interleukin-8 and Granulomatous-Disease--Chronic

Topics: Adolescent; Adult; Aged; Child; Cross-Sectional Studies; Fatigue; Female; Granulomatous Disease, Chronic; Humans; Interleukin-8; Middle Aged; NADPH Oxidases; Surveys and Questionnaires; United Kingdom; Young Adult

The innate immune response to bacterial infections includes neutrophil chemotaxis and activation, but regulation of inflammation is less well understood. Formyl peptides, byproducts of bacterial metabolism as well as mitochondrial protein biosynthesis, induce neutrophil chemotaxis, the generation of reactive oxygen intermediates (ROI), and the production of the neutrophil chemoattractant, IL-8. Patients with chronic granulomatous disease (CGD) exhibit deficient generation of ROI and hydrogen peroxide and susceptibility to bacterial and fungal pathogens, with associated dysregulated inflammation and widespread granuloma formation. We show in this study that in CGD cells, fMLF induces a 2- to 4-fold increase in IL-8 production and a sustained IL-8 mRNA response compared with normal neutrophils. Moreover, normal neutrophils treated with catalase (H(2)O(2) scavenger) or diphenyleneiodonium chloride (NADPH oxidase inhibitor) exhibit IL-8 responses comparable to those of CGD neutrophils. Addition of hydrogen peroxide or an H(2)O(2)-generating system suppresses the sustained IL-8 mRNA and increased protein production observed in CGD neutrophils. These results indicate that effectors downstream of the activation of NADPH oxidase negatively regulate IL-8 mRNA in normal neutrophils, and their absence in CGD cells results in prolonged IL-8 mRNA elevation and enhanced IL-8 levels. ROI may play a critical role in regulating inflammation through this mechanism.

Topics: Blotting, Northern; Cytokines; Enzyme Activation; Enzyme Inhibitors; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Interleukin-8; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Neutrophils; Reactive Oxygen Species; RNA, Messenger

We demonstrate here that neutrophils from chronic granulomatous disease (CGD) patients release larger amounts of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) than neutrophils from control subjects. Incremental cytokine production was observed under both basal and stimulated conditions in neutrophils from two CGD (gp 91phox) patients. The basal production of IL-8 was over seven-fold greater in CGD patients. The two samples assayed showed 3- and 10-fold increases in TNF-alpha. Basically, the same magnitude of increment was observed in lypopolysaccharide (LPS) and serum amyloid A protein (SAA)-stimulated cells. We also found that the levels of SAA and IL-8 were higher in the serum of CGD patients than the levels found in the serum of healthy donors. The increased responsiveness of neutrophils from CGD patients may be closely related with a deficiency in the assembly of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme system, or it may be due to a frequent inflammatory condition in these patients. In the latter case, the increased serum levels of systemic inflammatory factors, among them SAA, would contribute to the sustained accumulation and activation of phagocytes. Whatever the origin, the excessive production of cytokines may lead to inappropriate activation and tissue injury and even to increased susceptibility to invasive microorganisms, impairing the quality life of CGD patients.

Topics: Adult; Child; Female; Granulomatous Disease, Chronic; Humans; Interleukin-8; Lipopolysaccharides; Male; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidases; Neutrophils; Serum Amyloid A Protein; Tumor Necrosis Factor-alpha

Interleukin 8 (IL-8) is a recently described cytokine that functions as a potent neutrophil chemoattractant and activator. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) and the regulation of IL-8 gene expression to specifically test the hypothesis that ROI would induce production of IL-8 mRNA and protein. In lipopolysaccharide-stimulated human whole blood, the OH radical scavenger dimethyl sulfoxide (Me2SO) dramatically inhibited (approximately 90%) IL-8 production, but had minimal effects on the production of tumor necrosis factor, interleukin 1 beta (IL-1), and IL-6. To determine whether NADPH-oxidase-generated free radicals were critical in the regulation of IL-8, studies were performed using blood from patients with chronic granulomatous disease. In both normal individuals and patients with chronic granulomatous disease, production of IL-8 could be initiated with lipopolysaccharide, phytohemagglutinin, or aggregated immune complexes, and this production could be inhibited by Me2SO (1% v/v). To examine if oxidant stress represents a ubiquitous mechanism for the induction of IL-8, experiments were performed in cultured cell lines. In the human hepatoma cell line Hep-G2, Me2SO dose-dependently inhibited tumor necrosis factor-stimulated IL-8 production, with a 74 +/- 1% reduction observed at a Me2SO concentration of 1%. Direct exposure to ROI demonstrated that H2O2 stimulated IL-8 production in a dose-dependent manner in Hep-G2 cells, A549 pulmonary type II epithelial cells, and human skin fibroblasts; this induction could be prevented by addition of catalase. The production of IL-8 appeared to be specific to an oxidant stress since exposure of the cells to heat shock or chemical stress did not induce expression of IL-8. These studies demonstrate that oxidant stress is an important regulator of IL-8 gene expression and support the hypothesis that low levels of ROI may serve to initiate IL-8 production which then serves to recruit neutrophils to sites of inflammation.

Topics: Antioxidants; Base Sequence; Blotting, Northern; Carcinoma, Hepatocellular; Cell Line; Cell Survival; Dimethyl Sulfoxide; Fibroblasts; Free Radical Scavengers; Free Radicals; Gene Expression Regulation; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Infant, Newborn; Interleukin-8; Kinetics; Lipopolysaccharides; Liver Neoplasms; Lung; Male; Models, Biological; Molecular Sequence Data; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Oligonucleotide Probes; Oxidants; RNA, Messenger; Skin; Tumor Cells, Cultured

Human neutrophils stimulated with phorbol myristate acetate (PMA) were able to damage human erythroleukemic K-562 target cells as assessed by a 3-hr 51Cr-release assay. Neutrophils from a patient with chronic granulomatous disease of childhood were ineffective in mediating PMA-stimulated cytolysis. Cytotoxicity was inhibited under anaerobic conditions as well as by catalase and several free radical scavengers. Superoxide dismutase, azide, and cyanide failed to inhibit PMA-dependent cytotoxicity. The influence of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) on stimulus-dependent cytotoxicity was examined. Neutrophils preincubated with 1.0 X 10(-7) M FMLP did not display an increased cytotoxic response, but were markedly amplified in their ability to effect cytotoxicity with the addition of PMA. Enhancement of PMA-stimulated cytotoxicity resulted from a reversible cellular response to FMLP. The kinetics of the cytotoxic responses reflected the possibility that chemotactic peptide-treated neutrophils released cytotoxic mediators to a greater extent and at a faster rate than did untreated neutrophils. Although azide and superoxide dismutase did not inhibit cytotoxic responses of chemotactic peptide-activated neutrophils, the response was prevented by catalase and was markedly inhibited by several free radical scavengers. The ability of FMLP to enhance cytotoxic responses correlated well with its enhancement of PMA-stimulated chemiluminescence under a variety of conditions. In addition, the ability of PMA-stimulated neutrophils to mediate methane generation from dimethyl sulfoxide and ethylene generation from alpha keto-gamma-methiol-butyric acid (KMB)--assays that quantitate the generation of oxidizing radicals--was increased if the neutrophils were preincubated with FMLP. These results demonstrate that a chemotactic factor greatly potentiates the release of cytotoxic mediators from neutrophils upon stimulation with a nonchemotactic agent. The cytotoxic mediators appear to be products of oxidative metabolism. The cytotoxic potential of neutrophils that have responded to chemotactic stimuli to reach sites of inflammation may be activated in a similar manner.

Topics: Chemotactic Factors; Chromium Radioisotopes; Cytotoxicity, Immunologic; Granulomatous Disease, Chronic; Humans; Hydrogen Peroxide; Inflammation; Interleukin-8; Male; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Peroxidase; Tetradecanoylphorbol Acetate