Glucocorticoids potently block tumour necrosis factor-alpha- and lipopolysaccharide-induced apoptotic cell death in bovine glomerular endothelial cells upstream of caspase 3 activation

Abstract

1. Endothelial cell damage in glomeruli and kidney arterioles appears to play a pivotal role in glomerular inflammatory diseases. Glomerular endothelial cells, a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration, die by apoptosis in response to tumour necrosis factor-alpha (TNF-alpha), TNF-alpha/basic fibroblast growth factor (bFGF), TNF-alpha/cycloheximide, and bacterial lipopolysaccharide (LPS). Apoptotic cell death is characterized by extensive DNA cleavage, DNA ladder formation, and characteristic morphological alterations. 2. In search for apoptosis-preventing signals, we identified glucocorticoids as potent death preventing factors. Co-treatment of cells with 10 nM dexamethasone and TNF-alpha, TNF-alpha/bFGF, TNF-alpha/cycloheximide, or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. 3. Similarly to dexamethasone (TNF-alpha- and LPS-induced apoptosis are prevented with IC50 values of 0.8 and 0.9 nM, respectively), other synthetic and natural forms of glucocorticoids, such as fluocinolone, prednisolone, hydrocortisone, and corticosterone potently inhibited cell death with IC50 values of 0.2, 6, 50 and 1000 nM, for TNF-alpha and 0.7, 8, 100 and 500 nM for LPS, respectively. 4. Apart from glucocorticoids, mineralocorticoids such as aldosterone also blocked TNF-alpha/LPS-induced apoptosis (IC50 approximately 500 nM for TNF-alpha and approximately 500 nM for LPS), whereas sex hormones, i. e. beta-estradiol and testosterone remained without effect. 5. The protective effect of glucocorticoids (and mineralocorticoids) required glucocorticoid receptor binding as it could be antagonized by the glucocorticoid receptor antagonist RU-486. Concerning TNF-alpha and LPS signal transduction, we found that dexamethasone efficiently prevented TNF-alpha- and LPS-induced activation of caspase-3-like proteases. Therefore, we postulate inhibitory mechanisms upstream of terminal death pathways.

Figure 1

Dexamethasone suppressed apoptotic DNA fragmentation induced by TNF-α and bacterial LPS. (A) Bovine glomerular endothelial cells were incubated for 24 h with 25 ng ml⁻¹ TNF-α and 30 ng ml⁻¹ LPS in the absence or presence of increasing concentrations of dexamethasone. DNA fragmentation was quantitated by the diphenylamine reaction. Data are means±s.e. mean of four separate experiments. ^*P<0.05 (ANOVA and for multiple comparison the data were corrected by Dunn's Method). (B) The formation of oligonucleosomal DNA fragments was assessed by conventional agarose gel electrophoresis. Cells were incubated with vehicle (control), 10 nM dexamethasone (Dex), 25 ng ml⁻¹ TNF-α (TNF-α), 25 ng ml⁻¹ TNF-α/10 nM dexamethasone (TNF-α+Dex), 30 ng ml⁻¹ LPS (LPS), and 30 ng ml⁻¹ LPS/10 nM dexamethasone (LPS+Dex) for 24 h as indicated. The formation of a DNA ladder is a marker of apoptosis rather than necrosis. The results are representative of three independent experiments.

Figure 2

Effects of mineralocorticoids on apoptotic DNA fragmentation induced by TNF-α and bacterial LPS. Bovine glomerular endothelial cells were incubated for 24 h with 10 ng ml⁻¹ TNF-α (A) and 10 ng ml⁻¹ LPS (B) in the absence or presence of increasing concentrations of aldosterone or aldosterone-21-acetate. DNA fragmentation was quantitated by the diphenylamine reaction. Data are means±s.e.mean of four separate experiments. ^**P<0.05 (ANOVA and for multiple comparison the data were corrected by Dunn's Method).

Figure 3

The synergistic action of TNF-α and bFGF was blocked by co-treatment with dexamethasone. Incubations were done for 24 h with 10 nM dexamethasone (Dex), 10 ng ml⁻¹ TNF-α (TNF-α), 10 ng ml⁻¹ TNF-α+10 nM dexamethasone (TNF-α+Dex), 10 ng ml⁻¹ bFGF (bFGF), 10 ng ml⁻¹ TNF-α+10 ng ml⁻¹ bFGF (TNF-α+bFGF), and 10 ng ml⁻¹ TNF-α+10 ng ml⁻¹ bFGF+10 nM dexamethasone (TNF-α+bFGF+Dex). DNA fragmentation was quantitated using the diphenylamine reaction as described in the Methods section. Values are means±s.e.mean of three independent experiments. BFGF significantly potentiated TNF-α-induced apoptosis (^*P⩽0.01) and dexamethasone blocked apoptosis induction by TNF-α or TNF-α/bFGF (^**P⩽0.001) (ANOVA and for multiple comparison the data were corrected by Dunn's Method).

Figure 4

The effect of dexamethasone depends on glucocorticoid receptor binding. Cells were incubated for 24 h with 25 ng ml⁻¹ TNF-α and 10 nM dexamethasone (A) or with 30 ng ml⁻¹ LPS and 10 nM dexamethasone (B) in the absence or presence of increasing concentrations of the glucocorticoid receptor antagonist RU-486 as indicated. In parallel, control incubations were done for 24 h with 25 ng ml⁻¹ TNF-α (A) or 30 ng ml⁻¹ LPS (B) (bars). DNA fragmentation was determined using the diphenylamine reaction. Values are means±s.e.mean of at least four individual experiments.

Figure 5

The effect of aldosterone is antagonized by the glucocorticoid receptor antagonist RU-486. Cells were incubated for 24 h with 10 ng ml⁻¹ TNF-α, 10 ng ml⁻¹ TNF-α and 1000 nM aldosterone or with 10 ng ml⁻¹ TNF-α, 1000 nM aldosterone and either 100 or 1000 nM of the glucocorticoid receptor antagonist RU-486 as indicated. DNA fragmentation was determined using the diphenylamine reaction. Values are means±s.e.mean of at least four individual experiments. ^**P<0.05 compared to the incubations without RU-486 (ANOVA and for multiple comparison the data were corrected by Dunn's Method).

Figure 6

Fluocinolone suppressed apoptosis triggered by co-treatment with TNF-α and cycloheximide. Bovine glomerular endothelial cells were exposed to increasing concentrations of cycloheximide (control), 1 ng ml⁻¹ TNF-α plus cycloheximide, and 1 ng ml⁻¹ TNF-α plus cycloheximide plus 10 nM fluocinolone (A); or 5 ng ml⁻¹ LPS plus cycloheximide, and 5 ng ml⁻¹ LPS plus cycloheximide plus 10 nM fluocinolone (B). The amount of DNA fragmentation was determined with the diphenylamine reaction. Values are means±s.e.mean of four individual experiments.