fMLP-activated Neutrophils Research Articles

Galectin-1 modulation of neutrophil reactive oxygen species production depends on the cell activation state

Here we report the effects of exogenous and endogenous galectin-1 (Gal-1) in modulating the functional responses of human and murine neutrophils at different stages of activation, i.e. naive, primed, and activated. Exposure to Gal-1 did not induce ROS production in either naive or N-formyl-methionyl-leucyl-phenylalanine-primed (fMLP; 10−9 M) neutrophils. However, Gal-1 elicited a concentration-dependent ROS production in neutrophils activated with fMLP at concentrations ranging from 10-8 M to 10-6 M. Additional fMLP (10-7 M) stimulation of fMLP-activated neutrophils increased ROS production, whose intensity was inversely related to the fMLP concentration used in the first activation step (10-8 M to 10-6 M), and was not influenced by the presence of Gal-1. Naive neutrophils treated with Gal-1 and then exposed to fMLP (10-6 M) or phorbol-12-myristate-13-acetate (10-7 M) produced less ROS, as compared to naive neutrophils not treated with Gal-1. Interestingly, these in vitro Gal-1 effects were associated with Gal-1 carbohydrate-binding activity and the ability to decrease FPR-1 (formyl peptide receptor 1) expression in naive human neutrophils. Conversely, positive ROS modulation by Gal-1 in activated neutrophils was not associated with FPR-1 expression but it was related to its carbohydrate recognition. In vitro, fMLP stimulation of Gal-1-/- mouse neutrophils produced more ROS than fMLP stimulation of Gal-1+/+ neutrophils and this effect may be associated with increased FPR-1 expression. Exogenous Gal-1 induced ROS production in Gal-1-/- mouse neutrophils more effectively than in Gal-1+/+ mouse neutrophils. Compared to Gal-1+/+ mice, Gal-1-/- mice exhibited lower bacterial load in the peritoneal fluid and peripheral blood, thus indicating a greater bactericidal activity in vivo. These findings demonstrate that endogenous Gal-1 restricts ROS generation that correlates with bacterial killing capacity in inflammatory neutrophils. Thus, endogenous and exogenous Gal-1 may either positively or negatively modulate the effector functions of neutrophils according to the cell activation stage.

Synthetic leu-enkefalin analogue prevents activation of neutrophils induced by a bacterial component

Background: Neutrophil activation is a mandatory stage and a sensitive marker of systemic inflammatory response. The development of this condition is associated with subsequent multiple organ failure which is the main indication for the patients stay in the intensive care unit. The search for drugs that could prevent the development of systemic inflammatory response and reduce mortality remains an urgent task of anesthesiology/resuscitation.Aim: To study the anti-inflammatory effect of dalargin, a synthetic analogue of lei-enkephalin, on human neutrophils in vitro.Materials and methods: The study was performed on blood neutrophils isolated from 5 healthy donors. A proportion of neutrophils were activated by 10 mkM formil-Met-Leu-Pro (fMLP) and 100 ng/mL lipopolysaccharide (LPS) with subsequent assessment of their activity by fluorescent antibodies to the degranulation markers CD11b and CD66b. Thereafter intact and activated neutrophils were treated with dalargin solution at concentrations of 50 and 100 mcg/mL.Results: Dalargin at 100 mcg/mL reduced the expression of CD11b molecules on the surface of intact neutrophils by 5.5-fold (p=0.008). On the contrary, LPS at a dose of 100 ng/mL increased the expression of the same molecules by 46% (p=0.08). The addition of dalargin at 50 mcg/mL to LPS-activated neutrophils reduced the expression of CD11b molecules (p=0.016). The addition of dalargin at 50 mcg/mL to fMLP-activated neutrophils significantly (p=0.008) reduced the expression of CD11b molecules and reversed their expression virtually to the level of the control. The addition of dalargin at 100 mcg/mL to neutrophils activated by fMLP at 10 mkM reduced the expression of CD11b on their surface to a level below the control by 23% (p=0.08).Conclusion: Dalargin at the studied concentrations has an anti-inflammatory effect on both intact and pre-activated bacterial components of neutrophils, thus inhibiting the process of activation and degranulation in a dose-dependent manner.

Nitroxide Tempol down-regulates kinase activities associated with NADPH oxidase function in phagocytic cells and potentially decreases their fungicidal response

AimsThe identification of novel targets to control inflammation in humans is probably the primary challenge that impairs the development of new anti-inflammatory drugs. Therefore, the modulation of intracellular signaling pathways in phagocytes may be an interesting means of achieving this goal. However, this change to signaling can compromise the host's susceptibility to invading pathogens. We investigated whether the antioxidant nitroxide Tempol regulates the activity of kinases associated with the production of oxidants in neutrophils, which affects the fungicidal capability of these cells. Main methodsThe effects of Tempol on PMA- or fMLP-activated neutrophils were examined by oxygen consumption as an index of the oxidative burst, a release of extracellular and total Reactive Oxygen Species (ROS) by chemiluminescence, kinase activities through analysis of ATP consumption during enzyme activities and the dot blot immunoassay and, finally, by neutrophil capacity of killing Candida albicans. Key findingsTempol significantly inhibited the neutrophil oxidative burst in a concentration-dependent manner and decreased oxygen consumption (IC50 = 45 μM) and extracellular/total ROS formation with an increase on the lag period response. In addition, Tempol inhibited neutrophil kinase activities (i.e., a decrease in protein phosphorylation) elicited through different biochemical pathways and consequently impaired the fungicidal activity of these cells. SignificanceAlthough Tempol has potential anti-inflammatory activity that acts on different intracellular pathways (such as those involving kinases), researchers should be cautious, since this nitroxide down-regulated oxidants production and the fungicidal response of neutrophils.

Mitochondrial reactive oxygen species are involved in chemoattractant-induced oxidative burst and degranulation of human neutrophils in vitro

Activation of neutrophils is accompanied by the oxidative burst, exocytosis of various granule types (degranulation) and a delay in spontaneous apoptosis. The major source of reactive oxygen species (ROS) in human neutrophils is NADPH oxidase (NOX2), however, other sources of ROS also exist. Although the function of ROS is mainly defensive, they can also play a regulatory role in cell signaling. However, the contribution of various sources of ROS in these processes is not clear. We investigated a possible role of mitochondria-derived ROS (mtROS) in the regulation of neutrophil activation induced by chemoattractant fMLP in vitro. Using the mitochondria-targeted antioxidant SkQ1, we demonstrated that mtROS are implicated in the oxidative burst caused by NOX2 activation as well as in the exocytosis of primary (azurophil) and secondary (specific) granules. Scavenging of mtROS with SkQ1 slightly accelerated spontaneous apoptosis and significantly stimulated apoptosis of fMLP-activated neutrophils. These data indicate that mtROS play a critical role in signal transduction that mediates the major neutrophil functional responses in the process of activation.

The Effect of Sphingosine 1-Phosphate/Sphingosine 1-Phosphate Receptor on Neutrophil Function and the Relevant Signaling Pathway

Background/Aims: This study investigated the priming effect of sphingosine 1-phosphate (S1P) on formyl-Met-Leu-Phe-OH (fMLP)-activated neutrophils, by specific analysis of the neutrophil respiratory burst and the signaling pathway involved in S1P activity. Methods: The neutrophil respiratory burst was indirectly detected by the cytochrome c reduction method and the dihydrorhodamine 123 staining method. The signal transduction pathways of neutrophil respiratory burst primed by S1P were detected by Western blotting. Results: Our results showed that the S1P receptors (S1PRs) 1, 4 and 5 were the predominantly expressed neutrophil S1PRs at the cDNA level. After pretreatment with S1P, the fMLP-activated neutrophils released increased levels of superoxide anions. PI3K and Akt proteins were involved in the signaling pathway of the neutrophil respiratory burst primed by S1P. Conclusion: The results indicate that S1P is a new priming reagent for neutrophils and primes the respiratory burst of fMLP-activated neutrophils. S1P interacts with its receptors on neutrophils, resulting in NADPH oxidase activation by the PI3K-Akt cell signaling pathway and induction of the neutrophil respiratory burst.

Thiotaurine modulates human neutrophil activation.

In acute inflammation, neutrophils are activated in response to several agonists generating reactive oxygen species (ROS) by NADPH oxidase-dependent mechanisms. Thiotaurine, a thiosulfonate structurally related to hypotaurine and taurine, is a metabolic product of cystine and contains sulfane sulfur which can play several regulatory roles in biological systems. In the present study, the effects of thiotaurine on human leukocyte activation have been evaluated. Neutrophils were stimulated by phorbol 12-myristate 13-acetate (PMA) or chemotactic peptide (fMLP) and respiratory burst was assessed by luminol enhanced chemiluminescence. Superoxide generation was estimated by measuring cytochrome c reduction. Thiotaurine caused a decrease of the respiratory burst of PMA-stimulated neutrophils in a dose-dependent manner, while fMLP-stimulated cells were slightly affected by thiotaurine. Addition of 100 μM thiotaurine induced 64.4 % inhibition of ROS production by PMA-activated cells, as evaluated by luminol enhanced chemiluminescence. As thiotaurine can release sulfane sulfur producing hydrogen sulfide (H2S) and hypotaurine, the effects of these compounds on leukocyte functional responses were evaluated and compared with those exerted by thiotaurine, which had proved more effective in inhibiting respiratory burst than H2S, hypotaurine and taurine. However, fMLP-induced azurophilic degranulation as analyzed by elastase release was unaffected by thiotaurine up to 5 mM concentration. Compared with fMLP-activated neutrophils, PMA-activated neutrophils were more susceptible to thiotaurine inhibition, suggesting that thiotaurine may interfere with the PKC-dependent pathway of neutrophil activation.

Diosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via multiple pathways

Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)- activated neutrophils, with IC50 values of 0.50 ± 0.08 μM and 0.66 ± 0.13 μM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA2), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders.

Contribution by Polymorphonucleate Granulocytes to Elevated Gamma-Glutamyltransferase in Cystic Fibrosis Sputum

BackgroundCystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gamma-glutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail.Principal FindingsThe present study was aimed to evaluate the origin and the biochemical characteristics of the GGT detectable in CF sputum. We found GGT activity both in neutrophils and in the fluid, the latter significantly correlating with myeloperoxidase expression. In neutrophils, GGT was associated with intracellular granules. In the fluid, gel-filtration chromatography showed the presence of two distinct GGT fractions, the first corresponding to the human plasma b-GGT fraction, the other to the free enzyme. The same fractions were also observed in the supernatant of ionomycin and fMLP-activated neutrophils. Western blot analysis confirmed the presence of a single band of GGT immunoreactive peptide in the CF sputum samples and in isolated neutrophils.ConclusionsIn conclusion, our data indicate that neutrophils are able to transport and release GGT, thus increasing GGT activity in CF sputum. The prompt release of GGT may have consequences on all GGT substrates, including major inflammatory mediators such as S-nitrosoglutathione and leukotrienes, and could participate in early modulation of inflammatory response.

Effects of heparin and related drugs on neutrophil function

We have previously demonstrated that heparin inhibits neutrophil activation, but the precise mechanism of action remains to be elucidated. The current aim was to further investigate the effects of heparin at inducing apoptosis of neutrophils and whether this was related to antagonism at IP3 receptors. Furthermore, we investigated the ability of heparin and related molecules to inhibit acute neutrophil-induced injury to human bronchial epithelial cells (HBECs) in vitro.Neutrophils were isolated from human peripheral venous blood. Expression of annexin-V was determined in neutrophils following incubation with LMWH. The effects of LMWH and related molecules upon thapsigargin or m-3M3FBS (phospholipase C activator) induced neutrophil elastase (NE) release were also investigated. The cytotoxic effects of fMLP-activated neutrophils following co-incubation with HBECs were quantified through counting adherent cells before and after incubation.There was no detectable increase in annexin-V positive neutrophils following pre-incubation with LMWH at 30 min, 60 min or 16 h, but an increase was observed with Fas-activating antibody at 16 h. LMWH significantly inhibited NE release induced by either m-3M3FBS (73.4 ± 6.1%, 100 IU ml−1, P < 0.01) or thapsigargin (62.4 ± 6.9%, 100 IU ml−1, P < 0.01) in a sulphate-dependent manner. LMWH and related sulphated molecules all abrogated the cytotoxic effects of fMLP-activated neutrophils upon HBECs.In conclusion we were not able to demonstrate that heparin induces apoptosis and we did not find any evidence for heparin acting as an IP3 receptor antagonist in neutrophils. Nonetheless, the potent inhibitory effects of heparin and related molecules upon neutrophil-induced injury to HBECs provide further evidence of the therapeutic potential of heparin and related molecules in the treatment of chronic inflammatory diseases.

M-ficolin and leukosialin (CD43): new partners in neutrophil adhesion

M-ficolin specificity for sialylated ligands prompted us to investigate its interactions with the main membrane sialoprotein of human neutrophils, CD43. rM-ficolin bound CD43 and prevented the access of anti-CD43 mAb. Moreover, rM-ficolin reacted exclusively with CD43 on Western blots of neutrophil lysate. We confirmed that M-ficolin is secreted by fMLP-activated neutrophils, and this endogenous M-ficolin also binds to CD43 and competes with anti-CD43 mAb. Anti-CD43 antibody cross-linking or fMLP resulted in M-ficolin and CD43 colocalization on polarized neutrophils. The binding of rM-ficolin to resting neutrophils induced cell polarization, adhesion, and homotypic aggregation as anti-CD43 mAb. The M-ficolin Y271F mutant, unable to bind sialic acid, neither reacted with neutrophils nor modulated their functions. Finally, rM-ficolin activated the lectin complement pathway on neutrophils. These results emphasize a new function of M-ficolin, different from ficolin pathogen recognition, i.e., a participation to neutrophil adhesion potentially important in early inflammation, as nanomolar agonist concentrations are sufficient to mobilize M-ficolin to the neutrophil surface. This multivalent lectin could then endow the antiadhesive CD43, essentially designed to prevent leukocyte aggregation in the blood flow, with new adhesive properties and explain, at least in part, dual-adhesive/antiadhesive roles of CD43 in neutrophil recruitment.

2-(2-Fluorobenzamido)benzoate ethyl ester (EFB-1) inhibits superoxide production by human neutrophils and attenuates hemorrhagic shock-induced organ dysfunction in rats

Neutrophil activation after trauma–hemorrhagic shock (T/H) has been implicated in the development of multiple organ dysfunction (MOD). In this study, we report that a small chemical compound, 2-(2-fluorobenzamido)benzoic acid ethyl ester (EFB-1), exhibited a potent inhibitory effect on the formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-induced superoxide anion (O2•−) release and CD11b expression by human neutrophils. Additionally, administration of EFB-1 in rats subjected to T/H caused a significant improvement in MOD. EFB-1 treatment induced an increase in cAMP formation and protein kinase (PK) A activity in FMLP-activated neutrophils, which occurred through the selective inhibition of cAMP-specific phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function or cGMP-specific PDE activity. FMLP-induced phosphorylation of protein kinase B (AKT), but not calcium mobilization, was reduced by EFB-1. The inhibitory effects of EFB-1 on O2•− production, CD11b expression, and AKT phosphorylation were reversed by PKA inhibitors (H89 and KT5720). Significantly, administration of EFB-1 (1mg/kg body wt) attenuated the myeloperoxidase activity of the intestines, lungs, and liver and reduced the wet/dry weight ratio of the intestines and lungs and plasma alanine aminotransferase and aspartate aminotransferase levels in Sprague–Dawley rats after T/H. Therefore, EFB-1 is a new inhibitor of cAMP-specific PDE that potently suppresses O2•− release and CD11b expression by human neutrophils and attenuates T/H-induced MOD in rats.

Effects of reactive oxygen species and neutrophils on endothelium-dependent relaxation of rat thoracic aorta.

Reactive oxygen species (ROS) are produced in different metabolic processes including the respiratory burst of neutrophils accompanying local inflammation. The aim of this study was to analyze the effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP)-activated neutrophils, isolated from the guinea pig peritoneal cavity, on isolated rings of a large (conduit) artery, the rat thoracic aorta. FMLP-activated neutrophils enhanced the basal tension increased by α1-adrenergic stimulation. In phenylephrine-precontracted aortae, they elicited marked contraction, while in noradrenaline-precontracted rat aortal rings they caused a biphasic response (contraction-relaxation). To eliminate interaction of activated neutrophils with catecholamines, in the subsequent experiments the basal tension was increased by KCl-induced depolarization. Activated neutrophils evoked a low-amplitude biphasic response (relaxation-contraction) on the KCl-induced contraction. Not only the acetylcholine- and A23187-induced relaxations but also the catalase sensitive hydrogen peroxide (H2O2) elicited contractions were endothelium-dependent. Even though the acetylcholine-induced relaxation was changed by activated neutrophils and by the ROS studied, their effects differed significantly, yet none of them did eliminate fully the endothelium-dependent acetylcholine relaxation. The effect of activated neutrophils resembled the effect of superoxide anion radical (O2 •–) produced by xanthine/xanthine oxidase (X/XO) and differed from the inhibitory effects of Fe2SO4/H2O2-produced hydroxyl radical (•OH) and H2O2. Thus O2 •– produced either by activated neutrophils or X/XO affected much less the endothelium-dependent acetylcholine-activated relaxation mechanisms than did •OH and H2O2. In the large (conduit) artery, the effects of activated neutrophils and various ROS (O2 •–, •OH and H2O2) seem to be more dependent on muscle tension than on endothelial mechanisms.

Distribution of dystrophin- and utrophin-associated protein complexes during activation of human neutrophils

Dystrophins, utrophins, and their associated proteins are involved in structural and signaling roles in nonmuscle tissues; however, description of these proteins in neutrophils remained unexplored. Therefore we characterize the pattern expression, and the cellular distribution of dystrophin and utrophin gene products and dystrophin-associated proteins (i.e., beta-dystroglycan, alpha-syntrophin, and alpha-dystrobrevins) in relation to actin filaments in resting and activated with formyl-methionyl-leucyl-phenylalanine human neutrophils. Resting and fMLP-activated human neutrophils were analyzed by immunoblot and by confocal microscopy analysis. Immunoprecipitation assays were performed to corroborate the presence of protein complexes. Immunoprecipitation assays and confocal analysis demonstrated the presence of two dystrophin-associated protein complexes in resting and activated neutrophils: the former formed by Dp71d/Dp71Delta(110)(m) and dystrophin-associated proteins (beta-dystroglycan, alpha-syntrophin, alpha-dystrobrevin-1, and -2), while the latter contains Up400, instead of Dp71d/Dp71Delta(110)(m), as a central component of the dystrophin-associated protein complexes (DAPC). Confocal analysis also showed the subcellular redistribution of Dp71d/Dp71Delta(110)(m) approximately DAPC and Up400 approximately DAPC in F-actin-based structures displayed during activation process with fMLP. Our study showed the existence of two protein complexes formed by Dp71d/Dp71Delta(110)(m) or Up400 associated with DAPs in resting and fMLP-treated human polymorphonuclears. The interaction of these complexes with the actin cytoskeleton is indicative of their dynamic participation in the chemotaxis process.

Synthesis and evaluation of benzoxazinone derivatives on activity of human neutrophil elastase and on hemorrhagic shock-induced lung injury in rats

A new series of benzoxazinone analogs were designed, synthesized, and assayed to determine their effects on superoxide anion generation and neutrophil elastase (NE) release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils. Of these, compounds 6– 10 showed a potent dual inhibitory effect on NE release and superoxide anion generation. In contrast, compounds 11– 15 exhibited highly selective and potent inhibitory activities on NE release. These results indicate that the inhibitory activity on NE release in FMLP-activated human neutrophils depended on the position of chloro-substituent in the A ring. On the other hand, 13 significantly attenuated the increase in myeloperoxidase (MPO) activity and edema in the lung of rats after trauma-hemorrhagic shock. Therefore, these compounds could be developed as new NE inhibitors.

Inhibition by magnolol of formylmethionyl-leucyl-phenyl alanine-induced respiratory burst in rat neutrophils.

The influence of the plant product magnolol on neutrophil superoxide anion (O2-*) generation has been investigated in the rat. Intraperitoneal injection of magnolol (30mg kg(-1)) significantly inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat whole blood ex-vivo. Magnolol also inhibited the 02-* generation with an IC50 (concentration resulting in 50% inhibition) of 15.4+/-1.6 microM and O2 consumption in rat neutrophils in-vitro. Magnolol weakly inhibited the O2-* generation in the xanthine-xanthine oxidase system, decreased cellular cyclic AMP level and had no effect on cyclic GMP levels. It weakly inhibited neutrophil cytosolic protein kinase C activity but did not alter porcine heart protein kinase A activity. Magnolol attenuated fMLP-induced protein tyrosine phosphorylation with an IC50 of 24.0+/-1.9 microM and the phosphorylation of mitogen-activated protein kinase p42/44 with an IC50 of 28.5+/-4.5 microM. However, magnolol alone activated neutrophil phospholipase D activity as determined by the formation of phosphatidic acid and phosphatidyl-ethanol in the presence of ethanol. In the presence of NADPH, the arachidonate-activated NADPH oxidase activity in a cell-free system was weakly suppressed by magnolol. These results suggest that the inhibition of respiratory burst in fMLP-activated neutrophils by magnolol is probably attributable mainly to the attenuation of protein tyrosine phosphorylation and p42/44 mitogen-activated protein kinase activation, and partly to the suppression of protein kinase C and NADPH oxidase activities.

DSM-RX78, a new phosphodiesterase inhibitor, suppresses superoxide anion production in activated human neutrophils and attenuates hemorrhagic shock-induced lung injury in rats

Neutrophils are activated following hemorrhagic shock and the accumulation of neutrophils in the lung is associated with lung injury. This research investigated the effects of a semisynthetic 2-benzoylaminobenzoic acid derivative, methyl 2-(2-fluorobenzamido)benzoate (DSM-RX78), on superoxide anion (O 2 −) production in formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP)-activated human neutrophils, and on lung injury in Sprague–Dawley rats subjected to trauma-hemorrhage. DSM-RX78 concentration-dependently inhibited O 2 − production, but not elastase release, in FMLP-activated human neutrophils. DSM-RX78 displayed no superoxide-scavenging ability, and it failed to alter the subcellular NADPH oxidase activity. Significantly, DSM-RX78 increased cAMP formation and protein kinase (PK)A activity in FMLP-activated neutrophils, which occurred through the selective inhibition of cAMP-specific phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function or cGMP-specific PDE activity. These results show that DSM-RX78 is a new inhibitor of cAMP-specific PDE. Moreover, DSM-RX78 reduced FMLP-induced phosphorylation of protein kinase B (Akt), but not calcium mobilization. The inhibitory effects of DSM-RX78 on O 2 − production and Akt phosphorylation were reversed by PKA inhibitors, suggesting that DSM-RX78 regulates O 2 − production of human neutrophils by promoting cAMP/PKA-dependent inhibition of Akt activation. On the other hand, administration of DSM-RX78 significantly attenuated the increase in myeloperoxidase activity and edema in the lung, as well as protein concentrations in bronchoalveolar lavage fluid in rats after trauma-hemorrhagic shock. In summary, these results strongly suggest that DSM-RX78 exerts anti-inflammatory effects, which result from the elevation of cAMP levels and PKA activity through its inhibition of cAMP-specific PDE. Also, our findings show that DSM-RX78 attenuates hemorrhagic shock-induced lung injury in rats.

Potent inhibition of superoxide anion production in activated human neutrophils by isopedicin, a bioactive component of the Chinese medicinal herb Fissistigma oldhamii

Fissistigma oldhamii is widely used in traditional Chinese medicine to treat rheumatoid arthritis. Activation of neutrophils is a key feature of inflammatory diseases. Herein, the anti-inflammatory functions of isopedicin, a flavanone derived from F. oldhamii, and its underlying mechanisms were investigated in human neutrophils. Isopedicin potently and concentration-dependently inhibited superoxide anion (O 2 − ) production in formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP)-activated human neutrophils with an IC 50 value of 0.34 ± 0.03 μM. Furthermore, isopedicin displayed no superoxide-scavenging ability, and it failed to alter subcellular NADPH oxidase activity. The inhibitory effect of isopedicin on O 2 − production was reversed by protein kinase A (PKA) inhibitors. Moreover, isopedicin increased cAMP formation and PKA activity in FMLP-activated human neutrophils, which occurred through the inhibition of phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function. In addition, isopedicin reduced FMLP-induced phosphorylation of extracellular regulated kinase and c-Jun N-terminal kinase, which was reversed by the PKA inhibitor. In contrast, isopedicin failed to alter FMLP-induced phosphorylation of p38 mitogen-activated protein kinase and calcium mobilization. In summary, these results demonstrate that inhibition of O 2 − production in human neutrophils by isopedicin is associated with an elevation of cellular cAMP and activation of PKA through its inhibition of cAMP-specific PDE.

Ethanol alters neutrophil membrane mechanics and adhesion molecule expression

Ethanol (EtOH) has been linked to both protective and damaging effects in inflammatory and immune responses of leukocytes. To characterize EtOH effect on neutrophil mechanics, we studied neutrophil rolling, tethering, and firm arrest behavior at physiologically relevant EtOH concentrations achieved in humans by alcohol consumption (&lt;0.5 v/v%). Neutrophils were perfused over parallel‐plate flow chambers with P‐selectin‐coated beads or confluent HAEC (human aortic endothelial cells) monolayer at a venous shear rate of 100 s−1. EtOH decreased the adhesion efficiency of neutrophils from 0.085 to 0.031, increased the average tether length and tether growth velocity fourfold, and decreased bond forces, suggesting a lower degree of membrane‐cytoskeleton association. When perfused over IL‐1‐activated HAEC, the fraction of neutrophils converting to firm arrest decreased from 36% to 24% with EtOH, and average rolling length and rolling velocity both increased with EtOH (24.8 to 33.7 μm and 2.5 to 3.4 m/sec, respectively). EtOH resulted in ~20% increase in L‐selectin and PSGL‐1 expression in quiescent neutrophils as measured by flow cytometry, and significantly inhibited the upregulation of CD11b and shedding of L‐selectin in fMLP‐activated neutrophils. Our results indicate that EtOH affects bond mechanics and surface molecules to change neutrophil response. This work was supported by NIH R01 HL56621.

Reactive oxidants regulate membrane repolarization and store-operated uptake of calcium by formyl peptide-activated human neutrophils

Although the rapid and considerable membrane depolarization response which accompanies activation of the phagocyte NADPH oxidase is due to transmembrane electron fluxes, little is known about the involvement of reactive oxidant species (ROS) in the subsequent repolarization response. In the current study, we have investigated the effects of superoxide dismutase (SOD), catalase, methionine, and the myeloperoxidase (MPO) inhibitors, sodium azide and 4-aminobenzoyl hydrazide (ABAH), as well as those of H 2O 2 and HOCl (both at 100 μM) on the alterations in membrane potential which accompany activation of human neutrophils with the chemoattractant, FMLP (1 μM), and on store-operated uptake of Ca 2+. The generation of ROS by FMLP-activated neutrophils was monitored according to the magnitude of oxygen consumption and autoiodination, while spectrofluorimetric procedures were used to measure alterations in membrane potential and influx of Ca 2+. Treatment of the cells with H 2O 2, and HOCl, significantly impeded membrane repolarization, while sodium azide, ABAH, methionine, and catalase exerted the opposite effects, potentiating both the rates and the magnitudes of membrane repolarization and store-operated uptake of Ca 2+. These observations demonstrate that NADPH oxidase regulates neutrophil membrane potential and Ca 2+ influx not only via its electrogenic activity, but also as a consequence of the generation of ROS.

Maggot excretions/secretions inhibit multiple neutrophil pro-inflammatory responses

There is renewed interest in the use of maggots ( Lucilia sericata) to aid in healing of chronic wounds. In such wounds neutrophils precipitate tissue damage rather than contribute to healing. As the molecules responsible for the beneficial actions of maggots are contained in their excretions/secretions (ES), we assessed the effects of ES on functional activities of human neutrophils. ES dose-dependently inhibited elastase release and H 2O 2 production by fMLP-activated neutrophils; maximal inhibition was seen with 5–50 μg of ES/ml. In contrast, ES did not affect phagocytosis and intracellular killing of Candida albicans by neutrophils. Furthermore, 0.5 μg of ES/ml already inhibited neutrophil migration towards fMLP. ES dose-dependently reduced the fMLP-stimulated expression of CD11b/CD18 by neutrophils, suggesting that ES modulate neutrophil adhesion to endothelial cells. ES did not affect the fMLP-induced rise in [Ca 2+] i in neutrophils, indicating that ES act down-stream of phospholipase C-mediated activation of protein kinase C. In agreement, ES inhibited PMA-activated neutrophil functional activities. ES induced a rise in intracellular cAMP concentration in neutrophils and pharmacological activators of cAMP-dependent mechanisms mimicked their inhibitory effects on neutrophils. The beneficial effects of maggots on chronic wounds may be explained in part by inhibition of multiple pro-inflammatory responses of activated neutrophils by ES.