Activation Of Phagocyte NADPH Oxidase Research Articles

How Important are S100A8/S100A9 Calcium Binding Proteins for the Activation of Phagocyte NADPH Oxidase, Nox2

S100A8 and S100A9 are two soluble calcium-binding proteins highly expressed in myeloid cells, mainly neutrophils (45% of cytosolic proteins) or monocytes (1-5%) and also early differentiated macrophages. In neutrophils, they are believed to be expressed as a 1/1 non covalent heterodimer; the process of dimer and mainly tetramer formation is calcium dependent. The S100A8/S100A9 calcium loaded complex binds arachidonic acid and shuttles between cytosol and plasma membrane upon neutrophil stimulation. Neutrophils display, upon stimulation, a respiratory burst in which the cells catalyze NADPH oxidase activity through a redox membrane hemoprotein, cytochrome b558, which is constituted of 2 subunits: gp91-phox, the redox core and p22- phox the stabilizing partner. In neutrophils, this activity is transitory: to be active, regulatory cytosolic factors, p67-phox, p47-phox, p40-phox and Rac1/2 assemble with membrane cytochrome b558. Both S100A8 and S100A9 were recently introduced as partners for NADPH oxidase activation and associate with the cytosolic activating factors especially p67-phox and Rac1/2. Moreover, S100A8/S100A9 potentiates NADPH oxidase activity. This was observed ex vivo after co-transfection of genes encoding both S100A8 and S100A9 in B lymphocytes that express all the components of the phagocyte oxidase, but display a very low NADPH oxidase activity (in these cells, S100A8 and S100A9 are not present endogenously). In the biological function of S100A8/S100A9, S100A8 is a strategic protein that needs to be active in vivo as in vitro, its specific partner S100A9. New data introduce S100A8 and S100A9 as positive effectors in allosteric regulation of phagocyte NADPH oxidase activity.

Enzymatic measurement of phosphatidic acid in cultured cells

In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 microM, and the detection limit was 5 microM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes.

Losartan Metabolite EXP3179 Blocks NADPH Oxidase-Mediated Superoxide Production by Inhibiting Protein Kinase C

Oxidative stress plays a critical role in the pathogenesis of hypertension. The NADPH oxidase constitutes a major source of superoxide anion in phagocytic cells, and its activation is associated with matrix metalloproteinase (MMP)-9 secretion by these cells. We investigated the effects of the angiotensin II type 1 receptor antagonist losartan and its metabolites (EXP3174 and EXP3179) on NADPH oxidase activity and MMP-9 secretion in human phagocytic cells. EXP3179, but not losartan and EXP3174, dose-dependently inhibited (P<0.05) phorbol myristate acetate and insulin-stimulated NADPH oxidase activity. EXP3179 also inhibited phorbol myristate acetate-induced NADPH oxidase in endothelial cells. In addition, EXP3179 inhibited (P<0.05) both phorbol myristate acetate-stimulated p47phox translocation from cytosol to membranes and protein kinase C activity. Affinity experiments and enzymatic assays confirmed that EXP3179 inhibited several protein kinase C isoforms. EXP3179 also inhibited (P<0.05) phorbol myristate acetate-stimulated MMP-9 secretion. In a study performed in 153 hypertensive patients, phagocytic NADPH oxidase activity was lower (P<0.05) in losartan-treated compared with untreated patients and in patients treated with other angiotensin II type 1 receptor antagonists or with angiotensin-converting enzyme inhibitors. Plasma levels of MMP-9 were lower (P<0.05) in losartan-treated hypertensives compared with the other group of patients. Thus, EXP3179 acts as a blocker of the NADPH oxidase in phagocytic cells by a potential mechanism that targets the protein kinase C signaling pathway. This effect can be involved in reduced MMP-9 secretion by these cells. It is proposed that the EXP3179 metabolite may confer to losartan the specific capacity to reduce oxidative stress mediated by phagocytic cells in hypertensive patients.

Importance of the non-selective cation channel TRPV1 for microglial reactive oxygen species generation

Activated microglial cells generate reactive oxygen species (ROS), which have detrimental effects in neuroinflammatory and neurodegenerative diseases. In the present study, we have identified a novel mechanism involved in microglial NADPH oxidase-mediated ROS production. In PMA-stimulated microglia, ROS production was substantially reduced upon inhibition of the non-selective cation channel TRPV1 with La 3+, ruthenium red, capsazepine and 5-iodo-resinferatoxin. Furthermore, sustained membrane depolarization, a hallmark of NADPH oxidase activity in phagocytes, was found to induce non-selective cation/TRPV1 channel activity in microglia. Together, our data suggest that TRPV1 channels are involved in regulating NADPH oxidase-mediated ROS generation in microglia.

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH oxidase activity caused by defects in the gene encoding gp91phox. Here we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91phox expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared to lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen, and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared to fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.

Identification of Phosphorylation Sites that Activate Voltage Gated Proton Channels in Leukocytes

One of the best established functions of proton channels is facilitating NADPH oxidase activity in phagocytes. NADPH oxidase moves electrons across the membrane, leaving protons in the cytoplasm. Proton channels extrude most of this acid, simultaneously compensating for the electrogenic action of the oxidase and preventing cytoplasmic acidification. Agonists that activate NADPH oxidase also produce an “enhanced gating mode” in proton channels, characterized by faster activation, increased conductance, and a negative shift of the gH-V relationship, all of which increase the likelihood of proton channel opening. The enhanced gating mode is the result of PKC activity (Morgan et al, 2007, J. Physiol. 579:327-344). We assessed proton channel responses in a murine B cell line, LK35.2. Nontransfected LK35.2 cells have no detectable proton current; cells transfected with the human proton channel gene, HVCN1, have proton currents that respond to PMA and the PKC inhibitor, GF109203X (GFX), when studied in perforated-patch configuration. The response of proton currents in these cells to PMA or GFX was qualitatively like that of native leukocytes, but smaller in amplitude. In contrast, there was no detectable PMA response of human or murine proton channels expressed in HEK-293 or COS-7 cells (Musset et al, 2008, J. Physiol. 586:2477-2486). Here we mutated two putative PKC phosphorylation sites on the human proton channel. When the mutant channels were expressed in LK35.2 cells, the response to PMA or GFX was ablated. These studies indicate that PKC phosphorylates the proton channel directly.Supported by the NIH-HLBI (HL61437), Philip Morris, and the Schmidtmann Foundation.

Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox

Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47(phox), p67(phox), p40(phox), and Rac2) translocate to the membranes, where they associate with flavocytochrome b(558), which is composed of gp91(phox)/NOX2 and p22(phox), to form the active system. During neutrophil stimulation, p47(phox), p67(phox), p40(phox), and p22(phox) are phosphorylated; however, the phosphorylation of gp91(phox)/NOX2 and its potential role have not been defined. In this study, we show that gp91(phox) is phosphorylated in stimulated neutrophils. The gp91(phox) phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91(phox), which confirms that this phosphoprotein is gp91(phox). The protein kinase C inhibitor GF109203X inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of gp91(phox), and protein kinase C (PKC) phosphorylated the recombinant gp91(phox)- cytosolic carboxy-terminal flavoprotein domain. Two-dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91(phox)-cytosolic tail on the same peptides that were phosphorylated on gp91(phox) in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91(phox) flavoprotein cytosolic domain and its binding to Rac2, p67(phox), and p47(phox). These results demonstrate that gp91(phox) is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91(phox)/NOX2 is a novel mechanism of NADPH oxidase regulation.

Mechanisms of inflammatory neurodegeneration: iNOS and NADPH oxidase

Inflammation contributes to a wide variety of brain pathologies, apparently via glia killing neurons. A number of mechanisms by which inflammatory-activated microglia and astrocytes kill neurons have been identified in culture. These include iNOS (inducible nitric oxide synthase), which is expressed in glia only during inflammation, and PHOX (phagocytic NADPH oxidase) found in microglia and acutely activated by inflammation. High levels of iNOS expression in glia cause (i) NO (nitric oxide) inhibition of neuronal respiration, resulting in neuronal depolarization and glutamate release, followed by excitotoxicity, and (ii) glutamate release from astrocytes via calcium-dependent vesicular release. Hypoxia strongly synergizes with iNOS expression to induce neuronal death via mechanism (i), because NO inhibits cytochrome oxidase in competition with oxygen. Activation of PHOX (by cytokines, beta-amyloid, prion protein, ATP or arachidonate) causes microglial proliferation and inflammatory activation; thus PHOX is a key regulator of inflammation. Activation of PHOX alone causes no death, but when combined with expressed iNOS results in extensive neuronal death via peroxynitrite production.

A novel CYBA variant, the –675A/T polymorphism, is associated with essential hypertension

Oxidative stress is implicated in hypertension and the NADPH oxidase systems constitute the main source of superoxide in vascular wall. We searched for new polymorphisms within the CYBA promoter, the human gene that encodes the p22phox protein, and studied their potential association with essential hypertension. A case-control study in a random sample of the general population. CYBA polymorphisms were determined by restriction fragment length polymorphism and allelic discrimination. NADPH oxidase activity was quantified in phagocytic cells by chemiluminescence. We identified three novel polymorphisms, at positions -852, -675 and -536 from the ATG codon. Only the -675(A/T) polymorphism associated with essential hypertension. The prevalence of the TT genotype and the T allele frequency were significantly higher (P < 0.05) in hypertensives than in normotensives. Furthermore, TT hypertensives exhibited higher (P < 0.05) systolic blood pressure values than TA/AA hypertensives. Increased phagocytic NADPH oxidase activity was observed in TT subjects compared to TA and AA individuals (P < 0.05). Enhanced carotid intima-media thickness, a surrogate marker of atherosclerosis, was found in TT subjects compared to TA and AA individuals (P < 0.05). Finally, mutagenesis experiments demonstrated a functional role of this polymorphism on the CYBA promoter activity. The -675 (A/T) CYBA polymorphism may be a novel genetic marker associated with essential hypertension. Furthermore, TT subjects exhibit features of NADPH oxidase-mediated oxidative stress and asymptomatic atherosclerosis.

Role of NF-κB in transcriptional regulation of the phagocyte NADPH oxidase by tumor necrosis factor-α

Macrophages play an important role in the pathogenesis of chronic inflammatory disease. Activation of these phagocytes induces the production of proinflammatory cytokines, such as IL-1 and TNF-alpha and the generation of reactive oxygen species (ROS), such as superoxide anion (O2*-). Recently, we found that TNF-alpha treatment of human monocytic cells (MonoMac1) and isolated human monocytes resulted in up-regulation of the NADPH oxidase gene, neutrophil cytosolic factor 2 (NCF2). These results suggested that TNF-alpha, produced by activated macrophages, could serve as an autocrine/paracrine regulator of the oxidase, resulting in increased and/or prolonged production of O2*-. To gain a better understanding of the mechanisms involved in NADPH oxidase regulation by TNF-alpha, we evaluated transcriptional regulation of oxidase genes in MonoMac1 cells and human monocytes. We show that TNF-alpha-treated cells have increased levels of mRNA and up-regulated expression of NADPH oxidase subunits p47(phox), p67(phox), and gp91(phox), as well as increased oxidase activity. Pharmacological inhibitors of NF-kappaB activation blocked TNF-alpha-induced up-regulation of NCF1, NCF2, and CYBB message, which correlated with a reduction in expression of the corresponding oxidase proteins and decreased O2*- production. These data demonstrate that the increase in and/or maintenance of O2*- production in TNF-alpha-treated MonoMac1 cells and monocytes are a result, in part, of transcriptional up-regulation of three essential NADPH oxidase genes via the NF-kappaB pathway. This novel finding supports a model, whereby TNF-alpha-dependent activation of NF-kappaB up-regulates phagocyte NADPH oxidase activity, leading to enhanced ROS production and further NF-kappaB activation, potentially contributing to sustained oxidant production in chronic inflammation.

Regulation of phagocyte NADPH oxidase activity: identification of two cytochromeb558activation states

Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O2(-) x s(-1) x mol heme b(-1)), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (approximately 50 mol O2(-) x s(-1) x mol heme b(-1)). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states.

Protein kinase C-α and -δ are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts

The regulation of the activation of non phagocytic NADPH oxidase is poorly understood. Previously we demonstrated that in fibroblasts the exposure to WKYMVm induced p47 phox phosphorylation and translocation and that these effects were mediated by ERKs activation. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in polymorphonucleate cells stimulated via FPRL1 receptor, but its involvement in fibroblasts was not demonstrated. Therefore, we investigated in IMR90 cells exposed to WKYMVm the role of PKC isoenzymes in the activation of NADPH oxidase-like enzyme. Preincubation with general pharmacological inhibitors of PKC, before stimulation with WKYMVm, prevented the ERKs activation, p47 phox phosphorylation and translocation. The analysis of cellular partitioning of PKC isoenzymes demonstrated that PKCα and PKCδ translocated from the cytosolic to the membrane fraction upon stimulation with WKYMVm. Preincubation with Gö6976 or with rottlerin prevented the phosphorylation and translocation of NADPH oxidase regulatory subunit.

A Transverse Tubule NADPH Oxidase Activity Stimulates Calcium Release from Isolated Triads via Ryanodine Receptor Type 1 S -Glutathionylation

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.

Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent α-helix of p22phox

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151-160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161-164), adopting an a-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.

Suramin inhibits NADPH oxidase activity in cerebral arteries after subarachnoid hemorrhage

Excessive production of superoxide by NADPH oxidase in the cerebral artery wall after subarachnoid hemorrhage (SAH) is associated with endothelial dysfunction. We recently found that sulphonated aryls, such as suramin, display selectivity for inhibition of vascular vs phagocytic NADPH oxidase activity. Hence the aim of this study was to test whether treatment with suramin, either before or after SAH, could prevent changes in cerebral artery superoxide production and endothelium-dependent dilatation. Autologous blood (or saline) was injected into the cisterna magna of rats 2 days before measuring basilar artery superoxide production in vitro or dilator responses in vivo using a cranial window preparation. Rats were treated 5 days before, or at the time of SAH induction, with either saline or suramin (43 mg/kg/d sc). NADPH-induced superoxide production of isolated basilar arteries was increased by 70% after SAH (13122±2006 vs 22481±3960 counts/s/mg, P<0.05). Pre- or post-treatment with suramin normalized superoxide production by the basilar artery after SAH (12138±2079 and 10450±2510, both P>0.05 vs control). Similarly, SAH attenuated dilator responses to acetylcholine by up to 80%, whereas pre- or post-treatment with suramin fully normalized those responses. These findings are consistent with impaired endothelium-dependent cerebral vasodilatation after SAH being due to inactivation of nitric oxide by NADPH oxidase-derived superoxide. Furthermore, suramin may represent a novel therapeutic for vascular diseases involving oxidative stress, such as SAH.

Phagocytic NADPH Oxidase Overactivity Underlies Oxidative Stress in Metabolic Syndrome

Oxidative stress plays a critical role in the pathogenesis of atherosclerosis in patients with metabolic syndrome. This study aimed to investigate whether a relationship exists between phagocytic NADPH oxidase activity and oxidative stress and atherosclerosis in metabolic syndrome patients. The study was performed in 56 metabolic syndrome patients (metabolic syndrome group), 99 patients with one or two cardiovascular risk factors (cardiovascular risk factor group), and 28 healthy subjects (control group). NADPH oxidase expression and activity was augmented (P < 0.05) in metabolic syndrome compared with cardiovascular risk factor and control groups. Insulin was enhanced (P < 0.05) in metabolic syndrome patients compared with cardiovascular risk factor and control groups and correlated with NADPH oxidase activity in the overall population. Insulin stimulated NADPH oxidase activity; this effect was abolished by a specific protein kinase C inhibitor. Oxidized LDL and nitrotyrosine levels and carotid intima-media thickness were increased (P < 0.05) in the metabolic syndrome group compared with cardiovascular risk factor and control groups and correlated with NADPH oxidase activity in the overall population. These findings suggest that phagocytic NADPH oxidase overactivity is involved in oxidative stress and atherosclerosis in metabolic syndrome patients. Our findings also suggest that hyperinsulinemia may contribute to oxidative stress in metabolic syndrome patients through activation of NADPH oxidase.

The arachidonic acid‐binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phoxand Rac‐2

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

P47(phox) PX domain of NADPH oxidase targets cell membrane via moesin-mediated association with the actin cytoskeleton.

Activation of phagocytic NADPH oxidase requires association of its cytosolic subunits with the membrane-bound flavocytochrome. Extensive phosphorylation of the p47(phox) subunit of NADPH oxidase marks the initiation of this activation process. The p47(phox) subunit then translocates to the plasma membrane, bringing the p67(phox) subunit to cytochrome b558 to form the active NADPH oxidase complex. However, the detailed mechanism for targeting the p47(phox) subunit to the cell membrane during activation still remains unclear. Here, we show that the p47(phox) PX domain is responsible for translocating the p47(phox) subunit to the plasma membrane for subsequent activation of NADPH oxidase. We also demonstrate that translocation of the p47(phox) PX domain to the plasma membrane is not due to interactions with phospholipids but rather to association with the actin cytoskeleton. This association is mediated by direct interaction between the p47(phox) PX domain and moesin.

Cathelicidins, multifunctional peptides of the innate immunity.

Cathelicidins comprise a family of mammalian proteins containing a C-terminal cationic antimicrobial domain that becomes active after being freed from the N-terminal cathelin portion of the holoprotein. Many other members of this family have been identified since the first cathelicidin sequences were reported 10 years ago. The mature peptides generally show a wide spectrum of antimicrobial activity and, more recently, some of them have also been found to exert other biological activities. The human cathelicidin peptide LL-37 is chemotactic for neutrophils, monocytes, mast cells, and T cells; induces degranulation of mast cells; alters transcriptional responses in macrophages; stimulates wound vascularization and re-epithelialization of healing skin. The porcine PR-39 has also been involved in a variety of processes, including promotion of wound repair, induction of angiogenesis, neutrophils chemotaxis, and inhibition of the phagocyte NADPH oxidase activity, whereas the bovine BMAP-28 induces apoptosis in transformed cell lines and activated lymphocytes and may thus help with clearance of unwanted cells at inflammation sites. These multiple actions provide evidence for active participation of cathelicidin peptides in the regulation of the antimicrobial host defenses.

Long-term high-level reconstitution of NADPH oxidase activity in murine X-linked chronic granulomatous disease using a bicistronic vector expressing gp91phox and a Delta LNGFR cell surface marker.

A murine model of X-linked chronic granulomatous disease (X-CGD), an inherited immune deficiency with absent phagocyte NADPH oxidase activity caused by defects in the gp91(phox) gene, was used to evaluate a bicistronic retroviral vector in which expression of human gp91(phox) and a linked gene for Delta LNGFR, a truncated form of human low-affinity nerve growth factor receptor, are under the control of a spleen focus-forming virus long-terminal repeat (LTR). Four independent cohorts of 11-Gy irradiated X-CGD mice (total, 22 mice) were transplanted with or without preselection of transduced X-CGD bone marrow (BM). Transplanted mice had high-level correction of neutrophil gp91(phox) expression and reconstitution of NADPH oxidase activity. Expression lasted for at least 14 months in primary transplants, and persisted in secondary and tertiary transplants. Both gp91(phox) and Delta LNGFR were detected on circulating granulocytes, lymphocytes, lymphoid, and (for Delta LNGFR) red blood cells. Mice receiving transduced bone marrow [BM] preselected ex vivo for Delta LNGFR expression had high-level (= 80%) reconstitution with transduced cells, with an improved fraction of oxidase-corrected neutrophils posttransplant. Analysis of secondary and tertiary CFU-S showed that silencing of individual provirus integrants can occur even after preselection for Delta LNGFR prior to transplantation, and that persistent provirus expression was associated with multiple integration sites in most cases. No obvious adverse consequences of transgenic protein expression were observed.