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β1-Adrenergic Receptors on Immune Cells
Impair Innate Defenses against Listeria
Rebecca T. Emeny, Donghong Gao and David A. Lawrence
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Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
J Immunol 2007; 178:4876-4884; ;
doi: 10.4049/jimmunol.178.8.4876
http://www.jimmunol.org/content/178/8/4876
The Journal of Immunology
␤1-Adrenergic Receptors on Immune Cells Impair Innate
Defenses against Listeria
Rebecca T. Emeny, Donghong Gao, and David A. Lawrence1
S
tress is known to increase an organism’s susceptibility to
infection and disease progression, contributing to individual morbidity and mortality. Despite the enormous impact
of stress on our health (1) as well as rising health care expenditures
(2), precise molecular and cellular mechanisms responsible for
neuroimmunosuppression are uncertain. This study investigated
the role of sympathetic nervous system modulation of murine host
defenses against the well-defined intracellular pathogen Listeria
monocytogenes (LM).2 Primary infection with the Gram-positive
bacterium LM activates both innate and adaptive immune cells to
produce cytokines required for bacterial clearance (3). Successful
host resistance (measured by a decline in LM) is mediated by NK
and CD8⫹ T cells (4). This decline usually begins by 3 days after
infection with a relatively low dose (⬍104 CFU) of LM (5). Day
3 of infection is the time when innate immunity is initiating adaptive immune mechanisms (4, 6). Because host defense against LM
infection is dependent upon the coordination of innate and adaptive cell-mediated immune responses, it is a useful infectious
Laboratory of Clinical and Experimental Endocrinology and Immunology, Wadsworth Center, New York State Department of Health, Albany, NY 12201
Received for publication September 1, 2006. Accepted for publication February
1, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. David A. Lawrence, Wadsworth
Center, New York State Department of Health, Molecular Medicine, Empire State
Plaza, C419, Albany, NY 12201. E-mail address: [email protected]
2
Abbreviations used in this paper: LM, Listeria monocytogenes; ␤1AR, ␤1-adrenoceptor; ␤2AR, ␤2-adrenoceptor; ␤AR, ␤-adrenoceptor; CORT, corticosterone; CR,
cold restraint; DA, dopamine; DTH, delayed-type hypersensitivity; Epi, epinephrine;
LLO91–99, listeriolysin O91–99; NE, norepinephrine; RT, room temperature; WT,
wild type.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
model with which to analyze the influence of stress on host immunity. We have previously shown that neither cytokine profiles
nor depletion of B or T cells can explain our model of stress-induced
inhibition of host defenses (7, 8). In this study, we further investigated
cytotoxic mechanisms that may be involved in stress-altered early
host defenses, including LM-induced CD8⫹ T cell expansion, perforin expression, and anti-keyhole limpet hemocyanin (KLH) humoral and delayed-type hypersensitivity (DTH) responses.
The experimental model of 1-h cold (4°C) restraint (CR) is
known to elicit both physical and psychological stress (9, 10). We
have used CR treatment followed by a low-dose bacterial infection
in mice in an attempt to simulate a familiar human condition,
namely the experience of psychological or physical stressors and
exposure to common infectious agents. Psychological and physiological stresses elicit functional changes in many cell types, by
modifying the supply of oxygen and metabolites required for a
successful “fight or flight” response (11). In addition to controlling
cardiovascular functions, energy metabolism, and thermoregulation, stress factors are known to influence immune cell functions.
Numerous neuroendocrine factors, such as prostaglandins, glucocorticoids, catecholamines, and neuropeptides, have regulatory
influences on host-pathogen interactions (12). It has been proposed
that catecholamines provide a physiologic mechanism to prevent
an overactive cell-mediated immune response, by shifting the activity of APCs and Th1 cells from a Th1-promoting to a Th2promoting response via ␤2-adrenoceptor (␤2AR) (13). However,
the kinetics of stress-factor interactions with immune cells also
may be critical in determining whether stress-mediated neuroimmune interactions have beneficial or detrimental consequences.
We hypothesize that if immune cells encounter stress before they
begin to respond to a pathogen, the immunosuppressive effects of
stress-induced neuroendocrine factors can weaken host defenses
and increase the pathogenic burden. It is this acute, stress-induced
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Cold restraint (CR) for 1 h elicits a psychological and physiological stress that inhibits host defenses against Listeria monocytogenes
(LM). Previous analyses indicated that this inhibition is not due to depletion of B or T cells but is instead dependent on signaling
through ␤-adrenoceptors (␤ARs). We now show that impaired host resistance by CR cannot be accounted for by a decrease in
LM-specific (listeriolysin O91–99 tetramerⴙ) effector CD8ⴙ T cells; this result is consistent with previous observations that CRinduced effects are mainly limited to early anti-LM responses. ␤2-Adrenoceptor (␤2AR)ⴚ/ⴚ FVB/NJ and wild-type FVB/NJ mice
had equivalent anti-LM defenses, whereas ␤1-adrenoceptor (␤1AR)ⴚ/ⴚ FVB/NJ mice had lower levels of LM even when subjected
to CR treatment. Additionally, host-resistance competency of ␤1ARⴚ/ⴚ mice could be transferred to irradiated wild-type mice
reconstituted with ␤1ARⴚ/ⴚ bone marrow progenitors and spleen cells, indicating that ␤1AR signaling on immune cells reduces
anti-LM responses. ␤1ARⴚ/ⴚ mice had improved cellular (delayed-type hypersensitivity) responses while ␤2ARⴚ/ⴚ mice had
improved humoral responses (IgG1, IgG2, and IgM), a result that further explains the strain differences in LM defenses. CRinduced expression of ␤1AR and ␤2AR mRNA was assessed by real-time PCR. CR treatment significantly increased ␤AR mRNAs
in Ficoll-purified and F4/80ⴙ-enhanced liver but not splenic homogenates, demonstrating an organ-specific effect of stress that
alters host defenses. Finally, CR treatment induced early increases in perforin expression that may enhance immune cell apoptosis
and interfere with LM clearance. In conclusion, ␤1AR signaling has immunomodulatory effects on early cell-mediated immune
responses; a lack of ␤1AR signaling improves antilisterial defenses and cell-mediated immunity, in general. The Journal of
Immunology, 2007, 178: 4876 – 4884.
The Journal of Immunology
4877
Materials and Methods
Mice, CR treatment, and LM infection
All in vivo studies were performed using mice housed at the Wadsworth
Center Animal Production Unit in accordance with the Institutional Animal
Care and Use Committee Guidelines. The listeriolysin O91–99 (LLO91–99)
(I-Ad) tetramer study used BALB/c mice from Taconic Farms. The
␤1AR⫺/⫺ and ␤2AR⫺/⫺ mice (provided by Dr. B. Kobilka, Stanford University, Palo Alto, CA) were bred on the FVB/NJ background. As previously described, both ␤AR-deficient strains were derived by the insertion
of a neomycin resistance cassette into the genetic sequence of the fourth
transmembrane domain of the receptor, rendering the transcription of the
complete receptor impossible (19). Strain verification studies were performed using real-time PCR and RT-PCR to confirm the lack of ␤1AR and
␤2AR transcript in both liver and spleen of ␤1AR⫺/⫺ and ␤2AR⫺/⫺mice,
respectively (data not shown). Control mice were left in their original cages
undisturbed, while mice subjected to CR treatment were individually restrained in well-ventilated plastic 60-ml syringes at 4°C for 1 h in the dark.
CR represents a physical and a psychological stress. CR was performed
between 8 and 11 a.m. on day 0; the mice were infected immediately after
CR. LM was originally isolated from a meningitis patient and has been
maintained as previously described (20). Mice were i.v. injected with a
sublethal dose of LM (2–3 ⫻ 103 CFU/i.v. injection for FVB/NJ, and
3.5–10 ⫻ 103 CFU/i.v. for BALB/c mice) with or without CR administered
before the inoculation. Except for anti-KLH Ig and DTH responses, all
other measurements were obtained from different groups of mice depending upon the background strain or timing that was required for each assay.
Determination of viable LM burden in liver and spleen
To determine the bacterial load in mice following LM infection, we performed enumeration of viable LM as described previously (5). Briefly,
mice were sacrificed by lethal CO2 anesthesia, and the spleen and liver
were removed aseptically and homogenized in sterile 0.9% NaCl. Serial
dilutions of organ homogenates were plated on blood-agar plates and cultured overnight for enumeration of viable LM. Bacterial burdens are expressed as number of viable LM CFU per organ.
Flow cytometric analysis of T cells
Spleens were removed aseptically from 6- to 8-wk-old BALB/c male mice
as described above, and single-cell suspensions were prepared by grinding
the tissue between two frosted microscope slides. RBCs were lysed and
FIGURE 1. Listeria-specific CD8⫹ T cell expansion is unaltered by CR.
Memory (CD62L⫺) CD8⫹ T cells were quantified in spleens from CRstressed and nonstressed BALB/c mice at 8 and 10 days after LM infection
(i.v.; 7.3–9.5 ⫻ 103 CFU). A, Typical flow cytometric dot plots of CD8-gated
lymphocytes from an uninfected mouse and from a day 8-infected mouse. The
memory (CD62L⫺)/LM-specific (LLO91–99 tetramer⫹) cells were enumerated
by first gating the spleen cells by light scatter and CD8 expression, as described in Materials and Methods. B, Memory (CD62L⫺)/LM-specific
(LLO91–99 tetramer⫹) cells were enumerated in spleens from control and CRtreated mice. Results are representative of two repeated experiments; each bar
shown is the mean (SD) for two mice. C, The kinetics of LM killing, showing
the LM levels at time of CD8 analysis.
cells were washed twice with PBS, and total cell numbers were determined;
1 ⫻ 106 cells/tube were used for staining. MHC class I (H2-Kd) tetramers
containing an immunodominant LM epitope from the pore-forming toxin
listeriolysin (LLO91–99) were used to quantify LM-specific responses; the
tetramers were provided by Dr. E. G. Pamer (Sloan-Kettering Institute,
New York, NY). All mAbs against cell surface molecules were purchased
from BD Biosciences Pharmingen. Cells were treated with FcR-blocking
buffer containing 20 ␮g/ml streptavidin and anti-mouse CD16/CD32 FcR
(1 ␮g/tube) in 50 ␮l of staining buffer (0.5% BSA and 0.02% NaN3 in PBS
(pH 7.5)) for 20 min on ice. Further staining with PE-MHC class I tetramer
(LLO91–99), allophycocyanin-anti-mouse-CD62L, and FITC-anti-mouseCD8 was performed for 60 min on ice. Enumeration of LM-specific CD8⫹
populations was accomplished using TruCount tubes (BD Biosciences
Pharmingen). Regulatory T cell subsets following LM infection with or
without CR-treatment were analyzed using a combination of FITC-CD4/
PE-GITR/allophycocyanin-CD25 Abs. The acquisition and analysis of all
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immunosuppression before immune activation that is the focus of
our current studies.
Cell surface dopaminergic and adrenergic receptors, which are
expressed on immune cells (12), bind the catecholamines dopamine
(DA), epinephrine (Epi), and norepinephrine (NE). Pharmacologic
blocking studies provided the first evidence for an immunomodulatory role of ␤1-adrenoceptor (␤1ARs) in host immunity (7). Specifically, peripheral administration of atenolol (a ␤1AR-specific antagonist commercially available as Tenormin) blocked the CR-induced
delay in bacterial clearance whereas ␣AR- or ␤2AR-specific antagonists (phentolamine and ICI118,551, respectively) did not. It is well
documented that ␤2AR is ubiquitously expressed on Th0, Th1, and
B cells; ␤2AR is down-regulated on Th2 cells by histone deacetylation (14), thereby implicating this receptor in the regulation of
humoral and Th1 functions by catecholamines (15). Depending
upon the activation state and developmental stage of the Th cell,
the ␤2AR signal can enhance (16, 17) or suppress (18) IFN-␥
production. Despite a well-established role of sympathetic neuroimmune modulation and despite also the widespread clinical use of
“␤ blockers” and synthetic catecholamines, the immunologic relevance of ␤1ARs has been largely unexplored. The current in vivo
studies used wild-type (WT) mice (either BALB/c or FVB/NJ) and
FVB/NJ mutant strains with a deficiency of ␤1AR or ␤2AR
(␤1AR⫺/⫺ and ␤2AR⫺/⫺, respectively) to investigate the role of
stress-induced ␤-adrenoceptors (␤ARs) on host immunity, and to
evaluate overall in vivo immunity to KLH. Based on previous
studies, our a priori hypothesis was that CR stress-induced ␤1AR
signaling impairs host defenses against a low-dose LM infection
by altering innate immune mechanisms.
4878
␤1ARs ALTER HOST IMMUNITY
FIGURE 2. CR-induced modulation of ␤1AR⫺/⫺, ␤2AR⫺/⫺, and
FVB/NJ (WT) host defenses against
LM. These three strains (n ⫽ 5–11/
treatment/strain) were assessed for viable LM in the liver (A) and spleen (B)
at 3 days after infection with 2.5–
3.0 ⫻ 103 CFU immediately after CR
treatment. The ⴱ indicates a significant
difference (⬍0.05) compared with the
FVB/NJ LM-infected, nonstressed
control.
Perforin expression by Western blot analysis
Protein was isolated from frozen livers and spleens of 2- to 3-mo-old male
BALB/c mice and ␤1AR⫺/⫺ male and female mice were treated with or
without CR and infected with 3.6 ⫻ 103 LM by homogenization of a
portion of each organ in 0.5 ml of mammalian protein extraction reagent
(Pierce) with protease inhibitor mixture (Sigma-Aldrich). Lysed homogenates were centrifuged for 30 min at 15,000 ⫻ g. Protein concentrations
were measured using the BCA protein assay kit (Pierce), and 100 ␮g of
protein from each sample was diluted with one-half volume of sample
buffer containing 30% (v/v) glycerol, 10% (v/v) 2-ME, and 0.25% (w/v)
bromophenol blue in 62.5 mM Tris-HCl buffer (pH 6.8). SDS-PAGE was
performed in 4 –20% gradient separating gels; the gels were then placed
upon nitrocellulose paper and subjected to blot transfer. The blotted proteins were blocked with 5% fish gelatin in PBS 0.01% NaN3 for 1 h at room
temperature (RT) and washed in TBS-T (25 mM Tris-HCl, 125 mM NaCl,
and 1.0% Tween 20 (pH 8.0)). Blots were incubated with mAb to mouse
␤-actin (Sigma-Aldrich) and either rabbit anti-rat perforin (catalog no.
CPP100; Cell Sciences) or rabbit anti-mouse granzyme B (catalog no. RB9015-PO; NeoMarkers) in blocking buffer overnight at 4°C. Blots were
washed twice in TBS-T and then incubated with HRP-conjugated secondary Abs (goat anti-rabbit IgG for perforin and granzyme and goat antimouse IgG, for actin; Sigma-Aldrich) for 2 h at RT with rocking. Blots
were washed three times for 20 min each in TBS-T and then developed
with Super Signal/chemiluminescent substrate (Pierce) for 5 min and assayed with a LAS-1000plus (Fuji).
Splenocyte and liver preparation for RNA isolation
Male BALB/c mice were either CR-treated or left undisturbed in their
home cage. Immediately after CO2 administration, mice were perfused
through the right ventricle with 30 ml of PBS, followed by 10 ml of digestion buffer (0.05 M TES (C6H15NO6S) and 0.36 M CaCl (pH 7.5)).
Livers and spleens were placed into digestion buffer containing collagenase
IV (200 ␮g/ml; Sigma-Aldrich) and DNase I (50 ␮g/ml; Sigma-Aldrich)
and were coarsely chopped with a sterile razor blade before 37°C incubation for 20 min. Splenocytes were then homogenized as described above,
whereas livers were pipetted through a Teflon mesh. Single-cell suspensions were washed twice in PBS and then layered over mouse Ficoll (Ficoll:metrizoate 12:5, density 1.090 g/ml) (21) and centrifuged for 15 min
at RT at 1300 ⫻ g. The total Ficoll-purified cells were counted and pelleted
for immediate isolation of total RNA.
F4/80⫹ Kupffer cells were isolated from liver homogenates following
the above procedure with slight modifications. After single-cell suspensions had been obtained by passage through a Teflon mesh, liver homogenates were centrifuged (110 ⫻ g for 2 min at 4°C) to remove hepatocytes
(22). F4/80⫹ cells were enriched from the single-cell suspension by use of
a SpinSep mouse enrichment mixture, SpinSep mouse dense particles, and
density gradient centrifugation cell separation procedures per the manufacturer’s protocol (StemCell Technologies).
RNA isolation from immune cells and ␤AR mRNA determination
by TaqMan real-time PCR
Cell pellets containing 5 ⫻ 105–1 ⫻ 106 Ficoll-purified liver or spleen cells
or F4/80-enriched cells from liver homogenates were resuspended in 50 ␮l
FIGURE 3. ␤ARs increase in liver but not spleen immediately following CR stress. RNA was isolated from Ficoll-purified BALB/c liver (A) and spleen
(C) homogenates, and expression of ␤ARs was quantified by real-time TaqMan PCR (n ⫽ 5 and 4 for nonstressed, control and CR groups, respectively).
F4/80⫹cells (B) were enhanced from liver homogenates obtained from CR-treated and control mice (n ⫽ 3). The ⴱ indicates significant differences (p ⬍
0.05) measured between stressed and control mice. The # indicates significant differences (p ⬍ 0.005) between ␤1AR and ␤2AR expression in the spleen,
irrespective of CR treatment.
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specimens were performed on a BD FACSCalibur using CellQuest software at the Wadsworth Center Flow Cytometry Core.
The Journal of Immunology
4879
Table I. Serum corticosterone at baseline and following CR treatmenta
BALB/c
FVB/NJ
␤1AR⫺/⫺
␤2AR⫺/⫺
Baseline Serum
CORT (pg/ml)b
Average CORT Increase
in CR as % Controlc
269.7 ⫾ 163.4
272.0 ⫾ 85.0
221.0 ⫾ 52.1
283.9 ⫾ 97.6
208.8
155.2
130.9
128.8
n ⫽ 4 – 8 animals tested/strain.
No significant differences found between strains.
c
Significant increases in CR vs control observed for all strains ( p ⬍ 0.05).
a
b
Hemopoietic ablation and lymphocyte reconstitution
of recipient mice
FVB/NJ WT- and ␤AR-deficient mice that were used as recipient hosts for
lymphocyte reconstitution experiments received lethal 137Cs irradiation of
10 Gy (dose rate 2.5 Gy/min). On the following day, a mixture of 1 ⫻ 106
bone marrow progenitor cells and 10 ⫻ 106 splenocytes was injected i.v.
in 200 ␮l through the tail vein as described previously (23). Transplant
recipients were housed in pathogen-free facilities and given neomycin
(1 mg/ml) in drinking water for the first 2 wk. After 6 – 8 wk total, CR
administration, LM infection, and subsequent immunologic analysis of
host immune responses were performed as described above.
Splenocyte and bone marrow isolation
Spleens were aseptically harvested from euthanized FVB/NJ WT- and
␤AR-deficient donor mice into a sterile petri dish containing Dulbecco’s
PBS (without calcium and magnesium; Sigma-Aldrich) on ice. Inside a
biosafety hood, spleens were transferred into another sterile petri dish with
5 ml of Dulbecco’s PBS. Each spleen was homogenized between two
frosted microscope slides (Erie Scientific), and the cell suspension was
transferred into a 15-ml polypropylene tube with a Pasteur capillary pipette. The cell suspension was allowed to settle for 3 min at RT for separation of cellular debris, and the single-cell suspension in the supernatant
was then transferred to a new tube. Following centrifugation at 200 ⫻ g for
10 min, RBCs were eliminated by use of lysing buffer (0.15 M NH4Cl, 10
mM KHCO3, and 0.1 mM Na2 EDTA (pH 7.2–7.4)). After lysis (5 min at
RT), the cell suspension was centrifuged again, and the cell pellet was
resuspended in 10 ml of DPBS; 20 ␮l of this cell suspension was taken for
determination of total cell numbers (Coulter Counter). After centrifugation,
the cell pellet was resuspended to the appropriate concentration in sterile
PBS. Bone marrow progenitors were isolated as described previously (24).
KLH immunization
Female WT (FVB/NJ), ␤1AR⫺/⫺, and ␤2AR⫺/⫺ mice (age 2 mo) were
immunized with 100 ␮g of KLH (Calbiochem) plus TiterMax Gold adjuvant in 200 ␮l of saline at days 1 and 28.
Corticosterone (CORT) measurement
Serum preparation
The concentrations of serum CORT at baseline and immediately following
1-hr CR treatment were determined by enzyme immunoassay with CORT
EIA Ab (Assay Designs). The sensitivity of the CORT EIA was 32 pg/ml.
Peripheral blood was obtained by retro-orbital phlebotomy, into 1.7-ml
Eppendorf tubes. After it had been allowed to clot overnight at 4°C, serum
was collected following centrifugation.
FIGURE 4. CR stress increases perforin protein. Protein was obtained from livers (A) and spleens (B) of stressed (n ⫽ 3) or nonstressed, control BALB/c
mice (n ⫽ 4) in the absence of LM and from mice infected with LM with or without prior stress treatment (n ⫽ 3 for all time points examined following
LM infection (4, 24, and 48 h), except LM infected, nonstressed, control 48 h, n ⫽ 2). The ⴱ indicates significant increases (p ⬍ 0.05) in CR-treated mice
compared with the control group at that time point.
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of PBS and lysed with RLT/2-ME buffer from Qiagen. Cell lysates were
homogenized with the Shredder Spin Columns provide by Qiagen, and
RNA was isolated using the Qiagen RNA capture minicolumns, according
to the manufacturer’s protocol for isolation of RNA from cultured cells.
Isolated RNA was resuspended in 60 ␮l of RNase-free water and quantified
using a Beckman DU 640 spectrophotometer. After dilution of each RNA
preparation to a working concentration of 0.1 ␮g/␮l, cDNA was prepared
from 1 ␮g of total RNA using a High-Capacity cDNA Archive kit from
Applied Biosystems. Following the synthesis reaction, an aliquot of 100 ␮l
of PCR-grade water was added to each tube, and 5 ␮l was used for amplification of murine ␤1AR, ␤2AR, and GAPDH using TaqMan Gene Expression Assay kits from Applied Biosystems. Amplifications were conducted in quadruplicate in a 7500 Real Time PCR instrument (Applied
Biosystems). Amplification conditions used were specified by the manufacturer as follows: 2 min at 50°C, then 10 min at 95°C, then 40 cycles of
15 s at 95°C, and 1 min at 60°C. Data for the amplification plot was
collected during the 60°C step. Relative quantitation results were measured
using the comparative cycle threshold method, whereby the amplification
of the gene of interest is normalized to amplification of the gene encoding
GAPDH, measured from the same cDNA synthesis sample. Four APC
lines, RAW 264.7, PMJ2-PC, N9, and A20, were also used for RNA isolation and subsequent ␤AR amplification procedures.
␤1ARs ALTER HOST IMMUNITY
4880
Table II. Western blot analysis of perforina in ␤1AR⫺/⫺ mice
Control
CR
Baseline (0 h)
24 h after LM Infection
70.7 ⫾ 28.3
82.0 ⫾ 42.1
58.1 ⫾ 18.3
57.3 ⫾ 8.9
a
Perforin protein normalized to actin, n ⫽ 4/group; no significant differences were
detected between control and CR-treated mice.
ELISA for IgG isotype and IgM
IgG isotype and IgM were measured by a standard ELISA, as described
previously (25).
DTH assay
Statistical analysis
Data from two or more independent experiments were combined and differences between two experimental groups (where n ⱖ 3) were determined
using the t test SigmaStat (Jandel Scientific). Effects of CR stress and
␤1AR or ␤2AR deficiency on host immunity were considered significant
only if the p value was ⬍0.05.
Results
Ag-specific expansion is not altered by CR stress
The in vivo expansion of CD8⫹ Ag-specific lymphocytes of control or CR-treated BALB/c mice was measured using the immunodominant LLO91–99 tetramer. Tetramer-positive cells were detected by day 8 after LM-infection (Fig. 1A), but no differences
were detected between CR-treated and control mice (Fig. 1B).
Stress-related changes in the percentage of either CD4⫹/CD25⫹ or
CD4⫹/CD25⫹/GITR⫹ splenic subsets were not observed at 24 h
after LM infection (data not shown).
FIGURE 5. Adoptive transfer of
␤1AR⫺/⫺, ␤2AR⫺/⫺, or FVB/NJ
(WT) bone marrow progenitor cells
and splenocytes into lethally irradiated
WT, ␤1AR⫺/⫺, or ␤2AR⫺/⫺ recipient
mice. Six weeks after reconstitution,
WT (A and D), ␤1AR⫺/⫺ (B and E), or
␤2AR⫺/⫺ (C and F) host mice (n ⫽
3–5 for all groups, except n ⫽ 2 for
␤1AR⫺/⫺ donor cells into ␤2AR⫺/⫺
hosts, and n ⫽ 2 for ␤2AR⫺/⫺ donor
cells into ␤1AR⫺/⫺ hosts) were LM
infected (3 ⫻ 103) and 3 days later
spleens (top panels, A–C) and livers
(bottom panels, D–F) were quantified
for LM (CFU/organ). The ⴱ indicates
significant differences (p ⬍ 0.05)
compared with the WT host reconstituted with WT immune cells. ⴱⴱ, Differences in LM burden compared with
the ␤1AR⫺/⫺ host reconstituted with
␤1AR⫺/⫺ immune cells (p ⫽ 0.057).
The immunosuppressive effect of CR previously reported for
BALB/c mice (5, 7, 8, 26) was also observed in WT FVB/NJ mice
by day 3 of a low-dose LM infection. However, because FVB/N
mice were more sensitive than BALB/c mice to LM, we had to use
a lower LM inoculum (⬃3 ⫻ 103 CFU). Liver colonization was
significantly increased in CR-treated WT mice compared with control WT mice (Fig. 2A; p ⫽ 0.037). CR did not suppress host
resistance of ␤1AR⫺/⫺ mice. Relative to WT or ␤2AR⫺/⫺ mice,
␤1AR⫺/⫺ mice had improved host defenses against LM; CR did
not impair their resistance in livers (Fig. 2A) or spleens (Fig. 2B).
Splenic and overall body weights did not differ statistically between the LM-infected mice treated with CR and those not subjected to CR (data not shown).
CR stress increases ␤AR mRNA in immunocytes of liver but not
spleen
Because ␤1AR and ␤2AR appear to have substantially different
effects on host defenses against LM, it was critical that we assess
the expression of these receptors by immune cells. We prepared
immune cells from both liver and spleen homogenates of BALB/c
mice and analyzed them for mRNA expression of ␤1AR and
␤2AR transcripts (Fig. 3). Immediately following CR treatment
and in the absence of LM infection, significant increases in
␤1AR and ␤2AR expression were measured in livers (Fig. 3A;
p ⫽ 0.04 and p ⫽ 0.004, respectively). We further investigated
the expression of ␤1AR and ␤2AR on the Kupffer (F4/80⫹) cells
of livers obtained from stressed and control mice (Fig. 3B).
Although both receptor subtypes were identified by real-time
TaqMan PCR analysis, stress treatment did not significantly alter
the expression levels. Splenocytes expressed greater levels of
␤2AR than ␤1AR (Fig. 3C; p ⬍ 0.005); stress treatment did not
significantly alter the expression of either receptor. Although radioisotope-binding assays have provided evidence for ␤ARs on
monocyte-derived cells, we used real time PCR to confirm expression of ␤1AR and ␤2AR in two cell lines of monocyte origin
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
DTH assays were performed 21 days after the last immunization with
KLH. After measurement of the thickness of the left and right footpad of
each mouse, KLH (100 ␮g/25 ␮l saline) was s.c. injected into the right
footpad and saline only was injected into the left footpad. The DTH response was measured 24 h later with a Spi dial thickness gauge. The response was defined as the difference between the right and left footpad
swellings.
Mice lacking ␤1AR are more resistant to CR-induced
immunosuppression than are WT or ␤2AR⫺/⫺ mice
The Journal of Immunology
4881
(RAW 264.7 and PMJ2-PC), a microglial cell line (N9), and a B
cell line (A20) (data not shown).
Serum CORT is increased following CR treatment
Although CORT is known to play a significant role in the stress
response, both WT- and ␤AR-deficient strains appear to have
equally robust increases in CORT following CR treatment (Table
I). Furthermore, our previous research using pharmacological
blocking agents had suggested a protective role for CORT in this
model of stress and infection rather than an immunosuppressive
one (5).
Perforin expression in liver is increased following CR treatment
Suppression of host immunity is associated with ␤1AR⫹ immune
cells
␤AR-competent immune cells were reconstituted in ␤AR-deficient, irradiated mice, and ␤AR-deficient immune cells were reconstituted in ␤AR-competent, irradiated mice to aid us in assessing the effects of ␤AR nonimmune and immune cells on immune
defenses after CR. WT (FVB/NJ) recipient mice reconstituted with
␤1AR⫺/⫺ immune cells had a statistically significant decrease (by
⬃3-fold) in bacterial colonization in the liver, relative to mice
reconstituted with WT immune cells (Fig. 5D; p ⫽ 0.03). The
transfer of ␤2AR⫺/⫺ cells into WT mice produced a nonsignificant
decrease in liver colonization. The transfer of WT immune cells
into ␤1AR⫺/⫺ hosts or ␤2AR⫺/⫺ immune cells into ␤2AR⫺/⫺
hosts gave rise to a greater bacterial burden in liver than did the
transfer of ␤1AR⫺/⫺ immune cells into ␤1AR⫺/⫺ hosts (Fig. 5, E
and F; p ⫽ 0.05). ␤1AR⫺/⫺ mice reconstituted with their own
immune cells had significantly lower bacterial colonization of the
liver than did control mice reconstituted with their own immune
cells (Fig. 5, D and E; p ⫽ 0.04). ␤2AR⫺/⫺ hosts reconstituted
with either WT or ␤1AR⫺/⫺ immune cells had lower bacterial
loads in their livers and spleens on day 3 of the infection than did
the same ␤2AR⫺/⫺ hosts reconstituted with ␤2AR⫺/⫺ donor cells,
but these effects were not statistically significant.
The absence of ␤1AR enhances cell-mediated immunity, whereas
␤2AR deficiency improves humoral responses
The production of Abs specific for KLH was measured in
␤1AR⫺/⫺, ␤2AR⫺/⫺, and WT (FVB/NJ) mice (Fig. 6, A–C). IgG
subtypes and IgM were highest in serum from ␤2AR⫺/⫺ mice and
lowest in ␤1AR⫺/⫺ mice; the single exception was IgG1 for which
WT mice had the lowest production. The differences in production
FIGURE 6. Anti-KLH humoral and cellular responses measured in
␤1AR⫺/⫺(n ⫽ 5), ␤2AR⫺/⫺(n ⫽ 7), or FVB/NJ (WT, n ⫽ 9) mice; bars
indicate SE. Female mice were injected with 100 ␮g of KLH in TiterMax
adjuvant, and retro-orbital blood samples were obtained for evaluation of
KLH-specific Ab responses (A–C) at the end of week 1 (IgM) and week 2
(IgG1 and IgG2a). Ab data are presented as optical densities standardized
to an internal assay control. DTH responses (D) were measured 3 wk after
a secondary immunization given at week 4. E, The ratio of humoral to
cell-mediated responses as the product of the summed Ab response divided
by the DTH response. The ⴱ indicates a significant difference (p ⬍ 0.05)
compared with WT control.
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The expression of the cytotoxic protein perforin was analyzed in
livers and spleens of BALB/c mice (Fig. 4) and ␤1AR⫺/⫺ mice
(Table II). Early changes in perforin expression were evident in
livers of CR-treated BALB/c mice (Fig. 4A). The greatest increases in perforin were observed immediately after stress treatment, and 4 h after LM infection ( p ⬍ 0.05). No changes in
perforin expression were observed in the spleen (Fig. 4B), nor
were differences in granzyme B expression detected in either liver
or spleen (data not shown). No significant increases were observed
in liver homogenates of ␤1AR⫺/⫺ mice immediately following CR
treatment or 24 h after LM infection (Table II).
4882
of IgG1 between ␤2AR⫺/⫺ and WT mice were statistically significant (Fig. 6A). In contrast to humoral immunity, the DTH response in ␤1AR⫺/⫺ mice was more robust than the DTH response
in either WT or ␤2AR⫺/⫺ mice (Fig. 6D). The ratio of humoral to
cell-mediated responses was significantly higher in ␤2AR⫺/⫺ mice
than in WT mice (Fig. 6E).
Discussion
cells to NK cell perforin-dependent killing (33). Thus, we suggest
that the early CR-induced increase of the cytotoxic perforin
molecule plays an important role in our experimental model of
neuroimmunomodulation by increasing apoptosis, which consequently increases susceptibility to infection.
LM infection is known to induce lymphocyte apoptosis through
the immunodominant LLO protein (34), and type 1 IFNs increase
the susceptibility of lymphocytes to apoptosis (35). LM-induced
apoptosis is thought to attenuate innate antilisterial responses because the engulfment of apoptotic lymphocytes by macrophages
favors IL-10 production (36). It was recently shown that NE also
can induce apoptosis of lymphoid cells (37), and our previous studies have demonstrated a stress-induced increase in TNF-␣ production in the liver between days 1 and 3 of an LM infection (26);
such an increase could confer increased susceptibility of lymphocytes to apoptosis. Therefore, a possible explanation for the observed stress-induced immunosuppression of host defenses is that
stressed mice display premature release of cytotoxic factors, and
these factors increase lymphocyte apoptosis, thereby overwhelming the innate mechanisms required for both bacterial clearance
and activation of optimal innate and adaptive responses. Perforininduced death of LM-infected cells does not influence total bacterial burden (38). Therefore, our observation that LM burdens are
similar in both stressed and control animals until day 2–3 of the
infection is consistent with a possible role of perforin-induced apoptosis early in the infection. Although differences in regulatory T
cell subsets were not detected at 24 h after LM infection, it is
possible that other CD4⫹ T cell subsets (i.e., CXCR3⫹) induce
apoptosis in the liver via mechanisms not involving proinflammatory cytokines (39). Additionally, the stress-induced increase
in ␤AR receptor expression in the liver, specifically ␤1AR signaling on F4/80⫹ cells, implicates catecholamine-induced
changes in monocytes. It is known that catecholamines alter
macrophage redox state (40 – 42) and early inflammatory cytokine profiles (43– 45).
Although our research aim has been to elucidate stress-induced
mechanisms of immunosuppression, the improved host resistance
that we observe in ␤1AR⫺/⫺ infected mice, relative to WT (FVB/
NJ)-infected mice, exemplifies the importance of sympathetic nervous system activities on host-pathogen interactions. The role of
sympathetic neuronal activity in regulation of host susceptibility to
infectious organisms, in particular LM, is well documented (46).
Along with our laboratory, others have shown that early host defenses against LM are enhanced by treatment with the neurotoxicant 6-hydroxydopamine (6-OHDA). 6-OHDA selectively and
temporarily ablates peripheral dopaminergic nerves, thereby depleting peripheral tissues of catecholamines because DA is the
precursor to NE. In these studies, LM colonization of livers and
spleens in sympathectomized mice was significantly reduced at
days 3–5 after infection, as compared with the colonization of
control mice with innervated organs (5, 47– 49). Additionally, peripheral administration of 6-OHDA blocked stress-impaired immunity in spleen but not in liver (5), a pattern that differs from the
complete abrogation of stress-induced immunosuppression in both
organs of ␤1AR⫺/⫺ mice. It is possible that the livers of 6-OHDAtreated mice are still susceptible to stress-induced release of Epi
from the adrenal glands, and circulating Epi could influence liver
defenses through functional ␤1AR signaling.
The enhanced host resistance of mice with denervated peripheral organs had been correlated with increased numbers of splenic
neutrophils (48) and activated peritoneal macrophages (49) during
the first 3 days of an i.p. infection. However, between days 5 and
7 of LM infection, 6-OHDA treatment decreased splenic leukocyte
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Although there have been numerous reports that ␤2AR-signaling
modulates immunity, this is the first report to describe ␤1AR inhibition of a DTH response and cell-mediated host immune defenses. Additionally, we have shown that CR treatment inhibits
host defenses against LM in mice with functional ␤1AR signaling
(WT FVB/NJ and ␤2AR⫺/⫺ mice), whereas ␤1AR⫺/⫺ mice are
not immunologically impaired by the CR treatment. Previous studies have demonstrated normal immune functions in ␤2AR⫺/⫺
mice (27). Our results agree with these findings in that WT and
␤2AR⫺/⫺ mice coped similarly with Listeria infection. Moreover,
both WT and ␤2AR⫺/⫺ mice were immunocompromised by CR
treatment. The present data demonstrating improved host defenses
against LM in the absence of ␤1AR corroborate the findings of
previous pharmacological studies in which ␤1AR was identified as
a stress-signaling molecule that weakens host resistance (5, 7, 8,
26). The CR-induced increase in bacterial colonization of WT and
mutant FVB/NJ mice and mRNA expression of ␤ARs in the liver,
but not in the spleen of BALB/c mice, also delineates the existence
of stress-induced responses that are organ specific.
Based on the lack of CR-induced inhibition of host immunity in
CD4⫺/⫺ BALB/c mice (8), it had been suggested that CD4⫹ T
cells, possibly regulatory T cells, or downstream CD8⫹ T cell
effectors are involved in the stress-induced immunosuppression.
However, CR-induced early changes in the number or phenotype
of peripheral lymphocytes have not been detected (8). In this
study, we show that CR does not alter the expansion of Ag-specific
CD8⫹ lymphocytes of LM-infected BALB/c mice, indicating that
CR-treatment does not interfere with the priming of LM-specific
effectors, which occurs within the first 12 h of infection (28). The
observed LM-specific CD8 responses further confirm the previous
suggestion (5) that CR-treatment inhibits defenses only early (days
2–3) after infection. The investigation of LM-specific CD8⫹ T
cells in ␤AR-deficient mice was not performed because the
LLO91–99-specific class I (H2d) tetramers do not match the MHC
of the FVB/NJ strain (H2q). Additionally, an increase in the number of regulatory T cells does not appear to be able to account for
the inhibition of host defenses against LM.
Because an increase in perforin expression would likely be perceived as beneficial for handling early primary LM infection (29),
we were surprised that CR treatment actually increased perforin
expression in the liver of BALB/c mice. This stress-induced increase of perforin was still seen at 4 h after LM infection of the
CR-treated mice but declined by 24 h. Stress-mediated alteration
of NK activities in mice has been previously reported; the duration
and type of stress treatment had differential effects on the immunologic outcome in that 1 h of hyperthermic treatment (30) or the
stress treatment of electric foot shock for 1 h daily for 3 days (31)
both decreased perforin. NK perforin-dependent cytotoxic activities (YAC-1 cytotoxicity) of mononuclear liver preparations were
also diminished 24 h after Epi injection (20 ␮g i.p.); however,
NKT cell Fas-dependent killing of syngeneic thymocytes was increased (32). Interestingly, Oya et al. (32) found a significant 50%
decrease in T cells (NK1⫺CD3high) 24 h after a 12-h restraint
stress treatment, the decrease was posited to be due to stress-induced apoptotic mechanisms. In another study, stress (H2O2, heat,
or high-density growth) increased the susceptibility of activated T
␤1ARs ALTER HOST IMMUNITY
The Journal of Immunology
Acknowledgment
We thank the Immunology Core of Wadsworth Center for their assistance
with the flow cytometry.
Disclosures
The authors have no financial conflict of interest.
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␤1ARs ALTER HOST IMMUNITY