|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
IMMUNOBIOLOGY
From the Department of Clinical and Experimental
Medicine, Section of Pharmacology, University of Perugia, Italy.
Previously a novel gene was identified that encodes a
glucocorticoid-induced leucine zipper (GILZ) whose expression is
up-regulated by dexamethasone. This study analyzed the role of GILZ in
the control of T-cell activation and its possible interaction with nuclear factor Glucocorticoid hormones (GCHs) are commonly used as
therapeutic agents for many acute and chronic inflammatory and
autoimmune diseases, in transplant patients, and in the treatment of
leukemias and lymphomas.1-5 Their efficacy is in part due
to the effect of GCHs on T-cell activity. T-cell activation,
differentiation, and death are regulated by a number of stimuli that
can contribute either to positive or negative
selection.6-10 After antigen-T-cell receptor (TCR)
interaction, clonal expansion and differentiation contribute to
generate activated effectors and an immune response, followed by
elimination of most of the activated cells to end the immune response
and thus avoid a continuous clonal expansion and the generation of
autoimmunity.11,12 TCR-driven activation involves the
coordinated expression of a number of molecules involved in activation
and clonal expansion, such as interleukin-2/interleukin-2 receptor
(IL-2/IL-2R), as well as those involved in activation-induced cell
death (AICD), such as Fas/Fas ligand (Fas/FasL).12-18
Moreover, a number of genes coding for transcription factors are
involved as regulators of T-cell activation and death. In particular,
the role of nuclear factor Stimuli other than antigen-TCR interaction also regulate T-cell
activation and death, including cytokines and coaccessory molecules.25-27 Among different stimuli, GCHs are critical
regulators of T-cell development.28-32 In particular, GCHs
inhibit T-lymphocyte activation and death and also counter
activation-induced IL-2/IL-2R and Fas/FasL
expression.31,33 The inhibitory effect on T-cell activation and death relates in part to the glucocorticoid-induced inhibition of NF- Recently, DEX-induced genes able to inhibit the TCR-activated
death have been described.41,42 In particular, we have
previously identified a new gene, glucocorticoid-induced leucine zipper
(GILZ), encoding a novel member of the leucine zipper
family.42 GILZ is found to be expressed in normal T
lymphocytes present in the thymus, spleen, and lymph nodes, while low
or no expression was detected in other nonlymphoid tissues. Moreover,
GILZ protein is detected in the nucleus of transfected
clones.42 In thymocytes and peripheral T cells, GILZ gene
expression is induced by DEX, a synthetic GCH with high affinity for
the GR. Furthermore, GILZ expression protects T cells from
TCR-activated apoptosis but not by treatment with other apoptotic
stimuli. This antiapoptotic effect correlates with inhibition of
activation-induced Fas/FasL up-regulation. Thus, GILZ is a candidate
transcription factor involved in the regulation of T-cell activation.
In the present paper we have analyzed the role of GILZ in the control
of IL-2/IL-2R expression induced by T-cell activation and the possible
interaction with NF- Cell lines and animals
Human kidney epithelial carcinoma cell line 293 and embryonal carcinoma
cell line NTera-2 were cultured in Dulbecco modified Eagle medium
supplemented with 10% fetal calf serum and antibiotics.
Transfection of cultured cells and clone preparation
Luciferase assay Eukaryotic expression plasmids pMT2T-p65 and pMT2T-p52 were obtained from Dr Dalla Favera (Columbia University, NY). The pMT2T-GILZ was constructed by inserting the human GILZ cDNA coding sequences (405 base pairs) into pMT2T expression vector at the EcoRI site. (Human GILZ GenBank accession number AF228 339.) Reporter plasmids containing tandem repeats of the murine IgK/HIV- B (pBIIXLUC) site
linked upstream to a minimal murine c-fos promoter and
luciferase coding sequences were obtained from Dr Dalla Favera. The
pEGFP-N1 N-terminal protein fusion vector was purchased from Clontech
(Palo Alto, CA). Calcium phosphate-mediated transient DNA transfection and luciferase assays were performed as described.45
NTera-2 or 293 cells were plated at 2 × 106/100-mm Petri
dish 24 hours prior to an 8-hour transfection period. At 48 hours after
transfection, cells were harvested and transcription activation was
assayed as luciferase activity. The amounts of effector plasmids
reported in the figures were cotransfected with 15 µg reporter
plasmid (pBIIXLUC) and 3 µg pEGFP-N1. The pEGFP-N1 plasmid was used
to normalize the transfection efficiency of each sample.
Similar assays were performed using a luciferase reporter gene (pGL3-IL-2) containing an IL-2 promoter region (1.9 kilobases of 5'-flanking sequence of IL-2 gene) obtained from Dr L. D'Adamio (Albert Einstein College of Medicine, NY).46 Control vector pRc/RSV-luc containing the coding region for luciferase gene was obtained from Dr M. Cippitelli (University "La Sapienza," Rome). Each transfection was performed in triplicate. Cell lysis and luciferase quantification were performed using commercial reagents (Luciferase Reporter Gene Assay, Roche Diagnostics, Monza, Italy). The values are expressed as fold increases above the level of luciferase activity of cells transfected with the reporter plasmids. The values marked EC (endogenous control) represent the values obtained by transfection of the reporter plasmids along with the control pMT2T vector. Antibody cross-linking and cell treatment Hamster antimouse CD3 (clone 145-2C11; Pharmingen, San Diego,
CA) monoclonal antibody (mAb) at 1 µg/mL was allowed to adhere in
flat-bottomed, high-binding 96-well plates (Costar, Cambridge, MA) at
4°C in 100 µL phosphate-buffered saline (PBS). After 20 hours,
plates coated with mAb were washed and transfected clones were plated
at 1 × 105 cells per well and incubated at 37°C for
different times as indicated. Isotype-matched rat antimouse
immunoglobulin G2b (IgG2b) mAb (clone R 35-38, Pharmingen) was used as
control.47,48
IL-2 assay Supernatants from clones untreated or treated with anti-CD3 mAb for 18 hours were tested for IL-2 concentration by 2-site enzyme-linked immunosorbent assay (ELISA) using mAb JES6-1A12 as the primary reagent and biotinylated monoclonal S4B6 as the secondary reagent. Both antibodies were purchased from Pharmingen. The IL-2 titer (means ± SD of replicate samples) was expressed as pg/mL, calculated by reference to standard curves constructed with known amounts of IL-2. The sensitivity limit was approximately 20 pg/mL.Flow cytometry evaluation of IL-2R expression A single suspension (1 × 106cells/sample) was incubated for 30 minutes on ice in 50 µL staining buffer (PBS plus 5% fetal calf serum) containing 10 µg/mL hamster antimouse IL-2R mAb directly conjugated to R-phycoerytrin or phycoerytrin-hamster IgG (isotype control). Both mAbs were purchased from Pharmingen. The percentage values of IL-2R histograms were calculated using lysis II research software (Becton Dickinson, Mountain View, CA).Apoptosis evaluation by propidium iodide solution Apoptosis was measured by flow cytometry as described elsewhere.47 Briefly, cells were centrifuged and the pellets resuspended in 1.5 mL hypotonic propidium iodide (PI) solution. The tubes were kept at 4°C in the dark overnight. The PI fluorescence of individual nuclei was measured by flow cytometry with standard FACScan equipment (Becton Dickinson).Nuclear extracts and electrophoresis mobility shift assay Cells (2 × 107/group) were washed with ice-cold PBS, and packed cells were resuspended in 1 mL hypotonic buffer (25 mM HEPES, 50 mM KCl, 0.5% Nonidet P-40 [NP-40], 0.1 mM dithiothreitol, 10 mg/mL leupeptin, 20 mg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride [PMSF] solution in ethanol). Ten minutes after incubation on ice, the supernatants, containing cytoplasmic proteins, were separated from the nuclear pellets by centrifugation. Nuclear pellets were then washed with hypotonic buffer without NP-40 and resuspended in 10 mL lysis buffer (25 mM HEPES, 2 mM KCl, 0.1 mM dithiothreitol, 10 mg/mL leupeptin, 20 mg/mL aprotinin, and 1 mM PMSF). Fifteen minutes after incubation on ice, lysates were diluted with 10 vol dilution buffer (25 mM HEPES, 0.1 mM dithiothreitol, 10 mg/mL leupeptin, 20 mg/mL aprotinin, and 1 mM PMSF and 20% glycerol) and cleared in a precooled microfuge for 30 minutes at 14 000g. All DNA binding reactions were conducted for 20 minutes at room temperature in a final volume of 20 µL. The reactions were started by adding 10 µg nuclear protein extract to a reaction mix containing 1 µg poly [d(I-C)], 4 µL 5 × binding buffer (50 mM Tris, 250 mM NaCl, 5 mM ethyleneglycotetraacetic acid [EDTA], 25% glycerol, and 5 mM dithiothreitol), and approximately 20 000 cpm of the respective ( 32P)ATP-labeled double-stranded DNA (dsDNA)
oligonucleotide. Cold competitor oligonucleotides were added to the
reaction mix before the radiolabeled probe. The sample was then loaded
on 5% native polyacrylamide gel in Tris-borate-EDTA buffer. After
electrophoresis for 2.5 hours at room temperature and 10 V/cm, gels
were dried, and separated protein-DNA complexes were visualized by
autoradiography using Kodak XAR5 films. The following dsDNA
oligonucleotides (Promega, Milan, Italy) were used in electrophoresis
mobility shift assay (EMSA) analysis both as labeled or competitor cold
probe: 5'-AGAGGGGACTTTCCGAGAGGC-3' for NF-B,
5'-TATGTGTAATATGTAAAA-3' for OCT-1, and 5'-AAGAGGAAAATTTGTTTCATACAG-3' for NF-activated T cell (NF-AT). For antibody-induced supershift assays 2 µL (0.2 µg/mL) antibody, anti-p65 or anti-p52 (Santa Cruz
Biotechnology, Santa Cruz, CA), was incubated with 10 µg nuclear
extract for 30 minutes at room temperature after the addition of
radiolabeled probe.
Production of recombinant proteins Human GILZ was in-frame cloned into the pGEX-4T2 plasmid (Pharmacia, Uppsala, Sweden). The pGEX-4T2 plasmid is a glutathione-S-transferase (GST) fusion vector carrying a tac promoter for chemically (isopropyl- -D-thiogalactopyranoside [IPTG])
inducible high-level expression of the protein. GST fusion protein was
expressed in Escherichia coli, grown at 30° C, and induced
with 0.1 mM IPTG for 90 to 180 minutes. Following lysis by sonication,
most of the induced protein was found in the soluble material, which
was purified with Glutathione Sepharose 4B beads (Pharmacia)
following the manufacturer's instructions.
In vitro interaction studies Human GILZ, p65, p52, c-fos, and Fra-1 proteins were in vitro-translated with [35S]methionine by using the rabbit reticulocyte-coupled in vitro transcription translation system (Promega, Madison, WI) under the T7 promoter according to the manufacturer's instruction. In vitro-translated proteins were diluted with the binding buffer (final concentration: 25 mM HEPES, pH 7.5; 10% glycerol; 50 mM NaCl; 0.05% NP-40; 1 mM dithiothreitol) and precleared with glutathione beads for 45 minutes at 4°C. The labeled products were assayed for interaction with GST-GILZ fusion protein or GST protein, prepared by glutathione-coated bead purification from appropriate bacterial sonicates as previously reported.49,50 GST or GST protein bound to glutathione beads were incubated with in vitro-translated proteins for 20 minutes at 20°C. The beads were subsequently washed 5 times with 0.5 mL PBS, and bound proteins were recovered by boiling in SDS sample buffer and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For I-B competition experiments, GST was removed from
GST-GILZ fusion protein following the manufacturer's instructions
(Pharmacia) and GILZ (1 µg) was incubated overnight with in
vitro-translated p65 (5 µL) diluted in the binding buffer described
above, in the absence or in the presence of GST-I-B fusion
protein (100 µg, Santa Cruz). Immunoprecipitation was performed with
antibody anti-p65 (3 µL, Biomol Research Laboratories, Plymouth Meeting, PA), and Western blotting was performed with antibody anti-p65
(1:1000) or anti-GILZ (1:1000) or anti-I-B (1:1000) (Cell
Signaling Technology, Beverly, MA).
Immunoprecipitation and coimmunoprecipitation NTera-2 cells were transiently transfected as reported for the luciferase assay. Transfected cells were incubated for 30 minutes in medium lacking methionine and then metabolically labeled for 4 hours with [35S]methionine and immediately harvested. Whole-cell extracts were prepared and immunoprecipitations performed in radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1% NP-40; 0.5% deoxycholate; 0.1% SDS; and 5 mM EDTA) supplemented with 1 mM PMSF. Antigen-antibody complexes were precipitated with protein A bound to Sepharose beads (Pharmacia) prior to SDS-PAGE. A rabbit polyclonal antiserum recognizing GILZ was prepared with the use of a fusion protein containing the full GILZ amino acid sequence as previously reported.42Protein lysates (500 µg in radioimmunoprecipitation assay buffer) from untreated or DEX-treated thymocytes were immunoprecipiteted with 3 µL anti-p65 subunit rabbit polyclonal antibody. For coimmunoprecipitation experiments, Western blot was performed using both anti-p65 and anti-GILZ antibodies. Immunofluorescence staining for nuclear translocation To evaluate the nuclear translocation of NF-B or NF-AT,
clones untreated or treated with anti-CD3 mAb for 2 hours were
processed for immunofluorescence by the paraformaldehyde-saponin
procedure.51 After extensive washing in PBS with 1%
HEPES, cells were fixed in 4% formaldehyde for 20 minutes on ice,
washed again, and incubated at 4°C for 1 hour with blocking buffer
(PBS with 3% bovine serum albumin and 1% glycine). For staining,
cells were incubated for 45 minutes at 4°C with 100 ng polyclonal
rabbit anti-p65 antibodies (Santa Cruz) or 100 ng polyclonal rabbit
anti-NF-AT1 (Upstate Biotechnology, Lake Placid, NY) in buffer
containing 0.1% saponin, washed, and incubated for 45 minutes at 4°C
with Texas Red-conjugated goat antirabbit IgG in PBS-saponin. Cells
were then washed, stuck on slides coated with poly-L-lysine, and
mounted in buffered glycerol for fluorescence microscopic analysis.
Photographs were taken on a Leitz Dialux 20 microscope.
Northern blot assay Total RNA was extracted by the guanidium thiocyanate method and separated in 1.2% agarose gels containing 2.2 M formaldehyde and transferred to nitrocellulose filters (Scheicher and Schuell, Dassel, Germany). Nitrocellulose filters with 25 µg RNA were hybridized overnight with mouse GILZ cDNA 32P-labeled using the nick translation kit (Roche Diagnostics). Filters were washed 3 times in 1 × SSC with 0.05% SDS at room temperature, followed by 2 washes at 50°C in 0.1 × SSC with 0.1% SDS;-actin was used as control.
Western blot analysis Extracted proteins were separated on an SDS-polyacrylamide gel and studied by Western blotting as previously described.48 Primary antibody was a rabbit polyclonal antiserum recognizing GILZ,42 and the secondary antibody was a horseradish peroxidase-labeled goat antirabbit IgG (Pierce, Rockford, IL). Anti--tubulin mAb (Calbiochem, San Diego, CA) was used as control.
The antigen-antibody complexes were revealed by enhanced
chemiluminescence following the manufacturer's instructions
(SuperSignal, Pierce). The PhotoPlus I-B (Ser32)
antibody kit for analysis of I-B phosphorylation was
purchased from Cell Signalling Technology.
Statistical analysis Each experiment was performed at least 3 times. Representative experiments are shown. Due to the nonnormal distribution of the data, nonparametric tests (Kruskall-Wallis' analysis of variance) were adopted for statistical evaluation.
GILZ inhibits apoptosis and IL-2 and IL-2R expression We performed experiments to evaluate whether GILZ could counter the TCR-induced increase of IL-2 and IL-2R. For that purpose we used empty vector- and GILZ-transfected clones nonstimulated or stimulated by treatment with anti-CD3 mAb. After stimulation with cross-linked anti-CD3 mAb, GILZ- and empty vector-transfected clones were stained with anti-IL-2R mAb or labeled for PI staining for flow cytometry apoptosis evaluation. Furthermore, the supernatants were used for IL-2 detection in an ELISA assay. Results show that, upon activation by anti-CD3 mAb, empty vector-transfected clones express high levels of IL-2R and produce IL-2 (Figure 1A-B). In contrast, anti-CD3-activated GILZ-transfected clones express low or undetectable IL-2 and IL-2R (Figure 1A-B). Moreover, GILZ-transfected clones are resistant to anti-CD3-activated apoptosis (Figure 1C). These results confirm previous data showing that GILZ inhibits TCR-induced apoptosis42 and further indicate that GILZ inhibits IL-2 and IL-2R expression.
GILZ inhibits NF- B-driven transcription, we tested the ability of GILZ
overexpression to modulate the in vivo transcription using a reporter
gene linked to a B sequence in transient transfection assay. In
particular, eukaryotic expression plasmids pMT2T-p65, pMT2T-p52, and
pMT2T-GILZ were cotransfected in 293 cells with a target plasmid in
which an IgK/HIV-B (pBIIXLUC) sequence was linked upstream of a
minimal c-fos promoter and luciferase coding sequence.
Luciferase activity was measured as a function of B-dependent
transcription at 48 hours after transfection as previously
described.45 The values, reported in Figure
2, are expressed as fold increases of
luciferase activity. The value marked EC (endogenous control)
represents the value obtained by transfection of the target plasmid
along with the control pMT2T empty vector and documents the level of endogenous NF-B activity in 293 cells (Figure 2A-C,G). In
particular, Figure 2A indicates that 293 cells have an intrinsic
NF-B activity and that this transcriptional activity is inhibited by
GILZ expression (P < .01). Moreover, Figure 2B indicates
that p65 and p52 overexpression greatly enhances luciferase activity in
transfected cells and shows that GILZ has the capability to negatively
regulate NF-B-driven transcription (P < .01, column 3 versus column 2) but does not have an intrinsic capability of
transactivation when transfected alone (Figure 2B, column 4). This
negative effect is dose dependent and augments progressively with GILZ
transfectant concentrations ranging from 0.01 pmol to 1 pmol (Figure
2G). Furthermore, GILZ has the capability to negatively regulate
B-driven transcription mediated by p65 (Figure 2C).
Because undifferentiated NTera-2 cells have no detectable endogenous
NF- We also performed experiments to analyze whether GILZ could modulate
the NF-
GILZ prevents DNA binding of NF- B has been shown to be involved in T-cell activation,
induction of IL-2 and IL-2R, Fas and FasL gene expression, and
apoptosis.19,22,52,53 Because GILZ inhibits IL-2 and IL-2R
expression and NF-B transcriptional activity, we performed
experiments to analyze whether GILZ could also interact with the
NF-B-DNA binding.
EMSA performed using a specific sequence containing an NF-
GILZ prevents NF- B nuclear translocation. For
that reason empty vector-transfected and GILZ-transfected cells,
untreated or treated with anti-CD3 mAb, were stained and processed for
immunofluorescence analysis using anti-NF-B antibodies. Figure
5 indicates that anti-CD3 treatment
induces NF-B translocation in empty vector-transfected cells
(Figure 5Aii) as compared with untreated control (Figure 5Ai) and that
GILZ overexpression inhibits the anti-CD3-induced translocation
(Figure 5Aiv versus 5Aiii). In contrast, NF-AT nuclear
translocation occurs in both empty vector-transfected (Figure
5Av-Avi) and GILZ-transfected (Figure 5Avii-Aviii) clones. As
control, the levels of apoptosis (Figure 5B) and GILZ expression
(Figure 5C) were also determined.
These results indicate that GILZ inhibits anti-CD3-induced NF- GILZ coimmunoprecipitates with p65 and p52 To investigate the possible in vivo protein-protein interaction between GILZ and NF-B, we performed coimmunoprecipitation experiments in GILZ and p65/p52 cotransfected cells. For that purpose,
NTera-2 cells, which lack the NF-B complex including I-B
molecules, were cotransfected with expression vectors encoding GILZ,
p65, or p52 and labeled with [35S]methionine. As shown in
Figure 6, untransfected NTera-2 cells contain no detectable GILZ protein (lane 1), whereas cells transfected with GILZ clearly show the band of GILZ protein (lane 2). As control, the immunoprecipitation is blocked by addition of the fusion protein previously used to generate the GILZ antibody (lane 3).
Furthermore, anti-GILZ antibodies coimmunoprecipitate GILZ and p65
(lane 5) or GILZ and p52 (lane 9), and this again is blocked by
inclusion of the GILZ peptide (lanes 6 and 10). The identity of the
coimmunoprecipitated p65 and p52 in lanes 5 and 9 is confirmed by its
comigration with the p65 and p52 proteins immunoprecipitated with
anti-p65 and anti-p52 antibodies, respectively (lanes 7 and 11), and by
immunoprecipitation from unlabeled extracts followed by Western
blotting with p65 and p52 antibodies (data not shown). Finally,
immunoprecipitation with anti-GILZ antibodies of extracts from cells
cotransfected with GILZ, p65, and p52 results in the detection of GILZ,
p65, and p52 (lane 12).
GILZ binds p65 and p52 in vitro To further investigate if GILZ can physically associate with p65 and p52, partially purified GST-GILZ fusion protein was mixed with radiolabeled p65 or p52 and precipitated with glutathione-coated beads. Figure 7 shows that GST-GILZ fusion protein binds p65 (lane 2) and p52 (lane 5), whereas the GST protein alone (lanes 3 and 6) does not. As control, the in vitro-transcribed p65 (lane 1) and p52 (lane 4) were loaded. Furthermore, to analyze the binding specificity, we also performed similar experiments using other members of the leucine zipper family. In vitro-translated c-fos and Fra-1 proteins were incubated with GST-GILZ. Results indicate that neither Fra-1 (lane 8) nor c-fos (lane 11) proteins bind GILZ. As control, in vitro-translated Fra-1 (lane 7) and c-fos (lane 10) were loaded. These results indicate that there is a direct GILZ-NF-B
interaction.
GILZ, up-regulated by DEX treatment of thymocytes,
coimmunoprecipitates with NF- B transactivation, we performed EMSA analysis using nuclei from DEX-treated normal thymocytes. Figure
8 shows that an increased GILZ
expression, induced by DEX treatment (Figure 8B), correlates with the
inhibition of anti-CD3-driven NF-B activation (Figure 8A). In the
attempt to analyze if this effect could be due to a direct
GILZ-NF-B interaction we also performed, in the same experiment,
coimmunoprecipitation assay. Figure 8 indicates that anti-p65 antibody
immunoprecipitated p65 from untreated and DEX-treated thymocytes
(Figure 8C, lanes 1 and 2) and coimmunoprecipitated GILZ from
DEX-treated but not from untreated thymocytes (Figure 8D, lane 2 versus
lane 1). These data suggest a good correlation between DEX-induced GILZ
overexpression, NF-B inhibition, and GILZ-NF-B interaction.
GILZ does not interfere with I- B expression. Results indicate that there were
no differences in p65 or p52 expression between anti-CD3-treated empty
vector- and GILZ-transfected clones either at the protein level, as
evaluated by Western blotting (Figure
9Ai-Aii), or at the transcription level,
as evaluated by Northern blotting (data not shown).
Furthermore, because phosphorylation of I- TCR triggering inhibits GILZ expression We have previously shown that GILZ is normally expressed in freshly isolated thymocytes, spleen, and lymph node cells and that its expression is increased by treatment with DEX.42 Moreover, GILZ overexpression counters the TCR/CD3-activated Fas/FasL up-regulation and AICD,42 suggesting that GILZ could contribute to the DEX-induced inhibition of T-cell activation, Fas/FasL up-regulation, and death.In an attempt to determine the possible physiologic role of GILZ,
we performed experiments to analyze the effect of TCR/CD3 triggering on
GILZ expression, comparing resting and anti-CD3-treated freshly
isolated thymocytes. As shown in Figure
10, results obtained with normal thymus cells confirm previous data
showing that GILZ is expressed in resting cells and is up-regulated by
DEX (Figure 10A and 10B, lane 3). Moreover, the figure also indicates
that stimulation of the TCR/CD3 complex, through anti-CD3 mAb, inhibits
GILZ expression both at the mRNA and protein level (Figure 10A and 10B,
lane 2 versus lane 1) and that this inhibition is countered by DEX
treatment (Figure 10A and 10B, lane 4). Moreover, the anti-CD3-induced
GILZ inhibition is a consequence of T-cell activation in that it
is weakened by cyclosporin A (Figure 10C, lane 4 versus lane 2), a drug
that is able to inhibit the TCR-activated transduction
pathway.54 These results indicate that GILZ expression
inversely correlates with T-cell activation, being higher in
resting cells and lower in cells activated by TCR/CD3 triggering.
In the present paper we describe a new mechanism by which GCHs
inhibit T-cell activation. In particular, we show that the glucocorticoid-induced leucine zipper factor GILZ inhibits NF- GCHs induce T-cell apoptosis in different experimental systems, including hybridoma T cells, thymocytes, and peripheral lymphocytes.28,29,55 However, a protective effect of GCHs has also been described in T cells, and in particular it has been shown that DEX is able to counter TCR-activated death.33,56,57 This dual effect, apoptosis activation and inhibition, is not surprising because it has been reported that GCHs may activate a caspase cascade, a central mechanism involved in T-cell apoptosis, as well as weaken TCR-induced activation of the NF- |
B
Top
Abstract
Introduction
) and anti-CD3-treated clones
(
) for 18 hours on plates coated with anti-CD3 (1 µg/mL), empty
vector-transfected (PV5, PV6, PVT1, PVT2) clones, and GILZ-transfected
(SG11, ST7, SG5, ST5, GIRL19) clones as evaluated by fluorescent
anti-IL-2 mAb and flow cytometry analysis (see "Material and
methods"). (B) IL-2 production (pg/mL) in untreated (
