LIPOSOME-ENCAPSULATED HEMOGLOBIN MODULATES LIPOPOLYSACCHARIDE-INDUCEDTUMOR-NECROSIS-FACTOR-ALPHA PRODUCTION IN MICE

Objective: To investigate the effect of liposome-encapsulated hemoglob in, an experimental blood substitute, on the function of the mononucle ar phagocytic system in normovolemic mice, in ex vivo murine splenocyt es and in a transformed murine monocytic cell line, RAW 264.7. Design: Prospective, randomized trial, Setting: Center for Biomolecular Scien ce and Engineering, Naval Research Laboratory, and the Thomas Jefferso n University, Subjects: Female Balb/c mice (n = 27), Interventions: Mi ce were injected into the tail vein with liposome-encapsulated hemoglo bin or liposome vehicle and were killed at varying time points for blo od sampling and splenocyte isolation and culture, Measurements and Mai n 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 ser um tumor necrosis factor (TNF)-alpha concentrations at 2, 8, 15, and 2 4 hrs after administration, in the ex vivo procedure, lipopolysacchari de (1 mu g/mL)-induced TNF-alpha production by splenocytes from mice i njected with liposome-encapsulated hemoglobin was attenuated at 2 and 4 hrs (73%, p = .002 at 2 hrs), compared with TNF-or production by spl enocytes from sham animals challenged with the same concentration of l ipopolysaccharide, In the in vitro procedure, simultaneous exposure of liposome-encapsulated hemoglobin (0.88 to 8.8 mg/mL) and lipopolysacc haride (0.125 to 1 mu g/mL) to the murine-derived, peritoneal monocyti c RAW 264.7 cell line showed significantly reduced TNF-alpha, peptide, but not messenger RNA, 1 to 4 hrs after exposure as compared with cel ls challenged with lipopolysaccharide alone, This effect correlated wi th the rapid phagocytosis (1 hr to 4 hrs) of liposome-encapsulated hem oglobin by RAW 264.7 cells, Phagocytic activity in RAW 264.7 cells exp osed to both liposome-encapsulated hemoglobin and lipopolysaccharide s howed reduced uptake compared with uptake of liposome-encapsulated hem oglobin. The liposome-induced reduction in TNF-alpha, peptide producti on elicited by lipopolysaccharide was countered by extending the time period to an overnight delay between liposome-encapsulated hemoglobin exposure and lipopolysaccharide challenge, Liposome-encapsulated hemog lobin incubated with lipopolysaccharide in vitro, and subsequently was hed 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 t hrough binding of lipopolysaccharide, presentation to macrophages with subsequent phagocytosis, and modulation of cytokine response by a pos t-transcriptional mechanism, This effect is attenuated by extending th e period between exposure to liposome-encapsulated hemoglobin and endo toxin, The clinical relevance of these findings awaits further investi gation.