Effect of lipopolysaccharide and antidepressant drugs on glucocorticoid receptor-mediated gene transcription

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Short communication

Effect of lipopolysaccharide and antidepressant drugs on glucocorticoid receptor-mediated gene transcription

Bogus³awa Budziszewska^1,2, Agnieszka Basta-Kaim^1,2, Marta Kubera^1,2, Lucylla Jaworska^1,2, Monika Leœkiewicz¹, Magdalena Tetich¹,

Magdalena Otczyk^1,2, Alena Zajicova³, Vladimir Holan³, W³adys³aw Lasoñ¹

Department of Experimental Neuroendocrinology, Laboratory of Immunoendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

!Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague 6, Czech Republic

Correspondence: Bogus³awa Budziszewska, e-mail: budzisz@if-pan.krakow.pl

Abstract:

It has been hypothesized that pro-inflammatory response and hyperactivity of hypothalamic-pituitary-adrenocortical axis (HPA) are involved in the pathogenesis of depression. Hyperactivity of HPA axis results probably from deregulation of glucocorticoid receptor function and impairment of the control mechanism of glucocorticoid secretion. Previously, we found that antidepressants inhibited glucocorticoid receptor (GR) function under thein vitro condition. In order to study a role of some mediators of pro-inflammatory response in this process, presently, we investigated the effect of lipopolysaccharide (LPS) on imipramine- or fluoxetine-induced inhibition of GR-mediated gene transcription in fibroblast cells, stably transfected with mouse mammary tumor virus promoter (LMCAT cells). Two days of incubation of the cells with imipramine (3–10mM), fluoxetine (10 mM) or LPS (1 mg/ml) inhibited the corticosterone-induced gene transcription. Concomitant incubation of the cells with LPS and fluoxetine or imipramine had stronger inhibitory effect than that evoked by each compound alone. Moreover, we found that fluoxetine (10mM) but not imipramine (3–10 mM) significantly inhibited the LPS-stimulated interleukin-6 (IL-6) production in these cells. These data suggest that pro-inflammatory agents facilitate antidepressant-induced inhibition of glucocorticoid receptor function.

Key words:

glucocorticoid receptor, lipopolysaccharide, antidepressant drugs, interleukin-6

Introduction

Neurodegenerative processes and dysregulation of neuroendocrine and immune system contribute to the pathogenesis of depression. Signs and symptoms that are characteristic of depression include changes in the hypothalamic-pituitary-adrenocortical (HPA) system,

which in the majority of depressed patients is re- flected by altered regulation of corticotropin (ACTH) and cortisol secretion. More refined analysis of the HPA system revealed that corticosteroid receptor sig- naling is impaired in major depression resulting, among other things, in an increased production of corticotropin-releasing hormone (CRH) [8]. Immune activation and the acute phase immune response have

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also been shown to occur particularly in severely depressed patients. The latter included an increase in serum level of soluble interleukin-2 (IL-2) and IL-6 re- ceptors as well as production of IL-1 and IL-6 in mitogen-stimulated cells. The contribution of the inflammatory process to both hypercortisolemia and neurodegenerative processes has been suggested, the more so that IL-1 and IL-6 are potent stimulators of CRH secretion [1].

Recently, it has been shown that antidepressant drugs suppressed production of pro-inflammatory cytokines and corrected HPA axis activity, but mechanism of their action is poorly recognized [10]. High variability in depression symptoms and in clinical ef- fectiveness of antidepressants may depend on activity of endocrine and immune systems.

Our previous studies have shown that some antidepressant drugs are able to inhibit GR-mediated gene transcription under in vitro conditions [5]. Since the same pro-inflammatory cytokines also inhibit GR function in cell line [12], it is not unlikely that the effect of antidepressants on GR-mediated gene transcription depends on immune system activity.

In the present study, we evaluated the effect of imipramine and fluoxetine on the GR-mediated chloramphenicol acetyltransferase (CAT) gene expression in fibroblast (LMCAT) cell line under basal culture conditions and during activation by lipopolysaccharide (LPS). LPS, located in the outer membrane of Gram- negative bacteria, is a potent activator of the immune system. LPS is known to induce specific humoral immune response in infected animals by activation of B lymphocytes [6] and synthesis of various cytokines, especially IL-1, IL-6 and tumor necrosis factor (TNF) by many cells [7]. Therefore, additionally, the effect of co-incubation of LMCAT cells with imipramine or fluoxetine combined with LPS on cytokine (IL-1b and IL-6) production was estimated.

Materials and Methods

Cell culture conditions

Effects of LPS, fluoxetine and imipramine on the GR-mediated gene expression were determined in mouse fibroblast cells (L929), stably transfected with a mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT)-reporter plasmid (LMCAT

cells). The LMCAT cell line was generously provided by Dr E.R. Sanchez (Department of Pharmacology, Medical College of Ohio, Toledo, OH). The cells were grown in DMEM (Life Technologies, Paisley, U.K.) with a 10% heat-inactivated fetal bovine serum (Life Technologies, New Zealand) and a 0.02% ge- neticin (Life Technologies, Paisley, U.K.) at 37°C in a 5% CO₂/95% air atmosphere.

Drug treatments

LMCAT cells (final confluence: 90%) were treated with LPS (E.coli. 026:B6; Sigma, St. Louis, MO, USA; 1 mg/ml) or the drugs: imipramine hydrochlo- ride (Polfa, Kraków, Poland), and fluoxetine hydro- chloride (Farmacom, Kraków, Poland) both at doses 3, 5 and 10mM for 2 days. The control cells were cul- tured with the appropriate vehicle. Next the cells were treated with LPS (1 mg/ml) and imipramine hydro- chloride (3, 5 and 10 mM) or fluoxetine (10 mM) jointly for 2 days. CAT activity was stimulated by addition of 1mM corticosterone (Sigma, St. Louis, MO, USA) for 2 h before harvesting the cells. Imipramine, fluoxetine and LPS were dissolved in water, while corticosterone was dissolved in a small amount of ethanol and diluted with water (final concentration of ethanol was below 0.5%).

CAT activity

Cell lysates were prepared by a freezing/thawing pro- cedure. CAT activity was determined by the method described previously [5]. Aliquots of the lysate (after heating for 10 min at 60°C) were incubated in 0.25 M Tris-HCl buffer (pH 7.8) supplemented with 0.25mCi of D-threo-[dichloroacetyl-1-¹⁴C]-chloramphenicol (Am- ersham Pharmacia Biotech, Buckinghamshire, UK) and 0.2 mM n-butyryl Coenzyme A (Sigma) at 37°C for 1 h. The butyrylated forms of chloramphenicol (in direct proportion to the CAT gene expression) were extracted twice with xylene, washed with 0.25 M Tris-HCl buffer, and the radioactivity was measured in ab-counter (Beckmann LS 335 liquid scintillation counter). The results are presented as dpm of the butyrylated fraction of chloramphenicol per 10 mg of protein per one hour of incubation. Protein concentration in cell lysates was determined by a method of Lowry et al. [11].

LPS, antidepressants and GR-mediated gene transcription

Bogus³awa Budziszewska et al.

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Cell viability

LMCAT cells were treated with the vehicle, LPS (1 mg/ml) imipramine and fluoxetine (both at doses 3–10mM) and LPS with imipramine or fluoxetine for 2 days. The effect of these compounds on cell viability was determined by counting viable and non-viable (blue) cells in a hemocytometer. The cell suspensions were mixed (at the 1:1 ratio) with a 0.4% trypan blue, and the number of non-viable cells per a total of 100 cells was counted.

Cytokine detection and quantification

The presence of IL-1b and IL-6 in the medium col- lected after 2-day culture of LMCAT cells with imipramine, fluoxetine and/or LPS were measured by enzyme-linked immunosorbent assay (ELISA) using sets of cytokine-specific antibodies purchased from R&D System (Minneapolis, USA) for IL-1 and from PharMingen (San Diego, MA, USA) for IL-6. All as- says were performed according to the manufactures’

instructions. In order to quantify a cytokine level, a standard purchased from Genzyme (Boston, MA, USA) was included in all ELISA determinations. The intra-assay variations were lower than 8% for all as- says.

Addition of corticosterone at a concentration of 1mM for 2 h increased CAT activity about 30-fold. The CAT activity induced by corticosterone was com- pletely blocked by RU 38486, a specific antagonist of the type II GR (data not shown), which confirms involvement of the GR in the response [5]. Treatment of cells with antidepressant drugs or LPS did not change the low basal CAT activity (data not shown).

LPS at the concentration of 1mg/ml, imipramine at the concentrations of 3, 5 and 10mM and fluoxetine at the concentration of 10mM present in the medium for two days significantly inhibited corticosterone induced CAT activity (Fig. 1). Concomitant treatment of the cells with LPS (1 mg/ml) and imipramine or fluoxetine (both drugs at the 10 mM concentration) produced stronger inhibition of CAT activity than each of these compounds given alone (Fig. 1). The ex- posure of LMCAT cells to imipramine, fluoxetine, LPS, imipramine + LPS or fluoxetine + LPS at the concentrations mentioned above for 2 days produced no toxic effect as estimated by counting non-viable cells (data not shown).

ELISA study showed that LPS present in the medium for 2 days potently stimulated production of IL-6 (ca. 30-fold as compared to the control) in LMCAT cells (Fig. 2) but production of IL-1b was under detection limit (not shown). Imipramine and

0 10000 20000 30000 40000

Drug ( M ) –

LPS (1 g/ml) – + + + + +

CATactivity

3 3 5 5 10 10 10 10

–

– – –

Fig. 1. The effect of fluoxetine or imipramine and LPS on the CAT gene transcription induced by corticosterone in LMCAT cells. Drugs at the indicated concentrations and LPS (1 µg/ml) were present in medium (alone or jointly) for 2 days. Corticosterone (1 µM) was added 2 h before harvesting the cells for the assay of CAT activity. The data are presented as the mean ± SEM (dpm of the butyrylated fraction of chloramphenicol per 10 µg of protein per 1h of incubation); n = 68;

the significance of differences between the means was calculated by the Duncans test following a one-way analysis of variance. * p < 0.01 vs. vehicle/vehicle group; # p < 0.01 vs. respective drug/vehicle group, ^ p < 0.01 vs. vehicle/LPS group

0 50 100 150 200 250 300 350

Imipramine Fluoxetine Control

Drug ( M ) – – 3 3 5 5 10 10 10 10

LPS (1 g/ml) – + – + – + – + – +

IL- 6 (pg/ml)

–

Fig. 2. The effect of fluoxetine or imipramine and LPS on IL-6 production by LMCAT cells. Drugs at the indicated concentrations and LPS (1 µg/ml) were present in medium (alone or jointly) for 2 days. The data are presented as the mean ± SEM; n = 68; the significance of differences between the means was calculated by the Duncans test following a one-way analysis of variance. * p < 0.001 vs. vehicle/vehicle group; # p < 0.001 vs. respective drug/vehicle group, ^ p < 0.01 vs. vehicle/LPS group

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fluoxetine alone had no effect on IL-6 production.

Fluoxetine at the concentration of 10 mM (but not at 3 and 5 mM) significantly inhibited LPS-stimulated IL-6 production whereas imipramine at the concentrations of 3–10mM had no effect (Fig. 2).

The present data confirm our former observation that antidepressants and immune system activator – LPS, when given separately, are able to inhibit stimu- latory effect of corticosterone on the GR-mediated gene transcription [3–5]. The inhibitory effect of LPS on corticosterone-induced gene transcription is in agreement with the report showing that IL-1a reduced GR function [12]. LPS is known to induce synthesis of some pro-inflammatory cytokines also in other fibroblast cells [13], therefore, it is possible that this en- dotoxin affects GR functionvia influencing cytokine production. Moreover, pro-inflammatory cytokines not only stimulate HPA axis and increase serum glu- cocorticoids level but also can inhibit GR function.

Indeed, Bantel et al. [2] showed that high, local concentration of cytokines produced during inflammatory process induced resistance of glucocorticoid receptor.

In the present study, synthesis of IL-6 was more than 30 times increased by LPS stimulation so we may speculate that IL-6 plays a role in inhibition of GR function in LMCAT cells. On the other hand, we con- firmed our previous observation that LMCAT cells are not able to produce IL-1b [4] whereas secretion of other cytokines, which can be produced by these cell line, have not been studied yet.

The new finding of this study is that antidepressants imipramine and fluoxetine show the enhanced inhibitory effects on GR function in the presence of LPS. In fact, we expected an opposite effect because in several our former studies it was shown that antidepressants antagonized some effects of LPS. For exam- ple antidepressants inhibited LPS-stimulated production of pro-inflammatory cytokines by human blood cells [10]. If pro-inflammatory cytokines were re- sponsible for inhibitory activity of LPS on GR functions then antidepressant drugs by the diminution of the synthesis of these cytokines should decrease inhibitory activity of LPS on GR functions but instead in the present study we observed rather additive effect. Although, as mentioned above, IL-6 itself may play some role in inhibition of GR function by LPS, its involvement in antidepressant action is doubtful, because these drugs do not induce IL-6 synthesis, moreover, fluoxetine significantly (almost 50%) re-

duced LPS-induced IL-6 production. On the other hand, imipramine had no inhibitory effect on LPS- stimulated IL-6 production and had inhibitory effect on GR function in the presence of LPS at much lower concentration than fluoxetine did. Thus, it cannot be excluded that the lack of effect of imipramine on IL-6 production contributes to stronger inhibitory effect of imipramine on GR function than fluoxetine.

The molecular mechanism underlying the additive effect of antidepressants and LPS on GR function re- mains to be elucidated but it may involve several mitogen-activated protein kinase (MAPK) pathways:

extracellular signal regulated kinases (ERK) 1 and 2, c-Jun N-terminal kinase (JNK) and p38 kinase. Acti- vated MAP kinases are known to phosphorylate and decrease transcriptional activity of GR in some cell types [9]. In our former studies, it was shown that p38 and JNK-MAP kinases were involved in LPS-induced inhibition of GR function. SB-203580, an inhibitor of p38 kinase and at higher concentration (10mM) also an inhibitor of some isoforms of JNK-MAP kinase at- tenuated the inhibitory effect of LPS on GR function.

On the other hand, this inhibitor had no effect on chlorpromazine-induced decrease in corticosterone- mediated gene transcription [4].

In summary, these results showed that LPS facili- tated inhibitory effect of imipramine and fluoxetine on GR function and fluoxetine inhibited LPS- stimulated IL-6 production.

Acknowledgment:

This study was supported by the grant No. 6P05A076 20 from the State Committee for Scientific Research (KBN), Warszawa, Poland.

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Received:

April 1, 2005; in revised form: June 6, 2005.