Innate immune cells in cirrhosis.

Cirrhosis is a multisystemic disease wherein inflammatory responses originating from advanced liver disease and its sequelae affect distant compartments. Patients with cirrhosis are susceptible to bacterial infections, which may precipitate acute decompensation and acute-on-chronic liver failure, both of which are associated with high short-term mortality. Innate immune cells are an essential first line of defence against pathogens. Activation of liver macrophages (Kupffer cells) and resident mastocytes generate proinflammatory and vaso-permeating mediators that induce accumulation of neutrophils, lymphocytes, eosinophils and monocytes in the liver, and promote tissue damage. During cirrhosis progression, damage- and pathogen-associated molecular patterns activate immune cells and promote development of systemic inflammatory responses which may involve different tissues and compartments. The antibacterial function of circulating neutrophils and monocytes is gradually and severely impaired as cirrhosis worsens, contributing to disease progression. The mechanisms underlying impaired antimicrobial responses are complex and incompletely understood. This review focuses on the continuous and distinct perturbations arising in innate immune cells during cirrhosis, including their impact on disease progression, as well as reviewing potential therapeutic targets.

IL-33/ST2 pathway regulates neutrophil migration and predicts outcome in patients with severe alcoholic hepatitis.

BACKGROUND & AIMS: Severe alcoholic hepatitis (SAH) is associated with a high risk of infection. The IL-33/ST2 pathway is involved in sepsis control but data regarding its role in alcohol-related liver disease (ALD) are lacking. We aimed to characterize the role of IL-33/ST2 in the polymorphonuclear neutrophils (PMNs) of patients with ALD and SAH. METHODS: Serum and circulating neutrophils were collected from patients with SAH, alcoholic cirrhosis and healthy controls. We quantified IL-33/ST2 pathway activity and CXCR2 at baseline and after exposure to IL-33. We also determined the migration capacity of PMNs. RESULTS: The decoy receptor of IL-33 (soluble ST2 [sST2]) was increased in SAH vs. cirrhosis and controls, demonstrating the defect in this pathway during ALD. The sST2 level was associated with response to treatment, 2-month survival, infection-free survival and probability of infection in SAH. Endotoxemia was weakly correlated with sST2. GRK2, a negative regulator of CXCR2, was overexpressed in PMNs of patients with SAH and cirrhosis and was decreased by IL-33. CXCR2 levels on PMNs were lower in SAH vs. cirrhosis and controls. Treatment with IL-33 partially restored CXCR2 expression in SAH and cirrhosis. PMN migration upon IL-8 was lower in patients with SAH and cirrhosis vs. controls. Treatment with IL-33 partially restored migration in those with SAH and cirrhosis. Interestingly, the migration capacity of PMNs and the response to IL-33 were enhanced in responders to corticosteroids (Lille <0.45) compared to non-responders. CONCLUSION: The IL33/ST2 pathway is defective in SAH and predicts outcome. This defect is associated with decreased CXCR2 expression on the surface of PMNs and lower migration capacity, which can be corrected by IL-33, especially in patients responding to steroids. These results suggest that IL-33 has therapeutic potential for SAH and its infectious complications. LAY SUMMARY: The neutrophils of patients with severe alcoholic hepatitis are associated with a defect in the IL-33/ST2 pathway. This defect is associated with lower migration capacities in neutrophils and a higher probability of getting infected. Administration of IL-33 to the neutrophils at least partly restores this defect and may be effective at reducing the risk of infection in patients with severe alcoholic hepatitis.

Characterization of Blood Immune Cells in Patients With Decompensated Cirrhosis Including ACLF.

Background and Aims: Patients with cirrhosis and acute-on-chronic liver failure (ACLF) have immunosuppression, indicated by an increase in circulating immune-deficient monocytes. The aim of this study was to investigate simultaneously the major blood-immune cell subsets in these patients. Material and Methods: Blood taken from 67 patients with decompensated cirrhosis (including 35 critically ill with ACLF in the intensive care unit), and 12 healthy subjects, was assigned to either measurements of clinical blood counts and microarray (genomewide) analysis of RNA expression in whole-blood; microarray (genomewide) analysis of RNA expression in blood neutrophils; or assessment of neutrophil antimicrobial functions. Results: Several features were found in patients with ACLF and not in those without ACLF. Indeed, clinical blood count measurements showed that patients with ACLF were characterized by leukocytosis, neutrophilia, and lymphopenia. Using the CIBERSORT method to deconvolute the whole-blood RNA-expression data, revealed that the hallmark of ACLF was the association of neutrophilia with increased proportions of macrophages M0-like monocytes and decreased proportions of memory lymphocytes (of B-cell, CD4 T-cell lineages), CD8 T cells and natural killer cells. Microarray analysis of neutrophil RNA expression revealed that neutrophils from patients with ACLF had a unique phenotype including induction of glycolysis and granule genes, and downregulation of cell-migration and cell-cycle genes. Moreover, neutrophils from these patients had defective production of the antimicrobial superoxide anion. Conclusions: Genomic analysis revealed that, among patients with decompensated cirrhosis, those with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils (that had a unique phenotype) and macrophages M0-like monocytes, and depletion of several lymphocyte subsets (including memory lymphocytes). All these lymphocyte alterations, along with defective neutrophil superoxide anion production, may contribute to immunosuppression in ACLF, suggesting targets for future therapies.

Mast cell chymase protects against acute ischemic kidney injury by limiting neutrophil hyperactivation and recruitment.

Here we investigated the role of murine mast cell protease 4 (MCPT4), the functional counterpart of human mast cell chymase, in an experimental model of renal ischemia reperfusion injury, a major cause of acute kidney injury. MCPT4-deficient mice had worsened kidney function compared to wildtype mice. MCPT4 absence exacerbated pathologic neutrophil infiltration in the kidney and increased kidney myeloperoxidase expression, cell death and necrosis. In kidneys with ischemia reperfusion injury, when compared to wildtype mice, MCPT4-deficient mice showed increased surface expression of adhesion molecules necessary for leukocyte extravasation including neutrophil CD162 and endothelial cell CD54. In vitro, human chymase mediated the cleavage of neutrophil expressed CD162 and also CD54, P- and E-Selectin expressed on human glomerular endothelial cells. MCPT4 also dampened systemic neutrophil activation after renal ischemia reperfusion injury as neutrophils expressed more CD11b integrin and produced more reactive oxygen species in MCPT4-deficient mice. Accordingly, after renal injury, neutrophil migration to an inflammatory site distal from the kidney was increased in MCPT4-deficient versus wildtype mice. Thus, contrary to the described overall aggravating role of mast cells, one granule-released mediator, the MCPT4 chymase, exhibits a potent anti-inflammatory function in renal ischemia reperfusion injury by controlling neutrophil extravasation and activation thereby limiting associated damage.

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 gp91 phox 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 p47 phox (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.

Gadolinium chloride attenuates acetic acid-evoked colitis in mice by reducing neutrophil infiltration and pro-oxidative enzyme activity.

This study investigated the potential of gadolinium chloride (GdCl3), an inhibitor of kupffer cells on the myeloperoxidase (MPO) function, both in vivo on colon inflammation model and in vitro on thioglycollate-elicited peritoneal neutrophils. Colon inflammation was induced in mice (n = 7) by 4% acetic acid (AA) enema. GdCl3 (10 mg/kg) treatment was given 24 h before AA challenge. Clinical changes during the protocol were scored. Colons were segmented into distal and proximal parts for histological and biochemical assessment. Furthermore, myeloperoxidase (MPO) enzymes were extracted and analyzed by western blot. Short-term GdCl3 treatment inhibited dose-dependently superoxide anion (O2·-), alkaline phosphatase (ALP), and MPO release and promoted neutrophil apoptosis. In vivo, low-dose GdCl3 improved colitis scores and inhibited acute phagocyte recruitment and colon damage within the mucosa as revealed by the decrease in MPO, nitric oxide (NO), and malondialdehyde (MDA) levels. At the same time, GdCl3 restored catalase (CAT), superoxide dismutase (SOD) activities, and reduced glutathione (GSH) levels, thus reversing the MDA/GSH ratio in both distal and proximal colons. Compared to proximal, distal colon was more altered and displayed higher pathological manifestations. Lastly, the induction of apoptosis and regulation of the major nitrosative and oxidative functions of neutrophils by GdCl3 suggests its consideration as a beneficial tool in attenuating colon inflammation.

NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.

OBJECTIVE: Cirrhosis downregulates phagocyte oxidant production via their antibacterial superoxide-generating system, NADPH oxidase (NOX2) and increases patients' susceptibility to infection and mortality rate. To explore novel biochemical parameters that explain susceptibility to infections, we investigated the expression of NOX2 and partners in neutrophils of patients with severe alcoholic cirrhosis and have provided a novel approach to restore superoxide production capacity in patients' neutrophils and blood. DESIGN: Neutrophils were isolated from patients with decompensated alcoholic cirrhosis. NOX2 activity was assessed after stimulation of purified neutrophils or whole blood with the bacterial-derived peptide fMet-Leu-Phe. The expression of NOX2 and partners was studied by western blot analysis, flow cytometry and reverse transcription-PCR. RESULTS: The impaired superoxide production by patients' neutrophils was associated with a severe deficient expression of the NADPH oxidase catalytic core flavocytochrome-b558 (gp91 phox /NOX2 and p22 phox ), its cytosolic partner p47 phox but not p67 phox . NOX2 expression decreased rapidly by protein degradation involving elastase released during degranulation of healthy neutrophils stimulated with fMet-Leu-Phe, or highly present in patients' plasma. Interestingly, the deficient superoxide production was reversed by treatment of patients' neutrophils and whole blood with toll-like receptor 7/8 (TLR7/8) agonists. This treatment stimulated a rapid NOX2 transcription and translation through a process involving mammalian target of rapamycin (mTOR) whose expression was also deficient in patients' neutrophils. NOX2 expression was also increased by the TLR4 agonist lipopolysaccharide but with only a modest improvement of reactive oxygen species production. CONCLUSION: Impairment of neutrophil oxidants production in alcoholic cirrhosis is associated with NOX2 degradation and deficient mTOR-dependent translational machinery. The NOX2 depletion can be reversed via TRL7/8 activation and might be used to restore antimicrobial responses of immunocompromised patients.

Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis.

BACKGROUND & AIMS: Myeloperoxidase exocytosis and production of hydrogen peroxide via the neutrophil superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contribute to efficient elimination of bacteria. Cirrhosis impairs immune functions and increases susceptibility to bacterial infection. We recently showed that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a severe impairment of formylpeptide receptor (fPR)-mediated intracellular signaling and superoxide production. Here, we performed ex vivo studies with these patients' neutrophils to further investigate myeloperoxidase release, bactericidal capacity and signaling events following fPR stimulation by the formylpeptide formyl-met-leu-phe (fMLP). METHODS: Myeloperoxidase release was studied by measuring extracellular myeloperoxidase activity. Activation of signaling effectors was studied by Western blot and their respective contribution to myeloperoxidase release studied using pharmacological antagonists. RESULTS: fMLP-induced myeloperoxidase release was strongly impaired in patients' neutrophils whereas the intracellular myeloperoxidase stock was unaltered. The fMLP-induced phosphorylation of major signaling effectors, AKT, ERK1/2 and p38-MAP-Kinases, was also strongly deficient despite a similar expression of signaling effectors or fPR. However, based on effector inhibition in healthy neutrophils, AKT and p38-MAPK but not ERK1/2 upregulated fMLP-induced myeloperoxidase exocytosis. Interestingly, patients' neutrophils exhibited a defective bactericidal capacity that was reversed ex vivo by the TLR7/8 agonist CL097, through potentiation of the fMLP-induced AKT/p38-MAPK signaling axis and myeloperoxidase release. CONCLUSIONS: We provide first evidence that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a deficient AKT/p38-MAPK signaling, myeloperoxidase release and bactericidal activity, which can be reversed via TLR7/8 activation. These defects, together with the previously described severe deficient superoxide production, may increase cirrhotic patients' susceptibility to bacterial infections.

Inhibition of mammalian target of rapamycin aggravates the respiratory burst defect of neutrophils from decompensated patients with cirrhosis.

UNLABELLED: Cirrhosis is commonly accompanied by impaired defense functions of polymorphonuclear leucocytes (PMNs), increased patient susceptibility to infections, and hepatocellular carcinoma (HCC). PMN antimicrobial activity is dependent on a massive production of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) 2 (NADPH oxidase 2; NOX2), termed respiratory burst (RB). Rapamycin, an antagonist of mammalian target of rapamycin (mTOR), may be used in the treatment of HCC and in transplanted patients. However, the effect of mTOR inhibition on the PMN RB of patients with cirrhosis remains unexplored and was studied here using the bacterial peptide, formyl-Met-Leu-Phe (fMLP), as an RB inducer. fMLP-induced RB of PMN from patients with decompensated alcoholic cirrhosis was strongly impaired (30%-35% of control) as a result of intracellular signaling alterations. Blocking mTOR activation (phospho-S2448-mTOR) with rapamycin further aggravated the RB defect. Rapamycin also inhibited the RB of healthy PMNs, which was associated with impaired phosphorylation of the NOX2 component, p47phox (phox: phagocyte oxidase), on its mitogen-activated protein kinase (MAPK) site (S345) as well as a preferential inhibition of p38-MAPK relative to p44/42-MAPK. However, rapamycin did not alter the fMLP-induced membrane association of p47phox and p38-MAPK in patients' PMNs, but did prevent their phosphorylation at the membranes. The mTOR contribution to fMLP-induced RB, phosphorylation of p47phox and p38-MAPK was further confirmed by mTOR knockdown in HL-60 cells. Finally, rapamycin impaired PMN bactericidal activity, but not bacterial uptake. CONCLUSION: mTOR significantly up-regulates the PMN RB of patients with cirrhosis by p38-MAPK activation. Consequently, mTOR inhibition by rapamycin dramatically aggravates their PMN RB defect, which may increase patients' susceptibility to infection. Thus, concerns should be raised about the use of rapamycin in immuno-depressed patients.

Towards specific NADPH oxidase inhibition by small synthetic peptides.

Reactive oxygen species (ROS) production by the phagocyte NADPH oxidase is essential for host defenses against pathogens. ROS are very reactive with biological molecules such as lipids, proteins and DNA, potentially resulting in cell dysfunction and tissue insult. Excessive NADPH oxidase activation and ROS overproduction are believed to participate in disorders such as joint, lung, vascular and intestinal inflammation. NADPH oxidase is a complex enzyme composed of six proteins: gp91phox (renamed NOX2), p22phox, p47phox, p67phox, p40phox and Rac1/2. Inhibitors of this enzyme could be beneficial, by limiting ROS production and inappropriate inflammation. A few small non-peptide inhibitors of NADPH oxidase are currently used to inhibit ROS production, but they lack specificity as they inhibit NADPH oxidase homologues or other unrelated enzymes. Peptide inhibitors that target a specific sequence of NADPH oxidase components could be more specific than small molecules. Here we review peptide-based inhibitors, with particular focus on a molecule derived from gp91phox/NOX2 and p47phox, and discuss their possible use as specific phagocyte NADPH oxidase inhibitors.

Protein kinase B (AKT) mediates phospholipase D activation via ERK1/2 and promotes respiratory burst parameters in formylpeptide-stimulated neutrophil-like HL-60 cells.

Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (K(m) 1 µm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67(phox) and p47(phox), ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47(phox), p67(phox), and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase.

Peptide-based inhibitors of the phagocyte NADPH oxidase.

Phagocytes 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 over-production 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.

Phosphorylation of NADPH oxidase activator 1 (NOXA1) on serine 282 by MAP kinases and on serine 172 by protein kinase C and protein kinase A prevents NOX1 hyperactivation.

NADPH oxidase activator 1 (NOXA1) together with NADPH oxidase organizer 1 (NOXO1) are key regulatory subunits of the NADPH oxidase NOX1. NOX1 is expressed mainly in colon epithelial cells and could be involved in mucosal innate immunity by producing reactive oxygen species (ROS). Contrary to its phagocyte counterpart NOX2, the mechanisms involved in NOX1 activation and regulation remain unclear. Here we report that NOX1 activity is regulated through MAP kinase (MAPK), protein kinase C (PKC), and protein kinase A (PKA)-dependent phosphorylation of NOXA1. We identified Ser-282 as target of MAPK and Ser-172 as target of PKC and PKA in vitro and in a transfected human embryonic kidney 293 (HEK293) cell model using site directed mutagenesis and phosphopeptide mapping analysis. In HEK293 cells, phosphorylation of these sites occurred at a basal level and down-regulated constitutive NOX1 activity. Indeed, S172A and S282A single mutants of NOXA1 significantly up-regulated constitutive NOX1-derived ROS production, and S172A/S282A double mutant further increased it, as compared to wild-type NOXA1. Furthermore, phosphorylation of NOXA1 on Ser-282 and Ser-172 decreased its binding to NOX1 and Rac1. These results demonstrated a critical role of NOXA1 phosphorylation on Ser-282 and Ser-172 in preventing NOX1 hyperactivation through the decrease of NOXA1 interaction to NOX1 and Rac1.

CCR5 signaling through phospholipase D involves p44/42 MAP-kinases and promotes HIV-1 LTR-directed gene expression.

The chemokine receptor CCR5 plays an important role as an entry gate for the human immunodeficiency virus-1 (HIV-1) and for viral postentry events. Among signal transducers used by chemoattractant receptors, the phosphatidylcholine-specific phospholipase D (PLD) produces large amounts of second messengers in most cell types. However, the relevance of PLD isoforms to CCR5 signaling and HIV-1 infection process remains unexplored. We show here that CCR5 activation by MIP-1beta in HeLa-MAGI cells triggered a rapid and substantial PLD activity, as assessed by mass choline production. This activity required the activation of ERK1/2-MAP kinases and involved both PLD1 and PLD2. MIP-1beta also promoted the activation of an HIV-1 long terminal repeat (LTR) by the transactivator Tat in HeLa P4.2 cells through a process involving ERK1/2. Expression of wild-type and catalytically inactive PLDs dramatically boosted and inhibited the LTR activation, respectively, without altering Tat expression. Wild-type and inactive PLDs also respectively potentiated and inhibited HIV-1(BAL) replication in MAGI cells. Finally, in monocytic THP-1 cells, antisense oligonucleotides to both PLDs dramatically inhibited the HIV-1 replication. Thus, PLD is activated downstream of ERK1/2 upon CCR5 activation and plays a major role in promoting HIV-1 LTR transactivation and virus replication, which may open novel perspectives to anti-HIV-1 strategies.

A role of p44/42 mitogen-activated protein kinases in formyl-peptide receptor-mediated phospholipase D activity and oxidant production.

Phosphatidylcholine-specific phospholipase D (PLD) is a major cellular source of phosphatidic acid and choline, which regulate various physiopathological processes. PLD activation mediated by chemoattractants involves protein phosphorylation. This study provides pharmacological and biochemical evidence of a major role of p44/42 MAP kinases (ERK1/2) in PLD activation induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). ERK1/2 inhibition by the MEK1/2 antagonist U0126 in neutrophilic HL-60 cells or HEK 293T cells stably expressing fMLP receptors abolished fMLP-mediated PLD activity. Conversely, a constitutively activated MEK1 mutant expressed in HEK 293T cells potentiated fMLP-induced PLD activity. Expression of inactive PLD mutants showed that PLD2, but not PLD1, contributed to fMLP-mediated PLD activity. PLD2 co-immunoprecipitated with ERK1/2 and became phosphorylated on MAP kinase consensus sites in fMLP-stimulated cells. In cell-free systems, ERK2 gave rise to strong ATP-dependent PLD activity and directly phosphorylated PLD2 that generated two phosphopeptides only after tryptic digestion. Finally, pharmacological inhibition of ERK activation and the inhibition of PLD expression by antisense oligonucleotides in HL-60 cells suggest that the ERK/PLD2 pathway contributes to fMLP-mediated oxidant production. In conclusion, the fMLP-mediated PLD activity is regulated by ERK1/2, involving a predominant contribution of PLD2. The ERK/PLD2 coupling may provide potential pharmacological targets to control PLD-associated cellular dysfunctions.

Ginkgolide B stimulates signaling events in neutrophils and primes defense activities.

Ginkgolide B (GKB) is a bioactive component of the standardized extract from the leaves of the Ginkgo biloba tree (EGb 761), which is used in Chinese and in occidental medicine. GKB is known as a platelet-activating factor receptor antagonist. Here, we provide evidence that GKB per se (0.25-5 microM) stimulated tyrosine phosphorylation of proteins, phospholipase D activation, calcium transients, and activation of p38 but not p44/42 Map kinases in human polymorphonuclear leukocytes (PMN). These stimulatory effects remained relatively weak and primed PMN for subsequent stimulation of respiratory burst (RB) or directed locomotion by the chemoattractant fMet-Leu-Phe (fMLP) or complement-derived factor C5a. A similar RB priming was observed with rat exudate PMN after in vivo administration of EGb 761 (25 and 50 mg/kg) to rats before pleurisy induction. Thus, GKB primarily induces activation of intracellular signaling events and has the potential to prime cellular functions such as PMN defense activities.

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 (O2*-) 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). O2*- 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 O2*- 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 O2*- than those from controls. Nifedipine treatment considerably decreased O2*- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O2*- 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 O2*- 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.

Identification of the leukocyte cell-derived chemotaxin 2 as a direct target gene of beta-catenin in the liver.

To clarify molecular mechanisms underlying liver carcinogenesis induced by aberrant activation of Wnt pathway, we isolated the target genes of beta-catenin from mice exhibiting constitutive activated beta-catenin in the liver. Adenovirus-mediated expression of oncogenic beta-catenin was used to isolate early targets of beta-catenin in the liver. Suppression subtractive hybridization was used to identify the leukocyte cell-derived chemotaxin 2 (LECT2) gene as a direct target of beta-catenin. Northern blot and immunohistochemical analyses demonstrated that LECT2 expression is specifically induced in different mouse models that express activated beta-catenin in the liver. LECT2 expression was not activated in livers in which hepatocyte proliferation was induced by a beta-catenin-independent signal. We characterized by mutagenesis the LEF/TCF site, which is crucial for LECT2 activation by beta-catenin. We further characterized the chemotactic property of LECT2 for human neutrophils. Finally, we have shown an up-regulation of LECT2 in human liver tumors that expressed aberrant activation of beta-catenin signaling; these tumors constituted a subset of hepatocellular carcinomas (HCC) and most of the hepatoblastomas that were studied. In conclusion, our results show that LECT2, which encodes a protein with chemotactic properties for human neutrophils, is a direct target gene of Wnt/beta-catenin signaling in the liver. Since HCC develops mainly in patients with chronic hepatitis or cirrhosis induced by viral or inflammatory factors, understanding the role of LECT2 in liver carcinogenesis is of interest and may lead to new therapeutic perspectives.

Stroma cell-derived factor 1alpha mediates desensitization of human neutrophil respiratory burst in synovial fluid from rheumatoid arthritic patients.

Classical chemoattractants such as fMLP or the complement factor C5a use G protein (Gi)-coupled receptors to stimulate both chemotaxis and production of reactive oxygen species (respiratory burst, RB) by polymorphonuclear leukocytes (PMN). The chemokine stroma cell-derived factor 1alpha (SDF1alpha) and its Gi-coupled receptor, CXCR4, regulate leukocyte trafficking and recruitment to the synovial fluid of rheumatoid arthritic patients (RA-SF). However, the role of SDF1alpha in the RB is unknown and was studied in this work in vitro with healthy PMN in the absence and presence of RA-SF. In healthy PMN, SDF1alpha failed to stimulate the RB, even though the p38 mitogen-activated protein kinase was activated to a similar level as in fMLP-stimulated PMN. In contrast, the SDF1alpha-mediated calcium transients and activation of phosphatidylinositol 3-kinase/Akt were partially deficient, while p44/42 mitogen-activated protein kinases were not activated. SDF1alpha actually desensitized weakly the fMLP-mediated RB of healthy PMN. This cross-inhibitory effect was amplified in PMN treated with RA-SF, providing a protection against the exacerbation of RB induced by C5a or fMLP. This SDF1alpha beneficial effect, which was prevented by the CXCR4 antagonist AMD3100, was associated with impairment of C5a- and fMLP-mediated early signaling events. Thus, although SDF1alpha promotes leukocyte emigration into rheumatoid synovium, our data suggest it cross-desensitizes the production of oxidant by primed PMN, a property that may be beneficial in the context of arthritis.