Eva Stefanski

Groups IV, V, and X Phospholipases A2s in Human Neutrophils ROLE IN EICOSANOID PRODUCTION AND GRAM-NEGATIVE BACTERIAL PHOSPHOLIPID HYDROLYSIS

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A2 (PLA2) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA2 (GV and GX sPLA2) mRNA and contain GV and GX sPLA2 proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA2s are undetectable. GV sPLA2 is a component of both azurophilic and specific granules, whereas GX sPLA2 is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA2 and most, if not all, of the PLA2 activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA2. GV sPLA2 does not contribute to fMLP-stimulated leukotriene B4 production but may support the anti-bacterial properties of the neutrophil, because 10–100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA2-α (group IV PLA2α), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B4 in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.

Pathogenesis of hypotension in septic shock: correlation of circulating phospholipase A2 levels with circulatory collapse.

Circulating phospholipase A2 (PLA2) has been recognized as a mediator of circulatory collapse in experimental endotoxic shock. To assess the role of serum PLA2 in septic shock in man, we determined serum PLA2 profiles in a prospective study in 12 patients with septic shock. During the hypotensive phase of sepsis, serum PLA2 levels were consistently elevated as high as 33,428 U/ml (normal range 115 +/- 12 [SE]; n = 101). In all 12 patients, PLA2 levels correlated directly with the magnitude and duration of circulatory collapse (p less than .001), with a progressive fall of serum PLA2 levels during convalescence. In contrast, serum PLA2 levels in patients with cardiogenic shock secondary to myocardial infarction remained low. In pancreatitis, PLA2 levels paralleled fluctuations of serum amylase and lipase, whereas in septic shock without pancreatic involvement, PLA2 changes were discordant with changes in pancreatic enzymes. As well, septic shock serum PLA2 failed to crossreact by radioimmunoassay with antiserum against human pancreatic PLA2. These data are consistent with an extrapancreatic source of intravascular PLA2 release during sepsis. Since endogenous serum PLA2 levels correlate directly with the magnitude of hypotension in both experimental endotoxic shock and clinical septic shock, and since parenteral administration of purified exogenous PLA2 reproduces hypotension in experimental models, we conclude that high levels of intravascular PLA2 may contribute similarly to the circulatory collapse in septic shock in man.

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.

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.

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.

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.

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)

Potential therapeutic efficacy of inhibitors of human phospholipase A2 in septic shock

Soluble phospholipase A2 has been implicated in the pathogenesis of local and systemic inflammatory reactions. Elevated levels of circulating phospholipase A2 (PLA2) correlate with the severity of circulatory collapse and pulmonary dysfunction in gram-negative septic shock. Characterization of septic shock serum PLA2 revealed a calcium-dependent enzyme with absolute 2-acyl specificity with a pH optimum of 7.5. We tested a number of therapeutic agents for their ability to inhibit PLA2 from human septic shock serum. Chloroquine, chlorpromazine, dexamethasone base, dexamethasone sodium phosphate, indomethacin, lidocaine, oleic acid, palmitic acid, promethazine, trans-retinoic acid, rutin and dl-α-tocopherol were all studied over the range of 10−2 to 10−7 M. All agents, with the sole exception of dexamethasone base, inhibited PLA2 activity at concentrations greater than 10−3M. PLA2 inhibition by dexamethasone sodium phosphate was factitious, due to the formation of calcium-phosphate complexes. Of the 11 agents studied, chlorpromazine was the most effective, with an IC50 of 7.5×10−5 M, a membrane concentration achievable within its therapeutic range. Inhibition was non-competitive, with an apparent Ki of 5 nM. Since serum PLA2 levels correlate with mortality in both experimental endotoxemia and clinical gram-negative septic shock, and chlorpromazine was previously shown to improve survival in these conditions, we postulate that its therapeutic efficacy resides at least in part in its PLA2-inhibitory activity.

MAP Kinase Kinase 6–p38 MAP Kinase Signaling Cascade Regulates Cyclooxygenase-2 Expression in Cardiac Myocytes In Vitro and In Vivo

&NA; —Cyclooxygenase‐2 (COX‐2) catalyzes the rate‐limiting step in delayed prostaglandin biosynthesis. The purpose of this study was to evaluate the role of the MAP kinase kinase 6 (MKK6)‐p38 MAPK signaling cascade in the regulation of myocardial COX‐2 gene expression, in vitro and in vivo. RT‐PCR analysis identified p38&agr; and p38&bgr;2 MAPK mRNA in rat cardiac myocytes. Interleukin‐1&bgr; induced the phosphorylation of p38&agr; and p38&bgr;2 MAPK in cardiomyocytes and stimulated RNA polymerase II binding to the COX‐2 promoter, COX‐2 transcription, COX‐2 protein synthesis, and prostaglandin E2 (PGE2) release. Infecting cardiomyocytes with adenoviruses that encode mutant, phosphorylation‐resistant MKK6 or p38&bgr;2 MAPK inhibited interleukin‐1&bgr;—induced p38 MAPK activation, COX‐2 gene expression, and PGE2 release. Treatment with the p38&agr; and p38&bgr;2 MAPK inhibitor, SB202190, attenuated interleukin‐1&bgr;—induced COX‐2 transcription and accelerated the degradation of COX‐2 mRNA. Cells infected with adenoviruses encoding wild‐type or constitutively activated MKK6 or p38&bgr;2 MAPK, in the absence of interleukin‐1&bgr;, exhibited increased cellular p38 MAPK activity, COX‐2 mRNA expression, and COX‐2 protein synthesis, which was blocked by SB202190. In addition, elevated levels of COX‐2 protein were identified in the hearts of transgenic mice with cardiac‐restricted expression of wild‐type or constitutively activated MKK6, in comparison with nontransgenic littermates. These results provide direct evidence that MKK6 mediated p38 MAPK activation is necessary for interleukin‐1&bgr;—induced cardiac myocyte COX‐2 gene expression and PGE2 biosynthesis in vitro and is sufficient for COX‐2 gene expression by cardiac myocytes in vitro and in vivo. (Circ Res. 2003;92:757–764.)

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.