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Originally published online as doi:10.1189/jlb.0805466 on February 3, 2006

Published online before print February 3, 2006
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(Journal of Leukocyte Biology. 2006;79:818-827.)
© 2006 by Society for Leukocyte Biology

Divergent expression and function of glucocorticoid receptor ß in human monocytes and T cells

Ling-bo Li*, Donald Y. M. Leung*,{dagger},1, Clifton F. Hall* and Elena Goleva*

* Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado; and
{dagger} Department of Pediatrics, University of Colorado Health Sciences Center, Denver

1Correspondence: National Jewish Medical and Research Center, 1400 Jackson Street, Room K926i, Denver, CO 80206. E-mail: leungd{at}njc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucocorticoid (GC) insensitivity is a significant problem in the treatment of immune-mediated diseases. The current study examined whether T cells and monocytes differed in their response to GC and the potential molecular basis for their variation in response to steroids. Functional studies revealed that dexamethasone (DEX) inhibited phorbol 12-myristate 13-acetate/ionomycin-induced tumor necrosis factor {alpha} and interleukin-6 production to a significantly lesser extent in monocytes than T cells. In parallel, a significantly longer period of time was required for DEX to induce the steroid-responsive gene, mitogen-activated protein kinase phosphatase-1 (MKP-1), in human monocytes as compared with T cells. It is interesting that such differences were not observed between murine T cells and monocytes. GC receptor ß (GCRß) is a splicing variant of the classic GCR, GCR{alpha}, which functions as a dominant-negative inhibitor of GCR{alpha} in humans, not mice (as mice do not express GCRß mRNA as a result of a difference in the murine GCR 9b exon sequence). It was found that human monocytes had a significantly higher level of GCRß than T cells. Furthermore, GCRß was found in the cytoplasm and nucleus of monocytes, and GCRß was localized to the nucleus of T cells. This raised the possibility that GCRß in the cytoplasm could affect GCR{alpha} cellular shuttling in response to DEX. Indeed, we found that DEX-induced nuclear translocation of GCR{alpha} was decreased in monocytes as compared with T cells. Specific RNA silencing of GCRß in human monocytes resulted in enhanced steroid-induced GCR{alpha} transactivation and transrepression. Our data suggest that GCRß contributes to variation in the GC responses of monocytes versus T cells.

Key Words: steroid resistance • MKP-1 • dexamethasone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucocorticoids (GC) are commonly used in the treatment of inflammatory and immune-mediated diseases such as chronic asthma, inflammatory bowel disease, and collagen vascular diseases. However, a subset of these patients fails to respond to GC treatment. These patients have been referred to as "steroid-resistant" [1 2 3 ]. The molecular mechanism underlying steroid resistance is not well understood. The GC anti-inflammatory response is mediated through a 94-kDa intracellular receptor protein, the GC receptor {alpha} (GCR{alpha}), which is a ligand-dependent transcription factor [4 , 5 ]. At the cellular level, GC binding to GCR{alpha} causes translocation of GCR{alpha} from the cytoplasm to the nucleus, which binds as a homodimer to GC response elements (GRE) on the DNA to transactivate target genes [6 ]. The nuclear translocation of GCR{alpha} can also transrepress gene activation via direct interaction with proinflammatory transcription factors such as activator protein-1 and nuclear factor B (NF-{kappa}B) [4 , 5 ].

There are two highly homologous isoforms of GCRs in human cells: GCR{alpha} and GCRß, which are generated from alternative splicing of the human GCR gene. GCRß differs from GCR{alpha} in its carboxyl terminus, where the last 50 amino acids of GCR{alpha} are replaced by a nonhomologous, 15 amino acid sequence [7 ]. As a result of this difference, GCRß does not bind GC or transactivate promoter regions in GC-responsive genes [8 9 10 ]. Increased GCRß expression has been associated with steroid resistance of mononuclear cells in several inflammatory diseases such as asthma and ulcerative colitis [11 12 13 14 ]. However, the precise cellular localization and role of GCRß in controlling steroid responses are debated [15 , 16 ]. To better understand the physiologic role of GCRß, we examined normal T cells and monocytes for expression of GCRß and surprisingly, found that the cell distribution of GCRß in these two cell types was quite different. In the current study, we therefore examined whether T cells versus monocytes differed in their response to GC and the potential role that GCRß expression might have in controlling their response to GC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Fluorochrome-labeled CD3 (Clone SK7), CD14 (Clone M5E2), CD19 (Clone HIB19), and CD56 (Clone NCAM 16.2) antibodies were purchased from BD PharMingen (San Diego, CA). Human monocyte isolation kit II, human pan T cell isolation kit II, mouse monocyte isolation kit II, and mouse pan T cell isolation kit (Miltenyi Biotec, Auburn, CA) were used to purify the cells. Mitogen-activated protein kinase phosphatase-1 (MKP-1) primers and probe were purchased from Applied Biosystems (Foster City, CA). Human monocyte Nucleofector® kit was purchased from Amaxa, Inc. (Gaithersburg, MD).

GCR{alpha} and GCRß plasmids were made in our laboratory. In brief, the cDNA encoding the human GCRß isoform (base pairs 23–2296) was subcloned into the replication-defective murine stem-cell virus retroviral vector as a bicistronic coding unit containing green fluorescent protein, followed by the encephalomyelitis virus, internal ribosome entry site, and the GCRß-coding region as described [17 ]. Human GCR{alpha} cDNA was prepared from mRNA of human peripheral blood mononuclear cells (PBMC) using specific GCR{alpha} primers located in a poly-A tail, published by DeRijk et al. [18 ], by reverse transcription (all reverse transcription reagents were from Invitrogen, Carlsbad, CA). cDNA encoding GCR{alpha} poly-A tail was amplified by polymerase chain reaction (PCR) using the same primers and gel-purified. The PCR product was ligated into pCR2.1®-TOPO® vector using a TOPO-TA cloning kit (Invitrogen).

Isolation of human PBMC and mouse spleen cells and magnetic cell sorting
PBMC were isolated by Ficoll-Hypaque® density gradient centrifugation from heparinized, venous blood of healthy donors as described previously [19 ]. Cells were subsequently cultured on the slides to stain GCR or directly used to sort monocytes and T cells. Untouched CD3+ or CD14+ were isolated by negative depletion using magnetic cell sorting based on the manufacturer’s instructions (Miltenyi Biotec).

Four- to 6-week-old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in the certified animal facility of the National Jewish Medical and Research Center (Denver, CO). Six mice were killed, and isolated mouse cells were gathered from single-cell suspension of spleen. In the same way as human cell-sorting, mouse spleen cells were sorted to obtain the purified T cells and macrophages. The purity of the enriched cells was evaluated by flow cytometry on FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) and was always over 90% positive.

Intracellular staining for GCRß and GCR{alpha}
Subcellular localization and nuclear translocation studies were carried out by using fluorescent microscopy (Leica, Wetzlar, Germany) using a x63 objective. Human PBMC (1x106 cells/ml) were allowed to settle and adhere on poly-D-lysine-coated coverslips, fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in phosphate-buffered saline (PBS) for 5 min, and blocked in a commercial blocking solution (Superblock, Scytek, Logan, UT) for 15 min. For GCRß intracellular staining, cells were first incubated with allophycocyanin (APC)-conjugated antibodies APC-CD3, APC-CD 14, APC-CD19, or APC-CD56 for 1 h at 4°C and permeabilized for another 15 min in permeabilization solution [PBS containing 0.1% (v/v) Tween 20, 0.1% (w/v) bovine serum albumin (Sigma-Aldrich), and 0.01% (w/v) saponin (Sigma-Aldrich)] and then incubated with an affinity-purified polyclonal antibody to GCRß (Affinity Bio-Reagents, Golden, CO), diluted in permeabilization solution (1/750) overnight at 4°C. Purified, nonimmune rabbit immunoglobulin G (IgG; Southern Biotechnology Associates, Birmingham, AL) was used as an isotype control. After washing, the cells were incubated with a donkey anti-rabbit IgG, F(ab')2-cyanine (Cy)3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; 1/200), and the nucleus was counterstained with 300 nm 4',6'-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 45 min at room temperature and washed in PBS/0.1% Tween 20 for 15 min.

For GCR{alpha} nuclear translocation experiments, the cells were preincubated with dexamethasone (DEX) for 1–3 h or remained untreated and then were treated as described above. Intensity of GCR{alpha} or GCRß staining was assessed using image analysis software (Slidebook, Intelligent Imaging Innovations, Denver, CO) and expressed as mean fluorescence intensity (MFI).

The specificity of GCR antibodies was tested by preincubating GCR{alpha} or GCRß antibodies with 20-fold excess of its neutralizing peptide (Affinity Bio-Reagents). This resulted in a loss of reactivity (data not shown).

Immunoassay for tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin (IL)-6
Purified monocytes (1x106/ml) or T cells were stimulated with five different concentrations of phorbol 12-myristate 13-acetate (PMA; 1, 5, 10, 25, 50 ng/ml) with 1 µg/ml ionomycin in the 96-well plates for 24 h in the absence or presence of 10–9–10–6M DEX. The supernatants were collected, frozen, and stored in –80°C until use. For GCRß silencing experiments, 3 x 106/ml purified monocytes were transfected with 1 µg GCRß short interfering (si)RNA or control siRNA and cultured for 24 h. Then, cells were treated with PMA/ionomycin with or without DEX for an additional 24 h, and the supernatants were collected and analyzed as described above. Specific, commercially available enzyme-linked immunosorbent assays (ELISAs) were used to measure TNF-{alpha} and IL-6 (R&D Systems, Minneapolis, MN). The assays were performed according to the manufacturer’s instructions. Absorbance was transformed to cytokine concentration using standard curves.

Western blotting
Analysis of GCR{alpha} nuclear translocation and GCRß localization in monocytes and T cells was performed by Western blot. Nuclear and cytoplasmic extracts from purified monocytes and T cells were prepared with a NE-PER® nuclear and cytoplasmic extraction reagents kit (Pierce, Rockford, IL).

The Western blot was performed as described previously [20 ]. Same amounts of total protein or fractionated protein per condition were run on 4–15% gradient gel (BioRad, Hercules, CA) and transferred to the membrane. The membranes were blotted with anti-GCR{alpha} (P-20; Santa Cruz Biotechnology, CA) or anti-GCRß (Abcam Inc., Cambridge, MA) antibodies. To control the quality of nuclear and cytoplasmic protein preparations, the membranes were stripped and reprobed with anti-NF-1 and anti-ß-tubulin antibodies (Santa Cruz Biotechnology) as nuclear and cytoplasmic proteins, respectively.

Real-time PCR assay for GCR and MKP-1 mRNA
Total RNA from purified T cells or monocytes was extracted using the RNeasy mini kit (Qiagen, Valencia, CA), reverse-transcribed into cDNA using reverse-transcription reagents (Invitrogen), following the manufacturer’s instructions as described [18 , 21 ]. For DEX-induced MKP-1 experiments, the cells were incubated with or without 10–7 M DEX for 1–24 h before RNA extraction. Primers and probes for MKP-1, interferon-{gamma} (IFN-{gamma}), and glyceraldehyde 3-phosphate dehydrogenase (GADPH) were purchased from Applied Biosystems. GCR{alpha} and GCRß primers were custom-ordered from Applied Biosytems based on the sequences published by DeRijk et al. [18 ]. The quantitative real-time PCR was performed as described previously [21 ]. Briefly, the reactions were carried out using the dual-labeled fluorigenic probe method. ABI Prism 7000 sequence detector (Applied Biosystems) was used to run PCR and collect fluorescence data, and relative expression levels were calculated and normalized to the corresponding levels of the housekeeping gene (GADPH). Standard curves for MKP-1, IFN-{alpha}, IFN-ß, and GADPH were generated using the fluorescent data from twofold serial dilutions of 1000 ng total RNA of the target sample. Standard curves for GCR{alpha} and GCRß were generated from tenfold serial dilutions of the GCR{alpha} and GCRß plasmids.

Silencing of GCRß expression by specific siRNA
siRNAs were used to inhibit GCRß gene expression. Annealed siRNA (sense 5'-GGCUUUUCAUUAAAUGGGAtt-3', antisense 5'-UCCCAUUUAAUGAAAAGCCtc-3'), corresponding to the C-terminal in Exon 9ß, was used to inhibit GCRß RNA expression. The siRNA were purchased from Ambion (Austin, TX). A commercial Nucleofector human monocytes kit (Amaxa, Inc.) and special transfection program for human monocytes on the Nucleofector device (Amaxa, Inc.) were used. In brief, 3 x 106-purified monocytes were suspended in 100 µl transfection solution and transfected with 1 µg GCRß siRNA or nonsilencing, control siRNA using the Y001 program. Transfected cells were diluted immediately with prewarmed monocyte growth media (Amaxa, Inc.) and cultured in 24-well plates or on poly-D-lysine-coated coverslips. To monitor the transfection efficiency, the siRNA was labeled with the Cy5 siRNA labeling kit (Ambion) based on the manufacturer’s instruction. To make certain that GCRß siRNA reduced target mRNA level and to determine the exact RNA silencing time, the transgenic cells were serially assayed by real-time PCR and immunocytochemistry from 24 h to 72 h as described above. To determine the effect of siRNA delivery on cell viability, the trypan blue dye exclusion test was used, and IFN-{alpha} and IFN-ß mRNA expression was evaluated by real-time PCR 24 h after transfection with siRNA.

Statistical analysis
Immunofluorescence assay and the cell cytokine production data were analyzed using the Student’s t-test. P < 0.05 was considered significant. Data were expressed as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The functional response of monocytes versus T cells to GC
In these experiments, human monocytes versus T cells were stimulated for 24 h with different concentrations of PMA and ionomycin in the presence and absence of DEX; culture supernatants were analyzed for TNF-{alpha} by ELISA. Production of TNF-{alpha} by resting human monocytes and T cells was comparable: 150.0 ± 25.6 pg/ml by resting monocytes versus 42.7 ± 24.5 pg/ml by T cells. The PMA concentration was determined by preliminary dose-response experiments. A dose-dependent increase in cytokine production was found when cells were stimulated with 1–25 ng/ml PMA. The plateau effect was achieved with PMA concentrations above 25 ng/ml. Therefore, 1 ng/ml, 5 ng/ml, and 25 ng/ml PMA were the concentrations chosen for all subsequent experiments. It was found that DEX suppressed TNF-{alpha} secretion in a concentration-dependent manner in monocytes and T cells (Fig. 1A ). However, at concentrations of DEX between 10–8 and 10–9 M, TNF-{alpha} production by monocytes was inhibited by DEX to a significantly lesser extent as compared with T cells (P<0.05). These differences in response to GC could be overcome at 10–7M or higher concentrations of DEX.


Figure 1
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  		Figure 1. Inhibition of TNF-{alpha} (A) and IL-6 (B) secretion by purified human monocytes and T cells in the presence of DEX. Monocytes and T cells were stimulated with different concentrations of PMA/ionomycin for 24 h; culture supernatants from activated cells were assayed for TNF-{alpha} and IL-6 by ELISA. Values are mean ± SEM of six independent experiments. *, P < 0.05; **, P < 0.01, as compared with PMA/ionomycin-treated cells (control).

 
To determine if this differential effect of DEX on monocytes versus T cells is just limited to TNF-{alpha} production, we examined IL-6 production using the same culture supernatants. Similar amounts of IL-6 were produced by resting human monocytes (100.5±35.5 pg/ml) and T cells (80.5±34.1 pg/ml). IL-6 release was enhanced by PMA in a dose-dependent manner in monocytes and T cells (Fig. 1B) . It should be noted that compared with the effect of DEX on TNF-{alpha} production, IL-6 production by monocytes was more resistant to DEX, as even higher doses of DEX (i.e., 10–7 M and 10–6 M) had no effect on IL-6 secretion. As for IL-6 production in T cells, the suppression of IL-6 by DEX was concentration-dependent and significant (P<0.05). DEX-induced gene expression of MKP-1 (a steroid-responsive gene) was also measured by real-time PCR in monocytes versus T cells. There was no difference in MKP-1 mRNA expression between freshly isolated monocytes (11.05±1.36 ng MKP-1/ng GAPDH) and T cells (13.41±2.92 ng MKP-1/ng GAPDH). In human T cells, DEX induced MKP-1 rapidly. As shown in Figure 2A , incubation with DEX for 2 h induced a threefold increase of MKP-1 gene expression, and as the incubation time increased, the induction of MKP-1 gene expression increased further. However, in monocytes, it required a significantly (P<0.01) longer period of time to induce MKP-1 gene expression.


Figure 2
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  		Figure 2. DEX has different effects on MKP-1 gene induction in human (A) or murine (B) monocytes/macrophages and T cells. (A) Purified human monocytes and T cells were collected at various time-points after DEX treatment. MKP-1 mRNA induction by DEX in cell isolates as compared with media-treated cells was analyzed by real-time PCR (n=3; *, P<0.01, as compared with DEX-induced MKP-1 levels in human monocytes). (B) Purified murine spleen macrophages and T cells were collected at various time-points after DEX treatment. MKP-1 mRNA induction by DEX in the cell isolates as compared with media-treated cells was analyzed by real-time PCR (n=3).

 
We also compared the response of murine monocytes to murine T cells after incubation with DEX. As shown in Figure 2B , in contrast to human cells, there was no difference in MKP-1 induction between murine macrophages and T cells over a 6-h period. In contrast to the human GCR gene, mice lack the correct splice acceptor site in the rodent GCR{alpha} and do not express GCRß mRNA as a result of a difference in the murine GCR 9b exon sequence [22 ]. These data therefore suggested a potential role for GCRß in the divergent response of human monocytes versus T cells to DEX. Further studies were therefore be carried out to examine more precisely the expression of GCRß in monocytes versus T cells and the role of GCRß in determining steroid response.

GCRß protein and mRNA level in the normal human monocytes versus T cells
To examine GCRß expression and subcellular distribution in human monocytes and T cells, we compared GCRß expression in different populations of human PBMC by combining surface staining and intracellular staining. Figure 3A and 3B , shows staining results from a normal donor (representative of six normal individuals). In these experiments, APC-conjugated CD3 and CD14 antibodies were used to distinguish different cell populations in whole PBMC by cell-surface staining. The nucleus was defined by staining with the blue DNA-binding dye, DAPI, and GCRß localization was identified by indirect immunofluorescence using anti-GCRß antibody detected by Cy3-conjugated secondary antibody (red). Immunofluorescent microscopy revealed that GCRß protein was expressed at a significantly higher level (P<0.01) in the CD14+ monocytes compared with CD3+ T cells (Fig. 3) . Specificity of the immunoreaction for GCRß was demonstrated by the lack of reactivity of rabbit isotype control to the cells and the observation that anti-GCRß staining of monocytes and T cells was blocked by purified GCRß-immunizing peptide. Moreover, the distribution of GCRß protein was strikingly different in these two cell types: GCRß was found in the cytoplasm and the nucleus of monocytes, whereas GCRß was localized exclusively to the nucleus of T cells. In addition, GCRß protein expression in CD19+ B cells and CD56+ natural killer cells was similar to CD3+ lymphocytes (data not shown). Western blotting results also verified that GCRß was localized to the cytoplasm and nucleus of monocytes, and GCRß was just observed in the nucleus of T cells (Fig. 3D) .


Figure 3
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  		Figure 3. GCRß expression in different cell populations of human PBMC. Immunofluorescent microscopy was performed to visualize subcellular distribution of GCRß (original magnification, x630; blue, DAPI-nuclear staining; red, Cy3-GCRß; green, APC-CD3+ or -CD14+). Representative images of GCRß distribution in peripheral blood CD3+ lymphocytes (A) and CD14+ monocytes (B) are shown. The cytoplasmic and nuclear localization of the GCRß in CD14+ monocytes should be noted. The MFI of Cy3 staining (GCRß) in CD3+ and CD14+ cells (C) was assessed by the analysis software within the computer-generated masks corresponding to whole cells, which at least 50–100, were analyzed for each donor studied (n=6). (D) GCRß distribution in cellular lysates from puriefied human monocytes and T cells. Freshly isolated monocytes and T cells were fractionated to yield nuclear (N) and cytoplasmic (C) fractions and were analyzed by Western blot.

 
A quantitative real-time PCR assay was used to compare GCR{alpha}/GCRß mRNA level between purified monocytes and T cells. Primers and probes were designed to be complementary to the sequence within the region of exon 9{alpha} for GCR{alpha} or exon 9ß for GCRß. Our results demonstrated that there were a significantly greater number of GCRß mRNA copies in human monocytes than in human T cells (Table 1 ). In contrast, there was no significant difference in the GCR{alpha} mRNA expression level between monocytes and T cells (Table 2 ).


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  		Table 1. GCRß mRNA Expression between Human Monocytes and T Cells

 

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  		Table 2. GCR{alpha} mRNA Expression between Human Monocytes and T Cells

 
DEX-induced GCR{alpha} nuclear translocation differs in monocytes versus T cells
Previous studies have reported that GCRß forms heterodimers with GCR{alpha} [17 ]. We therefore hypothesized that GCRß in the cytoplasm might affect GCR{alpha} cellular shuttling in response to DEX. As there exist different levels of GCRß in monocytes versus T cells, and monocytes but not T cells have cytoplasmic GCRß, we compared DEX-induced GCR{alpha} nuclear translocation in these two cell types. GCR{alpha} nuclear translocation was assessed at serial time-points in response to 10–6 M DEX (0 h, 1 h, 3 h) by immunofluorescent staining. At baseline, GCR{alpha} was mainly localized to the cell cytoplasm of T cells. As shown in Figure 4A in T cells, DEX induced GCR{alpha} nuclear translocation within 1 h. Increasing the time of DEX treatment for greater than 1 h had no further effect on GCR{alpha} nuclear translocation.


Figure 4
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  		Figure 4. Different time requirements for DEX to induce GCR{alpha} nuclear translocation in human T cells and monocytes (original magnification, x630; blue, DAPI-nuclear staining; red, Cy3-GCR{alpha}). Representative images of GCR{alpha} cellular shuttling in human T cells (A) and monocytes (B) in response to 10–6 M DEX treatment in vitro are shown. Delayed GCR{alpha} nuclear translocation in human monocytes as compared with T cells in response to steroids was detected by immunofluorescent microscopy. The pictures are representative of three independent experiments in which 50–100 cells were analyzed for each donor studied. (C) Cy3-GCR{alpha} nuclear/cytoplasm ratio in the T cells or monocytes from three different donors. The MFI of Cy3 staining (GCR{alpha}) in cells was assessed by the analysis software within the computer-generated masks corresponding to cell nuclei (the DAPI region) and cytoplasm of T cells and monocytes before and after DEX treatment (*, P<0.01). (D) Western blot results of GCR{alpha} cellular shuttling in human monocytes versus T cells. Purified T cells and monocytes were treated with DEX for the indicated time. The specific GCR{alpha} antibody was used to quantify the GCR{alpha} in nuclear and cytoplasmic fractions. The purity of the subcellular fractions was verified using anti-ß-tubulin and anti-NF-1 antibodies for detection of cytoplasmic and nuclear-specific proteins, respectively. The data are representative of three independent experiments.

 
In contrast to T cells, 1 h of DEX treatment had no effect on monocyte GCR{alpha} nuclear translocation (Fig. 4B) . However, when monocytes were treated with DEX for 3 h, there was significant GCR{alpha} nuclear translocation (as measured by GCR{alpha} nuclear/cytoplasm ratio in the cells in Fig. 4C ). Thus, it took a significantly longer period of time for DEX to induce GCR{alpha} nuclear translocation in monocytes than in T cells.

To demonstrate more precisely the difference in GCR{alpha} cellular shuttling in monocytes versus T cells, we quantified GCR{alpha} in nuclear and cytoplasmic compartments of the cells by Western blotting using nuclear and cytoplasmic proteins fractionated from monocytes or T cells before and after DEX treatment. Western blotting was done with the same antibody against GCR{alpha}, which was used for immunostaining. The results from these experiments were consistent with microscopy data above (Fig. 4D) and revealed delayed GCR{alpha} nuclear translocation in monocytes as compared with T cells after DEX treatment.

The effect of GCRß RNA silencing on monocyte-steroid response
To provide more definitive evidence regarding the role of GCRß in GCR{alpha} transactivation, we silenced the GCRß gene in human monocytes and examined the effects of DEX on MKP-1 induction. Purified monocytes from normal donors were transfected by electroporation with GCRß siRNA or nonsilencing control siRNA or were treated with control culture medium. Real-time PCR (GCRß mRNA level was 0.009±0.002 fg/ml in the GCRß siRNA-transfected group, 0.052±0.006 fg/ml in the control siRNA group, 0.052±0.003 fg/ml in the medium control group) and microscopy results (Fig. 5A ) indicated that introduction of GCRß siRNA 24 h after transfection could specifically inhibit GCRß expression in the monocytes but had no effect on GCR{alpha} mRNA expression (GCR{alpha} mRNA level was 0.057±0.005 pg/ml in the GCRß siRNA-transfected group, 0062±0.007 pg/ml in the control siRNA group, 0.059±0.004 pg/ml in the medium control group). Therefore we selected this time-point to compare MKP-1 induction followed by incubation with DEX for 4 h. Figure 5B showed that silencing of GCRß resulted in a significant increase of DEX-induced MKP-1 production (MKP-1 mRNA fold induction was 5.010±1.963, n=3, P<0.05) compared with the nonsilencing siRNA or medium control, with values of 1.743 ± 1.021, 1.522 ± 0.430, respectively.


Figure 5
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  		Figure 5. Inhibition of GCRß gene expression using specific siRNA in human monocytes enhances GCR{alpha} transactivation. (A) Introduction of GCRß siRNA into monocytes resulted in specific inhibition of GCRß expression as shown by immunocytochemistry. The pictures are representatives of three independent experiments (original magnification, x630). (B) Silencing of GCRß resulted in a significant increase of DEX-induced MKP-1 production by human monocytes [n=3; P<0.05, as compared with untransfected cells (control) or nonspecific siRNA control group]. (C) Silencing of GCRß has no effect on IFN induction by real-time PCR. (D) Silencing of GCRß in monocytes resulted in a significant DEX inhibition of TNF-{alpha} and IL-6 production. *, P< 0.05, as compared with nonspecific siRNA control group within the same concentration of DEX.

 
As some reports indicate that exogenously delivered siRNA can activate the IFN pathway, and this may lead to cell death [23 , 24 ], we measured the mRNA level of IFN in our siRNA delivery system. Real-time PCR results showed that there were no difference in IFN-{alpha}1, IFN-{alpha}2, and IFN-ß mRNA among the GCRß siRNA, nonsilencing control siRNA, and control culture medium groups (Fig. 5C) . The cell viability assay indicated that greater than 65% cells were alive after siRNA transfection. Our results are consistent with other studies, which also showed no IFN activation after introduction of siRNA into human cells [25 , 26 ].

We also examined the effect of GCRß silencing of human monocytes on their reactivity to DEX by assaying TNF-{alpha} and IL-6 secretion. Figure 5D showed that the silencing of GCRß resulted in a significant DEX inhibition of PMA/ionomycin-induced TNF-{alpha} production. Percent inhibition of TNF-{alpha} production by DEX in the GCRß siRNA group was 44.6 ± 9.2% (10–9 M DEX), 57.8 ± 8.9% (10–8 M DEX), and 60.3 ± 9.6% (10–7 M DEX) as compared with 11.5 ± 8.5% (10–9 M DEX), 20.4 ± 7.0% (10–8 M DEX), and 35.6 ± 9.5% (10–7 M DEX) in the control siRNA group. A similar effect was also observed for IL-6 production.

These data support the concept that the relatively higher GCRß expression in the monocytes plays a functional role in reducing steroid responsiveness, despite the fact that GCR{alpha} is in excess of GCRß in this cell type.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current study assessed the steroid responsiveness of human peripheral blood monocytes as compared with T cells. We demonstrate here for the first time that monocytes and T cells differ in their distribution and expression of GCRß. Furthermore, increased GCRß in monocytes appears to functionally reduce the response of this cell type to steroids, as RNA silencing of GCRß increases steroid responses of monocytes.

Sensitivity to GC varies considerably among individuals and even within the same individual responsiveness to GC, differs among tissues [27 ] and cells [28 , 29 ]. Previous studies have suggested that macrophages are less sensitive to GC than other immune cells [30 31 32 33 34 35 ]. However, little is known about the molecular mechanism that determines steroid responsiveness in macrophages. Allergic rhinitis is associated with the local accumulation of various immune cells after contact of the nasal mucosa with allergen, and the efficacy of GC treatment depends on its ability to reduce the accumulation and activation of inflammatory cells. Several independent studies have reported that topical GC can significantly decrease the numbers of nasal inflammatory cells such as T cells and eosinophils; however, these same studies did not observe a change in the number of macrophages after steroid treatment [30 , 31 ]. Cigarette smoking can induce a striking increase in macrophage number in the respiratory tract; paralleling this, the anti-inflammatory response to GC is reduced in bronchoalveolar lavages from smokers compared with nonsmokers [32 ]. In addition, reduced GC efficacy in chronic obstructive pulmonary disease could be a result of the relative steroid insensitivity of macrophages in the respiratory tract [33 , 34 ]. We reported earlier that the airway macrophage might drive the reduction in steroid responsiveness at night in nocturnal asthma [35 ].

Our present studies confirm that peripheral blood monocytes are less sensitive to DEX as compared with T cells. Moreover, we extended this observation by investigating the possible molecular mechanism of the relative steroid insensitivity of monocytes/macrophages. In the present study, we demonstrate for the first time that peripheral blood monocytes have a higher level of GCRß mRNA expression than T cells by real-time PCR. Using intracellular staining for GCRß combined with cell-surface staining, we confirmed that there was a relatively higher level of GCRß protein in monocytes than T cells. Functional assays, including GCR{alpha} nuclear translocation, DEX-induced MKP-1 gene expression, and functional studies of cytokine inhibition after treatment with DEX, demonstrated that the increased expression of GCRß was associated with decreased GC responses in monocytes as compared with T cells.

However, the physiologic role of GCRß has been controversial. In contrast to GCR{alpha}, GCRß interacts weakly with heat-shock proteins, does not bind GC, and is transcriptionally inactive [6 , 7 ]. Various studies have shown that GCRß exerts a dominant-negative effect on GCR{alpha} transactivation of target genes, which may involve competition between GCR{alpha} and GCRß for binding to GRE, competition for coactivators in the nucleus, as well as formation of GCR{alpha}-GCRß heterodimers, which are transcriptional-inactive [8 , 36 , 37 ]. Increased expression of GCRß has also been found in several diseases, which are associated with GC insensitivity, suggesting that an increased ratio of GCRß to GCR{alpha} may induce GC insensitivity [11 12 13 ]. Conversely, the level of GCRß has been reported to be lower than GCR{alpha} in mixed populations of PBMC [38 39 40 41 ]. However, such populations of cells are predominantly T cells, which in the current study, were found to have little GCRß as compared with GCR{alpha}. There has also been controversy about GCRß subcellular distribution. Oakley et al. [10 , 42 ] found that GCRß was localized predominantly to the cell nucleus of long-term cell lines. Other reports detected GCRß in the nucleus and cytoplasm of other cell lines [43 , 44 ]. However, although there are studies about GCRß expression in primary cells, there were no reports about its subcellular location in mononuclear cells [11 12 13 14 ]. Our study showed for the first time that GCRß is located in the cytoplasm and nucleus of human monocytes, whereas GCRß is located exclusively in the nucleus of T cells. GCR{alpha} nuclear translocation studies showed that DEX can induce GCR{alpha} nuclear translocation in T cells and monocytes, but it takes a significantly longer period of time for nuclear translocation of GCRß in monocytes than in T cells using physiologic concentrations of steroids. This suggested that GCRß in the cytoplasm can influence GCR{alpha} nuclear translocation in response to steroids, most likely as a result of heterodimer formation between GCR{alpha} and GCRß.

Several studies have already demonstrated that these two GCR isoforms can interact when in the same cell compartment, but most studies in cell lines have reported the presence of GCRß in the nucleus, where it has been shown to interfere with GCR{alpha} transactivation [36 ]. Our observation that GCRß may interfere with GCR{alpha} nuclear translocation provides a new dimension by which GCRß may interfere with GCR{alpha} action, independent of its known inhibitory effects on transactivation.

To demonstrate that GCRß expression has an important physiologic role in monocytes, we used siRNA transfection technology to silence GCRß mRNA and examined the influence of GCRß elimination on GCR{alpha} function. Indeed, we found that specific GCRß siRNA, but not control siRNA, can significantly inhibit GCRß protein and mRNA expression in human monocytes after transfection. In GCRß-silenced cells, MKP-1 mRNA induction and cytokine inhibition by steroids were enhanced significantly. These data provide direct evidence supporting that the GCRß expression level is related directly to its inhibitory effect on GCR{alpha} activity, even in cells that have an excess of GCR{alpha} compared with GCRß.

Our current study demonstrates that differences in steroid responses by monocytes as compared with T cells are a result of divergent GCRß expression. We report that GCRß is distributed evenly between the cell cytoplasm and nucleus in human monocytes but not T cells. These data suggest that strategies aimed at GCRß modulation may play a role in restoring steroid responsiveness of chronic inflammatory diseases associated with chronic monocyte/macrophage activation.


   ACKNOWLEDGEMENTS
 
NIH Grants AR41256 and 5R21AR051634, NIH/NIAID Contracts N01 AI40029 and N01 AI40030, General Clinical Research Center Grant MO1 RR00051 from the Division of Research Resources, the Edelstein Family Chair in Pediatric Allergy and Immunology, and the University of Colorado Cancer Center supported, in part, the work of D. Y. M. L. The authors thank Maureen Sandoval for her help in preparing this manuscript.

Received August 19, 2005; revised September 13, 2005; accepted December 14, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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