Flow cytometry analysis of glucocorticoid receptor expression and binding in steroid-sensitive and steroid-resistant patients with systemic lupus erythematosus.
BACKGROUND
Glucocorticoid (GC) therapy is the main treatment for systemic lupus erythematosus (SLE). However, some patients are resistant to these agents. Abnormalities of glucocorticoid receptor (GR) seem to be related to steroid resistance. This study evaluated GRs in T lymphocytes and monocytes of SLE patients by flow cytometry (FCM) using a monoclonal antibody (mAb) and FITC-Dex probes.


METHODS
Thirty-five patients with SLE before treatment and 27 age- and sex-matched normal controls were studied. Disease activity scores were determined before and after treatment and used to divide the patients into steroid-resistant (SR) and steroid-sensitive (SS) groups. GRs in T lymphocytes (CD3+) and monocytes (CD14+) were examined by FCM with GR-mAb and FITC-Dex probes before treatment. Peripheral blood mononuclear cells (PBMCs) were isolated for in vitro GCs sensitivity assays. The validity of FCM analysis of intracellular staining for GR with GR-mAb and FITC-Dex probes was evaluated through comparison with western blot and radioligand binding assay (RLBA) in U937 and K562 cells in vitro. One-way ANOVA, student's t test, linear regression and spearman correlation were performed.


RESULTS
A significant decrease in GR binding and the expression in K562 and U937 cells with 10-6 M dexamethasone (Dex) was found compared with those without Dex. In addition, a positive correlation was found between FCM and RLBA as well as FCM and Western blot. The expression and binding of both CD3/GR and CD14/GR in SR patients with SLE, detected by FCM, were all lower than those in SS patients with SLE, whereas there was no significant difference in SS patients and controls. In vitro corticosteroid sensitivity assay indicated that PHA-stimulated tumour necrosis factor-alpha (TNF-alpha), IL-12 and interferon-gamma (IFN-gamma) secretion was significantly inhibited by 10-6 M Dexamethasone in all controls and SS patients, compared with that in SR group, which confirms patient classification as SR and SS by disease activity index (SLEDAI) score.


CONCLUSIONS
Abnormalities of expression and binding of the GR may be involved in tissue resistance to steroids in SLE patients. Determination of GR expression and binding by FCM may be useful in predicting the response to steroid treatment of SLE patients.
Du
Juan
J
Department of Integrative Medicine, Changhai Hospital, The Second Military Medicine University, No. 168 Changhai Road, Shanghai, PR China. dujuan714@163.com
Li
Min
M
Zhang
Denghai
D
Zhu
Xiaoyan
X
Zhang
Weiwei
W
Gu
Wei
W
Feng
Yinglu
Y
Zhai
Xiaofeng
X
Ling
Changquan
C
D000328:Adult; D000893:Anti-Inflammatory Agents / Q000627:therapeutic use; D015153:Blotting, Western; D017252:CD3 Complex / Q000378:metabolism; D003907:Dexamethasone / Q000627:therapeutic use; D004351:Drug Resistance / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D005938:Glucocorticoids / Q000627:therapeutic use; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000188:drug therapy / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D017252:CD3 Complex; D005938:Glucocorticoids; D018950:Lipopolysaccharide Receptors; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone