calcimycin and Granulomatous-Disease--Chronic

Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune and inflammatory diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating aberrant NET release. The initiation of NETosis after the recognition of pathogens by specific receptors is mediated by an increase in intracellular Ca

Topics: Adolescent; Calcimycin; Calcium; Cations, Divalent; Cells, Cultured; Child; Electron Transport; Extracellular Traps; Free Radical Scavengers; Granulomatous Disease, Chronic; Healthy Volunteers; Humans; Loss of Function Mutation; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; NADPH Oxidase 2; Neutrophils; Oxidation-Reduction; Plastoquinone; Primary Cell Culture; Reactive Oxygen Species; Respiratory Burst

Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin,

Topics: Calcimycin; Candida albicans; Extracellular Traps; Granulomatous Disease, Chronic; Healthy Volunteers; Humans; Metabolic Networks and Pathways; Neutrophils; Nigericin; Streptococcus; Tetradecanoylphorbol Acetate

Chronic granulomatous disease (CGD) is an inherited disease characterized by severe and recurrent bacterial and fungal infections. Phagocytic cells of CGD patients are unable to produce superoxide anion, and their efficiency in bacterial killing is significantly impaired. In these patients, the prophylactic and therapeutic validity of a long-term use of trimethoprim-sulfamethoxazole (TMP-SMX) has been well established. However a role of nitric oxide (NO) produced by phagocytic cells from CGD patients is unknown, and the mechanism of TMP-SMX in CGD is unclear. We have directly measured NO production in whole human blood by using 4,5-diaminofluorescein as a novel fluorescent indicator for intracellular NO. Intracellular NO production of gated neutrophils increased time dependently when stimulated by lipopolysaccharide (LPS) and calcium ionophore. Although all polymorphonuclear leukocyte (PMN) specimens from patients with CGD failed to generate hydrogen peroxide, NO production by CGD PMNs was significantly increased compared with that of control PMNs (p<0.05). TMP-SMX with LPS significantly increased compared with LPS-stimulated samples at clinical (n=5, p<0.05) and 10-fold clinical concentrations (n=5, p<0.01). TMP-SMX with LPS in CGD PMNs significantly increased the production of NO in comparison with the LPS stimulation at 10-fold clinical concentrations (n=5, p<0.05). In conclusion, our data indicate the possibility that NO production by neutrophils from patients with CGD treated with TMP-SMX has a role of bactericidal activity instead of O(2)(-) in host defense mechanism.

Topics: Adolescent; Adult; Anti-Infective Agents; Calcimycin; Child; Child, Preschool; Enzyme Inhibitors; Fluoresceins; Fluorescence; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Indazoles; Infant; Isothiuronium; Lipopolysaccharides; Male; Neutrophils; Nitric Oxide; Trimethoprim, Sulfamethoxazole Drug Combination

Recently, a superoxide-generating NADPH-oxidase system in human fibroblasts has been described. Therefore, we reassessed the possible use of this cell type for prenatal diagnosis of CGD patients comparing normal and CGD peripheral blood neutrophils (PMN) and skin fibroblasts in their reactive oxygen intermediate (ROI)-producing capacity. While PMN of the CGD patient showed a clearly reduced respiratory burst activity, which correlated well with the measured content of cytochrome b558, fibroblasts of the same individual showed no impaired production of superoxide anion or H2O2 upon stimulation by cytokines (TNF and IL-1) or other agents (Ca2+ ionophores and PAF, unpublished results). Furthermore, fibroblasts of the CGD patient or of normal donors could be inhibited in ROI production by diphenylene iodonium (DPI) and 2-iodobiphenyl. In contrast to PMN, no inhibition of the fibroblast NADPH-oxidase system was observed using staurosporin, an inhibitor of proteinkinase C. These data demonstrate, in contrast to previous studies, that fibroblasts are able to produce ROI. Nevertheless, since fibroblasts obtained from a CGD patient exhibited no difference in ROI production compared with fibroblasts obtained from healthy donors, they are not suitable for prenatal diagnosis of CGD.

Topics: Calcimycin; Cells, Cultured; Child; Cytochrome b Group; Female; Fibroblasts; Granulomatous Disease, Chronic; Humans; Interleukin-1; Kinetics; Male; NADPH Oxidases; Neutrophils; Platelet Activating Factor; Reference Values; Skin; Superoxides; Tumor Necrosis Factor-alpha; X Chromosome

Topics: Calcimycin; Cell Aggregation; Cytochrome b Group; Female; Granulomatous Disease, Chronic; Humans; Kinetics; Leukotriene B4; Male; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Neutrophils; Superoxides

We studied the effect of phorbol myristate acetate (PMA) on endogenous leukotriene B4 (LTB4) metabolism of calcium ionophore A23187-stimulated human neutrophils. Preincubation of normal neutrophils with PMA significantly suppressed the recovery of endogenous LTB4 induced by A23187. PMA did not suppress the recovery of LTB4 produced by neutrophils from patients with chronic granulomatous disease (CGD), which is known to be defective in NADPH oxidase activation to produce reactive oxygen species (ROS). PMA inhibited the formation of omega-oxidation products of LTB4, but enhanced arachidonic acid release in normal and CGD neutrophils. Furthermore, 5-lipoxygenase activity of 10,000 x g supernatants from normal neutrophils pretreated with PMA was equivalent to that of the controls. Decrease in LTB4 recovery was not attributed to the suppression of the intracellular Ca2+ increase. Thus, it is suggested that reactive oxygen species (ROS) produced by PMA may directly affect endogenous LTB4 and convert it into metabolite(s) distinct from omega-oxidation products.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Granulomatous Disease, Chronic; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Male; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; SRS-A; Superoxides; Tetradecanoylphorbol Acetate

Topics: Animals; Arachidonic Acids; Calcimycin; Calcium; Cytoplasmic Granules; Electric Conductivity; Granulomatous Disease, Chronic; Humans; Leukocytes; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; NADH, NADPH Oxidoreductases; NADPH Oxidases; Nucleotides, Cyclic; Oxygen Consumption; Peroxidase; Protein Kinases; Tetradecanoylphorbol Acetate

Polymorphonuclear leukocytes (PMNL) isolated from two patients with chronic granulomatous disease (CGD) were tested for their ability to metabolize arachidonic acid to lipoxygenase products including 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4). Analyses of incubations of these PMNL with arachidonic acid and the calcium ionophore A23187 did not differ from simultaneous controls in the production of LTB4, other 5,12-dihydroxy-eicosatetraenoic acids, or monohydroxy-eicosatetraenoic acids. The clinical diagnosis of CGD was confirmed in both cases by determination of PMNL chemiluminescence. Leukocytes from both patients failed to generate active oxygen species in response to either LTB4 or formyl-methionyl-leucyl-phenylalanine. The observation of arachidonic acid oxidation in the absence of superoxide anion precludes a role for the active oxygen species in this metabolic process. These studies clearly dissociate the ionophore-induced leukocyte respiratory burst from the oxidation of arachidonate to the leukotrienes. In addition, the defect of CGD appears to be unrelated to the ability of PMNL to carry out arachidonate oxygenation.

Topics: Adult; Arachidonic Acid; Arachidonic Acids; Calcimycin; Granulomatous Disease, Chronic; Humans; Leukotriene B4; Luminescent Measurements; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

The effect of the calcium ionophore A23187 on the release and metabolism of [3H]arachidonic acid was examined in normal polymorphonuclear leukocytes and those obtained from patients with chronic granulomatous disease. The ionophore A23187 which stimulates oxidative metabolism in normal polymorphonuclear leukocytes was ineffective in increasing oxidative metabolism (chemiluminescence) in polymorphonuclear leukocytes from patients with chronic granulomatous disease. However, the ionophore A23187 stimulated the release of [3H]arachidonic acid from chronic granulomatous disease neutrophil phospholipids and stimulated its metabolism into hydroxyeicosatetraenoic acids and leukotrienes.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Granulomatous Disease, Chronic; Humans; Hydroxyeicosatetraenoic Acids; Luminescent Measurements; Neutrophils; Phospholipids

A specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN) is induced by the synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), and this stimulation of tyrosinolation in PMN is completely inhibited in the presence of various reducing agents. Further studies to characterize the mechanism of stimulation of tyrosinolation in PMN have revealed that conditions that inhibited the respiratory burst in stimulated PMN, e.g., an anaerobic atmosphere, or addition of antioxidants such as cysteamine, azide, or 2,3-dihydroxybenzoic acid, also inhibited the peptide-induced stimulation of tyrosinolation in these cells. Moreover, the sulfhydryl reagent, N-ethylmaleimide, depressed tyrosinolation in resting PMN and completely inhibited the fMet-Leu-Phe-induced stimulation. In contrast, addition of diamide, which preferentially oxidizes cellular glutathione, significantly stimulated tyrosinolation both in resting and fMet-Leu-Phe-stimulated PMN. Furthermore, resting levels of tyrosinolation in seven patients with chronic granulomatous disease (CGD), whose oxidative metabolism is severely depressed, were 35-45% lower (P less than 0.01). Most strikingly, PMN from CGD patients failed to respond to fMet-Leu-Phe or the Ca2+-ionophore A23187, which also induced stimulation of tyrosinolation in normal resting PMN. Methylene blue normalized the depressed tyrosinolation in resting CGD PMN, although it did not increase tyrosinolation in stimulated PMN. These results are consistent with the idea that the characteristic activation of the oxidative metabolism and the associated changes in the redox state in stimulated PMN are coupled to the induction of stimulation of tubulin tyrosinolation in these cells.

Topics: Anaerobiosis; Antioxidants; Calcimycin; Diamide; Granulomatous Disease, Chronic; Humans; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Oxidation-Reduction; Sulfhydryl Reagents; Tubulin; Tyrosine

Studies were performed to elucidate further the phenomenon of secretagogue-mediated enhancement in the binding of the chemoattractant f-met-leu-[3H]phe to human neutrophils (PMN). Specific f-met-leu-[3H]phe binding to unstimulated PMN reached maximum levels after 10 to 15 min of incubation at 0 degrees C with a saturating concentration of peptide, and consisted of a readily displaceable and a nondisplaceable component. PMN, preexposed to A23187 (2.5 X 10(-8) M) or PMA (0.5 ng/ml) for 30 min at 37 degrees C to stimulate limited and preferential release of specific (secondary) granules (10 to 20% of total lysozyme, no beta-glucuronidase), demonstrated an approximate doubling in the displaceable component of f-met-leu-["3H]phe binding, accompanied by an increasing nondisplaceable component that could not be explained by bulk pinocytosis of extracellular fluid (assessed by [3H]sucrose uptake). The increase in f-met-leu-[3H]phe binding was not affected by inhibitors of protein synthesis, could not be attributed to the secreted products lysosyme or lactoferrin acting on the cell, and, on the basis of studies with PMN from patients with chronic granulomatous disease, could not be attributed to the effects of reactive oxygen species generated in low concentration during stimulation. Functional studies on PMN indicated that preexposure to secretagogues at concentrations demonstrated to increase receptor availability also enhanced subsequent f-met-leu-phe-mediated superoxide and hydrogen peroxide generation. The present data demonstrate that secretagogues may activate PMN to enhance their subsequent responses in f-met-leu-phe-mediated processes, and, combined with previous reports, support the concept that specific granules provide a source of preformed membrane and receptor material that is translocated to the cell surface during the secretion associated with directed locomotion.

Topics: 1-Butanol; Butanols; Calcimycin; Calcium; Chemotaxis, Leukocyte; Cycloheximide; Cytoplasmic Granules; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Lactoferrin; Methionine; Muramidase; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Pinocytosis; Puromycin; Receptors, Immunologic; Superoxides; Tetradecanoylphorbol Acetate

Topics: Adult; Calcimycin; Cells, Cultured; Female; Granulomatous Disease, Chronic; Humans; Infant; Macrophages; Male; Monocytes; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Oligopeptides; Opsonin Proteins; Oxygen; Superoxides; Tetradecanoylphorbol Acetate; Zymosan

Stimulation of normal granulocytes with chemotactic factor, phorbol myristate acetate, concanavalin A, and calcium ionophore results in rapid depolarization which precedes the 'respiratory burst'. Treatment of granulocytes in chronic granulomatous disease with these stimulants fails to generate chemiluminescence. This defect is associated with an absence of transmembrane potential shifts in response to treatment with chemotactic factor, phorbol myristate acetate, and concanavalin A while depolarization in response to A23187 is unaffected by this disease state.

Topics: Calcimycin; Chemotactic Factors; Concanavalin A; Granulocytes; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Membrane Potentials; Tetradecanoylphorbol Acetate

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.

Topics: Adolescent; Adult; Calcimycin; Carbocyanines; Chediak-Higashi Syndrome; Chemotactic Factors; Chemotaxis, Leukocyte; Child; Child, Preschool; Female; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Male; Membrane Potentials; Neutrophils; Onium Compounds; Superoxides; Tetradecanoylphorbol Acetate; Tetraphenylborate; Trityl Compounds; Valinomycin