Glycodelin-A interferes with IL-2/IL-2R signalling to induce cell ...

Human Reproduction, Vol.0, No.0 pp. 1–11, 2012
doi:10.1093/humrep/der477
ORIGINAL ARTICLE Early pregnancy
Glycodelin-A interferes with IL-2/IL-2R
signalling to induce cell growth arrest,
loss of effector functions and apoptosis
in T-lymphocytes
Chetna Soni and Anjali A. Karande*
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
*Correspondence address. Tel: +91-80-2293-2306; Fax: +91-80-2360-0814; E-mail: anjali@biochem.iisc.ernet.in
Submitted on September 5, 2011; resubmitted on November 14, 2011; accepted on December 19, 2011
background: The progesterone-regulated glycoprotein glycodelin-A (GdA), secreted by the decidualized endometrium at high concentrations
in primates, inhibits the maternal immune response against fetal antigens and thereby contributes to the tolerance of the semiallogenic
fetus during a normal pregnancy. Our earlier studies demonstrated the ability of GdA to induce an intrinsic apoptotic cascade in
CD4 + T-lymphocytes and suppress the cytolytic effector function of CD8 + T-lymphocytes. In this report, we investigated further into the
mechanism of action of GdA controlling perforin and granzyme B expression in CD8 + T-lymphocytes and the mechanism of action of GdA
leading to lymphocyte death.
methods: Flow cytometry analysis was performed to check for the surface expression of interleukin-2 receptor a (IL-2Ra) and intracellular
eomesodermin (Eomes) in activated T-lymphocytes, whereas quantitative RT–PCR analysis was used to find out their mRNA profile
upon GdA treatment. Western analysis was carried out to confirm the protein level of Bax and Bcl-2.
results: GdA reduces the surface expression of the high-affinity IL-2R complex by down-regulating the synthesis of IL-2Ra (CD25). This
disturbs the optimal IL-2 signalling and decreases the Eomes expression, which along with IL-2 directly regulates perforin and granzymes
expression. Consequently, the CD8 + T-lymphocytes undergo growth arrest and are unable to mature into competent cytotoxic
T-lymphocytes. In the CD4 + T-lymphocytes, growth factor IL-2 deprivation leads to proliferation inhibition, decreased Bcl-2/enhanced
Bax expression, culminating in mitochondrial stress and cell death.
conclusions: GdA spurs cell cycle arrest, loss of effector functions and apoptosis in different T-cell subsets by making T-lymphocytes
unable to respond to IL-2.
Key words: glycodelin-A / CD25 / eomesodermin / Bcl-2 / Bax
Introduction
Hum. Reprod. Advance Access published February 7, 2012
The ability of the maternal immune cells to tolerate the semi-allogenic
fetus is conferred by mechanisms which either suppress fetal allogenicity
or spatio-temporally inhibit maternal immune cells’ growth and
functions (Pearson, 2002; Veenstra van Nieuwenhoven et al., 2003;
Niederkorn, 2006; Trowsdale and Betz, 2006; Terness et al., 2007).
Glycodelin-A (GdA) or placental protein 14, a glycoprotein found specifically
in primates, is synthesized by the luminal and glandular epithelium
of the decidua and its concentration in the uterine compartment
reaches up to 2.5 mM between 14 and 16 weeks of pregnancy
(Seppala et al., 2002). It is one of the immunomodulatory factors
that augment fetal tolerance (Bolton et al., 1987; Tulppala et al.,
1995). GdA, a 162 amino acid secreted protein, is spatio-temporally
regulated by progesterone (Joshi, 1983; Taylor et al., 1998) and functions
as a galactose-specific lectin (SundarRaj et al., 2009) to bring
about its inhibitory effects on maternal immune cells which are activated
by the trophoblastic antigens at the fetal–maternal interface
during pregnancy (van Kampen et al., 2001).
The placenta, which is comprised of both fetal and maternal tissues,
is the site of rigorous immune cell activity. It contains a diverse population
of maternal immune cells; uterine natural killer (NK) cells, dendritic
cells, T-lymphocytes, monocytes and B cells (Starkey et al., 1988;
Williams et al., 2009). GdA exerts its inhibitory effect on both innate
and adaptive immune responses (Seppala et al., 2002). Subjects with
subnormal levels of GdA face unexplained infertility (Mackenna
et al., 1993), habitual abortions (Tulppala et al., 1995) and recurrent
miscarriages (Dalton et al., 1998). GdA inhibits the proliferation of
& The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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2 Soni and Karande
B cells, along with reduced immunoglobulin M (IgM) secretion and
major histocompatibility complex class II surface expression upon activation
(Yaniv et al., 2003; Alok and Karande, 2009). Experiments
carried out on peripheral NK cells and decidual large granular lymphocytes
confirm the ability of GdA to inhibit their cytolytic activity
(Okamoto et al., 1991) and to enhance interleukin-6 (IL-6), IL-13
and granulocyte-macrophage colony-stimulating factor production
from them, signifying its role in trophoblast invasion (Lee et al.,
2010). Survival and proliferation of monocytes are inhibited (Tee
et al., 2008; Alok et al., 2009) and dendritic cells derived from monocytes
acquire a tolerogenic phenotype on GdA treatment (Scholz
et al., 2008).
The most extensively studied effect of GdA is that on the
T-lymphocytes. GdA inhibits the proliferation of mitogen stimulated
T-cells (Bolton et al., 1987; Pockley and Bolton, 1989; Rachmilewitz
et al., 1999; Mukhopadhyay et al., 2001). Additionally, it induces apoptosis
in activated T-lymphocytes (Mukhopadhyay et al., 2001) and
the protein backbone is necessary and sufficient for this activity
(Jayachandran et al., 2004). Importantly, the N-linked glycosylation
on GdA modulates accessibility of the active site on the protein
(Mukhopadhyay et al., 2004; Jayachandran et al., 2006; Poornima
and Karande, 2007). GdA also shifts the Th1–Th2 cytokine balance
towards Th2 cytokines (Mishan-Eisenberg et al., 2004) by selectively
triggering apoptosis in Th1 cells (Lee et al., 2011). Interestingly, like
the Th2 cells, B cells (Alok and Karande, 2009) as well as CD8 + Tlymphocytes
are resistant to GdA-induced apoptosis (Soni and
Karande, 2010), though the proliferation (SundarRaj et al., 2009)
and cytolytic potential of CD8 + CTLs (cytotoxic T-lymphocytes) are
compromised upon GdA treatment (Soni and Karande, 2010).
Further exploring the regulatory effect of GdA on the effector functions
of CD8 + T-cells, which predominate the uterine T-lymphocyte
population during pregnancy (Williams et al., 2009), we reported
that GdA exerts its inhibitory effect on the cytolytic activity of CTLs
by down-modulating the synthesis and release of key cytolytic
factors perforin and granzyme B from them (Soni and Karande, 2010).
In this study, we have probed further into the mechanistic details of
the regulation of perforin and granzymes by GdA. In agreement with
the report by Pockley and Bolton (1989), we find that GdA induces
suboptimal IL-2 signalling, by down-regulating the surface expression
of an IL-2Ra subunit (CD25), which together with reduced eomesodermin
(Eomes) expression result into abated perforin and granzyme
B contributing to the debilitated cytolytic activity of CTLs. Interestingly,
our data suggest that the aberrant IL-2 signalling in the presence of
GdA translates into a dis-balance between the pro- and anti-apoptotic
proteins within T-cells. Thus, data reported in this study indicate that
growth factor depletion may be a potential mechanism not only for
the inhibition of proliferation but also for apoptosis and loss of effector
functions induced by GdA in T-lymphocytes at the feto-maternal
interface.
Materials and Methods
Cells and cell lines
Peripheral blood mononuclear cells (PBMCs) were isolated using ‘Ficoll’
density gradient centrifugation (Histopaque, Sigma-Aldrich, MO, USA),
from fresh whole blood of normal healthy donors (males and non-pregnant
females of the age group 25–30 years; Boyum, 1964). PBMCs were cultured
in Roswell Park Memorial Institute (RPMI)-1640 medium (Sigma-Aldrich)
supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA),
100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine,
unless mentioned otherwise. Ovarian carcinoma cell line OVCAR-3
(obtained from Prof. J. Hilgers, Vrije University, Amsterdam) was cultured
in Delbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented
with 10% FBS, 2 mM L-glutamine and antibiotics.
Antibodies and reagents
Anti-CD4 and CD8 monoclonal antibodies (mAbs) were purified from
culture supernatants of OKT4 and OKT8 hybridoma cells [National Facility
for Animal Tissue and Cell Culture (NFATCC), Pune, India] cultured in
Iscove’s modified Dulbecco’s medium (IMDM) with 10% FBS, using
protein-A affinity chromatography. Purified Abs were labelled with FITC
using the standard protocols (Bioconjugate Techniques by Greg
T. Hermanson, second edition). Anti-Eomes Alexa Fluor R 647, anti-human
CD25-PE, anti-human Bcl-2 and anti-human Bax antibodies were purchased
from eBiosciences (USA). Anti-human beta-actin horse-radish peroxidase
(HRP) conjugate, anti-human interferon (IFN)-g-PE conjugate, HISTOPA-
QUE, PHA (phytohaemagglutinin) and RNA isolation reagent (TRI reagent)
was purchased from Sigma-Aldrich. Rabbit anti-mouse HRP-conjugated antibody
was procured from Dako (Denmark). RevertAid reverse transcriptase,
Oligo-dT and dNTP mix was obtained from Thermo Fisher Scientific
(Canada). For real-time PCR analysis, iQ SYBR Green sensimix was procured
from Bio-Rad (CA, USA). CD4 + and CD8 + T-cells were isolated using
magnetic-activated cell sorting (MACS—Meltenyi Biotec, Germany), by the
MS-MACS columns using streptavidin–phycoerythrin microbeads.
Purification of GdA
GdA was purified from amniotic fluid by immunoaffinity chromatography,
using a Gd-specific mAb, D9D4 using the standard lab protocol (Mukhopadhyay
et al., 2001). Briefly, GdA was eluted with 100 mM gly–HCl, pH
2.5, and neutralized with 1 M Tris–HCl, pH 8.0, thereafter dialysed against
50 mM phosphate-buffered saline (PBS). The purity of the protein was
determined using sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE) followed by silver staining and specificity ascertained
by western blotting. The protein preparations were checked for
LPS (lipopolysaccharide) contamination using E-Toxate, LPS detection kit
(Sigma-Aldrich). Protein preparations free of LPS contamination were
used in all the assays. Also, the activity of GdA was ascertained by cell proliferation
and apoptosis assays (Soni and Karande, 2010). One of the most
abundant proteins of the amniotic fluid, namely human serum albumin
(HSA), was purified from amniotic fluid (Mukhopadhyay et al., 2001)
and has been used as the negative control throughout the study.
Alloactivation and T-cell stimulation
To activate PBMCs and to generate CTLs in vitro, a previously standardized
method of alloactivation was followed (Soni and Karande, 2010). Briefly,
PBMCs (responders) were alloactivated, using gOVCAR-3 (OVCAR-3
cells irradiated with 30 Gy of gamma rays using the BI-2000 gamma irradiator),
as stimulators. Stimulators were co-cultured with responders in a
ratio of 1:10 in IMDM supplemented with 10% FBS, 10% normal human
serum, 2 mM sodium pyruvate and 2 mM L-glutamine in 60 mm culture
grade Petri plates, at a cell density of 6 × 10 6 /ml for 96 h at 378C in
5% CO 2. About 5 U/ml of recombinant IL-2 (Sigma-Aldrich) was supplemented
at 0 and 48 h of the co-culture. Depending on the requirement of
the experiment, the alloactivation was either terminated after 18 h of
co-culture or continued for 96 h or both and has been mentioned wherever
necessary. Cells were treated with GdA for 24 h post-alloactivation.
Alternatively, purified PBMCs (2.5 × 10 6 cells/ml) were activated with
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Glycodelin-A dampens CD25 expression in T-cells 3
5 mg/ml of PHA for 18 h (or as mentioned) in RPMI supplemented with
10% FBS. PHA-stimulated cells were simultaneously treated with GdA
unless mentioned otherwise.
Cell surface/intracellular staining and flow
cytometry
For cell surface staining, activated cells (1 × 10 6 ) were stained with
anti-CD4-FITC/CD8-FITC and/CD25-PE direct conjugates (as indicated)
or anti-CD28 for 1 h on ice. Direct conjugates were washed twice with
fluorescence-activated cell sorting (FACS) wash buffer [0.2% bovine
serum albumin (BSA) (w/v) and 0.1% (w/v) sodium azide in 50 mM
PBS], fixed with 2% paraformaldehyde and stored at 48C until acquisition
on FACSCalibur/Canto II flow cytometer (Becton Dickenson, NJ, USA).
Acquired data were analysed using WinMDI 2.9 software/Cell Quest
Pro and plotted using Graph Pad Prism Ver 5.0. Anti-CD28 surfacelabelled
cells were washed with FACS wash buffer and then probed
with Rabbit-anti-mouse FITC conjugate (Dako), washed to remove
unbound secondary antibody and then fixed with 2% paraformaldehyde.
Data were acquired and analysed as aforementioned. In all cases, at
least 10 000 events were acquired per sample for analysis.
Intracellular staining: for staining secreted proteins like IFN-g, PBMCs
were cultured with 2 mM monensin (Sigma-Aldrich) for the last 12 h
during the 18-h alloactivation and also during 24 h of GdA treatment.
Thereafter, the same protocol was followed for secreted or intracellular
proteins. Cells (up to 1 × 10 6 ) were surface stained with desired antibodies
against various surface markers, fixed with 2% paraformaldehyde
(pHCHO), for 15 min at RT, washed to remove pHCHO followed by permeabilization
with 1 ml of 90% methanol in 50 mM PBS (pH 7.2) for
30 min on ice. Cells were then centrifuged at 1957 g for 15 min to
remove methanol and washed twice with FACS wash buffer, resuspended
in 100 ml of FACS wash buffer and incubated with either anti-IFN-g-PE or
anti-Eomes-Alexa Fluor for 1 h on ice. Washed to remove the excess antibody
and stored at 48C until acquisition by FACS Canto II (Becton
Dickenson). Data were analysed using BD FACSDiva 6.0/Cell Quest
Pro software. At least 20 000 events were acquired per sample.
RNA preparation and real-time quantitative
RT–PCR
Total RNA was isolated using TRI reagent (Sigma-Aldrich) from unactivated
PBMCs, alloactivated PBMCs (18 or 96 h) treated with control
protein (HSA) and alloactivated GdA-treated (24 h) PBMCs. 2.5 mg of
total RNA was reverse transcribed in a vol of 20 ml using random hexamers
(olig-dT) and RevertAid M-MuLV reverse transcriptase as per the manufacturer’s
instructions. Real-time quantitative PCR was performed using
the DNA-binding dye, the SYBR Green method (Morrison et al., 1998).
Reaction was carried out in triplicates in 20 ml on a BioRad iCycler iQ5.
The primers used for real-time PCR are listed in Table I. The amplification
conditions for housekeeping and test genes were: initial denaturation at
958C for 3 min followed by 40 cycles of denaturation at 948C for 10 s,
primer annealing at 638C for 30 s and extension at 728C for 45 s. The
final extension was carried out at 728C for 5 min. The fluorescence
emitted at each cycle was collected for the entire period of 45 s during
the extension step of each cycle. The homogeneity/specificity of the
PCR amplicons was verified by running 2% agarose gels and also by melt
curve analysis. Mean Ct values generated in each experiment using the
iCycler software (Bio-Rad) were used to calculate the relative fold change
in gene expression upon activation and GdA treatment. Briefly, data for
the test genes were normalized with reference gene (h18S rRNA) and calculated
manually as the fold change in test gene expression above control (unactivated
PBMCs) by the 2 2△△Ct method. Final fold change in gene expression
Table I List of primers for real time RT-PCR.
S.
No.
Gene Primer sequence
........................................................................................
1 h18s rRNA F: 5 ′ -CGC CGC TAG AGG TGA AAT TC-3 ′
R: 5 ′ -TTG GCA AAT GCT TTC GCT C-3 ′
2 hEOMES F: 5 ′ -GGG GAG GTC GAG GTT CTT ACC AGA-3 ′
R: 5 ′ -CTT GAA CAC AGT GGG GCT TGT TCT-3 ′
3 hT-Bet F: 5 ′ -GCC TGG GGT CTC CCT ACC CG-3 ′
R: 5 ′ -GGC TCC AAG GAA GCG GCT C-3 ′
4 hCD25 F: 5 ′ -TCG GCC TGG AGT GGT GTG TC-3 ′
R: 5 ′ -GGG CCC CTC CTT TTG GGG GA-3 ′
5 hBcl-2 F: 5 ′ -CCT GTC GAT GAC TGA GTA CC-3 ′
R: 5 ′ -GAG ACA GCC AGG AGA AAT CA-3 ′
6 hBax F: 5 ′ -ATG CGT CCA CCA AGA AGC TGA GC-3 ′
R: 5 ′ -CCC CGA TGC GCT TGA GAC ACT-3 ′
7 hCD28 F: 5 ′ -AGC AGG CTC CTG CAC AGT GAC-3 ′
R: 5 ′ -TAG GCT GCG AAG TCG CGT GG-3 ′
8 hCTLA4 F: 5 ′ -GCA TCG CCA GCT TTG TGT GTG AGT-3 ′
R: 5 ′ -TCC GAAGCA CTG TCA CCC GGA CC-3 ′
was plotted using Graph Pad Prism 5.0 software. Statistical analysis was performed
using the paired Student’s t-test.
Cell lysate preparation and immunoblotting
Resting or PHA-activated PBMCs, treated with control protein or GdA,
were washed with 50 mM PBS and pelleted at 2000 rpm/5 min. Cell
pellets were blotted to remove any remaining PBS. Pellets were tapped
and cells lysed for 30 min on ice in RIPA buffer (100 ml for 1 × 10 7
PBMCs) consisting of 50 mM Tris–HCl (pH 7.4), 1% Nonidet-P-40,
0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulphonyl
fluoride, 1 mg/ml each of aprotinin, leupeptin and pepstatin
(added freshly), 1 mM Na3VO 4 and 1 mM NaF. Samples were then
vigorously vortexed and centrifuged at 14 000 rpm for 10 min at 48C.
Pellet was discarded and protein estimated using Bradford’s method of
protein estimation. Equal protein from each sample was boiled in SDS
sample buffer and subjected to SDS–PAGE along with a molecular
weight marker. After equilibration in transfer buffer (25 mM Tris-base,
192 mM glycine and 20% methanol) gels were transferred onto nitrocellulose
membrane (Millipore, MA, USA) by semi-dry western blotting
(Bio-Rad). Non-specific binding was blocked with 5% non-fat dry milk
powder in Tris-buffered saline Tween-20 (TBS-T; 20 mM Tris–HCl, pH
7.4, 137 mM NaCl and 0.1% Tween-20) for 2–3 h at 48C with constant
rocking. The blots were thereafter incubated overnight with constant
rocking at 48C, with primary antibodies diluted in TBS-T with 5% BSA.
Thereafter, blots were thoroughly washed with TBS-T and then incubated
with anti-mouse IgG secondary antibodies conjugated with HRP for 1 h at
48C. After further washing in TBS-T, the immunoblots were developed
with an enhanced chemiluminiscence detection system (Millipore) as per
instructions. Data acquired using Fujifilm Las 3000 Imager and densitometric
analysis was done using Fujifilm Multigauge software.
Statistical analysis
All the data were evaluated using the paired Student’s t-test. P-value of
,0.05 was considered as statistically significant.
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4 Soni and Karande
Results
Activated PBMCs upon GdA treatment
express lower level of Eomes
In our previous study, we reported that GdA suppresses the cytolytic
activity of CTLs generated in vitro by impeding the transcription of
cytolytic genes viz. perforin and granzyme B (Soni and Karande,
2010). We went further to find out how GdA controls the expression
of perforin and granzyme B. T-box transcription factors T-Bet and
Eomes induced upon the CD8 + cell activation act as key regulatory
proteins in the development of competent effector and memory
CD8 + T-cells (Szabo et al., 2000, 2002; Pearce et al., 2003; Araki
et al., 2008). To test the possibility, if GdA was able to downmodulate
the cytolytic effector genes by affecting the induction of
Eomes and T-bet, we ascertained the transcriptional status of T-Bet
and Eomes by real-time RT–PCR analysis.
PBMCs alloactivated with gOVCAR-3 for 18 or 96 h were treated
with 1 mM GdA for 24 h (which is the time required for GdA to have
its inhibitory effect on CTLs). Two time points were chosen so as to
pick the message at the time of its optimal synthesis. RNA was isolated
and real-time RT–PCR was performed. Figure 1a and b depicts the
expression of T-Bet and Eomes mRNA, respectively, in alloactivated
PBMCs, expressed as fold change relative to the unactivated
PBMCs. As is evident from the graph, there was no significant
change in T-bet mRNA expression upon GdA treatment, but
Figure 1 GdA does not affect T-bet but down-modulates Eomes expression. Quantitative RT–PCR of the expression of T-Bet (a) and Eomes (b)in
18 or 96 h alloactivated PBMCs subsequently treated with GdA/HSA (Ctrl) for 24 h. Data normalized to 18 s rRNA and presented as fold change
relative to the gene expression in unstimulated PBMCs. Box plot shows median, interquartile range and range of four observations. (c) Flow cytometry
of the expression of Eomes in; unstimulated PBMCs (UA); alloactivated PBMCs treated with HSA [A (Ctrl)] and alloactivated PBMCs treated with
GdA [A (GdA)]. Unstained PBMCs (US) were used to define the P3 region. Numbers within the region P3 indicate the percent Eomes positive
cells among total PBMCs. At least 20 000 events were acquired per sample. Lymphocytes were gated on FSC/SSC. Data are representative of
five independent experiments. (d) Percentage of Eomes positive unactivated or activated PBMCs upon Ctrl or GdA treatment were quantified
using WinMDI and plotted using Graph Pad Prism 5.0, Box plot shows median, interquartile range and range of five observations *P ¼ 0.0139 and
**P ¼ 0.0171.
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Glycodelin-A dampens CD25 expression in T-cells 5
transcription of Eomes was significantly impeded by GdA (P ¼ 0.03).
The result prompted us to look at the intracellular protein profile of
Eomes. Unstimulated PBMCs, alloactivated PBMCs (24 h) followed
by control/1 mM GdA treatment for 24 h, were stained for Eomes.
Figure 1c shows representative histograms with the expression level
of Eomes in resting and alloactivated PBMCs. Figure 1d depicts the
% Eomes positive cells from five independent experiments. As is
evident from the plots, GdA treatment reduced the intracellular
protein level of Eomes. Interestingly, we did not observe any change
in the intracellular IFN-g protein expression in activated CD8 +
T-cells upon GdA treatment (Supplementary data, Fig. SI).
GdA dampens CD25 expression in
T-lymphocytes
We observed in stimulated CD8 + T-cells that upon GdA treatment,
they were cytolytically disabled and expressed relatively low levels
of perforin and granzyme B (Soni and Karande, 2010). Furthermore,
our observation that upon GdA treatment, Eomes expression in activated
T-cells was reduced (Fig. 1) indicated that GdA induced a lowperforin,
low-granzyme, low-Eomes profile, which was indicative of
weak IL-2 signalling (Belz and Masson, 2010). Most importantly, very
early in glycodelin research, it was reported that GdA suppresses IL-2
synthesis in mitogen-stimulated T-cells and that exogenous supplementation
of IL-2 can rescue T-cells from the GdA-induced inhibition of
proliferation (Pockley and Bolton, 1989) which was further confirmed
by a recent study (Lee et al., 2009). Building up on the available information,
we looked at the mRNA level (at an early and late time point of
activation) and surface expression of CD25 in stimulated PBMCs.
As expected, we observed a significant decrease in CD25 mRNA
expression in PBMCs alloactivated for 18 and 96 h (Fig. 2a), plate
bound anti-CD3-activated and PHA-activated PBMCs (data not
shown) upon treatment for 24 h with 1 mM GdA. Figure 2b shows
the kinetics of CD25 surface expression upon PHA stimulation. Its expression
started at around 6 h post-stimulation, peaked at around 18 h
(at 24–30 h in alloactivated PBMCs). Clearly a CD25 hi/bright and a
CD25 lo/dim population could be seen, as indicated in the figure. The
expression was fairly sustained until 48 h of stimulation.
To ascertain the effect of GdA on CD25 surface expression,
PBMCs were stimulated with PHA for 24 h and simultaneously
treated with GdA and surface stained for CD4/CD8 and CD4/
CD25. As can be seen in the representative dot plot in Fig. 2c,
GdA treatment resulted in a modest decrease in surface CD25 hi
population, with a concomitant increase in CD25 lo and CD25 neg
T-cells. The type of stimulation did not alter the result, only the percentage
of cells expressing CD25 was higher upon PHA stimulation
than upon alloactivation(data not shown). Data from six such independent
experiments have been compiled and presented in Fig. 2d.
There was ≏41.5% reduction in CD25 hi CD4 + T-cells, whereas a
≏32.3% reduction in CD25 hi CD8 + T-cells was observed in GdAtreated
samples.
GdA does not affect the expression
of CD28 or CTLA4
CD28 and CTLA4 (CTL-associated protein 4) are immunoglobulin
superfamily proteins expressed on the T-cell surface and are ligands
for the B7 molecules (B7-1 and B7-2), which are constitutively
expressed on antigen presenting cells and act antagonistically. CD28
(present on resting and activated cells) delivers a positive
co-stimulatory signal, while signalling through CTLA4 (present only
on activated T-cells) is inhibitory. We reasoned that GdA might
exert its inhibitory effect on T-cells by affecting CD28 or CTLA4
levels; hence, we looked at the mRNA and protein profile of CD28
and the mRNA expression of CTLA4 in 18 or 96 h alloactivated
PBMCs treated with GdA for 24 h. We did not observe any significant
variation in the CD28 mRNA/protein upon GdA treatment (Supplementary
data, Fig. S2a and b) Similarly, CTLA4 mRNA expression was
also found to be unaffected by GdA (Supplementary data, Fig. S3).
GdA signals to down-regulate Bcl-2 and
up-regulate Bax in activated PBMCs
Three distinct IL-2R signalling pathways are reported to exist,
mediated by c-myc, lck and Bcl-2 (Miyazaki et al., 1995). Resting
T-cells gain metabolic activity following the recognition of cognate
Ag by the T cell receptor (TCR); yet, they do not progress to
S-phase and instead undergo apoptosis, unless additionally stimulated
by IL-2 or other mitogenic cytokines (Akbar et al., 1996; Van Parijs
et al., 1997). Bcl-2 and Bcl-x have been proposed to augment cell viability
because constitutive expression of these genes in cytokinedependent
cell lines significantly delays the onset of the apoptotic
program induced by growth factor deprivation (Deng and Podack,
1993).
In the light of the above literature, we probed into the mRNA and
protein expression status of important anti-apoptotic protein Bcl-2 in
activated PBMCs upon GdA treatment. Supplementary data, Fig. S4
shows the fold induction of Bcl-2 mRNA in control-treated and GdAtreated
alloactivated (18/96 h) PBMCs. There was a modest downregulation
of Bcl-2 mRNA upon GdA treatment; however it did not
reach statistical significance (n ¼ 6). Interestingly though, the assessment
of total Bcl-2 protein levels from the cell lysates prepared
after PHA activation and simultaneous GdA treatment of PBMCs for
24 h showed a moderate but a significant decrease (n ¼ 6). Essentially,
the modest increase seen in Bcl-2 expression upon activation was
abrogated in the presence of GdA and the Bcl-2 levels were similar
to the resting PBMCs (Fig. 3a and b).
Bcl-2 and Bax proteins are known to heterodimerize that prevents
Bax oligomerization and subsequent permeabilization of the outer
mitochondrial membrane by Bax (Oltvai et al., 1993; Hengartner,
2000). Hence, we went ahead to check for the expression profile of
the pro-apoptotic protein Bax in alloactivated PBMCs upon GdA
treatment. For RNA analysis, PBMCs were allostimulated for 18 h,
treated with GdA for 24 h. For protein assessment, PBMCs were
PHA activated along with GdA for 24 h and processed for total
protein extraction. RNA analysis by real-time RT–PCR revealed an
almost 5-fold up-regulation in Bax mRNA in the presence of GdA
(n ¼ 5; Fig. 4a). In concurrence, there was a significant increase in
the total Bax protein upon GdA treatment of PHA-stimulated
PBMCs (n ¼ 4; Fig. 4b and c).
Discussion
Many studies have put forward mechanisms to explain the inhibitory
effect of GdA on T-lymphocytes. Rachmilewitz et al. show that the
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6 Soni and Karande
Figure 2 GdA treatment reduces the mRNA surface expression of CD25 in T-lymphocytes. (a). Total RNA was extracted and cDNA synthesized
from resting PBMCs or PBMCs activated with gOVCAR-3 cells for 18 or 96 h followed by treatment with HSA (Ctrl) or GdA for 24 h. 18S rRNA and
CD25 mRNA levels were determined by real-time quantitative RT–PCR. Result is expressed as fold change in CD25 mRNA in 18 or 96 h
alloactivated-GdA/Ctrl-treated PBMCs, when compared with unactivated PBMCs and normalized with 18s rRNA transcription. Samples were run
in triplicates. Box plot show median, interquartile range and range of six observations. (b) Flow cytometric analysis of CD25 surface expression
on resting (i) and PHA stimulated PBMCs at various time points indicated (ii–v). Panel vi represents dot plot for CD4 and CD25 double-stained
PBMCs after 18 h of PHA activation. Regions Q1 and Q2 represent CD25 hi population, Q3 and Q4 represent CD25 lo population and Q5 and
Q6 are CD25 neg . Numbers in i–v represent the percentage of CD25 hi and CD25 lo cells in the respective dot plot. (c) Resting PBMCs or PBMCs
activated using 5 mg/ml of PHA and simultaneously treated with HSA (Ctrl) or GdA were harvested; stained for surface CD4/CD8/CD25 and
data acquired using flow cytometry. Upper three panels show representative two colour dot plots showing FACS analysis of PBMCs stained for
CD4 and CD25; lower three panels show staining with CD8/CD25 Abs. CD25-PE acquired in FL-2 channel and CD4/CD8 in FL-1 channel. At
least 10 000 events acquired within the lymphocyte gate. Data analysed using winMDI software, Ver.5.0. (d) Percentage of CD4 + /CD8 + T-cells
with CD25 lo or CD25 hi expression upon Ctrl or GdA treatment were quantified using WinMDI and plotted using Graph Pad Prism 5.0. Data
expressed as box plots showing median, interquartile range and range from four independent experiments. *P ¼ 0.0128, **P ¼ 0.0261,
***P ¼ 0.0026 and ****P ¼ 0.007.
inhibitory function of GdA is dependent on its localization to the phosphatase
CD45 at the site of TCR triggering, where it negatively
regulates T-cell activation by reducing the half-life of TCR-triggered
phosphotyrosines (Rachmilewitz et al., 2001, 2003). Delineating
the mechanism of induction of apoptosis in T-lymphocytes,
SundarRaj et al. (2008) provide compelling evidences of a
TCR-independent pathway, wherein GdA induces a stress signal
which culminates into mitochondrial membrane potential loss and permeabilization,
causing T-lymphocytes to apoptose via the intrinsic
mitochondrial pathway. Corroborating these data, overexpression of
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Glycodelin-A dampens CD25 expression in T-cells 7
Figure 3 GdA inhibits the up-regulation of Bcl-2 expression in activated
PBMCs. (a) Immunoblot analysis of test protein Bcl-2 and reference
protein b-actin in resting PBMCs and PBMCs activated for
24 h with 5 mg/ml of PHA with simultaneous HSA (Ctrl) treatment
or GdA treatment. Proteins detected with anti-human Bcl-2 Ab and
anti-b-actin HRP in whole cell lysates. Data are representative of
five independent experiments. (b) Quantification of Bcl-2 protein expression.
Bcl-2 protein expression in PHA-activated (HSA-treated/
Ctrl) and PHA-activated (GdA-treated) PBMCs was quantified from
western blots using densitometric analysis with multigauge software,
normalized with b-actin expression and expressed relative to the
level in resting PBMCs. Data compiled from five independent experiments
and represented as box plots showing median, interquartile
range and range.
Bcl-2 and prevention of mitochondrial membrane permeabilization
are sufficient to rescue T-cells from death by GdA (SundarRaj et al.,
2008). Pockley and Bolton (1989) and Lee et al. (2009) attribute
the immunosuppressive effect of GdA to its ability to cause inhibition
of IL-2 synthesis in T-lymphocytes. We have reported earlier that
GdA induces functional ineptness in differentiated CD8 + T-cells by
blunting the synthesis and release of cytolytic molecules (Soni and
Karande, 2010).
We began this study with the simple objective to further unravel the
mechanism regulating the expression of perforin and granzyme B in
alloactivated CD8 + T-cells upon GdA treatment. From the available
literature, we knew that T-Bet, Blimp-1, Eomes and Bcl-6 are the
known transcription factors regulating CD8 + T-cell effector functions.
Among them, under antigen-specific stimulation conditions, T-Bet is
required for the differentiation of naïve CD8 + T-cells to effector
CTLs and also for the IFN-g synthesis by these cells (Sullivan et al.,
2003). Also, Eomes complements T-bet functions and plays a major
Figure 4 GdA signals to induce Bax mRNA and protein expression in
activated PBMCs. (a) Bax mRNA levels were analysed by quantitative
RT–PCR in unstimulated PBMCs or PBMCs stimulated with
gOVCAR-3 for 18 h followed by HSA (Ctrl) or GdA treatment for
24 h. Expression was normalized with 18s rRNA gene as control and
plotted as relative fold change in Bax expression upon alloactivation
with or without GdA treatment. Data are represented as a box plot
showing median, interquartile range and range of four individual experiments.
(b) Immunoblot analysis of test protein Bax and reference protein
b-actin in resting PBMCs and PBMCs activated for 24 h with 5 mg/ml of
PHA with simultaneous HSA (Ctrl) or GdA treatment. Proteins were
detected with anti-human Bax Ab and anti-b-actin HRP in whole cell
lysates. 50 mg protein was loaded per well. Data are representative of
four independent experiments. (c) Quantification of Bax protein expression.
Bax protein expression in PHA-activated (HSA-treated/Ctrl) and
PHA-activated (GdA-treated) PBMCs was quantified from western
blots using densitometric analysis with multigauge software, normalized
with b-actin expression and expressed relative to the level in resting
PBMCs. Data represented as box plots showing median, interquartile
range and range compiled from four independent experiments.
role in memory CD8 + T-cell development (Intlekofer et al., 2005).
So we looked at the effect of GdA on the induction of the two transcription
factors, T-Bet and Eomes upon activation. We found that
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8 Soni and Karande
with GdA treatment, T-bet was unaffected but Eomes was significantly
down-regulated both at the RNA and at the protein level (Fig. 1).
It is well established that the quality and magnitude of the CD8 +
T-cell responses is governed by the strength of the stimulatory
signal, cytokine milieu and availability of ‘CD4 help’. These factors
help shape the transcriptional program in the responding CD8 +
T-cells which in turn affect their phenotypic and functional attributes.
We reasoned that GdA could potentially hamper Eomes expression
by affecting the stimulatory signals to the cells during activation (attributable
to its property of being a lectin; SundarRaj et al., 2009) orby
altering the cytokine milieu through its inhibitory effect on CD4 +
T-cells, whose survival and functions are known to be affected
by GdA.
First, we looked at the effect of GdA on CD28 and CTLA4 which
are required for accelerating or braking T-cell proliferation upon activation,
necessary for T-cell homeostasis. GdA did not affect the expression
of CD28 on T-cells; CTLA4 was also not up-regulated
upon GdA treatment, suggesting that GdA (most likely) did not
perturb the pro- or anti-stimulatory signals generated upon T-cell
activation.
Our knowledge about the involvement of IL-2 in the development
of CD8 + T-cell effector and memory functions (Pipkin et al., 2010)
and that GdA suppresses IL-2 production by stimulated T-cells
(Pockley and Bolton, 1989) prompted us to check for CD25 expression
as the read out for IL-2 signalling in both CD4 + and CD8 + T-cell
subsets upon GdA treatment. It is well known that responsiveness to
IL-2 is controlled by the IL-2R complex, which is composed of the
high-affinity IL-2R a chain (CD25), the IL-2R b chain (CD122) and
the common g chain (CD132), and that signalling through IL-2Ra is
essential for protective immunity (Williams et al., 2006). We observed
that CD25 mRNA and protein expression were significantly reduced
upon GdA treatment in both the T-cell subsets, suggesting weak
IL-2 signalling in the presence of GdA (Fig. 2). Our finding was
strengthened by the observations made by other researchers, i.e.
high IL-2 signals induce transcription factors like STAT5 and Eomes
which directly bind to perforin and granzyme gene locus and the
amount of IL-2 signalling regulates the recruitment of polymerase II
to the perforin (high IL-2) or the IL-7R (low IL-2) loci and that lack
IL-2Ra expression on CD8 + T-cells causes aberrant killing of virally
infected cells (Pipkin et al., 2010). Additionally, a modest difference
exists in effector molecules expression between CD25 hi and CD25 lo
T-cells (Kalia et al., 2010), with CD25 lo CD8 + T-cells expressing
fewer cytolytic molecules.
Thus, these findings indicate that the interaction of GdA with activated
CD4 + and CD8 + T-cells suppresses IL-2/IL-2R signalling
induced upon T-cell activation, which seems to be the causative
agent for the loss of proliferation of T-cells upon GdA treatment. Additionally,
aberrant IL-2R-mediated signalling also contributes to
reduced expression of cytolytic genes (perforin and granzyme B) in
CD8 + T-cells, which are directly controlled by IL-2 as well (Janas
et al., 2005).
Consolidating data from our previous work (Soni and Karande,
2010) and this study, we can say with much confidence that in
CD8 + T-cells, GdA induces suboptimal IL-2 signalling that leads to
the inhibition of proliferation and suppression of perforin and granzyme
synthesis directly and also indirectly by suboptimal induction
of Eomes. T-bet levels were unaffected by GdA which explains the
moderate effects of GdA seen on CD8 + T-cells and the requirement
of a very high concentration of GdA to affect CTL activity (Soni and
Karande, 2010). It also explains why there was no significant effect
of GdA treatment on IFN-g production in CD8 + T-cells. We can envisage
that a reduction in Eomes expression by GdA might also contribute
to lowered number of CD8 + T-memory cells, further
contributing to fetal tolerance.
Studies on total T-lymphocytes clearly demonstrate that in addition
to promoting proliferation, IL-2Ra expression also prevents apoptosis
of T-cells, by up-regulating Bcl-2 expression (Gomez et al., 1998). We
observed a reduction in CD25 hi/bright surface expression on T-cells
upon GdA treatment (Fig. 2d). Previous work from our laboratory
attributed the apoptogenic activity of GdA on primary T-cells and
T-cell lines via the intrinsic mitochondrial pathway (Mukhopadhyay
et al., 2001; SundarRaj et al., 2008), which could be competed out
by the overexpression of Bcl-2 (SundarRaj et al., 2008). We questioned
whether suppression of CD25 expression by GdA was
causing a stoichiometric imbalance between the pro- and anti-survival
proteins within T-cells. Indeed, we found that GdA treatment did not
allow an increase in the Bcl-2 protein level within cells which is otherwise
observed in activated T-cells (Fig. 3), although at the mRNA level,
the decrease was not found to be statistically significant. Interestingly
though, pro-apoptotic protein Bax was up-regulated at both the
mRNA and the protein level (Fig. 4). At least four proteins Bcl-2,
14-3-3, humanin and Ku-70 have been shown to inhibit apoptosis
by binding to Bax and sequestering it from the mitochondria (Amsel
et al., 2008). With the data available on the activity of GdA and
from the present study, we can surmise that the perturbation in the
ratio of Bcl-2 and Bax leads to the availability of free Bax to oligomerize
and initiate mitochondrial membrane permeabilization leading to
apoptosis. This phenomenon seems to operate in CD4 + T-cells,
where the suboptimal IL-2R expression may lead to a reduced IL-2
level which is insufficient to support the optimal up-regulation of
Bcl-2 upon T-cell stimulation, necessary for survival. Unable to cope
with the mitochondrial stress, CD4 + T-cells undergo apoptosis. Intriguingly,
although the CD8 + T-lymphocytes have reduced CD25 expression
upon GdA treatment, they are resistant to GdA-induced
apoptosis (Soni and Karande, 2010). Similarly, the Th2 cells are also
resistant to GdA-induced apoptosis (Lee et al., 2011), suggesting
that the process of apoptosis induction may not be as simplistic and
there may be many more players involved that may vary in different
T-cell subsets.
The different mechanisms put forth by various researchers to
explain GdA activity on different cell types can be explained by the
fact that GdA functions as a galactose-specific lectin (SundarRaj
et al., 2009), and it can potentially bind to varied glycosylated proteins
on the T-cell surface (Rachmilewitz et al., 2003; Yaniv et al., 2003;
SundarRaj et al., 2009). Among the many potential candidates as the
receptor for GdA on T-cells, CD45 (Rachmilewitz et al., 2003) and
CD7 are the most extensively discussed. We observed that
GdA-induced apoptosis in T-cell lines in a CD7-dependent manner
(SundarRaj et al., 2009). GdA treatment itself did not affect the expression
of CD7 on both the T-cell subsets (data not shown). CD7
is known to augment T-cell proliferation/function by up-regulating
CD25 expression and IL-2 production (Wallace et al., 2000; Stillwell
and Bierer, 2001). Very interestingly, CD7-deficient mice show
decreased CTL effector functions (Lee et al., 1998). Until now, a
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Glycodelin-A dampens CD25 expression in T-cells 9
definitive ligand for CD7 is not known and only lectins have been proposed
to be potential CD7 ligands (Leta et al., 1996). Considering the
fact that glycodelin is also a lectin, it will be interesting to probe
whether glycodelin might exert its inhibitory effect on T-cells by interacting
with CD7 and inhibiting its normal co-stimulatory function.
Altogether, from the present study and available literature, it can be
proposed that the binding of GdA to CD4 + and CD8 + T-cells blocks
the co-stimulatory signals required for optimal CD25 expression and
hence IL-2 production. Resultant suboptimal IL-2 signalling has
varied effects; (i) cell cycle arrest as seen in both the major T-cell
subsets; (ii) reduced induction of Eomes that causes retarded synthesis
of cytolytic genes, causing suppression of CTL activity in CD8 + T-cells;
(iii) insufficient induction of anti-apoptotic genes upon T-cell activation,
which increases the ratio of pro- to anti-apoptotic proteins,
affecting mitochondrial integrity, finally leading to cell death in some
CD4 + T-cell subsets. The mechanism of GdA activity suggested
here seems to be operational in vivo and the expression of CD25
has also been found to be selectively reduced on decidual CD4 +
and CD8 + T-lymphocytes in normal pregnancies (Chao et al.,
2002). Nevertheless, it is important that similar studies be carried
out using decidual T-lymphocytes to further confirm the effects of
GdA seen on peripheral T-cells, as the localized abundance of progesterone
in the uterus during pregnancy may alter the activity of GdA on
decidual T-cells. It will also be pertinent to mention here that peripheral
T-cells from men and women (at different times in their menstrual
cycle), used as the model system for this study, are exposed to varying
concentrations of serum progesterone and oestrogen, which may have
a bearing on the effect of glycodelin on the T-cells and can be an interesting
factor to study in depth.
In conclusion, the results put forth in this study demonstrate a previously
unappreciated/underplayed effect of GdA on IL-2/IL-2R signalling.
IL-2 growth factor deprivation can explain effects of GdA on
T-cells, including the inhibition of proliferation, apoptosis and loss of
effector functions which contribute to the establishment, progression
and maintenance of primate pregnancy but the variations observed in
the responsiveness of different cell types to GdA indicate that the
understanding of the mechanistic details of GdA activity on hematopoietic
cells is still far from complete.
Supplementary data
Supplementary data are available at http://humrep.oxfordjournals.
org/.
Authors’ roles
C.S. has participated in the study design, execution of experiments,
data analysis, writing the manuscript and discussions. A.A.K. supervised
the study and was involved in the design of experiments and critical
analysis of the data, manuscript corrections and discussions.
Funding
This work is supported by grants from the Department of Biotechnology
(DBT), India and the Indian Institute of Science, (IISc) Bangalore,
India.
Conflict of interest
None declared.
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