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Housing Temperature−Induced Stress Is
Suppressing Murine Graft-versus-Host
Disease through β2-Adrenergic Receptor
Signaling
Nicholas D. Leigh, Kathleen M. Kokolus, Rachel E. O'Neill,
Wei Du, Jason W.-L. Eng, Jingxin Qiu, George L. Chen,
Philip L. McCarthy, J. David Farrar, Xuefang Cao and
Elizabeth A. Repasky
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2015 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2015; 195:5045-5054; Prepublished online 12
October 2015;
doi: 10.4049/jimmunol.1500700
http://www.jimmunol.org/content/195/10/5045
The Journal of Immunology
Housing Temperature–Induced Stress Is Suppressing Murine
Graft-versus-Host Disease through b2-Adrenergic Receptor
Signaling
Nicholas D. Leigh,*,1 Kathleen M. Kokolus,*,1 Rachel E. O’Neill,* Wei Du,*
Jason W.-L. Eng,* Jingxin Qiu,† George L. Chen,‡ Philip L. McCarthy,‡ J. David Farrar,x
Xuefang Cao,* and Elizabeth A. Repasky*
G
raft-versus-host disease (GVHD) limits the full therapeutic potential of allogeneic hematopoietic cell transplantation (alloHCT) for many patients (1). To better
understand the immunobiology underlying GVHD, preclinical
mouse models are often employed (2). However, a well-recognized
aspect of many mouse models is that they do not develop severe
GVHD when only bone marrow (BM) cells are used for transplantation (3). There are several plausible explanations for this resistance, including a smaller number of T cells in the murine BM
*Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263;
†
Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263;
‡
Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263; and
x
Department of Immunology and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390
1
N.D.L. and K.M.K. contributed equally to this work.
ORCID: 0000-0002-8219-2290 (J.W.-L.E.).
Received for publication March 24, 2015. Accepted for publication September 16,
2015.
This work was supported by National Institutes of Health Grants R01CA135368 (to
E.A.R.), R01CA184728 (to X.C.), and T32 CA085183 (to N.D.L., K.M.K., and
J.W.-L.E.). This work utilized shared resources supported by Roswell Park Cancer
Institute Comprehensive Cancer Center Support Grant CA016056.
Address correspondence and reprint requests to Dr. Xuefang Cao and Dr. Elizabeth
A. Repasky, Department of Immunology, Roswell Park Cancer Institute, Elm and
Carlton Streets, Buffalo, NY 14263. E-mail addresses: [email protected]
(X.C.) and [email protected] (E.A.R.)
Abbreviations used in this article: alloHCT, allogeneic hematopoietic cell transplantation; b-AR, b-adrenergic receptor; b2-AR, b2-adrenergic receptor; BM, bone marrow; GVHD, graft-versus-host disease; MLR, mixed lymphocyte reaction; TCD-BM,
T cell–deleted bone marrow; WT, wild-type.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500700
graft (4) or an increased proportion of suppressive populations of
T cells (3, 5). To offset the quantitative and/or qualitative dearth of
GVHD-capable murine BM-derived T cells, additional T cells
(from spleen or lymph nodes) are added to the BM graft to generate GVHD (3, 6).
Work from murine physiologists has long established that mice
housed at the relatively cool, currently mandated ambient temperature in research facilities (typically ∼22˚C) experience
a mild but chronic cold stress, yet they maintain normal body
temperature (7–9). Our previous findings have shown that standard housing temperatures impair maturation of APCs (10) and
inhibit T cell–dependent immune responses in tumor-bearing
mice while enhancing the frequency of immunosuppressive
cells (7). Our recent work demonstrated that cold stress–induced
phenotypes in tumor-bearing mice could be completely reversed
by using pharmacological blockade of adrenergic receptors
(b-blockers) involved with norepinephrine-dependent signaling
(11). Recently, these same drugs reveal an important role for
b-adrenergic receptor (b-AR) signaling in modulation of immune activity (12–15). Although acute stress–induced b-AR
signaling can benefit the immune system in a variety of ways
helping to protect the host from infections (16–19), other research demonstrates that long-term norepinephrine-driven
b-AR signaling can inhibit T cell function and increase immunosuppression. Notably, previous reports show that b-AR
signaling can decrease APC function (12, 13), influence CD4+
Th cell function (20), prevent lymphocyte egress from lymph
nodes (15), and increase the suppressive capacity of regulatory
T cells (21). GVHD is known to be both dependent on immune
effector activity (mediated by APCs and T cells) and influenced
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Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation, a potentially curative
therapy for hematologic diseases. It has long been thought that murine bone marrow–derived T cells do not mediate severe GVHD
because of their quantity and/or phenotype. During the course of experiments testing the impact of housing temperatures on
GVHD, we discovered that this apparent resistance is a function of the relatively cool ambient housing temperature. Murine bone
marrow–derived T cells have the ability to mediate severe GVHD in mice housed at a thermoneutral temperature. Specifically,
mice housed at Institutional Animal Care and Use Committee–mandated, cool standard temperatures (∼22˚C) are more resistant
to developing GVHD than are mice housed at thermoneutral temperatures (∼30˚C). We learned that the mechanism underlying
this housing-dependent immunosuppression is associated with increased norepinephrine production and excessive signaling
through b-adrenergic receptor signaling, which is increased when mice are cold stressed. Treatment of mice housed at 22˚C with
a b2-adrenergic antagonist reverses the norepinephrine-driven suppression of GVHD and yields similar disease to mice housed at
30˚C. Conversely, administering a b2-adrenergic agonist decreases GVHD in mice housed at 30˚C. In further mechanistic studies
using b2-adrenergic receptor–deficient (b2-AR2/2) mice, we found that it is host cell b2-AR signaling that is essential for
decreasing GVHD. These data reveal how baseline levels of b-adrenergic receptor signaling can influence murine GVHD and
point to the feasibility of manipulation of b2-AR signaling to ameliorate GVHD in the clinical setting. The Journal of Immunology, 2015, 195: 5045–5054.
5046
b2-ADRENERGIC RECEPTOR SIGNALING REGULATES MURINE GVHD
by immunosuppressive activity (mediated by regulatory T cells
and suppressive cytokines) (2, 22).
Based on these previous findings, we wondered whether the
long-recognized inability of murine BM-derived T cells to mediate
GVHD in many experimental models is actually a function of an
unrecognized variable, housing temperature. We hypothesized that
the cool standard housing temperature could be dampening antihost immune activity through a mechanism related to cold
stress. To test this, we investigated whether the inability of murine
BM-derived T cells to mediate GVHD could be related to increased
b-AR signaling in mice housed at standard temperatures. Our
findings indicate that standard housing temperature of laboratory
mice and the subsequent increase of b-AR signaling in the host
explain, in large part, the suppressed ability of murine BM-derived
T cells to mediate GVHD.
Materials and Methods
Animals
Male and female 8-wk-old C57BL/6 (H-2b), 8-wk-old male and female BALB/c
(H-2d), and 8-wk-old female 129/SvJ (H-2b) mice were obtained from the
National Cancer Institute and Charles River Laboratories and maintained under
specific pathogen-free conditions following guidelines established by
Institutional Animal Care and Use Committee at Roswell Park Cancer
Institute. b2-Adrenergic receptor–deficient (b2-AR2/2) BALB/c mice
were provided by J. David Farrar (University of Texas Southwestern
Medical Center). All mice were housed at either 22˚C or 30˚C in precision refrigerated plant-growth incubators (Thermo Fischer Scientific).
Mice were acclimated to indicated temperature for at least two weeks
prior to experiments and remained at their assigned temperature for the
entirety of every experiment. Humidity within incubators was controlled
using Top Fin air pump AIR 1000 and Top Fin airline tubing. Mice were
provided autoclaved water and Tekland Global 18% Protein Diet
(Harlan, Madison, WI) ad libitum. Mice were housed five or fewer per
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FIGURE 1. GVHD is exacerbated following alloHCT in mice maintained at 30˚C. (A–D) BALB/c mice were transplanted as described in Materials and
Methods. Following transplant of total BM alone (A and B) or TCD-BM plus PanT (C and D), weight loss and survival of BALB/c hosts was monitored. (E
and F) C57BL/6 mice were lethally irradiated (965 cGy) and transplanted on day 21 with 2 3 106 129/SvJ BM plus 7.5 3 106 splenocytes on day 0.
Weight loss (E) and survival (F) were subsequently monitored. (A, C, and E) Data are presented as mean 6 SEM (n = 3–10). ****p , 0.0001, two-way
ANOVA. (B, D, and F) Data are presented as percentage survival (n = 3–10). ##p , 0.01, Mantel–Cox test.
The Journal of Immunology
cage and Enrich-o’Cobs Premium Blended Enrichment Bedding (The
Andersons, Maumee, Ohio) was provided.
Acute GVHD models
b-adrenergic agonist/antagonist treatments
Starting on day 1 posttransplant, mice were injected daily i.p. with 10 mg/kg
body weight of propranolol, metoprolol, or salbutamol, 1 mg/kg body
weight ICI 118,551 or 200 ml PBS diluent as a control.
Histopathological analysis of GVHD target organs
Liver and large and small intestines were removed, formalin fixed, sectioned, and stained with H&E. Intestinal tissues were examined using
a previously established semiquantitative scoring system (23, 24). Blinded
assessments were made for the presence of crypt epithelial cell apoptosis,
crypt loss, surface colonocyte vacuolization, surface colonocyte attenuation, lamina propria inflammatory cell infiltrate, mucosal ulceration, and
luminal sloughing of cellular debris, leukocyte infiltration of the lamina
propria, and villous blunting. Liver samples were evaluated using the
clinical GVHD scoring system as previously described (25). Assessment
for the percentage of pathologic small bile ducts designated 0 as normal, 1
as ,25%, 2 as 25–49%, 3 as 50–75%, and 4 as .75%. Representative
images were captured at 3100.
Liver preparation
planted lethally irradiated BALB/c (H-2d) mice housed at either
22˚C or 30˚C with total BM from C57BL/6 (H-2b) mice. Consistent with the literature (3, 27), following alloHCT of BM with
no additional T cells obtained from the spleen or lymph node,
mice housed at standard temperature (22˚C) show no signs of
severe or lethal GVHD (Fig. 1A, 1B). This was evidenced by no
weight loss (Fig. 1A) or lethality (Fig. 1B) at 22˚C. In contrast, we
observed significant weight loss and decreased survival in mice
given the identical transplant, but housed at 30˚C (Fig. 1A, 1B).
We also followed a commonly accepted protocol by transplanting
TCD-BM plus purified splenic T cells (PanT). As expected,
adding T cells in the BM graft induced more severe GVHD, as
evidenced by aggravated weight loss. Additionally, we found that
mice housed at 30˚C showed acute and substantial lethality
whereas those housed at 22˚C showed no lethality despite weight
loss (Fig. 1C, 1D). Similar results were also obtained in an MHCmatched, minor histocompatibility Ag–mismatched alloHCT model
using 129/SvJ (H-2b) donor-derived BM plus total splenocytes and
C57LB/6 (H-2b) hosts (Fig. 1E, 1F). These data suggest that GVHD
is significantly decreased when mice experience chronic mild cold
stress.
Norepinephrine levels are significantly increased in
cold-stressed mice
Norepinephrine, a b-adrenergic agonist produced in response to
stress, is increased in cold-stressed mice (28). To assess the activation of a stress response associated with mild cold stress, we
measured serum norepinephrine in mice housed at 22˚C or 30˚C.
We found that naive mice housed at 22˚C had significantly higher
norepinephrine levels than did their counterparts at 30˚C (Fig. 2).
Norepinephrine levels were also elevated at 22˚C compared with
30˚C on day 13 following alloHCT (Fig. 2). Thus, pre- and postalloHCT mice housed at 22˚C are experiencing a greater degree of
cold stress compared with mice housed at 30˚C.
Stress-induced adrenergic signaling suppresses GVHD
mediated by allogeneic T cells
We next examined whether the resistance to GVHD observed in
mice housed at 22˚C was due to increased norepinephrine-driven
Livers were processed through a 70-mm filter and flow-through was passed
through a second 70-mm filter. The cell suspension was centrifuged and
then resuspended and mixed well in 35% Percoll in RPMI 1640. This
gradient was centrifuged for 20 min at 2000 rpm at 4˚C. Hepatocytes were
then removed and remaining cells were red cell lysed before flow
cytometry staining.
Flow cytometry
Single-cell suspensions were stained with Abs to H-2Kd (SF1-1.1), H-2Kb
(AF6-88.5.5.3), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD11b
(M1/70), and CD11c (N418) and with fixable Live/Dead Aqua (Invitrogen).
Cells were stained for 10 min at room temperature in 200 ml PBS and washed
once with FACS buffer. All samples were run on a LSRFortessa (BD
Biosciences). All data were analyzed with FlowJo.
Mixed leukocyte reaction
Splenocytes were harvested from WT C57BL/6 mice and WT or b2-AR2/2
BALB/c mice. Splenocytes from all three types of mice were red cell lysed
and either stained with 500 mM CFSE (responders) or given 3000 cGy
irradiation from a [137Cs] source (stimulators). CFSE+ responders (0.25 3
106) and 0.75 3 106 irradiated stimulators were plated into 96-well roundbottom plates and incubated for 96 h as previously described (26). CFSE
dilution in CD4+ and CD8+ T cells was analyzed by flow cytometry.
Results
Mild cold stress inhibits GVHD development
To determine whether ambient temperature affected GVHD, we
used a standard MHC-mismatched alloHCT model. We trans-
FIGURE 2. Mild cold stress increases circulating norepinephrine before
and after alloHCT. BALB/c mice were housed at 22˚C or 30˚C and
transplanted as described in Materials and Methods. Norepinephrine levels
were measured in BALB/c mice prior to transplant and on day 13 following alloHCT. Data are presented as mean 6 SEM (n = 5–8). *p , 0.05,
Student t test.
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MHC-mismatched transplants were performed using C57BL/6 and wild-type
(WT) or b2-AR2/2 BALB/c mice as donors and hosts. BALB/c mice received 792 cGy and C57BL/6 mice 965 cGy irradiation from a [137Cs] source
24 h prior to transplant. Unless otherwise indicated, BALB/c mice received
7 3 106 total BM cells i.v. from C57BL/6 mice. MHC-matched transplants
were performed using 129/SvJ mice as donors and C57BL/6 mice as hosts.
C57BL/6 mice received 965 cGy from a [137Cs] source 24 h prior to transplant.
Male C57BL/6 mice were then i.v. injected with 2 3 106 female 129/SvJ BM
plus 7.5 3 106 splenocytes and weight loss was tracked. For experiments with
T cell–depleted BM (TCD-BM) alone, mice received 3 3 106 TCD-BM cells.
Anti-CD90.2 microbeads were used to deplete T cells from BM (Miltenyi
Biotec). Syngeneic transplants were performed using C57BL/6 mice. C57BL/6
mice received 965 cGy from a [137Cs] source 24 h prior to transplant and were
given 7 3 106 total BM cells i.v. from C57BL/6 mice. Following transplant,
mice were monitored regularly for weight loss and survival. Mice were considered moribund once weight loss reached 20% of their initial weight. For
alloHCT involving purified T cells, 3 3 106 TCD-BM with or without 0.5 3
106 PanT splenic T cells sorted from C57BL/6 mice were injected into BALB/c
hosts. C57BL/6 hosts received 4 3 106 WT BALB/c TCD-BM with or without
3 3 106 WT or b-AR2/2 PanT cells.
5047
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b2-ADRENERGIC RECEPTOR SIGNALING REGULATES MURINE GVHD
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FIGURE 3. The adrenergic stress response reduces allogeneic T cell–mediated GVHD. (A and B) BALB/c mice were transplanted and treated as described in Materials and Methods. Mice were treated daily with propranolol or PBS, and weight (A) and survival (B) were monitored. (C and D) C57BL/6
mice were transplanted with syngeneic BM as described in Materials and Methods. Host mice were treated daily with propranolol or PBS, and weight loss
(C) and survival (D) were monitored. (E and F) BALB/c mice were transplanted with TCD-BM alone as described in Materials and Methods. Host mice
were treated daily with propranolol or PBS, and weight loss (E) and survival (D) were monitored. (A, C, and E) Data are presented as mean 6 SEM (n = 5–
10). ****p , 0.0001, two-way ANOVA. (B, D, and F) Data presented as percentage survival (n = 5–10). #p , 0.05, ##p , 0.01, Mantel–Cox test.
b-AR signaling. We transplanted mice housed at 22˚C and 30˚C
and blocked b-AR signaling with propranolol. Following
transplantation, GVHD was increased in propranolol-treated
mice housed at 22˚C (Fig. 3A, 3B). At 30˚C, there is minimal norepinephrine-driven signaling, and propranolol treat-
ment of these mice had no additional effect on the severity of
GVHD above that seen by 30˚C housing alone (Fig. 3A, 3B).
Thus, alleviating adrenergic stress by housing at 30˚C or
pharmacologically blocking b-AR signaling results in increased GVHD.
The Journal of Immunology
Previous work has demonstrated that either housing mice at 30˚C
(10) or treating with propranolol (29) could decrease body weight
compared with controls. Thus, we were concerned that weight loss
and possibly survival differences were not due to GVHD but to the
5049
specific experimental conditions. We therefore tested the likelihood that allogeneic graft-versus-host responses were responsible
for changes in weight and survival. C57BL/6 mice underwent
syngeneic transplant with BM from C57BL/6 donors. Neither
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FIGURE 4. Alleviating adrenergic stress increases liver GVHD. BALB/c mice were transplanted as described in Materials and Methods. Fifty-four days
following alloHCT, livers (A and B) and small (C) and large (D) intestines were excised and fixed in formalin, sectioned, and stained with H&E. GVHD was
scored using a previously described semiquantitative scoring system. (E–H) Livers were harvested day 5 after alloHCT as described in Materials and
Methods. Cells were counted by trypan blue exclusion using a hemocytometer and then analyzed by flow cytometry for the presence of donor CD3+CD4+
T cells (E), CD3+CD8+ T cells (F), CD11b+CD11c2 cells (G), and CD11b2CD11c+ cell (H). Data are pooled from two individual experiments. (A)
Representative areas of liver tissue. Scale bar, 100mm. (B–H) Data presented as mean 6 SEM (n = 6). *p , 0.05, **p , 0.01, Student t test.
5050
b2-ADRENERGIC RECEPTOR SIGNALING REGULATES MURINE GVHD
housing temperature nor propranolol treatment affected weight
loss or survival following syngeneic HCT (Fig. 3C, 3D). Thus, an
allogeneic response is required to increase GVHD severity in mice
housed at 30˚C or mice housed at 22˚C and treated with propranolol. Next, to determine the importance of donor allogeneic
T cells in the regulation of GVHD in mice with reduced b-AR
signaling, we performed an alloHCT with a depletion of donor
T cells from the BM graft. T cell depletion from the BM inoculum
completely eliminated symptoms of GVHD in mice either housed
at 30˚C or treated with propranolol (Fig. 3E, 3F). These data
suggest that adrenergic stress downregulates allogeneic T cell
responses, thereby diminishing GVHD severity.
Liver GVHD is significantly increased in mice with reduced
adrenergic signaling
In addition to weight loss and survival for assessing GVHD, we
performed histopathologic analyses to examine how b-AR signaling influence GVHD occurrence and severity in target organs.
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FIGURE 5. b2-AR but not b1-AR signaling reduces GVHD. BALB/c mice were transplanted and treated as described in Materials and Methods. Mice
were treated daily with metoprolol, ICI 118,551, salbutamol, or PBS, and weight (A, C, and E) and survival (B, D, and F) were monitored. (A, C, and E) Data
are presented as mean 6 SEM (n = 5–10). ****p , 0.0001, two-way ANOVA. (B, D, and F) Data are presented as percentage survival (n = 5–10). #p ,
0.05, ##p , 0.01, Mantel–Cox test.
The Journal of Immunology
b2- but not b1-adrenergic signaling influences GVHD severity
As a pan b-antagonist, propranolol inhibits norepinephrine binding of both b1-AR and b2-AR. We tested whether blockade of
either class of receptor with propranolol was sufficient to affect
GVHD severity. Using metoprolol, a b1-AR antagonist, we found
that GVHD severity was unaffected in mice undergoing alloHCT
housed at 22˚C (Fig. 5A, 5B). In contrast, treatment with ICI
118,551, a b2-AR antagonist, increased GVHD in mice housed at
22˚C (Fig. 5C, 5D). In line with our previous data using the pan
b-AR antagonist propranolol, specifically blocking b1-AR or b2AR signaling did not impact GVHD in mice housed at 30˚C
(Fig. 5A–D). Taken together, these data suggest that b2-AR signaling is responsible for decreased GVHD in mice housed at 22˚C.
Thus, we postulated that targeting b2-AR with a specific agonist
FIGURE 6. b2-AR signaling decreases T cell
proliferation in MLRs. (A and B) WT or b2-AR2/2
BALB/c responders were CFSE stained and
cocultured with irradiated WT C57BL/6 stimulators for 96 h. CFSE dilution in H-2Kd+CD4+ and
H-2Kd+CD8+ T cells was analyzed. (C and D) WT
C57BL/6 responders were CFSE stained and
cocultured with irradiated WT or b2-AR2/2 BALB/c
stimulators for 96 h. CFSE dilution in H-2Kb+CD4+
and H-2Kb+CD8+ T cells was analyzed. Data are
presented as mean 6 SEM (*p , 0.05, **p , 0.01,
Student t test). Representative data are from one of
two experiments.
would reduce GVHD in mice housed at 30˚C. We used the b2-AR
agonist salbutamol to determine whether it would lessen the severity of GVHD. As expected, mice housed at 22˚C, in which high
norepinephrine levels already decrease GVHD, salbutamol-treated
mice did not develop GVHD following alloHCT (Fig. 5E, 5F).
However, salbutamol did decrease GVHD severity in mice housed
at 30˚C (Fig. 5E, 5F). This demonstrates that specific modulation
of b2-AR signaling can control GVHD.
b2-AR signaling decreases T cell proliferation intrinsically and
extrinsically
Although it appears that b2-AR signaling can reduce GVHD, the
question remains as to what cells are mediating this phenotype. To
answer this question, we performed mixed lymphocyte reactions
(MLRs) to decipher whether b2-AR signaling on the T cell or
APC was responsible for decreasing GVHD. To determine the
importance of T cell b2-AR signaling, we cultured either WT or
b2-AR2/2 BALB/c T cell responders with irradiated WT C57BL/6
stimulators. We found that b2-AR2/2 T cells proliferated more
than WT T cells, suggesting that b2-AR signaling could directly
decrease T cell proliferation in vitro (Fig. 6A, 6B). We next asked
whether b2-AR signaling could influence the ability of APCs to
stimulate T cell proliferation. In this system, we mixed WT
C57BL/6 responders with irradiated WT or b2-AR2/2 BALB/c
stimulators. b2-AR2/2 stimulators showed an increased ability
to stimulate allogeneic T cell proliferation compared with WT
controls (Fig. 6C, 6D). Therefore, the presence of b2-AR signaling
on APCs dampens their ability to stimulate T cell proliferation.
Taken together, these data indicate that in vitro T cell proliferation
can be reduced by T cell (intrinsic) or APC (extrinsic) b2-AR
signaling stimulated by endogenous norepinephrine present in the
serum used for cell culture (11).
GVHD is increased in the absence of host, but not donor
T cell–derived, b2-AR signaling
Our in vitro data suggested that b2-AR signaling could affect T cell
responses by directly decreasing T cell proliferation and de-
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We harvested small intestine, large intestine, and liver on day 54
following alloHCT for histopathologic analyses. Blinded histopathologic quantification using an established clinical GVHD
scoring system (25) revealed significantly increased liver inflammation consistent with GVHD in the livers of mice housed at 30˚C
and/or treated with propranolol (Fig. 4A, 4B). However, using
a previously established semiquantitative scoring system (23, 24),
we found no major differences in GVHD score in the intestines
(Fig. 4C, 4D). These data suggest that blocking b-AR signaling
significantly increases liver GVHD.
We next characterized the inflammatory infiltrate in the livers of
mice housed at 22˚C or 30˚C. We evaluated an early time point
(day 5 after alloHCT) in which allogeneic donor T cells begin to
enter the liver (30). Consistent with our pathologic GVHD data,
both donor CD4+ and CD8+ T cells were substantially higher in
the livers of mice housed at 30˚C compared with those housed at
22˚C (Fig. 4E, 4F). Furthermore, we analyzed the infiltrate of
donor-type APCs and found that both CD11b +CD11c2 and
CD11b+CD11c+ cells were significantly increased in the livers of
mice housed at 30˚C versus 22˚C (Fig. 4G, 4H). Taken together,
these cellular infiltrates are consistent with the increased liver
pathology following alloHCT of mice housed at 30˚C.
5051
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b2-ADRENERGIC RECEPTOR SIGNALING REGULATES MURINE GVHD
creasing the ability of APCs to stimulate T cell proliferation. We
sought to test whether these findings could be recapitulated in our
in vivo GVHD model. To test the ability of T cell–intrinsic b2-AR
signaling, we isolated WT and b2-AR2/2 BALB/c PanT cells and
transplanted them with WT TCD-BM into WT C57BL/6 hosts.
However, it appeared that this donor/host combination did not
induce severe GVHD, and the elimination of b2-AR signaling on
donor T cells did not exacerbate GVHD as we expected (Fig. 7A,
7B). Because host APCs are essential for GVHD (22, 31, 32), we
reasoned that host APC-derived b2-AR was responsible for controlling GVHD after alloHCT. To test this, we transplanted WT
and b2-AR2/2 BALB/c mice with total BM from WT C57BL/6
donors. We found that b2-AR2/2 hosts had significantly more
GVHD than did WT controls (Fig. 7C, 7D). These data demonstrate that host cell b2-AR signaling suppresses GVHD.
Discussion
FIGURE 7. Host-derived, but not donor T cell–derived, b2-AR signaling is essential for controlling GVHD. (A and B) WT C57BL/6 host mice were
housed at 22˚C and transplanted with BALB/c-derived TCD-BM plus PanT as described in Materials and Methods. Weight loss (A) and survival (B) of
C57BL/6 hosts were monitored. (C and D) WT or b2-AR2/2 BALB/c host mice were housed at 22˚C and transplanted with C57BL/6-derived total BM cells
as described in Materials and Methods. Weight loss (C) and survival (D) of WT and b2-AR2/2 BALB/c hosts were monitored. Data are combined from two
individual experiments. (A and C) Data are presented as mean 6 SEM (n = 5–10; *p , 0.05, two-way ANOVA). (B and D) Data are presented as percentage
survival (n = 5–10). #p , 0.05, Mantel–Cox test.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
In this study, we show that both a physiologic stress (i.e., mild cold
stress) known to generate increased norepinephrine-induced adrenergic activity and pharmacologic stimulation of b2-AR signaling can significantly reduce GVHD severity. Importantly, the
cold stress imposed by standard housing conditions accounts, in
large part, for the long-standing observation that many mouse
models appear to be resistant to GVHD following alloHCT with
only BM-derived T cells. Whereas previous studies have posited
that the quantity or immunosuppressive nature of BM-derived
T cells explains the lack of GVHD seen in many murine models
(3–5), our data demonstrate that immune activity is highly dependent on the room temperature used to house mice. Cold stress
is countered by a norepinephrine-driven adrenergic response designed to generate heat to aid in body temperature maintenance
(33, 34). However, because adrenergic receptors are expressed on
myriad cell types, additional effects of norepinephrine signaling
are possible. In our model, we have shown one of these additional
effects to be the dampening of the immune response following
alloHCT.
In particular, our data using b2-AR2/2 mice demonstrate that
this effect is dependent on host-derived b2-AR signaling. Host
APCs participate in the initiation of T cell activation during
GVHD (22, 31, 32), and mild cold stress (10) and b-adrenergic
stimulation (12, 13) suppress the ability of APCs to activate
T cells. These data suggest that host APC-derived b2-AR signaling may decrease the ability of APCs to stimulate T cell–mediated
GVHD. These data are corroborated by the fact that b2-AR2/2
stimulators showed an enhanced ability to stimulate T cell proliferation in MLRs. Work from our group and others has shown
that antitumor activity of CD8+ T cells has been shown to be
impaired by mild cold stress (7) whereas CD4+ T cell activity can
be modulated by adrenergic signaling (14). In line with work from
others (35), it appears that in vitro b2-AR signaling can impair
The Journal of Immunology
data may lead to a more cautious use of nonspecific b-AR antagonists (i.e., commonly prescribed b-AR blockers) for other
unrelated medical indications during clinical alloHCT so as to avoid
the potential risk for increased GVHD severity.
Acknowledgments
We thank Drs. Sarah Holstein, Bonnie Hylander, Michelle Messmer, Kelvin
Lee, and Michael Nemeth for discussions and technical support. We thank
Jeanne Prendergast for laboratory and editorial assistance.
Disclosures
The authors have no financial conflicts of interest.
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T cell proliferation (Fig. 6), although in our in vivo model, GVHD
was no different between mice receiving WT or b2-AR2/2
PanT cells (Fig. 7). This implies that direct donor T cell–derived
b2-AR signaling is not as effective as host cell–derived b2-AR
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purified T cells do not induce severe GVHD in the BALB/c to
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Our data suggest that b2-AR signaling may decrease donor
T cell liver infiltration after alloHCT, which could account for less
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that increased b2-AR signaling can inhibit T cell egress from the
lymph node resulting in reduced autoimmunity (15). This inhibition of T cell egress could contribute to reduced symptoms of
GVHD. Furthermore, nonhematopoietic cells (i.e., hepatocytes)
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Our data highlight the importance of ambient temperature as
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