Flow cytometry analysis of glucocorticoid receptor expression and binding in steroid-sensitive and steroid-resistant patients with systemic lupus erythematosus

Juan Du, Min Li, Denghai Zhang, Xiaoyan Zhu, Weiwei Zhang, Wei Gu, Yinglu Feng, Xiaofeng Zhai, Changquan Ling, Juan Du, Min Li, Denghai Zhang, Xiaoyan Zhu, Weiwei Zhang, Wei Gu, Yinglu Feng, Xiaofeng Zhai, Changquan Ling

Abstract

Introduction: 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.

Trial registration: ClinicalTrials.gov NCT00600652.

Figures

Figure 1
Figure 1
Evaluation of FCM analysis of GR binding by RLBA. Analysis of GR binding in (a) K562 and (b) U937 with and without 10-6 M Dex by FITC-Dex-FCM and RLBA. Parallel FCM-FITC-Dex and radiometric assays were performed on the same day, using the same cell cultures to minimise variation. Specific FCM analysis was obtained as difference of mean channel number between total and nonspecific binding. By linear regression analysis, a positive correlation between results from the two methods was found. Dex = dexamethasone; FCM = flow cytometry; FITC = fluorescein isothiocyanate; GR = glucocorticoid receptor; RLBA = radiolabelled receptor ligand.
Figure 2
Figure 2
Evaluation of FCM analysis of GR expression by western blot. Analysis of GR repression in (left) K562 and (right) U937 with and without 10-6 M Dex by GR-mAb FCM and western blot. Data are expressed as described in 2 figures at the bottom of Figure 2. Parallel GR-mAb FCM and western blot assays were performed on the same day, using the same cell cultures to minimise variation. Specific FCM analysis was obtained as difference of mean channel number between total and nonspecific binding. By linear regression analysis, a positive correlation between results from two methods was found. Dex = dexamethasone; FCM = flow cytometry; GR = glucocorticoid receptor; mAb = monoclonal antibody.
Figure 3
Figure 3
Percentage of GR-positive T lymphocytes and monocytes in SLE patients and controls. (a) Percentage of T lymphocytes and monocytes presentsing GR. The percentage of positive T lymphocytes and monocytes presenting GR in SR group did not differ from those in SS and the normal control groups (P > 0.05). (b) Percentage of GR-binding positive T lymphocytes and monocytes. The percentage of positive GR-binding cells was also similar among the SR, SS and normal control groups (P > 0.05). GR = glucocorticoid receptor; SLE = systemic lupus erythematosus; SR = steroid-resistant; SS = steroid-sensitive.
Figure 4
Figure 4
FCM analysis of GR in SLE patients and controls. Comparison of GR (a) expression and (b) binding in lymphocytes (CD3/GR) and monocytes (CD14/GR) among the SR group (n = 17) and the SS group (n = 18) with SLE, and the normal control group (n = 27). Bars show the mean ± standard error of the mean average fluorescence intensity of CD3/GR or CD14/GR detected by FCM before treatment. GR expression and binding in the SR group were significantly lower than in the other two groups. No differences were found between the SS group and normal control group in the expression and the binding of GR, except for the GR binding in CD3, which in the SS group was significantly lower than that in the control group. *P < 0.01, vs normal group, △ P < 0.01, vs SS group. FCM = flow cytometry; GR = glucocorticoid receptor; MFI = mean fluorescence intensity; SLE = systemic lupus erythematosus; SR = steroid-resistant; SS = steroid-sensitive.
Figure 5
Figure 5
Inhibition of cytokine secretion in SR and SS groups. Percentage of inhibition of (a) TNF-α, (b) IL-12 and (c) IFN-γ cytokine secretion after PBMC incubation in RPMI with PHA plus 10-6 M dexamethasone in normal controls, SS patients and SR patients. Calculation formula of percentage inhibition of cytokine secretion by steroid was described in Materials and Methods. *P < 0.01, vs normal group, △ P < 0.01, vs SS group. (solid circle is outliers, and hollow circle is extreme values). IFN = interferon; IL = interleukin; PBMC = peripheral blood mononuclear cells; SR = steroid-resistant; SS = steroid-sensitive; TNF = tumour necrosis factor.
Figure 6
Figure 6
Correlation between GR binding and TNF-α, IL-12 and IFN-γ. Percentage inhibition of (a) TNF-α, (b) IL-12 and (c) IFN-γ cytokine secretion after PBMC incubation in RPMI with PHA plus 10-6 M dexamethasone were correlated to GR binding in (left) CD3+ and (right) CD14+ subpopulation in normal controls, SS patients and SR patients. (hollow circle represents normal group, triangle represents SS group, solid circle represents SR group). GR = glucocorticoid receptor; IFN = interferon; IL = interleukin; PBMC = peripheral blood mononuclear cells; SR = steroid-resistant; SS = steroid-sensitive; TNF = tumour necrosis factor.

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