Stimulation Of Superoxide Anion Production Research Articles

Vimentin is a target of PKCβ phosphorylation in MCP-1-activated primary human monocytes

We designed a study to detect downstream phosphorylation targets of PKCβ in MCP-1-induced human monocytes. Two-dimensional gel electrophoresis was performed for monocytes treated with MCP-1 in the presence or absence of PKCβ antisense oligodeoxyribonucleotides (AS-ODN) or a PKCβ inhibitor peptide, followed by phospho- and total protein staining. Proteins that stained less intensely with the phospho-stain, when normalized to the total protein stain, in the presence of PKCβ AS-ODN or the PKCβ inhibitor peptide, were sequenced. Of the proteins identified, vimentin was consistently identified using both experimental approaches. Upon (32)P-labeling and vimentin immunoprecipitation, increased phosphorylation of vimentin was observed in MCP-1 treated monocytes as compared to the untreated monocytes. Both PKCβ AS-ODN and the PKCβ inhibitor reduced MCP-1-induced vimentin phosphorylation. The IP of monocytes with anti-vimentin antibody and immunoblotting with a PKCβ antibody revealed that increased PKCβ becomes associated with vimentin upon MCP-1 activation. Upon MCP-1 treatment, monocytes were shown to secrete vimentin and secretion depended on PKCβ expression and activity. We conclude that vimentin, a major intermediate filament protein, is a phosphorylation target of PKCβ in MCP-1-treated monocytes and that PKCβ phosphorylation is essential for vimentin secretion. Our recently published studies have implicated vimentin as a potent stimulator of the innate immune receptor Dectin-1 as reported by Thiagarajan et al. (Cardiovasc Res 99:494-504, 2013). Taken together our findings suggest that inhibition of PKCβ regulates vimentin secretion and, thereby, its interaction with Dectin-1 and downstream stimulation of superoxide anion production. Thus, PKCβ phosphorylation of vimentin likely plays an important role in propagating inflammatory responses.

Intravenous isosorbide dinitrate inhibits neutrophil aggregation and plasma-mediated stimulation of superoxide anion production

Polymorphonuclear neutrophils are known to be activated during myocardial ischaemia causing release of free oxygen radicals and capillary plugging by cell aggregates and therefore to exacerbate ischaemic myocardial injury. Nitric oxide has been shown to modulate neutrophil activation within the ischaemic myocardium and therefore reduce myocardial injury during ischaemia. Drugs that act as nitric oxide donors may therefore modify neutrophil activation. We evaluated the effect of intravenous treatment with isosorbide dinitrate on neutrophil aggregation and plasmamediated stimulation of neutrophil superoxide anion production in patients with ischaemic heart disease. Samples were obtained from patients before treatment and 15 and 30 min after receiving intravenous isosorbide dinitrate. Isosorbide dinitrate decreased neutrophil aggregation visualised in whole blood (25.3 ± 3.6, 19.0 ± 2.6 and 18.5 ± 2.6 per 300 cells, respectively, P < 0.01). When patient's plasma was incubated with neutrophils obtained from healthy donors, superoxide anion release was 18.99 ± 6.23, 11.38 ± 2.79 and 11.49 ± 3.15 nmol O 2 − 10 6 cells, respectively ( P < 0.01). Therefore, intravenous isosorbide dinitrate inhibited both plasma-mediated stimulation of neutrophil superoxide anion production and neutrophil aggregation.

S-Thiolation of Individual Human Neutrophil Proteins Including Actin by Stimulation of the Respiratory Burst: Evidence against a Role for Glutathione Disulfide

Protein S-thiolation, a reversible modification of protein sulfhydryls resulting in formation of mixed-disulfides, was studied in human neutrophils stimulated with phorbol diester to produce superoxide anion. Rapid S-thiolation of several proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glutathione was identified as the primary protein-bound thiol by HPLC chromatography, contributing considerably more than 85% of the total. Minor amounts of homocysteine and/or cysteine were also detected as protein-bound thiols. During the first 30 min after stimulation, 10% of the cellular glutathione became protein bound (2 nmol/mg of protein). There was no increase in glutathione disulfide suggesting that S-thiolation of the proteins did not occur by thiol/disulfide exchange. Approximately 10 mol% of one heavily modified band (29 kDa) was S-thiolated after 30 min. A second major band of 42 kDa was identified as actin. It contained 1/10th of the total protein-bound glutathione and approximately 5 mol% was S-thiolated after 30 min. These experiments identify a subset of S-thiolated neutrophil proteins, including actin, whose modification is related to the phorbol diester stimulation of superoxide anion production in human neutrophils. Ten percent of the total glutathione pool became protein-bound without an appreciable change in non-bound concentration of glutathione or glutathione disulfide. These results suggest that glutathione was synthesized during initial phases of the respiratory burst, compensating for the amount of glutathione that became protein-bound. Since there was no significant increase in glutathione disulfide, it was probably not important in the observed protein S-thiolation.

Stimulation of superoxide anion production in guinea pig polymorphonuclear leukocytes by hypotonic conditions in combination with protein kinase C activators

Conditions for Superoxide anion (O − 2) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, O − 2 production was significantly enhanced by concurrent treatment with low concentrations of 1-oleoyl-2-acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced O − 2 production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic O − 2 production with hypotonicity. Phosphorylation of 46-kDa protein(s), which was commonly enhanced in parallel with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 μ m OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in O − 2 production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase.

Priming action of inositol hexakisphosphate (InsP 6) on the stimulated respiratory burst in human neutrophils

After priming by a number of different host, bacterial and chemical agents, human neutrophils may be stimulated to produce a greater respiratory burst than would be elicited by the stimulus alone. Other neutrophil functions may be similarly enhanced by pre-exposure to a priming agent. We describe here a new extracellular role for inositol hexakisphosphate (InsP 6) as a priming agent for a variety of human neutrophil functional responses. Preincubation of the cells with InsP 6 alone (up to 250 μM) has no stimulatory effect upon the basal production of reactive oxygen intermediates but the response to a subsequent stimulus (FMLP, PMA or phagocytic particles) is substantially enhanced. Levels 100–200% higher than ‘stimulus only’ controls have been recorded. Peak enhancement of the FMLP-induced oxidative response occurs after 1–2 min preincubation with InsP 6 and the effect is dose-dependent (maximum at approx. 100 μM InsP 6). As others have shown FMLP stimulation of superoxide anion production has no external Ca 2+ dependence but the presence of low levels of Ca 2+ and Mg 2+ (0.1 mM) during priming appears to be an essential requirement for full expression. Reports of intracellular concentrations of InsP 6 in mammalian cells in the 30–100 μM range suggest that the local release of this inositol polyphosphate from damaged or effete cells could have a physiologically important modulatory role on neutrophil functions.

Lung lining fluid modification of asbestos bioactivity for the alveolar macrophage.

It is likely that chrysotile fibers deposited in the lower respiratory tract become rapidly coated by components of lung lining fluid. Therefore, we have used lung lining fluid and its components as part of an in vitro model to study chrysotile stimulation of superoxide anion production by the alveolar macrophage. In terms of superoxide anion production, lung lining fluid-treated chrysotile was 50% as effective as the untreated fibers. Fractionated lung lining fluid components and pure phospholipids were tested individually for their effects on chrysotile bioactivity. Pretreatment of chrysotile with lung surfactant isolated from a 30,000g pellet of lung lining fluid decreased chrysotile-stimulated superoxide anion production by 90%. The inhibitory activity of lung surfactant was found to reside in a chloroform extract containing hydrophobic proteins and lipids. Total proteolysis of the proteins did not affect the inhibitory activity of the chloroform extract, but treatment with phospholipase C significantly decreased its inhibitory activity. The inhibitory effects of lung surfactant could be simulated with phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol at concentrations equivalent to those found in lung lining fluid. These results strongly suggest that phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol in lung lining fluid can modify chrysotile bioactivity for the alveolar macrophage. Together with previous results indicating that IgG enhances asbestos bioactivity, it would appear that lung lining fluid contains components that can either inhibit or enhance the bioactivity of asbestos and that it is the relative amounts of these components that determines the overall bioactivity of the fiber.

Possible mechanism of chrysotile asbestos-stimulated superoxide anion production in guinea pig alveolar macrophages

Excessive production of active oxygen radicals by macrophages is proposed to play an important role in asbestos-related diseases. The purpose of this study was to examine the capacity and mechanisms of action of various forms of asbestos to stimulate superoxide anion production by guinea pig alveolar macrophages. Chrysotile, but not the amphiboles (crocidolite, anthophyllite, or amosite), stimulated a rapid (<1 min) and dose-dependent (2.5–50 μg/ml) production of superoxide anion at noncytotoxic doses (2.5 to 25 μg/ml). The stimulation of superoxide anion production by chrysotile could be blocked by putative protein kinase C inhibitors (staurosporine, sphingosine, and fluphenazine). Chrysotile also stimulated phosphatidylinositol turnover as measured using 32 P i incorporation into phospholipids, [ 3H]-diacylglycerol levels, and intracellular calcium mobilization as measured using fura-2 and 45Ca. In addition, pertussis toxin partially blocked chrysotile-stimulated superoxide anion production. We conclude that the mechanism of guinea pig alveolar macrophage stimulation by chrysotile, but not the amphibole asbestos forms, is consistent with a mechanism which is similar to that used by agonists such as N-formyl-Nle-Leu-Phe resulting in stimulated phosphatidylinositol turnover, calcium mobilization, and activation of protein kinase C.

Dehydroepiandrosterone and two structural analogs inhibit 12-O-tetradecanoylphorbol-13-acetate stimulation of prostaglandin E2 content in mouse skin.

Dehydroepiandrosterone, a naturally occurring adrenal steroid, is a highly effective tumor chemopreventive agent in laboratory mice and rats, inhibiting spontaneous breast cancer and chemically induced tumors of the lung, colon, skin, liver and thyroid. Dehydroepiandrosterone blocks three processes that have been implicated in experimental tumorigenesis: (i) carcinogen activation through the mixed-function oxidases, (ii) 12-O-tetradecanoylphorbol-13-acetate stimulation of superoxide anion production in neutrophils, and (iii) 12-O-tetradecanoylphorbol-13-acetate stimulation of [3H]thymidine incorporation in mouse epidermis. All of these effects of dehydroepiandrosterone very likely result from glucose-6-phosphate dehydrogenase inhibition and a lowering of the NADPH cellular pool. It is now reported that oral administration of dehydroepiandrosterone (0.2% in the diet) for two weeks inhibits the stimulation in prostaglandin E2 content in mouse epidermis produced by topical application of 12-O-tetradecanoylphorbol-13-acetate. Two synthetic steroids, 16 alpha-fluoro-5-androsten-17-one and 16 alpha-fluoro-5 alpha-androstan-17-one, which are more potent inhibitors of the above three processes in tumorigenesis and are also more effective than dehydroepiandrosterone in inhibiting skin papilloma development in the mouse, are more active in suppressing prostaglandin E2 induction by 12-O-tetradecanoyl-phorbol-13-acetate. These two structural analogs, which also lack specific side-effects associated with dehydroepiandrosterone treatment, may find application as cancer chemopreventive drugs in humans.

A requirement for cholesterol for phorbol ester-induced adhesion of a human monocyte-like cell line

The human monocyte/macrophage-like cell line U937, which is a cholesterol auxotroph, is nonadherent. However, it becomes adherent after treatment with phorbol 12-myristate 13-acetate (phorbol ester). We investigated the effects of cellular cholesterol depletion and repletion on the effectiveness of phorbol ester to induce adhesion to substratum. Almost 70% of cellular cholesterol is depleted by incubation of the cells for 24 hrs in the growth medium in which delipidated fetal calf serum is substituted for fetal calf serum without affecting viability or the rate of growth. The use of delipidated fetal calf serum inhibited phorbol ester-induced adhesion by 40%. If the cells were preincubated in the medium containing delipidated fetal calf serum 6 hrs prior to addition of phorbol ester, adhesion was inhibited by 90%. Addition of cholesterol to the medium containing delipidated fetal calf serum, which replenishes cellular cholesterol, restored the ability of phorbol ester to induce adhesion to levels seen in cells cultured in the medium containing fetal calf serum. Epicholesterol was not as effective as cholesterol in supporting adhesion. Cholesterol depletion did not inhibit phorbol ester stimulation of superoxide anion production. These observations indicate a function for cholesterol in phorbol ester-induced adhesion that is independent of phorbol ester-induced superoxide anion production. It is proposed that cholesterol is required for synthesis and/or proper orientation and distribution, in the plasma membrane, of macromolecule(s) that mediate phorbol ester-induced adhesion.

Change in digitonin-stimulated superoxide anion production by guinea pig polymorphonuclear leukocytes in response to the presence of calcium ion.

Treatment of guinea pig polymorphonuclear leukocytes with digitonin in the presence of Ca2+ induced marked stimulation of superoxide anion production. Removal of Ca2+ from the incubation medium by addition of ethyleneglycolbis (β-aminoethyl ether) N, N, N', N'-tetraacetate (EGTA) either before or after the digitonin addition suppressed the stimulation of the superoxide anion production. Furthermore, readdition of Ca2+ in excess of the added EGTA restored the stimulatory effect of digitonin on the superoxide anion production in the leukocytes. These findings suggest the existence of reversible regulation of the digitonin-stimulated superoxide anion production by Ca2+.