Bradykinin stimulates phosphoinositide turnover and phospholipase C but not phospholipase D and NADPH oxidase in human neutrophils

In response to formyl-Met-Leu-Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2-) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP-stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2- production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2- production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK-induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2-PLC measured as the release of [3H]IP3. On the contrary, a PC-PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA-derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2- production by fMLP-activated PMN in a time-dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO- formation. These data show that BK plays an important role in inflammation by modulating the PMN function.     

Analysis of signal transduction pathways in human eosinophils activated by chemoattractants and the T-helper 2-derived cytokines interleukin-4 and interleukin-5

Activation and recruitment of eosinophils in allergic inflammation is in part mediated by chemoattractants and T-helper 2 (Th2)-derived cytokines. However, little is known concerning the signal transduction mechanisms by which this activation occurs. We have investigated tyrosine kinase-mediated activation of phosphatidylinositol 3-kinase (PI3K) and compared this with the activation of the p21ras-ERK signaling pathway in human eosinophils. The related cytokines interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF), all induced PI3K activity detected in antiphosphotyrosine immunoprecipitates. Furthermore, the chemoattractants platelet-activating factor (PAF), RANTES, and C5a were also able to induce phosphotyrosine-associated PI3K activity. Protein kinase B (PKB) is a downstream target of PI3K activation by growth factors. Induction of PKB phosphorylation in human eosinophils was transiently induced on activation with the cytokines IL-4 and IL-5, as well as the chemoattractants PAF, C5a, and RANTES showing a broad activation profile. Surprisingly, analysis of the activation of the mitogen-activated protein (MAP) kinases p44(ERK1) and p42(ERK2), showed that ERK2, but not ERK1, was transiently activated in human eosinophils after stimulation with IL-5 or PAF. Activation kinetics correlated with activation of p21ras by both cytokines and chemoattractants as measured by a novel assay for guanosine triphosphate (GTP)-loading. Finally, using specific inhibitors of both the p21ras-ERK and PI3K signaling pathways, a role was demonstrated for PI3K, but not p21ras-ERK, in activation of the serum-treated zymosan (STZ)-mediated respiratory burst in IL-5 and PAF-primed eosinophils. In summary, these data show that in human eosinophils, Th2-derived cytokines differentially activate both PI3K and MAP kinase signal transduction pathways with distinct functional consequences showing complex regulation of eosinophil effector functions.     

Oncostatin M, but not interleukin-6 or leukemia inhibitory factor, stimulates expression of alpha1-proteinase inhibitor in A549 human alveolar epithelial cells

Alpha-1 proteinase inhibitor (A1-Pi) is the main serine proteinase inhibitor found in human plasma and is a potent elastase inhibitor in various tissues, including lung. A1-Pi is expressed and induced in liver during inflammatory responses but can also be produced by epithelial cells. Since hepatocyte A1-Pi production is stimulated by interleukin-6 (IL-6) and other gp130-cytokines, such as leukemia inhibitory factor (LIF) and oncostatin M (OM), we investigated the role of these cytokines in regulating A1-Pi in lung epithelial cells. We show that OM, a monocyte and T cell product, can specifically and potently induce A1-Pi production in lung-derived A549 alveolar (epithelial) cells, as well as in liver-derived HepG2 cells. Both A1-Pi protein (as detected by ELISA and Western blots) and mRNA levels were enhanced 20-fold to 30-fold in A549 cells. OM was also able to stimulate the expression of tissue inhibitor of metalloproteinase-1 in these cells. Interestingly, other members of the IL-6 family (IL-6 and LIF) had little or no effect on A549 cells, and proinflammatory cytokines, such as IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) also had no stimulatory effect on A1-Pi synthesis in A549 cells. Costimulation with IL-1 beta resulted in a decrease in A1-Pi production from OM-stimulated A549 cells. However, IL-6 production was synergistically enhanced. OM was also able to stimulate A1-Pi production from a bronchial epithelial primary cell line, whereas an intestinal epithelial cell line HT29 responded to IL-6 but not OM. These results suggest that lung levels A1-Pi could be derived not only from liver and inflammatory cells but also from epithelial cells, which can be upregulated on stimulation by OM. This may have implications for regulation of local activity of human neutrophil elastase (HNE) in such diseases as emphysema and cystic fibrosis.     

Oestradiol-17 beta modulates PAF-evoked phospholipase D activity but not inositide-lipid hydrolysis in human endometrial cell line, HEC-1B

The skeleton of a middle-aged adult male, found in the Late Epigravettian necropolis of Arene Candide cave (Italy) and dated to the XII millennium B.C., exhibits abnormal changes, including bowing deformities, stunted growth, enthesopathies, and increased bone density. The pattern of the observed changes is less consistent with diagnoses of metaphyseal chondrodysplasias, hypophosphatasia, dietary rickets, or diffuse idiopathic skeletal hyperostosis (DISH) than with X-linked hypophosphatemic rickets, which is the most likely etiological factor. This diagnosis may explain other abnormalities (exceptional elongation of the skull and bilateral absence of the lesser trochanter) displayed by other individuals from the same site.     

Formation of eosinophilic and monocytic intradermal inflammatory sites in the dog by injection of human RANTES but not human monocyte chemoattractant protein 1, human macrophage inflammatory protein 1 alpha, or human interleukin 8

Equilibrium binding studies on canine mononuclear and granulocytic cells allow the identification of a single high affinity receptor for the human C-C chemokine RANTES (dissociation constant, 14 +/- 8 pM), that, in contrast to the human RANTES receptor, has no affinity for human macrophage inflammatory protein 1 alpha (hMIP-1 alpha). A single intradermal injection of hRANTES in dog resulted in eosinophil- and macrophage-rich inflammatory sites within 4 h. Cell infiltration peaked at 16-24 h after hRANTES injection. There was histological evidence of intravascular activation of eosinophils at 4 h, although eosinophils in the vasculature and interstitium contained apparently intact granules. Monocytes were the predominant cells adherent to venular endothelium at 16-24 h. Human MIP-1 alpha elicited no response in canine dermis, whereas monocyte chemoattractant protein 1 caused mild perivascular cuffing with monocytes. In contrast, human interleukin 8 induced a neutrophilic dermal infiltrate that was maximal by 4 h after challenge. This provides the first direct evidence in vivo that RANTES has significant proinflammatory activity and, in addition, could be a mediator in atopic pathologies characterized by eosinophilic and monocytic inflammatory responses.     

Possible involvement of mitogen-activated protein kinase in phospholipase D activation induced by H2O2, but not by carbachol, in rat pheochromocytoma PC12 cells

We have previously reported that hydrogen peroxide (H2O2) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen-activated protein (MAP) kinase in PC12 cells. H2O2-induced PLD activation and MAP kinase phosphorylation were dose-dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol-mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H2O2, but not by carbachol. In the present study, H2O2 also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H2O2-induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor, almost completely prevented the release of [3H]OA by H2O2 treatment. From the preferential release of OA and sensitivity to other PLA2 inhibitors, the involvement of a Ca2+-independent cytosolic PLA2-type enzyme was suggested. In contrast to OA release, MAFP did not inhibit PLD activation by H2O2. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H2O2-induced PLD activation may be mediated by MAP kinase and also that H2O2-mediated OA release, which would be catalyzed by a Ca2+-independent cytosolic PLA2-like enzyme, is not linked to the PLD activation in PC12 cells.     

Prostaglandin f2alpha treatment in vivo, but not in vitro, stimulates protein kinase C-activated superoxide production by nonsteroidogenic cells of the rat corpus luteum

Luteal regression is associated with the generation of reactive oxygen species (ROS). To determine the nature of the ROS generator, cells isolated from luteinized rat ovaries were examined for ROS production using luminol-amplified chemiluminescence (LCL). Cells cultured for 2-48 h exhibited minimal LCL, but there was a significant (30- to 50-fold), rapid (maximum at 3-5 min), and dose-dependent increase in LCL in response to phorbol ester (phorbol 12-myristate 13-acetate; TPA; ED50 = 0.03 microM) and diacylglycerol (1,2-dioctanoyl-glycerol; ED50 = 30 microM). The TPA-induced response was cell number dependent and was virtually abolished by superoxide dismutase, freezing, or heating (95 degrees C for 5 min). Zymosan, known to induce a phagocytic response in leukocytes, stimulated a superoxide (O2-.) response with a slow onset (maximum at 40 to 60 min) and a maximum about one third of that observed for TPA. The response to TPA and zymosan was inhibited by the NADPH/NADH-oxidase inhibitor, diphenylene iodonium (ID50 = 5 microM for TPA), but not by the mitochondrial inhibitors, potassium cyanide, rotenone, or sodium azide. Fractionation of cells by centrifugal elutriation showed that TPA-stimulated O2-. production coeluted with the nonsteroidogenic cells and that little, if any, O2-. generation coeluted with the steroidogenic cells. Cells isolated 1, 2, and 4 h after in vivo treatment with a luteolytic dose of prostaglandin F2alpha (PGF2alpha) showed a significant increase in TPA-stimulated O2-. production at 2 h, whereas luteal cells or corpora lutea incubated directly with 1 microM PGF2alpha did not show any increase in response. Corpora lutea isolated from naturally regressed ovaries (18 days after ovulation) showed a significantly elevated level of TPA-stimulated O2-. production. In conclusion, there is a superoxide generator in luteinized ovaries that is activated through a protein kinase C pathway, localized in nonsteroidogenic cells, transiently increased during PGF2alpha-induced luteolysis in vivo, and elevated during natural luteal regression.     

Activation of phospholipase C and phospholipase D by stimulation of adenosine A1, bradykinin or P2U receptors does not correlate well with protein kinase C activation

Gradient-enhanced, two-dimensional, homonuclear correlation techniques (GCOSY) of carbohydrates provide numerous correlations based on 4J and 5J long-range interactions. Intraresidue correlations, involving all 1H resonances of a given pyranose ring with its anomeric proton, are consistently observed in alpha-pyranosyl residues at approximately 5 to 10 times lower intensities than vicinal 3J correlation cross peaks. beta-Anomers, pyranosyl residues with axial H1 protons, show very few such effects. Both alpha and beta anomers do, however, exhibit interresidue 4J correlations across the glycosidic linkage as shown for several linear and branched oligosaccharides ranging from three to five residues and are especially useful for spectral assignments in the envelope of pyranosyl ring protons located in the typically very crowded 3 to 4 ppm region. These effects depend on the strength and duration of the applied gradients.     

Enhancement of excessive lever-pressing after post-training signal attenuation in rats by repeated administration of the D? antagonist SCH 23390 or the D? agonist quinpirole, but not the D? agonist SKF 38393 or the D? antagonist haloperidol.

The authors have recently shown that attenuation of an external response feedback leads to excessive lever-pressing that is not associated with attempts to collect reward, and they have suggested that this may be an analogue to "unreasonable" excessive behavior characteristic of obsessive–compulsive disorder. The present study shows that repeated administration of SCH 23390 or quinpirole, but not SKF 38393 or haloperidol, enhances this behavioral pattern. On the basis of data regarding the enduring effects of chronic treatment with dopaminergic agents, these results suggest that overstimulation of striatal D? receptors underlies enhanced response to signal attenuation. These results may link the hypothesis that obsessions and compulsions result from a deficient response feedback mechanism with findings implicating dopaminergic abnormalities in the production of obsessions and compulsions. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Stimulation of primed neutrophils by soluble immune complexes: priming leads to enhanced intracellular Ca2+ elevations, activation of phospholipase D, and activation of the NADPH oxidase

Soluble immune complexes activate a rapid burst of reactive oxidant secretion from neutrophils that have previously been primed with GM-CSF. Binding of these complexes to the cell surface of unprimed neutrophils results in the generation of intracellular Ca2+ transients, but the NADPH oxidase fails to become activated. No phospholipase D activity was observed following the addition of soluble immune complexes to unprimed cells. Upon priming with GM-CSF, the intracellular Ca2+ signal generated following soluble complex binding was greatly extended and phospholipase D was activated: there was also increased phosphorylation of proteins on tyrosine residues and the NADPH oxidase was activated. When Ca2+ influx was prevented, this phospholipase D activity was not observed. This primed oxidase activity was completely inhibited by erbstatin. Treatment of unprimed neutrophils with pervanadate (to inhibit protein tyrosine phosphatases) mimicked the effects of priming in that pervanadate-treated neutrophils secreted reactive oxidants in response to soluble immune complexes. The data indicate that during priming a new signaling pathway is activated that involves Ca2+ influx, phosphorylation on tyrosine residues, phospholipase D activity, and NADPH oxidase activation.     

Appetite suppressant drugs as inhibitors of human cytochromes P450: in vitro inhibition of P450-2D6 by D- and L-fenfluramine, but not phentermine

BACKGROUND: We compared our results with bullous vs diffuse emphysema by performing a bilateral thoracoscopic stapled volume reduction technique in 15 patients (age 45-80, 10 males, five females). METHODS: Eight patients demonstrated bullous emphysema and seven patients diffuse emphysema. Lung reduction was performed with a bilateral thoracoscopic stapled technique utilizing bovine pericardium in the supine position. RESULTS: Comparison of the bullous versus diffuse groups revealed no significant differences in means for the following variables: length of air leak (7.5 vs 3.3 days); length of stay (8.1 vs 6.5 days); pre-op FEV1, (23% vs 22%); pre-op dyspnea index (3.4 vs 3.6). At 3 months the bullous subset had a highly significant improvement (p < 0.007) in FEV1 (88%) compared with the diffuse subset FEV1 (59%). CONCLUSIONS: These early results suggest that patients with bullous emphysema are at no greater risk and demonstrate a significantly greater improvement in FEV1 than patients with diffuse emphysema.     

Superantigen activation of immune cells evokes epithelial (T84) transport and barrier abnormalities via IFN-gamma and TNF alpha: inhibition of increased permeability, but not diminished secretory responses by TGF-beta2

Superantigens (SAgs) are extremely potent stimulants of T cell activity that have been implicated in the etiopathophysiology of inflammatory disease. Here, we tested the hypothesis that Staphylococcus aureus enterotoxin B (SEB), a model SAg, can alter epithelial transport and/or barrier functions via immune stimulation. Confluent monolayers of the human colonic T84 epithelial cell line, grown on filter supports, were cocultured with SEB +/- PBMC. Subsequently, T84 transport (consisting of baseline short-circuit current (Isc, indicates net ion transport) and secretory responses to carbachol and forskolin) and barrier functions (consisting of transepithelial resistance and fluxes of 3H-labeled mannitol and 51Cr-EDTA) were examined in Ussing chambers. T84 monolayers cocultured with SEB-activated PBMC displayed a time- and dose-dependent decrease in secretory responses to carbachol and forskolin and a significant increase in permeability. These dramatic changes in epithelial function were not due to reduced epithelial viability. Neutralizing Abs to IFN-gamma partially prevented the transport abnormalities, and Abs to TNF-alpha inhibited the increase in epithelial permeability. Abs to IL-1beta and IL-6 did not modulate the SEB-activated PBMC-induced T84 pathophysiology. Addition of TGF-beta2 to conditioned medium from SEB-activated PBMC partially inhibited the increase in T84 permeability but did not affect the transport abnormalities. We conclude that SAgs can elicit epithelial irregularities characteristic of enteric inflammation and that IFN-gamma and TNF-alpha are key mediators in this coculture model of epithelial dysfunction. Additionally, we would highlight the role that TGF-beta2 may play in preventing prolonged increases in epithelial permeability.