Complement activation by apoptotic endothelial cells following hypoxia/reoxygenation

Reperfusion of ischaemic tissue initiates an inflammatory reaction that inc reases tissue injury. Complement activation at the endothelium contributes to this inflammation. This study investigated the mechanism of complement a ctivation following reoxygenation of hypoxic human umbilical vein endotheli al cells (HUVEC) as a model for complement activation observed on endotheli um in reperfused ischaemic tissue. HUVEC cultured in 1% oxygen followed by reoxygenation activated the classical complement pathway resulting in C3 de position. There was an increase in apoptotic cells in these cultures that w as demonstrated by binding of fluorescein isothiocyanate-Annexin V and stai ning for hypodiploid nuclei. To determine if apoptotic HUVEC activate compl ement, uniformly apoptotic cells were produced by serum and growth factor d eprivation. These cells, but not the control HUVEC. activated the classical complement pathway in the absence of antibody or other serum factors. To d etermine if apoptotic cells in the reoxygenated cultures were activating co mplement. fluorescent analysis was done. Annexin V binding and C3d depositi on on cells from reoxygenated cultures showed complete concordance on the s ubpopulation of apoptotic cells. In addition, complement activation followi ng reoxygenation of HUVEC was eliminated by treatment of the cultures with a caspase inhibitor during reoxygenation. These results suggest that oxidat ive damage to endothelial cells during reoxygenation initiates apoptosis wi th exposure of phosphatidylserine. Apoptotic cells directly activate the cl assical pathway of complement by binding C1. Activation of complement at th e endothelium may contribute to the inflammatory response as well as cleara nce and repair.