Therapeutic and adverse effects of a non-steroidal glucocorticoid receptor ligand in a mouse model of multiple sclerosis

Abstract

Background: Dissociating glucocorticoid receptor (GR) ligands hold great promise for treating inflammatory disorders since it is assumed that they exert beneficial activities mediated by transrepression but avoid adverse effects of GR action requiring transactivation. Here we challenged this paradigm by investigating 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (CpdA), a dissociating non-steroidal GR ligand, in the context of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).

Methodology/principal findings: CpdA inhibited pro-inflammatory mediators in myelin-specific T cells and fibroblasts in a GR-dependent manner while gene activation was abolished. However, it also induced massive apoptosis in various cell types even in the absence of the GR by engaging a Bcl-2- and caspase-dependent pathway. (1)H NMR spectroscopy corroborated these findings by revealing that CpdA dissolved in buffered solutions rapidly decomposes into aziridine intermediates known to act as alkylating pro-apoptotic agents. Importantly, the dichotomy of CpdA action also became evident in vivo. Administration of high-dose CpdA to mice was lethal while treatment of EAE with low to intermediate amounts of CpdA dissolved in water significantly ameliorated the disease. The beneficial effect of CpdA required expression of the GR in T cells and was achieved by down regulating LFA-1 and CD44 on peripheral Th cells and by repressing IL-17 production.

Conclusions/significance: CpdA has significant therapeutic potential although adverse effects severely compromise its application in vivo. Hence, non-steroidal GR ligands require careful analysis prior to their translation into new therapeutic concepts.

Figure 1. CpdA mediates transrepression in myelin-specific T effector cells via the GR but not transactivation.

(A) T_enc cells were cultured in the presence of APCs and 10 µg/ml gpMBP, either treated with 10⁻⁶ M Dex or 10⁻⁵ M CpdA for 6 hrs or left untreated (con). IFNγ and IL-17 production was assessed by intracellular flow cytometry and normalized to each control (100%) based on the percentage of cells positively staining for the respective cytokines. n = 3. (B) T_enc cells were cultured in the presence of 10⁻⁶ M Dex or 10⁻⁵ M CpdA for 6 hrs followed by the analysis of GILZ mRNA expression by quantitative RT-PCR. The expression levels were normalized to the housekeeping gene β-actin and the results are depicted as fold induction relative to untreated control cells. n = 3. (C) MEFs derived from GRN^+/+ mice (wildtype cells) and GRN^−/− mice (GR-deficient cells) were preincubated with 5 ng/ml PMA for 1 hr. Subsequently, they were stimulated with 10⁻⁶ M Dex or 10⁻⁵ M CpdA for 5 hrs or left untreated (con). MMP-13 mRNA expression was determined by quantitative RT-PCR and normalized to β-actin. MMP-13 levels in the medium controls were set as 100%. n = 3. *: p<0.05, **: p<0.01, ***: p<0.001, n.s.: p>0.05.

Figure 2. CpdA exerts pro-apoptotic activity in various cell-types.

(A) Thymocytes were cultured in the presence or absence of 10⁻⁶ M Dex or 10⁻⁵ M CpdA for 24 hrs. Apoptosis was assessed at different time points by flow cytometry based on staining for AnnexinV/7-AAD. Survival of untreated cells was set as 100% for each time point to correct for spontaneous apoptosis. n = 3. (B) Thymocytes were treated with Dex or CpdA for 24 hrs as in panel A, either in the absence (con) or the presence of 100 µM Z-VAD-fmk (pan-caspase inhibitor). n = 3. (C) SK-N-SH neuroblastoma cells were treated with Dex or CpdA at concentrations of 10⁻⁶ M and 10⁻⁵ M for 24 hrs or left untreated (con). To test whether CpdA action depends on caspase-activity, the experiment was additionally performed in the presence of 100 µM Z-VAD-fmk. n = 6. (D) 5×10⁴ MEFs generated from GRN^+/+ and GRN^−/− fetuses were cultured in the absence (con) or presence of 10⁻⁶ M Dex or 10⁻⁵ M CpdA. After 48 hrs cell numbers were determined by microscopic counting. n = 3. *: p<0.05, **: p<0.01, ***: p<0.001, n.s.: p>0.05.

Figure 3. CpdA-induced apoptosis is independent of the GR.

(A,B) Thymocytes were isolated either from GRN^+/+ or GRN^−/− E18.5 embryos and cultured in the absence (con) or presence of different concentrations of Dex (A) or CpdA (B) for 24 hrs. Apoptosis was assessed by flow cytometry based on AnnexinV/7-AAD staining. Survival of untreated cells was set as 100% for each time point to correct for spontaneous apoptosis. n = 3. (C,D) WEHI 7.1 cells stably transduced with a retrovirus encoding a GR-specific shRNA (GR-siRNA), Bcl-2 or the empty retroviral vector LMP, were cultured in the absence (con) or presence of different Dex concentrations (C) or 10⁻⁵ M CpdA (D) for 24 hrs. Apoptosis was assessed by flow cytometry based on AnnexinV/7-AAD staining. Survival of untreated cells was set as 100% for each time point to correct for spontaneous apoptosis. n = 3−5. *: p<0.05, **: p<0.01, n.s.: p>0.05.

Figure 4. Decay of CpdA into aziridine derivatives and synephrine depends on the solvent and pH.

(A) Schematic depiction of the in vitro decay of CpdA in pure water (upper part) and PBS (pH = 7.6) or Tris-HCl (pH = 7.6) buffer (lower part) based on ¹H NMR spectroscopic analysis. When dissolved in water, CpdA directly decomposes into acetyl synephrine within approximately 5 days, followed by ester hydrolysis into synephrine after several weeks to months. In contrast, dissolving CpdA in buffered solutions leads to the formation of a mixture of the corresponding cis and trans aziridines, which can be detected by ¹H NMR spectroscopy within minutes. With a half-life (t_1/2) of several days the aziridines then become hydrolyzed into acetyl synephrine and subsequently synephrine. (B) The half-life of CpdA dissolved in phosphate-buffered solutions with pH values of 6.7, 7.1, 7.7 and 8.1 was determined by ¹H NMR spectroscopy and its logarithm was plotted against the pH value. The exact values and the linear regression curve are depicted in the graph. r² indicates the correlation coefficient.

Figure 5. Treatment of MOG_35–55 induced EAE by CpdA.

(A) EAE was induced in C57Bl/6 mice followed by treatment with 15 mg/kg CpdA (dissolved in 20% ethanol), 100 mg/kg Dex or PBS as a control (con) on 3 consecutive days starting at an average clinical score of 2 (marked by arrows); n = 11; statistical analysis: days 11–22. The cross indicates that all animals of this group either died or had to be sacrificed for ethical reasons. The experiment was repeated twice with similar results. (B) CpdA was dissolved in water and therapeutically applied at doses of 5 mg/kg or 1.5 mg/kg for 3 consecutive days after the mice had developed an average clinical score of 3 (marked by arrows); treatment with the solvent alone (con) served as a control; n = 18/6/19; statistical analysis: days 13–23. The experiment was repeated five times with similar results. (C) EAE was induced in C57Bl/6 and GR^lckCre mice followed by treatment with CpdA dissolved in water at a dose of 5 mg/kg or the solvent alone (con). Therapy was started at an average score of 2 (defined as day 0); n = 6; statistical analysis: days 1–10. The experiment was repeated twice with similar results. (D) C57Bl/6 wildtype or GR^lckCre mice were treated with 5 mg/kg or 15 mg/kg CpdA dissolved in water or PBS as a control on 3 consecutive days. On the following morning surviving mice were sacrificed and the cellularity of the spleen was determined by microscopic counting. n = 3/12. **: p<0.01, ***: p<0.001, n.s.: p>0.05.

Figure 6. Mechanism of CpdA action in the treatment of EAE.

(A) EAE was induced in C57Bl/6 mice followed by treatment with 1.5 mg/kg or 5 mg/kg CpdA dissolved in water for 3 consecutive days after the mice had developed an average clinical score of 3. On the following morning the mice were sacrificed and the leukocytes isolated from the spinal cord. Cellularity was determined by microscopic counting; staining for AnnexinV binding and surface expression of LFA-1 on CD3⁺CD4⁺ Th cells was performed by flow cytometry. n = 3−7. (B) The spleen was isolated from the same animals as in panel A and the leukocytes analyzed for AnnexinV binding as well as LFA-1 and CD44 surface expression on CD3⁺CD4⁺ Th cells by flow cytometry. n = 3−8. (C, D) Splenocytes from the same animals as in panels A and B were cultured in the presence or absence of ConA or MOG_35–55 peptide. Proliferation was measured by ³[H]-thymidine incorporation assay and expressed as a proliferation index relative to the values obtained in the absence of any stimulus (C); IL-17 and IFNγ levels in the supernatant were determined by ELISA (D); n = 7−10. *: p<0.05, ***: p<0.001, n.s.: p>0.05.