AB0157 IGG FROM PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND SYSTEMIC SCLEROSIS HAVE AN INFLUENCE ON COAGULATION FACTORS IN HUMAN CEREBRAL MICROVASCULAR ENDOTHELIAL CELLS IN-VITRO

Abstract

Background:Endothelial cells from the microvasculature (hBMEC) of the brain show significant morphological and functional differences compared to EC from other anatomical areas. They are characterized by tight junctions, are not fenestrated and show less active transport mechanisms. On the other hand, the mitochondrial density is relatively high in hBMEC due to the high cerebral glucose metabolism.It could be already observed that interferon-α from SLE-sera induces the expression of MHC class I molecules on human dermal microendothelial cell line, but it is not known whether this also occurs on hBMEC. hBMECs can synthesize pro-inflammatory cytokines and chemokines such as IL-1β, but in lower concentrations than human umbilical vein endothelial cells.Patients suffering from systemic lupus erythematosus (SLE) or systemic sclerosis (SSc) show a wide spectrum of central nervous symptoms. Both, SLE and SSc, are characterised by different autoantibodies and endothelial vascular damage, especially in microvessels. 10-40% of patients with SLE suffer from lupus vasculopathy. Vascular dysfunction is one of the earliest pathological changes in SSc. Anti-endothelial autoantibodies (AECA) appear in SLE as well as in SSc and other connective tissue diseases. Research within the last years revealed that AECA play a critical role within the vascular pathogenesis of SLE and SSc. So far there is no evidence that AECA bind to hBMEC and it is not clear whether they have an effect on this special endothelial class.Objectives:In this project, we investigated if autoantibodies against hBMEC are detectable in SLE and SSc patients and if they have an influence on the activation of the endothelium by inducing adhesion molecules and on haemostasis by inducing factors of the clotting cascade.Methods:HiTrap Protein G HP antibody purification columns were used to purify IgG antibodies. Flow cytometry was used for analysis of autoantibodies against human cerebral microvascular endothelial cell line (hCMEC/D3). 26 sera of patients with SLE and 29 sera of patients with SSc were tested for presence of autoantibodies against hCMEC/D3. To analyse in vitro effects on hCMEC/D3, we measured changes in the expression of the following surface proteins: ICAM-1, VCAM-1, MHC class I and II, tissue factor, von-Willebrand-Factor, E-Selectin, P-Selectin, Thrombomodulin, CD73 and t-PA, each before and after three- and 24-hours incubation with IgG-fractions. IgG fractions of 12 SLE patients, 13 SSc patients and 13 healthy control persons (HC) were tested.Results:Autoantibodies against hCMEC/D3 were found in 21 of 26 patients with SLE (81%) and in 19 of 29 patients with SSc (66%) (p > 0.05) but not in healthy donors. After three hours incubation of hCMEC/D3 IgG-fractions, an upregulation of tissue factor by SSc-IgG (6.7% ± 5.2%) compared to HC-IgG (1.1% ± 2.8%, p < 0.01) and to SLE-IgG (1.6% ± 3.9%, p < 0.05), was detectable.There was no significant correlation between changes in surface protein expression and detection of ANA or of anti-hCMEC/D3 antibodies (p > 0.05).No change in expression of ICAM-1, VCAM-1, MHC class I and II, von-Willebrand-Factor, E-Selectin, P-Selectin, Thrombomodulin, CD73 and t-PA could be detected after incubation with IgG-fractions.Conclusion:Both, patients with SLE and patients with SSc showed autoantibodies against hBMEC. IgG fractions of patients with SSc, but not with SLE, induced an upregulation of tissue factor on the cell surface of hCMEC/D3. This could be an indicator for a direct pathogenic effect of AECA on hBMEC and might have an influence on haemostasis by activating the clotting cascade. Inhibition of these antibodies could reduce cerebral involvement of SSc.