α‐haemolysin from Escherichia coli induces shrinkage and phagocytosis of erythrocytes by THP‐1 monocytes

The lysis produced by α‐haemolysin (HlyA) from by E. coli is surprisingly preceded by a volume reduction.(1) This volume reduction is a consequence of an increase in the intracellular Ca 2+ concentration, which triggers activation of the K + channel K Ca 3.1 and Cl − channel TMEM16A. These events are associated with phosphatidyl serine (PS)‐exposure, which is a signal for clearance of erythrocytes (RBCs) by monocytes from the blood stream. Calcein loaded RBCs were incubated with or without HlyA, and added to THP‐1 cells on coverslips. Phagocytosis was measured as calcein positive THP‐1 cells after 60 min. RBCs exposed to ionomycin was used as positive controls. Addition of HlyA and ionomycin both significantly increased the phagocytosis of RBCs with 126 % and 120% respectively. The uptake of RBCs in THP‐1 cells was verified by immunocytochemistry using an anti‐haemolglobin antibody. By this method we could visualize the volume reduced RBC inside the THP‐1 cell. HlyA is known to trigger ATP release, but the HlyA and ionomycin induced phagocytosis was not affected by extracellular ATP, neither did ATP by itself trigger RBC phagocytosis by THP‐1 cells. These data suggest that HlyA is a strong signal for phagocytosis, which potentially will allow clearance of RBCs before substantial hemolysis is evident during gram‐negative sepsis. This project was funded by The Danish Council for Independent Research, Medical Sciences.