Endotoxin-induced adhesion of human red blood cells to vascular endothelium does not depend on the presence of leukocytes but is modified by different flow pattern

Introduction

Endotoxin-induced (ETX) adhesion of leucocytes to vascular endothelial cells is a well investigated phenomenon. However, little is known about the effects of endotoxin on erythrocyte-endothelial cell interactions . The objective of this study was to investigate the effects of ETX on adhesion of human red blood cells (RBC) to human vascular endothelial cells (HUVEC) in the presence or absence of leukocytes and under different conditions of flow.

Methods

Endothelial cells were obtained from human umbilical veins and cultured as first and second passage monolayers and then grown to complete confluency on cover slips. RBC were harvested from fresh blood donated by healthy volunteers, washed with isotonic NaCl and resuspended to a hematocrit of 30% in medium (M199, GIBCO, Canada). Group A (n = 7) served as a control, whereas in group B, C and D (n = 7 in each) both RBC and HUVECs were incubated with ETX (75 μg/ml) at 37°C for 2 h. In group E whole blood was incubated with ETX and thereafter RBC were isolated as described above. The HUVEC coated cover slips were placed in a flowing chamber and perfused with the RBC suspensions at a flow rate of 0.65 ml/min in group A, B and E and 1.3 ml/min in group C. In group D stop and go flow patterns found in the microcirculation of septic animals were mimicked by applying a flow rate of 0.65 ml/min with four stops (4–6 s) per minute. The flow chambers were arranged on a microscope and connected to a video recording system. In each experiment 15–20 sites of a defined area were recorded and analysed.

Results

The control group showed an adhesion of 71 ± 8 cells/mm². Incubation of HUVECs and RBC with ETX increased RBC adhesion in group B to 172 ± 25 cells/mm² (P < 0.05). Incubation of whole blood including leucocytes with ETX did not exhibit a different degree of adhesion compared to group B. When flow rate was elevated to 1.3 ml/min, the number of adherent RBC decreased to 89 ± 20/mm² comparable to the control group with a flow rate of 0.65 ml/min. In group D with intermittent stops of flow, RBC adhesion increased to 274 ± 35 cells/mm² (P < 0.05) compared to control, group B and group C.

Conclusion

Incubation of RBC and HUVECs with endotoxin promoted erythrocyte adhesion to human vascular endothelium. The presence of leukocytes during the endotoxin exposure did not affect the degree of adhesion. Elevating flow rate, however, reduced erythrocyte adhesion, while stop and go flow pattern favoured erythrocyte adhesion. These findings suggest that altered RBC may contribute to the microcirculatory injury observed in sepsis.