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.

Interleukin-1 alpha stimulates the release of prostaglandin E2 and phospholipase A2 from fetal rat calvarial cells in vitro: relationship to bone nodule formation.

We have shown previously that interleukin-1 (IL-1) has biphasic effects on the formation of bone nodules in long-term cultures of fetal rat calvarial (RC) cells (Ellies and Aubin, Cytokine 2:430-437, 1990). To determine the role of arachidonic acid metabolism in this process, we have examined the release of prostaglandin E2 (PGE2) and phospholipase A2 (PLA2) from RC cells under conditions that allowed concomitant analysis of the formation of bone nodules. Recombinant human IL-1 alpha (rhIL-1 alpha) stimulated PGE2 and PLA2 release in a time- and dose-dependent manner. PGE2 release was highest in preconfluent cultures (days 1-6) and was stimulated up to 8.5-fold in response to 50 U/ml of rhIL-1 alpha. In contrast, extracellular PLA2 activity was maximal in postconfluent cultures, with 50 U/ml of rhIL-1 alpha causing a 20-fold increase by day 15. PLA2 release by RC cells was not significantly affected by PGE2, the glucocorticoid dexamethasone, or the cyclooxygenase inhibitor indomethacin. Indomethacin partially blocked the inhibition of bone nodule formation caused by rhIL-1 alpha, and exogenous PGE2 reversed this effect. Addition of group I PLA2 from Naja naja venom to RC cells had no effect on bone nodule development; however, group II PLA2 from Crotalus adamanteus venom inhibited the formation of bone nodules in a dose range similar to that induced by rhIL-1 alpha. These results indicate that PGE2 release does not have a direct temporal correlation with increases in PLA2 activity. In addition, the data show that only part of the inhibition of bone formation seen with rhIL-1 alpha is mediated by PGE2 and suggest that extracellular PLA2 also accounts for part of the inhibition.

Induction of circulating phospholipase A2 activity by intravenous infusion of endotoxin in patients with neoplasia.

The purpose of this study was to evaluate the impact of repeated intravenous infusions of endotoxin (EN) in patients with cancer on the systemic release of extracellular proinflammatory phospholipase A2 (PLA2) and its relationship to the release of tumor necrosis factor (TNF) and interleukin-6 (IL-6). Six patients received 15 infusion of EN isolated from Salmonella abortus equi at a dose of 4 ng/kg. Marked increase in the activity of circulating PLA2 was noted within 3 h after the first EN infusion and reached a maximal level of 20.4-fold greater than baseline 24 h after infusion. In five patients challenged with EN 2 weeks later, PLA2 reached peak levels 15.5-fold greater than baseline. In two patients who received three sequential daily infusions, the incremental increase in PLA2 activity after the second and third challenge reached maximum levels 6 h after EN infusion. PLA2 response followed those of TNF and IL-6 but was quantitatively different. Whereas maximal levels of TNF and IL-6 declined substantially after repeat EN challenges, no such decline occurred in PLA2 activity. Since, in the clinical setting of gram-negative sepsis, there is recurrent increase in circulating EN, our study approximates this clinical situation and shows that extracellular release of PLA2 follows temporally that of proximal cytokines such as TNF and IL-6. These cytokines may be related to PLA2 release and sustained high activity in the systemic circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular phospholipase A2 secretion is a common effector pathway of interleukin-1 and tumour necrosis factor action.

Inflammatory processes are characterized by increased levels of extracellular phospholipase A2 (PLA2) and cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF). IL-1, TNF and PLA2 share a number of proinflammatory, arthritogenic effects. The sequential induction, first of the cytokines followed by PLA2, suggests that these cytokines may regulate synthesis and secretion of PLA2. To test this postulate, foetal rat calvarial bone-forming cells (FRCC) were treated with recombinant human IL-1 and TNF and extracellular PLA2 release was quantitated. Both IL-1 and TNF induced the de novo synthesis of PLA2 in a concentration-dependent manner. Continuous exposure of FRCC in primary culture to IL-1 (50 units/ml) over 15 days resulted in as much as 100-fold increase in PLA2 secretion. IL-1 (50 units/ml) added to post-confluent cultures for a 48-h pulse increased PLA2 activity 9.4-fold. The combination of IL-1 (50 units/ml) and TNF (500 units/ml) was synergistic with an observed increase in extracellular PLA2 secretion of 146-fold following a 48-h pulse. Interleukin-6, alone or in combination with IL-1 or TNF, did not further enhance PLA2 synthesis of secretion. Cytokine-induced synthesis of PLA2 was inhibited 80% by 10 microM cycloheximide but not by dexamethasone over the range of 10(-6) to 10(-8) M. FRCC-derived PLA2 was neutral-active with a pH optimum of 6-7.5 and was calcium-dependent with optimal activity in the presence of 2-7 mM calcium. It had absolute 2-acyl specificity using micellar phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline.

Extracellular phospholipases A2 play an important role in articular and extra-articular inflammatory processes. Secretory non-pancreatic phospholipase A2 (PLA2) has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to the tissue damage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines such as minocycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were markedly inhibitory to both pancreatic and non-pancreatic PLA2. Using [14C]oleic acid labeled Escherichia coli membrane phospholipids as substrate, the IC50 values for minocycline and doxycycline were 3.6 x 10(-5) M (18 micrograms/mL) and 0.98 x 10(-4) M (47 micrograms/mL), respectively. In a scooting mode assay using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as substrate, IC50 values for minocycline were 5 microM (2.47 micrograms/mL) for non-pancreatic PLA2 and 8 microM (3.95 micrograms/mL) for pancreatic PLA2. Addition of excess calcium up to 50 mM did not reverse the inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA2, probably by interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions in which PLA2 is implicated pathogenetically.

Purification of a soluble phospholipase A2 from synovial fluid in rheumatoid arthritis.

A soluble phospholipase A2 (PLA2) was purified 4,500-fold from human rheumatoid synovial fluid. Preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two bands of PLA2 activity of molecular weights 15,000 and 17,000 and pl 4.2-5.0. Purified PLA2 had absolute 2-acyl specificity, and hydrolyzed phosphatidylcholine with optimal activity at pH 7.5-8.0 and phosphatidylethanolamine with optimal activity at pH 7.0. Human synovial fluid PLA2 did not cross-react with anti-human pancreatic PLA2, as tested by radioimmunoassay.

Multiple forms of phospholipase A2 in arthritic synovial fluid.

Phospholipase A2 (PLA2) has been purified to homogeneity from human arthritic synovial fluid. The activity resolved into multiple peaks by preparative HPLC. The most abundant peak (A) was present in synovial fluid from patients with rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. A second major peak (B) was variable and lower in relative abundance, but was distinguishable from peak A by its stimulated activity in the presence of either 0.5 M Tris or 0.1% sodium deoxycholate (DOC), in addition to its longer HPLC column retention time. Both peaks required Ca2+ and showed optimal activity in DOC/phosphatidylcholine (PC) mixed micelle assays between pH 8.0 and 9.0. Both peaks showed higher activity with PC as substrate than with PI, however peak A exhibited higher activity with PE than PC. Upon preparative SDS-polyacrylamide gel electrophoresis, both peaks of PLA2 activity were resolved as proteins of approximately 14,000 Da. The N-terminal sequence obtained from purified peak A material matched that of a recent similar isolate (Hara et al. (1988) J. Biochem. 104, 326-328).

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.

Characterization of extracellular phospholipase A2 in rheumatoid synovial fluid.

Phospholipase A2 (PLA2) activity has now been identified in rheumatoid synovial fluids. This PLA2 is a calcium-requiring protein of MW 11,000 with a neutral pH optimum. Its activity was inhibited by high concentrations of Mg2+, and by the active site-directed histidine reagent p-bromophenacyl bromide. Ionic and nonionic detergents, or the sulfhydryl reagent dithiothreitol caused loss of enzyme activity. Synovial fluid PLA2 did not interact with sulphated mucopolysaccharides such as heparin or chondroitin sulphate. Release and sequestration of PLA2 in the joint space may contribute to the characteristic rheumatoid inflammatory changes.

Enzymatic activity and distribution of phospholipase A2 in human cartilage.

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.

Serum phospholipase A2 enzyme activity and immunoreactivity in a prospective analysis of patients with septic shock.

Massive elevations of serum phospholipase A2 activity have been documented in patients with septic shock. Serum PLA2 activity correlated to the degree and duration of circulatory collapse, while purified native PLA2 reproduced hypotension in experimental animals. In a prospective study of patients with septic shock, we have determined the relationship of PLA2 enzyme activity to PLA2 immunoreactivity using radiolabelled E. coli phospholipid substrate and an ELISA specific for group II human nonpancreatic PLA2. In all patients, there was a clear concordance of the two assays. Maximal PLA2 concentration was increased a mean of 554-fold over normal levels. We found no evidence to support the presence of activating or inhibitory proteins. These data confirm that the observed increase in serum PLA2 activity in septic shock is due to intravascular release of group II nonpancreatic PLA2.

Influence of plasma proteins on activity of proinflammatory enzyme phospholipase A2.

The influence of plasma proteins on quantitation of the proinflammatory enzyme phospholipase A2 (PLA2) from rheumatoid synovial fluid was studied using two different assays. Human and bovine serum albumins increased the rate of PLA2 hydrolysis of membrane-associated phospholipid substrates. In contrast, albumin profoundly inhibited the PLA2 hydrolysis of synthetic phospholipids in micellar dispersion. Other plasma proteins (alpha, beta and gamma-globulins) had minimal effect on PLA2 activity in either assay system. Since the presence of albumin may compromise estimation of PLA2 and of phospholipase-inhibitory proteins, the appropriate selection of assay conditions is obligatory for the accurate quantitation of their respective activities.

Comparison of group I and II soluble phospholipases A2 activities on phagocytic functions of human polymorphonuclear and mononuclear phagocytes.

Soluble phospholipase A2 (PLA2) purified from rheumatoid synovial fluid (group II) and repurified Naja naja venom PLA2 (group I) were compared for their influence on phagocytic activity of human polymorphonuclear (PMN) and mononuclear (MO) phagocytes. Group II PLA2 reduced chemotaxis, adhesiveness, and intracellular bactericidal activity (ICBA) and induced release of muramidase from PMNs. Group I PLA2 suppressed chemotaxis, and enhanced ICBA but had no influence on other phagocytic functions. Group II PLA2 purified from synovial fluid or from placenta caused marked spontaneous superoxide generation followed by inhibition of phagocytosis-induced burst of energy. Group I Naja naja and porcine pancreatic PLA2 had no effect on superoxide generation. Group II but not group I PLA2 reduced markedly ICBA of monocytes. It may be concluded that human group II soluble PLA2, in concentrations comparable to those present in inflamed joints or in sera of patients with active arthritis or septic shock, causes spontaneous formation of the oxygen radical superoxide and release of lysosomal enzymes, and suppresses conventional phagocytic activities of PMNs and monocytes. Marked differences between group I and group II PLA2s may mean that these enzymes exert different influences on cell membrane.

Compartmental heterogeneity of soluble phospholipases A.

Multiple forms of soluble phospholipase A2 (PLA2) are known to coexist in venoms of individual reptilian species. While similar observations in several mammalian species suggest that this is a common phenomenon, the functional implications are not yet understood. In attempting to devise therapeutic strategies for treatment of inflammatory disorders by inhibition of PLA2, it is imperative to define the various PLA2 species in the relevant compartments. Herein, we report the presence of three PLA2 isotypes in rheumatoid arthritis serum, one pancreatic and two nonpancreatic phospholipases A2. The pancreatic and one of the nonpancreatic forms were optimally active in 7 mM calcium at pH 7.5. The other nonpancreatic form was calcium-independent and optimally active at pH 7.0. Only the calcium-dependent nonpancreatic form was observed in rheumatoid synovial fluid. Of the three serum isotypes, only the calcium-dependent nonpancreatic form correlated with markers of disease activity, such as the joint count and Landsbury index. Therefore, not all soluble or circulating phospholipases A2 are relevant to inflammatory processes. Selective inhibition of the proinflammatory form of PLA2 may prove to have some therapeutic benefit while minimizing the possible adverse effects of this form of intervention.

Enzymatic activity and immunoreactivity of extracellular phospholipase A2 in inflammatory synovial fluids.

Synovial fluid PLA2 concentration was measured by an ELISA technique using monoclonal antibodies raised against human recombinant "synovial-type" group II phospholipase A2. This ELISA was specific for synovial-type PLA2 and did not detect pancreatic (group I) PLA2. In all synovial fluids examined, including rheumatoid, osteoarthritic, psoriatic, and gouty fluids, synovial fluid PLA2 enzyme activity significantly correlated with PLA2 immunoreactivity (P < 0.001). Within the limits of the ELISA technique, there was no evidence for the presence of specific or nonspecific modulation of PLA2 activity by either putative PLA2 activating or inhibitory proteins.

Hyperphospholipasemia A2 in human volunteers challenged with intravenous endotoxin.

Phospholipase A2 (PLA2) activity was measured in the serum of 23 individuals infused intravenously with endotoxin (EN) at a dose of 4 ng/kg body weight. A marked increase in PLA2 was noted 3 h after EN challenge (mean 828 +/- 513 units/ml), reached its maximum at 24 h after the challenge (mean 2667 +/- 2442 units/ml), and was still evident at 48 h (mean 763 +/- 366 units/ml). In contrast, TNF levels were maximal (mean 712 +/- 375 pg/ml) 90 min after the EN challenge and subsided to very low values (5 +/- 5 pg/ml) 5 h after the challenge. There was a positive correlation between the maximum response of TNF and that of PLA2 (r = 0.82, P < 0.01). Administration of ibuprofen or pentoxifylline did not alter the PLA2 response. EN challenge did not affect serum pancreatic PLA2 concentration or that of the lysosomal cationic enzyme, lysozyme. Neutralizing antibody against human recombinant (synovial type) PLA2 completely abolished PLA2 activity in the sera tested. We conclude that EN infusions cause marked intravascular release of nonpancreatic secretory PLA2 and that the magnitude of this response seems to be related to the prior generation of TNF.

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.