Moreover, T cell responses to nucleosomes were increased in SLE p

Moreover, T cell responses to nucleosomes were increased in SLE patents [14]. If Fas-mediated apoptosis of T cells is defective, activated T cells reactive to self-antigens may escape apoptosis and proliferate abnormally, resulting in the destruction of target tissues. Given that oestrogen triggers SLE activity, which correlates with

an apoptotic defect of T cells [15], it can be postulated that oestrogen may affect the survival of activated T cells and their associated molecules, although the direct effects of oestrogen on SLE T cells have not yet been tested. The aim of this study was to determine whether oestrogen acts as a regulator of AICD and FasL expression in SLE T cells. This work was approved by the institutional review committees of the Catholic Medical Center

(Seoul, Republic of Korea). Heparinized peripheral blood (100 ml) was collected aseptically from SLE patients. Informed consent for usage of cells was obtained from all the SLE patients included in this study. Peripheral blood mononuclear cells were isolated by density gradient centrifugation on a Ficoll-Hypaque. Sorting of CD3+, CD4+ and CD8+ T cells (1 × 105 cells) was performed using anti-CD3, anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec, Auburn, CA, USA), respectively. T cells were then cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA), 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine. Each culture was performed this website in triplicate in 96-well plates. Cells were incubated for the predetermined times at 37°C in a 5% CO2 atmosphere and then stimulated

with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml) plus ionomycin (5 µg/ml) in the absence or presence of 17β-oestradiol (Sigma, Carbachol St Louis, MO, USA), ranging from 10−8 M to 10−6 M. Assessment of T cells undergoing apoptosis was accomplished using a cellular DNA fragmentation enzyme-linked immunosorbent assay (ELISA), as described previously [16]. Briefly, an anti-DNA antibody was fixed in the wells of a microtitre plate. The bromodeoxyuridine (BrdU)-labelled DNA fragments contained in the sample were then bound to the immobilized anti-DNA Ab. Following this, the immune-complexed BrdU-labelled DNA fragments were denatured and fixed on the surface of the plate through microwave irradiation. In the final step, the anti-BrdU peroxidase conjugate was reacted with the BrdU incorporated into the DNA. After removing the unbound peroxidase conjugates, the quantity of peroxidase bound in the immune complex was determined photometrically with 3,3,5′,5′-tetramethylbenzidine dihydrochloride (TMB) as a substrate.

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