Oxidative stress in sepsis. Possible production of free radicals through an erythrocyte-mediated positive feedback mechanism

Abstract

Background: Sepsis is an illness with a high morbidity for which no effective treatment exists. Its treatment has a high cost because it usually requires an intensive care unit and expensive antibiotics. The present study focus in the production of reactive oxygen species in the early stages of sepsis. This study aimed at investigating the production of reactive oxygen specie during the inflammatory response in patients with sepsis.

Methods: Reactive oxygen specie production and insoluble myeloperoxidase obtained from fresh whole blood were measured by photon counting chemiluminescence in the blood of 18 septic patients and 12 healthy individuals. Modified red blood cells were evaluated by staining of blood smears. The production of reactive oxygen species by macrophages and polymorphonuclear leukocytes put into contact with modified red blood cells were also assessed by photon counting chemiluminescence.

Results: The appearance of oxidatively modified erythrocytes, which is an evidence of oxidative stress, was supported by the detection of reactive oxygen species and insoluble myeloperoxidase in the whole blood of all septic patients. Peroxynitrite was the main reactive oxygen species found in the whole blood. Oxidatively modified erythrocytes activated phagocytic cells in vitro, leading to the considerable production of free radicals.

Conclusion: It was found that sepsis led to a high oxidative stress and to extensive modification of erythrocytes. It is proposed that a positive feedback mechanism, involving the activation of circulating leukocytes by these modified erythrocytes would maintain the pro-oxidative state even after the disappearance of bacteria.

Keywords: Inflammation; Modified erythrocytes; Oxidative stress; Sepsis.

Fig. 1

View by optical microscopy of erythrocytes in blood smears. (A) Modified erythrocytes forms from a septic patient with the predominance of echinocytes. (B) Intact erythrocytes from a healthy volunteer.

Fig. 2

Levels of ROS in septic patients’ and healthy volunteers’ blood samples. The peroxynitrite inhibitor hydralazine was added to some patients’ samples (p <0.001, Kruskal–Wallis followed by Dunn's test).

Fig. 3

Effects of specific inhibitors of peroxynitrite on the detection of ROS in whole blood from septic patients. The results are from two representative patients.

Fig. 4

Detection of insoluble myeloperoxydase (MPO) in septic patients’ and blood. The MPO inhibitor of sodium azide was added to some samples (p < 0.0001, Kruskal–Wallis followed by Dunn's test).

Fig. 5

ROS production by J774 macrophages during contact with erythrocytes, as measured by chemiluminescence. (A) ROS production by macrophages in the presence of erythrocytes from septic patients. The SOD inhibitor of superoxido was added to some samples. (B) Basal photon counts/second observed when J774 macrophages were put into contact with erythrocytes of healthy volunteers. (C) ROS production by macrophages during contact with erythrocytes that had been oxidatively modified in vitro by treatment with peroxynitrite. The results are representative of three patients.

Fig. 6

ROS production by neutrophils during contact with erythrocytes, as measured by chemiluminescence. (A) ROS production by neutrophils in the presence of erythrocytes from septic patients. The SOD inhibitor of superoxido was added to some samples. (B) ROS production by neutrophils in the presence of erythrocytes from healthy volunteers. (C) ROS production by neutrophils during contact with erythrocytes that had been oxidatively modified in vitro by treatment with peroxynitrite. The SOD inhibitor of superoxido was added to some samples. The results are representative of three patients.

Fig. 7

Visualization by scanning electron microscopy of the phagocytosis of erythrocytes by J774 macrophages. Macrophages and erythrocytes were incubated for 1 h at 37 °C and then fixed with Karnovsky. (A) Adhesion and phagocytosis of oxidatively modified erythrocytes from a septic patient by J774 macrophages. (B) Erythrocytes from healthy volunteer in presence of J774 macrophages.

Fig. 8

Visualization by scanning electron microscopy of phagocytosis of erythrocytes by neutrophils. The cells were incubated for 1 h at 37 °C and then fixed with Karnovsky. (A) Phagocytosis of oxidatively modified erythrocytes by neutrophils from a septic patient. (B) Erythrocytes from healthy volunteer in presence of neutrophils.