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Abstract
While hemolysis in patients with sepsis is associated with increased mortality its mechanisms are unknown and Toll-like receptor (TLR)-4 mediated effects, complement-mediated hemolysis, or direct cell membrane effects are all conceivable mechanisms. In this study, we tested the hypotheses that toxic lipopolysaccharide (LPS) as well as non-toxic RS-LPS evokes hemolysis (1) by direct membrane effects, and (2) independent of the complement system and TLR-4 activation. We found, that incubation with LPS resulted in a marked time and concentration dependent increase of free hemoglobin concentration and LDH activity in whole blood and washed red cells. Red cell integrity was diminished as shown by decreased osmotic resistance, formation of schistocytes and rolls, and a decrease in red cell membrane stiffness. Non-toxic RS-LPS inhibited the LPS-evoked increase in TNF-α concentration demonstrating its TLR-4 antagonism, but augmented LPS-induced increase in supernatant hemoglobin concentration and membrane disturbances. Removal of plasma components in washed red cell assays failed to attenuate hemolysis. In summary, this study demonstrates direct physicochemical interactions of LPS with red cell membranes resulting in hemolysis under in vitro conditions. It might thus be hypothesized, that not all effects of LPS are mediated by TLR and may explain LPS toxicity in cells missing TLR.
Highlights
While hemolysis in patients with sepsis is associated with increased mortality its mechanisms are unknown and Toll-like receptor (TLR)-4 mediated effects, complement-mediated hemolysis, or direct cell membrane effects are all conceivable mechanisms
Since most of the increases occurred within the initial 6 hours, we chose a 6 h period in subsequent experimental series to assess the relationship between hemoglobin concentration/lactate dehydrogenase (LDH) activity and LPS concentration
In whole blood samples exposed to increasing concentrations of LPS (100, 250, 500, 750, or 1000 μg/ml) but not in controls, free hemoglobin concentrations and LDH activity significantly increased with each LPS concentration in an apparently concentration dependent manner (Fig. 1C/D)
Summary
While hemolysis in patients with sepsis is associated with increased mortality its mechanisms are unknown and Toll-like receptor (TLR)-4 mediated effects, complement-mediated hemolysis, or direct cell membrane effects are all conceivable mechanisms. This study demonstrates direct physicochemical interactions of LPS with red cell membranes resulting in hemolysis under in vitro conditions. It might be hypothesized, that not all effects of LPS are mediated by TLR and may explain LPS toxicity in cells missing TLR. We and others recently demonstrated that hemolysis in both humans and animals, carries a poor prognosis[5,6,7], possibly mediated by the toxicity of hemoglobin and its degradation products[8] Along this line, greater haptoglobin concentrations were associated with improved survival[9]. We tested the hypotheses that LPS-induced hemolysis (1) is complement or cytokine-dependent, and (2) can be triggered by direct membrane interactions, without co-stimulatory mediators required
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