Reactive oxygen species produced up- or downstream of calcium influx regulate proinflammatory mediator release from mast cells: role of NADPH oxidase and mitochondria

Earlier studies have demonstrated that mast cells produce reactive oxygen species (ROS), which play a role in regulating Ca(2+) influx, while in other cell types ROS are produced in a Ca(2+)-dependent manner. We sought to determine whether ROS are produced downstream of the extracellular Ca(2+) entry in mast cells. Thapsigargin (TG), a receptor-independent agonist, could evoke a robust burst of intracellular ROS. However, this response was distinct from the antigen-induced burst of ROS with respect to time course and dependence on Ca(2+) and phosphatidylinositol-3-kinase (PI3K). The antigen-induced ROS generation occurred immediately, while the TG-induced ROS generation occurred with a significant lag time (~2 min). Antigen but not TG caused extracellular release of superoxide (O(2)(*-))/hydrogen peroxide (H(2)O(2)), which was blocked by diphenyleneiodonium, apocynin, and wortmannin. A capacitative Ca(2+) entry resulted in the generation of O(2)(*-) in the mitochondria in a PI3K-independent manner. Blockade of ROS generation inhibited TG-induced mediator release. Finally, when used together, antigen and TG evoked the release of leukotriene C(4), tumor necrosis factor-alpha, and interleukin-13 as well as ROS generation synergistically. These results suggest that ROS produced upstream of Ca(2+) influx by NADPH oxidase and downstream of Ca(2+) influx by the mitochondria regulate the proinflammatory response of mast cells.     

Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse PT-18 mast cell.

A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.     

Mast cell mediators prostaglandin-D2 and histamine activate human eosinophils.

Airway damage secondary to eosinophil activation is thought to contribute to the development of asthma. Using the fluorescent dye FURA-2 to measure the concentration of cytosolic calcium, we found that supernatants from anti-IgE-stimulated human lung mast cells increased cytosolic calcium in human eosinophils. We then examined the major mast cell mediators (histamine, PGD2, platelet-activating factor (PAF), eosinophil chemotactic factor of anaphylaxis (ECF-A), leukotriene (LT)C4 and LTB4) for their ability to increase cytosolic calcium in eosinophils. We found that both PAF (5 x 10(-9) to 5 x 10(-6) M) and PGD2 (two of five donors responsive at 1 x 10(-9) M) were potent stimuli for calcium mobilization. LTB4 (10(-8), 10(-7) M) and histamine were also active, although higher concentrations of histamine were required to see a response (3 x 10(-7) to 10(-5) M). LTC4, val-ECF-A, and ala-ECF-A were inactive. The effects of PGD2 and histamine were specific for eosinophils, although LTB4 and PAF increased calcium in both neutrophils and eosinophils. The histamine-induced increase in intracellular calcium was not blocked by the H1 or H2 antagonists pyrilamine or cimetidine (10(-4) M), respectively; however, the response to 10(-6) M histamine was completely blocked by the specific H3 antagonist thioperamide (10(-6) M). To evaluate the relative contribution of these stimulatory mast cell mediators on the calcium mobilizing activity in supernatants from anti-IgE-stimulated human lung mast cell (HLMC), we examined the effect of supernatants from HLMC pretreated with indomethacin and/or the 5-lipoxygenase pathway inhibitor MK886. These supernatants were added to FURA-2-loaded eosinophils that had been preincubated with thioperamide and/or the PAF antagonist WEB-2086. We found that the increase in eosinophil calcium in response to supernatants from anti-IgE-stimulated-HLMC was totally inhibited only when the mast cells were challenged in the presence of indomethacin and MK886, and the eosinophils were preincubated with thioperamide. WEB-2086 had little effect. When we examined the effect of these mediators on eosinophil secretory function, we found that PGD2 (not histamine) primed eosinophils for enhanced release of LTC4 in response to the calcium ionophore A23187. We conclude that the activation of eosinophils by PGD2 and other mast cell products may contribute to airways inflammation that is characteristic of asthma.     

A proteomic analysis of the wound response in Medicago leaves reveals the early activation of a ROS-sensitive signal pathway

Wounded Medicago truncatula leaves produce a burst of O(2)(-) (phase I) between 1 and 15 min, then of O(2)(-) and H(2)O(2) (phase II) between 1 and 3 h. Our previous results suggest reactive oxygen species (ROS) may provide signals to mobilise early (6 h), apoplastic, wound-responsive proteins (WRPs). 2DE and MALDI-TOF/TOF were used to analyse how the suppression of ROS production at different time points by diphenyleneiodonium (DPI), affects the expression of WRPs. Rapid (≤3 min) DPI inhibition of phase I O(2)(-) production suppressed the differential regulation of 7 out of 19 WRPs, which were consequently classified as ROS-dependent WRPs. DPI inhibition of only phase II ROS production failed to suppress the wound regulation of 18 out of 19 WRPs, but led to the altered expression of 1 ROS-dependent WRP and 2 non-WRPs (Group B). The data indicates Group B proteins are alternatively targeted via the modulation of phase II ROS production. This reinforces an important role for phase I O(2)(-) signalling in the early wound response, but indicates that this response is partly regulated by phase II of the oxidative burst. This data provides an informed basis for further proteomic studies aimed at identifying early activated O(2)(-) signalling components in wounded Medicago.     

Effects of arachidonic acid analogs on FcepsilonRI-mediated activation of mast cells

Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) have been shown to modulate a number of inflammatory disorders. Mast cells play a critical role in the initiation and maintenance of inflammatory responses. However, the effects of PUFAs on mast cell functions have not been fully addressed. We here-in examined the effects of PUFAs on the high affinity IgE receptor (FcepsilonRI)-mediated mast cell activation using RBL-2H3 cells, a rat mast cell line, that were cultured in the medium containing palmitic acid (PA), AA, or the AA analogs mead acid (MA) and eicosapentaenoic acid (EPA). In AA-supplemented cells, the FcepsilonRI-mediated beta-hexosamidase and TNF-alpha release, calcium (Ca(2+)) influx, and some protein tyrosine phosphorylations including Syk and linker for activation of T cells (LAT) were enhanced, whereas, in MA- or PA-supplemented cells, they were not changed when compared with cells cultured in control medium. In EPA-supplemented cells, the enhancements of beta-hexosamidase release and protein tyrosine phosphorylations were observed. Furthermore, in AA- or EPA-supplemented cells, FcepsilonRI-mediated intracellular production of reactive oxygen species (ROS) that is required for the tyrosine phosphorylation of LAT and Ca(2+) influx were enhanced when compared with the other cells. Thus, preincubation of AA or EPA augmented FcepsilonRI-mediated degranulation in mast cells by affecting early events of FcepsilonRI signal transduction, which might be associated with the change of fatty acid composition of the cell membrane and enhanced production of ROS. The results suggest that some PUFAs can modulate FcepsilonRI-mediated mast cell activation and might affect FcepsilonRI/mast cell-mediated inflammation, such as allergic reaction.     

Diphenyleneiodonium prevents reactive oxygen species generation, tyrosine phosphorylation, and histamine release in RBL-2H3 mast cells

Mast cells play a central role in immediate allergic reactions mediated by immunoglobulin E. It has recently been reported that mast cells generate intracellular reactive oxygen species (ROS) in response to stimulation with divergent physiologically relevant stimulants. However, the physiological role of ROS is poorly understood. Here we demonstrate that mast cell model rat basophilic leukemia (RBL-2H3) cells generate ROS in response to antigen and the calcium-ionophore A23187 via activation of diphenyleneiodonuim (DPI)-sensitive enzyme and that blockade of ROS generation by DPI suppresses histamine release induced by either stimulant. Increased tyrosine phosphorylation of pp125(FAK) and a 77-kDa protein coprecipitating specifically with the kinase occurred in parallel with the secretion, and blockade of ROS generation by DPI also suppressed the tyrosine phosphorylation of both proteins. These findings suggest that ROS generated by a flavoenzyme-dependent mechanism may be involved in histamine release through the pp125(FAK) pathway.     

Selective activation of Fyn/PI3K and p38 MAPK regulates IL-4 production in BMMC under nontoxic stress condition

Mast cells have the ability to react to multiple stimuli, implicating these cells in many immune responses. Specific signals from the microenvironment in which mast cells reside can activate different molecular events that govern distinct mast cells responses. We previously demonstrated that hydrogen peroxide (H(2)O(2)) promotes IL-4 and IL-6 mRNA production and potentates FcepsilonRI-induced cytokine release in rat basophilic leukemia RBL-2H3 cells. To further evaluate the effect of an oxidative microenvironment (which is physiologically present in an inflammatory site) on mast cell function and the molecular events responsible for mast cell cytokine production in this environment, we analyzed the effect of H(2)O(2) treatment on IL-4 production in bone marrow-derived, cultured mast cells. Our findings show that nanomolar concentrations of H(2)O(2) induce cytokine secretion and enhance IL-4 production upon FcepsilonRI triggering. Oxidative stimulation activates a distinct signal transduction pathway that induces Fyn/PI3K/Akt activation and the selective phosphorylation of p38 MAP kinase. Moreover, H(2)O(2) induces AP-1 and NFAT complexes that recognize the IL-4 promoter. The absence of Fyn and PI3K or the inhibition of p38 MAPK activity demonstrated that they are essential for H(2)O(2)-driven IL-4 production. These findings show that mast cells can respond to an oxidative microenvironment by initiating specific signals capable of eliciting a selective response. The findings also demonstrate the dominance of the Fyn/p38 MAPK pathway in driving IL-4 production.     

Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst

Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.     

Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.     

Gold activates mast cells via calcium influx through multiple H2O2-sensitive pathways including L-type calcium channels

Heavy metals, including gold, induce severe contact hypersensitivity and autoimmune disorders, which develop through an initial Th2-independent process followed by a Th2-dependent process. It has been shown that mast cell activation plays a role in the Th2-independent process and that gold stimulates histamine release in vitro. However, the mechanisms of the gold-induced mast cell activation remain largely unclear. Here we report that gold directly activates mast cells in a Ca2+-dependent manner. HAuCl4 [Au(III)] at nontoxic concentrations (≤50 μM) induced substantial degranulation and leukotriene C4 secretion in an extracellular Ca2+-dependent manner. Au(III) induced a robust Ca2+ influx but not Ca2+ mobilization from internal stores. Au(III) also stimulated intracellular production of reactive oxygen species, including H2O2, and blockade of the production abolished the mediator release and Ca2+ influx. Au(III) induced Ca2+ influx through multiple store-independent Ca2+ channels, including Cav1.2 L-type Ca2+ channels (LTCCs) and 2-aminoethoxydiphenyl borate (2-APB)-sensitive Ca2+ channels. The 2-APB-sensitive channel seemed to mediate Au(III)-induced degranulation. Our results indicate that gold stimulates Ca2+ influx and mediator release in mast cells through multiple H2O2-sensitive Ca2+ channels including LTCCs and 2-APB-sensitive Ca2+ channels. These findings provide insight into the roles of these Ca2+ channels in the Th2-independent process of gold-induced immunological disorders.     

O-methylated catechins from tea leaves inhibit multiple protein kinases in mast cells

Tea contains a variety of bioactive compounds. In this study, we show that two O-methylated catechins, (-)-epigallocatechin-3-O-(3-O-methyl) gallate and (-)-epigallocatechin-3-O-(4-O-methyl) gallate, inhibit in vivo mast cell-dependent allergic reactions more potently than their nonmethylated form, (-)-epigallocatechin-3-O-gallate. Consistent with this, these O-methylated catechins inhibit IgE/Ag-induced activation of mouse mast cells: histamine release, leukotriene release, and cytokine production and secretion were all inhibited. As a molecular basis for the catechin-mediated inhibition of mast cell activation, Lyn, Syk, and Bruton's tyrosine kinase, the protein tyrosine kinases, known to be critical for early activation events, are shown to be inhibited by the O-methylated catechins. In vitro kinase assays using purified proteins show that the O-methylated catechins can directly inhibit the above protein tyrosine kinases. These catechins inhibit IgE/Ag-induced calcium response as well as the activation of downstream serine/threonine kinases such as Akt and c-Jun N-terminal kinase. These observations for the first time have revealed the molecular mechanisms of antiallergic effects of tea-derived catechins.     

Insulin-induced activation of hypoxia-inducible factor-1 requires generation of reactive oxygen species by NADPH oxidase

Hypoxia-inducible factor (HIF)-1 activation in response to hypoxia requires mitochondrial generation of reactive oxygen species (ROS). In contrast, the requirement of ROS for HIF-1 activation by growth factors like insulin remains unexplored. To explore that, insulin-sensitive hepatic cell HepG2 or cardiac muscle cell H9c2 cells were pretreated with NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) or apocynin and HIF-1 activation was tested by electrophoretic mobility shift and reporter gene assay. Antioxidants DPI or apocynin completely blocked insulin-stimulated HIF-1 activation. The restoration of HIF-1 activation by H(2)O(2) in DPI-pretreated cells not only confirmed the role of ROS but also identified H(2)O(2) as the responsible ROS. The role of NADPH oxidase was further confirmed by greater stimulation of HIF-1 during simultaneous treatment of suboptimal concentration of insulin along with NADPH but not by NADH. The role of oxidant generated by insulin is found to inhibit the protein tyrosine phosphatase as suggested by the following observations. First, tyrosine phosphatase-specific inhibitor sodium vanadate compensates DPI-inhibited HIF-1 activity. Second, sodium vanadate stimulates HIF-1 activation with suboptimal concentration of insulin. Third, DPI and pyrrolidene dithiocarbamate (PDTC) blocks insulin-receptor tyrosine kinase activation. The activity of phosphatidylinositol 3-kinase as evidenced by Akt phosphorylation, involved in HIF-1 activation, is also dependent on ROS generation by insulin. Finally, DPI pretreatment blocked insulin-stimulated expression of genes like VEGF, GLUT1, and ceruloplasmin. Overall, our data provide strong evidence for the essential role of NADPH oxidase-generated ROS in insulin-stimulated activation of HIF-1.     

Ebselen, a glutathione peroxidase mimetic seleno-organic compound, as a multifunctional antioxidant. Implication for inflammation-associated carcinogenesis.

Ebselen, a seleno-organic compound showing glutathione peroxidase-like activity, is one of the promising synthetic antioxidants. In the present study, we investigated the antioxidant activities of ebselen using a 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated mouse skin model. Double pretreatments of mouse skin with ebselen significantly inhibited TPA-induced formation of thiobarbituric acid-reacting substance, known as an overall oxidative damage biomarker, in mouse epidermis, suggesting that ebselen indeed acts as an antioxidant in mouse skin. The antioxidative effect of ebselen is attributed to its selective blockade of leukocyte infiltration and activation leading to attenuation of the H(2)O(2) level. In in vitro studies, ebselen inhibited TPA-induced superoxide generation in differentiated HL-60 cells and lipopolysaccharide-induced cyclooxygenase-2 protein expression in RAW 264.7 cells. In addition, we demonstrated for the first time that ebselen potentiated phase II enzyme activities, including NAD(P)H:(quinone-acceptor) oxidoreductase1 and glutathione S-transferase in cultured hepatocytes and in mouse skin. These results strongly suggest that ebselen, a multifunctional antioxidant, is a potential chemopreventive agent in inflammation-associated carcinogenesis.     

Hypoxic reperfusion of the ischemic heart and oxygen radical generation

Postischemic myocardial contractile dysfunction is in part mediated by the burst of reactive oxygen species (ROS), which occurs with the reintroduction of oxygen. We hypothesized that tissue oxygen tension modulates this ROS burst at reperfusion. After 20 min of global ischemia, isolated rat hearts were reperfused with temperature-controlled (37.4 degrees C) Krebs-Henseleit buffer saturated with one of three different O2 concentrations (95, 20, or 2%) for the first 5 min of reperfusion and then changed to 95% O2. Additional hearts were loaded with 1) allopurinol (1 mM), a xanthine oxidase inhibitor, 2) diphenyleneiodonium (DPI; 1 microM), an NAD(P)H oxidase inhibitor, or 3) Tiron (10 mM), a superoxide scavenger, and were then reperfused with either 95 or 2% O2 for the first 5 min. ROS production and tissue oxygen tension were quantitated using electron paramagnetic resonance spectroscopy. Tissue oxygen tension was significantly higher in the 95% O2 group. However, the largest radical burst occurred in the 2% O2 reperfusion group (P < 0.001). Recovery of left ventricular (LV) contractile function and aconitase activity during reperfusion were inversely related to the burst of radical production and were significantly higher in hearts initially reperfused with 95% O2 (P < 0.001). Allopurinol, DPI, and Tiron reduced the burst of radical formation in the 2% O2 reperfusion groups (P < 0.05). Hypoxic reperfusion generates an increased ROS burst originating from multiple pathways. Recovery of LV function during reperfusion is inversely related to this oxygen radical burst, highlighting the importance of myocardial oxygen tension during initial reperfusion.     

Phospholipid scramblase 1 modulates a selected set of IgE receptor-mediated mast cell responses through LAT-dependent pathway

Engagement of the IgE receptor (FcepsilonRI) on mast cells leads to the release of preformed and newly formed mediators as well as of cytokines. The signaling pathways responsible for these responses involve tyrosine phosphorylation of multiple proteins. We previously reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after FcepsilonRI aggregation. Here, PLSCR1 expression was knocked down in the RBL-2H3 mast cell line using short hairpin RNA. Knocking down PLSCR1 expression resulted in significantly impaired degranulation responses after FcepsilonRI aggregation and release of vascular endothelial growth factor, whereas release of MCP-1 was minimally affected. The release of neither leukotriene C4 nor prostaglandin D2 was altered by knocking down of PLSCR1. Analysis of FcepsilonRI-dependent signaling pathways revealed that whereas tyrosine phosphorylation of ERK and Akt was unaffected, tyrosine phosphorylation of LAT was significantly reduced in PLSCR1 knocked down cells. Tyrosine phosphorylation of phospholipase Cgamma1 and consequently the mobilization of calcium were also significantly reduced in these cells. In nonactivated mast cells, PLSCR1 was found in part in lipid rafts where it was further recruited after cell activation and was constitutively associated with Lyn and Syk but not with LAT or Fyn. Altogether, these data identify PLSCR1 as a novel amplifier of FcepsilonRI signaling that acts selectively on the Lyn-initiated LAT/phospholipase Cgamma1/calcium axis, resulting in potentiation of a selected set of mast cell responses.     

Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species

Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS). This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H(2)O(2), because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H(2)O(2), acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.     

Oxidative stress augments the production of matrix metalloproteinase-1, cyclooxygenase-2, and prostaglandin E2 through enhancement of NF-kappa B activity in lipopolysaccharide-activated human primary monocytes

The excessive production of reactive oxidative species (ROS) associated with inflammation leads to a condition of oxidative stress. Cyclooxygenase-2 (COX-2), PGE(2), and matrix metalloproteinases (MMPs) are important mediators during the process of inflammation. In this paper we report on studies examining how the ROS hydrogen peroxide (H(2)O(2)) affects the production of MMP-1, COX-2, and PGE(2). Addition of H(2)O(2) to LPS-activated monocytes, but not naive monocytes, caused a significant enhancement of the LPS-induced production of MMP-1, COX-2, and PGE(2). The mechanism by which H(2)O(2) increased these mediators was through enhancement of IkappaBalpha degradation, with subsequent increases in NF-kappaB activation and NF-kappaB p50 translocation to the nucleus. The effects of H(2)O(2) on IkappaBalpha degradation, NF-kappaB activation, and NF-kappaB p50 localization to the nucleus were demonstrated through studies of coimmunoprecipitation of IkappaBalpha with p50, ELISA of NF-kappaB p65 activity, and Western blot analysis of the nuclear fraction extract for p50. The key role for NF-kappaB in this process was demonstrated by the ability of MG-132 or lactacystin (proteasome inhibitors) to block the enhanced production of MMP-1, COX-2, and PGE(2). In contrast, indomethacin, which inhibited PGE(2) production, partially blocked the enhanced MMP-1 production. Moreover, although PGE(2) restored MMP-1 production in indomethacin-treated monocyte cultures; it failed to significantly restore MMP-1 production in proteasome inhibitor-treated cultures. Thus, in the presence of LPS and H(2)O(2), NF-kappaB plays a dominate role in the regulation of MMP-1, COX-2, and PGE(2) expression.     

Regulation of rat basophilic leukemia-2H3 mast cell secretion by a constitutive Lyn kinase interaction with the high affinity IgE receptor (Fc epsilon RI)

Signaling through the high affinity IgE receptor is initiated by noncovalently associated Lyn kinase, resulting in the secretion of inflammatory mediators from mast cells. A fraction of the total cellular Lyn is associated via its N-terminal unique domain with the cytoplasmic domain of the Fc epsilonRI beta subunit before receptor aggregation. In the current study, we stably transfected the unique domain of Lyn into rat basophilic leukemia-2H3 mast cells and examined the consequences on Fc epsilonRI-induced signal transduction and mediator secretion to further define the role of the unique domain of Lyn in mast cell secretion. Tyrosine phosphorylation of Fc epsilonRI beta and gamma subunits was partially inhibited in the Lyn unique domain transfectants after Ag stimulation. Ag stimulation of Lyn unique domain transfectants was accompanied by enhanced phosphorylation of MEK and ERK-2, which are required for leukotriene C4 (LTC4) release, and production of LTC4 was increased 3- to 5-fold, compared with cells transfected with vector alone. Conversely, tyrosine phosphorylation of the adaptor protein Gab2, which is essential for mast cell degranulation, was inhibited after Ag stimulation of Lyn unique domain transfectants, and Ag-induced release of histamine was inhibited up to 48%. In rat basophilic leukemia-2H3 cells, Lyn thus plays a dual role by positively regulating Fc epsilonRI phosphorylation and degranulation while negatively regulating LTC4 production. This study provides further evidence that the constitutive interaction between the unique domain of Lyn and the Fc epsilonRI beta subunit is a crucial step in the initiation of Fc epsilonRI signaling and that Lyn is limiting for Fc epsilonRI-induced secretion of inflammatory mediators.     

TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.