Complement activation by apoptotic endothelial cells following hypoxia/reoxygenation

• Published in: Immunology Vol. 102; no. 3; pp. 359 - 364
• Main Authors: Mold, C., Morris, C. A.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.03.2001; Wiley Subscription Services, Inc; Blackwell Science Inc
• Subjects: Amino Acid Chloromethyl Ketones - pharmacology; Apoptosis - immunology; Caspase Inhibitors; Cell Culture Techniques; Cell Hypoxia - immunology; Complement C3 - metabolism; complement component C3; Complement Pathway, Classical - drug effects; Complement Pathway, Classical - immunology; Cysteine Proteinase Inhibitors - pharmacology; Endothelium, Vascular - immunology; Endothelium, Vascular - pathology; Humans; Immunoglobulin M - metabolism; Original; Reperfusion Injury - immunology
• ISSN: 0019-2805; 1365-2567
• DOI: 10.1046/j.1365-2567.2001.01192.x

Summary Reperfusion of ischaemic tissue initiates an inflammatory reaction that increases tissue injury. Complement activation at the endothelium contributes to this inflammation. 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 fluorescein 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, fluorescent 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 inflammatory response as well as clearance and repair.