Acute stress differently modulates β1, β2 and β3 adrenoceptors in T cells, but not in B cells, from the rat spleen.

Abstract

Stress-induced rise in circulating catecholamines (CAs), followed by modulation of β-adrenergic receptors (adrenoceptors, ARs), is one of the pathways involved in the stress-mediated effects of immune functions. The spleen is an organ with a high number of lymphocytes and provides a unique microenvironment in which they reside. Thus, lymphocytes may respond differently to CAs in the spleen than in the circulation. No reports exist concerning the involvement of β-ARs in stress-mediated effects on T and B cells isolated from the spleen. Therefore, our aim was to investigate the effect of single stress exposure on gene expression and cellular localization of β-adrenoceptor subtypes in splenic T and B cells. We tried to correlate changes in adrenoceptors with the expression of apoptotic proteins.
Immobilization (IMMO) was used as a stress model. T and B cells were isolated from rat spleen using magnetically labeled antibodies. The gene expression of individual adrenoceptors and apoptotic proteins was evaluated by real-time PCR. Immunofluorescence was used to evaluate localization and adrenoceptor expression.
We have found T cells to be more vulnerable to stress compared to B cells, because of increased β₁-, β₂- and β₃-ARs after a single IMMO. Moreover, β₂-ARs translocated from the nucleus to the plasma membrane in T cells after IMMO. The rise in β-ARs most probably led to the rise of Bax mRNA and Bax to Bcl-2 mRNA ratio. This might suggest the induction of an apoptotic process in T cells.
Higher susceptibility of T cells to stress via modulation of β-ARs and apoptotic proteins might shift the immune responsiveness in the spleen.

Full text

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Fax +41 61 306 12 34
E-Mail karger@karger.ch
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Original Paper
Neuroimmunomodulation 2012;19:69–78
DOI: 10.1159/000329002
Acute Stress Differently Modulates Beta 1, Beta 2
and Beta 3 Adrenoceptors in T Cells, but Not in B
Cells, from the Rat Spleen
Marcela Laukova a Peter Vargovic a Lucia Csaderova c Lucia Chovanova a
Miroslav Vlcek a Richard Imrich c Olga Krizanova b Richard Kvetnansky a
a Institute of Experimental Endocrinology, Center of Excellence CENDO and Center of Excellence for Cardiovascular
Research, b Institute of Molecular Physiology and Genetics, Center of Excellence for Cardiovascular Research, and
c Molecular Medicine Center, Slovak Academy of Sciences, Bratislava , Slovakia
evaluate localization and adrenoceptor expression. Results:
We have found T cells to be more vulnerable to stress com-
pared to B cells, because of increased ? 1 -, ? 2 - and ? 3 -ARs
after a single IMMO. Moreover, ? 2 -ARs translocated from the
nucleus to the plasma membrane in T cells after IMMO. The
rise in ? -ARs most probably led to the rise of Bax mRNA and
Bax to Bcl-2 mRNA ratio. This might suggest the induction
of an apo ptotic process in T cells. Conclusion: Higher sus-
ceptibility of T cells to stress via modulation of ? -ARs and
apoptotic proteins might shift the immune responsiveness
in the spleen.
Copyright © 2012 S. Karger AG, Basel
Introduction
Stress is considered an important regulator of many
physiological functions, including the immune system.
While acute stress has been shown to act immunostimu-
latory, prolonged or excessive stress shifts the immune
reaction towards Th2-mediated humoral response and/
or has immunosuppressive effects [1, 2] . On the other
hand, stress hormones may be necessary for optimal im-
mune responsiveness during an infection [3] and for in-
creased resistance to squamous cell carcinoma [4] . Stress
Key Words
Stress ? Spleen ? T cells ? B cells ? ? -Adrenergic receptors ?
Apoptosis
Abstract
Objectives: Stress-induced rise in circulating catechol-
amines (CAs), followed by modulation of ? -adrenergic re-
ceptors (adrenoceptors, ARs), is one of the pathways in-
volved in the stress-mediated effects of immune functions.
The spleen is an organ with a high number of lymphocytes
and provides a unique microenvironment in which they re-
side. Thus, lymphocytes may respond differently to CAs in
the spleen than in the circulation. No reports exist concern-
ing the involvement of ? -ARs in stress-mediated effects on
T and B cells isolated from the spleen. Therefore, our aim
was to investigate the effect of single stress exposure on
gene expression and cellular localization of ? -adrenoceptor
subtypes in splenic T and B cells. We tried to correlate chang-
es in adrenoceptors with the expression of apoptotic pro-
teins. Methods: Immobilization (IMMO) was used as a stress
model. T and B cells were isolated from rat spleen using
magnetically labeled antibodies. The gene expression of in-
dividual adrenoceptors and apoptotic proteins was evalu-
ated by real-time PCR. Immunofluorescence was used to
Received: February 18, 2011
Accepted after revision: April 29, 2011
Published online: January 11, 2012
Marcela Laukova
Institute of Experimental Endocrinology, Center of Excellence CENDO and
Center of Excellence for Cardiovascular Research, Slovak Academy of Sciences
Vlarska 3, SK–83306 Bratislava (Slovakia)
Tel. +421 2 5477 2800, E-Mail marcelalau   @   email.cz
© 2012 S. Karger AG, Basel
1021–7401/12/0192–0069$38.00/0
Accessible online at:
www.karger.com/nim

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Laukova et al.  
Neuroimmunomodulation 2012;19:69–78
70
also evokes tissue-specific changes in immune cell distri-
bution [5, 7] . However, the exact mechanism by which
stress affects the immune system as well as the conse-
quences of stress exposure on specific immune compart-
ments is not yet sufficiently investigated. Rinner et al. [6]
demonstrated that immobilization (IMMO) stress regu-
lated immune response in the circulation differently than
in the spleen. Stress-induced rise in circulating catechol-
amines (CAs), followed by modulation of ? -adrenergic
receptors (adrenoceptors, ARs), is one of the pathways
involved in the stress-mediated effects on immune func-
tions. Increased levels of CAs were observed in the rat
spleen after single and repeated IMMO stress [8] and re-
mained increased after 3 h of rest [9] . Moreover, besides
circulating CAs and CAs released from sympathetic
nerve terminals, the rat spleen also contains the ‘machin-
ery’ to produce CAs de novo that might contribute to the
CA ‘pool’ in the spleen [9, 10] . Nevertheless, function of
the endogenously produced CAs in the spleen is not yet
fully understood. The rise in splenic CAs was associated
with changes in AR expression that could play a role in
altered gene expression of several cytokines [8, 11] . The
spleen is also an organ with a high number of lympho-
cytes and provides a unique microenvironment in which
these cells reside. Splenic lymphocytes are localized in
direct apposition to sympathetic nerve terminals, and
therefore, are exposed to higher CA concentrations, espe-
cially during stress, compared to the circulation [12] .
Therefore, lymphocytes may respond differently to CAs
in the spleen than in the circulation, which might lead to
different functional changes in these immune cells [12] .
There is evidence that the ? 2 -AR is the main immu-
noregulatory subtype on lymphocytes, which regulates
physiologically different immune functions like inhibi-
tion of proinflammatory cytokine production, adhesion,
chemotaxis and many others [13] . Nevertheless, ? 2 -AR-
mediated functions may differ according to the cell type,
tissue localization and conditions. On contrary, little is
known about ? 1 -ARs in the immune system. Emeny et al.
[14] showed that splenic ? 1 -ARs reduced a defense against
Listeria monocytogenes, while ? 1 -AR knockout (–/–) mice
had increased cell-mediated immunity. It was reported
that a small number of ? 1 -ARs ( ! 10%) exist directly on
peripheral lymphocytes compared to predominantly
( 1 90%) expressed ? 2 -ARs [15, 16] . The ? 1 -AR gene ex-
pression was higher in regulatory T cells compared to
other T-cell populations [17] . The overexpression of ? 1 -
AR has been detected in peripheral lymphocytes from
hypertensive patients and patients with congestive heart
disease after drug treatment [18, 19] . The ? 1 -AR mRNA
level in lymphocytes directly correlated with urinary al-
bumin excretion in microalbuminuric patients [19] .
Nevertheless, sparse data exist concerning ? 3 -ARs
and immunity. Administration of the ? 3 -AR agonist in-
creased the number but not the proliferation of cluster of
differentiation 4 positive (CD4+) and CD8+ T lympho-
cytes in the spleen of obese animals [20] . The ? 3 -AR
mRNA was detected in human peripheral lymphocytes
[18, 19, 21] , but functional significance has not yet been
investigated.
Based on this information, the aim of our study was to
analyze the gene expression and cellular localization of
different ? -adrenoceptor subtypes in T and B cells iso-
lated from rat spleen during stress. Since correlation of
the ? -AR signaling in the process of apoptosis was al-
ready suggested [22, 23] , we also correlated changes ob-
served in the ? -ARs with the expression of pro- and an-
tiapoptotic proteins.
Material and Methods
Animals
Male Sprague-Dawley rats (250–300 g, Charles River, Sulzfeld,
Germany) were used in our experiments. Animals were housed
3–4 per cage in a controlled environment (22 8 2   °   C, 12-hour
light-dark cycle, lights on at 6.00 a.m.). Food and water were avail-
able ad libitum. Immobilization (IMMO) stress was performed to
induce stress reaction, as described previously [24] . Rats were im-
mobilized once for 2 h and decapitated immediately after termi-
nation of the stress stimulus. Control rats were sacrificed im me-
diately after removal from their home cages. The Ethics Commit-
tee of the Institute of Experimental Endocrinology (Slovak
Academy of Sciences, Bratislava, Slovakia) approved all experi-
mental procedures with the animals used in this study in protocol
No. RO-2804/07-221/3. After decapitation, spleens were rapidly
extirpated and placed into cold phosphate-buffered saline (PBS).
Isolation of T and B Cells from Rat Spleen
Each spleen was placed into a special tube (M tube; Myltenyi
Biotech, Germany) and gently homogenized in PBS solution using
the gentleMACS Dissociator (Myltenyi Biotech) according to the
manufacturer’s protocol. Cell suspension was devoid of cell debris
by passing through the 30- ? m nylon mesh (Myltenyi Biotech).
Red blood cells, platelets and polynuclear cells were depleted by
Ficoll gradient centrifugation (Sigma Aldrich) at 300 g for 30 min
at room temperature. After two subsequent washes in PBS (300 g,
10 min) T and B lymphocytes were isolated from the mononucle-
ar cell fraction using magnetically labeled antibodies – Anti-T
Cell (OX52) MicroBeads (Myltenyi Biotech, order No. 130-090-
320) and CD45RA MicroBeads (Myltenyi Biotech, order No. 130-
090-494) – on autoMACS Pro Separator (Myltenyi Biotech) ac-
cording to the manufacturer’s protocol. The Trypan blue test re-
vealed 1 95% cell vitality in each fraction. Cells were pelleted
(300 g, 7 min) and used for further analysis.

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Stress Differently Regulates
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Neuroimmunomodulation 2012;19:69–78
71
RNA Isolation and Relative Quantification of mRNA Levels
by Real-Time RT-PCR
Total RNA from T- and B-cell-enriched fractions was isolated
by TRI Reagent (MRC Ltd., Cincinnati, Ohio, USA). The purity
and integrity of isolated RNAs was checked on GeneQuant Pro
spectrophotometer (Amersham Biosciences, Piscataway, N.Y.,
USA). Reverse transcription was performed using 1.5 ? g of total
RNAs and Ready-To-Go You-Prime First-Strand Beads (Amer-
sham) with pd(N)6 primer (Amersham), according to the manu-
facturer’s protocol. To verify the purity of each fraction, expres-
sion of T- and B-cell-specific CD markers was evaluated, using
primers for CD3 ? for T cells, CD14 for monocytes/macrophages
and CD19 for B-cell evaluation. The PCR amplification and de-
tection was carried out on the ABI Prism 7900HT Sequence De-
tection System (Applied Biosystems, Inc., Foster City, Calif., USA)
with 10% of the reverse transcription product, primers at 125 n M ,
and SYBR Green Master Mix with ROX reference dye (Fermentas,
Germany) in a final volume of 20 ? l. Master Mix with primers
and template was separately loaded onto 96-well plates (Applied
Biosystems Inc.). Plates were centrifuged to remove any air bub-
bles in the wells. Each sample was run in duplicates and with 1 ‘no
template’ control. Each cycle consisted of 15 s at 95   °   C, 30 s at
60–65   °   C and 30 s at 72   °   C for 40 cycles after the initial activating
step for 2 min at 50   °   C and a denaturing step for 10 min at 95   °   C.
Primers and annealing temperatures used for specific genes are
described in table 1 . To exclude the presence of nonspecific prod-
ucts, a routine melting curve analysis was performed after finish-
ing the amplification. This was done by high resolution data col-
lection during an incremental temperature increase from 60 to
95   °   C. Data were analyzed with SDS software version 2.3 (Applied
Biosystems) and inspected to determine artifacts (loading errors,
threshold errors). Baseline levels for each gene were computed au-
tomatically.
Count numbers were exported to an Excel spreadsheet and
analyzed according to the ? ? CT method described in ABI User
Bulletin No. 2 (Applied Biosystems). The copy number of the tar-
get genes was normalized to glyceraldehyde 3-phosphate dehy-
drogenase as an endogenous reference, which was not affected by
stress exposure. The fold change of controls was set at 1, and nor-
malized fold change of genes was calculated.
Table 1. Primers and annealing temperatures used in real-time PCR
GenePrimerOligonucleotide sequenceAnnealing
° C/time, s
Size, bp
?1-AR
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
5?-CATCATGGGTGTGTTCACGCTCTG-3?
5?-GCGTAGCCCAGCCAGTTGAAGAA-3?
5?-ACATCACTCAGGAACGGGACGAAG-3?
5?-CAGCACACGCCAAGGAGGTTATG-3?
5?-GCAACCTGCTGGTAATCACA-3?
5?-GGATTGGAGTGACACTCTTG-3?
5?-GCAGCGTGTGTTGGATTTGACTC-3?
5?-TGTTGAGATGATGCTTTGACAGATGGC-3?
5?-GGCTTCCAGGGTGCTTCGCAAAT-3?
5?-TGTGAGCGTGGACTCATTCACGG-3?
5?-GTGCTCGTAGTGGTCCTCATC-3?
5?-CAGCAAGGGTAGCGTCATCT-3?
5?-ACCGACACTGCGAACTCAA-3?
5?-GCAGGGTTGGCAGACAGTA-3?
5?-AGATCCACAACGGATACATT-3?
5?-TCCCTCAAGATTGTCAGCAA-3?
5?-TTGGCTGGCCTCTTCCTGGTGAT-3?
5?-TGCGACTTCTTCTTTGCTCCTCGG-3?
5?-AATCTGACGACCCAGAGCCAGCA-3?
5?-TTTCCCAGTTTGTGCAGGGGTGC-3?
5?-AACAACGGATACCTGGCTCGCA-3?
5?-CGCTAAAACTTGGAGGGTCGGGAA-3?
5?-CAAGAAGCTGAGCGAGTGTC-3?
5?-GCAAAGTAGAAGAGGGCAACC-3?
5?-ACTTCTCTCGTCGCTACCGT-3?
5?-GTTCCACAAAGGCATCCCAG-3?
5?-ACGAACGGACCTGTGGACCTGAA-3?
5?-CCGGGTGCGGTAGAGTAAGCATA-3?
60/30120
?2-AR
60/30168
?3-AR
60/30418
IL-263/30199
IL-463/30157
CD6961/30232
CD40 63/15200
GAPDH60/60 308
CD3?
65/30 266
CD1965/30503
CD1465/30220
Bax61/30183
Bcl-261/30294
Caspase 360/30218
GAPDH = Glyceraldehyde 3-phosphate dehydrogenase.

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Laukova et al.  
Neuroimmunomodulation 2012;19:69–78
72
The purity of isolated fractions according to CD products
from real-time PCR revealed 1 97% purity of each fraction. To il-
lustrate the purity of cell-specific fractions, real-time PCR prod-
ucts were visualized on 2% agarose gel. The intensity of individ-
ual bands was evaluated by Gel Logic 200 Imaging System
(Carestream Health, Inc., N.Y., USA) and PCBASE 2.08e software
(Raytest, Inc., Düsseldorf, Germany).
Absolute Quantification of mRNA Levels by Real-Time
RT-PCR
Absolute mRNA levels of ? 1 -, ? 2 - and ? 3 -AR in T and B cells
were evaluated by quantitative real-time PCR, as described previ-
ously [25] . Standards for absolute quantification of ? 1 -, ? 2 - and
? 3 -AR were prepared by PCR amplification, purification by gel
extraction and subsequent dilution to concentrations of 30 to 30
! 10 6 copies/ ? l using ddH 2 O containing 50 ng/ ? l of sonicated
salmon sperm DNA (Sigma-Aldrich) as a DNA carrier.
Immunofluorescence
After the isolation procedure, T and B cells were fixed in ice-
cold methanol. Nonspecific binding was blocked by incubation
with PBS containing 3% bovine serum albumin (Merck Biosci-
ences, Germany) for 1 h at 37   °   C. Afterwards, cells were incubated
with corresponding primary antibodies diluted 1:
at 37   °   C. We used primary rabbit polyclonal antibodies to ? 1 -AR
(Santa Cruz Biotechnology, Inc., USA), ? 2 -AR (Abnova, USA) and
? 3 -AR (Alpha Diagnostic International, USA). Cross-reactivity
data and catalog numbers are described in table 2 . Thereafter,
cover slips were washed in PBS and incubated with CFTM488
goat anti-rabbit IgG (H+L) secondary antibody (Biotium, Hay-
ward, Calif., USA) for 60 min at 37   °   C. Finally, cells were mounted
onto slides in mounting medium with Citifluor (Agar Scientific
Ltd., UK), analyzed by fluorescent microscope Leica DM450B
with Leica DFC 480 and software Leica IM 500 also by inverted
confocal microscope Zeiss Axiovert 200M with an LSM510 ex-
pert mode program. Micrographs were taken at 63 ! magnifica-
tion with optical zoom 2. Micrographs were deconvolved in Huy-
gens Essential software (SVI, The Netherlands) and analyzed in
ImageJ software Volume Viewer.
500 for 60 min
Statistical Analysis
Each value represents the average of 5–6 animals, from which
both T and B cells were isolated. Results are presented as means
8 SEM. Statistical differences among groups were determined by
one-way analysis of variance (ANOVA). p ! 0.05 was considered
to be significant. For multiple comparisons, an adjusted t test with
p values corrected by the Bonferroni method was used (SigmaStat,
version 3.1, Systat Software, Inc., USA).
Results
Real-time PCR for immune cell-specific CD markers
(CD3 ? , CD14 and CD19), performed in each fraction af-
ter cell separation, revealed very good purity of T and B
cells isolated from rat spleen ( fig. 1 ). Negative control
groups after T- and B-cell separation (TBM-T, TBM-B;
fig. 1 ) confirmed the accuracy and successful separation
procedure.
Real-time PCR further revealed that specific AR sub-
types are differently expressed in T and B cells of control
groups ( fig. 2 ). In both lymphocyte populations, ? 2 -AR
mRNA levels were much higher than ? 1 - and ? 3 -AR
mRNA. In addition, ? 3 -AR gene expression was approx-
imately 3-times higher in B than in T cells.
Single IMMO stress increased the gene expression of
? 1 -ARs (p ! 0.001; fig. 3 a, white columns), ? 2 -ARs (p !
0.05; fig.  3 b, white columns) and ? 3 -ARs (p ! 0.001,
fig. 3 c, white columns) in T cells with the most prominent
increase in ? 3 -AR mRNA. In the B-cell fraction, only
changes in ? 3 -AR mRNA were observed, which signifi-
cantly declined after IMMO (p ! 0.05; fig. 3 c, grey col-
umns). No significant changes were found in ? 1 - and ? 2 -
ARs in B cells after stress exposure (p 1 0.05; fig. 3 a, b,
grey columns).
Protein level and subcellular distribution of the ? 1 -,
? 2 - and ? 3 -ARs were shown by immunofluorescent
staining ( fig. 4 ). Specificity of antibodies was verified by
negative control, where primary antibody was omitted
(data not shown). The ? 1 -ARs were mostly present in the
plasma membrane in both T and B cells ( fig. 4 ). The ? 1 -
AR protein correlated with changes found in ? 1 -AR
mRNA in both cells and was increased in T cells after
single IMMO. ? 2 -ARs were localized mostly in the nucle-
us of control groups and also correlated well with ? 2 -AR
mRNA in both cell types. While single IMMO did not
affect the mRNA, nor the protein of ? 2 -AR in B cells, an
increase in ? 2 -AR mRNA as well as its protein was found
in T cells. Moreover, some of the ? 2 -AR translocated
from the nucleus to the plasma membrane as a result of
Table 2. C ross-reactivity of antibodies used in immunofluorescence
Primary antibodyCross-reactivity ProviderCatalogue No.
?1-AR
?2-AR
?3-AR
mouse, rat, human
mouse, rat, human
mouse, rat
Santa Cruz Biotechnology, USA
Abnova, USA
Alpha Diagnostic International, USA
Sc-568
PAB2838
B3AR13-S

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Stress Differently Regulates
Adrenoceptors in T and B Cells
Neuroimmunomodulation 2012;19:69–78
73
stress exposure ( fig. 4 , white arrows). The ? 3 -ARs were
observed in the plasma membrane of T and B cells.
Stress-induced rise in ? 3 -AR mRNA correlated with up-
regulated ? 3 -AR protein in T cells. However, decreased
? 3 -AR mRNA did not reflect the same changes observed
in the protein level in B cells.
To explain functional significance of changes found in
? -ARs, we investigated whether changes in ? -ARs may
play a role in cell apoptosis. Therefore, we performed an-
other real-time PCR for proapoptotic Bax and caspase 3
mRNA as well as antiapoptotic Bcl-2 protein mRNA in T
and B cells ( fig. 5 ). In T cells, Bax mRNA (p ! 0.05) and
TB TBM-T TBM-B TBM
TB
TBM-T TBM-B
TBM
TB
TBM-T TBM-B
TBM
a
b
c
Fig. 1. Relative evaluation of cell purity in each fraction. Real-time
PCR for cell-specific CD markers in each fraction after cell sepa-
ration from the rat spleen. a T-lymphocyte-specific CD3 ? mRNA.
b B-lymphocyte-specific CD19 mRNA. c Monocyte/macrophage-
specific CD14 mRNA in each fraction. T = T-cell fraction; B = B-
cell fraction; TBM = mononuclear cells isolated from the rat
spleen; TBM-T = mononuclear cell fraction depleted of T cells
(negative control); TBM-B = mononuclear cell fraction depleted
of B cells (negative control).
0
1
2
3
4
5
6
7
8
9
10
T cells
B cells
11
104 mRNA/µg total RNA
?1-AR
?2-AR
Absolute ?-AR mRNA in lymphocytes
?3-AR
Fig. 2. Comparison of ? 1 -, ? 2 - and ? 3 -AR absolute mRNA levels
in T and B cells isolated from the rat spleen. In both T and B cells,
? 2 -AR gene expression was markedly higher than ? 1 - and ? 3 -AR
mRNA. In T cells, the ? 3 -AR mRNA level was lower than in B
cells. Each column shows the mean 8 SEM and represents an av-
erage of 6 control animals, from which both T and B cells were
isolated.
0
1
2
3
4
5
6
7
8
T cells
B cells
?1-AR mRNA/
GAPDH mRNA (AU)
Control
a
1 × IMMO
?1-AR mRNA in lymphocytes
***
0
1
2
3
4
?2-AR mRNA/
GAPDH mRNA (AU)
Control
b
1 × IMMO
?2-AR mRNA in lymphocytes
*
0
1
2
3
15
20
25
30
35
?3-AR mRNA/
GAPDH mRNA (AU)
Control
c
1 × IMMO
?3-AR mRNA in lymphocytes
***
*
Fig. 3. The mRNA levels of ? 1 - ( a ), ? 2 - ( b ) and ? 3 -ARs ( c ) in T and
B cells isolated from the rat spleen after acute IMMO stress for
2 h. In T cells, acute IMMO significantly increased the mRNA of
? 1 - ( a , p ! 0.001), ? 2 - ( b , p ! 0.05) and ? 3 -ARs ( c , p ! 0.001). In B
cells, only a decrease in ? 3 -ARs was observed ( c , p ! 0.05) after
exposure to IMMO compared to the control group. Each column
shows the mean 8 SEM and represents an average of 5 indepen-
dent cell fractions, each isolated from 1 animal sacrificed at the
same day. Statistical significance of IMMO versus the control
group: *  p ! 0.05, * * *  p ! 0.001. GAPDH = Glyceraldehyde 3-phos-
phate dehydrogenase; AU = arbitrary units.

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74
the Bax/Bcl-2 mRNA ratio (p ! 0.01) increased signifi-
cantly, while caspase 3 mRNA did not change after
IMMO (p 1 0.05; fig. 5 , white columns), though we ob-
served a nonsignificant rising trend in caspase 3 mRNA
after stress exposure. In B cells, no significant changes of
pro- and antiapoptotic parameters were found (p 1 0.05;
fig. 5 , white columns). We further investigated changes in
the gene expression of T- and B-cell parameters produced
in the cytoplasm [interleukin (IL)-2 and IL-4] or located
in the plasma membrane (CD69 and C40) that play an
important role in immune response. The gene expression
of IL-2 and IL-4 was not affected by stress in T cells (p 1
0.05; table 3 ), while the mRNA of both genes significant-
ly declined in B cells after IMMO (p ! 0.05; table 3 ). Sim-
ilarly, the T-cell-specific CD69 gene expression was not
affected by stress (p 1 0.05), but the B-cell-specific CD40
mRNA was decreased after single IMMO (p ! 0.05; ta-
ble 3 ).
10 µm
B-cell cont.B-cell IMMO T-cell IMMOT-cell cont.
?1-AR
?2-AR
?3-AR
Fig. 4. Subcellular distribution and protein levels of ? 1 -, ? 2 - and
? 3 -ARs in T and B cells isolated from the rat spleen after acute
IMMO stress for 2 h. Immunofluorescence was done using pri-
mary antibody against ? 1 -, ? 2 -, and ? 3 -ARs and fluorescently
labeled corresponding secondary antibody, as described in
Material and Methods. Nuclei were stained by DAPI. The ? 1 -
and ? 3 -ARs were localized primarily in the plasma membrane,
whereas ? 2 -ARs were present in the nucleus of both lymphocyte
populations. In T cells, protein levels of ? 1 -, ? 2 - and ? 3 -ARs in-
creased after IMMO and correlated with changes in mRNA lev-
els. Nevertheless, some of the ? 2 -ARs translocated from the nu-
cleus to the plasma membrane (white arrows). In B cells, protein
levels of ? 1 - and ? 2 -AR were not changed while ? 3 -AR protein
increased after IMMO. Each image represents the observation
from 10 independent micrographs. cont. = Control group;
IMMO = IMMO group.
Table 3. m RNA levels of IL-2, IL-4, CD69 and CD40 in T and B
cells isolated from the rat spleen after 2-hour IMMO stress
Group T-cell mRNA, AUB-cell
mRNA AU
IL-2control
1!IMMO
control
1! IMMO
control
1! IMMO
control
1! IMMO
1.0480.15
0.7280.29
1.1980.28
1.4280.09
1.1480.28
0.8080.28
–
–
1.1380.22
0.3980.11*
1.2780.37
0.5280.24*
–
–
2.1680.67
0.8880.33*
IL-4
CD69
CD40
I n T cells, no significant changes were found (p > 0.05). In B
cells, a significant decrease in IL-2, IL-4 and CD40 (p < 0.05) was
observed after IMMO, compared to the control group. Values are
displayed as the mean 8 SEM and represent an average of 5–6
independent samples. Statistical significance of the IMMO group
versus the control group: * p < 0.05.
Color version available online

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Neuroimmunomodulation 2012;19:69–78
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Discussion
Stress-induced rise in circulating CAs, followed by
modulation of ? -ARs, is one of the pathways involved in
the stress-mediated effects on immune functions. In-
creased CA levels were observed in the rat spleen after
single and repeated IMMO stress [6, 8] . This rise was as-
sociated with changes in AR expression that might play a
role in altered gene expression of several cytokines [6, 11] .
Until recently, CA-mediated effects were mainly ascribed
to ? 2 -ARs [2, 12, 13] . However, few reports have indicated
that other ? -AR subtypes coexist in the immune system
and might facilitate some of the effects of CAs in the
spleen [14] or directly on peripheral lymphocytes [18, 19] .
Moreover, exercise-induced CAs, acting predominantly
through the ? 1 -AR were responsible for suppression of
plasma tumor necrosis factor- ? after lipopolysaccharide
administration [26] . ? 1 -AR was shown to be involved also
in the modulation of inflammatory cytokines and in
interferon- ? -inducible gene expression in the skin and
lungs [27, 28] .
Our study confirmed the expression of all 3 ? -ARs on
splenic T and B cells, as determined by real-time PCR and
immunofluorescence. Additionally, the gene expression
of ? 2 -AR was predominantly found in both lymphocyte
populations and was approximately 4 to 5-times higher
than ? 1 - and ? 3 -AR mRNA. Moreover, the expression of
? 3 -AR was lower in T cells compared to B cells. We fur-
ther observed T cells appearing more vulnerable to acute
stress compared to B cells. The gene expression and pro-
tein level of all 3 ? -ARs significantly increased in T cells
after acute IMMO stress. Similarly, upregulation of ? -
ARs after agonist treatment was already reported in
many papers. It was shown that after short-term exhaus-
tive exercise, characterized by increased plasma CAs, in-
creased ? 2 -AR expression and density was observed in
human peripheral lymphocytes [29, 30] . In addition, ? 3 -
ARs were shown to be upregulated in rat neonatal cardio-
myocytes following chronic exposure to noradrenaline
[31] . The ? 1 -AR overexpression has been detected in pe-
ripheral lymphocytes of hypertensive and cardiac pa-
tients [18, 19] that are often associated with higher plasma
CAs [32, 33] . All 3 ? -ARs increased following a 3-hour
exposure to phenylephrine ( ? -adrenergic agonist) in
PC12 cells in vitro [22] .
We have shown that in both T and B cells, ? 1 - and ? 3 -
ARs are localized in the plasma membrane, while ? 2 -ARs
are mainly found in the nucleus of both cell types and
translocate to the plasma membrane of T cells after
exposure to IMMO. Several studies showed that ? 2 -AR is
localized not only in the membrane and cytoplasm, but
also in the nucleus [22, 34] . It is suggested that some of
0
1
2
3
4
5
T cells
B cells
Bax mRNA/
GAPDH mRNA (AU)
Control
a
1 × IMMO
Bax mRNA in lymphocytes
*
0
1
2
3
4
5
6
7
Bcl-2 mRNA/
GAPDH mRNA (AU)
Control
b
1 × IMMO
Bcl-2 mRNA in lymphocytes
0
1
2
3
4
5
Bax/Bcl-2 mRNA
Control
c
1 × IMMO
Bax/Bcl-2 mRNA in lymphocytes
**
0
1
2
3
4
Caspase 3 mRNA/
GAPDH mRNA (AU)
Control
d
1 × IMMO
Caspase 3 mRNA in lymphocytes
Fig. 5. The mRNA levels of proapoptotic
Bax and caspase 3 mRNA and antiapo-
ptotic Bcl-2 mRNA in T and B cells iso-
lated from the rat spleen after acute IMMO
stress for 2 h. In T cells, acute IMMO sig-
nificantly increased Bax mRNA and the
Bax to Bcl-2 mRNA ratio ( a , c white col-
umns, p ! 0.05), while Bcl-2 and caspase
3 mRNA did not change significantly
( b , d white columns, p 1 0.05). No signifi-
cant changes of all these parameters were
observed in B cells after IMMO (grey col-
umns, p 1 0.05) compared to the control
group. Each column shows the mean 8
SEM and represents an average of 5–6 an-
imals, from which both T and B cells were
isolated. Statistical significance of IMMO
versus the control group: *  p ! 0.05; * *  p !
0.01. AU = Arbitrary units.

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Neuroimmunomodulation 2012;19:69–78
76
? 2 -ARs synthesized in the cytoplasm are trafficked onto
the membrane to mediate adrenergic response, whereas
other ? 2 -ARs are translocated into the nucleus to regulate
gene expression [35] . Increased gene expression of all 3
? -ARs together with ? 2 -AR translocation after phenyl-
ephrine treatment has been shown to modulate apoptosis
of PC12 cells in vitro [22] . As we observed similar chang-
es in ? -AR expression and translocation in T cells, fol-
lowed by exposure to CA, we proposed that a similar
mechanism might modulate T-cell functions during
stress.
Therefore, we investigated whether all changes ob-
served have any functional significance, especially in reg-
ulation of apoptosis in T and B cells. The level of proapo-
ptotic Bax mRNA and the ratio of Bax to Bcl-2 mRNA
were raised, while caspase 3 mRNA remained unchanged
in T cells after stress. This suggests an induction of apo-
ptotic process in T cells. However, the 2-hour IMMO was
not long enough to stimulate the gene expression of other
apoptotic proteins, e.g. caspase 3. Most probably, a pro-
longed or more excessive stress stimulus would induce
further downstream changes in the process of T-cell
apoptosis. A similar observation was described in PC12
cells, where all ? -AR subtypes increased after short incu-
bation with an apoptotic agent or phenylephrine treat-
ment and more pronounced when both stimuli were
combined [22] . In addition, the adrenoceptor signaling
pathway was also involved in lymphocyte apoptosis [23] .
Therefore, we can propose that the ? 1 -, ? 2 - and ? 3 -AR
interplay and ? 2 -AR translocation play an important role
in T-cell apoptosis induced by CAs. We cannot exclude
that heterodimerization of ? 1 - and ? 2 -ARs or between
? 2 - and ? 3 -ARs may occur in lymphocytes and lead to
distinct signaling with unique functional and pharmaco-
logical properties as described in cardiomyocytes and
HEK 293 cells [36–38] . Nevertheless, whether the in-
crease in all ? -ARs is a consequence or inducer of apo-
ptotic processes remains to be elucidated. Compared to T
cells, no changes were found in the gene expression of
apoptotic proteins in B-cell fractions.
There is evidence that chronic stress might suppress
and/or shift the immune response towards Th2-mediat-
ed humoral immune reaction. IL-2 is considered a Th1
cytokine, while IL-4 induces the Th2 response. Addition-
ally, IL-2 production is usually decreased, while that of
IL-4 is increased following stress exposure [2, 39, 40] .
IL-4 also has the potency to inhibit monocyte/macro-
phage activation when released by lymphocytes [41, 42] .
These changes favor many immune diseases. Several pa-
pers demonstrated that among other cytokines, IL-2 and
IL-4 are regulated by ? -adrenergic mechanism [43] .
CD69 production increases in activated T cells and CD40
appears during B-cell activation and differentiation [44,
45] . Induction of CD40 on B cells was also reported in
experimental autoimmune neuritis [46] . We wanted to
know whether acute stress might evoke changes in the
transcription level of these parameters in lymphocytes
and stimulate Th2 polarization. We also investigated
whether stress per se is able to stimulate the process of
lymphocyte activation, independent of any specific im-
mune challenge.
Nevertheless, we observed no significant alterations in
T cells. We propose that without apoptosis, upregulation
of ? -ARs may probably affect different immune param-
eters important for T-cell function, e.g. adhesive recep-
tors involved in lymphocyte trafficking [4, 47] . For ex-
ample, co-operation of ? 2 - and ? 3 -ARs was found in he-
matopoietic progenitor cell mobilization in the bone
marrow [48] .
Compared to T cells, we found a significant drop in
IL-2, IL-4 and CD40 mRNAs in B cells. A ? -AR-mediat-
ed suppression of IL-2 receptors has been demonstrated
in human lymphocytes [43] . Similarly, in phytohemaglu-
tinin-stimulated peripheral blood mononuclear cells,
IL-4 synthesis was decreased by a high dose of CAs. A
? -blocking agent attenuated most of the CA effects in
lymphocytes [49] . Although we found changes in the ex-
pression of these immune parameters in B cells, it is com-
plicated to correlate them with changes in ? -ARs, espe-
cially as we did not observe significant changes in ? -ARs
in B cells.
In summary, this is the first evidence on the expres-
sion of all 3 ? -AR subtypes on T and B cells. Further-
more, acute IMMO stress increases the expression of ? 1 -
? 2 - and ? 3 -ARs and translocates ? 2 -ARs to the plasma
membrane of T cells isolated from the rat spleen. These
changes are most probably involved in the regulation of
T-cell apoptosis during stress exposure. Nevertheless, de-
tailed mechanism and functional importance of all ? -AR
subtypes in lymphocyte populations during the stress re-
main to be elucidated.
Acknowledgements
This work was supported by VEGA grants 2/0036/11, 2/0188/09
and VEGA 2/0049/10, NFM/EEA SK0095 grant and Nadacia In-
tenda. The authors thank Ms. Marta Sirova for her excellent tech-
nical help with immunostaining.

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Adrenoceptors in T and B Cells
Neuroimmunomodulation 2012;19:69–78
77
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