Review of Defective NADPH Oxidase Activity and Myeloperoxidase Release in Neutrophils From Patients With Cirrhosis

Patients with decompensated cirrhosis are highly susceptible to develop bacterial infections and these can trigger multiorgan failure associated with high in-hospital mortality. Neutrophils from patients with decompensated cirrhosis exhibit marked alterations that may explain the susceptibility of these patients to develop bacterial infections. These neutrophil alterations include marked defects in intracellular signaling pathways involving serine/threonine kinases such as protein kinase B (AKT), p38-mitogen-activated protein kinase (MAPK), and the MAP kinases1/2; activation of the NADPH oxidase complex; myeloperoxidase (MPO) release; and bactericidal activity of neutrophils stimulated by the bacterial peptide formyl-Methionine-Leucine-Phenylalanine (fMLF). Impaired activity of the NADPH oxidase 2 (NOX2) complex is also related to reduced levels of expression of its major components through post-transcriptional mechanisms. In addition, the catalytic NOX2 component gp91phox is subject to degradation by elastase highly present in patients' plasma. A defect in the protein kinase B (AKT) and p38 MAPK-mediated signaling pathways may explain the decrease in phosphorylation of p47phox (an important component of the NADPH oxidase complex) and MPO release, in response to neutrophil stimulation by fMLF. Most of these alterations are reversible ex vivo with TLR7/8 agonists (CL097, R848), raising the possibility that these agonists might be used in the future to restore neutrophil antibacterial functions in patients with cirrhosis

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O(2)(•-)) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O(2)(•- )production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O(2)(•- )production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O(2)(•- )than those from controls. Nifedipine treatment considerably decreased O(2)(•- )production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O(2)(•- )production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O(2)(•- )overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress

Peptide-Based Inhibitors Of The Phagocyte Nadph Oxidase

International audiencePhagocytes such as neutrophils, monocytes and macrophages play an essential role in host defenses against pathogens. To kill these pathogens, phagocytes produce and release large quantities of antimicrobial molecules such as reactive oxygen species (ROS), microbicidal peptides, and proteases. The enzyme responsible for ROS generation is called NADPH oxidase, or respiratory burst oxidase, and is composed of six proteins: gp91phox, p22phox, p47phox, p67phox, p40phox and Rac1/2. The vital importance of this enzyme in host defenses is illustrated by a genetic disorder called chronic granulomatous disease (CGD), in which the phagocyte NADPH oxidase is dysfunctional, leading to life-threatening recurrent bacterial and fungal infections. However, excessive NADPH oxidase activation and ROS overproduction can damage surrounding tissues and participate in exaggerated inflammatory processes. As ROS production is believed to be involved in several inflammatory diseases, specific phagocyte NADPH oxidase inhibitors might have therapeutic value. In this commentary, we summarize the structure and activation of the phagocyte NADPH oxidase, and describe pharmacological inhibitors of this enzyme, with particular emphasis on peptide-based inhibitors derived from gp91phox, p22phox and p47phox

Innate immune cells in cirrhosis

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Immunochemical identification and translocation of protein kinase C zeta in human neutrophils

AbstractWestern blots of human polymorphonuclear leukocyte (PMN) extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. Two bands corresponding to PKC type ξ with apparent molecular masses of 81 kDa and 76 kDa were identified in the cytosolic fraction of resting cells, in addition to PKC types α and β. PKCξ was apparently abundant, like PKCβ, whereas PKCδ, -ε, and -γ were not detectable. Following short stimulation (5 min) of PMN with phorbol-12-myristate-13-acetate (1 μ/ml), physical translocation of PKCξ from the cytosol to the plasma membrane fraction occurred, although this isoform does not bind phorbol esters. These data show that, in addition to the two calcium-dependent isoenzymes α and β, human PMN express a calcium-independent isoenzyme ξ which translocates in stimulated cells, suggesting a role in the regulation of antibacterial activities

Differential nucleocytoplasmic shuttling of beta-arrestins. Characterization of a leucine-rich nuclear export signal in beta-arrestin2

International audiencebeta-arrestins (betaarrs) are two highly homologous proteins that uncouple G protein-coupled receptors from their cognate G proteins, serve as adaptor molecules linking G protein-coupled receptors to clathrin-coat components (AP-2 complex and clathrin), and act as scaffolding proteins for ERK1/2 and JNK3 cascades. A striking difference between the two betaarrs (betaarr1 and betaarr2) is that betaarr1 is evenly distributed throughout the cell, whereas betaarr2 shows an apparent cytoplasmic localization at steady state. Here, we investigate the molecular determinants underlying this differential distribution. betaarr2 is constitutively excluded from the nucleus by a leptomycin B-sensitive pathway because of the presence of a classical leucine-rich nuclear export signal in its C terminus (L395/L397) that is absent in betaarr1. In addition, using a nuclear import assay in yeast we showed that betaarr2 is actively imported into the nucleus, suggesting that betaarr2 undergoes constitutive nucleocytoplasmic shuttling. In cells expressing betaarr2, JNK3 is mostly cytosolic. A point mutation of the nuclear export signal (L395A) in betaarr2, which was sufficient to redistribute betaarr2 from the cytosol to the nucleus, also caused the nuclear relocalization of JNK3. These data indicate that the nucleocytoplasmic shuttling of betaarr2 controls the subcellular distribution of JNK3

Effects of nifedipine (NIF) on phorbol myristate acetate (PMA)-induced protein phosphorylation in monocytes as detected with the fluorescent probe ProQ-Diamond

<p><b>Copyright information:</b></p><p>Taken from "Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis"</p><p>Arthritis Research & Therapy 2004;7(1):R93-R100.</p><p>Published online 16 Nov 2004</p><p>PMCID:PMC1064885.</p><p>Copyright © 2004 Allanore et al.; licensee BioMed Central Ltd.</p> Representative gels of phosphorylated protein from homogenates of monocytes from healthy donors, treated with the indicated concentrations of nifedipine (a, b) or calphostin (CAL) (b) for 30 min and then stimulated with 100 nM PMA for 10 min. Right panels show a representative densitometric analysis of 10 protein bands (molecular masses from 15 to 60 kDa) that were strongly phosphorylated in the presence of PMA. Effects of nifedipine on the cytosolic fraction of control monocytes treated with the indicated concentrations of nifedipine for 30 min before stimulation of protein kinase C with a mixture of calcium and diacylglycerol. Results are the net phosphorylation induced by PMA, expressed relative to the PMA lane (taken as 100%)