Activated granulocyte induced alterations in red blood cells and protection by antioxidant enzymes

Abstract

Activated leukocytes generate oxygen free radicals and cause injury in a variety of surrounding cells. Red blood cells (RBC) are among the most susceptible cells to this injury. Deformability, lipid peroxidation and membrane proteins were investigated in RBC after incubation with activated granulocytes (2 hours, at 37 QC), obtained from guinea pigs with experimental sepsis. A second group of experiments was conducted with non-activated granulocytes isolated from blood of healthy guinea pigs. RBC transit time through S 11m pores measured by a cell transit analyzer was 3.2S±0.12 msec and 2.86±0.02 msec in the suspensions containing activated and non-activated granulocytes, respectively (p<O.OS). Addition of superoxide dismutase (SOD) (20 Ilg/ml) and catalase (40 Ilg/rnl) inhibited the effect of activated granulocytes on RBC deformability. Lipid peroxidation was also increased under the influence of activated granulocytes (31.S0±3.36 nmol/grHb versus 20.02±2.97 nmol/grHb); SOD failed to prevent this increment, while catalase was found to be effective. A widening of Band 1 (spectrin) was observed in SDS-PAGE electrophoresis of membrane proteins of RBC incubated with activated granulocytes, indicating spectrin-hemoglobin crosslinking. This alteration was prevented by both SOD and catalase, as in the case of transit time. The superoxide radical seems to be responsible for the membrane protein and mechanical alterations, while lipid peroxidation was induced by a different oxidative mechanism.