title: Influence of hypoxia and glucose deprivation on tumour necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor expression in human cultured monocytes.
abstract: Ischaemia in wounds may modulate the expression of tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The release of these and other cytokines by stimulated macrophages influences wound healing. Our aim was to examine the separate and combined effects of hypoxia and glucose deprivation on TNF-alpha and GM-CSF mRNA levels in human monocytes isolated from peripheral blood by density gradient centrifugation and purified by adherence. Cells were incubated for a 16-hour period in a hypoxic (3% O2) or normoxic (21% O2) environment in the presence or absence of glucose followed by a further 4 h under normoxic conditions in the presence or absence of lipopolysaccharide (LPS, 100 pg/ml). These different incubation conditions had no effect on cell viability, cell number, lactate dehydrogenase release or superoxide anion generation (n = 5, p > 0.05, paired t test). However, Northern hybridisation showed that hypoxia decreased the expression of GM-CSF mRNA in LPS-stimulated human monocytes by 46% (n = 9, p < 0.05, paired t test) and increased the expression of TNF-alpha by 102% (n = 7, p < 0.05, paired t test). The increase in the level of immunoreactive TNF-alpha in the cell supernatants paralleled the increase in TNF-alpha mRNA. The combination of glucose deprivation and hypoxia decreased the expression of both GM-CSF and TNF-alpha mRNA in LPS-stimulated human monocytes. Similarly, a decrease in the level of TNF-alpha in the cell supernatants was observed (n = 3-5, p < 0.05, two-way ANOVA). These data suggest that incubation conditions simulating ischaemia reduce LPS-induced cytokine expression.
authors.affiliation: Bernard O'Brien Institute of Microsurgery, St Vincent's Hospital, Melbourne, Australia.
authors.forename: E
authors.initials: E
authors.lastname: Guida
authors.forename: A
authors.initials: A
authors.lastname: Stewart
mesh_terms: D002452:Cell Count; D015687:Cell Hypoxia; D002470:Cell Survival; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D007770:L-Lactate Dehydrogenase; D005947:Glucose; D010100:Oxygen