Liposome-encapsulated hemoglobin modulates lipopolysaccharide-induced tumor necrosis factor-alpha production in mice

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Abstract
Objective To investigate the effect of liposome-encapsulated hemoglobin, an experimental blood substitute, on the function of the mononuclear phagocytic system in normovolemic mice, in ex vivo murine splenocytes and in a transformed murine monocytic cell line, RAW 264.7. Design Prospective, randomized trial. Setting Center for Biomolecular Science and Engineering, Naval Research Laboratory, and the Thomas Jefferson University. Subjects Female Balb/c mice (n = 27). Interventions Mice were injected into the tail vein with liposome-encapsulated hemoglobin or liposome vehicle and were killed at varying time points for blood sampling and splenocyte isolation and culture. Measurements and Main Results Injection of liposome-encapsulated hemoglobin in mice (2.2 g of lipid/kg and 0.56 g of hemoglobin/kg, n = 9) did not increase serum tumor necrosis factor (TNF)-alpha concentrations at 2, 8, 15, and 24 hrs after administration. In the ex vivo procedure, lipopolysaccharide (1 micro g/mL)-induced TNF-alpha production by splenocytes from mice injected with liposome-encapsulated hemoglobin was attenuated at 2 and 4 hrs (73%, p = .002 at 2 hrs), compared with TNF-alpha production by splenocytes from sham animals challenged with the same concentration of lipopolysaccharide. In the in vitro procedure, simultaneous exposure of liposome-encapsulated hemoglobin (0.88 to 8.8 mg/mL) and lipopolysaccharide (0.125 to 1 micro g/mL) to the murinederived, peritoneal monocytic RAW 264.7 cell line showed significantly reduced TNF-alpha peptide, but not messenger RNA, 1 to 4 hrs after exposure as compared with cells challenged with lipopolysaccharide alone. This effect correlated with the rapid phagocytosis (1 hr to 4 hrs) of liposome-encapsulated hemoglobin by RAW 264.7 cells. Phagocytic activity in RAW 264.7 cells exposed to both liposome-encapsulated hemoglobin and lipopolysaccharide showed reduced uptake compared with uptake of liposome-encapsulated hemoglobin. The liposome-induced reduction in TNF-alpha peptide production elicited by lipopolysaccharide was countered by extending the time period to an overnight delay between liposome-encapsulated hemoglobin exposure and lipopolysaccharide challenge. Liposome-encapsulated hemoglobin incubated with lipopolysaccharide in vitro, and subsequently washed to remove free lipopolysaccharide, stimulated TNF-alpha expression by RAW 264.7 cells. Incubation with liposome-encapsulated hemoglobin alone did not evoke TNF-alpha production in these cells. Conclusions These data suggest that liposome-encapsulated hemoglobin modulates the response of the mononuclear phagocyte system to endotoxin, possibly through binding of lipopolysaccharide, presentation to macrophages with subsequent phagocytosis, and modulation of cytokine response by a posttranscriptional mechanism. This effect is attenuated by extending the period between exposure to liposome-encapsulated hemoglobin and endotoxin. The clinical relevance of these findings awaits further investigation. (Crit Care Med 1997; 25:460-468)