Comparison of cell-ELISA, flow cytometry and Western blotting for the detection of antiendothelial cell antibodies.

OBJECTIVE: There is still great uncertainty in the detection of antiendothelial cell antibodies (AECA). The aim of our study was to compare the results obtained using different methods. METHODS: Sera were obtained from 71 patients with a variety of vasculitides. Three assay methods were used: cell ELISA, flow cytometry (FACS) and Western blot (WB). RESULTS: In the ELISA 12/17 patients with systemic lupus erythematosus (SLE), 1/12 with Churg Strauss (CS) disease, 3/12 with micropolyarteritis (MPA) and 5/30 with Wegener's granulomatosis (WG) tested positive. Most of the sera that were positive on ELISA were not by FACS. Among the negative sera, 50% of WG, 40% of MPA, 20% of CS and 40% of SLE became positive on WB. There were some specific patterns of reactivity for a given disease, so that some bands could be assigned to a disease. CONCLUSION: The discrepancies in the results may most probably be accounted for by differences between the antigenic preparations. Caution must thus be exercised when interpreting the results of any of these three tests.

Anti-endothelial cell antibodies (AECA) in systemic sclerosis--increased sensitivity using different endothelial cell substrates and association with other autoantibodies.

OBJECTIVE: One of the main features of systemic sclerosis (SSc) is vascular damage, the mechanism of which is not understood. In the present study we examined whether screening of SSc patients for different anti-endothelial cells antibodies (AECA) of various origins increase the sensitivity of AECA detection in SSc patients. Secondary aim was an attempt to correlate AECA with other common autoantibodies. MATERIALS & METHODS: 478 SSc patients were studied for the presence AECA, anti-cardiolipin (aCL), anti-dsDNA, anti-heparin (AHA), anti-pyruvate dehydrogenase (PDH) and anti-PDC-E2 autoantibodies. AECA levels were detemined using human umbilical vein EC (HUVEC), bone marrow EC (BMEC), EC hybridoma (EA.hy 926) and Kaposi sarcoma EC (KS). RESULTS: Positive AECA were found in 49.5% of SSc patients (27.1% HUVEC; 34.3% BMEC; 26.3% EaHy 926 and 22.7% KS). The highest percent reactivity of AECA was obtained using microvascular BMEC. When combining BMEC and either other cell lines the reactivity ranged from 41.4% to 46%. A significant association between AECA on the one hand and AHA (p<0.001)) and anti-PDH (p<0.05) on the other was secn. Cross-reactivity with anti-PDC-E2 was excluded by inhibition tests, but AHA and anti-PDH may be part of the spectrum of AECA. CONCLUSIONS: Since false-negative AECA may result from lack of expression of various antigens on a specific EC, analysis of AECA in SSc patients requires using several EC types, including microvascular EC.

Functional heterogeneity of anti-endothelial cell antibodies.

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.

Anti-endothelial cell reactivity, the unresolved enigma.

Not only are some anti-endothelial cell antibodies (AECA) directed to thus far unidentified cell membrane structures, but some ot them recognize "planted" antigens and possibly ligand-receptor complexes. The functional heterogeneity of AECA is widely acknowledged: part of them activate the complement, mediate antibody-dependent cell cytotoxicity of trigger the production of tissue procoagulant factor. It has also recently been established that a proportion of AECA have the capacity to induce apoptosis of their target cells. In fact, the most direct demonstration of the pathogenicity of AECA is the autoantibody-induced murine model of vasculitis.

False positivity in a cyto-ELISA for anti-endothelial cell antibodies caused by heterophile antibodies to bovine serum proteins.

BACKGROUND: ELISAs with fixed endothelial cells or cell lines are widely used screening tests for anti-endothelial cell antibodies (AECAs), but spurious increases occur. We examined interferences by heteroantibodies and means to eliminate them. METHODS: AECAs were measured by ELISA on fixed layers of the human endothelial cell line, EA.hy 926, in a panel of 60 patient serum samples diluted in bovine serum albumin. Heteroantibodies against fetal calf serum (FCS) proteins were demonstrated and characterized in an ELISA-the interference assay-that used FCS-coated plates and Tween 20-containing buffer as blocking agent and sample diluent, as well as by immunoblotting. RESULTS: In 12 of 60 patient serum samples, spurious increases of AECA titers were produced by endogenous antibodies reacting with FCS proteins from culture medium that were coated onto the solid-phase at the time of cell plating. This mechanism of interference was supported experimentally by exposing extracellular matrix, varying cell density, and incubating wells with FCS alone. The heterophile antibodies were mainly IgG and IgA, and in inhibition experiments, they recognized serum proteins from goat, sheep, and horse. Washing cells free of FCS before plating, or adding FCS (100 mL/L) to the patient sample diluent eliminated spurious signals from all 30 tested sera, but the latter method had practical advantages. CONCLUSIONS: Antibodies against animal serum proteins are a frequent cause of erroneous results in cyto-ELISAs. The interference can be eliminated by simple antibody absorption in FCS-containing dilution buffer.

Anti-endothelial cell auto-antibodies in hepatitis C virus mixed cryoglobulinemia.

BACKGROUND/AIM: Hepatitis C virus (HCV) infection is often associated with mixed cryoglobulins (MC) and may manifest as small-vessel vasculitis. It has been suggested that antibody (Ab) or sensitized T cells to HCV-containing endothelial cells may initiate the vasculitis process. Anti-endothelial cell antibodies (AECA) have been found in various connective tissue disorders, with a high prevalence in systemic vasculitis. The aim of the study was to determine the prevalence of AECA in HCV patients with or without MC-associated vasculitis, and to identify associations with clinical, immunological, virological and liver characteristics. METHODS: Sixty-nine HCV patients (Group 1), 46 of whom had MC (type II=30, type III=16), and 23 without MC, were prospectively studied. HCV-MC-associated vasculitis was noted in 25 patients who had at least one of the following clinical features: peripheral neuropathy, glomerulonephritis, skin purpura, cerebral vasculitis. Group 2 included 20 patients with non-HCV viral diseases: HHV8 (10), miscellaneous (10). Group 3 included 25 patients with biopsy-proven non-HCV chronic liver diseases: hepatitis B virus (10), miscellaneous (15). Controls were 100 blood donors (Group 4). Sera were adsorbed onto a pellet of A549/8 epithelial cells before being evaluated. AECA were then searched using a cellular ELISA, with a permanent cell line (EA.hy 926) as the substrate. All sera were also examined for the presence of cryoglobulin, antinuclear Ab, anticardiolipin Ab, and rheumatoid factor. RESULTS: AECA were more frequently found in HCV patients than in blood donors (41% vs 5%, p=0.0001). The prevalence of AECA was lower in non-HCV than in group 1 patients [group 2=15%, p=0.03; group 3=16%, p=0.01]. There was no significant difference in AECA prevalence between groups 2, 3 and 4. In HCV patients, AECA were associated with age (p<0.001), the presence of MC (p=0.008), cryoglobulin level (p=0.016), HCV-associated vasculitis (p=0.04), genotype 1b (p=0.005) and severity of liver histologic damage. AECA isotypes were not different in the 4 groups. AECA were not associated with antinuclear Ab, anticardiolipin Ab, rheumatoid factor or interferon alpha treatment. CONCLUSION: AECA are a common finding in HCV patients (41%), but not in other viral diseases or in non-HCV chronic liver diseases. In HCV patients, AECA are associated with MC-vasculitis, suggesting that AECA may be a marker for HCV-induced vasculitis.

Two populations of endothelial cell antibodies cross-react with heparin.

As endothelial cells (EC) express heparin-like glycosaminoglycans, such as heparan sulfate, it was essential to investigate the relation of anti-EC antibody (AECA) to heparin reactivity. AECA were detected in 43 of 131 autoimmune sera and anti-heparin antibodies (AHA) in 25. These autoimmune reactivities were significantly associated (P corrected < 0.0005). Seven AECA-positive/AHA-positive and three AECA-negative/AHA-positive sera were affinity-purified using protein G column followed by a heparin-Sepharose column. Two populations of AECA were recovered from the second column. One was eluted with 0.4 M NaCl which bound to EC and to solid-phase heparin with low affinity, but not to soluble heparin. The second population of AECA, which was eluted with 4 M guanidine HCl/2 M NaCl, recognized EC and solid-phase heparin with high affinity, but also soluble heparin. The latter population of AECA might thus be an important cause of autoimmune vascular thrombosis in systemic diseases.