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, Reduces Glucocorticoid Receptor Translocation and Function1
Section of Clinical Neuropharmacology, Institute of Psychiatry (C.M.P.), London, United Kingdom SE5 8AF; and the Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia 30322
Address all correspondence and requests for reprints to: Andrew H. Miller, M.D., Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, 1639 Pierce Drive, Suite 4000, Atlanta, Georgia 30322. E-mail: amill02{at}emory.edu
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Abstract |
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(IL-1), on 1) GR translocation from cytoplasm to
nucleus using GR immunostaining, 2) cytosolic radioligand GR binding,
and 3) GR-mediated gene transcription in L929 cells stably transfected
with the mouse mammary tumor virus-cholamphenicol acetyltransferase
reporter gene. L929 cells were treated with IL-1 (100 and 1000 U/ml)
for 24 h in the presence or absence of dexamethasone (Dex; 10
nM to 1 µM). IL-1 inhibited Dex-induced GR
translocation and alone induced GR up-regulation. Pretreatment with
IL-1 followed by Dex treatment for 1.5 h led to about 20%
inhibition of Dex-induced GR-mediated gene transcription, whereas
coincubation of IL-1 plus Dex for 24 h inhibited Dex-induced
GR-mediated gene activity up to 42%. The latter effect was reversed by
the IL-1 receptor antagonist. These results suggest that cytokines
produced during an inflammatory response may induce GR resistance in
relevant cell types by direct effects on the GR, thereby providing an
additional pathway by which the immune system can influence the
hypothalamic-pituitary-adrenal axis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Glucocorticoids mediate their effects on target immune tissues via two distinct receptor subtypes, the mineralocorticoid receptor and the glucocorticoid receptor (GR). Although the mineralocorticoid receptor has a higher affinity for circulating glucocorticoids than the GR, the GR is expressed in much higher amounts in immune tissues (6). In addition, there is a great degree of heterogeneity in GR expression among immune cells and tissues, allowing for cell- and tissue-specific responses to glucocorticoids (6, 7, 8). Interestingly, studies have shown that aside from activating glucocorticoid release, cytokines can also influence the expression and function of GR. For example, in vivo and in vitro treatment with cytokines and cytokine inducers (e.g. endotoxin) has been found to alter GR expression and function in a number of cells and tissues, including T cells (9), monocytes/macrophages (10, 11), bronchial cells and lung (12, 13), and liver (11, 14, 15, 16, 17, 18). A number of these studies have demonstrated that treatment with cytokines induces a decrease in GR function (GR resistance), as evidenced by decreased sensitivity to the effects of glucocorticoids on functional end points (9, 15, 17, 18) and decreased GR affinity for ligand (9, 10, 11, 12). Studies performed on peripheral cells and tissues of patients with inflammatory diseases such as asthma, ulcerative colitis, acquired immunodeficiency syndrome, and rheumatoid arthritis, especially those showing resistance to the therapeutic effects of glucocorticoids, also have demonstrated reductions in GR function and affinity that are similar to those induced by in vivo and in vitro treatment with cytokines (19, 20, 21, 22, 23, 24, 25). Thus, converging evidence suggests that cytokines produced during an inflammatory response may directly modulate the capacity of glucocorticoids to transmit signals to target tissues and induce GR resistance in relevant cell types. Moreover, given the role of GR in mediating feedback inhibition on the HPA axis, the effects of cytokine on GR function may provide an additional pathway by which the immune system can influence HPA axis activity.
To investigate potential mechanism(s) by which cytokines might inhibit
GR function, we examined whether the proinflammatory cytokine, IL-1,
disrupts translocation of the GR from the cytoplasm to the nucleus.
According to the nucleocytoplasmic traffic model, the GR in its
unactivated form resides primarily in the cytoplasm, and after being
bound by steroid undergoes a conformational change (activation),
dissociates from a multimeric complex including several heat shock
proteins, and translocates from the cytoplasm to the nucleus, where it
either binds to hormone response elements (HREs) on DNA or interacts
with other transcription factors (26, 27). Experiments were conducted
in a mouse fibroblast cell line (L929 cells), as fibroblasts are an
important stromal cell type involved in the acute phase response and
have been shown to express receptors for IL-1 at a high density
(
5000 sites/cell) (28). IL-1 was chosen based on our previous
studies in experimental murine viral infections, where we have found
that IL-1 (along with IL-6) plays a pivotal role in the induction of
glucocorticoids during viral infection (29). Cells were treated with
IL-1 for 24 h in the presence or absence of the synthetic
steroid, dexamethasone (Dex), and GR translocation was investigated
using GR immunostaining of the receptor in the cytoplasm and nucleus.
In addition, evaluation of GR number was performed using cytosolic
radioligand receptor binding. Finally, GR function was measured by
means of GR-mediated gene transcription in L929 cells stably
transfected with the mouse mammary tumor virus-chloramphenicol
acetyltransferase (MMTV-CAT) reporter gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was purchased from R&D Systems
(Minneapolis, MN). The LMCAT cell line was provided by E. R.
Sanchez (Department of Pharmacology, Medical College of Ohio, Toledo,
OH). IL-1 receptor antagonist (IL-1ra) was a gift from Synergen
(Boulder, CO).
Cell culture conditions and drug treatments
Mouse fibroblast cells (L929) and the stably transfected CAT
reporter cell line LMCAT (derived from L929, see Ref. 30) were
maintained in 175-cm2 flasks (Becton Dickinson and Co.) at 37 C with a 5% CO2 and 95% air atmosphere.
L929 cells were maintained in DMEM with 50 U/ml penicillin, 50 µg/ml
streptomycin, and 10% heat-inactivated (56 C, 30 min) calf serum.
LMCAT cells were maintained in DMEM with 0.2 mg/ml G418 (geneticin)
antibiotic and 10% heat-inactivated (56 C, 30 min) newborn calf serum
that had been charcoal extracted (1% activated charcoal-0.1% dextran)
to remove endogenous steroids. For immunostaining, cells were
subcultured in fibronectin-coated chamber slides (Nunc, Naperville, IL)
in DMEM with 50 U/ml penicillin, 50 µg/ml streptomycin, and 10%
charcoal-extracted bovine calf serum for 12 h to obtain a final
confluence of approximately 70% and then treated. For binding assays,
L929 cells were subcultured in fibronectin-coated 175-cm2
flasks in DMEM with 50 U/ml penicillin, 50 µg/ml streptomycin, and
10% heat-inactivated calf serum for 48–72 h (final confluence, 95%)
before treatment. For the CAT assay, LMCAT cells were subcultured in
their usual growth medium in fibronectin-coated six-well plates and
grown for 12 h (final confluence, 95%) before treatment.
Treatment of both L929 cells and LMCAT for all assays consisted of
incubation with fresh medium containing final concentrations of IL-1
(100 and 1000 U/ml) and Dex (10 nM to 1 µM). This range
of concentrations has been used in previous studies examining the
impact of cytokines on the GR.
Immunostaining procedures and fluorescence quantitation
Immunostaining for the GR was performed as described previously
(31). Cells were fixed/permeabilized with methanol at -20 C for 10
min, followed by 30-min incubation with 5% BSA to block nonspecific
antibody binding. Cells were then incubated with the rabbit polyclonal
antibody, clone 57 (GR57), against the human GR (catalog no. PA1–511,
Affinity BioReagents, Inc., Golden, CO) (32) in 2% normal
donkey serum (NDS) in PBS for 30 min at room temperature, followed by
overnight incubation at 4 C. The following day, cells were incubated
with biotin-conjugated donkey antirabbit antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in NDS for
1 h, followed by incubation with the fluorescein
isothiocyanate-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.) in PBS for 1 h (in darkness). Two
washes with PBS buffer were performed between all steps. Slides were
mounted with a glass coverslip using the Slowfade-Light Antifade
reagent in glycerol buffer (Molecular Probes, Inc.,
Eugene, OR). Microscopic examination was performed using a
Nikon Microphot-SA microscope with a Nikon
PlanApo 20/0.75 objective (Nikon, Melville, NY). The
protocol for the quantitative analysis of fluorescence in the cytoplasm
and the nucleus was developed in consultation with Dr. David E. Wolf
(Cell Biology Group, Worcester Foundation for Biomedical Research,
Worcester, MA) and has been previously validated and described
(31). A digital processing system including a computer station, NIH
Image analysis software, and two devices for image acquisition (a
camera and a scanner) was used to acquire, store, and process the
microscopic images as well as to perform the quantitative analyses.
Microscopic fields were captured under both light and fluorescence
illuminations and transformed into digital images to be shown on a
computer screen. Sampling was performed on two to four different areas
of each well. The microscope and the camera settings were maintained
constant between all experimental conditions, and no adjustment of the
gray scale was performed in the images. A region of interest (ROI) was
selected in the cytoplasm and nucleus of each cell of the section. The
intensity value of each pixel within the ROI ranged between 0–255 and
was proportional to the number of fluorescent photons emitted from the
corresponding point in the specimen. After subtraction of the
background (no cells), the mean intensity of the ROI was calculated.
This value represented a measure of the fluorescence detected from each
ROI and was used to make comparisons between the same compartments
under different conditions. Several steps were included to have an
objective and accurate series of measurements. First, the ROI was
initially outlined blind to the fluorescent signal (and to the
treatment condition) using light microscopy images. Afterward the
selections were superimposed onto the corresponding fluorescent image,
and the fluorescence intensity in the region was quantified. Second,
the ROI was defined using the oval tool of the image software, and its
width was kept constant. Third, the entire cell and the nucleus were
manually demarcated, and the resulting areas (number of pixels) were
calculated to control for possible changes in the shape of the
cells.
GR binding assay
GR binding was determined using a previously described in
vitro cytosolic exchange assay (33). After incubation with
cytokine in 175-cm2 flasks, cells were washed and scraped
in cold HBSS, transferred to 50-ml tubes, pelleted at 700 x
g for 10 min, resuspended in HBSS for three consecutive
washes, and then fractionated using a freeze/thaw procedure in a volume
of 0.7 ml binding buffer (10 mM Tris, 1 mM
EDTA, 20 mM molybdic acid, 5 mM dithiothreitol,
and 10% glycerol in double distilled water, pH 7.4 at 4 C), yielding
an approximate final protein concentration of 0.5–1.5 mg/ml cytosol.
After centrifugation at 105,000 x g for 60 min at 4 C,
the supernatant-cytosol was added to incubation solutions containing
radiolabeled (3H) Dex with or without unlabeled competitor.
Bound radiolabeled steroid was separated from unbound steroid by
filtration through minicolumns containing 1.25 ml LH-20 Sephadex
(Pharmacia Biotech, Piscataway, NJ). Scintillation fluor
(Ultima Gold, Packard, Meridien, CT) was added to eluate containing the
bound fraction of steroid, and tritium (3H) radioactivity
was determined in a Wallac, Inc. LKB 1209
liquid scintillation counter (LKB, Uppsala, Sweden). For
single point assays, GR receptor binding was defined as the amount of
total [3H]DEX (10 nM) binding displaced by
the selective GR agonist, RU28362 (0.5 µM). Incubations
with [3H]Dex were performed (at 14-fold above its
Kd) at 4 C for 18–22 h.
Specific binding was expressed as femtomoles per mg cytosolic protein. Protein content for all samples was determined by the method of Bradford (34) with use of BSA as a standard.
CAT reporter cell line and CAT assay
The LMCAT cell line (derived from L929 cells) is stably
transfected with the MMTV-CAT reporter plasmid. Expression of CAT
activity by these cells is under hormonal control by virtue of several
HREs residing within the MMTV promoter, which lies upstream of the CAT
reporter gene (30). Measurement of CAT enzyme activity was performed
using a liquid scintillation counting detection system according to the
manufacturer’s instructions (Promega Corp., Madison, WI).
Briefly, cell extracts were obtained using a Tris buffer (0.25
M Tris-HCl, pH 8.0), freeze/thaw procedure, followed by
heating to 60 C for 10 min to inactivate endogenous deacetylase
activity. After centrifugation (20,000 x g for 2 min),
supernatants were transferred to fresh tubes and processed for CAT
enzyme activity. Each reaction was initiated by adding the cofactor
n-butyrl coenzyme A to tubes containing cell extracts and
radiolabeled [3H]chloramphenicol. The CAT reaction was
stopped, and the butyrylated forms of [3H]chloramphenicol
were separated by three consecutive extractions with mixed xylene. The
extracts were transferred to vials for liquid scintillation counting.
The counts per min measured in each sample represents the butyrylated
fraction of the enzyme (as determined by a standard curve) and is
directly proportion to the CAT gene expression.
Protein content for all samples was determined by the method of Bradford (34) with use of BSA as a standard.
Statistical analysis
Data are presented as the mean ± SEM
(SEM) and were analyzed using one-way ANOVA. When ANOVA
revealed a significant main effect of treatment condition, both a
conservative (Student-Newman-Keuls test) and a powerful (Student’s
t test) post-hoc test were used for between-group
comparisons.
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Results |
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on GR immunostaining on GR nucleocytoplasmic traffic,
we employed a fluorescence/immunostaining procedure using an anti-GR
polyclonal antibody coupled with quantitative analysis of fluorescence
in the cytoplasm and nucleus of cells using digital image analysis (see
Materials and Methods). Cells were grown in steroid-free
conditions (charcoal-extracted serum) and treated with vehicle, IL-1
(1000 U/ml) for 24 h, Dex (10 nM) for 1.5 h, or
IL-1 (1000 U/ml) for 24 h followed by coincubation of IL-1
(1000 U/ml) plus Dex (10 nM) for 1.5 h. L929 cells
stained for the GR after the various treatments are presented in Fig. 1
. After treatment with vehicle (Fig. 1A), the pattern of staining was heterogeneous among cells, with a
diffuse staining in both the cytoplasm and nucleus. In the majority of
cells, the fluorescent signal was more intense in the cytoplasm than in
the nucleus, although a few cells with brighter nuclei were present.
Nucleoli were never stained. After treatment with Dex (10
nM; Fig. 1B), cells presented more intense nuclear
staining, confirming that treatment with Dex induced translocation of
the GR from the cytoplasm to the nucleus [these results are consistent
with our previous observation of GR immunostaining in L929 cells
(31)]. Cells treated with IL-1 alone for 24 h showed more
intense and diffuse brightness in both cytoplasm and nucleus, but there
was no apparent effect on GR translocation (Fig. 1C). Finally,
treatment with IL-1 and Dex (10 nM; Fig. 1D) showed a
reduced effect (less bright nuclei) compared with Dex alone.
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). Quantitative analysis showed that Dex
(10 nM) alone induced a decrease in the GR fluorescent
signal in the cytoplasm of approximately 18%, whereas coincubation of
IL-1 plus the same dose of Dex completely blocked the decrease in
the cytoplasmic fluorescence. In the nucleus, Dex alone induced an
increase in the signal of approximately 47%, whereas coincubation of
IL-1 plus Dex led to a partial blocking of this effect, leading only
to a 16% increase in nuclear fluorescence. Treatment with IL-1
alone induced an increase in both cytoplasmic (+23%) and nuclear
(+16%) fluorescence, thus confirming the visual results of increased
diffuse brightness.
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Effect of IL-1 on cytosolic GR binding
We evaluated [3H]Dex-binding sites using an in
vitro exchange assay of cytoplasmic homogenates. When most of the
receptor is in the unactivated state, cytosolic GR binding is a measure
of the amount of receptor in the cells, and therefore, its evaluation
may be useful to investigate expression of GR protein. L929 cells were
treated with vehicle or IL-1 (1000 U/ml) for 24 h. As shown in
Fig. 3A, IL-1 induced an increase in
cytosolic GR binding (up-regulation). The increase in cytosolic binding
(+23%) is consistent with the increase in the cytoplasmic fluorescent
signal (+23%), thus suggesting that both of these techniques reveal
increased levels of GR protein induced by treatment with IL-1 for
24 h. Protein concentrations were examined in every sample, and no
effect of IL-1 treatment on protein content was detected [vehicle,
0.676 (SE, 0.157) mg/ml; IL-1, 0.607 (SE,
0.122) mg/ml; P = NS].
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-induced changes
in nucleocytoplasmic traffic and protein expression, we examined
GR-mediated gene transcription in L929 cells stably transfected with a
CAT enzyme reporter gene that is under hormonal control by virtue of
several HREs residing in the upstream MMTV promoter (LMCAT cells).
According to our data on GR nucleocytoplasmic traffic and GR binding,
IL-1 alone induced GR up-regulation in the absence of steroids, and
cells treated with IL-1 and Dex showed less GR translocation than
cells treated with the same dose of Dex alone. Therefore, to determine
whether receptor up-regulation was accompanied by increased functional
activity and whether blockade of Dex-induced GR translocation was
associated with a relative decrease in functional activity, the impact
of the same treatment protocols on GR-mediated gene transcription in
the presence or absence of various doses of Dex was determined. LMCAT
cells were grown in steroid-free medium and treated with vehicle,
IL-1 (100 and 1000 U/ml) for 24 h, Dex (10 nM to 1
µM) for 1.5 h, or IL-1 (10–1000 U/ml) for
24 h followed by incubation of IL-1 (10 and 1000 U/ml) plus Dex
(10 nM to 1 µM) for 1.5 h. Results are
presented in Figs. 3B
and 4 and are
expressed as relative CAT activity (percentage of vehicle-treated
control samples). Treatment with IL-1 (1000 U/ml) alone for 24
h induced an increase in GR-mediated gene transcription (+24%). This
increase (+24%) was consistent with the +23% up-regulation induced by
the same dose of IL-1 described previously in the immunostaining and
binding data (Figs. 1, 2, and 3A). In contrast, at every concentration
of Dex, pretreatment with IL-1 (1000 U/ml) caused a decrease in
Dex-induced GR-mediated gene transcription compared with that in cells
treated with the same dose of Dex alone (Fig. 4). Dex-induced
GR-mediated gene transcription was reduced by 4% at the
10-nM dose of Dex and about 20% at the 100-nM
and 1-µM Dex doses (Fig. 4). As under physiological
conditions, hormone and cytokine would occur together rather than in
tandem as represented in the previous experiment, cells were
coincubated with IL-1 and Dex for 24 h and compared with cells
treated with Dex alone for 24 h. Previous experiments have
demonstrated that under these conditions (Dex treatment for 24 h),
GR down-regulation does not occur and therefore would not confound
interpretation of the results (35). Coincubation of cells with Dex plus
IL-1 (100 and 1000 U/ml) led to marked reduction (42%) in relative
CAT activity compared with treatment with Dex alone (Fig. 5A). In addition, to confirm the
specificity of the effects of IL-1 on GR-mediated gene
transcription, IL-1ra was added to the combination of IL-1 plus Dex
(10 nM). To optimize the stochiometry of the antagonist
treatments in these experiments, the lower dose of IL-1 (100 U/ml)
was used in combination with a 1000-fold excess of IL-1ra calculated on
a mg per mg basis. As shown in Fig. 5B, IL-1ra completely eliminated
the inhibitory effects of IL-1 on Dex-induced GR-mediated gene
transcription.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inhibits GR translocation and function and induces GR
up-regulation, on GR translocation and function. We
used an in vitro experimental system employing the mouse
fibroblast cell line L929, a cell line that has been previously
described by our group and others to be sensitive to the effects of GR
agonists, antagonists, and nonsteroid GR modulators, including
antidepressants (31), heat shock (30), and cytokines (36). We also used
a panel of quantitative assays to investigate GR nucleocytoplasmic
traffic, GR expression, and GR-mediated gene transcription. Our results
demonstrate that 24-h treatment with IL-1 inhibits Dex-induced
translocation of the GR from cytoplasm to nucleus. Moreover, 24-h
pretreatment with IL-1 or 24-h coincubation with IL-1 led to
significant reduction in Dex-induced GR-mediated gene
transcription, an effect that was reversed by IL-1ra. Finally,
evaluation of both immunocytochemical staining of the GR and cytosolic
GR binding in cells treated with IL-1 for 24 h provided
evidence of GR up-regulation.
These results contribute to the identification of a possible mechanism
by which cytokines can induce glucocorticoid resistance. As previously
noted, several studies have shown that in vivo and in
vitro treatment with proinflammatory cytokines, including IL-1,
decreases GR function (GR resistance). For example, in vivo
treatment of mice or in vitro treatment of a hepatoma cell
line with IL-1 and IL-1ß has been found to decrease
glucocorticoid-stimulated induction of the gluconeogenesis enzyme,
phosphenolpyruvate carboxykinase (15, 17, 18). Of note is that the
degree of maximum inhibition of enzyme induction in these previous
studies (25%) is comparable to the percent inhibition of
GR-mediated gene transcription found in the present study
(20–40% inhibition). Other studies have reported similar results.
For example, in vitro treatment with the IL-1 inducers,
endotoxin and lipopolysaccaride (LPS), or with IL-1ß have all been
shown to decrease GR affinity for ligand, and in the case of endotoxin
has also led to resistance to the inhibitory effects of Dex on
corticosterone secretion (11, 12, 13, 37). In contrast, one study has
reported that in vitro treatment with IL-1ß has a
stimulating effect on GR function (36), however the experiments on IL-1
involved cell lines that were transiently transfected with an MMTV-CAT
plasmid and chemically shocked before treatment with cytokine. Chemical
shock is known to be associated with increased GR function; therefore,
such manipulation confounds interpretation of the result (30).
In contrast to the above studies on GR function and affinity, studies
examining the effects of IL-1 or IL-1 inducers on GR expression have
yielded more mixed results. In fact, although the majority of the
above-mentioned studies demonstrated reduced GR function and/or reduced
GR affinity for ligand, in those studies that additionally examined GR
expression, some found GR down-regulation (12, 15, 17) and, like the
present study, others found GR up-regulation (11, 13, 36). To further
complicate attempts to integrate the available data, a number of
studies have described changes in GR expression in the absence of
functional data, and both GR down-regulation by endotoxin (14) and LPS
(16) and up-regulation by LPS (38) have been described. Of note is that
patients with ongoing immune activation and GR resistance have been
shown to exhibit decreased GR affinity and GR up-regulation (see
below). One explanation for these discrepant findings regarding
receptor expression is the specific characteristics of the different
treatments (IL-1 vs. IL-1ß vs. IL-1
inducers) and of the various cell lines and/or experimental conditions
in each study. The regulation of transcription of GR messenger RNA
(mRNA), for example, appears to be under the control of several
different promoters, the utilization of which is a function of cell and
tissue phenotype (Seckl, J., personal communication). Moreover, changes
in GR expression (as well as function) occur as a cumulative result of
a number of distinct steps, including receptor assembly,
phosphorylation, interactions with heat shock proteins, and regulation
by transcription factors. Therefore, it is very likely that different
cytokines may have tissue-specific effects on GR expression and
eventually function. In addition, among the studies showing
cytokine-induced GR resistance, those examining cells treated with IL-1
for 24–48 h or, notably, tissues from patients with chronic
inflammatory diseases consistently show GR up-regulation (11, 20, 21, 25, 36, 39), whereas shorter in vitro or in vivo
treatments with IL-1 or IL-1 inducers (4–6 h) are associated with GR
down-regulation (12, 14, 15, 16, 17). It is noteworthy that the majority of the
studies showing receptor down-regulation after short term treatments
have also measured cytosolic GR binding; therefore, decreased cytosolic
GR binding after short term cytokine treatments may be a function of
the kinetics and compartmentalization of the receptor during the acute
response to cytokines, as opposed to being a qualitatively different
effect. Interestingly in this regard, a study by Verheggen et
al. (13) found LPS- and IL-1ß-induced GR up-regulation in the
absence of increased GR mRNA, suggesting that increased GR mRNA
translation or half-life or alterations in GR compartmentalization were
involved.
IL-1 alone increased CAT activity by about 25% after 24 h of
treatment. The magnitude of this increase is similar to that seen after
1.5 h of treatment with 10 nM Dex, but much smaller
than that seen after 24 h of treatment with 10 nM Dex,
which induces CAT activity 700-2500% above that with vehicle.
IL-1-induced increases in CAT activity may represent the results of
up-regulated GR. Nevertheless, as these experiments employed media
stripped of endogenous steroids, the mechanism underlying the increased
CAT activity remains unclear. We suggest that continuous recirculation
of GR may occur in the unstimulated condition, thus leading to a
baseline activation of CAT activity, which is influenced mostly by the
number of GRs. GR trafficking under these conditions may be insensitive
to the inhibitory effects of IL-1. On the contrary, GR activation by
steroids, such as Dex, even at low doses involves several distinct
steps, and one or more of these may be affected by the presence of
IL-1. Clearly, this mechanism needs further elaboration.
Mechanisms of the effects of IL-1 on GR
IL-1 exerts its effects by interacting with specific membrane
receptors, followed by activation of second messenger pathways,
regulation of protein kinases or phosphatases, and, finally, activation
of transcription factors such as nuclear factor-
B (40). Fibroblasts
contain mRNA for both the type I and the type II IL-1 receptor, but
only the type I receptor appears to be biologically active (41).
Although the exact IL-1 signal transduction pathway has not been
established for fibroblasts, interactions with G proteins to activate
adenylate cyclase as well as stimulation of Ca2+ influx
have been suggested (42, 43). Furthermore, there is evidence that
protein phosphorylation (by mitogen-activated, tyrosine, or
ß-casein kinases) also plays a role in fibroblast responses to
IL-1 (40, 43, 44). There is, however, no consensus as to how proximal
the kinase cascade is to the IL-1 receptor. Of note, responsiveness of
human fibroblasts to IL-1 in vitro occurs when cells are
plated on fibronectin, as in our experiments, as opposed to
poly-L-lysine (43). Glucocorticoid hormones, in turn,
modulate the effects of cytokines by specifically targeting the
induction of transcription factors. For example, activated GR has been
shown to either inhibit cytokine-induced synthesis of these
transcription factors or to increase the levels and activity of
transcription factors inhibitors, such as inhibitory factor B
(45, 46). These pathways are believed to be crucial to the
immunomodulatory and immunosuppressive effects of glucocorticoid
hormones. However, activation of protein kinases and transcription
factors by IL-1 is probably a relevant mechanism by which these
compounds might modify GR function and expression. First,
phosphorylation of the GR and/or other nuclear substrates from
cAMP-dependent protein kinase may have a relevant role in the
regulation of GR function. For example, both adenyl cyclase and protein
kinase A activators have been found to influence GR function (47).
Moreover, cell lines containing a defective cAMP-dependent protein
kinase give rise to glucocorticoid-resistant variants at a high
frequency (48). Second, protein-protein interaction has been described
to occur between GR and nuclear factor-B (49) and between GR and
c-Jun, one of the components of activating protein-1
(50, 51), and reciprocal functional antagonism has been described.
Alternatively, cytokines may act on heat shock proteins, one of the
constituents of the GR complex. In fact, cytokines, including IL-1,
have been shown to induce heat shock proteins (52), and stabilization
of GR/90-kDa heat shock protein interaction by sodium molybdate has
been associated with inhibition of Dex-induced GR translocation (53)
much like that produced by IL-1.
Clinical relevance
The experimental evidence suggesting that cytokines decrease GR
function is consistent with the well known presence of glucocorticoid
resistance in subpopulations of patients with acute or chronic
inflammatory diseases, such as sepsis, asthma, ulcerative colitis,
acquired immunodeficiency syndrome, rheumatoid arthritis, and allogenic
organ transplantation (24). Specifically, studies performed in
peripheral tissues of patients with the above-mentioned diseases,
especially those showing resistance to therapeutic effects of
glucocorticoids, have demonstrated changes in GR number and function
similar to those induced in vivo and in vitro by
treatment with cytokines and immune system activators, i.e.
reduced effects of glucocorticoids on functional endpoints (19, 23, 37, 54), reduced GR affinity for the ligands (20, 21, 25), and GR
up-regulation (20, 21, 25, 39). Interestingly, these GR abnormalities
revert to normal after in vitro tissue culture (21).
Moreover, these GR abnormalities recover when patients are treated with
very high doses of glucocorticoids, possibly due to the effects of
these hormones on glucocorticoid-sensitive immune cells leading to a
decrease in ongoing immune activation (22). Finally, GR resistance
seems to be tissue specific, as suggested by the clinical evidence that
asthmatic patients who fail to show therapeutic responses to high doses
of glucocorticoids nevertheless exhibit features of glucocorticoid
excess (Cushingoid features) in other tissues (24). Therefore,
converging evidence suggests that local concentrations of cytokines
produced during an inflammatory response may produce an acquired,
localized GR resistance. Inhibition of GR translocation, as shown
in vitro in the present report, may be a relevant step in
this process. It is also of note that the psychiatric disorder, major
depression, has also been associated with GR resistance
(hypercortisolemia, reduced feedback inhibition on the HPA axis, and
decreased sensitivity of peripheral tissues to the effects of
glucocorticoids) as well as evidence of inflammation, including
increased levels of proinflammatory cytokines and acute phase reactants
(55, 56). Moreover, we have recently demonstrated that the tricyclic
antidepressant desipramine increases GR translocation and Dex-induced
GR-mediated gene transcription (31), an effect that is virtually the
opposite of the effects of IL-1 described here. Therefore, it is
intriguing to speculate that cytokines produced in the brain or
periphery may contribute to the pathogenesis of GR resistance described
in patients with major depression, and antidepressants may overcome
these receptor alterations.
In summary, our results show that IL-1 inhibits Dex-induced GR
translocation and Dex-induced GR-mediated gene transcription and
induces up-regulation of the cytosolic receptor. This impact on the GR
may be an important mechanism by which cytokines produced during an
inflammatory response may directly modulate the capacity of
glucocorticoids to transmit signals to target tissues and induce GR
resistance in relevant cell types, therefore providing an additional
pathway by which the immune system can influence the HPA axis and,
ultimately, disease expression.
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Received October 23, 1998.
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) or IL-1
) for 24
h. Cell lysates were centrifuged, and the supernatant-cytosol was added
to incubation solutions containing radiolabeled (3H) Dex
for the exchange assay. Values are presented as the mean ±
SEM GR binding in vehicle- and IL-1
), or
Dex (10 nM), IL-1
); in
panel B only). Cells were fractionated, and lysates were analyzed for
relative CAT enzyme activity (presented as a percentage of vehicle
activity). A, Results depicted are from a representative experiment in
which the mean ± SEM of five or six separate
determinations per treatment group are shown. There was a significant
main effect of treatment condition on GR-mediated gene activity using
one-way ANOVA [F(