IgG Anti-endothelial Cell Antibodies Research Articles

Immunoglobulin G anti-endothelial cell antibodies: inducers of endothelial cell apoptosis in pulmonary arterial hypertension?

Endothelial cell (EC) apoptosis seems to play an important role in the pathophysiology of pulmonary arterial hypertension (PAH). We aimed to test the hypothesis that circulating anti-endothelial cell antibodies (AECA) of PAH patients induce EC apoptosis. Immunoglobulin (Ig)G was purified from sera of PAH patients (n = 26), patients with systemic lupus erythematosus (SLE) nephritis without PAH (n = 16), patients with systemic sclerosis (SSc) without PAH (n = 58) and healthy controls (n = 14). Human umbilical vein endothelial cells (HUVECs) were incubated with patient or healthy control IgG for 24 h. Thereafter, apoptosis was quantified by annexin A5 binding and hypoploid cell enumeration by flow cytometry. Furthermore, real-time cell electronic sensing (RT-CES™) technology was used to monitor the effects of purified IgG from patient and healthy control IgG on HUVECs. As demonstrated previously, IgG of AECA-positive SLE nephritis patients (n = 7) induced a higher percentage of apoptosis of HUVECs compared to IgG of AECA-negative SLE nephritis patients and healthy controls. Furthermore, IgG of AECA-positive SLE nephritis patients induced a marked decrease in cell index as assessed by RT-CES™ technology. IgG of AECA-positive PAH patients (n = 12) and SSc patients (n = 13) did not alter the percentage of HUVEC apoptosis or cell index compared to IgG of AECA-negative PAH and SSc patients and healthy controls. AECA-positive PAH patients, in contrast to SLE nephritis patients, do not have circulating IgG AECA that enhances apoptosis of HUVECs in vitro. Further studies should focus on other mechanisms by which AECA may enhance EC apoptosis in PAH, such as antibody-dependent cell-mediated cytotoxicity.

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Aim To determine the changes in gene expression in Endothelial Precursor Cells (EPC) exposed to Anti Endothelial Cell Antibodies (AECA) that are Non-HLA-specific. Methods Magnetic-bead column isolation followed by XM-One crossmatch procedure using reagents from Absorber Inc. was employed to identify serum samples with non-HLA AECA. For Gene expression assays, EPCs were obtained from T and B cell depleted PBL using reagents from Absorber Inc. The endothelial cell gene expression was performed on total RNA using Real-Time RT-PCR profiler reagents from QIAGEN Inc. Results EPCs exposed to a combination of IgM and IgG AECAs, affected the expression of more endothelial cell genes than EPCs exposed to IgM or IgG alone. Exposure of EPCs to IgM AECA alone caused changes in a few genes while exposure to IgG AECA alone did not significantly alter expression of any endothelial cell genes. In all, the genes which were up-regulated (ANXA5; APOE; BAX; BCL2L1; CALCA; CFLAR; COL18A1; EDN1; EDN2; EDNRA; FLT1; HIF1A; HMOX1; ICAM1; ITGAV; ITGB1; KDR; OCLN; PF4; SELE; THBS1) were those associated with pro-apoptotic, thrombocytic and or hemolytic events. One of the highly down-regulated genes was the Tissue inhibitor of Matrix Metalloproteinase (TIMP) that inhibit the matrix metalloproteinases which leads to increased extracellular protein deposition resulting in excessive extracellular connective tissue deposition, which is a hallmark of fibrosis which in turn is associated with chronic allograft rejection. Conclusions Human EPCs seem to respond to non-HLA antibodies by altering the gene expressions of endothelial cell biology which might be associated with events leading to allograft injury in a complement independent way. More detailed gene expression studies with organ specific endothelial cells and IgM/IgG specific non-HLA endothelial cell antibodies are needed to assess the full significance of AECA in allograft rejection.

Functional implications of IgG anti-endothelial cell antibodies in pulmonary arterial hypertension

The objective of this study was to research the functionality of anti-endothelial cell antibodies (AECA) in pulmonary arterial hypertension (PAH) by assessing the effects of IgG from AECA-positive PAH patients on the induction of adhesion molecules on human umbilical vein endothelial cells (HUVECs) and on the production of pro-inflammatory cytokines and chemokines by HUVECs. To achieve this purified IgG from 28 PAH patients were included. IgG from systemic sclerosis (SSc) (n = 58) and systemic lupus erythematosus (SLE) (n = 16) patients without PAH were included as disease controls. Intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression on HUVECs, incubated with patient IgG, were quantified by flow cytometry. Production of interleukin (IL)-1β, -6, -8, and CC chemokine ligand 2 (CCL2) by HUVECs, incubated with patient IgG, were quantified by multiplex flow cytometry. Our results showed that IgG from AECA-positive PAH, SSc and SLE patients induced significantly higher expression of ICAM-1, VCAM-1, and E-selectin and production of IL-6, -8, and CCL2 compared to IgG from AECA-negative patients and IgG from healthy controls. Like in SLE and SSc, IgG from AECA-positive PAH patients can activate endothelial cells to a pro-adhesive and pro-inflammatory state. Therefore, IgG AECA could play a pathogenic role by inducing inflammatory injury of vascular endothelium which is considered a key player in the initiation and progression of PAH.

Editorial Comment: The clinical relevance of antibody-mediated rejection: a new era of heart transplantation

This issue reports a case where the authors can be congratulated on having saved the life of a patient who originally suffered from giant cell myocarditis and had been treated with cyclosporine (CyA), azathioprine (AZA) and corticosteroids (PDN) for 4 years before clinically deteriorating and been subjected to heart transplantation (HTx) [1]. The pretransplant work-up included human leukocyte antigen (HLA)-typing of both patient and donor, screening for anti-HLA antibodies and cross-match with regard to T- and B-cells being negative. Postoperatively, conventional immunosuppression (CyA, AZA and PDN) was introduced. There were three HLA Class I and one HLA Class II allele mismatches between donor and recipient. The first endomyocardial biopsy (EMB) showed mild rejection (ISHLT 1R) without C4d deposition in immunohistochemistry. The second EMB (3 weeks postoperatively) revealed severe macrophage (CD68+), and eosinophilic and lymphocytic infiltration, again without C4d deposition. The patient developed fever without clinical signs of infection and was treated under the presumption of acute cellular rejection (ACR). After initial stabilization, the patient developed cardiogenic shock 2 days later, which required urgent treatment with extracorporeal membrane oxygenation, followed by the implantation of a biventricular assist device the next day. At that time, EMB showed severe ACR (ISHLT 3R) and antibodymediated rejection (AMR), strongly positive for C4d [2, 3]. The cytotoxic T- and B-cell cross-match tests remained negative. The endothelial precursor cells cross-match test (EPC-XM; XM-ONE ® , AbSorber AB, Sweden) was synchronously performed with EMBs, measuring serum levels of the amount of antiendothelial-cell antibodies (AECAs), either against donorspecific EPC’s or against autoreactive EPC’s (the patient’ so wn EPCs). After initially being negative, donor-specific AECA became positive at the first EMB (ISHLT 1R), but autoreactive AECAs were still negative. At the time of acute graft failure, the test was surprisingly positive for both donor-specific and autoreactive IgM antiendothelial AECA, but not for donor IgG AECA. The authors conclude that severe AMR was caused by non-HLA, AECAs. Epicrisis was successfully treated by plasmapheresis, intravenous immunoglobulin (IVIgG), rituximab, muronomab-CD3, bortezomib and re-HTx. One month after re-HTx, the patient had ACR (ISHLT 2R), but remained free of AMR. During the last 15 years, AMR became a topic of major clinical relevance. Potential explanations are improved diagnostic tests and the rising population of patients having undergone VAD implantation before HTx. Its pathophysiology remains unknown to date. AMR is often associated with haemodynamic compromise, increased mortality and development of graft coronary artery disease (CAD). Younger age, congenital heart disease, positive donor-specific cross-match, positive panel reactive antibody (PRA) titres, sensitization to OKT3, CMV seropositivity, previous blood transfusions and female gender have been identified as risk factors for AMR [4].

Antiendothelial cell antibodies in patients with coronary artery ectasia

The mechanisms involved in the pathogenesis of coronary artery ectasia (CAE) have not been elucidated. Circulating antiendothelial cell antibodies (AECA) are often detectable in systemic vasculitis and have been implicated in the pathogenesis of endothelial injury. Their prevalence in CAE is not known. Out of 475 consecutive patients subjected to coronary angiography, 27 patients were diagnosed with CAE. Thirty patients matched for age, body mass index, sex, and coronary artery disease prevalence, served as controls. Serum AECA of IgG, IgM, and IgA isotypes were detected using a cell-based enzyme-linked immunosorbent assay (ELISA). Antinuclear antibodies (ANA) and antineutrophil cytoplasmic antibodies (ANCA) were detected using indirect immunofluorescence. IgG and IgM anticardiolipin antibodies (aCL) were detected using commercial ELISA. The prevalence of ANA and ANCA was similar in CAE patients and controls (33.3 vs. 43.3%, and 3.3 vs. 7.4%, respectively). There was no significant difference in IgG or IgM aCL reactivity between patients and controls. Both CAE patients and controls were negative for IgG AECA. The frequency of IgM AECA positivity was similar in CAE patients and controls. The prevalence of AECA of the IgA isotype was significantly higher in CAE patients (37.0 vs. 10%, P<0.05). There is increased prevalence of circulating AECA of the IgA isotype in patients with CAE. This provides evidence for a role of autoimmunity in the pathogenesis of certain cases of CAE.

Multicenter Evaluation of a Novel Endothelial Cell Crossmatch Test in Kidney Transplantation

Despite their clinical importance, clinical routine tests to detect anti-endothelial cell antibodies (AECA) in organ transplantation have not been readily available. This multicenter prospective kidney transplantation trial evaluates the efficacy of a novel endothelial cell crossmatch (ECXM) test to detect donor-reactive AECA associated with kidney allograft rejection. Pretransplant serum samples from 147 patients were tested for AECA by a novel flow cytometric crossmatch technique (XM-ONE) using peripheral blood endothelial progenitor cells as targets. Patient enrolment was based on acceptance for transplantation determined by donor lymphocyte crossmatch results. Donor-reactive AECA were found in 35 of 147 (24%) patients. A significantly higher proportion of patients with a positive ECXM had rejections (16 of 35, 46%) during the follow-up of at least 3 months compared with those without AECA (13 of 112, 12%; P<0.00005). Both IgG and IgM AECAs were associated with graft rejections. Mean serum creatinine levels were significantly higher in patients with a positive ECXM test at 3 and 6 months posttransplant. XM-ONE is quick, easy to perform on whole blood samples and identifies patients at risk for rejection and reduced graft function not identified by conventional lymphocyte crossmatches.

Vascular damage in Wegener’s granulomatosis and microscopic polyarteritis: presence of anti-endothelial cell antibodies and their relation to anti-neutrophil cytoplasm antibodies

To define mechanisms of vascular injury in Wegener's granulomatosis and microscopic polyarteritis, anti-endothelial cell antibodies (AECA) were sought in serum from 168 patients, all of whom had anti-neutrophil cytoplasm antibodies (ANCA) detectable by indirect immunofluorescence. Using an ELISA with human umbilical vein endothelial cells (HUVEC), IgG AECA were demonstrated in 59% and IgM AECA in 68% of patients. Pretreatment of HUVEC with tumour necrosis factor (TNF), IL-1 or interferon-gamma (IFN-gamma) led to increased binding. Adsorption of AECA/ANCA-containing serum with HUVEC or neutrophils demonstrated that AECA and ANCA recognized different targets. von Willebrand factor (vWf) antigen levels in the patient samples were markedly elevated, with a mean of 3.10 +/- 1.89 U/ml (control population mean 1.04 +/- 0.36 U/ml), suggesting widespread endothelial cell damage. Studies using an 111In-labelled HUVEC release assay with 29 AECA-containing sera did not demonstrate complement-mediated cytotoxicity, even following activation of HUVEC with TNF. Four out of 16 AECA-containing sera tested showed antibody-dependent cellular cytotoxicity with unfractionated peripheral blood mononuclear cells. These data suggest that patients with Wegener's granulomatosis or microscopic polyarteritis can develop AECA to constitutively expressed but cytokine modulated determinants on HUVEC. These antibodies do not appear to support complement-mediated cytotoxicity, but a proportion can support antibody-dependent cellular cytotoxicity, suggesting that they may contribute to vascular injury.

Antiendothelial cell antibodies can be cytotoxic to endothelial cells without cytokine pre-stimulation and correlate with ELISA antibody measurement in Kawasaki disease.

To confirm the presence of antiendothelial cell antibodies (AECA) and study the clinical value of their measurement in Kawasaki disease (KD), sera from patients with KD were assessed for binding to human umbilical vein endothelial cells (HUVEC) using a cellular based ELISA, and for complement-mediated cytotoxicity using 111In-labelled HUVEC. In the ELISA assay, IgM AECA were detected in 16/22 sera, whereas IgG AECA were only present in one out of 19 sera. There was a significant difference in IgM AECA titres when comparing patients with controls. Eight out of 16 sera showed cytotoxicity against HUVEC, and this was significantly enhanced when HUVEC were pretreated with tumour necrosis factor (TNF). IgM AECA titres measured by ELISA were positively correlated with their cytotoxicity. The findings suggest that IgM class AECA in sera from patients with KD are detectable by ELISA method, and that some of these antibodies, 50% in this study, can mediate complement-dependent cytotoxicity against endothelial cells.

Antiendothelial Cell Antibody Levels in Healthy Normotensives with High Normal Blood Pressure

High normal blood pressure (HNBP) seems to be related to an increased cardiovascular risk in healthy normotensive subjects. According to the literature, elevated levels of antibodies against endothelial cell surface antigen antiendothelial cell antibodies (AECA) play an important role in the early stages of atherosclerosis process and in borderline hypertension. The aim of this study was to compare AECA levels of healthy normotensives with HNBP to those of healthy normotensives with normal blood pressure (NBP), matched for age, sex, and body mass index (BMI).Methods. Ninety healthy normotensives with HNBP (43M, 47F; mean age, 48 ± 2.6 yrs; BMI 23.6 ± 1.5 Kg/m2) (Group A) and 80 healthy normotensives with NBP (41M, 39F; mean age, 46 ± 3 yrs; BMI 24 ± 1.8 Kg/m2) (Group B) were studied. Both group subjects were matched for sex, age, and BMI. AECA levels were determined in each subject using an enzyme-linked immunosorbent assay (ELISA). AECA levels were expressed as mean values. Results. Twenty-five subjects from group A (28%) showed elevated IgG antiendothelial cell antibodies levels vs. three from group B (3.75%, p < 0.001). IgM AECA levels were elevated in 18 subjects from group A (20%) vs. two from group B (1.5%, p < 0.001). Conclusions. The present findings suggest that healthy normotensives with HNBP have significantly higher AECA levels of both classes (IgG, IgM) compared to healthy normotensives with NBP. This may have prognostic significance for the future development of essential hypertension in this group of healthy subjects.

Antibodies to endothelial cells in Kawasaki disease lyse endothelial cells without cytokine pretreatment.

Kawasaki disease (KD) is characterized by diffuse vasculitis and marked immune activation. To confirm the presence of antiendothelial cell antibodies (AECA) and cytotoxicity of AECA, we investigated the presence of AECA using ELISA and cytotoxicity of AECA in KD. Sera from patients with acute KD were assessed for binding to human umbilical vein endothelial cells (HUVEC) using a cell-based ELISA, and for cytotoxicity against HUVEC as indicated by the conversion of a tetrazolium salt (MTT) into a coloured product. IgM AECA were detected in 8/19 KD sera, IgG AECA were detected in 5/19 KD sera. Significant differences in both AECA titres existed between patients and febrile and afebrile controls. Six out of 19 patients showed complement-dependent cytotoxicity against HUVEC. Cytotoxicity was significantly enhanced by pretreating HUVEC with tumour necrosis factor (TNF), and reduced by incubation with gammaglobulin. Serum titres of IgM AECA in the KD patients were positively correlated with cytotoxicity. Findings suggest that IgM AECA mediates complement-dependent cytotoxicity against endothelial cells in patients with KD, and gammaglobulin may reduce complement-dependent cytotoxicity of AECA against endothelial cells.

IgG anti-endothelial cell antibodies (AECA) in type I diabetes mellitus; induction of adhesion molecule expression in cultured endothelial cells.

AECA were detected in 25 of 71 patients with type 1 diabetes mellitus and in two of 33 healthy subjects. Patients with diabetes of < 1 year duration and those with long-standing disease had the highest levels of these antibodies. Inhibition studies suggest that at least part of the AECA reactivity is due to cross-reactive anti-ssDNA antibodies. AECA-positive sera were able to increase intercellular adhesion molecule-1 (ICAM-1) and E-selectin on human umbilical vein endothelial cells (HUVEC). Increased binding of polymorphonuclear (PMN) cells was also found to accompany raised E-selectin expression. Soluble ICAM-1 and E-selectin were also found to be increased in the sera of AECA-positive patients. An effect of AECA on endothelial cell function is suggested in diabetes mellitus.

Human monoclonal anti-endothelial cell IgG-derived from a systemic lupus erythematosus patient binds and activates human endothelium in vitro.

Our objectives were to obtain monoclonal anti-endothelial cell antibodies (AECA) from systemic lupus erythematosus (SLE) patients, to characterize their antigen specificity, and their capability to induce a pro-inflammatory and pro-adhesive endothelial phenotype, and to investigate the mechanism of endothelial cell (EC) activation in vitro. Monoclonal IgG AECA were generated by hybridoma formation with human SLE B cells. Antigen specificity was characterized by immunoblotting with enriched cell membrane fractions, by cytofluorimetry and by cell solid-phase ELISA. Endothelial activation was evaluated by measuring increases in U937 cell adhesiveness, adhesion molecule (E-selectin and ICAM-1) expression and IL-6 production. In addition, mechanisms of endothelial activation were investigated by assessment of NF-kappaB by measuring the loss of its inhibitor I-kappaB. mAb E-3 bound live EC and recognized a 42 kDa EC membrane protein, it enhanced U937 adhesiveness, E-selectin and ICAM-1 expression and IL-6 production, and caused the loss of I-kappaB. We conclude this is the first in vitro demonstration that a human monoclonal AECA from a SLE patient reacts with a constitutive endothelial membrane antigen and induces a pro-inflammatory endothelial phenotype through NF-kappaB activation.

Anti-endothelial cell antibodies to the endothelial hybridoma cell line (EAhy926) in systemic lupus erythematosus patients with antiphospholipid antibodies.

The endothelial hybridoma (EAhy926) cell line was employed to clarify whether antiphospholipid antibodies (aPA) [lupus anticoagulant (LA), antiprothrombin antibody (aPT) and/or anticardiolipin antibody (aCL)] and anti-endothelial cell antibodies (AECA) are identical, and establish whether beta2-glycoprotein I (beta2-GPI) is needed for reactivity of aPA to endothelial cells. Ig-G AECA was positive in 9/30 SLE patients with aPA (30.0%) and 10/22 SLE patients negative for aPA (45.5%). Ig-M AECA was positive in one SLE patient with aPA and one SLE patient without aPA. AECA-positivity was not significantly different among unfixed, TNF-stimulated and fixed EAhy926. The influence of beta2-GPI on the reactivity of serum to EAhy926 was minimal, and absorption experiments of serum with cardiolipin-liposome/beta2-GPI or phosphatidylserine-liposome/prothrombin gave little evidence of cross-reactivity of aPA and AECA. The results of our study suggest that aPA and AECA may have partially cross-reacted, but were different antibodies. However, further study is needed to clarify the clinico-pathological significance of AECA.

Characterization of Anti-endothelial Cell Antibodies in the Patients with Systemic Lupus Erythematosus: A Potential Marker for Disease Activity

To investigate the prevalence and possible role of anti-endothelial cell antibodies (AECA) in the pathogenesis of systemic lupus erythematosus (SLE), cell membrane antigen was prepared from cultured human umbilical vein endothelial cells and immunoblotting performed to detect AECA in SLE sera. IgG–AECA could be detected in 41 (86%) of 47 SLE patients. They were highly specific and failed to react with membrane antigens of human peripheral blood mononuclear cells or granulocytes. IgG–AECA reacted with endothelial membrane antigens which ranged from 15 to 200 kDa in molecular size. Further analysis of the antigens reacting with IgG–AECA revealed some interesting correlations between specific species of antibodies with certain clinical manifestations. Thus, patients having lupus nephritis, vasculitis, and hypocomplementemia had IgG–AECA against a 66-kDa membrane antigen; those with thrombocytopenia had IgG–AECA against a 55-kDa antigen; those with pleuritis had IgG–AECA against an 18-kDa antigen. These results indicate that IgG–AECA in the sera of SLE patients consist of heterogenous species.

A Prospective Study on Anti–endothelial Cell Antibodies in Patients with Systemic Lupus Erythematosus

IgG anti–endothelial cell antibodies (AECA) were detected in 48.5% of patients with active systemic lupus erythematosus (SLE) and in 7% of patients during remission and were associated with the development of diffuse proliferative lupus nephritis. Sixteen AECA-positive patients were prospectively studied for 25.2 ± 2.9 months. Serial AECA levels correlated with disease activity in 10 (62.5%) patients. Seven (43.8%) of 16 patients remained AECA positive during clinical remission. Among four episodes of disease exacerbation and 16 instances of clinical improvement, 85% (17 episodes) were accompanied by corresponding changes in the level of AECA, while corresponding changes in C3, anti–nuclear antibodies, and anti–double-stranded DNA antibodies were noted in 60, 60, and 80% of cases, respectively (p= not significant). AECA served as the only serologic marker of altered disease activity in five episodes, when C3, ANA, and anti–double-stranded DNA levels remained unaltered. We conclude that the level of AECA can serve as a marker of disease activity in SLE and that serial monitoring of AECA can complement other serologic parameters in the management of patients.

Sera from patients with systemic lupus erythematosus demonstrate enhanced IgG binding to endothelial cells pretreated with tumour necrosis factor alpha.

Fluorescence flow cytometry and indirect immunofluorescence were used to detect circulating IgG antiendothelial cell antibodies (IgG-AECA) in the sera of patients suffering from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS). Pretreatment of endothelial cells with tumour necrosis factor alpha (TNF alpha), but not with interferon gamma (IFN gamma), increased the IgG binding from sera of some patients with active SLE. In contrast, no change in binding activity to cytokine-stimulated endothelial cells was observed in the RA and PSS sera. The results of this study suggested that the enhanced binding of IgG from the sera of patients with SLE to endothelial cells stimulated with TNF alpha may be due to the ability of this cytokine to increase the expression of potential antigens on the surface of these cells. Hence, TNF alpha may play a role in the immune-mediated vascular damage associated with SLE.

Antibodies to Endothelial Cells in Patients with Behçet's Disease

Autoantibodies that bind to endothelial cells have been identified in patients with several forms of vasculitis. Behçet's disease—a multisystem inflammatory disorder of unknown etiology—is associated with thrombosis in addition to systemic manifestations resulting from small and large vessel vasculitis. We studied 72 Turkish patients (33 female, 39 male) with Behçet's disease in order to investigate the prevalence of antiendothelial cell antibodies (AECA) and to examine their possible relationship with clinical and laboratory features of the illness. Sera from 30 healthy Turkish people were used as controls. Human umbilical vein endothelial cells were cultured and used unfixed in a cellular ELISA to detect AECA. IgG and/or IgM AECA were found in 13 (18.1%) patients but not in healthy controls. Antiendothelial cell antibodies did not induce complement-mediated cytotoxicity as assessed by 51Cr release assay and the binding was not due to immune complexes. The prevalences of acute thrombotic events and retinal vasculitis at the time of the AECA assay among patients with AECA were significantly higher than those in patients without AECA. Laboratory parameters of active disease were higher in patients with AECA. There was no correlation between other clinical and laboratory features of Behçet's disease and AECA. Anticardiolipin and antineutrophil cytoplasmic antibodies were negative in our series, excluding a possibility of crossreaction with AECA. Our results suggest a possible role of AECA in association with thrombosis and vasculitis in patients with Behçet's disease.

IgG and IgM anti-endothelial cell antibodies in patients with collagen-vascular disorders

A cellular enzyme-linked immunosorbent assay (ELISA) was used to detect circulating anti-endothelial cell antibodies (AECA) in sera of patients suffering from rheumatoid arthritis (RA). Felty's syndrome (FS), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS) and those with a "lupus anticoagulant" (LA). IgG AECA were detected in RA, FS, SLE and LA sera, while IgM AECA were only detected in RA and FS. AECA were not specific for endothelial cells. However, IgG binding to endothelial cells and dermal fibroblasts was F(ab) mediated while in all other cell types tested, nonspecific binding most likely via the Fc region occurred. In RA and FS rheumatoid factor was shown to augment immunoglobulin binding to endothelial cells. Additional studies revealed that some of these pathological sera also contained cytotoxic IgG autoantibodies which fixed complement and damaged human umbilical vein endothelial cells in vitro. These studies suggest that a group of autoantibodies, present in a variety of collagen vascular disorders, react with endothelial cells and thus may be important aetiopathogenic factors in the vasculopathies associated with these disorders.

Anti-endothelial cell antibodies: detection and characterization using a cellular enzyme-linked immunosorbent assay.

Anti-endothelial cell antibodies (AECA) have been detected in autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma (PSS) but their role in pathogenesis is unknown. Immunofluorescence, immunohistochemistry, complement-dependent antibody lysis, and radioimmunoassay have been used in the past to detect AECA. We have developed a rapid, sensitive, and quantitative cellular enzyme-linked immunosorbent assay (ELISA) to detect and characterize AECA. Sera were obtained from 28 normal volunteers, 28 patients with SLE, and 14 patients with PSS. We also performed studies in 47 patients with various monoclonal gammopathies. Endothelial cells (EC) were obtained from human umbilical veins by standard methods and subcultured on 96-well tissue culture plates without fixation. EC were then sequentially incubated with sera, peroxidase-conjugated goat anti-human Ig (IgG, IgM, or IgA), and substrate. Optical density readings were converted to arbitrary units by developing a standard curve. Heavy-chain specific antibodies were used to determine the class of AECA binding to EC. IgG was purified by using protein A columns and digested with pepsin to obtain F(ab')2 fragments. The mean units of AECA from normals were 19.3 for IgG and 12.5 for IgM. SLE sera showed significant levels of IgM AECA (37 units, P less than 0.001) but not IgG (29 units, P less than 0.1). PSS sera showed significant levels of both IgM AECA (38 units, P = 0.001) and IgG AECA (42.7 units, P less than 0.005). IgA AECA were not detected in normal, SLE, or PSS sera. Blocking Fc receptors with rabbit IgG did not affect the titer of IgG or IgM AECA.(ABSTRACT TRUNCATED AT 250 WORDS)