Anti-endothelial cell IgG fractions from systemic lupus erythematosus patients bind to human endothelial cells and induce a pro-adhesive and a pro-inflammatory phenotype in vitro.

Affinity purified immunoglobulin G (IgG) fractions from systemic lupus erythematosus (SLE) patients positive for anti-endothelial cell antibodies (AECA) bind human umbilical vein endothelial cell (HUVEC) monolayers. In vitro incubation of serial protein concentrations of SLE AECA IgG induces a dose-dependent endothelial activation: i) increase of functional adhesion of the monocytic cell line U937; ii) upregulation of E-Selectin, ICAM-1, VCAM-1 expression evaluated by a cell solid-phase enzyme linked immunoassay; and iii) increased secretion of interleukin (IL)-6 in the culture supernatants. Control experiments carried out with HUVEC monolayers incubated with IgG fractions from normal healthy controls or from AECA negative SLE sera do not affect at all endothelial adhesion molecule expression or pro-inflammatory cytokine secretion. The AECA IgG effects are not related to both anti-phospholipid or anti-DNA activities. Taken together the findings suggest that these autoantibodies might be important in recruiting and in activating mononuclear leukocytes responsible for vessel wall infiltration and raise the possibility that AECA might display a pathogenic role in SLE vessel damage.     

Anti-endothelial cell antibodies from lupus patients bind to apoptotic endothelial cells promoting macrophage phagocytosis but do not induce apoptosis

OBJECTIVE: Anti-endothelial cell antibodies (AECA) have been reported to induce apoptosis. We investigated the induction of apoptosis by these autoantibodies and their involvement in the removal of apoptotic cells. METHODS: AECA isolated from patients with active systemic lupus erythematosus (SLE) were incubated with human umbilical vein endothelial cells (HUVECs). AECA-positive sera were identified using a cell-based ELISA. Apoptosis was measured by morphology and phosphatidylserine externalization using flow cytometry with fluorescein isothiocyanate (FITC)-conjugated annexin V. Flow cytometry was used to investigate AECA binding to apoptotic cells using FITC-conjugated anti-human immunoglobulin G (IgG). Apoptotic endothelial cells were stained with a red dye (PKH26) and co-cultured with macrophages, and phagocytosis was visualized under phase contrast microscopy. RESULTS: AECA from patients with SLE did not induce apoptosis compared with normal IgG (nIgG) at any time point, as assessed by morphology (at 24 h, P = 0.167) or phosphatidylserine externalization (at 24 h, P = 0.098). However, there was increased binding of AECA to apoptotic endothelial cells (48.8 +/- 11.9 compared with 25.8 +/- 6.7% AECA binding to freshly isolated cells, P< 0.001). These opsonized endothelial cells showed greater phagocytosis by macrophages (mean phagocytic index 24.9 +/- 4.5%) when cells opsonized with nIgG were compared with AECA (34.8 +/- 3.4% n = 5, P = 0.01). CONCLUSION: In conclusion, AECA bind to apoptotic endothelial cells but do not induce endothelial cell apoptosis. Macrophage phagocytosis is increased by opsonization of apoptotic endothelial cells by AECA, a proinflammatory mechanism of cell removal.     

Regulation of adhesion molecule expression by human synovial microvascular endothelial cells in vitro

Objective. To examine the in vitro expression of E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1), ICAM-2, vascular cell adhesion molecule 1 (VCAM-1), and platelet–endothelial cell adhesion molecule 1 (PECAM-1) by synovial microvascular endothelial cells (SMEC) in comparison with microvascular neonatal foreskin endothelial cells (FSE) and macrovascular human umbilical vein endothelial cells (HUVE). Methods. Cultured endothelial cells were treated for 4 hours with medium alone or tumor necrosis factor α (TNF α). The expression of endothelial adhesion molecules was evaluated by flow cytometry, cell enzyme-linked immunosorbent assay, and Northern blot analysis. Results. SMEC continuously expressed E-selectin under basal culture conditions, whereas FSE and HUVE did not. TNF α treatment of rheumatoid arthritis (RA) SMEC resulted in sustained peak expression of E-selectin for up to 24 hours, which subsequently declined but remained elevated even at 72 hours. In contrast, peak E-selectin expression in FSE and HUVE occurred between 4 hours and 16 hours after TNF α treatment and then declined to near basal levels by 24–48 hours. SMEC expressed significantly higher levels of ICAM-1 compared with HUVE under basal culture conditions. There was no difference between SMEC, FSE, and HUVE in the expression of P-selectin, VCAM-1, ICAM-2, or PECAM-1. Northern blot analysis demonstrated that the levels of E-selectin expression by TNF α-stimulated endothelial cells correlated with their respective messenger RNA levels. Conclusion. Regulation of E-selectin and ICAM-1 expression in RA synovial endothelium is different from that in neonatal foreskin and human umbilical vein endothelium. The augmented expression of adhesion molecules in RA synovial endothelium may facilitate the recruitment of leukocytes to this site.     

Anti-endothelial cell IgG from patients with chronic arsenic poisoning induces endothelial proliferation and VEGF-dependent angiogenesis

An endemic peripheral vascular disorder due to chronic arsenic poisoning, named Blackfoot disease (BFD), occurs in Taiwan. BFD causes destruction of vascular endothelial cells, and an anti-endothelial cell IgG antibody was found in the sera of BFD patients. We studied the role of this IgG antibody (BFD-IgG) in modulating proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs) and found that a low concentration of BFD-IgG (200 microg/mL) stimulated endothelial cell growth and increased expressions of vascular cell adhesion molecule-1 (VCAM-1), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF). The apoptosis events appeared not altered by addition of BFD-IgG. An in vitro neoangiogenesis assay demonstrated that BFD-IgG promoted the formation of tube-like structures, which was completely abrogated by anti-VEGF neutralizing antibody and partially by NOS inhibitor, L-NAME. We conclude that BFD-IgG at 200 microg/mL results in cell proliferation and enhanced VEGF-dependent angiogenesis in vitro. Those results suggested that a low concentration of BFD-IgG plays a protective role in the pathogenesis or the progression of BFD.     

Anti-endothelial cell IgG from patients with chronic arsenic poisoning induces endothelial proliferation and VEGF-dependent angiogenesis

An endemic peripheral vascular disorder due to chronic arsenic poisoning, named Blackfoot disease (BFD), occurs in Taiwan. BFD causes destruction of vascular endothelial cells, and an anti-endothelial cell IgG antibody was found in the sera of BFD patients. We studied the role of this IgG antibody (BFD-IgG) in modulating proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs) and found that a low concentration of BFD-IgG (200 μg/mL) stimulated endothelial cell growth and increased expressions of vascular cell adhesion molecule-1 (VCAM-1), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF). The apoptosis events appeared not altered by addition of BFD-IgG. An in vitro neoangiogenesis assay demonstrated that BFD-IgG promoted the formation of tube-like structures, which was completely abrogated by anti-VEGF neutralizing antibody and partially by NOS inhibitor, L-NAME. We conclude that BFD-IgG at 200 μg/mL results in cell proliferation and enhanced VEGF-dependent angiogenesis in vitro. Those results suggested that a low concentration of BFD-IgG plays a protective role in the pathogenesis or the progression of BFD.     

Tibolone inhibits leukocyte adhesion molecule expression in human endothelial cells

Tibolone is a synthetic steroid with mixed estrogenic and progestogenic/androgenic activity used for post-menopausal hormone replacement therapy. Since its cardiovascular effects are still not clear, and no data have been published on possible direct actions on the vessel wall, we studied the effects of tibolone and its metabolites on lipopolysaccharide (LPS)-induced expression of leukocyte adhesion molecules on human endothelial cells. Tibolone and its two estrogenic 3alpha-OH and 3beta-OH metabolites, but not the progestogenic/androgenic Delta(4)-isomer, concentration-dependently decreased LPS-induced vascular cell adhesion molecule-1 protein expression. This effect was estrogen receptor dependent, since it was completely blocked by the pure estrogen receptor antagonist ICI 182780. Furthermore, only tibolone, the 3alpha-OH and the 3beta-OH metabolites decreased endothelial expression of E-selectin, while none of the compounds changed the levels of intercellular adhesion molecule-1. These findings were associated with parallel changes in mRNA levels for the three adhesion molecules. Our data show that tibolone and its estrogenic metabolites exert direct actions on the vascular wall, decreasing the expression of endothelial-leukocyte adhesion molecules, thus producing potentially important direct anti-atherogenic effects.     

Anti-endothelial cell antibodies in patients with sarcoidosis

BACKGROUND: Anti-endothelial cell antibodies (AECA) are circulating antibodies that bind to endothelial antigens and induce endothelial cell damage. These antibodies have been detected in patients with collagen vascular disease and systemic vasculitis. Sarcoidosis is a multiple granulomatous disorder of unknown etiology, and its clinical presentation, organ involvement, and prognosis are highly diverse. In this study, we aimed to investigate the expression of AECA in patients with sarcoidosis. We also examined whether these antibodies could serve as a marker for the activity, severity, and prognosis of sarcoidosis. METHODS: Forty sarcoidosis patients, whose diagnosis was established by clinicoradiologic findings and histologic confirmation of noncaseating epithelioid cell granulomas, were studied. Serum and BAL samples were examined for AECA by cellular enzyme-linked immunosorbent assay (ELISA) using human umbilical vein endothelial cells. The findings were expressed in terms of an ELISA ratio (ER). Fifty-seven subjects without any clinical, radiologic, or serologic evidence of pulmonary disease or autoimmune disorders served as the reference population to judge the positivity of AECA. RESULTS: Patients with sarcoidosis had a significantly higher positivity rate for and levels of AECA in both serum and BAL fluid than the reference population. In addition, the ER of AECA was significantly elevated in patients with multiple lesions or who required corticosteroid therapy compared with that in patients without multiple lesions or who did not need corticosteroid therapy. CONCLUSION: Expression of AECA might be a useful marker to predict the course of sarcoidosis.     

Inability of plasma high-density lipoproteins to inhibit cell adhesion molecule expression in human coronary artery endothelial cells

High-density lipoproteins (HDL) have several antiatherogenic actions, including the ability to sequester cellular cholesterol, to protect low-density lipoproteins from oxidation and to inhibit platelet aggregation. An early event in atherogenesis is the adhesion and recruitment of blood monocytes, a process mediated by cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1) which is rapidly synthesized by endothelial cells in response to cytokines. It has been reported that HDL limits CAM expression in cultured human umbilical vein endothelial cells (HUVECs), implying that HDL also protects at an early stage in lesion development. Here, we have studied HDL suppression of CAM induction in human coronary artery endothelial cells (HCAECs), a model directly relevant to blood vessels susceptible to atherosclerosis. Arterial endothelial cells were preincubated with increasing amounts of total HDL, or different subfractions, and then activated with the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). Flow cytometric analysis failed to detect any downregulation of VCAM-1 or E-selectin expression by HDL in this model of vascular endothelium. Moreover, we were unable to confirm that HDL could suppress CAM induction in well-characterized, low-passage HUVECs, even though positive controls, 17beta-estradiol or a nitric oxide donor, did cause downregulation and factors such as variability in donors and HDL preparation, or culture conditions, were excluded. We tentatively conclude that, as isolated HDL did not downregulate CAM expression in cultured HCAECs or HUVECs, attenuation of CAM induction in arterial endothelium is unlikely to contribute to HDL antiatherogenic actions in vivo.     

Simvastatin alters human endothelial cell adhesion molecule expression and inhibits leukocyte adhesion under flow

Hypercholesterolaemia is implicated as an independent risk factor in the pathogenesis of atherosclerosis. HMG-CoA reductase inhibitors (statins) are prescribed for their lipid-lowering effects but recent evidence suggests they have pleiotropic effects independent of lipid balance regulation that may explain their role in dramatically decreasing cardiovascular mortality and morbidity. The mechanisms responsible are unclear but endothelial cell (EC) dysfunction is critical. To investigate potential anti-inflammatory properties of statins on EC, functional responses of human umbilical vein endothelial cells (HUVEC) and human neutrophils under physiological flow conditions were studied. These interactions were quantified in response to inflammatory mediators following pre-treatment with statin.Histamine stimulation resulted in significant (p < 0.001) increases in transient interactions between neutrophils and EC (tethering). These effects were significantly reduced (p < 0.001) on pre-treatment with statin. TNF-α stimulation resulted in significant (p < 0.001) increases in rolling interactions. These effects were significantly (p < 0.001) reduced following pre-treatment of EC with statin. Mevalonate pre-treatment of EC significantly reversed the effects of statin pre-treatment on both tethering and rolling (p < 0.001).Reductions in surface expression of P- and E-selectin were confirmed by ELISA. EC exposed to histamine demonstrated significantly increased (p < 0.01) levels of P-selectin, abrogated (p < 0.001) by pre-treatment with statin. EC exposed to TNF-α demonstrated a significant increase (p < 0.001) in levels of E-selectin, reduced (p < 0.05) by pre-treatment with statin.     

Anticardiolipin antibodies and antibodies to beta(2)-glycoprotein I in patients with Wegener's granulomatosis

SIR, In primary systemic vasculitides such as Wegener's granulomatosis(WG), vasculitis involving veins has been implicated as a contributoryfactor for thrombosis, although systematic studies of this problemare still lacking [1]. Other determinants for thrombosis havenot been evaluated in WG so far. Conventional anticardiolipin antibody (ACLA) assays detect bothß₂-glycoprotein I (ß₂GPI)-dependent antibodiesas well as ß₂GPI-independent antibodies. ß₂GPIacts as a cofactor for the interaction between ACLA and phospholipids.Antibodies solely directed to ß₂GPI have     

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 β₂-glycoprotein I (β₂-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 β₂-GPI on the reactivity of serum to EAhy926 was minimal, and absorption experiments of serum with cardiolipin-liposome/β₂-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.     

Anti-endothelial cell antibodies in patients with interstitial lung diseases

BACKGROUND: Anti-endothelial cell antibodies (AECA) are circulating antibodies that bind to endothelial antigens and induce endothelial cell damage. AECA have been detected in patients with collagen vascular disease (CVD) and their presence is associated with interstitial lung disease (ILD) in cases of CVD. However, the prevalence of AECA in patients with idiopathic interstitial pneumonias (IIPs) is not known. METHODS: We investigated the prevalence of AECA in patients with IIPs. We also examined whether the expression of AECA differed among the histologic subgroups usual interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP), and compared the values with those of CVD-associated ILD (CVD-ILD). Twenty patients with IIPs and 24 patients with CVD-ILD were studied. Serum samples were examined for AECA by cellular enzyme-linked immunosorbent assay (ELISA) using human umbilical vein endothelial cells. Values are expressed as ELISA ratios (ER). RESULTS: All sera from patients with idiopathic pulmonary fibrosis (IPF)/UIP were negative for AECA, whereas 5 out of 10 with idiopathic NSIP, 5 out of 14 with CVD-UIP and 4 out of 10 with CVD-NSIP tested positive (p<0.05). ER values were significantly lower in patients with IPF/UIP than idiopathic NSIP, CVD-UIP or CVD-NSIP (p<0.05). Among idiopathic NSIP, CVD-NSIP and CVD-UIP patients, the ER values did not differ. CONCLUSIONS: Among IIP patients, only those with idiopathic NSIP, not IPF/UIP, tested positive for AECA. The prevalence of AECA in idiopathic NSIP patients was similar to that in CVD-ILD patients. These results may provide important information to understand the distinct pathophysiology of each form of IIPs.     

Antiendothelial cell antibodies in patients with Wegener's granulomatosis: prevalence and correlation with disease activity and manifestations

OBJECTIVE: Previous studies in small cohorts of patients with Wegener's granulomatosis (WG) or antineutrophil cytoplasmic antibody (ANCA) associated vasculitis have yielded conflicting data regarding the prevalence of antiendothelial cell antibodies (AECA), ranging from 8% to 100%, and the use of AECA as a measure of disease activity. We examined a large, well-characterized cohort of patients with WG and active disease for the presence of AECA. METHODS: Serum from subjects with WG who participated in a clinical therapeutic trial was collected at baseline, when all subjects had active disease. Clinical manifestations and disease activity were documented using the Birmingham Vasculitis Activity Score for WG (BVAS/WG). Serum AECA (IgG) was measured by cyto-ELISA using unfixed human umbilical vein endothelial cells (HUVEC). The AECA positivity cutoff was determined using 71 healthy control samples. Statistical analyses utilized Student's t test, chi-square and Fisher's exact tests, and linear regression. RESULTS: AECA were detected in 34 of 173 (20%) evaluated serum samples. Mean BVAS/WG did not differ between patients with (7.3 +/- 3.2) or without AECA (7.0 +/- 3.3) (p = 0.58). Among the 34 patients positive for AECA, the antibody titer did not correlate with disease activity (BVAS/WG; r = 0.09, p = 0.60). There were no statistically significant differences in the frequency of major clinical manifestations between patients with or without AECA. CONCLUSION: AECA, as measured using HUVEC, are not highly prevalent among patients with active WG, are not associated with specific clinical manifestations, and do not correlate with level of disease activity.     

Human endothelial cells express proteinase 3, the target antigen of anticytoplasmic antibodies in Wegener's granulomatosis

Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (PR-3), have proved to be a useful clinical tool confirming the diagnosis or monitoring disease activity of Wegener's granulomatosis (WG). Although several concepts concerning the pathophysiologic potentials of ANCA have been discussed, only sparse data about ANCA-endothelium interactions have been available. In this study, we have investigated the expression of PR-3 in cytokine- treated human endothelial cells using purified anti-PR-3 antibodies of patients with WG, murine and human monoclonal anti-PR-3 antibodies as probes. We were able to show that tumor necrosis factor-alpha, interleukin-1 alpha/beta, and interferon-gamma led to an increased PR-3 expression in the cytoplasm of endothelial cells by performing polymerase chain reaction analysis, Western blot, cyto-enzyme-linked immunosorbent assays, and confocal laser scanning microscopy. Moreover, PR-3 was also translocated into the cell membrane, becoming accessible to ANCA. Our data suggest a possible direct pathogenic effect of anti- PR-3 antibodies in WG and other vasculitides. Anti-PR-3 antibodies represent an important missing link in ANCA-endothelial interactions.     

Lack of association between antiphospholipid antibodies and thrombocytopenia in patients with Wegener's granulomatosis

BACKGROUND AND OBJECTIVE: In patients with Wegener's granulomatosis (WG), thrombocytopenia is less common than thrombocytosis. An increased prevalence of antiphospholipid antibodies (aPL), which is associated with thrombocytopenia, has been noted in patients with WG. The aim of this study was to examine the relationship between thrombocytopenia and aPL in patients with WG. METHODS: Thrombocytopenic episodes were searched for in a random sample of 83 patients with WG. Stored sera obtained during thrombocytopenia, which was defined as platelet count below 130 x 10(9)/L, were examined by 2 different enzyme-linked immunosorbent assays (ELISA) for IgG and IgM anticardiolipin antibodies (aCL) and for IgG antiphosphatidylserine antibodies (aPS). Screening for lupus anticoagulant was performed by use of activated partial thromboplastin time (aPTT). Results were compared with the prevalence of aPL in 20 consecutive nonthrombocytopenic patients with WG. RESULTS: Six cases with thrombocytopenic episodes were found in the group of 83 patients with WG. Increased IgG and IgM aCL were detected in 1 patient, who also had elevated IgG aPS. A positive test result solely for IgM aCL was found in another patient. These findings were consistent in both ELISA for aPL. Five patients were being treated with cyclophosphamide when thrombocytopenia occurred. In the group of nonthrombocytopenic patients with WG, elevated IgG aCL and IgG aPS were consistently detected in 1 patient in both ELISA. Three other patients had positive results in single tests, which were not confirmed by the second assay. In all patients, aPTT was normal. CONCLUSIONS: Thrombocytopenia is a rare finding in patients with WG. A similar prevalence of aPL in thrombocytopenic and nonthrombocytopenic patients with WG provides no evidence that aPL play a major role in the pathogenesis of these events. Thrombocytopenia in WG is more likely caused by the myelotoxic effect of preceding cyclophosphamide treatment. We found a frequency of aPL in WG that exceeds frequencies seen in the general population but does not approximate those detected in systemic lupus erythematosus and closely related disorders. Semin Arthritis Rheum 31:4-11.     

Anti-endothelial cell antibodies: detection and characterization in sera from patients with autoimmune hypoparathyroidism

In a previous report, we described antibodies in autoimmune hypoparathyroidism (AHP) that are cytotoxic for cultured bovine parathyroid cells. In the present study, we show that sera from six AHP patients, but not from 26 patients with other autoimmune diseases or from 7 healthy subjects, react with bovine endothelial cells in culture (by flow cytometry and fluorescence microscopy) and in tissue sections (by immunohistology). We found uniformly that the immunoglobulin class reacting is IgM. Adsorption experiments showed that the antigenic determinants reacting with AHP sera were similar on bovine cultured endothelial cell membranes and in tissue sections of bovine parathyroid glands. The AHP sera also reacted with endothelial cells cultured from bovine adrenal medulla and pulmonary artery. Immunoblot analysis showed antibody binding to two major bands of 200 and 130 kDa solubilized from the membrane fraction of bovine parathyroid endothelial cells. Only one AHP serum consistently recognized endothelium-related structures on frozen sections of three different human parathyroid adenomas; two other sera reacted with one adenoma each; and three did not react with human adenomas. This indicates that human material is less suitable than bovine in detecting endothelium-related immune phenomena in AHP sera. The anti-endothelium IgM antibodies appear to be disease-specific but are not organ- or species-specific. The identification of endothelial cells as the target for antibodies in AHP raises the possibility that the endothelium subserves an important local function for endocrine epithelium.