ECs were incubated with AECAs categorized on the basis of their ability to activate (act +) and/or trigger apoptosis (apo+), and cocultured with monocyte-derived macrophages (M?s) for 3 h at 37C. with apoptosis, rather than activation. Caspase-3 was implicated in the two models of apoptosis, the ratios of several survival proteins to decreased, regardless of the ability of apo+ AECA to activate the cells, while radical oxygen species did not appear to be involved. Furthermore, it occurred that macrophages engulfed EC treated with apoptosis-promoting AECA, but not those incubated with AECA that did not induce apoptosis. Hence, AECA represent an extremely heterogeneous family of autoantibodies, not only because of the variety of their target antigens, but also the subsequent diversity of their effects. pAbs were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other reagents and cells Annexin V coupled with FITC was from Immunotech, and interleukin (IL)-1 from Genzyme (Cambridge, MA, USA), while saponin, propidium iodide (PI), PK67-GL, cycloheximide (CHX) and polymyxin B were all from Sigma. K12D31m4-derived lipopolysaccharide (LPS) was a kind gift of S. Chesne (Grenoble, France). EA.hy926 cells were grown in Dubelcco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, plus 2 m glutamine, and containing 100 m hypoxanthine, 04 m aminopterine and 16 m AN3199 thymidine (Sigma). HUVECs were isolated as described previously , and suspended in Iscove’s DMEM (Gibco, Paisley, UK). To evaluate the effects of AECA, these HUVECs were passaged twice and used as target cells instead of the EA.hy926 cells. Induction and assessment of activation HUVECs were harvested from the second-passage culture, and suspended at AN3199 a concentration of 104 cells in 100 l Iscove’s DMEM supplemented with 10% FCS and 100 IU/ml polymyxin B. They were distributed into three duplicate wells of a 96-well microtitre plate (Nunc, Roskilde, Denmark). AECA IgG or control IgG were all adjusted to a concentration of 320 g/ml in the culture medium. One hundred l AN3199 was added to the wells to reach a final concentration of 160 g IgG per ml. On each plate, separate wells contained 100 l of medium plus 100 l of 100 IU/ml of IL-1, as an internal positive control for activation. After a 6-h incubation at 37C for E-selectin and ICAM-1 Actb expression and a 24-h incubation for that of TM, cells were harvested using 025% trypsin-EDTA (Sigma). Those from duplicate wells were pooled and washed with PBS supplemented with 1% bovine serum albumin (PBS-BSA). There were thus three aliquots of cells. The first and the second were stained with saturating amounts of FITC-conjugated anti-ICAM-1 and FITC-conjugated anti-TM mAbs, respectively, while the third aliquot was treated with unconjugated anti-E selectin mAb, washed twice with PBS-BSA, and the mAb binding revealed with FITC-conjugated goat F(ab)2 anti\mouse IgG. Following two final washes with PBS-BSA, all the cell preparations were examined in a flow cytometer (Coulter Immunology, Hialeah, FL, USA). Irrelevant FITC-conjugated mouse IgG mAbs were used as a negative control. ICAM-1, E-selectin and TM antigen densities were indirectly measured by assessing the mean fluorescence intensity (MFI) of cells analysed in each test. The results were expressed as an MFI variation which was equal to 100 (AECA IgG-treated cells MFI C control IgG-treated cells MFI)/control IgG-treated cells. Identification of apoptotic cells Time-course and dose-effect curves were conducted to settle the optimal conditions for AECA-induced apoptosis, as reported previously [11,18]. Following a 24-h incubation of ECs with 320 g/ml of control or AECA IgG, apoptosis was documented by four methods. The phosphatidylserine (PS) translocation to the outer face of the membrane was established through the binding of annexin V coupled with FITC. This first procedure has been also described elsewhere [11,18]. PI was used to exclude dead cells, diluted to 10 g/ml. Percentages of annexin V-positive cells were thus calculated within the PI-negative AN3199 population of cells The cut-off level was set at 22%, i.e. the mean +?3 SD of five normal sera. Hypoploid cell enumeration was the second method. The technique was that described by Nicoletti . Briefly, the cell suspension was washed in citrate buffer (01 m sodium citrate, 01% Triton X-100), and incubated in 250 l citrate buffer made up of 10 g/ml PI overnight at 4C in the dark. Reduction in PI staining intensity compared.