGFbeta. Cycloheximide abolished completely the release of sPLA2 and markedly reduced the release of PGE2 from cytokine-stimulated FRCO, regardless of whether growth factors were present or not. NS-398, a specific inhibitor of Cox-2 abolished almost completely the release of PGE2 from cytokine-stimulated cells, regardless of the presence of growth factors. Thus, different signalling mechanisms are involved in the impact of growth factors on mRNA expression of sPLA2, cPLA2 and Cox-2. The differences between the impact on FRCO sPLA2 and that reported in other cells, imply that endogenous control of arachidonic acid cascade is cell-specific.

Induction of secretory phospholipase A2 confirms the systemic inflammatory nature of adjuvant arthritis.

Adjuvant arthritis (AA) is an accepted model of inflammatory arthritis. Until now, however, there is little information about inflammatory mediators, specifically in relation to the arachidonic acid cascade in AA. Our objective was to study the expression of secretory (sPLA2) and cytosolic (cPLA2) phospholipases A2 in various organs during the course of AA. AA was induced in Lewis rats which were sacrificed at days 0, 7, 14, 21, 28 and 42. Expression of sPLA2 mRNA and protein and mRNA of cPLA2 in paws, regional lymph nodes, spleen, liver, lungs and aorta was investigated. Serum sPLA2 activity increased from 15213 +/- 1131 to a maximum of 32,455 +/- 4109 nmol/30' on day 21. Maximal increase in sPLA2 mRNA in paws, lung and aorta was observed on day 14, and in the lymph nodes and spleen on day 28. In the liver, trace levels were found with no corresponding protein expression. In paws, lung, aorta and lymph nodes maximum increase in sPLA2 protein was noted on day 14 whereas the spleen showed constant sPLA2 protein level during AA. cPLA2 mRNA detected in all organs, did not significantly change during the course of AA, with the exception of regional lymph nodes where the message increased between 14 and 28 day. Induction of mRNA and protein of sPLA2 in several organs is an evidence that AA is a systemic inflammatory process. The parallelity of the sPLA2 expression to the severity of inflammatory process, implies that sPLA2 may play pathogenic role in AA. Lack of enhancement of cPLA2 mRNA may mean that this enzyme is either not induced in AA, or it increases earlier in the course of the inflammatory process.

Inhibition of extracellular release of proinflammatory secretory phospholipase A2 (sPLA2) by sulfasalazine: a novel mechanism of anti-inflammatory activity.

Sulfasalazine is widely used in rheumatoid arthritis and inflammatory bowel diseases. The mechanisms of its activity have not been elucidated. In leukocytes, sulfasalazine and its analogue, CL 42A, inhibited the formation of leukotrienes and possibly of the second messenger compounds at the level of phospholipase C. Partial inhibition of interleukin-lbeta (IL-1beta), IL-6 and tumor necrosis factor-alpha (TNF-alpha) was also found. Since the synthesis of eicosanoids is induced by phospholipase A2 and since secretory phospholipase A2 (sPLA2) is proinflammatory, we investigated the impact of sulfasalazine and related compounds on mRNA, protein synthesis, and release of sPLA2 from osteoblasts. Sulfasalazine and CL 42A markedly inhibited extracellular release of sPLA2. The impact of sulfasalazine was evident at 50 microM (P < 0.001) and maximal at 400 microM, and that of CL 42A at 10 microM (P < 0.001) and 200 microM, respectively. Split products of sulfasalazine, 5-aminosalicylic acid (400 microM) and sulfapyridine (400 microM), had no impact. The effect of sulfasalazine and CL 42A was evident regardless of whether the cells were stimulated with IL-1beta/TNF-alpha, lipopolysaccharide/forskolin, or dibutyryl-cAMP. Sulfasalazine and CL 42A did not alter the level of sPLA2 mRNA. Exposure of stimulated fetal rat calvaria osteoblasts (FRCO) to sulfasalazine did not show accumulation of the intracellular sPLA2 protein as tested by western blot; however, enzymatic activity of PLA2 in disrupted cells was definitely increased. Thus, the impact is on the post-transcriptional release of sPLA2 rather than on the synthesis. There was also an increase in the extracellular release of prostaglandin E2 from FRCO exposed to sulfasalazine or to CL 42A. In contrast, sulfasalazine had no effect on the extracellular release of gelatinase from the cells or on mRNA of cytosolic PLA2 or cyclooxygenase 2. We conclude that the anti-inflammatory activity of sulfasalazine may be related, in part, to the selective inhibition of the extracellular release of proinflammatory sPLA2.

Coordinate expression of group II phospholipase A2 and the acute-phase proteins haptoglobin (HP) and alpha1-anti-chymotrypsin (ACH) by HepG2 cells.

The early response to inflammation is characterized by the synthesis of a variety of proteins under cytokine and glucocorticoid control. During episodes of infection or inflammation, a secretory phospholipase A2 (sPLA2) appears in the circulation along with a variety of acute-phase proteins (APP), suggesting possible common regulatory elements amongst sPLA2 and APP. Using the human hepatoma line, HepG2, regulation of sPLA2 expression was examined in relation to synthesis of HP and ACH. The patterns of induction of sPLA2, HP and ACH were distinct for each of IL- 1, tumour necrosis factor (TNF) and IL-6, oncostatin M, IL-11 and leukaemia inhibitory factor. Dexamethasone had an enhancing effect on IL-6-induced expression of HP and ACH, but inhibited sPLA2 expression by 50%. Both 8-bromo-cAMP and dibutyryl cAMP increased sPLA2 expression (48.8-fold and 64.2-fold, respectively), whereas KT5720, an inhibitor of protein kinase A, down-regulated cytokine-induced sPLA2 synthesis by 51%. These data show that a panel of cytokines induced varying patterns of up-regulation of sPLA2, ACH and HP. Although dexamethasone potentiated IL-6-induced APP expression in HepG2 cells, it suppressed sPLA2 expression in a dose-dependent manner. In several respects, sPLA2 regulation is similar to that of HP and ACH, but a notable difference is the reciprocal effect of glucocorticoids on sPLA2 expression compared with that of ACH and HP.

Microtubule depolymerization selectively down-regulates the synthesis of proinflammatory secretory nonpancreatic phospholipase A2.

Microtubule depolymerizing agents (MTD) diminish the expression of cell surface receptors for TNF-alpha. Because TNF-alpha along with IL-1 beta markedly enhance the gene expression and extracellular release of proinflammatory secretory nonpancreatic phospholipase A2 (sPLA2), we tested the impact of MTD on the expression of sPLA2. We report that MTD markedly inhibit the expression and release of sPLA2 by fetal rat calvarial osteoblasts (FRCO), which synthesize and release sPLA2. When FRCO were pretreated with colchicine and then stimulated with IL-1 beta 0.2 ng/ml and TNF-alpha 25 ng/ml (IL-1/TNF), minute quantities of colchicine (1.25 nM) reduced the released sPLA2 activity to 11% of that in controls. IC50 was 0.75 nM. When IL-1/TNF and colchicine were added simultaneously, similar inhibition (8% of that in controls) required higher concentrations of colchicine (0.125 microM). IC50 was 68.75 nM. When FRCO were prestimulated by IL-1/TNF, much higher concentrations of colchicine were required to reduce sPLA2 activity. MTD inhibited the expression of sPLA2 by a mechanism(s) different from the way in which they impact TNF surface receptors, because they inhibited sPLA2 expression in FRCO stimulated by IL-1 beta or by cell-permeable cAMP analogs. Colchicine (1 microM) reduced the expression of sPLA2 induced by dibutyryl cAMP (2 mM) and 8-bromo-cAMP (4 mM) to 38% and 58% of that n controls, respectively. Photoinactivated lumicolchicines beta and gamma were noninhibitory. Microtubular stabilizer taxol (5 microM) abolished inhibitory activity of colchicine, increasing the expression of sPLA2 3.2-fold compared with that in control cells cultured without taxol. Other MTD, such as vinblastine (0.01 microM), inhibited sPLA2 release to 27% of the controls, whereas nocodazole (10 microM) was less inhibitory. Northern blot analysis of FRCO showed that sPLA2 mRNA was greatly induced by IL-1/TNF. The induction of sPLA2 mRNA by IL-1/TNF was nearly completely abolished by colchicine in a dose-related manner. Western blot analysis of intra- and extracellular sPLA2 protein showed complete inhibition of the synthesis by MTD. To determine whether the inhibition of sPLA2 is selective, mRNA levels of cytosolic PLA2 and of inducible cyclooxygenase-2 were investigated. Colchicine had no effect on the mRNA levels of these two enzymes, which suggests that the inhibitory effect of MTD on sPLA2 expression is selective and occurs at the transcriptional level. Thus, the microtubular system plays a significant role in the synthesis of proinflammatory sPLA2, a fact that may explain in part the anti-inflammatory activity of microtubular disrupters.

Secretory non-pancreatic phospholipase A2 and cyclooxygenase-2 expression by tracheobronchial smooth muscle cells.

Lipid mediators of inflammation, contribute to airway hyper-reactivity in asthma. Since production of lipid mediators is largely regulated by phospholipase A2 (PLA2), and since PLA2 expression in mesenchymal cells is induced by cytokines and other signals, we examined PLA2 expression by rat tracheobronchial smooth muscle cells (TBSMC). PLA2 expression in TBSMC cultures was markedly increased by tumour-necrosis factor (TNF) alpha (130-fold) and interleukin-1beta (IL-1beta) (7.4-fold). Lipopolysaccharide (LPS;100 ng/ml) resulted in a 51-fold increase in extracellular PLA2 activity. PLA2 expression by LPS-stimulated or cytokine-stimulated cells was downregulated by dexamethasone. Whereas forskolin or dibutyrl cAMP increased PLA2 activity, inhibition of protein kinase A but not tyrosine kinase reduced PLA2 expression. Northern blot analysis showed that TNF alpha and IL-1beta increased both PLA2 and inducible cyclooxygenase (Cox-2) mRNA transcription. Addition of dexamethasone substantially blunted the increase in PLA2 and Cox-2 mRNA. In contrast, the level of Cox-1 mRNA was very low and did not change with the various treatments. Since proinflammatory lipid mediators have been implicated in the pathogenesis of asthma and PLA2 activity regulates generation of these lipid mediators, cytokine-stimulated synthesis and release of PLA2 by airway smooth cells may contribute to the potentiation of airway inflammation in asthma.

Inhibition of the activity of pro-inflammatory secretory phospholipase A(2) by acute phase proteins.

Pro-Inflammatory non-pancreatic phospholipase A(2) (sPLA(2)) is markedly over-expressed in acute systemic and chronic local inflammatory processes. Since in acute phase reaction sPLA(2) is often over-expressed simultaneously with acute phase proteins (APP), it is important to determine whether APP interacts with sPLA(2). We tested ten APPs for interaction with sPLA(2) using as a substrate multilamellar Hposomes composed either of PC:Lyso PC or PE:Lyso PE. Using PC:Lyso PC substrate, CRP, lactoferrin and SAP were found to inhibit sPLA(2) activity with an IC(50) of 25 mug/ml, 7.5 mug/ml and 50 mug/ml, respectively, corresponding to 0.21 muM, 0.1 muM and 0.21 muM respectively. Using PE:Lyso PE substrate only SAP was inhibitory, with an IC(50) of 10 mug/ml (0.04 muM). Phosphorylcholine abolished the inhibitory activity of CRP but not of SAP or lactoferrin. Addition of phosphorylethanolamine or of excess calcium had no effect on the inhibitory activity of APP. Limulin, lysozyme, transferrin, beta(2)-microglobulin, alpha(2)-macroglobulin, human and bovine albumins had no effect on sPLA2 activity. Therefore neither the structure of pentraxins, or ironbinding, bacteriostatic property or amyloidogenic property preclude whether APP modulates sPLA(2) activity. Inhibition of pro-inflammatory sPLA(2) by APP may be one of the protective mechanisms of the acute phase reaction.

Induction of release of secretory nonpancreatic phospholipase A2 from human articular chondrocytes.

OBJECTIVE: Secretory nonpancreatic phospholipase A2 (sPLA2) is a known inducer/promoter of the inflammatory process in the joints. It correlates with disease activity in adult and juvenile rheumatoid arthritis. Synovial fluids contain high concentrations of sPLA2. We discovered that human articular cartilage contains large quantities of sPLA2 and that culture chondrocytes constitutively synthesize and release sPLA2. To test the mechanism controlling the release of sPLA2, we exposed cultured human articular chondrocytes to cytokines and other agents, known to induce sPLA2 in other cells. METHODS: Chondrocytes obtained from cartilage of normal appearance from rheumatoid and osteoarthritic joints, and from normal, neonatal joints were compared to rabbit articular chondrocytes. Radiolabeled Escherichia coli derived phospholipid assay and ELISA technique using monoclonal antibodies against recombinant human synovial type sPLA2 were employed. The cells were grown as monolayers as well as in alginate beads. RESULTS: Human articular chondrocytes from both arthritic and neonatal joints released sPLA2 constitutively but could not be further stimulated with interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), IL-6, oncostatin M, lipopolysaccharide (LPS), or forskolin. Marked stimulation was observed when the cells were exposed to 8-bromo cyclic adenosine monophosphate (cAMP). Growing the cells as monolayers or in alginate beads did not change the results. In contrast to human cells, rabbit chondrocytes responded to IL-1 beta and IL-1/TNF, but not to TNF-alpha alone, with a very marked increase in extracellular sPLA2 activity. CONCLUSION: Human articular chondrocytes synthesize and constitutively release sPLA2. Such continuous release is most probably responsible for the high concentration of sPLA2 in articular cartilage and may be the source of synovial fluid sPLA2. To our knowledge, human articular chondrocytes are the only sPLA2 producing cells tested to date that do not respond to cytokine stimulation with increased sPLA2 activity; yet enhancement was seen with 8-bromo cAMP. It seems therefore that, human articular chondrocytes possess signalling mechanisms for the release of sPLA2 unlike those from other mammalian cells. The significance of this observation remains to be elucidated.

Serum amyloid A protein enhances the activity of secretory non-pancreatic phospholipase A2.

The acute-phase proteins serum amyloid A protein (SAA) and secretory phospholipase A2 (sPLA2) are simultaneously expressed during inflammatory conditions. SAA associates with high-density lipoprotein (HDL) altering its physicochemical composition. We found that purified acute-phase SAA, but not the constitutive form, markedly enhances the lipolytic activity of sPLA2 in a dose-related manner with phosphatidylcholine/lysophosphatidylcholine or phosphatidylethanolamine/lysophosphatidylethanolamine liposomal substrates. Normal HDL was found to reduce activity of sPLA2 in a dose-dependent manner, but when acute-phase HDL containing 27% SAA was tested, it enhanced sPLA2 activity. Immunopurified monospecific antibodies against SAA completely abolished the enhancing activity of SAA and acute-phase HDL. Given the central role of HDL in lipoprotein metabolism, the interaction between HDL, SAA and sPLA2 may account for changes detected in lipoprotein metabolism during the acute phase.

Inhibition of human group II phospholipase A2 by C-reactive protein in vitro.

Endotoxin or cytokine-induced expression of a secretory non-pancreatic phospholipase A2 (sPLA2) has been implicated in the pathogenesis of multisystem organ dysfunction or failure in patients with septic shock. Circulating sPLA2 levels increase as much as 1000-fold during the course of septic shock. However, the mode of regulation of the activity of this lipolytic enzyme is unknown, since circulating inhibitors have not been identified. We investigated the potential inhibitory activity of the acute phase reactant, C-reactive protein (CRP), a phospholipid-binding protein whose expression increases as much as 1000-fold during severe infections. Serum CRP and sPLA2 profiles were highly concordant in patients with septic shock. In studies in vitro, human CRP inhibited hydrolysis of PC-lyso-PC (2:1) multilamellar liposomes by human recombinant sPLA2 in a dose-dependent manner, with an apparent IC50 of 50 micrograms/ml CRP and maximal inhibition at 100 micrograms/ml. Inhibition of sPLA2 activity by CRP was substrate concentration-dependent, and increasing substrate concentrations reversed the inhibitory effect of CRP using the PC-lyso-PC system. Preincubation of CRP with phosphorylcholine led to a concentration-dependent loss of CRP-induced inhibition of substrate hydrolysis. These observations are consistent with a substrate-depletion model of inhibition of sPLA2 activity by CRP.

Tenidap sodium inhibits secretory non-pancreatic phospholipase A(2) synthesis by foetal rat calvarial osteoblasts.

Tenidap (TD) was initially defined as a dual inhibitor of cyclooxygenase and lipoxygenase. This study was designed to assess its inhibitory activity against proinflammatory phospholipase A(2). This study shows that TD inhibits the synthesis of pro-inflammatory secretory non-pancreatic phospholipase A(2) (sPLA(2)). Concentrations as low as 0.25 mug/ml (0.725 muM) reduced the release of sPLA(2) by 40% from foetal rat calvarial osteoblasts stimulated with IL-1beta and TNFalpha, whereas a concentration of 2.5 mug/ml (7.25 muM) reduced the release by over 80%. TD also markedly reduced the release of sPLA(2) from unstimulated cells. There was no direct inhibition of sPLA(2) enzymatic activity by TD in vitro. Northern blot analysis showed that TD did not affect the sPLA(2) mRNA levels; however, immunoblotting showed a dose-dependent reduction in sPLA(2) enzyme. These results, together with a marked reduction in sPLA(2) enzymatic activity, suggest that TD inhibits sPLA(2) synthesis at the post-transcriptional level. Therefore TD seems to inhibit the arachidonic acid cascade proximally to cyclooxygenase and lipoxygenase and its anti-inflammatory activity may be related at least in part to the inhibition of sPLA(2) synthesis.

Phospholipase A2 in juvenile rheumatoid arthritis: correlation to disease type and activity.

OBJECTIVE: Secretory nonpancreatic phospholipase A2 (snpPLA2) is a proinflammatory enzyme and its activity in serum correlates with disease activity in adults with rheumatoid arthritis. Juvenile rheumatoid arthritis (JRA) may be stratified into 3 clinical types with differing degrees of disease activity. Since in JRA there are no reliable indices of disease activity, our objective was to find whether the level of circulating snpPLA2 correlates with the severity of inflammation and with JRA activity. METHODS: PLA2 enzymatic activity was assayed using E. coli membranes labelled with (14C)-oleic acid. SnpPLA2 immunoreactivity was tested by ELISA technique using monoclonal antibodies against recombinant human (rh) snpPLA2. SnpPLA2 activity was determined in sera of 127 children including 25 with systemic (S-JRA), 50 with polyarticular (Po-JRA) and 52 with pauciarticular (Pa-JRA) types of JRA. Twenty-five patients with active disease, were subsequently restudied in an inactive phase. RESULTS: Markedly increased snpPLA2 (> mean + 2 SD of normal mean, i.e., > 575 U/ml) was found during the active disease in 100% S-JRA, 57% Po-JRA and 25% Pa-JRA patients. The differences in the mean and median PLA2 activity among these 3 subtypes of JRA were highly significant (p < 0.001) with the highest levels found in S-JRA and the lowest in Pa-JRA. Presence of rheumatoid factor and/or of antinuclear antibody had no relation to the level of snpPLA2. SnpPLA2 activity became markedly lower when active inflammation became quiescent. In the whole group, snpPLA2 activity correlated highly with the Lansbury index, number of involved joints and number of effusions. A significant positive correlation was also found between snpPLA2 and erythrocyte sedimentation rate (ESR) and neutrophil count, while a significant negative correlation was noted with the level of albumin and hemoglobin. With the exception of snpPLA2, other laboratory variables did not correlate with the number of effusions or the number of active joints. However a negative correlation was noted between both hemoglobin and albumin, and Lansbury index. CONCLUSION: Circulating snpPLA2 significantly correlates with JRA activity and may serve as an index of activity in JRA especially in patients with systemic type of disease.

Increased serum phospholipase A2 activity in Malawian children with falciparum malaria.

Some clinical manifestations of severe malaria resemble those of sepsis and there may be mediators of the host response that are common to both sepsis and malaria. Phospholipase A2 (PLA2), a proinflammatory enzyme whose expression is induced by tumor necrosis factor (TNF), has been implicated in the pathogenesis of complications of the sepsis syndrome. We examined levels of circulating PLA2 in Plasmodium falciparum malaria and studied the association of PLA2 with disease severity. Plasma PLA2 and TNF were measured in 75 Malawian children with P. falciparum malaria. The mean (SD) plasma PLA2 activity in children with acute malaria was 53,804 (37,256) units/ml as compared with 424 (349) units/ml in 34 healthy controls (P < 0.00001). The mean PLA2 activity in 45 convalescent patients was 2,546 (7,372) units/ml (P < 0.00001). In 48 patients with pretreatment PLA2 activity less than 60,000 units/ml, mortality was 8.3%, while in 27 patients with pretreatment PLA2 levels greater than 60,000 units/ml, mortality was 33.3% (P = 0.008). There were significant correlations between PLA2 and TNF (r = 0.471, P < 0.01), density of parasitemia (r = 0.443, P < 0.0001) and a decrease in hematocrit (r = 0.352, P < 0.005). These data show that P. falciparum malaria is associated with a markedly increased circulating PLA2, especially in patients with severe disease, as manifested by high parasite burden, anemia, coma, and death.