Characterization of neutrophil NADPH oxidase factors p47-phox and p67-phox from recombinant baculoviruses

Superoxide production by phagocytic blood cells involves assembly of an active NADPH oxidase complex from components found both in membrane and cytosolic locations in resting cells. We recently cloned cDNAs encoding two cytosolic components (p47-phox and p67-phox) of the oxidase that are deficient in distinct forms of autosomal recessive chronic granulomatous disease. The precise roles of p47-phox and p67-phox were explored further using purified factors produced in large quantities using recombinant baculoviruses to infect cultured Sf9 insect cells. Neither p47-phox nor p67-phox are thought to represent the flavoprotein components of the oxidase, since neither of the purified recombinant factors contained or bound FAD. Recombinant p47-phox and p67-phox are capable of restoring the deficient cytosol from chronic granulomatous disease patient neutrophils to nearly normal levels in a cell-free reconstitution system. Both p47-phox and p67-phox, used together in the absence of neutrophil cytosol, are incapable of supporting cell free production of superoxide, confirming the involvement of other soluble factor(s) in the assembly of an active oxidase in vitro.     

Complementation of NADPH oxidase in p67-phox-deficient CGD patients p67-phox/p40-phox interaction.

Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.     

Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane.

Human neutrophil respiratory burst oxidase (NADPH-oxidase) activity can be reconstituted in a cell-free system consisting of plasma membrane, cytosol and an anionic amphiphile [e.g., sodium dodecyl sulfate (SDS) or arachidonate]. Herein, we report reconstitution of oxidase activity using isolated neutrophil plasma membrane together with purified recombinant p47-phox and p67-phox which had been produced using a baculovirus expression system. Activity required an anionic amphiphile (SDS or arachidonate) and was potentiated by diacylglycerol and GTP gamma S. Serial washes of the plasma membrane failed to affect its ability to reconstitute activity, indicating that a dissociable membrane component was not present. The Km for NADPH, 43 microM, was the same as that determined using cytosol in place of recombinant factors. The EC50 values for p47-phox and p67-phox under optimal activation conditions were 220 nM and 80 nM, respectively, indicating a relatively high affinity of these components in an activation complex. Since neither cytosolic component contains a nucleotide binding consensus sequence, these data indicate that the NADPH binding component of the oxidase resides in the plasma membrane.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

Abstract

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rap1a) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2 is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2 is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2 is transported to the plasma membrane to be released and to ensure host defense against infection.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2*-) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rapla) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2*-. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2*- is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2*- is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2*- is transported to the plasma membrane to be released and to ensure host defense against infection.     

New insights into the membrane topology of the phagocyte NADPH oxidase: characterization of an anti-gp91-phox conformational monoclonal antibody

Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b(558) conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b(558) conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b(558). Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.     

P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.     

Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.

We reported previously that diacylglycerol (diC8) and GTP gamma S synergize with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to produce high rates of superoxide generation in a cell-free system consisting of neutrophil plasma membrane plus cytosol [Burnham, D. N., Uhlinger, D. J., & Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559]. Here we investigate the effects of these activating factors on the plasma membrane association in an in vitro translated radiolabeled recombinant p47-phox protein. Apparent translocation, assayed by cosedimentation with plasma membranes, required the presence of excess cytosol and an anionic amphiphile, was enhanced by both GTP gamma S and diC8, and was inhibited by high salt, correlating qualitatively with activation; up to 70% cosedimentation was observed with the combination of activators (compared with less than 20% in their absence). Similar results were obtained using heat-inactivated cytosol, wherein another oxidase component, p67-phox, has been inactivated. Unexpectedly, from 50 to 80% of the apparent translocation occurred in the absence of membranes, indicating that protein aggregation accounted for a significant part of the observed translocation. Nevertheless, the percent translocation was increased in all cases by the presence of membranes, indicating some degree of protein-membrane interaction. While a control in vitro translated protein failed to translocate, cosedimentation of p47-phox occurred equally well when red blood cell or neutrophil plasma membranes lacking cytochrome b558 were used. Also, the peptide RGVHFIF, which is contained within the C-terminus of the large subunit of cytochrome b558, failed to inhibit translocation/aggregation of p47-phox, despite its ability to inhibit cell-free activation of the oxidase. The data are consistent with the following: (a) SDS, diC8, and GTP gamma S all act on cytosolic components to alter protein-protein and/or protein-membrane associations, and these changes are necessary (but not sufficient) for activation; (b) these altered associations are likely to function by increasing the local concentration of p47-phox and other components at the plasma membrane; (c) a high background of nonspecific associations in the cell-free activation system is likely to obscure any specific, functionally relevant associations (e.g., with cytochrome b558); and (d) the mechanism of translocation in the cell-free system differs from that seen in intact neutrophils.     

Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components.

Activated human polymorphonuclear neutrophils (PMNs) convert molecular oxygen into superoxide anion, a process known as the respiratory burst, through the activity of a latent multicomponent NADPH-dependent oxidase. Components of this respiratory burst oxidase include the membrane-bound cytochrome b558 and the cytosolic factors p47-phox and p67-phox. We initiated these studies based on three observations: 1) that stimulation of PMN oxidase activity is associated with translocation of the cytosolic oxidase components to the plasma membrane; 2) that p47-phox is phosphorylated during PMN activation and that there is a sequential relationship between phosphorylation of p47-phox in the cytosol and appearance of the phosphoprotein in the membran; and 3) that the predicted amino acid sequences of p47-phox and of p67-phox contain regions of homology to the SH3 or A domain of the src family of tyrosine kinases, a region found in a variety of proteins which interact with the cytoskeleton or the subplasmalemmal cytoskeleton. Thus the purpose of our studies was to examine the role of protein kinase C (PKC)-dependent phosphorylation in the stimulus-induced association of p47-phox and p67-phox with the plasma membrane and the cytoskeleton. Using the PKC activator phorbol myristate acetate (PMA) as the agonist, we found that activation of the respiratory burst oxidase was associated with translocation of cytosolic p47-phox and p67-phox to the plasma membrane as well as redistribution of p47-phox to the Triton-insoluble cytoskeleton. Furthermore, the PKC inhibitor staurosporine inhibited phosphorylation of p47-phox, interrupted the redistribution of cytosolic oxidase factors, and blocked PMA-induced generation of superoxide anion. Taken together these results indicate that PKC-dependent phosphorylation of p47-phox correlates with association of p47-phox with the cytoskeleton and with translocation of p47-phox and p67-phox to the plasma membrane, with the ensuing assembly of an active superoxide-generating NADPH-dependent oxidase.     

Phosphorylation of p67phox in the neutrophil occurs in the cytosol and is independent of p47phox.

p67phox and p47phox are phosphorylated in the course of stimulation of the NADPH oxidase in neutrophils. Isolated neutrophil cytosol can phosphorylate both of these proteins in vitro. Phosphoamino acid analysis showed that isolated membranes can tyrosine-phosphorylate p67phox in vitro. Further experiments with anti-phosphotyrosine antibodies did not support a role for tyrosine phosphorylation of p67phox in the cell. A phosphopeptide analysis showed that the phosphorylation of p67phox is unchanged in the absence of p47phox. These results further characterise the phosphorylation of p67phox and provide evidence that this is a cytosolic event independent of interaction with p47phox and the membrane.     

Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins.

The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.     

Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells.

The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components to the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca(2+)-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation to the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants.     

Fatty-acid-induced activation of NADPH oxidase in plasma membranes of human neutrophils depends on neutrophil cytosol and is potentiated by stable guanine nucleotides

Both cis and trans unsaturated fatty acids and sodium dodecyl sulfate activated NADPH oxidase in plasma membranes of human neutrophils in the presence of neutrophil cytosol. In contrast, 5,8,11,14-icosatetraynoic acid, saturated fatty acids, esters, peroxides and 4 beta-phorbol 12-myristate 13-acetate, a potent activator of protein kinase C, were inactive. 5,8,11,14-icosatetraynoic acid inhibited superoxide formation elicited by fatty acids. Guanosine 5'[gamma-thio]triphosphate (GTP[gamma S]), a potent activator of guanine-nucleotide-binding proteins (N-proteins) enhanced superoxide formation elicited by fatty acids up to fourfold, supporting our previous suggestion that NADPH oxidase is regulated by an N-protein [Seifert, R. et al. (1986) FEBS Lett. 205, 161-165]. Cytosols from various tissues, soybean lipoxygenase and protein kinase C, purified from chicken stomach, did not substitute neutrophil cytosol. The activity of neutrophil cytosol was destroyed by heating at 95 degrees C. Superoxide formation was not affected by the inhibitor of protein kinase C 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). Removal of cytosolic ATP by preincubation with hexokinase and glucose, dialysis of neutrophil cytosol or chelation of calcium with EGTA did not abolish the stimulatory effect of arachidonic acid and GTP[gamma S]. Thus, the cytosolic cofactor appears to be a neutrophil-specific and heat-labile protein, which is neither a lipoxygenase nor protein kinase C.     

Statistical and mutational analysis of chronic granulomatous disease in Japan with special reference to gp91-phox and p22-phox deficiency

Chronic granulomatous disease (CGD) is a group of inherited disorders of host defense caused by a mutation in any of the four components of phagocyte NADPH oxidase, namely gp91-, p22-, p47-, and p67-phox. We have made a precise statistical analysis of 229 registered patients from 195 families in Japan and mutation analysis of 28 and 5 independent patients, respectively, with gp91- and p22-phox deficiency. The gp91- and p22-phox proteins form the membrane cytochrome b558, which plays important roles in the assembly of the active oxidase and electron-transfer reaction, and the lesions in either subunit account for more than 80% of cases. The ratio of male to female patients was 6.6/1, the incidence was calculated to be about 1 out of 220,000 birth, and the life expectancy of the patients born in the 1970s was estimated to be 25-30 years old. For the X-linked gp91-phox deficiency, we found five missense and nine nonsense mutations, seven deletions, three insertions, and four splice site mutations, which included the following novel mutations: four missense, five nonsense, six deletions, one insertion, and two splice site abnormalities. With regard to p22-phox deficiency, two homozygous nonsense mutations and one homozygous deletion, a missense mutation together with a splice site mutation, and two different missense mutations were found. These mutations have not been reported before. Based on the present and reported data from Japan, we discuss the molecular defects of the disease and the difference in statistics between western countries and Japan.     

A carboxy-terminal peptide from p47-phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase.

Agonist-activated phosphorylation of neutrophil proteins including p47-phox, a cytosolic component of the respiratory burst oxidase, has been implicated in the signal transduction cascade which leads to activation of the superoxide generating respiratory burst. We have previously reported (J. Biol. Chem. 265, 17550-59) that in a cell-free activation system consisting of cytosol plus plasma membrane from human neutrophils, diacylglycerol acts synergistically with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to augment superoxide generation and assembly of the oxidase, and that p47 phosphorylation can occur under these conditions. Herein, we show that a peptide corresponding to a carboxy terminal sequence of p47-phox is a substrate for phosphorylation both by purified protein kinase C (a mixture of alpha, beta, and gamma forms) and by a distinct kinase or kinases present in neutrophil cytosol. Based on its activator requirements, the neutrophil kinase differs from classical protein kinase C, but may be a protein kinase C variant, based on inhibition by a protein kinase C peptide. Although in the cell-free system phosphorylation occurs under conditions which are similar to those for activation of superoxide generation, phosphorylation is not required for activation (1). Rather, protein assembly or aggregation which occurs under activation conditions may also promote phosphorylation.     

Determination of flavin contents in neutrophils by a sensitive chemiluminescence assay: evidence for no translocation of flavoproteins from the cytosol to the membrane upon cell stimulation.

A sensitive and specific chemiluminescence (CL) method with bacterial luciferase was adapted for accurate measurement of the flavins FAD and FMN in the membrane and cytosolic fractions of neutrophils prepared from pig and human blood. The FAD and FMN contents (FAD/FMN = 100:2) in the membranes were essentially the same in resting (R) and myristate-stimulated (S) cells, although O2(-)-generation was markedly enhanced exclusively in S membranes. The O2(-)-forming activity of S samples remained unchanged or even increased after washing the membranes with buffer, although one-third of the FAD was lost during washing (a decrease from 140 to 95 pmol/10(8) cell-equivalent (CE) during washing). The cytosol is known to contain at least three components that are essential for O2- production (p47-phox, p67-phox, and a G-protein), and that are translocated to membranes upon activation, but its flavin content was one tenth of that of the membranes. The cytosol was treated with fatty acids in the absence of membranes to induce substantial precipitation of p47-phox, p67-phox and a protein of 32 kDa. No difference relative to a solvent-control was noted in the low flavin content of the precipitate indicating that these cytosolic components are not flavoproteins. These results do not support the possibility of translocation of a cytosolic flavoprotein to the membrane upon activation of the respiratory burst.     

Expression of p22-phox and gp91-phox, essential components of NADPH oxidase, increases after myocardial infarction

Recent studies have shown that oxidative stress plays an important role in cardiovascular diseases. NADPH oxidase is one of the major sources of superoxide anions and a candidate for the initiation and development of atherosclerosis, which involves the remodeling of vasculature. However, the relevance of NADPH oxidase in ventricular remodeling has not been well-characterized. This is the first report showing that the expression of p22-phox and gp91-phox, essential components of NADPH oxidase, are increased in the infarcted sites after myocardial infarction. The levels of thiobarbituric acid reactive substance, which indicates the lipid peroxidation level, and nuclear factor-kappaB (NF-kappaB) DNA binding activity are also increased in infarcted sites. Our results suggest that the increased expression of NADPH oxidase may have an effect on left ventricular remodeling by increasing the redox-sensitive NF-kappaB DNA binding activity as well as the lipid peroxidation level.     

Induction of the respiratory burst in HL-60 cells. Correlation of function and protein expression

Differentiation of myeloid cells is associated with the gradual acquisition of functional capacity to produce a respiratory burst. In our study HL-60 cells were differentiated to the monocyte phenotype with IFN-gamma or 1,25-dihydroxyvitamin D3, or to the neutrophil phenotype with retinoic acid or DMSO to compare the time-course of expression of membrane and cytosolic oxidase components, and to correlate this with the appearance of a functional oxidase. Over a 6-day period of induction the rank order of the ability of these agents to induce expression of PMA-stimulated superoxide production was: IFN-gamma greater than 1,25(OH)2D3 greater than retinoic acid greater than DMSO. Immunoblot analysis of HL-60 membranes and cytosol was used to assess the amount of specific phagocyte oxidase factors (91 and 22 kDa subunits of membrane cytochrome b558 (gp91 and p22), and 47 and 67 kDa cytosol oxidase factors (p47 and p67)). HL-60 cell membranes or cytosol were tested in a cell-free assay of superoxide production by mixing with normal neutrophil cytosol or membranes, respectively. p47 was first detected at 16 h of differentiation, increasing similarly thereafter with all induction regimens and reaching a maximum by 3 to 4 days. The earliest detection of p67 varied from 2 to 6 days depending on the inducing agent and appeared to be the limiting cytosol component. Small amounts of both subunits of cytochrome b558 were detected in uninduced HL-60 membranes, but were sufficient to support substantial superoxide production when combined with normal neutrophil cytosol. Both cytochrome b558 subunit proteins and membrane oxidase activity increased during differentiation in parallel. We conclude that membrane and cytosol components of the NADPH oxidase complex appear at different times and increase differently during HL-60 differentiation. The production of p67 is the major factor limiting the respiratory burst during HL-60 differentiation.     

Production of myeloid cell cytosols functionally and immunochemically deficient in the 47 kDa or 67 kDa NADPH oxidase cytosolic factors

Professional phagocytes contain a unique NADPH oxidase responsible for the production of microbicidal oxidants. Activation of this oxidase requires participation of cytosolic and membrane proteins, but the interactions of these components are incompletely understood. Patients with autosomal recessive Chronic Granulomatous Diseases (CGD) are characterized by functional defects in phagocyte oxidase activity resulting from a deficiency of either a 47 kDa (p47) or a 67 kDa (p67) cytosolic oxidase component. Cytosols from such patients are valuable for biochemical studies of the oxidase, but are not generally available because CGD is a rare disorder. The present study illustrates means of producing cytosols functionally and immunochemically deficient in either p47 or p67. Cytosol from monocytes cultured for 6 days is immunochemically deficient in p47 but not p67, while cytosol from HL-60 cells induced with retinoic acid for 3 days is deficient in p67 but not p47. Each of these cytosols fail to generate superoxide when added to neutrophil membranes in a cell-free assay but complement each other when combined. Complementation studies in which these cytosols were mixed in the cell-free assay with p47- or p67- deficient CGD cytosol established the functional characteristics of the experimentally produced cytosols.