Inhibition of the production of mediators of inflammation by corticosteroids is a glucocorticoid receptor-mediated process

In order to find an explanation for corticosteroid resistance we assessed whether inhibition by dexamethasone (DEX) of the stimulated production of TNF-∝, IL-6, PGE2 and LTB4 by peripheral blood mononuclear cells (MNC) depends on binding to the glucocorticoid receptor (GR), and whether it is determined by the number or the affinity of the GR of these cells. GR number and affinity of MNC were determined by means of a whole cell DEX binding assay. MNC were incubated with DEX and LPS or A23187 in the absence or presence of RU486, a potent steroid antagonist. DEX caused a concentration dependent inhibition of TNF-∝, IL-6 and PGE2 production but had no effect on LTB4 production. RU486 significantly blocked the effect of DEX, but no correlations were found between the inhibition of mediator release and the Kd or receptor number.


Introduction
Corticosteroids decrease the severity of inflammation and cause a subjective improvement in the majority, but not in all, patients with noninfectious inflammatory disorders such as inflammatory bowel disease (IBD), asthma 2 and rheumatoid arthritis. 3 The anti-inflammatory effects of corticosteroids are ascribed to inhibition of the production of mediators of inflammation, including eicosanoids and cytokines. [4][5][6] Several control studies have shown that topical or systemic corticosteroids are much more effective than placebo controls, but that not all patients improve. Depending on the dose and route of application, duration of the treatment, severity of the exacerbation and the parameters under investigation, approximately 10-40% of patients do not respond or respond insufficiently to corticosteroids. 7 '8 Stimulation of mononuclear cells (MNC) by lipopolysaccharide (LPS) or Ca-ionophore enhances the production of a range of inflammatory mediators by these cells such as the cytokines tumour necrosis factor-0 (TNF-z) and interleukin 6 (IL-6), and the eicosanoids prostaglandin E2 (POt2) and leukotriene B4 (LTB4). 9 This provides an in vitro model for studying the anti-inflammatory properties of corticosteroids.
In order to clarify some aspects of the mechanism through which corticosteroids exert their anti-inflammatory effects we examined the effect of dexamethasone (DEX) on the produc-tion of mediators of inflammation by human MNC in vitro and assessed whether this effect depends on binding of corticosteroids to the glucocorticoid receptor (GR).
The hypothesis of this study was that the inhibition of the production of mediators of inflammation by corticosteroids is a glucocorticoid receptor-mediated process. We furthermore tested whether the ability of DEX to inhibit the LPS-stimulated production of TNF-z, IL-6, PGE2 and the Ca 2 +-ionophore enhanced LTB 4 production by MNC is determined by the number or affinity of glucocorticoid receptors (GR) on these cells.

Materials and Methods
Healthy volunteers.. Approval for this study was obtained from the Medical Ethics Committee of the University Hospital Rotterdam. Venous blood was obtained from nine healthy adults, ranging in age from 22 to 37 years, who joined voluntarily after full explanation of the nature, significance and scope of the study. None of the subjects had taken corticosteroids or any other anti-inflammatory drug for a period of at least 4 weeks prior to donating blood.
Isolation of human MNC. Mononuclear cells were isolated from the heparinized venous blood immediately after blood sampling. The method used was a modification of the technique originally described by Boyum. 1 Briefly, the blood was diluted 1.1 with phosphate buffered Statistics.. The effect of DEX on the stimulated saline (PBS; Oxoid, UK) before fractionating it release of each mediator was studied in nine by a one-step Ficoll-Paque gradient (Pharmacia, separate experiments, with blood from nine Sweden) centrifugation at 1100 x g for 15 min different donors. Values are expressed as mean at 20C. The interphase was washed in PBS and _ S.E.M. Control and DEX-treated cell cultures resuspended in Dulbecco's Modified Eagle's were compared by paired t-test. /values < Medium (DMEM) containing HEPES and foetal 0.05 were considered significant. The effect of calf serum (Gibco, UK), supplemented with DEX alone versus the effect of DEX after prepenicillin and streptomycin (Flow Lab, UK). incubation with RU486 was compared by Cells were stained by Hemacolor (Merck, Manova. Germany), the final yield of MNC was greater than 95% and the cell viability (tested by Trypan blue exclusion) was over 97%.
Culturing of MNC and measurement J cytokines Mediator production by MNC: After the incuba- atmosphere of 5% CO2 and 95% air. DEX was inhibitory effect of DEX on TNF-a production dissolved in culture medium, and the concentra-was of comparable magnitude to that of IL-6 tion of DEX varied from 10 -9 to 10-5M. and PGE2, with an IC50 value (concentration of TNF-a and IL-6 were measured by commer-DEX that causes a 50% decrease in mediator cially available ELISA kits (Central Laboratory for release) of 65nM in comparison to 145 and Blood Transfusion, The Netherlands), whereas 140nM for IL-6 and PGE2 respectively. There PGE2 and LTB4 were determined by specific was a considerable inter-individual variation in radioimmunoassays (standards, Sigma, USA; H-IC50 values in response to DEX with a 95% labels, Amersham, UK; and antibodies, Advanced confident interval of 10-400nM for TNF-a, 15-Magnetics Inc., USA).
1095 nM for IL-6 and 30-690nM for PGE2. The The effects of different concentrations of DEX effects of DEX on the production of the studied on secretion of TNF-a, IL-6 and PGE2 were mediators are shown in Fig. 1. DEX had no expressed as percentage inhibition of production effect on Ca 2 + ionophore-stimulated LTB 4 in control cultures (cells incubated with DMEM release. plus supplements and LPS).
Antagonism of the effects of DEX in MNC by Assessment of glucocorticoid receptor number by RU486: Incubation of MNC with RU486, a steroid a whole cell assay.. The method used was that receptor antagonist, for 2 h prior to the addition described by Lamberts et al., 11 but a 1000-fold of DEX diminished the inhibitory effect of the excess of unlabelled RU486 (Roussel, France), a glucocorticoid in a dose dependent manner corticoid receptor antagonist, was used instead of (Fig. 2). S.E.M.) and the I 9.5 _ _ _ 0.7nM. In Fig. 3 a In order to enhance the dissociation of endorepresentative Scatchard p_lot from one experigenously bound corticoids, the MNC used in the ment of the binding of H-DEX to glucocortiassay were washed three times in DMEM, each cold receptors of MNC is shown. The slope is time allowing for an equilibration period of equal to the negative reciprocal of the dissocia-12 30min at 37C in a shaking waterbath, tion constant (-1/K), and the intercept on the Specific binding was determined by subtractabscissa equal to the total concentration of ing the amount of nonspecifically bound radioreceptors (Bmax). NO correlations were found ligand from the total amount bound. Data were between the inhibition of mediator release and analysed by constructing a Scatchard plot. 1 I or receptor number.

Discussion
The inhibitory effect of DEX in this in vitro experiment varied considerably from donor to donor. The effect of DEX on the release of TNFa, IL-6 and PGE2 cannot be explained by the effect of DEX on the viability of the cells, otherwise the same effects should have been found for LTB4 release. Instead, no significant differences were found between LTB4 production in the presence versus the absence of DEX. This might indicate that corticosteroids do not inhibit 5-1ipoxygenase in MNC as they do the gene expression of .cytokine-induced cycloxygenase 2 in monocles. 4 Furthermore, culture of MNC with DEX, in the concentrations used in this study, did not affect the viability of these cells as determined by trypan blue exclusion. The fact that the effect of DEX could be strongly suggests a glucocorticoid receptor mediated process. Mononuclear cells play a central role in chronic inflammation. They possess the capacity to produce eicosanoids and cytokines which modulate the inflammatory response. [5][6][7] Corticosteroids exert their anti-inflammatory effects partly by inhibiting the production of inflammatory mediators, through a glucocorticoid receptor-mediated process. This means that the GR found, however, between the IC50 of the mediators under investigation and the receptor number or I. Thus, the degree of response of MNC of healthy donors to DEX does not seem to be determined by the characteristics of the GR, and other factors must play a role. This finding, however, does not exclude the possibility that changes in GR number or affinity are important in glucocorticoid resistance in inflammatory diseases.
The MNC used in this study were isolated from blood obtained from healthy volunteers, who presumably have a normal response to corticosteroids. It is to be expected that comparison of MNC obtained from patients who have proven to be non-responders to corticosteroids, with MNC of responders may reveal differences in GR characteristics more clearly. Studies with MNC isolated from blood obtained from inflammatory bowel disease patients who respond to corticosteroids and patients who do not respond have been initiated.