leukotriene-b4 and Granulomatous-Disease--Chronic

The leukocyte NADPH oxidase 2 (NOX2) regulates inflammation independent of its antimicrobial activity. Inherited defects in NOX2 lead to chronic granulomatous disease (CGD), associated with recurrent bacterial and fungal infections, often with excessive neutrophilic inflammation that results in significant inflammatory burden and tissue damage. We previously showed that excessive leukotriene B4 (LTB4) production by NOX2-deficient mouse neutrophils was a key driver of elevated lung neutrophil infiltration in the initial response to pulmonary challenge with the model fungal particle zymosan. We now identify interleukin-1β (IL-1β) and downstream granulocyte colony-stimulating factor (G-CSF) as critical amplifying signals that augment and sustain neutrophil accrual in CGD mice. Neutrophils, delivered into the lung via LTB4, were the primary source of IL-1β within the airways, and their increased numbers in CGD lungs led to significantly elevated local and plasma G-CSF. Elevated G-CSF simultaneously promoted increased granulopoiesis and mobilized the release of higher numbers of an immature CD101- neutrophil subset from the marrow, which trafficked to the lung and acquired a significantly more proinflammatory transcriptome in CGD mice compared with wild-type mice. Thus, neutrophil-produced IL-1β and downstream G-CSF act sequentially but nonredundantly with LTB4 to deploy neutrophils and amplify inflammation in CGD mice after inhalation of zymosan. NOX2 plays a critical role in dampening multiple components of a feed-forward pipeline for neutrophil recruitment, and these findings highlight NOX2 as a key regulator of neutrophil number, subsets, and function at inflamed sites.

Topics: Animals; Granulocyte Colony-Stimulating Factor; Granulomatous Disease, Chronic; Inflammation; Interleukin-1beta; Leukotriene B4; Mice; NADPH Oxidase 2; NADPH Oxidases; Neutrophils; Pneumonia; Zymosan

Topics: Cell Wall; Granulomatous Disease, Chronic; Humans; Leukotriene B4; NADPH Oxidases; Neutrophil Infiltration; Neutrophils

Chronic granulomatous disease is the manifestation of genetic defects of the leukocyte NADPH oxidase resulting in the absence of a respiratory burst. Patients with chronic granulomatous disease can develop chronic granulomas in many locations of the body, including the skin. Using an established murine model of X-linked chronic granulomatous disease (X-CGD) created by homologous recombinant disruption of the gene encoding the gp91phox component of the NADPH oxidase, in this study we examined cutaneous reactivity to sterile Aspergillus fumigatus hyphae. Injection of Aspergillus fumigatus into the dorsal ears of X-CGD mice resulted in an enhanced inflammatory response by 24 h, consisting of neutrophils, which developed into suppurative granulomas by 10 d. Intradermal injection of Aspergillus fumigatus into wild-type mice only resulted in a transient inflammatory response that resolved by 10 d. Injection of Aspergillus fumigatus into female carrier mice resulted in an acute inflammatory response that was similar to that of wild-type mice, but, at higher doses of Aspergillus fumigatus, many carriers subsequently developed granulomatous lesions that were qualitatively similar but smaller than those seen in X-CGD mice by 30 d. Consistent with the ability of X-CGD mice to mount an enhanced neutrophil-rich inflammatory response to Aspergillus fumigatus, significant levels of the potent neutrophil activator/chemoattractant leukotriene B4 were measured by mass spectrometry in skin biopsies at 24 and 72 h. In contrast to the exaggerated inflammatory response to intradermal Aspergillus fumigatus in X-CGD mice compared to their wild-type counterparts, similar levels of inflammation were seen in a model of delayed-type hypersensitivity using 2,4-dinitrofluorobenzene. This study represents the first report of a cutaneous granuloma model in mice with X-CGD, which may also prove useful as a functional test to evaluate the efficacy of gene therapy protocols being developed for chronic granulomatous disease.

Topics: Animals; Aspergillosis; Aspergillus fumigatus; Dermatitis; Female; Granulomatous Disease, Chronic; Heterozygote; Hypersensitivity, Delayed; Leukotriene B4; Mice; Mice, Inbred C57BL; Reference Values

Activated polymorphonuclear neutrophil granulocytes (PMN) from patients with chronic granulomatous disease (CGD) show reduced electron-proton shifts and an inability to acidify the cell. We studied whether this impaired pH-regulating capacity affected PMN membrane potential changes and the kinetics of homotypic aggregation by changing the extracellular pH over a wide range. At pH 7.4 normal PMN showed a rapid, transient membrane depolarization to leukotriene B4 (LTB4) and a slower response to N-formyl-methionyl-leucyl-phenylalanine. In contrast, PMN from 13 patients with CGD exhibited no or minute depolarization to these stimuli and 77% of tested patients with CGD displayed absence or marked reductions of the disaggregation to LTB4. On acidification of pH 5.0 to 6.4, PMN membrane depolarization appeared in six of nine tested patients. Likewise, disaggregation became evident in all of three patients. On alkalinization of normal PMN to pH 8.0 to 9.0, membrane depolarization and disaggregation to LTB4 disappeared, and cells reacted as CGD PMN. This change was not due to inefficient signal transduction, because normal PMN enhanced the superoxide ion production to N-formyl-methionyl-leucyl-phenylalanine on this alkalinization. Cytosolic pH changes in resting and LTB4-activated CGD cells at pH 6.0, 7.4, and 8.5 were similar those in control cells but for absence of an initial acidification. Thus neutrophil membrane potential changes and aggregation kinetics to LTB4 are abnormal in patients with CGD and return toward normal on extracellular acidification.

Topics: Adolescent; Adult; Anions; Cell Aggregation; Child; Female; Granulomatous Disease, Chronic; Humans; Hydrogen-Ion Concentration; Kinetics; Leukotriene B4; Luminescent Measurements; Male; Membrane Potentials; Neutrophils; Nitroblue Tetrazolium; Reference Values; Superoxides

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

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

Appropriately stimulated neutrophils release peroxidase and undergo a respiratory burst to form hydrogen peroxide (H2O2) and hydroxyl radicals (OH). We report here that both the myeloperoxidase-H2O2-halide system and OH released in this way can degrade the leukotrienes (LT) formed by neutrophils. More LTB4 and LTC4 were recovered from the supernatants of chronic granulomatous disease neutrophils (which are unable to respond to stimulation with a respiratory burst) than from normal or myeloperoxidase-deficient neutrophils when stimulated with the calcium ionophore A23187. When radiolabeled LTC4 was added, 72% of the LTC4 was recovered from the chronic granulomatous disease cells in contrast to 0% from the myeloperoxidase-deficient and normal cells. Inhibitor studies using catalase, superoxide dismutase, azide, mannitol, or ethanol suggested that LTC4 degradation was mediated primarily by the myeloperoxidase system in normal cells and by OH in myeloperoxidase-deficient cells. LTC4 degradation by the cell-free myeloperoxidase-H2O2-halide system and the OH -generating acetaldehyde-xanthine oxidase-Fe2+ system had inhibitor profiles comparable to normal and myeloperoxidase-deficient neutrophils, respectively. LTC4 degradation products formed by the stimulated neutrophils and model systems included the 5-(S), 12-(R)- and 5-(S), 12-(S)-6-trans-isomers of LTB4. Thus phagocytes may modulate LT activity in inflammatory sites by the inactivation of these potent biologic mediators by at least two oxidative mechanisms.

Topics: Female; Granulomatous Disease, Chronic; Humans; Kinetics; Leukotriene B4; Male; Neutrophils; Peroxidase; Peroxidases; SRS-A