Influence of hypoxia and glucose deprivation on tumour necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor expression in human cultured monocytes

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% O-2) or normoxic (21% O-2) 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.