Stobadine-modulated human neutrophil functional responsiveness: effect on particular and soluble stimulation

The effect of stobadine on degranulation (myeloperoxidase release) and on oxidative burst, measured as superoxide anion production, was investigated in human neutrophils activated with receptor-specific (fMLP, opsonized zymosan) and nonreceptor stimuli (PMA, A 23187). Wortmannin, a specific inhibitor of 1-phosphatidylinositol 3-kinase, significantly inhibited fMLP-stimulated generation only. This effect was pronounced by stobadine. Stobadine dose-dependently decreased superoxide generation and myeloperoxidase release after receptor-specific stimuli, with the highest effect on fMLP stimulation of superoxide generation and on opsonized zymosan stimulation of myeloperoxidase release.     

Differential inhibition of histamine release from mast cells by protein kinase C inhibitors: staurosporine and K-252a.

Pretreatment of rat peritoneal mast cells with either staurosporine or an analog K-252a [(8R*,9S*,11S*)-(-)-9-hydroxyl-9-methoxycarbonyl-8-methyl-2,3, 9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11-atrizadibenzo- [a,g]cycloocta[cde]trinden-1-one] led to a concentration-related inhibition of histamine release when the cells were stimulated with anti-IgE (IC50: staurosporine = 110 nM; K-252a = 100 nM). In contrast, the two protein kinase C (PKC) inhibitors (1-1000 nM) partially (less than 15%) inhibited histamine release induced by compound 48/80 (0.5 to 1 micrograms/mL). Furthermore, prostaglandin E2 (PGE2) synthesis mediated by anti-IgE from rat peritoneal mast cells was also inhibited by staurosporine and K-252a (IC50 = 100 nM). Exposure of anti-arsenate IgE (anti-Ars-IgE) sensitized mouse bone marrow derived mast cells to arsenate-bovine serum albumin (Ars-BSA) led to the release of both histamine (510 +/- 12.6 ng/10(6) cells) and immunoreactive leukotriene C4 (LTC4) (27.0 +/- 2.6 ng/10(6) cells). Both histamine and LTC4 release was inhibited by staurosporine and K-252a with an IC50 of 50 nM for both compounds. We also characterized a 45K molecular weight protein which is phosphorylated by PKC after Ars-BSA or phorbol, 12-myristate, 13-acetate (PMA) stimulation. This protein is phosphorylated in a broken cell preparation in which PKC is activated by phosphatidylserine/Diolein and Ca2+. Peptide mapping by V8 protease of the phosphorylated 45K protein revealed that the 45K protein phosphorylation patterns induced by IgE or PMA or in the broken cell preparation are identical. Pretreatment of 32P-labeled mouse bone marrow derived mast cells with either staurosporine or K-252a led to a concentration-related inhibition of 45K protein phosphorylation induced by PMA or Ars-BSA. This inhibition of protein phosphorylation correlated well with the inhibition of histamine and leukotriene release in bone marrow derived mast cells.     

Neutrophil priming by granulocyte colony stimulating factor and its modulation by protein kinase inhibitors.

Upon stimulation by various ligands, freshly isolated human peripheral neutrophils (PMN) respond in a variety of ways, such as superoxide (O2-.) generation, phagocytosis enzyme release, migration etc. Chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and opsonized zymosan activate neutrophils by a receptor-mediated mechanism, while phorbol myristate acetate and dioctanoylglycerol activate the cells by a mechanism involving Ca(2+)-and phospholipid-dependent protein kinase (PKC). Receptor-mediated but not PKC-mediated O2-. generation in PMN was enhanced by the priming of recombinant human granulocyte colony stimulating factor (G-CSF). FMLP-dependent luminol chemiluminescence was also enhanced by G-CSF. However, no appreciable enhancement was observed in FMLP-induced intracellular calcium ion concentration ([Ca2+]i). Enhancement of FMLP-induced generation of O2-. by G-CSF was inhibited by genistein or alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was stimulated by staurosporine and 1-(5-isoquinolinesulfonyl)-3-methyl-piperazine (H-7), inhibitors of PKC. The ED50 values of genistein and ST 638 for the inhibition of the FMLP-induced O2-. generation from G-CSF were 0.5 and 5 microM, respectively. In contrast, O2-. generation by PKC activation without G-CSF priming was inhibited by stauroporine and H-7, but was stimulated by genistein and ST 638. These results suggested that the enhancing effect of G-CSF on receptor-mediated generation of the O2-. might be regulated by protein kinases, such as TK and PKC, and that the TK inhibitor selectively inhibited the G-CSF-primed receptor-mediated O2-. generation of neutrophils.     

Activation of human neutrophils by chlordane: induction of superoxide production and phagocytosis but not chemotaxis or apoptosis

We have recently reported that human neutrophils are important targets of different xenobiotics, including chemicals of environmental concern. In the present study, we found that chlordane was not toxic for human neutrophils incubated for up to 24 h with concentrations ranging from 0.1 to 50 microg/ml. Chlordane was found to induce neutrophil superoxide production (O2-) in a concentration-dependent fashion and its potency to induce this response was found to be similar to phorbol 12-myristate 13-acetate (PMA), a classical neutrophil agonist. The use of different transduction signal inhibitors (genistein, pertussis toxin, staurosporine, and calphostin C) indicates that, as for PMA, chlordane induces O2- production via protein kinase C (PKC). In this respect, staurosporine and calphostin C were found to inhibit chlordane- and PMA-induced O2- production by 65% and 72%, and by 83% and 85%, respectively. Chlordane was also found to significantly enhance neutrophil phagocytosis of opsonized sheep red blood cells (SRBCs). Despite these effects, chlordane did not alter neutrophil apoptosis as assessed by cytology (Diff-Quick staining) and by flow cytometry (CD-16 expression). In addition, chlordane did not alter neutrophil chemotaxis (48-well Boyden chamber). Cells were, however, responsive as they were activated by the well-characterized interleukin (IL)-8 chemokine. We conclude that chlordane can activate O2- production by a PKC-dependent mechanism and induce phagocytosis without altering chemotaxis and apoptosis. Chlordane must be added to a growing list of environmental contaminants that share some pro-inflammatory properties.     

Antioxidant effect of hydroxytyrosol, a polyphenol from olive oil: scavenging of hydrogen peroxide but not superoxide anion produced by human neutrophils

Hydroxytyrosol (HT) (also known as dihydroxyphenylethanol (DPE)) is a polyphenol extracted from virgin olive oil. HT is known to exert an antioxidant effect but the mechanism of action and the identity of the reactive oxygen molecule(s) targeted are not known. In this study, we show that HT inhibits luminol-amplified chemiluminescence of human neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA) and opsonized zymosan. This effect was dose-dependent and occurred immediately after the addition of HT. However, HT had no effect on lucigenin-amplified chemiluminescence, suggesting that it does not inhibit NADPH oxidase activation or scavenge superoxide anions. Furthermore, HT inhibited H(2)O(2)-dependent-dichlorofuoroscein (DCFH) fluorescence of activated neutrophils, as measured by flow cytometry. Finally, HT inhibited luminol-amplified chemiluminescence in a cell-free system consisting of H(2)O(2) and HRPO. These results suggest that HT, a polyphenol derived from olive oil, could exert its antioxidant effect by scavenging hydrogen peroxide but not superoxide anion released during the respiratory burst.     

Effect of sphingosine and its N-methyl derivatives on oxidative burst, phagokinetic activity, and trans-endothelial migration of human neutrophils.

Neutrophils display three major functions: (i) oxidative burst, (ii) phagokinetic activity, and (iii) trans-endothelial migration. Sphingosine (SPN) is known to inhibit oxidative burst in human neutrophils via inhibition of protein kinase C (PKC). SPN is metabolically converted into N,N-dimethylsphingosine (DMS) in some tissues and cell lines. In previous studies, we have demonstrated that the PKC-inhibitory effect of DMS is stronger than that of SPN, and that of the synthetic analogue N,N,N-trimethylsphingosine (TMS) is even stronger. Therefore, in the present study, we compared the effects of SPN, DMS, and TMS on the neutrophil functions mentioned above. These three compounds, at 10-20 microM, showed equal inhibition of phorbol 12-myristate 13-acetate (PMA)-dependent superoxide (O2-) production and O2 consumption. They and other known PKC inhibitors (H-7, staurosporine, calphostin C), at 1-5 microM, showed equal inhibition of the phagokinetic activity of neutrophils. On the other hand, trans-endothelial migration of neutrophils was suppressed by SPN, DMS, and TMS at 5-10 microM, but was relatively unaffected by the other PKC inhibitors. All of these compounds inhibited PMA-induced phosphorylation of major neutrophil proteins with a M(r) of 60 and 47 kDa; this effect is ascribable to inhibition of PKC. Despite the similar effects of SPN, DMS, and TMS on neutrophil function, TMS was considerably less cytotoxic to neutrophils under the same experimental conditions. Furthermore, SPN and DMS at 10-20 microM caused obvious morphological changes of the endothelial cells, but TMS did not. SPN undergoes rapid metabolic conversion to various sphingolipid compounds, but TMS is stable. In view of all these findings, TMS appears to be a superior pharmacological agent, compared to SPN derivatives or other PKC inhibitors, for suppression of neutrophil overfunction associated with inflammatory processes and tissue injury.     

Zymosan induces NADPH oxidase activation in human neutrophils by inducing the phosphorylation of p47phox and the activation of Rac2: Involvement of protein tyrosine kinases,PI3Kinase,PKC, ERK1/2 and p38MAPkinase

Reactive oxygen species (ROS) production by the neutrophil NADPH oxidase plays a key role in host defense against pathogens, such as bacteria and fungi. Zymosan a cell-wall preparation from Saccharomyces cerevisiae is largely used to activate neutrophils in its opsonized form. In this study, we show that non-opsonized zymosan alone induced ROS production by human neutrophils. Zymosan-induced ROS production is higher than the formyl-methionyl-leucyl-phenylalanine (fMLF)- or the phorbol myristate acetate (PMA)-induced ROS production but is lower than the one induced by opsonized zymosan. Most of the zymosan-induced ROS production is intracellular. Interestingly, zymosan induced the phosphorylation of the NADPH oxidase cytosolic component p47phox on several sites which are Ser315, Ser328 and Ser345. Zymosan induced also the activation of the small G-protein Rac2. Phosphorylation of the p47phox as well as Rac2 activation were inhibited by genistein a broad range protein tyrosine kinase inhibitor and by wortmannin a PI3Kinase inhibitor. GF109203X a PKC inhibitor inhibited phosphorylation of p47phox on Ser315 and Ser328. SB203580 and UO126, inhibitors of p38MAPK and ERK1/2-pathway, respectively, inhibited phosphorylation of p47phox on Ser345. Zymosan-induced ROS production was completely inhibited by genistein and wortmannin and partially inhibited by SB203580, UO126 and GF109203X. These results show that zymosan alone is able to activate NADPH oxidase in human neutrophils via the phosphorylation of p47phox and Rac2 activation and that a protein tyrosine kinase, PI3Kinase, p38MAPK, ERK1/2 and PKC are involved in this process. These pathways could be potential pharmacological targets to treat zymosan- and S. cerevisiae-induced inflammation.     

Inhibitory effects of zafirlukast on respiratory bursts of human neutrophils

The effects of zafirlukast, a cysteinyl-leukotriene receptor antagonist, on the generation of the reactive oxygen species (ROS) released during respiratory bursts of human polymorphonuclear neutrophils (PMNs) is still unknown. The aim of this study was to investigate the ability of zafirlukast to interfere with the respiratory burst of PMNs. Respiratory burst responses of PMNs were investigated by luminol-amplified chemiluminescence (LACL) using particulate (Candida albicans and zymosan) and soluble stimulants [N-formyl-methionylleucyl-phenylalanine (fMLP) and phorbol 12 myristate 13 acetate (PMA)]. When incubated with PMNs for 10 min at concentrations ranging from 5 x 10(-9) M to 5 x 10(-6) M, zafirlukast did not significantly affect the respiratory bursts of PMNs induced by either the particulate or soluble stimuli. However, after incubation for 60 min, it did reduce the respiratory bursts of PMNs in a concentration-related fashion when the PMNs were stimulated with fMLP, and at a concentration of 5 x 10(-6) M when the stimulus was PMA. No significant effects were seen when the PMNs were challenged with particulate stimuli. Zafirlukast is able to interfere with the activation of the PMNs respiratory burst induced by soluble stimulants. The different behavior determined by different times of contact and different stimuli opens the way to interpretations concerning the antioxidant effect of zafirlukast.     

Selective inhibition of protein kinase C, mitogen-activated protein kinase, and neutrophil activation in response to calcium pyrophosphate dihydrate crystals, formyl-methionyl-leucyl-phenylalanine, and phorbol ester by O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470)

The effect of O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470) was investigated on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation in neutrophils stimulated by plasma-opsonized crystals of calcium pyrophosphate dihydrate (triclinic) [CPPD(T)], formyl-Met-Leu-Phe (fMLP), and phorbol 12-myristate 13-acetate (PMA). Neutrophil respiratory burst responses also were determined in AGM-1470-pretreated cells stimulated with the same agonists, using chemiluminescence and superoxide anion generation assays. AGM-1470 (5 microM) effectively inhibited PKC activation in cells treated with CPPD(T) crystals (50 mg/mL, 2 min) and fMLP (1 microM, 1 min), but had no effect on PMA-treated cells (0.5 microM, 5 min). AGM-1470 blocked MAPK activity completely and reduced neutrophil activation induced by fMLP and PMA but not by CPPD(T). The degree of inhibition of the respiratory burst plateaued at approximately 46+/-9 and 54+/-3% in fMLP- and PMA-treated cells, respectively. These data indicate that activation of neutrophil respiratory burst activity may be mediated through the MAPK pathway. AGM-1470 pretreatment did not inhibit CPPD(T) crystal- or fMLP-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity. These findings, coupled with further observations that the PI 3-kinase inhibitor wortmannin (10 nM) inhibited fMLP- and CPPD(T) crystal-induced but not PMA-induced chemiluminescence, indicate that at least two distinct signaling pathways (mediated by PI 3-kinase or MAPK) lead to neutrophil respiratory burst responses. PKC may also be required in the MAPK-stimulated pathway. We propose that the inhibitory effect of AGM-1470 on the neutrophil respiratory burst may be due to its ability to inhibit PKC and MAPK activation.     

Acetaminophen inhibits the human polymorphonuclear leukocyte function in vitro.

The aim of the study is to investigate the effect of Acetaminophen (Am) on the oxidative respiratory burst of isolated human polymorphonuclear leukocytes (PMNs). Acetaminophen inhibited the luminolchemiluminescence (CL) peak response of PMNs stimulated with phorbol myristate acetate (PMA) or opsonized zymosan in a concentration dependent manner. The inhibitory effect of Am on PMA-stimulated PMNs-CL response was partially reversible. The level of CL inhibition with Am plus the hydroxyl radical scavengers allopurinol, dimethyl sulfoxide (DMSO) or superoxide dismutase (SOD) is greater than that with Am alone. Generation of superoxide (O2-) by stimulated PMNs, as assayed by superoxide dismutase inhibitable reduction of Ferricytochrome c, was markedly inhibited by Am. Furthermore, the phagocytic activity of PMNs as tested for by the ingestion of opsonized dead yeast was significantly reduced in Am-treated cells. These results indicate clearly that Am causes significant inhibition of the human PMNs function in vitro.     

Hydrogen peroxide modulation of the respiratory burst of human neutrophils.

Addition of micromolar concentrations of hydrogen peroxide (H2O2) to human neutrophils resulted in a dose-dependent luminol-enhanced chemiluminescent response. Pretreatment of neutrophils with micromolar concentrations of H2O2 altered their response to the surface acting stimulants serum-treated zymosan (STZ) and formyl-methionyl-leucyl-phenylalanine (fMLP), but not to the intracellular stimulant phorbol myristate acetate (PMA). The alterations were partially reversible by catalase, but exacerbated by superoxide dismutase. These results suggest a modulatory role for H2O2 in the respiratory burst of neutrophils.     

Superoxide production by human eosinophils can be inhibited in an agonist-selective manner.

This paper focuses on eosinophil activation and its selective inhibition. Superoxide anion (O2-) production by human eosinophils, an indicator of their activation, was induced by a variety of activators. Several compounds which are known to inhibit protein kinase C (staurosporine, K252a, sphingosine) inhibited O2- production induced by phorbol ester (PMA) but failed to inhibit O2- production induced by IgG coupled to Sepharose beads. Inhibition of O2- production by other agents (plasma-activated zymosan, fMLP, and leukotriene B4 (LTB4), was intermediate. By contrast, wortmannin, a compound which has been previously reported to inhibit O2- production in neutrophils via a protein kinase-independent pathway, potently inhibited O2- production in eosinophils which had been activated by IgG and by Platelet-Activating Factor but was virtually inactive against PMA-induced O2- production. Taken together, the results indicate that, as a minimum, there must be two pathways of induction of O2- production in eosinophils. Moreover, the intermediate levels of inhibition in cells which had been activated with serum-activated zymosan, FMLP, and LTB4 suggest that these agents may either be acting via both of these pathways or that yet other pathways may exist.     

Inhibitors of protein kinase C. 1. 2,3-Bisarylmaleimides.

The design and synthesis of a series of novel inhibitors of protein kinase C (PKC) is described. These 2,3-bisarylmaleimides were derived from the structural lead provided by the indolocarbazoles, staurosporine and K252a. Optimum activity required the imide NH, both carbonyl groups, and the olefinic bond of the maleimide ring. 2,3-Bisindolylmaleimides were the most active, and the potency of these was improved by a chloro substituent at the 5-position of one indole ring (compound 28, IC50 0.11 microM). In a series of (phenylindolyl)maleimides, nitro compound 74 was most active (IC50 0.67 microM). Naphthalene 19 and benzothiophene 21 showed greater than 100-fold selectivity for inhibition of PKC over the closely related cAMP-dependent protein kinase (PKA).     

Studies of cellular mechanisms for the generation of superoxide by guinea-pig eosinophils and its dissociation from granule peroxidase release

Guniea-pig peritoneal eosinophils generated superoxide anions in response to opsonized zymosan, platelet activating factor, sodium fluoride, digitonin, phorbol ester and calcium ionophore, but were refractory to fMLP. These agonists did not stimulate release of eosinophil peroxidase. The phospholipase inhibitor, mepacrine, and the protein kinase inhibitor, trifluoperazine, were effective inhibitors of superoxide production. Activators of protein kinase C, such as exogenously added phorbol ester and endogenously derived diacylglycerol, stimulate superoxide production, which is therefore proposed to be via pathways dependent on phospholipase and protein kinase activity.     

Protein kinase C inhibitors decrease endothelin ET(B) receptor mRNA expression and contraction during organ culture of rat mesenteric artery

The effect of protein kinase C (PKC) inhibitors on the induction of endothelin ET(B) receptors during organ culture was examined in isolated segments of the rat mesenteric artery. After 24 h of organ culture, the endothelin ET(B) receptor agonist sarafotoxin 6c (S6c) induced a strong contraction compared to fresh segments. The contractile response after 24-h organ culture to S6c was studied in presence (30-min preincubation) or absence, after 24-h treatment, of the PKC inhibitors staurosporine, K252a and Ro31-7549. Exposure to staurosporine or K252a in presence and after 24-h treatment reduced the S6c contraction. In contrast, presence of 2-1[1-3(aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide (Ro31-7549), did not affect the S6c-induced contraction, whereas 24-h treatment abolished the increase of contraction. The PKA inhibitor N-(2-[bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H89) did not affect the S6c responses. The mRNA expressions of endothelin ET(B) receptors (analysed with real-time PCR) were abolished after 24-h treatment with the PKC inhibitors. These results suggest that PKC is involved in the endothelin ET(B) receptor upregulation following organ culture.     

Tetrahydrofuran lignans from Illicium floridanum and their activity in a luminol enhanced chemiluminescence assay

Two lignans of the tetrahydrofuran type, di-O-methyltetrahydrofuroguaiacin B (1) and (+)-veraguensin (2) were isolated from fruits and leaves of Illicium floridanum Ellis (Illiciaceae). These compounds are the first genuine lignans isolated from the genus Illicium. We investigated their radical scavenging potency towards 1,1-diphenyl-2-picrylhydrazyl radical reagent and their influence on luminol enhanced chemiluminescence (CHL) induced by different stimuli in human polymorphonuclear neutrophils (PMN). Both compounds were inactive in the TLC assay for chemical radical scavenge. In the PMN assay, the symmetric lignan 1 at concentrations below 1.0 microM displayed a strong inhibition of CHL induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP). At concentrations above 5 microM, 1 led to a pronounced increase of fMLP induced CHL. When CHL was stimulated with opsonized zymosan, both compounds were completely inactive. Thus, 1 must interfere selectively with a step in the signal cascade evoked by fMLP. In addition to their known PAF-receptor antagonism tetrahydrofuran lignans may thus also interfere with inflammatory responses by inhibition of free radical formation.     

Staurosporine inhibits protein kinase C and prevents phorbol ester-mediated leukotriene D4 receptor desensitization in RBL-1 cells

The aim of the present study was to investigate the effects of staurosporine on phorbol-myristate acetate (PMA)-induced activation of protein kinase C (PKC) and the desensitization of leukotriene D4 (LTD4)-stimulated Ca2+ mobilization in rat basophilic leukemia (RBL-1) cells. Staurosporine, one of the most potent PKC inhibitors known to date, markedly inhibited partially purified PKC from RBL-1 cells with an IC50 of 3 nM. Exposure of RBL-1 cells to PMA resulted in inhibition of LTD4-stimulated Ca2+ mobilization. However, prior treatment of the cells with staurosporine completely prevented PMA-induced desensitization of LTD4-stimulated Ca2+ mobilization. This reversal of Ca2+ desensitization by staurosporine was dose dependent with an IC50 of 0.1 microM. Treatment of RBL-1 cells with PMA resulted in translocation and activation of PKC from the cytosol to the membrane fraction. Pretreatment of RBL-1 cells with staurosporine inhibited the PMA-induced activation of PKC in the membrane fraction. The inhibition of PKC activity by staurosporine was time and dose dependent with an IC50 of 0.9 microM. These results show that PMA-induced heterologous desensitization is mediated by PKC and staurosporine prevented this process by directly inhibiting PKC in intact RBL-1 cells.     

Arachidonic acid release and platelet-activating factor formation by staurosporine in human neutrophils challenged with n-formyl peptide.

Staurosporine, a putative protein kinase C (PKC) inhibitor, increased the release of [14C]arachidonic acid dose dependently between 100 nM and 1000 nM in human neutrophils challenged with 100 nM N-formyl-methionine-leucine-phenylalanine (FMLP). Staurosporine also increased the formation of leukotriene B4 (LTB4) and platelet-activating factor (PAF) in a dose-dependent manner. In addition, exogenously added lyso-PAF further augmented [3H]PAF formation in staurosporine-pretreated human neutrophils stimulated by FMLP, thus suggesting an activation of acetyl-CoA: lyso-PAF acetyltransferase by staurosporine. The potentiation of [14C]arachidonic acid release and [3H]PAF formation by staurosporine was further enhanced in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA), which pinpoints a mechanism other than the modulation of PKC in this process, inasmuch as staurosporine antagonizes PMA-induced O2- production and [3H]PAF formation. Additional studies with other putative PKC inhibitors also revealed the potentiating effects of 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7, 20 microM) and sphingosine (2.5 microM) on FMLP-induced [14C]arachidonic acid release and [3H]PAF formation. We therefore conjecture that staurosporine-sensitive protein kinases including PKC are not involved in the activation of phospholipase A2 and acetyl-CoA:lyso-PAF acetyltransferase.     

Effect of the protein kinase C inhibitor GF 109 203X on elastase release and respiratory burst of human neutrophils

• 1.1. The effects of bisindolylmaleimide GF 109 203X, reported to be a potent and highly selective inhibitor of protein kinase C (PKC), have been investigated on some human neutrophil functions.
• 2.2. GF 109 203X prevented O₂⁻ production by NADPH-oxidase whatever the stimulus used for polymorphonuclear neutrophil (PMN) activation: directs PKC activators like phorbol myristate acetate (PMA) and dioctanoylglycerol, calcium ionophore (A23187), or receptor agonists like fMet-Leu-Phe (fMLP) and opsonized zymosan.
• 3.3. The effect of GF 109 203X was also examined on elastase exocytosis by neutrophils. PMA-mediated elastase release was prevented by GF 109 203X. However, GF 109 203X had no effect on exocytosis induced by A23187 and the effect of this compound on the fMLP response changed according to its concentration.
• 4.4. These data suggest that PKC might be essential for stimulus-mediated O₂⁻ production and also that PKC plays only a minor role in elastase secretion as compared to the role of the cytosolic calcium level.

     

Effect of butylated hydroxyanisole and some of its derivatives on human neutrophil oxidative burst: chemiluminescence evaluation

An acute inflammatory response begins during the reperfusion phase following an ischemic insult in which polymorphonuclear neutrophils (PMNs) play an important role and the release of reactive oxygen species (ROS) causes further damage and a reduction in endogenous antioxidant storage. The ability of butylated hydroxyanisole (BHA) and some phenolic, aliphatic and aromatic BHA derivatives to reduce the human PMN oxidative burst evoked by particulate (Candida albicans and zymosan) or soluble stimulants [N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA)] was investigated using luminol-amplified chemiluminescence. BHA and the derivative dt-BHA [3,5-di-t-butyl-4-hydroxyanisole] significantly reduced the PMN oxidative burst at concentrations ranging from 5 x 10(-6) to 5 x 10(-5) mol/l for C. albicans stimulation, while for zymosan stimulation, reduction was seen at concentrations ranging from 5 x 10(-6) to 5 x 10(-5) mol/l for BHA, and at concentrations ranging from 5 x 10(-7) to 5 x 10(-5) mol/l for dt-BHA, with dt-BHA being the most active. Another BHA derivative, Bu GAM 1, was active at 5 x 10(-5) mol/l for C. albicans and at 5 x 10(-6) to 5 x 10(-5) mol/l for zymosan. The findings obtained with fMLP and PMA were very similar to those previously reported. ROS release is related to PMN killing activity, but the inhibition of the PMN oxidative burst induced by BHA and BHA derivatives did not significantly modify PMN phagocytosis or killing. It has recently been observed that dt-BHA has a spasmolytic action by inhibiting the influx of Ca(2+) into cells through L-type Ca(2+) channels, which means that a single molecule is capable of counteracting two major steps in the sequence of events triggered by ischemia-reperfusion injury, i.e. free radical release and Ca(2+) overload.