Multiple responses to administration of liposome-encapsulated hemoglobin (LEH): Effects on hematopoiesis and serum IL-6 levels.

Liposome-encapsulated hemoglobin (LEH) has been tested in animals as an oxygen-carrying red cell substitute and has been shown to be beneficial in the treatment of hemorrhagic shock. The effects of LEH on immune responses have not been studied thoroughly in any well-controlled model. Using a murine model, we evaluated nephrotoxicity and hepatotoxicity as well as immune function parameters following LEH administration. Following intravenous administration of LEH, 1) a serum spike of interleukin-6 (IL-6) occurred in mice at 4-8 hours, with no elevation of IL-1, tumor necrosis factor (TNF), or interferon-gamma (IFN-gamma); 2) the serum liver function enzymes SGOT (AST, aspartate aminotransferase) and SGPT (ALT, alanine aminotransferase) were elevated at 48 hours; 3) only a slight increase in serum antibody to bovine hemoglobin was observed; and 4) increased hematopoietic activity was observed in the spleen and bone marrow. The finding that only IL-6 but not the associated TNF, IL-1, or IFN-gamma is secreted in vivo following LEH administration is novel and may have significance in defining the mechanisms underlying specific adverse responses observed with LEH administration in animals.

Kinetics of cytokine gene expression in macrophage and endothelial cell lines following liposome encapsulated haemoglobin (LEH) treatment in vitro.

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 (P388D1) 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-1 beta, IL-6, TGF-beta, TNF-alpha 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-1 beta, TGF-beta, TNF-alpha 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.