Functional heterogeneity of anti-endothelial cell antibodies

Abstract

While it has been claimed that some anti-endothelial cell antibodies (AECA) activate EC, there is also evidence that others trigger apoptosis. To address the issue of whether activation is a prerequisite for AECA-mediated apoptosis of EC, 23 AECA-positive sera were evaluated for their ability to induce activation and/or apoptosis. Activation was defined as an over-expression of E-selectin and intercellular adhesion molecule 1. Optical microscopy, annexin V binding, hypoploid cell enumeration, and determination of poly (ADP-ribose) polymerase cleavage-related products were used to assess apoptosis. Four functional profiles were defined: 10 sera promoted activation and apoptosis (act+/apo+), one was act+/apo-, six act-/apo+, and the remaining six act-/apo-. The reduced membrane expression of thrombomodulin was associated with apoptosis, rather than activation. Caspase-3 was implicated in the two models of apoptosis, the ratios of several survival proteins to Bax 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.

Fig. 1

Endothelial cells (ECs) undergo apoptosis following incubation with some, but not all, anti-EC antibodies (AECAs). Human umbilical vein ECs were incubated with 160 µg/ml of AECAs or control IgG. a, A first aliquot was stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI). Apoptotic ECs were identified as annexin V+/PI-. b, A second aliquot was treated with PI following permeabilization. Hypoploid cells were considered apoptotic. Representative examples of apoptosis-inducing (patient 1) and nonapoptosis-inducing AECAs (patient 9) are presented.

Fig. 2

Apoptosis-inducing anti‐endothelial cell (ECs) antibodies show the potential to cleave poly (ADP) ribose polymerase (PARP). Following a 6-h incubation with patient or control IgG, EC were disrupted, and the cellular extracts loaded on a 10% polyacrylamide gel, electrophoresed and transferred to a nitro-cellulose membrane. PARP (116 kDa) and its 85 kDa apoptosis-related fragment were blotted using a specific monoclonal antibody.

Fig. 3

Phagocytosis of endothelial cells (ECs) incubated with anti-EC antibodies (AECAs). 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 (MØs) for 3 h at 37°C. (A) In one aliquot, MØs were stained with phycoerythrin (PE)-conjugated anti-CD11b monoclonal antibody (mAb), and ECs dyed green with PKH67-GL coupled with fluorescein isothiocyanate (FITC) before examination by flow cytometry. (B) In another aliquot, MØs were stained with unconjugated anti-CD11b mAb revealed by a tetramethyl rhodamine isothiocyanate-labelled goat F(ab′)₂ antimouse IgG, while ECs were dyed green as above. These cells were examined by confocal laser microscopy.