title: Anti-endothelial cell antibodies from lupus patients bind to apoptotic endothelial cells promoting macrophage phagocytosis but do not induce apoptosis.
abstract: OBJECTIVE
  Anti-endothelial cell antibodies (AECA) have been reported to induce apoptosis. We investigated the induction of apoptosis by these autoantibodies and their involvement in the removal of apoptotic cells.
  
  
  METHODS
  AECA isolated from patients with active systemic lupus erythematosus (SLE) were incubated with human umbilical vein endothelial cells (HUVECs). AECA-positive sera were identified using a cell-based ELISA. Apoptosis was measured by morphology and phosphatidylserine externalization using flow cytometry with fluorescein isothiocyanate (FITC)-conjugated annexin V. Flow cytometry was used to investigate AECA binding to apoptotic cells using FITC-conjugated anti-human immunoglobulin G (IgG). Apoptotic endothelial cells were stained with a red dye (PKH26) and co-cultured with macrophages, and phagocytosis was visualized under phase contrast microscopy.
  
  
  RESULTS
  AECA from patients with SLE did not induce apoptosis compared with normal IgG (nIgG) at any time point, as assessed by morphology (at 24 h, P = 0.167) or phosphatidylserine externalization (at 24 h, P = 0.098). However, there was increased binding of AECA to apoptotic endothelial cells (48.8 +/- 11.9 compared with 25.8 +/- 6.7% AECA binding to freshly isolated cells, P< 0.001). These opsonized endothelial cells showed greater phagocytosis by macrophages (mean phagocytic index 24.9 +/- 4.5%) when cells opsonized with nIgG were compared with AECA (34.8 +/- 3.4% n = 5, P = 0.01).
  
  
  CONCLUSIONS
  In conclusion, AECA bind to apoptotic endothelial cells but do not induce endothelial cell apoptosis. Macrophage phagocytosis is increased by opsonization of apoptotic endothelial cells by AECA, a proinflammatory mechanism of cell removal.
authors.affiliation: Division of Immunity and Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: J M
authors.initials: JM
authors.lastname: Williams
authors.forename: R
authors.initials: R
authors.lastname: Colman
authors.forename: C J
authors.initials: CJ
authors.lastname: Brookes
authors.forename: C O
authors.initials: CO
authors.lastname: Savage
authors.forename: L
authors.initials: L
authors.lastname: Harper
mesh_terms: D000293:Adolescent; D000328:Adult; D017209:Apoptosis / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D004306:Dose-Response Relationship, Immunologic; D042783:Endothelial Cells / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D008264:Macrophages / Q000276:immunology; D008297:Male; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D010587:Phagocytosis / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D007074:Immunoglobulin G; C087589:anti-endothelial cell antibody