A need for standardization of the anti-endothelial-cell antibody test

Pierre Youinou, Pier-Luigi Meroni, Munther A. Kharnashta and Yehuda Shoenfeld

Anti-endothelial-cell antibodies (AECAs) were first described in the serum of patients with systemic lupus erythcmatosus (SLE) almost 25 years ago’**, and have been sub- sequently characterized by Cines and colleagues”. A variety of clini- cal conditions are now known to he associated with AECAs, but the re- sults obtained in a given disease vary markedly from one study to another (reviewed in Ref. 4). A standardi- zation programme of the AECA test has thus been launched, which should shed light on this complex group of autoantibodies,

Prevalence of AECAs A consensus on the prevalence of

AECAs has not yet been reached. The proportion of AECA’ sera ranges from 16% (TM. Chan, Hong Kong) to 63% in SLE (R. Cervera, Barcelona), from 0% to 57% in rheumatoid arthritis (RA) and from 45% to 77% in mixed connective- tissue disease (L.L.F. Mendoqa, SBo Pauio). Interestingly, type IV neph- ritis is observed ‘in 100% of SLE patients with AECAs but only in 76% of patients without AECAs. In addition, RA patients with vascuiitis have higher titres of AECAs than those with uncomplicated disease (0. Meyer, Paris). Chan described a clinical follow-up of 16 patienrs with SLE for 8-43 months, in which sequential serum samples were ert- amined for the presence of AECAs every other month: four disease ex- acerbations occurred, three of which were antedated by increased levels of AECAs, whereas 14 out of 16 epi- sodes of clinical improvement were associated with a reduction of the AECA titres.

Vascular damage is a common feature of all the above presumed

Anti-endothPliili-cell arttibodies (AECAs) have been described in a number of r-onditions. However, the twct prevalente of AEC;As is not yet kmswn because of t/w coiz- siderable variation i?z results ob- tained by different investigators. A recent meeting* discussed the requirement to standardize the

AECA test.

autoimmune conditions but, again, extreme variations between results have been obtained in vasculitic syndromes by different investiga- tors. For example, AECAs have been detected in 19-8 I % of patients with Wegener’s granulomatosis (WG), 2-60% of patients with microscopic polyarteritiq and O-26% of patients with Behset syndrome (R. Cervera).

AECAs have not been detected in the serum of patients with Churg- Strauss syndrome (W.H. Schmitt, Liibeck) or malignant atrophic papulosis (C. Franc& Paris), a rare and potentially lethal disease. By contrast, AECAs have been shown to be present in the serum of 35% of patients with Sneddon’s syn- drome (C. Franc&.), a disorder that may be associated with vasculitides. Although there have been claims of elevated AECA titres in a variety of conditions, including diabetes mellitus, early kidney transplant rejection, coronary artery disease following hrarr transplantation, and nephropathia epidemica, these findings have not been confirmed in any control study. There have also been reports that AECAs are el- evated in spinal cord sarcoidosis, ulcerative colitis. Crohn’s disease.

“The Workshop ‘Anti-Endochelial-C:ell Antibodies’ was part of rhe 4th Inter- national Conference on SLE, and was held in Jerusalem, Israel, on Z-J1

March 199.5.

endometriosis and primary biliary cirrhosis.

Detection of AECAs The discrepancies in the detec-

tion of AECAs may be ascribed to differences in the tests used in dif- ferent laboratories. A number of methods have been used, including indirect immunofluorescence (IlF), immunochemistry, complement-de- pendent lysis, radioimmunoassays and ELISA. Each of these methods has its limitations, but ELTSA has finally been selected by experts in the field.

ELlSA The initial substrate is a mono-

layer of human umbilical vein endothelial cells (HUVECs) eluted by collagenase digestion from hu- man umbilical cords. Cells are grown to confluence, harvested with trypsin-EDTA and passaged three or four times. These are checked visually by phase-contrast light microscopy (the cells should have a typical cobblestone appearance) and by IIF using mouse anti-factor VIII monodonal antibody. Confluent cells are usually fixed with glutaral- dehyde or paraformaldehyde to dis- courage endothelial cell (EC) detach- ment during the test and to avoid nonspecific IgG binding through the Fc.

All assays include the same posi- tive and negative control sera, and reproducibility is established bv cal- culating the variation coefficients. Values as high as 13% are found, and these may be the result of inter- cord quantitative. as well as quali- tative, differences owing to donor variability. Furthermore, ceils can- not be accurately counted, so num- bers vary from one microtitre plate

well to another. Therefore, results are expressed as binding indexes, which are derived from the optical densities. Inevitably, the number of cells is restricted, which constitutes an additional limitation in this tedious procedure.

Use of EC lines To overcome the variation in

EC numbers, a permanent cell line EA.hy926 has been established by Edge11 and co11eagues5, who fused HUVECs with the permanent epi- thelial cell line A549-8 (Ref. 6). In- deed, this was used by several par- ticipants in the Workshop to detect AECAs by ELISA. Although one may argue that immortalization and chromosomic segregation may have resulted in loss of native endothelial determinants and, thereby, in false- negative results, there were strong correlations between the AECA titres obtained on fixed HUVECs and fixed EA.hy926 cell monolayers. Conversely, sera may contain not only AECAs, but also antibodies specific for epithelial antigens. To exclude this background, all test sera must be pre-absorbed with epi- thelial cells, preferably the mother epithelial cells A549-8, before being evaluated on the EA.hy926 hybrid cell line.

There is widespread awareness of the limitations of the tests cur- rently in use, and therefore a multi- centre standardization programme has now been established. Twenty- five well-characterized sera have been sent to 11 investigators to be tested with commercial HUVECs, in-house HUVECs and EA.hy926 cells. This consensus study is still in progress and should allow us to gain further insights into the analy- sis of antigens targeted by AECAs.

New EC lines have been estab- lished by M. Le Tonqdze (Brest) by fusing HUVECs with A549-8, and cloned by limiting dilution. Preliminary results using a panel of these clones suggest that they would be helpful in defining several AECA patterns that might be specific for different diseases.

Autoantigens recognized by AECAs Immunoblotting techniques have

been used for some time to identify autoantigens recognized by AECAs, again leading to considerable dis-

crepancies. One group identified 11 proteins ranging in size from 56-133 kDa, a second group de- scribed two bands of 60 and 62 kDa, and a third group described I2 proteins ranging from 16-88 kDa. However, it is likely that two auto- antigen candidates may be con- firmed: proteinase 3 (PR3) and P,-glycoprotein I (&-GPI).

Autoanttgen PR3 W.J. Mayet and colleagues

(Mainz) have been able to show that treatment with tumour necro- sis factor (Y, interleukin la and lb, and interferon y leads to increased PR3 expression within the cyto- plasm-. Further experiments have provided data suggesting that EC injury is due to a PR3-antibody- dependent cellular cytotoxicity in WG and other vasculitides related to anti-cytoplasmic antibodies.

Autoantigen &-Cl’1 It is possible that P,-GPI is cross-

reactive with anti-phospholipid IPL) antibodies, which include those re- acting with negatively charged PL, such as cardiolipin (CL), and those reacting with neutral PL. P,-GPI may bind to CL and, hence, differ- entiate autoimmune from infection- induced anti-CL antibodies. Anti-CL antibodies have indeed been found to be associated with AECAs (Ref. 8), and antibodies eluted from ECs were shown to react with CL and platelets (N. Lanir, Tel-Hashomer). Furthermore P,-GPI might influence the binding of AECAs to PL on the membrane of ECs (P-L. Meroni, Milan; P. Youinou, Brest). The level of anti-EC activity is reduced if IgGs are tested on ECs maintained in serum-free mediumy, is abro- gated by the removal of P,-GPI and is restored by its re-introduction’“.

Significance of AECAs The pathogenicity of AECAs re-

mains unclear. Speculation on the mechanism of vascular damage in conditions associated with these autoantibodies has focused on en- hanced expression of adhesion mol- ecules by ECs following interaction with AECAs (N. Del Papa, Milan). This phenomenon, combined with the production of chemotactic cyto- kines, would facilitate recruitment and adhesion of leukocytes, leading

to endothelial damage (P-L. Meroni). The mechanisms of production and maintenance of AECAs are poorly understood. However, studies have shown that murine AECAs are induced in mice injected three months earlier with human AECAs (M. Damjanovic, Tel-Aviv), sug- gesting that these antibodies are under idiotypic control.

Conclusion In conclusion, the main message

emerging from the Workshop is the crucial need for standardization. Such a programme is currently in progress, and this should provide

new insights into understanding the AECA-EC system.

We gratefully acknowledge Annie Paul for secretarial assistance, and also thank Peter Lydyard.

Pierre Youinou is at the Laboratory of Immunology, Brest Universtity Medical School, 29609 Brest, France; Pier-Luigi Meroni is at the Istituto di Medicina Interna, Ospedale Policlinico, 20122 Milano, Italy; Munther Khamashta is at the Rayne Institute, St Thomas’ Hospital, London, UK SE1 7Ell; Yehuda Shoenfeld is at the Sheba Medical Centre, Tel-Hashomer 5262 2, Israel.

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