Submitted on February 17, 2004
Accepted on June 16, 2004
REDOX ACTIVE HEMOGLOBIN ENHANCES LIPOPOLYSACCHARIDE-INDUCED INJURY TO CULTURED BOVINE ENDOTHELIAL CELLS
Felice D'Agnillo1*
1 Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA
* To whom correspondence should be addressed. E-mail: dagnillo{at}cber.fda.gov.
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 co-incubated with purified bovine hemoglobin (BvHb), human hemoglobin (Hb) or alpha cross-linked human hemoglobin (
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 hydrogen peroxide (H2O2)-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 to 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.
