Redox active hemoglobin enhances lipopolysaccharide-induced injury to cultured bovine endothelial cells

Abstract

D'Agnillo, Felice. Redox active hemoglobin enhances lipopolysaccharide-induced injury to cultured bovine endothelial cells. Am J Physiol Heart Circ Physiol 287: H1875-H1882, 2004. First published June 17, 2004 10.1152/ajpheart.00164.2004.-The interaction of cell-free hemoglobin with lipopolysaccharide (LPS) is thought to aggravate the pathophysiology of sepsis and/or septic shock. This study examines the possible modulatory role of cell-free hemoglobin on LPS-induced apoptosis of cultured bovine aortic endothelial cells. Experiments were performed with or without fetal bovine serum, a source of LPS-binding protein and soluble CD14. In the absence of serum, LPS alone or coincubated with purified bovine hemoglobin (BvHb), human hemoglobin (Hb), or ␣-cross-linked Hb (␣␣Hb) did not induce apoptosis. In the presence of serum, LPS induced significant apoptosis. LPS combined with BvHb, Hb, or ␣␣Hb produced the same extent of apoptosis as LPS alone. To examine whether the H 2O2-driven redox activity of hemoglobin alters LPS-induced apoptosis, glucose oxidase was added to the system to generate a subtoxic flux of H2O2. The combined treatment of LPS, glucose oxidase, and BvHb, Hb, or ␣␣Hb enhanced apoptosis compared with LPS alone. These findings support a possible mechanism whereby the redox cycling of hemoglobin, and not its direct interaction with LPS, contributes to the hemoglobin-mediated enhancement of LPS-related pathophysiology.apoptosis; ferryl hemoglobin; hydrogen peroxide; glucose oxidase CELL-FREE HEMOGLOBIN resulting from intravascular hemolysis or the administration of hemoglobin-based oxygen therapeutics is thought to exacerbate the pathophysiology of sepsis or endotoxemia. Previous studies implicate the interaction of hemoglobin with lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria and the primary mediator of gram-negative sepsis (3, 26, 39 -41). Understanding how hemoglobin enhances the effects of LPS may be critical in the management of sepsis and/or septic shock given the likely occurrence of hemoglobin-LPS interactions during trauma, infection, injury, or surgery.Several studies have demonstrated that hemoglobin enhances the toxicity or lethality of LPS. Rabbits infused with hemoglobin containing LPS exhibited increased mortality compared with rabbits infused with hemoglobin or LPS (41). Intraperitoneal injection of LPS combined with an intravascular infusion of unmodified or cross-linked hemoglobins induced greater mortality in mice compared with the hemoglobins or LPS alone (39,40). Intravenous administration of ␣-cross-linked human hemoglobin (␣␣Hb) and LPS caused significantly greater mortality and cardiovascular dysfunction than ␣␣Hb or LPS alone in a rabbit model of nonlethal endotoxemia (26). Coinjection of purified hemoglobin and LPS enhanced the LPS-induced ocular inflammatory response in rabbits (30). Increased LPS sensitivity in hypophysectomized rats was linked to an increase in circulating plasma hemoglobin levels (3). Another recent study (20) reported increased ...

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Hemoglobin Enhances the Biological Activity of Synthetic and Natural Bacterial (Endotoxic) Virulence Factors: A General Principle

“…These data are in accordance with data from Roth et al [11] who demonstrated also clear changes in electron micrographs after addition of Hb compatible with a disaggregation. D'Agnillo [33] has studied the effect of various Hb structures and LPS on apoptosis, and found only in the absence of serum any effect. These data were interpreted by an effect of redox active Hb, driven by the low level of oxidative stress, thus exacerbating the LPS-induced apoptosis.…”