Effects of spironolactone on human blood mononuclear cells: mineralocorticoid receptor independent effects on gene expression and late apoptosis induction

Abstract

1 Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone antagonist. Recently, the drug was shown to have an early suppressive effect on several immunoactive and proinflammatory cytokines. 2 To elucidate the mechanism behind this, the four MR-binding steroids SPIR, canrenone, 7alpha-thiomethyl-spironolactone and aldosterone (ALDO) were investigated for effects on lipopolysaccharide- and phytohemagglutinin-A-activated human blood mononuclear cells. Gene expression was examined after 4 h using microarrays, and SPIR affected 1018 transcripts of the (=) 22,000 probed. In contrast, the SPIR-related steroids affected 17 or fewer transcripts. Combining SPIR and ALDO resulted in 940 affected transcripts, indicating that SPIR has an early gene-regulatory effect independent of MR. 3 The affected genes encode a large number of signalling proteins and receptors, including immunoinflammatory response genes and apoptosis and antiapoptosis genes. Apoptosis was evident in CD3-, CD14- and CD19-positive cells, but only after 18 h of exposure to SPIR. 4 The transcriptional network involving the differentially regulated genes was examined and the results indicate that SPIR affects genes controlled by the transcription factors NF-kappaB, CEBPbeta and MYC. 5 These observations provide new insight into the non-MR-mediated effects of SPIR.

Figure 1

SPIR-induced apoptosis in MNC in vitro. MNC from three healthy individuals were incubated with SPIR, at 10 and 100 μM, 7TS, CAN, ALDO, or SPIR+ALDO. Apoptosis and cell death were measured at various times using annexin-V/PI; the TUNEL assays were carried out after 22 h of incubation. Apoptotic cells are annexin-V positive and dead cells are annexin-V+PI double-positive. The cells were not challenged with LPS+PHA unless noted. Data represent mean values±s.d., n=3. A paired Student's t-test was used for comparisons. ^*P<0.05 versus cells added with PBS alone.

Figure 2

SPIR-induced apoptosis in MNC in vitro. MNC from the same individuals as in Figure 1 were incubated with SPIR, and apoptosis was measured at various times using the annexin-V assay together with phenotype markers specific for monocytes (CD14 positive), T cells (CD3 positive) and B cells (CD19 positive). Data are shown as in Figure 1.

Figure 3

SPIR effects on production of selected cytokines. MNC from six healthy individuals were incubated with PBS, SPIR or 7TS and challenged with LPS+PHA. After 22 h at 37°C, supernatant cytokine concentrations were measured. The extracellular levels of cytokines in cultures of LPS+PHA-activated MNC without SPIR (controls) were set to 100%, and the experimental data were in each setup normalised to these controls. The control levels in pg ml⁻¹±s.d. were: 19,173±1342 (TNF-α), 173±65 (TNF-β), 1500±1255 (IL-1α), 3619±3301 (IL-1β), 4218±2899 (IL-2), 18,143±2567 (IL-6), 3644±2549 (IL-10), 1531±1153 (IFN-γ) and 115±25 (GM-CSF). Data represent mean values±s.d., n=6. The Wilcoxon signed rank test was used for comparisons. ^*P<0.05 versus control.