Liposome-encapsulated hemoglobin modulates lipopolysaccharide-induced tumor necrosis factor-alpha production in mice.
OBJECTIVE
To investigate the effect of liposome-encapsulated hemoglobin, an experimental blood substitute, on the function of the mononuclear phagocytic system in normovolemic mic, in ex vivo murine splenocytes and in a transformed murine monocytic cell line, RAW 264.7.


METHODS
Prospective, randomized trial.


METHODS
Center for Biomolecular Science and Engineering, Naval Research Laboratory, and the Thomas Jefferson University.


METHODS
Female Balb/c mice (n = 27).


METHODS
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.


RESULTS
Injection of liposome-encapsulated hemoglobin in mice (2.2 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 microgram/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 microgram/mL) to the murine-derived, 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 expressed 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.
Rudolph
A S
AS
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USA.
Cliff
R
R
Kwasiborski
V
V
Neville
L
L
Abdullah
F
F
Rabinovici
R
R
D000818:Animals; D001802:Blood Substitutes / Q000494:pharmacology*; D002461:Cell Line, Transformed / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D005260:Female; D006454:Hemoglobins / Q000494:pharmacology*; D008070:Lipopolysaccharides; D008081:Liposomes; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000187:drug effects; D011897:Random Allocation; D013154:Spleen / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects* / Q000378:metabolism*
D001802:Blood Substitutes; D006454:Hemoglobins; D008070:Lipopolysaccharides; D008081:Liposomes; D014409:Tumor Necrosis Factor-alpha