KINETICS OF CYTOKINE GENE EXPRESSION IN MACROPHAGE AND ENDOTHELIAL CELL LINES FOLLOWING LIPOSOME ENCAPSULATED HAEMOGLOBIN (LEH) TREATMENT IN VITRO

Abstract

Liposome encapsulated haemoglobin (LEH), a blood substitute, has been shown to increase serum IL-6 levels in mice, but the cellular source of this cytokine is unknown. These experiments were conducted to determine cytokine gene expression by macrophages (P388Dl) and endothelial cells (EOMA) after LEH treatment in vitro. P388D1 and EOMA cells were incubated with LEH, final concentration of 10%, for 0, 0.5, 1, 2, 4, 8 and 24 h. Total RNA was extracted at the above times and semi-quantitative RT-PCR was carried out to detect cytokine mRNA expression in the presence of competitive internal standards specific for mouse IL-1β, IL-6, TGF-β, TNF-α and GM-CSF. The results demonstrated that although constitutive cytokine gene expression exists in both cell lines, the level of IL-6 mRNA was prominently elevated by incubation with LEH, rapidly reaching a peak at about 4 h and gradually declining over the next 20 h after LEH treatment. In contrast, there was no significant change in mRNA accumulation of IL-1β, TGF-β, TNF-α or GM-CSF at all times. These in vitro findings suggest that macrophages and endothelial cells may be the cellular sources of the elevated levels of IL-6 found in serum after in vivo LEH administration.