Ginger phenylpropanoids inhibit IL-1beta and prostanoid secretion and disrupt arachidonate-phospholipid remodeling by targeting phospholipases A2

The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A(2) (iPLA(2))-triggered maturation and the cytosolic phospholipase A(2) (cPLA(2))-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA(2) assay, most major ginger phenylpropanoids directly inhibited i/cPLA(2) from U937 macrophages, but not hog pancreas secretory phospholipase A(2). The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA(2) and methyl arachidonyl fluorophosphonate for cPLA(2), with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE(2) and thromboxane B2 (>50%) and partially also leukotriene B(4) in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA(2) and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA(2), the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.

Human group II 14 kDa phospholipase A2 activates human platelets

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.

FTY720 suppresses interleukin-1beta-induced secretory phospholipase A2 expression in renal mesangial cells by a transcriptional mechanism

BACKGROUND AND PURPOSE: FTY720 is a potent immunomodulatory prodrug that is converted to its active phosphorylated form by a sphingosine kinase. Here we have studied whether FTY720 mimicked the action of sphingosine-1-phosphate (S1P) and exerted an anti-inflammatory potential in renal mesangial cells. EXPERIMENTAL APPROACH: Prostaglandin E(2) (PGE(2)) was quantified by an enzyme-linked immunosorbent-assay. Secretory phospholipase A(2) (sPLA(2)) protein was detected by Western blot analyses. mRNA expression was determined by Northern blot analysis and sPLA(2)-promoter activity was measured by a luciferase-reporter-gene assay. KEY RESULTS: Stimulation of cells for 24 h with interleukin-1beta (IL-1beta) is known to trigger increased PGE(2) formation which coincides with an induction of the mRNA for group-IIA-sPLA(2) and protein expression. FTY720 dose-dependently suppressed IL-1beta-induced IIA-sPLA(2) protein secretion and activity in the supernatant. This effect is due to a suppression of cytokine-induced sPLA(2) mRNA expression which results from a reduced promoter activity. As a consequence of suppressed sPLA(2) activity, PGE(2) formation is also reduced by FTY720. Mechanistically, the FTY720-suppressed sPLA(2) expression results from an activation of the TGFbeta/Smad signalling cascade since inhibition of the TGFbeta receptor type I by a specific kinase inhibitor reverses the FTY720-mediated decrease of sPLA(2) protein expression and sPLA(2) promoter activity. CONCLUSIONS AND IMPLICATIONS: In summary, our data show that FTY720 was able to mimic the anti-inflammatory activity of TGFbeta and blocked cytokine-triggered sPLA(2) expression and subsequent PGE(2) formation. Thus, FTY720 may exert additional in vivo effects besides the well reported immunomodulation and its anti-inflammatory potential should be considered.

Role of calcium-independent phospholipase A2 in cortex striatum thalamus cortex circuitry-enzyme inhibition causes vacuous chewing movements in rats

RATIONALE: High levels of calcium independent phospholipase A2 (iPLA2) are present in certain regions of the brain, including the cerebral cortex, striatum, and cerebellum (Ong et al. 2005). OBJECTIVES: The present study was carried out to elucidate a possible role of the enzyme in the motor system. METHODS: The selective iPLA2 inhibitor bromoenol lactone (BEL), the nonselective PLA2 inhibitor methyl arachidonyl fluorophosphonate (MAFP), and an antisense oligonucleotide were used to interfere with iPLA2 activity in various components of the motor system. Control animals received injections of carrier (phosphate buffered saline, PBS) at the same locations. The number of vacuous chewing movements (VCM) was counted from 1 to 14 days after injection. RESULTS: Rats that received BEL and high-dose MAFP injections in the striatum, thalamus, and motor cortex, but not the cerebellum, showed significant increase in VCM, compared to those injected with PBS at these locations. BEL-induced VCM were blocked by intramuscular injections of the anticholinergic drug, benztropine. Increased VCM was also observed after intrastriatal injection of antisense oligonucleotide to iPLA2. The latter caused a decrease in striatal iPLA2 levels, confirming a role of decreased enzyme activity in the appearance of VCM. CONCLUSIONS: These results suggest an important role for iPLA2 in the cortex-striatum-thalamus-cortex circuitry. It is postulated that VCM induced by iPLA2 inhibition may be a model of human parkinsonian tremor.

A direct role for secretory phospholipase A2 and lysophosphatidylcholine in the mediation of LPS-induced gastric injury.

Endotoxemia from sepsis can injure the gastrointestinal tract through mechanisms that have not been fully elucidated. We have shown that LPS induces an increase in gastric permeability in parallel with the luminal appearance of secretory phospholipase A2 (sPLA2) and its product, lysophosphatidylcholine (lyso-PC). We proposed that sPLA2 acted on the gastric hydrophobic barrier, composed primarily of phosphatidylcholine (PC), to degrade it and produce lyso-PC, an agent that is damaging to the mucosa. In the present study, we have tested whether lyso-PC and/or sPLA2 have direct damaging effects on the hydrophobic barriers of synthetic and mucosal surfaces. Rats were administered LPS (5 mg/kg, i.p.), and gastric contents were collected 5 h later for analysis of sPLA2 and lyso-PC content. Using these measured concentrations, direct effects of sPLA2 and lyso-PC were determined on (a) surface hydrophobicity as detected with an artificial PC surface and with intact gastric mucosa (contact angle analysis) and (b) cell membrane disruption of gastric epithelial cells (AGS). Both lyso-PC and sPLA2 increased significantly in the collected gastric juice of LPS-treated rats. Using similar concentrations to the levels in gastric juice, the contact angle of PC-coated slides declined after incubation with either pancreatic sPLA2 or lyso-PC. Similarly, gastric contact angles seen in control rats were significantly decreased in sPLA2 and lyso-PC-treated rats. In addition, we observed dose-dependent injurious effects of both lyso-PC and sPLA2 in gastric AGS cells. An LPS-induced increase in sPLA2 activity in the gastric lumen and its product, lyso-PC, are capable of directly disrupting the gastric hydrophobic layer and may contribute to gastric barrier disruption and subsequent inflammation.

Rationale for reading fluconazole MICs at 24 hours rather than 48 hours when testing Candida spp. by the CLSI M27-A2 standard method.

We investigated if CLSI M27-A2 Candida species breakpoints for fluconazole MIC are valid when read at 24 h. Analysis of a data set showed good correlation between 48- and 24-h MICs, as well as similar outcomes and pharmacodynamic efficacy parameters, except for isolates in the susceptible dose-dependent category, such as Candida glabrata.

Thromboxane A2 acts as tonic immunoregulator by preferential disruption of low-avidity CD4+ T cell-dendritic cell interactions

Interactions between dendritic cells (DCs) and T cells control the decision between activation and tolerance induction. Thromboxane A2 (TXA2) and its receptor TP have been suggested to regulate adaptive immune responses through control of T cell-DC interactions. Here, we show that this control is achieved by selectively reducing expansion of low-avidity CD4(+) T cells. During inflammation, weak tetramer-binding TP-deficient CD4(+) T cells were preferentially expanded compared with TP-proficient CD4(+) T cells. Using intravital imaging of cellular interactions in reactive peripheral lymph nodes (PLNs), we found that TXA2 led to disruption of low- but not high-avidity interactions between DCs and CD4(+) T cells. Lack of TP correlated with higher expression of activation markers on stimulated CD4(+) T cells and with augmented accumulation of follicular helper T cells (TFH), which correlated with increased low-avidity IgG responses. In sum, our data suggest that tonic suppression of weak CD4(+) T cell-DC interactions by TXA2-TP signaling improves the overall quality of adaptive immune responses.

Phospholipase A2 group IIA is elevated in endometriomas but not in peritoneal fluid and serum of ovarian endometriosis patients.

Our previous gene expression analysis identified phospholipase A2 group IIA (PLA2G2A) as a potential biomarker of ovarian endometriosis. The aim of this study was to evaluate PLA2G2A mRNA and protein levels in tissue samples (endometriomas and normal endometrium) and in serum and peritoneal fluid of ovarian endometriosis patients and control women. One-hundred and sixteen women were included in this study: the case group included 70 ovarian endometriosis patients, and the control group included 38 healthy women and 8 patients with benign ovarian cysts. We observed 41.6-fold greater PLA2G2A mRNA levels in endometrioma tissue, compared to normal endometrium tissue. Using Western blotting, PLA2G2A was detected in all samples of endometriomas, but not in normal endometrium, and immunohistochemistry showed PLA2G2A-specific staining in epithelial cells of endometrioma paraffin sections. However, there were no significant differences in PLA2G2A levels between cases and controls according to ELISA of peritoneal fluid (6.0 ± 4.4 ng/ml, 6.6 ± 4.3 ng/ml; p = 0.5240) and serum (2.9 ± 2.1 ng/ml, 3.1 ± 2.2 ng/ml; p = 0.7989). Our data indicate that PLA2G2A is implicated in the pathophysiology of ovarian endometriosis, but that it cannot be used as a diagnostic biomarker.