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.

Endogenous circadian regulation of pro-inflammatory cytokines and chemokines in the presence of bacterial lipopolysaccharide in humans

“…Previous studies suggest that rhythmic changes in cell number may be one of the mechanisms controlling the circadian oscillations in cytokine and chemokine rhythms ( Born et al, 1997 ). Endogenous glucocorticoid levels may also modulate the daily oscillations in chemokine and cytokine expression since glucocorticoid administration attenuates proinflammatory cytokine and chemokine expression ( Cechin and Buchwald, 2014 ). The aim of the present study was to investigate whether daily rhythms in cytokine and chemokine expression are under endogenous regulation.…”

Design, Synthesis, and Evaluation of Novel Immunomodulatory Small Molecules Targeting the CD40–CD154 Costimulatory Protein-Protein Interaction

“…CD40 expressing NF-κB sensor cells (HEK Blue, InvivoGen, San Diego, CA, USA) were used to assess the ability of the present compounds to block CD154-induced activation as described before [ 56 , 76 , 77 ]. Briefly, cells were maintained in DMEM at 80% confluence for each experiment.…”

Small-Molecule Inhibitors of the CD40–CD40L Costimulatory Protein–Protein Interaction

“…CD40 expressing NF-κB sensor cells (HEK Blue, InvivoGen, San Diego, CA) were used to assess the ability of the present compounds to block CD40L-induced activation as described before 70, 108 . Briefly, cells were maintained in DMEM at 80% confluence for each experiment.…”