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Clin Exp Immunol. Jun 2001; 124(3): 492–501.
PMCID: PMC1906088

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.

Keywords: endothelial cell, anti-endothelial cell antibody, apoptosis, activation

INTRODUCTION

Endothelial cells (EC), lining the vasculature, have long proved to be a target for immune-mediated assaut [1], conceivably through the so-called anti-EC antibodies (AECA). These have since been detected in such an impressive diversity of conditions associated with widespread vasculitis (reviewed in [2]) that they certainly represent a heterogeneous family of autoantibodies. As a corollary, the antigens recognized by AECAs may be infered to be multiple, despite discrepancies between the results possibly due to differences between the assays.

Obviously, the presence of AECAs in vasculitides does not necessarily imply causation since their production may follow rather than precede EC damage [3]. There is, however, compelling evidence that, in a way, they contribute to the pathophysiology of these diseases, though no definite demonstration sustaining such a hypothesis has emerged. At this time, the most persuasive argument has indeed come from the development of an idiotypic experimental model of systemic vasculitis [4]. In fact, there seems to be numerous, although not mutually exclusive, possibilities. Recent speculation has focused on EC activation type II [5]. That is up-regulation of adhesion molecules, such as E-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule-1 [6,7]. Owing to the concomitant production of chemokines by EC, this enhanced adhesion molecule expression facilitates the recruitement and the ensuing attachment of monocytes as well as polymorphonuclear neutrophils to the inflamed vessel walls [7,8]. Interestingly, thrombomodulin (TM) which is a high-affinity thrombin receptor present on the EC membranes has also been found to be released in the culture supernatant [9] or the serum [10] following endothelial inflammatory damage. This might be associated with activation and reinforced by apoptosis. Another appealing property of some but not all AECAs is to encourage ECs to undergo programmed cell death (PCD), also referred to as apoptosis [11]. Presumably, this process leads to phagocytosis of apoptotic ECs (reviewed in [12]). Yet, it is not appreciated to what extent activation constitutes a prerequisite for AECA-mediated apoptosis of ECs.

These combined observations provide support for the postulate that AECAs are also functionnaly heterogeneous, most probably depending on their specificity. This is the standpoint from which we investigated whether 23 AECA-positive sera displayed any distinguishing effects. AECA-induced activation of the cells, and AECA-mediated apoptosis were evaluated, and the AECAs categorized on the basis of positivity for the related assays.

MATERIALS AND METHODS

Source of sera

Serum samples were forwarded to us by the Department of Internal Medicine and the Department of Rheumatology of the Brest University Hospital to be routinely tested for the presence of AECAs, using a cell-ELISA recently developed in our laboratory and described in great detail elsewhere [13]. The EA.hy926 hybrid cells (kindly donated by C.J.S. Edgell, University of North Carolina, Chapel Hill, NC) were used as the substrate, and 100 µl/l foetal calf serum (FCS) added to the patient diluent samples to obviate artefactual recognition by heterophile antibodies of bovine serum proteins bound to the cells. Because of the notorious limitations of all the AECA assays [14], positive results were confirmed by fluorescence-activated cell sorter (FACS) analysis [15] with unfixed human umbilical vein ECs (HUVECs), and EA.hy926 cells as substrates. Fluorescein isothiocyanate (FITC)-conjugated rabbit F(ab′)2 antihuman IgG (Dakopatts, Glostrup, Denmark) was the revealing agent for FACS analysis.

A total of 23 AECA-containing sera were selected on the basis of positivity in the three assays (cell-ELISA, FACS with HUVECs and FACS with EA.hy926 cells) and maintained at − 70°C until used. These were from eight patients with systemic lupus erythematosus (SLE), three with primary Sjögren's syndrome (pSS), three with venous thrombosis, and eight with a variety of disorders, one each, primary antiphospholipid syndrome, multiple sclerosis, rheumatoid arthritis, thrombotic thrombocytopenic purpura, Evans' syndrome, multiple myeloma and idiopathic purpura. All those patients with SLE and pSS fulfilled the proposed criteria for the respective diseases [16,17]. Additionaly, four patients were evaluated twice, one at 2-month interval, another at 5-month interval, and the remaining two at 2-year interval. Sera were also obtained from healthy control subjects, consisting of Departmental staff.

Polyclonal and monoclonal antibodies

To remove any contaminating antiphospholipid (PL) antibody (Ab), all the AECA-containing sera also found to be positive in the anti-PL IgG-specific ELISA were passed through a column of PLs, as previously reported [11]. The flow-through sera were collected and dialysed against phosphate-buffered saline (PBS). IgG fractions from these sera, as well as those of anti-PL Ab-negative patients and normal controls, were purified with protein G-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden) and recovered with HCl-glycine. The concentration of IgG was determined by a an in-house sandwich ELISA and equalized in all preparations.

Unconjugated monoclonal Abs (mAbs) against E-selectin, FITC-conjugated mAb against ICAM-1, phycoerythrin (PE)-conjugated and unconjugated mAbs against CD11b and control FITC-conjugated mouse mAbs were from Immunotech (Marseille, France), while, FITC-conjugated and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat F(ab′)2 antimouse IgG were obtained from Tago (Burlingame, CA, USA), FITC-conjugated anti-Bcl-2 and PE-conjugated anti-TM mouse mAbs from Dakopatts, FITC-conjugated F(ab′)2 goat anti‐rabbit polyclonal Ab (pAb) and control unconjugated polyclonal rabbit IgG from Jackson ImmunoResearch (West Grove, PA, USA), FITC-conjugated anti‐goat IgG mouse mAb from Sigma Chemical Co. (St Louis, MO, USA), and unconjugated goat anti‐human A1, rabbit anti‐human BclXL, rabbit anti‐human Mcl-1 and rabbit antihuman Bax 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. Escherichia coli 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 µm glutamine, and containing 100 µm hypoxanthine, 0·4 µm aminopterine and 16 µm thymidine (Sigma). HUVECs were isolated as described previously [11], 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 a concentration of 104 cells in 100 µl 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 µg/ml in the culture medium. One hundred µl was added to the wells to reach a final concentration of 160 µg IgG per ml. On each plate, separate wells contained 100 µl of medium plus 100 µl of 100 IU/ml of IL-1, as an internal positive control for activation. After a 6-h incubation at 37°C for E-selectin and ICAM-1 expression and a 24-h incubation for that of TM, cells were harvested using 0·25% 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 – 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 µg/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 µg/ml. Percentages of annexin V-positive cells were thus calculated within the PI-negative 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 et al. [19]. Briefly, the cell suspension was washed in citrate buffer (0·1 m sodium citrate, 0·1% Triton X-100), and incubated in 250 µl citrate buffer containing 10 µg/ml PI overnight at 4°C in the dark. Reduction in PI staining intensity compared with control cells by flow cytometry was taken as a measure of hypoploidy. Again, the threshold was set at the mean ± 3 SD of five normal sera, i.e. 13%. AECA IgG found to induce both the exposure of PS and hypoploidy were defined as pro-apoptotic.

Using the 23 sera, apoptosis was confirmed by quadruplicate analysis of the morphology of the cells centrifuged for 1 minute at 300 g on a microscope slide and stained in a May-Grünwald-Giemsa solution (Merck, Darmstadt, Germany) as a third method for assessment by morphology of apoptotic cells. Percentages of cells exhibiting a typical morphology of apoptosis were evaluated and the cut-off level established at 14% (mean + 3 SD of the five normal sera). Because poly (ADP-ribose) polymerase (PARP) is one of the earliest substrate to be cleaved in apoptotic cells [20], the fourth method we used was Western blotting using anti-PARP mAb (Biomol Research Laboratories, Plymouth Meeting, PA) to identify 116 kD PARP and the 85 kD apoptosis-related cleavage fragment. Following incubation with AECA IgG from five sera or control IgG, and after three washes with PBS-BSA, HUVECs were suspended at 107 cells/ml in sample buffer (62·5 mm Tris HCl, pH 6·8, 6 m urea, 10% glycerol, 2% sodium dodecyl sulphate, SDS, 0·00125% bromophenol blue, and 5% β–mercaptoethanol), and incubated for 15 min at 65°C. Cellular extracts were loaded on a 10% polyacrylamide gel, and electrophoresed at 20 mA in a running buffer (0·2 m glycine, 0·025 m Tris, and 0·1% SDS). Proteins were transferred to a nitro-cellulose membrane by semidry electroblotting. The sheet was blocked for 1 h with 5% nonfat dry milk in Tris buffer (50 mm Tris HCl, pH 7·4, 150 mm NaCl, and 0·1% Tween 20). Strips of nitrocellulose were then incubated with 1 µg/ml anti-PARP mAb, washed three times in Tris buffer, incubated with horseradish peroxidase-conjugated goat antimouse IgG (Dakopatts) diluted 1/3000 in the same buffer for one hour, and washed another three times. Colour was developed by incubation with diaminobenzidine and H2O2.

Characterization of AECA-induced apoptosis

To evaluate involvement of protein synthesis in apoptosis, CHX was added to the cell culture at a concentration of 2 µg/ml. This concentration had been previously set in pilot experiments using 10 µg/ml LPS to activate ECs, along with increasing amounts of CHX to define the dose noncytotoxic but still able to prevent the synthesis of E-selectin.

Among a number of cystein proteases called caspases [21], caspase-3 appears to stand at the crossroads of most of the cell death effector pathways. Our approach to assess the contribution of this molecule in AECA-triggered apoptosis was the use of Z-DEVD-FMK (R & D Systems Europe, Abingdon UK) which is a peptide binding preferentially to caspase-3, such that its activity is inhibited. Following two passages, HUVECs were further incubated with 100 µm Z-DEVD-FMK. The cells were then trypsinized, washed in PBS, and examined for apoptotis. This was carried out using the same annexin V binding method as above.

Given that the generation of reactive oxygen species (ROS) has also been shown to mediate apoptosis in numerous cells including EC [22], hydrogen peroxide and superoxide anion were then measured in EC following incubation with AECA. The method described by Carter et al. [23] was applied with minor modifications of our own. Briefly, dihydroethidium and 2′,7′-dichlorodihy-drofluorescein diacetate (Molecular Probes, Leiden, The Netherlands) were both used at 5 µm final concentration. HUVEC were incubated with either probe in several rows of triplicate wells containing AECA at 160 µg/ml final concentration. After various periods of time (15, 30, 60, 120 and 240 min), cells were collected by trypsin-EDTA, and washed with PBS for flow cytometry analysis. A dose of 200 µm H2O2 was used as a positif control.

Assessment of apoptosis regulators

Next, several apoptosis regulators [24] were evaluated. Following a 24-h treatment with AECAs or control IgG, HUVEC were collected and thoroughly washed in PBS. Aliquots of 2·104 cells were permeabilized by incubation in 250 µl PBS containing 1% FCS, 0·1% NaN3 and 0·25% saponin for 30 min at 4°C. Cells were then washed twice in PBS containing 1% FCS, 0·1% NaN3 and 0·1% saponin.

Fifty µl of HUVEC suspension was mixed with 5 µl rabbit anti-BclXL, anti-Bax, anti-Mcl-1 pAb, or with 10 µl goat anti-A1 pAb, or control rabbit IgG. After a 30-minute incubation at 4°C and two washes, FITC-conjugated antirabbit IgG goat pAb or FITC-conjugated antigoat mouse mAb were utilized as second-layer Abs. Another two aliquots of permeabilized cells were directly stained with FITC-conjugated anti-Bcl-2 mAb, incubated with isotype-matched non reactive FITC-labelled mAb, FITC-conjugated antirabbit IgG goat pAb or FITC-conjugated antigoat mouse mAb. After two rinses in the same buffer, all these cell preparations were analysed by flow cytometry, and the MFI of the intracellular proteins was evaluated.

Measurement of phagocytosis

Peripheral blood mononuclear cells were isolated from the blood of healthy volunteers by Ficoll-Hypaque density gradient centrifugation. Monocyte-derived macrophages (MØ) were prepared as described by Musson [25]. Briefly, mononuclear cells were suspended in Iscove's DMEM, and 8·104 cells distributed into Sonic Seal wells (Poly Labo, Strasbourg, France). They were allowed to adhere to polystyrene for 2 h at 37°C. Non-adherent cells were washed away using the same medium, whereas adherent cells were cultured for another 10 days in Iscove's DMEM supplemented with 10% autologous serum. This culture medium was renewed on the third day.

In parallel, 6 × 104 fresh HUVECs were suspended in 100 µl Iscove's DMEM supplemented with 10% FCS, and the wells flooded with 100 µl of 320 µg/ml AECAs or control IgG (final concentration 160 µg/ml). Following a 24-h incubation, HUVECs were harvested, washed three times with serum-free Iscove's DMEM, dyed green with PKH67-GL for 5 min at room temperature, and washed again three times with Hank's balanced salt solution (HBSS).

Each AECA-treated HUVEC preparation was transferred into two MØ wells and cocultured with these MØ at a ratio of 1 : 1 for 3 h at 37°C. Non-adherent cells were then removed by three washes with HBSS. The rest were collected from the first well and washed three times with PBS-BSA. MØ were stained with PE-conjugated anti-CD11b mAb for 30 min at 4°C. Phagocytosis was evaluated by flow cytometry, and determined as the percentage of HUVEC-containing MØs, according to the formula: 100 × (CD11b-positive/PKH67-GL-positive cells, divided by CD11b-positive/PKH67-GL-positive + CD11b-positive/PKH67-GL-negative cells). The MØs cultured in the second well were stained with unconjugated anti-CD11b mAb, followed by a TRITC-conjugated goat antimouse IgG. After two washes with PBS-BSA, they were examined by confocal laser microscopy, using a Leica TCS 4D (Leica Lasertechnik, Heidelberg, Germany).

Statistics

All the figures quoted below are individual values or arithmetic means (standard deviation (SD). Comparisons were made using the Mann–Whitney U-test for unpaired data, and correlations established using the Spearman's rank correlation test.

RESULTS

Effects of AECA

Activation of EC

IgG from 11 of the 23 AECA-containing sera (47·8%) reproducibly enhanced the expression of E-selectin (Table 1) from 61% (serum 21) through 217% (serum 9), as well as that of ICAM-1 from 29% (serum 21) through 115% (serum 1), compared with 620% for E-selectin and 103% for ICAM-1 when the cells had been incubated with IL-1. Taking the positive AECA IgG as a whole, the increased MFI of E-selectin paralleled that of ICAM-1 (r = 0·70, P < 0·01), suggesting that the cells were activated. It is notable that the AECA IgG-induced activation of the cells was transient, and the expression of E-selectin returned to normal after 24 h in culture (the MFI declined from 11·70 to 0·21 in patient 9, and from 11·40 to 0·62 in patient 23, compared with a reduction from 17·50 to 7·35 following a 12-h incubation with IL-1). In contrast, IgG from the remaining 12 sera failed to influence the adhesion molecule expression. There was no delayed effect in the sera originally characterized as nonactivating (sera 8 and 16 were evaluated after six hours and after 24 h in culture).

Table 1
Modulation of E-selectin, intercellular adhesion molecule (ICAM)-1 and thrombomodulin (TM) membrane expression following incubation of endothelial cells (ECs) with anti-EC antibodies (AECAs)

Apoptosis of EC

AECA IgG from 17 sera induced the translocation of PS, as established by the binding of annexin V, in comparison with control IgG (Table 2). Following interaction with these autoAb, the EC monolayer was disrupted, whereas it remained intact after exposure to control IgG. Apoptotic individual cells assumed a rounded appearance and were floating in the medium. Apoptosis was further confirmed by morphological features of stained HUVEC. There were correlations between the percentages of morphologically apoptotic cells on the one hand, those of annexin V-binding (P < 0·02) and hypoploid cells (P < 0·01) on the other. Sixteen of the 17 sera found positive in the annexin V-test committed 13–42% of the cells to become hypoploid. As a positive example, the effect of apoptosis-inducing AECA IgG from patient 1 is presented (Fig. 1), while that of AECA IgG from patient 9 was close to the level reached by the control IgG. Overall, 16 sera (69·6%) fulfilled the three criteria for the induction of apoptosis.

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 ...
Table 2
Some anti‐endothelial cell (EC) antibodies induce apoptosis of the cells

PARP is one of many proteins of which the cleavage completes the apoptotic process. The 85 kD apoptosis-related cleavage fragment of PARP was indeed identified by Western blotting, following incubation of the cells with act+/apo+ (see for example patient 1 in Fig. 2), as well as act-/apo+ AECA (patient 14 in Fig. 2), indicating that caspases operate whether HUVECs are activated or not by AECA prior to apoptosis.

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 ...

Expression of membrane TM

Since EC activation results in the loss of membrane TM (reviewed in [26]), variation of its expression was evaluated. As depicted in Table 1, the MFI of this molecule was reduced by 9 (sera 9 and 16) to 65% (serum 1). It remains unclear whether this expression declined due to the activation or apoptosis of AECA-injured EC, given that only one AECA-sample was able to induce activation but not apoptosis. Three converging arguments might go, however, to suggest that the reduction of TM was associated with apoptosis, rather than activation of the cells. Firstly, apoptosis-inducing IgG-treated HUVECs (n = 10) expressed less TM (MFI = 5·2 ± 2·7; 37·0 ± 11·3% stained cells; mean (SD) than those exposed to control IgG (n = 4; MFI = 10·9 ± 3·8, P < 0·02; 65·5 ± 6·6% stained cells, P < 0·001). In contrast, the amount of membrane TM (MFI = 10·7 ± 2·9; 66·6 ± 5·4% stained cells) was not modified by treatment of the cells with nonapoptotic AECAs (n = 3). Secondly, the lowered expression of TM correlated with annexin V binding (r = 0·79, P < 0·01) and hypoploidy (r = 0·61, P < 0·05), but not with up-regulation of E-selectin and ICAM-1 (r = 0·17 and 0·12, respectively). Thirdly, double staining of AECA-treated HUVECs with PE-conjugated anti-TM mAb and FITC-coupled annexin V (five experiments) revealed that as few of 17·0±11·1% of annexin V-positive cells carried detectable levels of TM, compared with 65·5±4·6% of annexin V-negative cells (P < 0·005).

Classification of AECA

Four AECA functional profiles could thus be defined (Table 3): 10 sera (referred to as act+/apo+) triggered activation and apoptosis, one was act+/apo-, six were act-/apo+, and the remaining six act-/apo-. Of two AECA-containing sera, originally categorized as act+/apo+ (patient 3) and act+/apo-(patient 9), both became act-/apo+ after a 2-year evolution. AECAs from patients 7 and 21, first classified as act+/apo+, remained unchanged four and five months later. Due to the extreme variability of the disorders, no relationship could be established between these functional profiles and the disease. Compare for example AECAs from eight patients with SLE: patients 2, 21 and 22 were act+/apo+, patient 9 was act+/apo-, patients 6 and 14 were act-/apo+, and patients 12 and 13 were act-/apo-. A clue to the clinical relevance of these autoAbs, however, that of five SLE patients with apoptotic AECAs, three had a severe vasculitis, while none of the three lupus patients without apoptotic AECAs suffered this complication.

Table 3
Four groups of anti‐endothelial cell autoantibodies

Mechanisms for induction of apoptosis

Several experiments were conducted, in an effort to determine whether there were different pathways of apoptosis for act+/apo+ AECAs and those act-/apo+ 0. The protein synthesis blocker CHX was applied to HUVECs sensitized with AECAs. The percentages of apoptotic and the subsequent proportions of intact cells (annexin V-negative/PI-negative) of these two samples were similar in the absence (49·3 ± 1·4 and 44·8 ± 1·4%; mean (SD of three separate experiments) and the presence of CHX (35·0 ± 2·5 and 39·0 ± 2·8%), indicating that protein synthesis was required in neither of these two models of apoptosis.

To address the issue of caspase-independent apoptosis [27], commitment of caspase-3 in act+/apo+ AECA-induced cell death, was compared with that due to act-/apo+ AECAs. The activity of this protease was blocked by a specific inhibitor. The percentages of annexin V-binding ECs were reduced by 26·5 ± 1·6 and 36·9 ± 5·1% when the cells had been incubated with IgG from patients 3 and 23, respectively (act+/apo+ sera). Similar results were obtained using act-/apo+ sera: 26·7 (1·5 and 13·3 ± 2·8% following incubation with IgG from patients 6 and 14). We then addressed whether hydrogen peroxide and superoxide anion were generated. Irrespective of their activating ability, AECAs did not favour at all the production of these ROS, compared with control values (data not shown).

Next we calculated the ratios of the cytosolic concentrations of Bcl-2, A1, BclXL and Mcl-1 which are survival proteins, to Bax which is a death promoter. As detailed in Table 4, Bcl-2/Bax, A1/Bax, BclXL/Bax and Mcl-1/Bax were similarly and significantly reduced, following incubation of HUVEC with act+/apo+ AECAs (n = 5) and act-/apo+ AECAs (n = 5), compared with act-/apo-AECA (n = 3) and control IgG (n = 5).

Table 4
Differential expression of the Bcl-2 family members in the cytosol of endothelial cells (ECs) following incubation of these cells with anti-EC antibodies (AECAs) of the activation (act)1/apoptosis (apo)+, the act-/apo+ and the act-/apo-categories, or ...

Phagocytosis of cells treated with AECA

To further specify the particularities of different populations of AECAs, engulfment of AECA-sensititized HUVEC by monocyte-derived MØs was investigated. Flow cytometric evaluation revealed that phagocytosis was related to the autoAb ability to trigger apoptosis, rather than activation or their binding to the cells (Fig. 3a). Following treatment with act+/apo+ or act-/apo+ IgG, HUVECs were detected in 91, 89, 87 and 86%, i.e. 88·3 ± 1·9% of MØs. In striking contrast, incubation with act+/apo-or act-/apo-IgG resulted in phagocytosis close to that induced by control IgG (47, 52, 51 and 50%, i.e. 50·0 ± 1·9 of MØs compared with 50, 51, 33 and 57% were positive, i.e. 47·8 ± 8·9: P < 0·05, compared with act ± /apo+, and p NS, compared the control IgG). To distinguish between binding and uptake of the HUVECs by MØs, the preparations were analysed by confocal laser microscopy (Fig. 3b). This method established that HUVECs had unequivocally been ingested by the phagocytes.

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 ...

DISCUSSION

The main message emerging from this study is that the diversity of AECA-associated conditions, not only refers to the hitherto unidentified antigen specificity of these autoAbs, but also denotes their different deleterious effects. Incubation of ECs with IgG from 11 of 23 sera caused the cells to display a proadhesive phenotype over a limited period of time. This phenomenon cannot be attributed to contaminating LPS [28] because all the experiments were performed in the presence of polymyxin B. Our finding that the excess of adhesion molecules was transient accords with the view that their expression declines in advanced vasculitis [29].

The ability of some AECAs to activate the cells was clearly irrelevant to the nature of the underlying disorder, given that, out of 11 patients, four had SLE, two presented with venous thrombosis, and the remainder suffered from various other diseases. Similar events have been described in AECAs from patients with a variety of conditions, e.g. Wegener's granulomatosis [6], SLE [7], scleroderma [8], and Takayasu arteritis [30]. These vasculitic syndromes represent, however, dissimilar entities inasmuch as Wegener's granulomatosis involves predominantly small blood vessels, whilst Takayasu arteritis affects primarily large arteries [31]. The fact that the vascular endothelium from vessels of different sizes and anatomical compartments exhibits distinct phenotypic properties [32] suggests that a vast array of EC membrane molecules might be critical targets for AECA-mediated activation. Furthermore, noteworthy is that four of the above AECA-positive SLE sera encouraged this occurrence, while another four samples failed to alter HUVEC adhesion molecule expression. In keeping with previous reports [7], it follows that AECAs vary markedly between patients with one and the same disease.

The same holds true regarding the induction of PCD, though as many as 16 of 23 AECA IgG promoted cell death. This effect appeared to be more commun that previously considered [11]. Intriguingly, two samples (sera 11 and 13) showed the potential to cause hypoploidy, but favoured only 22% of EC to bind annexin V. Though both had in common to be nonactivating, such a discrepancy awaits elucidation. It might result from some uncharacterized process, possibly resulting from the penetration of the Abs into the cells [33]. Conversely, serum 12 initiated the translocation of PS to the outer face of the plasma membrane, while little DNA fragmentation was detectable. Another argument sustaining this hypothesis is the finding that, once apoptosis has been triggered off, membrane phospholipid redistribution precedes nuclear changes [34]. Therefore, it is not unreasonable to assume that ECs may enter different apoptotic programmes, implying that distinct categories of AECAs do exist. Again, AECA-containing sera from SLE patients were functionally diverse in that four triggered apoptosis, whereas the remaining four did not. The possibility exists that, as recently described [35], EC apoptosis may be induced by Ab-dependent cell-mediated cytotoxicity.

On the basis of positivity for the two batteries of assays, those evaluating activation and those measuring apoptosis, AECAs were classified into four groups: act+/apo+ (10 patients), act+/apo-(one patient), act-/apo+ (six patients), and act-/apo-AECA (six patients). Two sera, originally identified as act+/apo+ and act+/apo-, respectively, became act-/apo+ after two years. It is possible that, in a given population of AECAs potential markers for disease acticity and/or certain complications [36] are represented by some but not all AECAs. In addition, the reduced expression of TM on cultured HUVECs was associated with apoptosis, rather than activation, suggesting that this receptor was internalized, as is the case for TM complexed with anti-TM Ab [37]. In fact, a subset of AECAs, among others in a positive serum, might recognize TM. Superimposed to this interpretation remains, however, the fact that activation suppresses TM expression of ECs [38].

Whether activation plays a role in the advent of apoptosis remains elusive. Accordingly, the main thrust of the following experiments was to differentiate apoptosis set off by act+/apo AECAs from that due to act-/apo AECAs. Protein synthesis is required in a number of models of PCD, but this is not always a prerequisite in view that at least two distinct mechanisms for induction of apoptosis have thus far been described [39]. New proteins were not synthesized following incubation of ECs with either group of AECAs, since CHX did not prevent cell death. Nonetheless, the first group of sera induced also activation type II which implies protein synthesis, demonstrating that these sera contained at least two independent subpopulations of AECAs, one responsible for activation and another for apoptosis. Although the caspase-dependent cascade is being challenged, caspase-3 is still regarded as a key executioner molecule in the apoptosis machinery [21]. This protease was found to be implicated in act+/apo+ AECA-induced apoptosis, as well as act-/apo+ AECAs. It should, however, be noted that the effect of the inhibitor was far from being complete. This observation indicates that alternative agents might operate in the AECA model. Given that some stimuli need further involvement of ROS to result in EC death [22], the production of hydrogen peroxide and superoxide anion was measured under the same conditions. No ROS were actually detected in the presence of AECAs. Furthermore, it was found that, albeit signicantly lower than in the control cells, the ratios of the survival proteins Bcl-2, A1, BclXL and Mcl-1 to the PCD-promoting factor Bax in the cytosol of ECs undergoing apoptosis were similar in ECs treated with act+/apo+ AECAs, and act-/apo+ AECAs. In fact, the levels of survival proteins were diminished, while those of Bax were raised. Such a finding suggests that there may be a relationship between these two events, so that AECAs commit cells to apoptosis by dysregulating manifold interactions between suppressors and inducers (reviewed in [40]). A major concern remains the specificity of the autoAbs used in our study, since we have already demonstrated [11] that AECAs do not react with the Fas receptor.

In some cases, the process was completed by the removal of damaged cells. Here, AECA IgG may act as an opsonin or require apoptosis to task successfully. Because some AECAs did not promote apoptosis and might be and epiphenomenon of vascular injury, it was impossible to normalize the number of apoptotic cells added to MØ. Nonetheless, our finding that the increased phagocytosis was restricted to those AECAs that induced apoptosis favour the second interpretation. Furthermore PS is widely accepted as being one of the most efficient marker for phagocytosis [41], and its cognate receptor seems to be defective in connective tissue diseases [42], thus setting in motion events that leads to inflammation. Hence, it is consequent to conclude that these findings raise the issue of the physiological role of natural AECAs [43] in the removal of unwanted damaged ECs, thereby preventing vasculitis.

Acknowledgments

We are indebted to Drs S. Chesne (Grenoble, France) and C.-J.S. Edgell (Chapel Hill, NC, USA) for the generous donation of reagents, to Prs P. Le Goff, Y.-L. Pennec and D. Mottier (Brest, France) for providing the patient sera, and to Drs J.-J. Chabaud and H. Le Bos (Brest) for their help in collecting umbilical cords. We gratefully acknowledge Dr J. Arvieux (Brest, France) and Pr P.M. Lydyard (London, UK) for their extremely valuable help. The expert secretarial assistance of Mrs. S. Hamon and S. Forest is greatly appreciated. Finally, A. Bordron and R. Révélen are recipients of a fellowship from the ‘Communauté Urbaine de Brest’.

REFERENCES

1. Petty RG, Pearson JD. Endothelium, the axis of vascular health and disease. J Roy Coll Phys London. 1989;23:92–102. [PubMed]
2. Meroni PL, Youinou P. Endothelial cell antibodies. In: Peter JB, Shoenfeld Y, editors. Autoantibodies. Amsterdam: Elsevier; 1996. p. 245.
3. Meroni PL, D'Cruz D, Khamashta M, Youinou P, Hughes GRV. Anti-endothelial cell antibodies: only for scientists or for clinicians too ? Clin Exp Immunol. 1996;104:111–9. [PMC free article] [PubMed]
4. Damianovich M, Gilburd B, George J, et al. Pathogenic role of anti-endothelial cell antibodies in vasculitis. An idiotypic experimental model. J Immunol. 1996;156:4946–51. [PubMed]
5. Hunt BJ, Jurd KM. Endothelial cell activation: a central pathophysiological process. Br Med J. 1998;316:1328–9. [PMC free article] [PubMed]
6. Del Papa N, Guidali L, Sironi M, et al. Anti-endothelial cell IgG antibodies from patients with Wegener's granulomatosis bind to human endothelial cells in vitro and induce adhesion molecule expression and cytokine secretion. Arthritis Rheum. 1996;39:758–66. [PubMed]
7. Carvalho D, Savage CO, Isenberg D, Pearson JD. IgG anti-endothelial cell autoantibodies from patients with systemic lupus erythematosus or systemic vasculitis stimulate the release of two endothelial cell-derived mediators, which enhance adhesion molecule expression and leukocyte adhesion in an autocrine manner. Arthritis Rheum. 1999;42:631–40. [PubMed]
8. Carvalho D, Savage CO, Black CM, Pearson JD. IgG anti-endothelial cell autoantibodies from scleroderma patients induce leukocyte adhesion to human vascular endothelial cells in vitro. Induction of adhesion molecule expression and involvement of endothelium-derived cytokines. J Clin Invest. 1996;97:111–9. [PMC free article] [PubMed]
9. Ishii H, Yama H, Kazama M. Soluble thrombomodulin antigen in conditioned medium is increased by damage of endothelial cells. Thromb Haemostas. 1991;65:618–23. [PubMed]
10. Boehme MWJ, Schmitt WH, Youinou P, Stremmel WG, Gross WL. Clinical relevance of elevated thrombomodulin and soluble E-selectin in patients with Wegener's granulomatosis and other systemic vasculitides. Am J Med. 1996;101:387–94. [PubMed]
11. Bordron A, Dueymes M, Levy Y, et al. The binding of some anti-endothelial cell antibodies induces endothelial cell apoptosis. J Clin Invest. 1998;101:2029–35. [PMC free article] [PubMed]
12. Savill J. Recognition and phagocytosis of cells undergoing apoptosis. Br Med J. 1997;53:491–508. [PubMed]
13. Revelen R, Bordron A, Dueymes M, Youinou P, Arvieux J. False positivity in a cyto-ELISA for anti-endothelial cell antibodies caused by heterophile antibodies to bovine serum proteins. Clin Chem. 2000;46:273–8. [PubMed]
14. Youinou P, Meroni PL, Khamashta MA, Shoenfeld Y. A need for standardization of the anti-endothelial cell antibody test. Immunol Today. 1995;16:363–4. [PubMed]
15. Westphal JR, Boerbooms AMTh, Schalkwijk CJM, et al. Anti-endothelial cell antibodies in sera of patients with autoimmune diseases: comparison between ELISA and FACS analysis. Clin Exp Immunol. 1994;96:444–9. [PMC free article] [PubMed]
16. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982;25:1271–7. [PubMed]
17. Vitali C, Bombardieri S, Moutsopoulos HM, et al. Preliminary criteria for the classification of Sjögren's syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum. 1993;36:340–7. [PubMed]
18. Bordron A, Dueymes M, Levy Y, et al. Anti-endothelial cell antibody binding makes negatively charged phospholipids accessible to antiphospholipid antibodies. Arthritis Rheum. 1998;41:1738–47. [PubMed]
19. Nicoletti I, Migliorati G, Pagliacci MC, et al. A rapid and simple method for measuring thymocyte apoptotis by propidium iodide and flow cytometry. J Immunol Methods. 1991;139:271–9. [PubMed]
20. Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earsnshaw WC. Cleavage of poly (ADP-ribose) polymerase by a proteinase with properties like ICE. Nature. 1991;371:346–7. [PubMed]
21. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998;281:1312–6. [PubMed]
22. Haendeler J, Zeiher AM, Dimmeler S. Vitamins C and E prevent lipopolysaccharide-induced apoptosis in human endothelial cells by modulation of Bcl-2 and Bax. Eur J Pharmacol. 1996;317:407–11. [PubMed]
23. Carter WO, Narayanan PK, Robinson JP. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol. 1994;55:253–8. [PubMed]
24. Fadeel B, Zhivotovsky B, Orrenius S. All along the watchover: on the regulation of apoptosis regulators. FASEB J. 1999;13:1647–57. [PubMed]
25. Musson RA. Human serum induces maturation of human monocytes in vitro. Changes in cytolytic activity, intracellular lysosomal enzymes, and non-specific esterase activity. Am J Pathol. 1993;11:331–40. [PMC free article] [PubMed]
26. Boffa MC. Considering cellular thrombomodulin distribution and its modulating factors can facilitate the use of plasma thrombomodulin as a reliable endothelial marker. Haemostasis. 1996;26:233–43. [PubMed]
27. Olie RA, Durrieu F, Cornillon S, et al. Apparent caspase independence of programmed cell death in Dictyostelium. Curr Biol. 1998;8:955–8. [PubMed]
28. Maeda K, Abello PA, Abraham MR, Wetzel RC, Robotham JL, Buchman TG. Endotoxin induces organ-specific endothelial cell injury. Shock. 1995;3:46–50. [PubMed]
29. Coll-Vincent B, Cebrian M, Cid MC, et al. Dynamic pattern of endothelial cell adhesion molecule expression in muscle and perineural vessels from patients with classic polyarteritis nodosa. Arthritis Rheum. 1998;41:435–44. [PubMed]
30. Blank M, Krause I, Goldkorn T, et al. Monoclonal anti-endothelial cell antibodies from a patient with Takayasu arteritis activate endothalial cells from large vessels. Arthritis Rheum. 1999;42:1421–32. [PubMed]
31. Lie JT. Illustrated histopathologic classification criteria for selected vasculitis syndromes. Arthritis Rheum. 1990;33:1074–87. [PubMed]
32. Page C, Rose M, Yacoub M, Pigott R. Antigenic heterogeneity of vascular endothelium. Am J Pathol. 1992;141:673–83. [PMC free article] [PubMed]
33. William RC, Jr, Peen E. Apoptosis and cell penetration by autoantibody may represent linked processes. Clin Exp Rheumatol. 1999;17:643–7. [PubMed]
34. Verhoven B, Schlegel RA, Williamson P. Mechanisms of phophatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med. 1995;182:1597–601. [PMC free article] [PubMed]
35. Sgonc R, Gruschwitz MS, Boeck G, Sepp N, Gruber J, Wick G. Endothelial cell apoptosis in systemic sclerosis is induced by antibody-dependent cell-mediated cytotoxicity via CD95. Arthritis Rheum. 2000;43:2550–62. [PubMed]
36. D'Cruz DP, Houssiau FA, Ramirez G, et al. Antibodies to endothelial cells in systemic lupus erythematosus: a potential marker for nephritis and vasculitis. Clin Exp Immunol. 1991;85:254–61. [PMC free article] [PubMed]
37. Brisson C, Archipoff G, Hartmann ML, et al. Antibodies to thrombomodulin induce receptor-mediated endocytosis in human saphenous vein endothelial cells. Thromb Haemostas. 1992;68:737–43. [PubMed]
38. Moore KL, Andreoli SP, Esmon NL, Esmon CT, Bang NU. Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest. 1987;79:124–30. [PMC free article] [PubMed]
39. Gräfe M, Steinheider G, Desaga V, et al. Characterization of two distinct mechanisms for induction of apoptosis in human vascular endothelial cells. Clin Chem Lab Med. 1999;37:505–10. [PubMed]
40. O'Connor R. Survival factors and apoptosis. Adv Biochem Eng Biotechnol. 1998;62:137–66. [PubMed]
41. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PL. Exposure of phophatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol. 1992;148:2207–16. [PubMed]
42. Herrmann M, Voll RE, Zoller OM, Hagenhofer M, Ponner BB, Kalden JR. Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus. Arthritis Rheum. 1998;41:1241–50. [PubMed]
43. Ronda N, Haury M, Nobrega A, Kaveri SV, Coutinho A, Kazatchkine MD. Analysis of natural and disease-associated autoantibody repertoires: anti-endothelial cell IgG autoantibody activity in the serun of healthy individuals and patients with systemic lupus erythematosus. Int Immunol. 1994;6:1651–60. [PubMed]

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