Effects of representative glucocorticoids on TNFα- and CD40L-induced NF-κB activation in sensor cells

Abstract

Glucocorticoids are an important class of anti-inflammatory/immunosuppressive drugs. A crucial part of their anti-inflammatory action results from their ability to repress proinflammatory transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) upon binding to the glucocorticoid receptor (GR). Accordingly, sensor cells quantifying their effect on inflammatory signal-induced NF-κB activation can provide useful information regarding their potencies as well as their transrepression abilities. Here, we report results obtained on their effect in suppressing both the TNFα- and the CD40L-induced activation of NF-κB in sensor cells that contain an NF-κB-inducible SEAP construct. In these cells, we confirmed concentration-dependent NF-κB activation for both TNFα and CD40L at low nanomolar concentrations (EC50). Glucocorticoids tested included hydrocortisone, prednisolone, dexamethasone, loteprednol etabonate, triamcinolone acetonide, beclomethasone dipropionate, and clobetasol propionate. They all caused significant, but only partial inhibition of these activations in concentration-dependent manners that could be well described by sigmoid response-functions. Despite the limitations of only partial maximum inhibitions, this cell-based assay could be used to quantitate the suppressing ability of glucocorticoids (transrepression potency) on the expression of proinflammatory transcription factors caused by two different cytokines in parallel both in a detailed, full dose-response format as well as in a simpler single-dose format. Whereas inhibitory potencies obtained in the TNF assay correlated well with consensus glucocorticoid potencies (receptor-binding affinities, Kd, RBA, at the GR) for all compounds, the non-halogenated steroids (hydrocortisone, prednisolone, and loteprednol etabonate) were about an order of magnitude more potent than expected in the CD40 assay in this system.

Keywords: CD154; Corticosteroids; Hill equation; TNF; Transrepression.

Figure 1

Semi-log plots of the TNFα- and CD40L-induced NF-κB activations in the sensor cells of the present study quantified via SEAP (symbols) and fitted after normalization with standard one-site specific binding models (lines) indicating median effective (EC50) concentrations of 0.14 nM (2.7 ng/mL) and 2.3 nM (41 ng/mL) (top and bottom figures, respectively).

Figure 2

Concentration-dependence of the percent suppression of the TNFα-induced NF-κB activation caused by selected glucocorticoids in sensor cells. Data are average of five independent experiments in triplicates or quadruplicates using TNFα stimulation (3 ng/mL) and were fitted by a standard specific binding model (eq. 1) with shared E_max and n_H values. The corresponding median inhibitory concentration (IC₅₀) values are summarized in Table 1, and they are in good agreement with published consensus K_d data [16] being about 3–5 fold lower here for all compounds.

Figure 3

Percent suppression of the TNFα-induced NF-κB activation caused by glucocorticoids at 1 nM concentration as a function of consensus glucocorticoid receptor affinity (log Kd, equivalent to the log of the commonly used relative receptor binding affinity, rRBA, scales). Data are average of five independent experiments in quadruplicates using TNFα stimulation in the presence of the listed glucocorticoids (1 nM).

Figure 4

Concentration-dependence of the percent suppression of the CD40L-induced NF-κB activation caused by selected glucocorticoids in sensor cells. Data are average of five independent experiments in triplicates or quadruplicates using CD40L stimulation (25 ng/mL) and were fitted by a standard specific binding model (eq. 1) with shared E_max and n_H values.

Figure 5

Percent suppression of the CD40L–induced NF-κB activation caused by glucocorticoids at 1 nM concentration as a function of consensus glucocorticoid receptor affinity (log K_d, equivalent to the log of the commonly used relative receptor binding affinity, rRBA, scales). Data are average of five independent experiments in quadruplicates using CD40L stimulation in the presence of the listed glucocorticoids (1 nM). Data for the three non-halogenated (HC, PRED, and LE) and the four halogenated steroids (DEX, TA, BDP, and CP) were fitted with different trend-lines.

Figure A.1

(A) Normalized effects produced by compounds of different potencies in the same assay system that is assumed to follow a typical Hill-type response function with unity Hill slope (eq. A.1, n_H = 1) shown as a classic concentration-dependent response on a semi-log scale. (B) Normalized effects produced by the compounds of different potencies from A used at a single test concentration (here, C_t = 1) shown this time as a function of log K_d. The effects used here corresponds to the intersection of the various curves with the vertical axis in A. (C) and (D) are the same as A and B, respectively, but for a system with a less abrupt response function (i.e., a lower Hill slope, n_H = 0.5).