Studies on the gene expression of several NADPH oxidase components.
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Cell Line↗
Biomedical subjects
F Bazzoni
Publications and source records attributed to F Bazzoni.
Molecular basis of interferon-gamma and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components.
In this study, we analyzed the expression of genes encoding for components of the phagocyte superoxide anion-generating system in human phagocytes treated with interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS). Human neutrophils express high levels of the 47-kDa cytosolic factor (p47-phox), which are down-regulated after treatment with IFN-gamma, but not with LPS. On the contrary, the steady-state levels of the heavy chain subunit of cytochrome b558 (gp91-phox) were increased by IFN-gamma and LPS in human monocyte-derived macrophages and neutrophils in a time- and dose-dependent fashion, whereas cytochrome b558 light chain subunit (p22-phox) mRNA was not influenced by either agent. Studies on post-transcriptional regulation at the level of mRNA stability indicate that, in neutrophils, IFN-gamma has no influence on gp91-phox and p47-phox mRNA half-lives. The content of the two cytochrome b558 subunits was quantified by enzyme-linked immunosorbent assay, which revealed that, in neutrophils, gp91-phox levels doubled after 4 h of treatment with IFN-gamma or LPS. Monocyte/macrophage maturation was associated with a gradual decrease in gp91-phox mRNA and protein levels, which were both restored by treatment with IFN-gamma for 24-48 h. These results suggest that induction of the gp91-phox gene and protein product by IFN-gamma or LPS is an important requirement in the mechanism of the enhancement of neutrophil and macrophage oxidative metabolism.
Cells, Cultured↗
Interferon gamma induces in human neutrophils and macrophages expression of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I or CD64).
Immune interferon (IFN-gamma) induces in human neutrophils accumulation of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I, CD64) with a mechanism that is independent from de novo protein synthesis and from activation of the Na+/H+ antiporter. Monocyte-derived macrophages can also be induced to express high levels of Fc gamma R-I mRNA by IFN-gamma, without requirement of protein synthesis. Unlike what is observed in neutrophils, induction by IFN-gamma of macrophage Fc gamma R-I mRNA was significantly depressed by the Na+/H+ antiporter inhibitor amiloride. These results indicate that phagocytes' Fc gamma R-I mRNA induction by IFN-gamma is regulated by different mechanisms depending on the target cells.
Amiloride↗
Isolation and characterization of a cDNA clone for a novel serine-rich neutrophil protein.
A cDNA expression library from pig blood neutrophils was immunoscreened with a rabbit antiserum raised against a 32 kDa neutrophil membrane phosphoprotein. Previous work indicated this protein as a component of the superoxide-forming NADPH oxidase enzyme complex (1,2). Only one cDNA clone (B+) was highly positive. The B+ clone contained a 1109 bp insert, with an open reading frame encoding for 284 amino acids. The deduced B+ amino acid sequence contained a 72 amino acid domain with proline and glutamine repeats and two domains extremely enriched with serine residues. The isolated cDNA hybridizes with a 3.1 kb mRNA expressed in pig and human leukocytes.
Amino Acid Sequence↗
Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release.
The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.
Arachidonic Acids↗
Activation of human neutrophils by substance P. Effect on oxidative metabolism, exocytosis, cytosolic Ca2+ concentration and inositol phosphate formation.
The neuropeptide substance P (SP), which has been suggested to mediate neurogenic inflammation, induces in human neutrophils the activation of the respiratory burst measured as O2 consumption and H2O2 production, and a cytochalasin B-dependent secretion of specific and azurophilic granules. The SP(4-11) fragment is much more stimulant than the entire molecule, whereas the SP(1-4) fragment is inactive. The respiratory and secretory response to SP are associated with an activation of phosphoinositide turnover, of Ca2+ influx and release from intracellular stores. Pertussis toxin inhibits 70% of the respiratory response and the residual 30% activity remains, even increasing 10-fold the concentration of the toxin. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine, a putative inhibitor of protein kinase C, does not modify the respiratory response to SP. Cytochalasin B significantly depresses the activation of the respiration by SP, whereas it moderately enhances the activation of phosphoinositide turnover and potentiates the increase of intracellular Ca2+ concentration. The results are discussed in relation to the receptor apparatus involved in SP activity, the signal transduction sequence activated by SP for the stimulation of NADPH oxidase, and the role of cell response to SP in the inflammatory process.
Calcium↗