Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide

Recombinant human granulocyte colony-stimulating factor (G-CSF) by itself was not an effective stimulus for inducing the release of superoxide (O-2) in human granulocytes. However, G-CSF was able to prime human granulocytes, and enhanced O-2 release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). The preincubation with G-CSF for 5-10 min at 37 degrees C was sufficient for priming the cells. The optimal enhancing effect was obtained at 25 ng/ml of G-CSF. The enhancement of O-2 release by G-CSF was observed over the complete range of effective concentrations of FMLP (10(-8)-10(-6) M). These findings indicate that G-CSF is a potent activator of mature granulocyte functions.     

Human endothelial cells modulate granulocyte adherence and chemotaxis

Although human endothelial cells (EC) and granulocytes interact in several ways, the factors that regulate such interactions are not well defined. In this study we found that EC and their products directly altered granulocyte adherence (GA) and chemotactic activity. The spontaneous adherence of granulocytes to human umbilical vein EC monolayers was significantly reduced at 5, 15 and 30 min if the granulocytes were preincubated with EC that were stimulated by rocking. At 30 min the spontaneous adherence of EC-preincubated granulocytes was 36% of that of control granulocytes (P = 0.004). The augmented adherence stimulated by FMLP was also decreased (54% of control) by preincubation of the granulocytes with rocked EC. The ability of stimulated EC to inhibit GA was attenuated when the EC monolayers that were used for preincubation with the granulocytes were pretreated with the cyclooxygenase inhibitor indomethacin. GA to unstimulated EC was not significantly altered by indomethacin or aspirin, suggesting that cyclooxygenase products do not influence GA under resting conditions. Preincubation of granulocytes with rocked EC monolayers or supernatant media from rocked EC monolayers diminished their chemotactic response to FMLP by 45 to 65. This inhibition was also attenuated by pretreatment of the EC with indomethacin. EC supernatant medium caused a rapid increase in granulocyte intracellular cyclic AMP, with a maximum increase to 200% of control at 1 min. These data indicate that stimulated EC release one or more arachidonic acid products that alter spontaneous and inflammatory mediator-stimulated granulocyte activity. Prostacyclin, a major cyclooxygenase product of EC arachidonate, depressed inflammatory mediator-augmented GA to EC monolayers and chemotaxis when present in nanomolar concentrations. We conclude that EC-derived prostacyclin, alone or in combination with other EC products, alters GA and chemotaxis stimulated by inflammatory mediators. This provides a mechanism by which EC may modulate granulocyte distribution as well as granulocyte responses that are influenced by adherence, such as the release of toxic oxygen metabolites and granular enzymes.     

An immunosuppressive retrovirus-derived hexapeptide interferes with intracellular signaling in monocytes and granulocytes through N-formylpeptide receptors.

We have previously found that the retroviral p15E-derived hexapeptide LDLLFL is a potent inhibitor of the FMLP-induced polarization response that is an early event in chemotaxis of monocytes and granulocytes. We investigated the mechanism of action of LDLLFL. LDLLFL inhibited the changes in [Ca2+]i in response to FMLP, but not to C5a or leukotriene B4. The reverse peptide LFLLDL was not inhibitory. In the presence of LDLLFL, the FMLP dose-response curve shifted to higher concentrations, indicating that LDLLFL interfered with binding of FMLP to its receptor. Indeed, binding of [3H]FMLP to neutrophilic granulocytes was inhibited in the presence of LDLLFL. Furthermore, immunosuppressive LDLLFL homologs also inhibited binding of FMLP to granulocytes, whereas noninhibitory LDLLFL homologs did not. Our results suggest that retroviral p15E and p15E-like factors, which can be found in serum of patients with cancer or chronic upper airway infections, may interfere with the interaction of N-formylpeptides derived from (opportunistic) bacteria, with monocytes and granulocytes. This receptor interference may impair monocyte and granulocyte reactivity toward these agents.     

Interleukin-2 induces chemotactic deficiency in patients with onco hematologic malignancies and autologous bone marrow transplantation.

Unusual gram positive bacteremia has been reported in non granulopenic patients receiving recombinant human interleukin-2 (IL-2) suggesting a beneficial effect of anti gram positive prophylaxis in such patients. We report here studies on granulocyte functions examined during the course of high dose IL-2 therapy (16 to 24 million IU/m2/days for 11 to 18 days) administered during a period of 35 days in 14 patients including 4 solid tumors, 5 chronic myeloid leukemias, 4 recipients of autologous bone marrow transplant (ABMT) and 1 recipient of syngeneic bone marrow transplant. Neutrophils functions were studied before IL-2 administration (d 0), after the first cycle (d 8) and after the third cycle (d 36). Nylon fiber adherence, superoxide production, random migration, phagocytosis, nitroblue tetrazolium reduction, lysozyme and elastase release were not impaired significantly throughout therapy. However N-Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) stimulated chemotaxis of granulocytes, normal before therapy, was significantly impaired as early at d 8 and severely inhibited at d 36 (p less than 0.001). Three septicemia, one corynebacteria parvum septicemia and two gram-negative septicemia despite normal neutrophil counts and oxacillin or Penicillin G plus Pefloxacin prophylaxis, occurred among the 14 patients studied. Although neutrophil functions were not more depressed in transplanted patients than in the other non transplanted patients, special attention should be paid to such patients in whom delayed immune reconstitution could increase the risk of sepsis.     

Human granulocyte surface molecules identified by murine monoclonal antibodies

We have investigated the nature of the antigens recognized by four classes of mouse anti-human monoclonal antibodies that characteristically reacted with neutrophilic granulocytes and their precursor cells, but not with monocytes or other normal hemopoietic cells. The antigenic targets of the majority (9/12) of the independently isolated monoclonal antibodies were present on two surface glycoproteins (Mr 145,000 and 105,000) and glycolipids. This antigen(s) was also detected on granulocyte precursor cells, including the bone marrow granulocyte/monocyte progenitor cells (CFU-GM). The same antigen(s) detected by these monoclonal antibodies was also present in non-hemopoietic cell lines (colon carcinoma and neuroblastoma). Three other antigens, defined by monoclonal antibodies AHN-8, L12.2, and L13.1 and present on granulocytes and their mid-late precursor cells, could not be identified as proteins but were detected in a protein-free glycolipid extract of these cells. The diversity of the antigens was confirmed by cross-competition experiments and by the identification of their different patterns of reactivity with cell lines and bone marrow cells.     

Two functional domains in the phagocyte membrane glycoprotein Mo1 identified with monoclonal antibodies

Granulocytes from patients genetically deficient in the leukocyte glycoprotein family, Mo1, LFA-1, and Leu-M5 (P150,94), have defective complement receptor type III (CR3) activity as well as abnormal adhesion-dependent functions such as spreading, chemotaxis, and phagocytosis. To determine the contribution of the Mo1 heterodimer deficiency to the various functional aberrations observed in deficient granulocytes, we mapped the functional domains of Mo1 using several monoclonal antibodies to this molecule. In addition to iC3b binding, two granulocyte adhesion functions were examined: Cell spreading on plastic coverslips and chemotaxis. One monoclonal antibody to Mo1, 44, inhibits all three functions. Other monoclonal antibodies (903, Leu-15, and OKM10) inhibit iC3b binding to granulocytes but have no effect on cell spreading and/or chemotaxis. Another antibody, 904, has no significant inhibition of iC3b binding but inhibits spreading on plastic and chemotaxis. These studies suggest the presence of two functional domains in Mo1: one involved in iC3b binding and the other in enhancing certain granulocyte adhesion-dependent functions.     

Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes

The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response.Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E₂ (PGE₂) by human granulocytes, release of PGE₂ and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of anti-inflammatory agents.     

Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor

Tumor necrosis factor (TNF) is a 17,000-Da protein which is produced by mononuclear cells upon exposure to endotoxin. Increases in adherence, phagocytosis, hydrogen peroxide release, and lysozyme secretion have been demonstrated after prolonged incubation of human neutrophils with TNF. In this study, the ability of highly purified recombinant human TNF to modulate neutrophil responses to soluble stimuli was evaluated. Tumor necrosis factor alone (0.1 to 10,000 units/ml) failed to induce neutrophil superoxide anion (O2-) production, granule release, or aggregation when incubated for up to 25 min at 37 degrees C. TNF did, however, stimulate a significant time-, dose-, and temperature-dependent increase in neutrophil F-actin content. Although exposure of neutrophils to TNF alone caused no superoxide anion production, it enhanced the O2- production in response to the chemotactic peptide, f-methionyl-leucyl-phenylalanine (FMLP) or the tumor promotor, phorbol myristate acetate, by as much as 278%. The enhancement was time-, dose-, and temperature-dependent and was due to a more rapid initial rate of O2- production. The TNF enhancement of FMLP-induced O2- production was blocked when an anti-TNF monoclonal antibody 241-1H11, is present during the preincubation period. TNF preincubation also enhanced FMLP-induced lysozyme release, but had no effect on aggregation and actin polymerization by FMLP. The kinetics of NADPH oxidase activation by arachidonic acid was unaltered by TNF. These results indicate that brief exposures to recombinant human TNF are able to enhance or prime the neutrophil oxidative burst in response to a second stimulus.     

Mg2+-ATPase as a membrane ecto-enzyme of human granulocytes. Inhibitors, activators and response to phagocytosis.

(1) The Mg2+-ATPase of purified human granulocytes is located at the plasma membrane. Thus, no additional enzyme activity was detected when the cells were disrupted. Moreover, the Mg2+-ATPase activity of intact cells was inhibited by such poorly permeant reagents as diazotized sulfanilic acid and suramin. Finally, the enzyme activity of cell homogenates was recovered in particulate fractions. (2)The surface Mg2+-ATPase of human granulocytes had an apparent Km of 50 microns for ATP and displayed substrate inhibition. (3) The enzyme was not affected by ouabain, but was inhibited by N-ethyl malemide, sodium meta-periodate, suramin and diazotized sulfanilic acid. The enzyme was activated by cytochalasins B and D and by UDP. Activation by UDP was characterized by changes in the enzyme's apparent Km and V and by belief of substrate inhibition. (4)Internalization of surface membranes subsequent to phagocytosis of suitable particles did not result in depletion of Mg2+-ATPase from the cell surface. The enzyme activity did not decrease after exposure to several varieties of paraffin oil emulsion particles, even if the challenged cells had been pretreated with colchicine of cytochalasin B. (5) Since suramin, which inhibited Mg2+-ATPase, had no effect upon other granulocyte functions such as chemotaxis, superoxide anion generation, or phagocytosis, it is unlikely that the enzyme plays a major role in these functions.     

A derivative of wheat germ agglutinin specifically inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis by blocking re-expression (or recycling) of receptors

We examined the mechanism of action of a derivative of wheat germ agglutinin (WGA-D) which specifically and irreversibly inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte (PMN) chemotaxis. At a concentration that completely inhibited PMN chemotaxis, WGA-D had no effect on either the uptake or release of [3H]-FMLP by PMN. Similarly, WGA-D did not affect either the short-term binding to, or internalization by, PMN of a fluoresceinated FMLP analog. WGA-D did interfere, however, with the re-expression (or recycling) of FMLP receptors by PMN that had been preincubated with 1 microM FMLP for 10 min at 4 degrees C. This effect was specific for WGA-D, because it was not observed when concanavalin A was used. Scatchard plot analysis of FMLP binding to PMN after receptor re-expression demonstrated that WGA-D-treated PMN had a significant diminution in the number of high affinity receptors. WGA-D-mediated inhibition of FMLP receptor re-expression was associated with inhibition of FMLP-induced PMN chemotaxis, but had no effect on either FMLP-induced PMN superoxide anion generation or degranulation. Studies using [125I]-WGA-D demonstrated that PMN did not internalize WGA-D spontaneously. PMN did internalize [125I]-WGA-D, however, when stimulated with FMLP. Internalization of WGA-D by FMLP-stimulated PMN was rapid, dependent on the concentration of FMLP, and specific. Internalization of [125I]-WGA-D by PMN did not occur when highly purified human C5a, instead of FMLP, was used as a stimulus. Subcellular fractionation studies demonstrated that [125I]-WGA-D and [3H]-FMLP were co-internalized by PMN, and segregated to a compartment co-migrating with Golgi markers. Western blot analysis, using PMN plasma membranes, demonstrated that WGA-D bound to a single membrane glycoprotein that migrated with an apparent m.w. of 62,000. The data indicate that WGA-D, perhaps by binding to the FMLP receptor, inhibits FMLP-induced PMN chemotaxis by blocking the re-expression (or recycling) of a population of receptors required for continuous migration.     

Effects of pepstatin A on neutrophils; cross-deactivation with FMLP

The ability of pepstatin A, a protease inhibitor produced by Streptomyces testaceus, to elicit a number of responses by the human PMN has been studied. In lysozyme and beta-glucuronidase release, pepstatin A 10(-5)M is equivalent to the synthetic oligopeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) 10(-7)M. In superoxide release, pepstatin A 10(-5)M produces 80% of that originated by FMLP 10(-7). After two minutes of incubation the superoxide release is important, there being no further increase after 10 minutes. Preincubation of the cells with cytochalasin B before stimulation with pepstatin A elicits a noticeable increase in O2- release. In chemotaxis, pepstatin A 10(-6) originates the same cell motility as FMLP 10(-9). Pepstatin A produces a cross deactivation with FMLP which adds further evidence to the hypothesis that both stimuli compete for the same receptor in the PMN.     

Transmembrane potential changes associated with superoxide release from human granulocytes

Treatment of human granulocytes with concanavalin A, phorbol myristate acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (FMLP), and A23187 (a calcium ionophore) stimulates the release of superoxide anion and the generation of chemiluminescence. The fluorescent probe, Di-S-C₃(5), has been used to monitor shifts in membrane potential in response to these stimulants which precede the secretion of superoxide. Concanavalin A, PMA, and FMLP induce a biphasic shift in transmembrane potential (E_m), i.e., a rapid depolarization followed by a prolonged hyperpolarization. This depolarization is dependent on both external sodium and calcium while the hyperpolarization is inhibited by ouabain which blocks the electrogenic Na-K pump. In contrast, A23187 induces a rapid and prolonged depolarization. This monophasic shift in E_m is dependent on external calcium. These results suggest that depolarization acts as a signal to initiate events associated with the “respiratory burst” of these phagocytes.     

Activation of normal genes in malignant cells: activation of chemotaxis in relation to other stages of normal differentiation in myeloid leukemia.

Genetically differerent clones of myeloid leukemic cells have been used to study the activation of normal genes in these malignant cells by the normal physiological inducer of myeloid cell differentiation, the protein MGI. In appropriate clones, MGI induced the normal differentiation-associated property of chemotaxis to a variety of compounds including the steroid hormone dexamethasone. The induced cells could also distinguish among different steroids by chemotaxis, suggesting that there are specific membrane interaction sites for steroids. The sequence of differentiation in these cells was the formation of C3 and Fc rosettes leads to phagocytosis of these rosettes and chemotaxis leads to synthesis and secretion of lysozyme leads to mature macrophages or granulocytes. The use of appropriate mutants and the comparison of induction by MGI and dexamethasone has shown that chemotaxis to casein can be dissociated from: chemotaxis to dexamethasone, ATP, and bacterial factor; formation of C3 or Fc rosettes; phagocytosis of these rosettes; synthesis of lysozyme; and the formation of mature cells. It is suggested from this dissection of normal differentiation that there are different membrane changes for specific chemotaxis, formation of these rosettes, and their phagocytosis, and that induction of each of these properties requires activation of different genes.     

Impaired neutrophil functions in the pathogenesis of an outbreak of recurrent furunculosis caused by methicillin-resistant Staphylococcus aureus among mentally retarded adults

Staphylococcus aureus is an important cause of skin infections. We recently described an outbreak of recurrent furunculosis involving methicillin-resistant S. aureus among mentally retarded adults. We sought to determine the role of impaired neutrophil functions in its pathogenesis. Blood neutrophil functions were determined during both the outbreak (1997) and a disease-free period (2000). Chemotaxis was measured by migration toward formyl-methionyl-leucyl-phenylalanine (FMLP), specifically and randomly; phagocytosis of opsonized zymosan (OZ) was assessed by microscopy; superoxide production was determined by cytochrome c reduction in unstimulated neutrophils and after stimulation with 50 ng/ml phorbol myristate acetate, 1 mg/ml OZ or 5 x 10(-7)M FMLP. Functions were compared between recurrent furunculosis (n=10) and non-recurrent furunculosis patients (n=13). During 2000, functions were normal among the 23 subjects, except for specific/nonspecific chemotaxis (mean 68%+/-26 and 69%+/-28). During infection, recurrent furunculosis patients had a significantly increased basal superoxide production as compared to disease-free period (10.5+/-4.7 vs. 4.9+/-1.9 nmol O(-)(2)/10(6) cells/min, p=0.003). During the disease-free period, recurrent furunculosis patients had lower basal superoxide production (4.9+/-1.9 vs. 7.7+/-3.5, p=0.067) and impaired specific chemotaxis (57%+/-28 vs. 76%+/-21, p<0.05) as compared to non-recurrent furunculosis patients. Only specific chemotaxis was an independent risk factor for recurrent furunculosis. Mentally retarded adults have impaired chemotaxis, with recurrent furunculosis cases having an even greater impairment. Abnormal specific chemotaxis is an independent risk factor for recurrent furunculosis. Impaired neutrophil functions thus have a role in the pathogenesis of outbreaks of recurrent furunculosis.     

Bimodal effect of exogenous hydrogen peroxide on human neutrophils: cytotoxic effect and modulation of respiratory burst in response to an agonist]

It has been found that high concentrations of exogenous hydrogen peroxide kill human neutrophils, the range of toxic concentrations being 100 times as high as that for human endothelial cells. Whereas the H2O2 doses of 30-100 mM induce a fast massive death of neutrophils, 10 mM hydrogen peroxide induces appreciable death only within several hours after treatment. H2O2 used at 30 mM decreases superoxide anion generation by neutrophils stimulated with PMA or FMLP. This decrease is commensurate in value with cell death, thus indicating a high functional resistance of survived cells. In the dose of 10 mM hydrogen peroxide potentiates FMLP (but not PMA-)-induced generation of superoxide anions. Augmentation of superoxide anion generation by H2O2-primed neutrophils in response to FMLP amounts to 200% of the control value. Hydrogen peroxide alone is incapable of inducing superoxide anion generation. It is concluded that exogenous oxidants can alter the functional activity of leukocytes freshly recruited in inflammatory and ischemic tissues.     

Granulocyte-macrophage colony-stimulating factor is a chemoattractant cytokine for human neutrophils: involvement of the ribosomal p70 S6 kinase signaling pathway

GM-CSF stimulates proliferation of myeloid precursors in bone marrow and primes mature leukocytes for enhanced functionality. We demonstrate that GM-CSF is a powerful chemotactic and chemokinetic agent for human neutrophils. GM-CSF-induced chemotaxis is time dependent and is specifically neutralized with Abs directed to either the ligand itself or its receptor. Maximal chemotactic response was achieved at approximately 7 nM GM-CSF, and the EC(50) was approximately 0.9 nM. Both concentrations are similar to the effective concentrations of IL-8 and less than the effective concentrations of other neutrophil chemoattractants such as neutrophil-activating peptide-78, granulocyte chemotactic protein-2, leukotriene B(4), and FMLP. GM-CSF also acts as a chemoattractant for native cells bearing the GM-CSF receptor, such as monocytes, as well as for GM-CSF receptor-bearing myeloid cell lines, HL60 (promyelomonocyte leukemic cell line) and MPD (myeloproliferative disorder cell line), following differentiation induction. GM-CSF induced a rapid, transient increase in F-actin polymerization and the formation of focal contact rings in neutrophils, which are prerequisites for cell migration. The mechanism of GM-CSF-induced chemotaxis appears to involve the cell signaling molecule, ribosomal p70 S6 kinase (p70S6K). Both p70S6K enzymatic activity and T(421)/S(424) and T(389) phosphorylation are markedly increased with GM-CSF. In addition, the p70S6K inhibitor hamartin transduced into cells as active protein, interfered with GM-CSF-dependent migration, and attenuated p70S6K phosphorylation. These data indicate that GM-CSF exhibits chemotactic functionality and suggest new avenues for the investigation of the molecular basis of chemotaxis as it relates to inflammation and tissue injury.     

Phagocytosis of enteric Campylobacter by human and murine granulocytes

The phagocytosis of enteric Campylobacter strains by murine and human granulocytes was studied in vitro. The number of fluorescein isothiocyanate-labelled bacteria per granulocyte was determined microscopically. The phagocytic index is strain-dependent, ranging from 0.05 to 0.4 bacteria per granulocyte. Human granulocytes phagocytose Campylobacter sp. with a twofold higher effectivity than murine cells. Opsonization with immune sera increased phagocytosis 11.6-fold; flagella-defective mutants were phagocytosed without opsonization with 3.3-fold higher effectivity than the isogenic mother strain. Stimulation and phagocytosis of granulocytes by 14 clinical isolates of Campylobacter sp. was monitored by measuring the oxidative burst and phagocytosis. Stimulation of granulocytes varied from 0.4 to 1.8 (relative units) and phagocytosis ranged from 0.03 to 0.68 bacteria per granulocyte. No statistically significant correlation among bio- or serovars and the degree of stimulation and phagocytosis was observed.     

Induction of granulocyte histaminase release by particle-bound complement C3 cleavage products (C3b, C3bi) and IgG

The interaction of opsonized particles with human granulocytes promotes a number of important biologic functions, including phagocytosis, superoxide generation, and release of a variety of enzymes, including histaminase. We have previously determined that histaminase release occurs via a C3-dependent process. Although fluid-phase C3b dimers can mediate release, the relative effects of particle-bound C3b and C3bi and of IgG have not been examined. In this report we demonstrate that particle-bound C3 deposited on activators of the alternative C pathway effected histaminase release in the absence of IgG. Particle-bound C3bi and C3b were both effective as mediators of histaminase release. The extent of release varied as a function of the activating surface on which C3 was deposited (zymosan C3b was considerably more potent than C3b bound to rabbit erythrocytes, which was slightly more potent than C3b bound to neuraminidase-treated sheep erythrocytes). In contrast, C3b or C3bi deposited on nonactivating surfaces (such as sheep erythrocytes) at inputs of up to 2,000,000 molecules per granulocyte failed to induce histaminase release unless IgG was also present. The ability of C3b bound to particles that serve as activators of the alternative pathway to induce histaminase release is apparently not the result of decreased susceptibility of C3b to proteolysis or to an increased binding affinity to the C3b receptor, but may relate to the interaction of other surface structures on activating particles with the PMN membrane.     

A step sensitive to pertussis toxin and phorbol ester in human neutrophils regulates chemotaxis and capping but not phagocytosis

Treatment of human neutrophils with pertussis toxin (PT) abolishes chemotaxis in response to either platelet-activating factor (PAF) or f-Met-Leu-Phe (FMLP), and capping induced via the concanavalin A (Con A) receptor. These functional effects are accompanied by the inhibition of calcium mobilization by PAF, FMLP and Con A. The agent phorbol 12-myristate-13-acetate (PMA) also inhibits chemotaxis and capping as well as calcium mobilization by these receptors. In sharp contrast, neither PT, cholera toxin (CT), nor PMA, inhibits the phagocytosis of non-opsonized and opsonized Candida albicans, sheep erythrocytes or fluorescent latex beads. Our results suggest that receptor-initiated chemotaxis and capping involve a step that is sensitive to PT and PMA, and that phagocytosis is not regulated in a similar fashion.     

The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils.

NADPH-dependent superoxide production by intact human neutrophils is inhibited by DPI (diphenyleneiodonium), when stimulated by either FMLP (N-formylmethionyl-leucyl-phenylalanine) or PMA (phorbol 12-myristate 13-acetate). Addition of 10 microM-DPI abolished the reduction of both the FAD and the cytochrome b components of the NADPH oxidase. DPI inhibition of the oxidase was associated with defective aerobic killing of staphylococci by human neutrophils. Anaerobic killing, phagocytosis, chemotaxis and motility were relatively unaffected by 10 microM-DPI. Degranulation of the azurophil and specific granules, induced by the soluble stimuli FMLP or PMA, and by particulate stimuli was decreased by the presence of DPI. The above effects of DPI on human neutrophils are similar to those found in chronic granulomatous disease.