Human Neutrophil Membranes Research Articles

Human neutrophil membrane fluidity after incubation in structuraily different lipid emusions

Human neutrophil membrane fluidity after incubation in structuraily different lipid emusions

Identification of immunoreactive tissue prokallikrein on the surface membrane of human neutrophils.

Putative binding sites for prokallikrein, the endogenous zymogen of the vasoactive and pro-inflammatory tissue kallikrein-kinin system, were recently demonstrated on human neutrophils. However, the occurrence and distribution of neutrophil-bound prokallikrein itself have so far not been examined. In this study, a specific anti-peptide antibody directed against the propart of the zymogen was used to localize the kallikrein precursor by confocal laser-scanning microscopy on unstimulated human blood neutrophils. Our results describe, for the first time, the presence of tissue prokallikrein on the membrane of circulating neutrophils. Immunoreactive prokallikrein was associated into punctate clusters occupying the external surface of the neutrophil membrane and, after addition of exogenous zymogen, immunolabeling was enhanced four-fold. In contrast, only moderate immunoreactivity to prokallikrein was observed intracellularly. These results suggest that resting neutrophils provide a circulating platform for tissue prokallikrein whose surface density may be upregulated as part of the inflammatory process.

Organization and Mobility of CD11b/CD18 and Targeting of Superoxide on the Surface of Degranulated Human Neutrophils

A monoclonal IgM, specifically recognizingbothCD11b and CD18 of human neutrophils, was used to examine the organization and mobility of CD11b/CD18 in the plasma membrane of human neutrophils degranulated by dihydrocytochalasin B (dhCB) treatment and fMet-Leu-Phe (fMLF) stimulation. Subcellular fractionation analysis of untreated or dhCB-treated control neutrophils indicated that 20% of CD11b/CD18 cosedimented with plasma membrane and the remainder with specific granules. In contrast, fMLF stimulation of dhCB-treated cells caused a major reorganization of CD11b/CD18, in which 60–70% of CD11b/CD18 sedimented in dense plasma membrane fractions that were also enriched in superoxide-generating NADPH oxidase activity. Similarly pretreated neutrophils were fixed, immunogold labeled, and examined by scanning electron microscopy. Immunogold particles were distributed uniformly over the symmetrically ruffled surface of unstimulated neutrophils. On dhCB-treated cells, immunogold was mostly uniformly distributed on a smooth membrane with a small percentage of particles lining up into linear arrays. After fMLF + dhCB stimulation, CD11b/CD18 gold label was more abundant on the cell surface and formed large aggregates on polarized membrane protrusions. However, when cells were adhered to an albumin-coated quartz surface and stimulated with fMLF in the presence of dhCB, immunogold was excluded on the articulated and rounded cell body but concentrated on the periphery of adherent lamellae. Fluorescence photobleaching recovery indicated that in unstimulated cells 38 ± 3% of CD11b/CD18 was mobile (R) with a diffusion constantDof 3.1 ± 0.3 × 10−10cm2/s. Treatment with dhCB raisedRandD24 and 74%, respectively. Stimulation using 1 μM fMLF with dhCB loweredDandRto near control levels. Since NADPH oxidase and CD11b/CD18 cosediment in high-density plasma membrane domains after fMLF + dhCB stimulation, we speculate that a stimulus-induced reorganization of CD11b/CD18 and NADPH oxidase to common membrane domains may occur in fMLF + dhCB-degranulated neutrophils.

3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes.

1. The present study describes the direct labelling of A2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[l,5-c]pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.34 nM and 75 fmol mg(-1) protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [3H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments. 3. Thermodynamic data indicated that [3H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A2A adenosine receptors. 4. It was concluded that in human neutrophil membranes, [3H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A2A adenosine receptors of other tissues. The receptors labelled by [3H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

The staphylococcal pore-forming leukotoxins open Ca2+ channels in the membrane of human polymorphonuclear neutrophils.

The ability of leukotoxins secreted by Staphylococcus aureus to modify the permeability of the membrane of human polymorphonuclear neutrophils has been studied by spectrofluorometry and appropriate fluorescent probes. This family of bicomponent leukotoxins is constituted by, at least, three pairs of proteins: LukS-PV/LukF-PV, HlgA/HlgB, HlgC/HlgB. After binding of both components to the membrane, each pair induces influxes of divalent cations and ethidium in polymorphonuclear neutrophils, although with different intensities. The influx of divalent cations appears sooner than the influx of ethidium. The pathway for divalent cations is not permeable to monovalent cations (Na+, K+, ethidium+) and is blocked by Ca2+ channel inhibitors that do not block the fluxes of ethidium and monovalent cations. It is concluded that the leukotoxins bind to a receptor linked to a divalent cation-selective channel or to the channel itself which is activated. Then, the leukotoxins open a second pathway by insertion into the membrane and subsequent formation of aspecific pores allowing an influx of ethidium.

Polyphosphoinositide Synthesis in Human Neutrophils: Effects of a Low Metabolic Energy State

Phosphatidylinositol (PtdIns) synthesis and polyphosphoinositide (PPI) formation were measured as the incorporation of [ 32P]orthophosphate ([ 32P]Pi) or [ 3H]inositol into non-stimulated intact human neutrophil membrane phospholipids. The rate of PtdIns “de novo” synthesis appeared to be a slow mechanism when compared to the rapid incorporation of [ 32P]Pi into PPIs. Of the “de novo” synthesized [ 3H]PtdIns, 70% was further phosphorylated to PPI. Nevertheless, this PPI pool represented less than 0.01% of the total nmols of PPIs formed evaluated as [ 32P]Pi labeling, indicating that PPI formation mainly involves a no “de novo” synthesized phosphatidylinositol pool. When evaluated at short incubation times, oscillations in the formation of PPIs were detected. A rapid phase was characterized after 30 s of incubation with [ 32P]Pi. Phosphorylation levels returned to an equilibrium state within a minute, and the second phase peaked at 5 min., returning to equilibrium at 15 min. The fluctuant kinetics though not the equilibrium level of PPI formation, could be abolished by neomycin. On the other hand, a selective inhibition of the rapid phase of PPI synthesis occured in the presence of the tyrosine kinase inhibitor genistein. When the incorporations of [γ- 32P]-adenosine triphosphate (ATP) or [ 32P]Pi into human neutrophil particulate fraction membranes were evaluated, PPIs synthesis showed fluctuations independently of the precursor used. Noticeably, [ 32P]from [ 32P]Pi was incorporated more efficiently into PPIs than that from [γ- 32P]ATP, when evaluated in parallel using equal specific activities for both radiolabeled precursors and under non-ATP synthesizing conditions. Moreover, the incorporation of [ 32P]Pi into particulate fraction PPIs was not abolished by high concentrations of non-radiolabeled ATP, and metabolically inhibited PMNs showed high rates of PPI syntesis. These data suggest that PPI formation is not necessarely a futile cycle in PMNs.

Anionic Amphiphile-independent Activation of the Phagocyte NADPH Oxidase in a Cell-free System by p47phox and p67phox, Both in C Terminally Truncated Forms: IMPLICATION FOR REGULATORY Src HOMOLOGY 3 DOMAIN-MEDIATED INTERACTIONS

Anionic amphiphiles, such as arachidonate, activate the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane, which contains cytochrome b558 comprising gp91(phox) and p22(phox), and three cytosolic proteins: p47(phox) and p67(phox), each harboring two SH3 domains, and the small GTPase Rac. Here we show that, even without the amphiphiles, the oxidase is activated in vitro by a C terminally truncated p47(phox), retaining the N-terminal and the two SH3 domains, and the N terminus of p67(phox). When either truncated p47(phox) or p67(phox) is replaced by the respective full-length one, the activation absolutely requires the amphiphiles. The results indicate that both p47(phox) and p67(phox) are the primary targets of the amphiphiles, and that their C-terminal regions play negative regulatory roles. We also find that the truncated p47(phox), but not the full-length one, can bind to p22(phox), a binding required for the oxidase activation. The N-terminal SH3 domain of p47(phox) is responsible for the binding not only to p22(phox), but also to the p47(phox) C terminus. Thus the SH3 domain is accessible in the active p47(phox), but is normally masked in the full-length one probably via intramolecularly interacting with the C terminus. The present findings support our previous proposal of regulatory SH3 domain-mediated interactions.

Roles for Proline-Rich Regions of p47phoxand p67phoxin the Phagocyte NADPH Oxidase Activationin Vitro

The cytosolic proteins p47phoxand p67phox,each containing two SH3 domains, are required for activation of the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane and the small GTPase Rac. Here we focus on roles of proline-rich regions (PRRs) that reside in p47phoxand p67phox.Deletion of the p47phoxPRR, to which the C-terminal SH3 domain of p67phoxbinds, results in three-fold decreased activation of the enzyme in the cell-free system with the full-length p67phox,suggesting a modulatory role of the p47phoxPRR. The modulation is likely mediated via the C-terminal region of p67phox,since the p47phoxmutant protein fully activates the oxidase in combination with the N-terminus of p67phox.Neither deletion of the p67phoxPRR nor substitutions for prolines in the region affects the ability to support superoxide production under the cell-free conditions, indicating that the PRR of p67phoxhas no primary function in the oxidase activation.

Fc receptor-triggered insertion of secretory granules into the plasma membrane of human neutrophils: selective retrieval during phagocytosis.

We studied the kinetics of secretion in human neutrophils stimulated by IgG-opsonized zymosan. Secretion of azurophilic and specific granules was quantified measuring the appearance of the granule markers CD63 and CD66b, respectively, at the cell surface. The kinetics of secretion was compared with the course of phagocytosis, revealed by the trapping of the fluid phase marker, Lucifer Yellow, in vacuoles containing zymosan particles. We found that secretion of both azurophilic and specific granules precedes phagosome sealing. An initial rapid phase of secretion was followed by a decrease in the amount of CD63 and CD66b at the cell surface. This subsequent disappearance of surface CD63 and CD66b was inhibited by cytochalasin B and probably represents internalization of the granular markers into the forming phagosome. The decrease in the amount of CD63 and CD66b exposed at the cell surface was not accompanied by a commensurate reduction in cell surface area, measured with the amphiphilic fluorescent dye FM1-43. These findings imply that CD63 and CD66b are selectively retrieved from the plasma membrane following secretion. Evidence is also presented that calcium is not the sole mediator of the rapid secretion of azurophilic and specific granules triggered by IgG-opsonized particles and that cytochalasin does not impair signaling of the calcium transient elicited by Fc receptors. Instead, actin disassembly appears to reduce the efficiency of the interaction between opsonized particles and their receptors, an effect that can be overcome by increasing the concentration of the stimulating particles.

Carbohydrate binding specificity of the neutrophil-activating protein of Helicobacter pylori.

The possible interaction of the neutrophil-activating protein of Helicobacter pylori with target cell glycoconjugates was investigated by the binding of 125I-labeled recombinant protein to glycosphingolipids from human neutrophils in solid phase assays. Thereby, a distinct binding of the neutrophil-activating protein to four bands in the acid glycosphingolipid fraction from human neutrophils was detected, whereas no binding to the non-acid glycosphingolipids or polyglycosyl ceramides from these cells was obtained. When using glycosphingolipids not present in the cell membrane of human neutrophils, it was found that the neutrophil-activating protein also bound to sulfated glycosphingolipids as sulfatide and sulfated gangliotetraosyl ceramide. Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources suggested that in human granulocytes, the neutrophil-activating protein of H. pylori preferentially recognizes glycoconjugates with a terminally unsubstituted NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta sequence.

The nucleoside diphosphate kinase of human neutrophils.

Nucleoside diphosphate kinase (NDP kinase) catalyses the phosphate transfer between nucleoside triphosphates and nucleoside diphosphates. As formation of guanosine triphosphate could be dependent on ATP in neutrophils, the presence of NDP kinase was tested in these phagocytic cells. Both membrane and cytosolic fractions of human neutrophils were found to contain NDP kinase activity. The specific activity measured in the cytosol appeared 10-fold higher than in the membrane and was not modified when the cells were activated with phorbol 12-myristate 13-acetate. Interestingly, stimulation with N-formylmethionyl leucylphenylalanine in the presence of cytochalasin B showed an increase in membrane NDP kinase activity together with the translocation of the enzyme from the cytosol to the membrane, suggesting a possible role of NDP kinase in regulating G-proteins as previously reported. In addition, activation with opsonized zymosan induced an increase in cytosolic activity, suggesting different regulation depending on the signal transduction pathway. The neutrophil enzyme consisted of two subunits of 21 kDa (NDPKA) and 18 kDa (NDPKB) again essentially present in the cytosol of the cell. Separation of proteins by two-dimensional PAGE demonstrated that each subunit consisted of at least four isoforms, indicating post translational modifications. A characteristic of this family of enzymes is the stability of the phosphorylated intermediate. In neutrophils, only one acidic isoform of each NDPKA and NDPKB was labelled in the presence of EDTA. In addition, non-denatured complexes were apparent between 91 and 130 kDa, suggesting a hexameric structure as was also proposed for NDP kinases from other eukaryotic cells. These complexes were found to differ in their isoelectric points, indicating the existence of various isoenzymes probably resulting from combination between several isoforms of each subunit.

Identification of neutrophil NADPH oxidase proteins gp91-phox, p22-phox, p67-phox, and p47-phox in mammalian species

Abstract Objective To identify and characterize 4 components of the NADPH oxidase complex, gp91-phox, p22-phox, p67-phox, and p47-phox, in common laboratory animal species. Animals 2 clinically normal animals from each of the following species: rabbit, sheep, cow, pig, and macaque (Macaca nemistrina). A pool of 8 rats. Procedure Neutrophils were harvested from blood. Membrane and cytosol fractions were isolated and separated by sodium dodecyl sulfate-polyacrylamide gels. Gels were transferred, and immunoblots were probed with antibodies directed against individual human NADPH oxidase proteins. Human neutrophil membrane and cytosol fractions served as controls. Results Immunoreactive bands were observed in all species for gp91-phox, p67-phox, and p47-phox proteins. Immunoreactive bands for p22-phox protein were observed in cells from rats, rabbits, pigs, and macaques. Conclusions The NADPH oxidase and its component proteins have been highly conserved across mammalian species. Lack of immunoreactivity to p22-phox in sheep and cows can be explained by sequence divergence and epitope variability at the p22-phox C-terminus. Clinical Relevance The high degree of NADPH oxidase protein conservation may allow the existing knowledge of the human neutrophil NADPH oxidase to be applied to the study of animal disease. Am J Vet Res 1996; 57:672–676

Near membrane Ca 2+ changes resulting from store release in neutrophils: detection by FFP-18

FFP-18 was incorporated into the inner face of the plasma membrane of human neutrophils by incubation with its acetoxymethyl ester. Conversion to the Ca 2+ sensitive intracellular indicator was monitored by the change in excitation spectra. The fluorescence from extracellularly facing FFP-18 was quenched by the membrane impermeant ion Ni 2+. Ratio fluorescence measurement of FFP-18 under these conditions permitted the detection of near membrane Ca 2+ changes resulting from the release of Ca 2+ from intracellular stores. Near membrane and cytosolic Ca 2+ changes were measured under conditions in which store release and Ca 2+ influx were triggered by FMLP, thapsigargin or immune complexes. There were significant differences in the timing and magnitude of Ca 2+ changes near the plasma membrane and bulk cytosolic Ca 2+ concentration, which were consistent with a Ca 2+ storage site deep within the neutrophil released by thapsigargin and FMLP, but Ca 2+ release sites with immune complex stimulation being close to the membrane. The use of FFP-18 to monitor Ca 2+ near the inner face of the plasma membrane thus provides evidence for the existence of two distinct Ca 2+ storage locations in neutrophils.

Effect of cetirizine on plasma membrane of human eosinophils, neutrophils and platelets: a dose response study

The effect of different concentrations of cetirizine (0.1, 0.3, 0.5, 0.7, 0.9 ug/ml) on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils and platelets were investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results demonstrate that cetirizine induced a significant increase in the lipid order in the exterior part of the membrane and a decrease in membrane heterogeneity of eosinophils at concentrations of 0.7 ug/ml and higher and that at a concentration of 0.9 ug/ml it does likewise in neutrophils and platelets. Moreover, cetirizine abolished the PAF induced changes in membrane fluidity in these cells. These data suggest that cetirizine interacts with the plasma membrane of eosinophils, neutrophils and platelets inducing functional changes.

Stoichiometry of p22-phox and gp91-phox in phagocyte cytochrome b558.

The phagocyte NADPH oxidase complex is an unusual electron transfer system. Its principal component, cytochrome b558, is a heme-containing integral membrane protein consisting of two subunits, gp91-phox and p22-phox. We used a novel method to measure precisely the gp91-phox:p22-phox stoichiometry. Cytochrome b558 was isolated in high purity from human neutrophil membrane preparations using a novel affinity purification method. We performed direct peptide sequencing of purified cytochrome b558 and detected two amino acid sequences which matched predicted sequences for gp91-phox and p22-phox. We quantitated amounts of both amino acids released from p22-phox and gp91-phox in each sequencing cycle. Averaging over 25 cycles, the mean p22-phox:gp91-phox ratio of released amino acids was 0.93 +/- 0.01. To correct for recovery differences between individual amino acids, we measured individual p22-phox:gp91-phox ratios for the eight different amino acids common to both p22-phox and gp91-phox in the first 25 positions. The mean of individual p22-phox:gp91-phox ratios for the eight common amino acids was 0.96 +/- 0.05. The p22-phox:gp91-phox ratios for each of the eight common amino acids varied from 0.81 to 1.20. Taken together, measured ratios for total and individual amino acids are consistent with a predicted ratio of 1.0 for 1:1 p22-phox:gp91-phox stoichiometry in cytochrome b558.

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons.

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10(-8) M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10(-8) M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.

S-Prenylated Cysteine Analogs Inhibit Receptor-Mediated G Protein Activation in Native Human Granulocyte and Reconstituted Bovine Retinal Rod Outer Segment Membranes

We have previously shown that the S-prenylated cysteine analogue N-acetyl-S-trans,trans-farnesyl-L-cysteine (L-AFC) inhibits basal and formyl peptide receptor-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) to and hydrolysis of GTP by membranes of HL-60 granulocytes and have presented evidence suggesting that this inhibition was not caused by reduced protein carboxyl methylation [Scheer, A., & Gierschik, P. (1993) FEBS Lett. 319, 110-114]. We now report a detailed analysis of the structural properties of S-prenylated cysteine analogues required for this inhibition and demonstrate that S-prenylcysteines also suppress basal and receptor-stimulated GTP[S] binding to human peripheral neutrophil and HL-60 granulocyte membranes when stimulated by formyl peptide and complement C5a, respectively. S-Prenylcysteines did not affect pertussis toxin-mediated [32P]ADP-ribosylation of Gi proteins. The inhibitory effect of L-AFC was reversible and was not mimicked by farnesylic acid. L-AFC also interfered with GTP[S] binding to retinal transducin when stimulated by light-activated rhodopsin in a reconstituted system. This inhibitory effect was fully reversed upon increasing the concentration of either the G protein beta gamma dimer or the activated receptor. On the basis of these results, we suggest that S-prenylated cysteine analogues like L-AFC inhibit receptor-mediated G protein activation by specifically and reversibly interfering with the interaction of activated receptors with G proteins, most likely with their beta gamma dimers, rather than by inhibiting alpha.beta gamma heterotrimer formation.

Leukotriene B4 plays a critical role in the progression of collagen-induced arthritis.

Leukotriene B4 (LTB4) is a product of the 5-lipoxygenase pathway of arachidonic acid metabolism. LTB4 is a potent chemotactic factor for neutrophils and has been postulated to play an important role in a variety of pathological conditions including rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease. The role of LTB4 in such diseases has not yet been defined but in this paper we provide direct evidence that LTB4 plays a critical role in a murine model of RA. CP-105,696, (+)-1-(3S,4R)-[3-(4-phenylbenzyl)- 4-hydroxychroman-7-yl]cyclopentane carboxylic acid, is an LTB4 receptor antagonist that inhibits LTB4 binding to human neutrophil membranes with an IC50 of 3.7 nM and inhibits LTB4-induced chemotaxis of these cells with an IC50 of 5.2 nM. CP-105,696 inhibits LTB4-induced neutrophil influx in mouse skin when administered orally with an ED50 of 4.2 mg/kg. CP-105,696 had a dramatic effect on both the clinical symptoms and histological changes of murine collagen-induced arthritis when administered at doses of 0.3-10 mg/kg. Inhibition was not associated with suppression of the humoral immune response to collagen and was equally effective if drug treatment was commenced just prior to the onset of arthritis or throughout the experiment. These results suggest that LTB4 receptor antagonists may be effective therapeutic agents for the treatment of RA.

Synthesis of an 125I analog of MK-0591 and characterization of a 5-lipoxygenase activating protein binding assay

The 125I analog of MK-0591, 1, has been prepared for use as a radioligand for developing a 5-lipoxygenase activating protein (FLAP) binding assay. The radiosynthesis involves a two step oxidative iododestannylation-saponification procedure. A FLAP binding assay has been developed in human neutrophil membranes. The binding of 1 to human neutrophil FLAP is rapid, reversible, of high affinity, saturable and selective for FLAP inhibitors.

The functional expression of p47- phox and p67- phox may contribute to the generation of superoxide by an NADPH oxidase-like system in human fibroblasts

Recent evidence suggests that a number of non-phagocytic cell types may contain a superoxide generating NADPH oxidase. Studies to date on cultured human fibroblasts have primarily concerned the identification of cytochrome b 558, whilst expression of other NADPH oxidase components have not been addressed. In this study we have investigated the expression of NADPH oxidase with particular reference to the cytosolic factors p47- phox and p67- phox. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that human fibroblasts express mRNA for p47- phox, p67- phox and p22- phox. Expression of the gp91- phox transcript was not detected, indicating that human fibroblasts may possess an NADPH oxidase isoenzyme. Western blot analysis of human fibroblast cytosol, using an anti-p47- phox antibody (JW-1), identified a 47 kDa protein. Cell-free reconstitution assays showed that fibroblast cytosol could initiate superoxide generation when mixed with either human fibroblast membranes (0.16 nmol superoxide/min/μg membrane protein), or resting human neutrophil membranes (0.20 nmol superoxide/min/μg membrane protein). These data indicate that the expression of p47- phox and p67- phox by human fibroblasts may contribute to the cells' generation of superoxide.