Complement activation by apoptotic endothelial cells following hypoxia/reoxygenation

Abstract

SUMMARYReperfusion of ischaemic tissue initiates an in¯ammatory reaction that increases tissue injury. Complement activation at the endothelium contributes to this in¯ammation. This study investigated the mechanism of complement activation following reoxygenation of hypoxic human umbilical vein endothelial cells (HUVEC) as a model for complement activation observed on endothelium in reperfused ischaemic tissue. HUVEC cultured in 1% oxygen followed by reoxygenation activated the classical complement pathway resulting in C3 deposition. There was an increase in apoptotic cells in these cultures that was demonstrated by binding of¯uorescein isothiocyanate-Annexin V and staining for hypodiploid nuclei. To determine if apoptotic HUVEC activate complement, uniformly apoptotic cells were produced by serum and growth factor deprivation. These cells, but not the control HUVEC, activated the classical complement pathway in the absence of antibody or other serum factors. To determine if apoptotic cells in the reoxygenated cultures were activating complement,¯uorescent analysis was done. Annexin V binding and C3d deposition on cells from reoxygenated cultures showed complete concordance on the subpopulation of apoptotic cells. In addition, complement activation following reoxygenation of HUVEC was eliminated by treatment of the cultures with a caspase inhibitor during reoxygenation. These results suggest that oxidative damage to endothelial cells during reoxygenation initiates apoptosis with exposure of phosphatidylserine. Apoptotic cells directly activate the classical pathway of complement by binding C1. Activation of complement at the endothelium may contribute to the in¯ammatory response as well as clearance and repair.

Defects in the disposal of dying cells lead to autoimmunity

“…The importance of complement in the scavenging process is becoming more and more evident. Complement activation by apoptotic cells, eg, endothelial cells, lymphocytes, and polymorphonuclear cells (PMN) was just recently reported by several groups independently [23,40,41]. Furthermore, an impaired uptake of apoptotic cells by human macrophages was to be observed in human serum depleted of specific complement components (C1q, C3) [20,42,43].…”

Preservation of Amputated Extremities by Extracorporeal Blood Perfusion; a Feasibility Study in a Porcine Model

“…Shedding of the endothelial glycocalyx, the anticoagulant and anti-inflammatory surface coating of healthy endothelial cells, has been identified as an important factor in endothelial I/R injury [49]. In addition, numerous studies have shown that the I/R-related endothelial damage is essentially dependent on complement [50,51], which is activated either directly by ischemiainduced changes on the endothelial surface, including apoptotic EC [52] or, according to data from animal models, by natural antibodies that bind to the ischemically modified EC surface [53,54]. The above presented results indicate that vascular leakage due to endothelial cell activation and damage was low, and that the endothelial function was essentially preserved throughout the perfusion.…”

The non-classical functions of the classical complement pathway recognition subcomponent C1q

“…Dormant cultured ECs do not have the potential to activate complement but the endothelium does exhibit anti- inflammatory properties that are maintained by cell surface and plasma complement inhibitors [68,69]. Trophoblasts are cells that facilitate the implantation of the embryo into the endometrium.…”