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Propranolol Attenuates Surgical Stress−
Induced Elevation of the Regulatory T Cell
Response in Patients Undergoing Radical
Mastectomy
Lei Zhou, Yunli Li, Xiaoxiao Li, Gong Chen, Huiying
Liang, Yuhui Wu, Jianbin Tong and Wen Ouyang
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Copyright © 2016 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2016; 196:3460-3469; Prepublished online 11
March 2016;
doi: 10.4049/jimmunol.1501677
http://www.jimmunol.org/content/196/8/3460
The Journal of Immunology
Propranolol Attenuates Surgical Stress–Induced Elevation of
the Regulatory T Cell Response in Patients Undergoing
Radical Mastectomy
Lei Zhou,*,1 Yunli Li,*,1 Xiaoxiao Li,* Gong Chen,* Huiying Liang,† Yuhui Wu,‡
Jianbin Tong,*,x and Wen Ouyang*,{,‖
B
reast cancer is by far the most common female malignancy and is the leading cause of cancer-related death
among women worldwide (1). Although surgery is a
mainstay of treatment for most breast cancer patients, studies have
suggested that perioperative immunosuppression may promote the
growth of pre-existing micrometastases, thereby playing a key role
in postoperative metastatic recurrences (2–4).
*Department of Anesthesiology, Third Xiangya Hospital of Central South University,
Changsha 410013, Hunan, China; †Department of Endocrinology, Second Xiangya
Hospital of Central South University, Changsha 410008, Hunan, China; ‡Department of
Breast Surgery, Xiangya Hospital of Central South University, Changsha 410008,
Hunan, China; xMedical Central Laboratory, Third Xiangya Hospital of Central South
University, Changsha 410013, Hunan, China; {Seniors Anesthesia and Perioperative
Management Research Center, Central South University, Changsha 410013, Hunan,
China; and ‖State Key Laboratory of Medical Genetics, School of Life Sciences, Central
South University, Changsha 410013, Hunan, China
1
L.Z. and Y.L. contributed equally to this study.
Received for publication July 30, 2015. Accepted for publication February 11, 2016.
This work was supported by National Natural Science Foundation of China Grant
NSFC No. 81172200 and Natural Science Foundation of Hunan Province Grant
12JJ3079.
Address correspondence and reprint requests to Prof. Wen Ouyang and Prof. Jianbin
Tong or Dr. Yuhui Wu, Department of Anesthesiology, Third Xiangya Hospital of
Central South University, 138 Tongzipo Road, Changsha 410013, Hunan, China
(W.O. and J.T.) or Department of Breast Surgery, Xiangya Hospital of Central
South University, 87 Xiangya Road, Changsha 410008, Hunan, China (Y.W.).
E-mail addresses: [email protected] (W.O.), [email protected]
(J.T.), or [email protected] (Y.W.).
Abbreviations used in this article: b-AR, b-adrenergic receptor; CA, catecholamine;
CMI, cell-mediated immunity; COX, cyclo-oxygenase; E, epinephrine; End-OP, end
of operation; NE, norepinephrine; POD, postoperative day; Pre-OP, preoperation;
SFC, spot-forming cell; TA, tumor Ag; Treg, regulatory T cell.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501677
It has been well documented that surgery profoundly suppresses
many components of cell-mediated immunity (CMI), including
decreasing the number and activity of NK cells, helper-inducer
T cells, and CTLs, as well as increasing the number of suppressor T cells. Immunosuppression begins immediately after surgery
and lasts for hours to several days postoperatively (5–8). Regulatory T cells (Tregs), characterized by the classical marker proteins CD25 and FOXP3 (9), can exert a broad suppressive effect
on antitumor immunity by direct or indirect mechanisms, ultimately resulting in immunological tolerance and escape of tumor
cells (10, 11). Elevation of CD4+CD25+FOXP3+ Tregs in the
peripheral blood is associated with a higher risk of tumor recurrence and a poor prognosis in breast cancer patients (11–13). It is
reasonable to speculate that the number and activity of Tregs
perioperatively may be predictive of clinical outcomes.
Surgery induces the release of catecholamines (CAs) and PGs at
levels that correspond to the extent of surgical stress and tissue
trauma. Recent data have demonstrated that CAs and PGs may be
key factors that mediate the suppressive effects on antimetastatic
CMI perioperatively (14–18). Both hormones have been repeatedly
demonstrated to suppress most aspects of CMI as well as promote
the Treg response in vitro (19–22). However, their effects on Tregs
in breast cancer patients undergoing surgery are not clear.
Surgical stress promotes increases in epinephrine (E) and norepinephrine (NE) levels. The suppressive effects of E and NE on
CMI are primarily mediated through their specific binding to the
b-adrenergic receptors (b-AR) of immune cells, which promote
tumor survival, migration, and resistance to anoikis (19, 23).
These effects are thought to be inhibited by the nonselective
b-adrenergic antagonist propranolol (24, 25). Interestingly, several
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Surgical stress and inflammatory response induce the release of catecholamines and PGs, which may be key factors in facilitating
cancer recurrence through immunosuppression. Animal studies have suggested the efficacy of perioperative blockades of catecholamines and PGs in reducing immunosuppression. In this study, to our knowledge, we present the first report of the effects of
perioperative propranolol and/or parecoxib on peripheral regulatory T cells (Tregs) in breast cancer patients. Patients were
randomly assigned to control, propranolol, parecoxib, and propranolol plus parecoxib groups. We demonstrated that levels of
circulating epinephrine, norepinephrine, and PGE2 increased in response to surgery. Meanwhile, peripheral FOXP3 mRNA level
and Treg frequencies were elevated on postoperative day 7. Propranolol administration, rather than parecoxib, attenuated such
elevation of Tregs, indicating the critical roles for catecholamines in surgery-induced promotion of Tregs. Besides, propranolol
plus parecoxib treatment demonstrated no additive or synergistic effects. Furthermore, a study of Treg activity on CD4+ T cell
responses to specific tumor Ags was performed in the control and propranolol groups. Propranolol abrogated the increased Treg
activity and accompanying suppression of CD4+ T cell responses after surgery. Finally, we conducted ex vivo experiments on the
effects of varying concentrations of epinephrine and/or propranolol on Treg proliferation over PBMCs from breast cancer
patients, to provide further direct evidence strengthening our clinical observations. Epinephrine markedly promoted Treg proliferation, whereas propranolol prevented such enhancement effect. In conclusion, our study highlights beneficial roles for
propranolol in inhibiting Treg responses in vivo and in vitro, and demonstrates that propranolol could alleviate surgical
stress–induced elevation of Tregs in breast cancer patients. The Journal of Immunology, 2016, 196: 3460–3469.
The Journal of Immunology
Materials and Methods
Patients
In the clinical trial part, the current study enrolled 154 women between the
ages of 25 and 65 y who underwent a modified radical mastectomy for the
treatment of primary breast cancer at the Third Xiangya Hospital of Central
South University. Patients with a previous radical mastectomy, inflammatory breast cancer, preoperative chemotherapy or radiotherapy, an
American Society of Anesthesiologists Physical Status of III–IV or greater,
immunologic or endocrinologic disease, or any contraindications to propranolol or parecoxib were excluded from participating in the study. Ultimately, 106 patients were enrolled in this section (Fig. 1). Clinical data
regarding patient characteristics and surgical procedures were recorded
(Table I).
For the in vitro study, we further recruited 17 women diagnosed with
primary breast cancer who newly registered for hospitalization at the Third
Xiangya Hospital of Central South University, and they met the same
exclusion criteria as described above.
The trial was approved by the Medical Ethics Committee of the Third
Xiangya Hospital of Central South University and was registered with the
Chinese Clinical Trial Registry (ChiCTR-IPR-14005271). All patients were
required to provide written informed consent.
Treatment program
In the clinical trial, patients were randomly assigned to control, propranolol,
parecoxib, and propranolol plus parecoxib groups. In the propranolol group,
patients were treated with oral propranolol (20 mg three times daily) from
the day of surgery until the third postoperative day. In the parecoxib group,
patients received i.v. parecoxib (40 mg once daily) from the day of surgery
until the second postoperative day. In the propranolol plus parecoxib group,
patients received propranolol and parecoxib by the oral and i.v. routes,
respectively. Peripheral blood samples (10 ml) were collected from each
patient early on the morning of surgery (preoperation [Pre-OP]) and at the
end of surgery (end of operation [End-OP]), as well as in the early morning
on the first (postoperative day [POD] 1), third (POD3), and seventh (POD7)
postoperative days.
ELISA
For each time point, ELISA was used to measure the plasma levels of E, NE,
and PGE2 in 2 ml blood samples that were collected from patients in the
control group (n = 10), the propranolol group (n = 10), the parecoxib group
(n = 10), and the propranolol plus parecoxib group (n = 10). The ELISAs
were performed in 96-well microtitration plates according to the manufacturer’s instructions. Commercial ELISA kits were purchased from R&D
Systems (Minneapolis, MN) for the E and NE assays and from Assay
Designs (Ann Arbor, MI) for the PGE2 assay. All samples were assayed in
triplicate.
Real-time PCR
Blood samples (2 ml) were collected from all patients at the Pre-OP, POD1,
POD3, and POD7 time points, and PBMCs were isolated thereafter. Realtime PCR (RT-PCR) was used to determine the mRNA levels of FOXP3 and
CTLA-4. Total RNA was extracted using TRIzol (Life Technologies),
according to the manufacturer’s instructions. The RNA (1 mg) was then
reverse transcribed with RT random primers using a High Capacity cDNA
Reverse Transcription Kit; the cDNA products were used for RT-PCR
amplification with the following primers: 59-CTGACCAAGGCTTCATCTGTG-39 and 59-ACTCTGGGAATGTGCTGTTTC-39 for the FOXP3;
59-TACCCACCGCCATACTACCT-39 and 59-AACAACCCCGAACTAACTGC39 for the CTLA4; and 59-TGACGTGGACATCCGCAAAG-39 and 59CTGGAAGGTGGACAGCGAGG-39 for the b-actin.
Quantitative real-time PCR was performed on a PRISM 7500 Real-Time
PCR System (Applied Biosystems) using SYBR Select Master Mix (Applied Biosystems), according to the manufacturer’s protocols. The transcript generated from the b-actin gene was used as an internal control. The
relative level of gene expression was represented as DCt = Ctgene 2
Ctreference, and the fold change of gene expression was calculated by the
22DDCt method. Experiments were repeated in triplicate.
Flow cytometry
PBMCs were isolated from 2-ml blood samples using standard Ficoll
density gradient centrifugation. Then, the PBMCs were incubated with
FITC-conjugated anti-CD4, PE-Cy7–conjugated anti-CD25, allophycocyanin-conjugated anti–CTLA-4, and the appropriate fluorochromeconjugated mouse IgGs as isotype controls for 20 min at room temperature in the dark. Intracellular staining of FOXP3 was performed with a PEconjugated FOXP3 Ab, according to the manufacturer’s instructions. All of
the Abs were purchased from BD Biosciences (San Jose, CA). Data were
acquired with a BD FACS Canto II Flow Cytometry system. Results were
analyzed with FACSDiva 6.1.3 software.
ELISPOT assays
The assays were conducted, as described previously (29, 30). Blood (10 ml)
was obtained from 29 patients at the Pre-OP and POD7 time points and
divided into duplicate aliquots (5 ml 3 2). PBMCs were extracted from the
blood by centrifugation. In one of the aliquots of the PBMC samples,
CD25high cells were depleted using magnetic separation with MACS CD25
microbeads (Miltenyi Biotec), following the manufacturer’s instructions,
and the efficacy of depletion was determined by flow cytometry (Fig. 4G,
4H). Thereafter, the cell viability of the two aliquots of PBMCs was determined by a trypan blue exclusion cell-counting assay. The cell densities
were adjusted to 3.5 3 106 cells/ml with AIM-V serum-free medium (Life
Technologies). CD25high-depleted or undepleted cells (100 ml) were cultured in triplicate in the presence of both CD3 mAb (2.5 mg/ml) and IL-2
(1 mg/ml) for nonspecific stimulation or exposed to a pool of purified
MUC1 (1 kU/ml), P53 (1 mg/ml), and HER2/neu (1 mg/ml) for specific Ag
stimulation in 96-well ELISPOT plates (Multiscreen IP plate; Millipore)
that had been precoated with IFN-g mAb (U-Cytech; Dutch). Wells containing PHA (1 mg/ml) or serum-free medium were used as positive or
blank controls, respectively. The culture plates were placed in a humidified
5% CO2 incubator at 37˚C for 48 h, and the Ag-specific IFN-g secretion
was determined by an ELISPOT assay using an ImmunoSpot analyzer
(Cellular Technology). The activated CD4+ T cells secreting IFN-g were
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epidemiological studies have demonstrated that the chronic use of
b-blockers improves the long-term prognosis of breast cancer
patients (26, 27). The proinflammatory PGs induced by surgical
inflammatory responses are also known to suppress CMI and to
protect tumors from immune destruction (28). In addition, animal
studies indicate that the combined use of propranolol and etodolac
(a selective cyclo-oxygenase [COX]-2 inhibitor), rather than either
treatment alone, could improve NK activity and potentially reduce
the postoperative recurrence and metastasis of tumors (16, 17). To
date, the effect of short-term perioperative regimen of b-blockers
and COX-2 inhibitors on suppressive Tregs has not been clinically
assessed in breast cancer patients.
In this study, we initially investigated the perioperative effects of
propranolol and parecoxib (another selective COX-2 inhibitor that
is used more frequently in the perioperative period) on the level of
CD4 + CD25 + FOXP3 + Tregs in the peripheral blood of breast
cancer patients. An ex vivo IFN-g ELISPOT assay was used to
measure the suppressive activity of Tregs on the responses of
CD4+ T cells to tumor Ags (TAs) perioperatively. The present
work indicated that propranolol administration alone was able to
abrogate the increased Treg level or activity observed in response
to mastectomy. Hence, to further investigate whether similar
changes would occur in Tregs exposed to stress hormones in vitro,
we extended our experiments on the effects of differing concentrations of E and/or propranolol on Tregs using an established
PBMC culture model. The PBMC culture system was previously
used to study the effects of stress hormones (cortisol or catecholamines) on immune balance, including the transcription of
regulatory FOXP3, IL-10, and TGF-b mRNA, as well as on the
Th1/Th2 cytokine balance (22). In this work, we chose to detect
Treg proliferation to more directly study the effects of catecholamines on Tregs over PBMCs from breast cancer patients. E,
acting as an in vitro representative for surgical stress, promoted
Treg proliferation, and propranolol treatment prior to E exposure
conferred a protective effect in the prevention of Treg elevation.
These results were consistent with our clinical observations. Thus,
our study demonstrates the beneficial effect of propranolol in the
inhibition of Treg responses in vivo and in vitro, and suggests that
clinical treatment with propranolol during the perioperative period
could improve CMI in breast cancer patients.
3461
3462
REDUCING REGULATORY T CELL ELEVATION AFTER MASTECTOMY
Table I. Patient characteristics and surgical procedures
Patient characteristics
Age (y)
Weight (kg)
ASA gGrade I/II (n)
TNM gGrade 0/I/II/III (n)
Surgical procedures
Duration of surgery (min)
Heart rate, Pre-OP (bpm)
Heart rate, Intra-OP (bpm)
Heart rate, End-OP (bpm)
Blood pressure, Pre-OP (mm Hg)
Blood pressure, Intra-OP (mm Hg)
Blood pressure, End-OP (mm Hg)
Control
Propranolol
Parecoxib
Propranolol + Parecoxib
46.42 6 7.57
54.73 6 8.11
17/18
8/5/19/3
45.91 6 9.24
55.62 6 7.86
13/21
6/8/15/5
44.84 6 10.33
57.21 6 11.44
8/10
5/3/10/0
48.19 6 12.51
52.86 6 10.52
7/12
2/6/9/2
114.82
75.16
65.83
81.05
127.32
106.37
120.13
6
6
6
6
6
6
6
23.58
13.42
8.11
20.78
19.75
9.24
19.47
118.40
80.39
57.09
73.26
121.18
112.84
125.46
6
6
6
6
6
6
6
26.73
18.90
5.26#
18.15
23.07
11.29
20.77
120.52
68.41
61.43
70.89
117.64
103.32
118.63
6
6
6
6
6
6
6
29.25
20.37
5.71
13.42
15.39
6.41
16.23
109.73
72.09
55.26
67.22
123.58
111.04
116.38
6
6
6
6
6
6
6
27.76
15.29
6.37#
16.10
17.26
7.15
18.19
Data are shown as means 6 SD.
#
p , 0.05 versus the control group.
ASA, American Society of Anesthesiologists; bpm, beats per minute; End-OP, the end of the operation; Hg, mercury; Intra-OP, 1 h after the beginning of the operation; PreOP, the morning on the day of the operation; TNM, tumor, node, metastases grading system.
Cell culture
In the in vitro study, PBMCs were separated from 20 ml blood samples of 17
patients by centrifugation and resuspended at 106 cells/ml in AIM-V serumfree medium (Life Technologies) containing 1% supplemental L-glutamine
and 100 U/ml human rIL-2 (R&D Systems). Twenty-four–well, flat-bottom
microtiter plates (Costar 3596, Cambridge, MA) were coated overnight at
4˚C with anti-CD3 mAb (Santa Cruz; clone PC3/188A) (10 mg/ml) and
anti-CD28 mAb (Santa Cruz; clone M-20) (5 mg/ml) in 0.5 ml PBS per
well. Thereafter, 1 ml PBMCs (106 per well) were cultured in the 24-well
plates precoated with anti-CD3/CD28 with various concentrations of E
(Sigma-Aldrich, St. Louis, MO) (0, 1, 5, 10 mM) or propranolol (SigmaAldrich) (1, 10, 20 mM), respectively. Each concentration was repeated in
triplicate. The effect of propranolol was assessed at an E concentration of
FIGURE 1. Two flow diagrams illustrating the clinical trial protocol. In (A), 101 qualified patients enrolled in this trial were randomly allocated to the
control, propranolol, parecoxib, and propranolol plus parecoxib groups. Blood samples were collected at the Pre-OP, End-OP, POD1, POD3, and POD7
time points. ELISA, RT-PCR, and flow cytometry were subsequently performed. In (B), 36 patients were randomized and assigned to the control and
propranolol groups. Blood samples were extracted from patients at the Pre-OP and POD7 time points for ELISPOT assays. Patients who received chemotherapy postoperatively or who had sample or data deficiencies during the trial were excluded.
Downloaded from http://www.jimmunol.org/ by guest on May 8, 2017
measured as spots per well at the single-cell level. The number of spots
in the negative control, subtracted from the number of spots in the experimental wells, was recorded as the number of final spot-forming cells
(SFC) per 106 PBMCs, and positive responses were defined as having .5
SFC per 106 PBMCs after subtracting the control value and at least a
50% increase compared with the control. Patients who had positive responses in the total CD4+ T cells were classified as responders (Fig. 7B).
The number of SFC in CD25high-depleted experimental wells after
subtracting the value of the undepleted wells was considered to represent
the inhibitory effects of CD4+CD25high Treg on total CD4+ T cells; an
increase of at least 50% over the undepleted wells was defined as a
positive effect of depletion. Similarly, patients who had positive depletion effects of Tregs were identified as suppressors (Fig. 7B).
The Journal of Immunology
3463
5 mM. Propranolol was administered 30 min before E treatment. These
concentrations were determined after a trial of different doses (0.1–50
mM for E and 0.1–100 mM for propranolol). We determined that high
concentrations of E (50 mM) or propranolol (100 mM) caused marked
cell death and the cell viability was too poor, whereas low concentrations
(0.1 mM) seemed to have little effect on cell proliferation. After 3-d incubation at 37˚C and 5% CO2 in a humidified incubator, cells were
washed and continuously expanded with anti-CD3/CD28 plus 100 U/ml
IL-2 for another 4 d. On day 7, PBMCs were harvested, and the percentage of CD4+CD25+FOXP3+ Tregs was determined by flow cytometry, as described above.
Statistical analysis
Statistical analyses were performed using the SPSS statistical package
(version 18.0; SPSS, Chicago, IL). Continuous variables were defined as
means 6 SD if they were normally distributed; otherwise, median values
and interquartile ranges (25th–75th percentile) were represented. We used
ANOVA, two-way ANOVA, paired or unpaired t tests, Mann–Whitney
tests, Wilcox signed ranks tests, Fisher’s exact tests, and McNemar tests, as
appropriate. The p values ,0.05 were considered statistically significant in
all studies, and all p values were two tailed.
Perioperative E, NE, and PGE2 levels in the peripheral blood
The perioperative levels of E, NE, and PGE2 in breast cancer
patients with or without propranolol and parecoxib treatment
were measured by ELISA (Figs. 1, 2, Table II). As shown in
Fig. 2A and 2B, there were no significant differences among the
three groups at the corresponding time points for either E or NE
levels. The End-OP and POD1 time points showed sharply increased levels of E and NE compared with the Pre-OP time point
(p , 0.05). E and NE then returned to levels that were similar to
the Pre-OP time point at POD3 and POD7. Even though PGE2
level increased at End-OP and POD1 in the control group (p ,
0.05), but not in the parecoxib or the propranolol plus parecoxib
groups, no significant differences were observed among the
groups (Fig. 2C). These results suggested that the levels of E,
NE, and PGE2 all increased after surgery; moreover, parecoxib
or propranolol plus parecoxib treatment had modest inhibitory
effects on postsurgical PGE2 level, whereas propranolol or propranolol plus parecoxib treatment had no obvious effects on the
production of E and NE.
Effects of propranolol and parecoxib on FOXP3 and CTLA-4
mRNA levels in the peripheral blood
We then investigated the effects of propranolol and parecoxib on
two Treg-related functional markers (FOXP3 and CTLA-4)
(Fig. 3, Table III). Both in the control and parecoxib groups,
the peripheral FOXP3 mRNA level decreased temporarily at
POD1 (p , 0.05); these levels then increased to a much higher
level at POD7 compared with the Pre-OP time point (p , 0.05).
However, no significant time-dependent changes in the propranolol or propranolol plus parecoxib group were observed
(p . 0.05); in these groups, FOXP3 mRNA level was significantly lower than in the control group at POD7 (p , 0.05)
(Fig. 3A). As shown in Fig. 3B, no significant changes in the
CTLA-4 mRNA level were observed between the control group
and the treatment groups at any time point. These results suggested that propranolol treatment (alone or in combination with
parecoxib), instead of parecoxib alone, was able to decrease the
FOXP3 mRNA level, but not the CTLA-4 mRNA level, in the
peripheral blood of patients at POD7.
Effects of propranolol and parecoxib on perioperative
peripheral Treg levels in breast cancer patients
Flow cytometry was used to measure the perioperative levels
of circulating CD4+CD25+FOXP3+ and CD4+CD25highFOXP3+
FIGURE 2. The perioperative E, NE, and PGE2 levels in the breast
cancer patients in each group were measured by ELISA. The peripheral
levels of E (A) and NE (B) in all three groups sharply increased at the
End-OP and POD1 time points, whereas the level of PGE2 (C) showed
only slight increases at End-OP and POD1 in the control group. No
significant differences were observed among the groups for all three
hormone levels. Data are presented as the mean 6 SD of three independent experiments. *p , 0.05 versus the Pre-OP time point.
Tregs (as percentages in total CD4+ T cells) (Figs. 4, 5, Table III).
Both CD25+ and FOXP3+ were gated accordingly using blank
controls and isotype controls (Fig. 4B, 4C). The CD4+CD25high
was defined by a CD25-staining intensity in the CD4+ T population that was greater than in CD42 cells (29). As shown in
Fig. 5, the percentages of both Treg subsets decreased at POD1
compared with the Pre-OP values (p , 0.05); moreover, they
began to increase and finally reached a significantly higher level
at POD7 than the Pre-OP level in the control group (p , 0.05). A
similar result was observed in patients treated with parecoxib
alone. However, the percentage of Tregs in the propranolol group
and the propranolol plus parecoxib group did not increase at
POD7 compared with the control group. There were some typical examples, presented in flow cytometry images, of CD4+
CD25+FOXP3+ Treg percentages at both Pre-OP and POD7 time
points for all four groups (Fig. 6). We also labeled the surface
molecule CTLA-4 and detected the frequency of CD4+CD25+
CTLA-4+FOXP3+ Tregs, but no significant changes were observed (data not shown). These observations indicated that propranolol treatment alone or in combination with parecoxib could
block surgical stress–induced increases in circulating Tregs after
the seventh postoperative day.
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Results
3464
REDUCING REGULATORY T CELL ELEVATION AFTER MASTECTOMY
Table II. Perioperative E, NE, and PGE2 levels in the peripheral blood
E (pg/ml)
Control
Propranolol
Propranolol + parecoxib
NE (pg/ml)
Control
Propranolol
Propranolol + parecoxib
PGE2 (pg/ml)
Control
Parecoxib
Propranolol + parecoxib
n
Pre-OP
End-OP
POD1
POD3
POD7
10
10
10
69.66 6 29.84
62.02 6 26.57
74.03 6 30.55
117.26 6 51.60*
110.25 6 49.39*
130.51 6 47.70*
97.85 6 41.80*
90.24 6 35.60*
103.63 6 38.31*
77.44 6 36.71
68.97 6 32.08
79.66 6 26.70
63.56 6 24.97
64.71 6 23.83
73.86 6 24.11
10
10
10
185.00 6 55.06
192.79 6 60.18
176.01 6 53.92
285.30 6 68.05*
292.19 6 76.66*
302.79 6 78.93*
246.85 6 52.29*
278.37 6 73.44*
274.67 6 51.99*
199.79 6 65.51
204.89 6 67.36
191.19 6 57.02
190.09 6 55.32
187.58 6 56.66
177.40 6 49.28
10
10
10
39.81 6 13.28
44.83 6 17.44
41.84 6 17.94
50.87 6 18.57*
45.42 6 18.35
44.84 6 16.31
56.66 6 19.46*
45.62 6 18.00
45.94 6 14.73
48.87 6 24.22
46.21 6 20.88
43.02 6 16.63
34.38 6 11.59*
38.81 6 14.64*
36.50 6 13.99*
Data are shown as means 6 SD of three independent experiments. Perioperative E, NE, and PGE2 levels of breast cancer patients with or without propranolol and/or
parecoxib treatment were measured by ELISA. No significant differences among the groups at each time point.
*p , 0.05 versus the Pre-OP time point.
Because propranolol treatment alone exerted an obvious effect on
Tregs, we further tested the effects of propranolol on the TA sp. act.
of perioperative Tregs with ex vivo highly sensitive ELISPOT assays
(Fig. 7, Table IV). IFN-g secretion by CD4+ T cells was measured
perioperatively, and the impact of CD25high Tregs on the responses
of CD4+ T cells was analyzed in the control and propranolol groups.
We observed no differences in each group at each time point after
nonspecific stimulation by CD3 plus IL-2 (Fig. 7A).
We next tested the CD4+ T cell responses to TA-specific Ags;
the magnitude of TA-specific experimental wells ranged from 6 to
328 SFC/106 PBMC. At POD7, decreased CD4+ T cell function
was observed compared with the Pre-OP time point in the control
group (p , 0.05) (Fig. 7C, Table IV), whereas in the propranolol
group the total CD4+ T cell function at POD7 did not change
significantly (Fig. 7D). In addition, the total CD4+ T cell
function was significantly higher in the propranolol group
than in the control group at POD7 (p , 0.05). We also found a
decrease in the percentage of TA-specific responders (percentage of responses to tumor Ags) at POD7 in the control group
(p , 0.05), but not in the propranolol group (Fig. 7E). However,
CD25high Treg-induced suppression increased at POD7 compared with the Pre-OP time point in the control group (p ,
0.05), but no difference between the two time points was observed in the propranolol group (p . 0.05, Table IV). Accordingly, the proportion of suppressors (percentage of suppressed
responses) significantly differed between the two groups at
POD7 (p , 0.05, Fig. 7F). Interestingly, we observed six patients whose TA-specific responses of CD4+ T cells were
completely inhibited by CD25high Tregs at POD7 in the control
group (6 of 15), whereas only two patients with such responses
were found in the propranolol group (2 of 14). These data
suggested that the TA-specific Treg activity was increased after
surgery, thereby suppressing the CD4+ T cell response. However, the immunosuppressive effect exerted by postsurgical Treg
responses could be alleviated by propranolol.
Effects of E and propranolol on Treg proliferation in vitro
FIGURE 3. Perioperative peripheral FOXP3 (A) and CTLA-4 (B)
mRNA levels in the breast cancer patients in each group. Data are presented as the mean 6 SD of three independent experiments. *p , 0.05
versus the Pre-OP time point, #p , 0.05 versus the control group.
PBMCs were exposed to varying doses of E (0, 1, 5, 10 mM) for 3 d
and sequentially cultured for an additional 4 d in vitro. On day 7,
the percentage of CD4+CD25+FOXP3+ Tregs was analyzed by
flow cytometry to determine the effect of E on Treg proliferation.
At that time, as shown in Fig. 8A, the percentage of CD4+CD25+
FOXP3+ Tregs significantly increased in the presence of E (1, 5,
10 mM) relative to the absence of E (0 mM) (p , 0.05). In addition, there is a trend for increased proliferation with high concentrations (5, 10 mM) in comparison with the low one (1 mM)
(p , 0.05). Interestingly, no significant differences were observed
between the two high doses (5, 10 mM) (p . 0.05).
Second, to assess the ability of propranolol to block the Einduced elevation of CD4+CD25+FOXP3+ Tregs, three increasing doses of propranolol (1, 10, 20 mM) were administered 30 min
prior to 5 mM E treatment, respectively. As shown in Fig. 8B,
although the low dose of propranolol (1 mM) did not affect the
upregulating effect of E (5 mM) on CD4+CD25+FOXP3+ Treg
proliferation (p . 0.05), both the 10 and 20 mM doses effectively
attenuated the E-induced promotion (p , 0.05), also with a significant dose-dependent reduction of the percentage of CD4+
CD25+FOXP3+ Tregs (p , 0.05). Besides, propranolol treatment
(10, 20 mM) alone had no direct effect on Treg proliferation relative to the unexposed control (0 mM) (data not shown).
Downloaded from http://www.jimmunol.org/ by guest on May 8, 2017
The effect of perioperative propranolol on Ag-specific IFN-g
production by CD4+ T cells in breast cancer patients, using ex
vivo ELISPOT assays
The Journal of Immunology
3465
Table III. Effects of propranolol and/or parecoxib on perioperative FOXP3 and CTLA-4 mRNA levels and Treg frequencies in the peripheral blood
n
POD1
POD3
POD7
1.31
1.34
1.31
1.33
6
6
6
6
0.69
0.71
0.50
0.55
0.86
1.01
0.67
0.96
6
6
6
6
0.44*
0.47
0.37*
0.60
1.04
1.04
1.16
1.07
6
6
6
6
0.66
0.78
0.66
0.38
1.92
1.09
1.87
1.15
6
6
6
6
0.90*
0.51#
0.63*
0.63#
1.04
1.13
1.13
1.03
6
6
6
6
0.41
0.56
0.50
0.44
0.85
0.85
0.84
0.83
6
6
6
6
0.44
0.33
0.64*
0.54
0.86
0.83
0.91
0.83
6
6
6
6
0.42
0.50
0.62
0.53
1.13
0.91
1.09
0.95
6
6
6
6
0.72
0.52
0.64
0.47
6.06
6.09
6.30
6.26
6
6
6
6
1.79
1.12
1.28
1.56
5.03
5.52
5.29
5.72
6
6
6
6
1.27*
1.34
1.13*
1.67
5.56
5.70
6.16
5.91
6
6
6
6
1.35
1.89
1.37
1.47
7.21
5.98
7.10
6.15
6
6
6
6
1.85*
1.73#
1.12*
1.31#
3.37
3.25
3.54
3.36
6
6
6
6
1.21
1.03
0.95
0.90
2.8
2.92
2.84
3.04
6
6
6
6
0.92*
0.75
0.86*
1.37
3.23
2.96
3.64
3.11
6
6
6
6
1.06
0.97
1.28
0.83
4.27
3.24
4.11
3.26
6
6
6
6
1.47*
0.76#
1.08*
0.80#
Values are expressed as means 6 SD of three independent experiments. The peripheral FOXP3 and CTLA-4 mRNA levels were determined by real-time PCR. The
frequencies of Tregs were measured by flow cytometry.
*p , 0.05 versus the Pre-OP time point, #p , 0.05 versus the control group.
These results revealed that 3-d treatment with E generated an
enhancement of Treg proliferation that could be inhibited by the
addition of propranolol.
Discussion
For patients with primary breast cancer, surgery is the crucial and
necessary treatment for eradicating the primary tumor. However,
an increased risk exists for promoting pre-existing micrometastases in the perioperative period (4), which is attributed to
the immunosuppressive effects caused by physiological mechanisms, such as excess postoperative release of CAs and PGs (2,
3, 6, 7). Therefore, it is critical to provide perioperative drug
treatment to block CAs or PGs with the goal of alleviating their
inhibitory effects on the antitumor immune response. To the best
of our knowledge, the current study is the first to demonstrate the
effect of the perioperative use of propranolol or parecoxib on the
number and activity of peripheral Tregs in breast cancer patients
who underwent a radical mastectomy.
FIGURE 4. Analysis of Treg percentages in the breast cancer patients by flow cytometry. PBMCs were labeled with fluorescence-conjugated Abs against
CD4, CD25, and FOXP3. All of the CD25, CD25high, and FOXP3 expressions were analyzed in the subsets of CD4+ T cells (A). CD25+ and FOXP3+ cells
were gated according to the blank control (B) and isotype control (C). The percentages of CD4+CD25+FOXP3+ (D), CD4+CD25high (E), and CD4+CD25high
FOXP3+ (F) Tregs within the population of CD4+ T cells were shown, as indicated. In the ELISPOT assays, CD25high cells were depleted from PBMCs
using magnetic separation, and the efficacy of depletion was determined by flow cytometry (G and H).
Downloaded from http://www.jimmunol.org/ by guest on May 8, 2017
FOXP3 mRNA
Control
18
Propranolol
17
Parecoxib
15
Propranolol + parecoxib
17
CTLA-4 mRNA
Control
16
Propranolol
15
Parecoxib
15
Propranolol + parecoxib
16
Percentage of CD4CD25 FOXP3 Tregs (%)
Control
20
Propranolol
20
Parecoxib
18
Propranolol + parecoxib
19
high
Percentage of CD4CD25
FOXP3 Tregs (%)
Control
20
Propranolol
20
Parecoxib
18
Propranolol + parecoxib
19
Pre-OP
3466
REDUCING REGULATORY T CELL ELEVATION AFTER MASTECTOMY
In this study, perioperative drug treatment with propranolol or
parecoxib (alone or in combination) was administered on the
morning of surgery and was terminated 2 or 3 d after surgery.
Although it has been demonstrated that immunosuppression begins
even before surgery due to psychological distress and other related
mechanisms (5), we chose this short-term perioperative drug
treatment to target immunosuppression that was induced by the
surgery itself. It has been reported that the stress response and
FIGURE 6. Contrasts of perioperative changes in CD4+CD25+FOXP3+ Treg percentages among the four groups. Examples of CD4+CD25+FOXP3+ Treg
percentages at both Pre-OP and POD7 time points in the control group (A), the propranolol group (B), the parecoxib group (C), and the propranolol plus
parecoxib group (D) are presented in flow cytometry images acquired by FACSDiva 6.1.3 software.
Downloaded from http://www.jimmunol.org/ by guest on May 8, 2017
FIGURE 5. Perioperative frequencies of peripheral CD4 + CD25 +
FOXP3 + (A) and CD4 + CD25 high FOXP3+ Tregs (B) in the breast
cancer patients in each group. Data are presented as the mean 6 SD
of three independent experiments. *p , 0.05 versus the Pre-OP time
point, # p , 0.05 versus the control group.
inflammatory response markedly increase postoperatively and last
for 2–3 d (7). In addition, short-term perioperative propranolol or
parecoxib administration has been clinically proven to be safer
than chronic drug administration and has additional benefits, such
as reduction of the dosage of anesthetic and analgesic agents and
protection of patients from stroke and cardiac complications (31).
Although both CAs and PGs increased postoperatively, propranolol,
rather than parecoxib, appeared to alleviate the increase in the surgeryinduced Treg response at POD7. It has been suggested that the surgical
stress response has a greater influence on peripheral Tregs than the
inflammatory response. It was important to show that the percentages of
peripheral Tregs and FOXP3 mRNA expression decreased temporarily
at POD1 and then increased markedly at the very time when the stress
response abated. Ogawa et al. (7) reported that CD8+ suppressor T cells
increased on the second day after surgery (when elevated CA levels
had already returned to baseline) in gastrointestinal cancer patients.
Additionally, MacConmara et al. (32, 33) demonstrated a significant
increase in circulating Tregs on day 7 in trauma patients, but no increase on the first day following injury. The following mechanism
may be responsible for the aforementioned changes in Treg levels.
Augmented CAs levels (induced by surgical stress) cause gentle and
temporary CMI activation in the very early phase; however, sustained
stimulation can result in immunosuppression by elevating Treg levels.
Indeed, convincing evidence suggests that it is possible for CAs
(stress) to affect Tregs in in vivo environment and in ex vivo cultures
(22, 34, 35). The effects of CAs on Tregs are achieved through both
indirect and direct mechanisms. Indirect mechanisms are exemplified
partly by the fact CAs regulate TGF-b generation; TGF-b is crucial
to the induction and proliferation of FOXP3+ Tregs. One in vitro study
has shown that NE can decrease or increase hepatocyte production of
TGF-b in a dose-dependent manner (36). Bhowmick et al. (34)
suggested that the number of peripheral Tregs is mediated by sympathetic nervous system signals, and TGF-b acts as a bridge between
the sympathetic nervous and immune systems. And TGF-b was decreased in PBMCs by short-term exposure to E at 24 h, whereas it
was increased at day 11 in long-term E cultures; a significant increase
in the expression of FOXP3 mRNA in cultures subjected to 11 d of E
The Journal of Immunology
treatment with 20 mM propranolol and concomitant E (5 mM) may
represent a fallback effect that occurs following sufficient blockade
of the b-AR. Our in vitro study demonstrated that CAs could promote Treg proliferation and that propranolol treatment was capable
of mediating preventive effects for E-induced elevation of Tregs.
CTLA-4, as a CD28-family receptor that binds to B7, is a central
mediator of Treg function (39). We did not observe obvious surface
CTLA-4 expression in the population of CD4+CD25+FOXP3+ Tregs.
One possible explanation is that the presence of functional CTLA-4
on the cell surface is infrequent (40) and undetectable in flow
cytometry (,0.1%). It has been suggested that CTLA-4 expression
was detectable early after activation by allogeneic dendritic cells
(16 h) on the surface of CD4+CD25+ T cells (41). Additionally, peripheral CTLA-4 mRNA expression was not significantly different in
the pre- and postoperative periods and did not differ among the groups.
Furthermore, the combination of propranolol and parecoxib did
not exhibit higher efficacy than propranolol alone, indicating that
these two drugs may not be synergistic. This finding differs from
earlier animal studies demonstrating that only the combined use of
propranolol and etodolac had a significant effect on postoperative
immunosuppression and the recurrence-free survival rate (14–18).
We hypothesize that two main reasons may account for this discrepancy. First, previous animal research studied the nonspecific
cytotoxicity of NK cells on tumor cells, as all animals were injected with tumor cells after the surgical procedure and therefore
FIGURE 7. Analysis of perioperative CD4+ T cell responses from PBMCs and CD25high-depleted PBMCs in the breast cancer patients, using ex vivo IFN-g ELISPOT
assays with nonspecific stimulation of CD3 mAb plus IL-2 and TA-specific stimulation of a pool of MUC1, P53, and HER2/neu. No differences in the SFC among the
time points or the groups were observed with nonspecific stimulation (CD3 + IL-2) (A). Examples of patients who had a nonresponse, response, and suppressed response
with positive effects of CD25high depletion to TA-specific stimulation are shown; +Tregs, CD25high undepleted cultures; 2Tregs, CD25high depleted cultures (B). The
number of TA-specific SFC per 106 PBMC (the total CD4+ T cell function) as determined at the Pre-OP and POD7 time points in the control group (C) and the
propranolol group (D) and the percentages of patients who had a positive response to TAs (responders) at the Pre-OP and POD7 time point in the two groups (E) are
shown. Whole PBMCs and CD25high Treg-depleted PBMCs were stimulated with TAs, and the IFN-g release was measured. The percentages of patients with partially or
completely suppressed responses (suppressors) are shown at both time points in both groups (F). Data are presented as the mean 6 SD of three independent experiments
(A) and as the median (interquartile ranges) of three independent experiments (C and D). *p , 0.05 versus the Pre-OP time point, #p , 0.05 versus the control group.
Downloaded from http://www.jimmunol.org/ by guest on May 8, 2017
exposure has also been observed (22). These stress hormones are also
believed to exert their effects directly on Tregs through surface b-AR
stimulation and intracellular cAMP/protein kinase A signal activation (19, 37, 38). It has been shown in a human trial that acute
psychological stress (lasting for 12 min) directly decreased the
percentage of peripheral CD4+FOXP3+ regulatory T cells, mainly by
activating b1-ARs (35). The present work indicated b-AR involvement in CA-induced Treg alterations by demonstrating that the administration of propranolol, a b-adrenergic antagonist, prevented the
elevation of Tregs observed following surgery or in vitro E exposure.
While acknowledging the b-AR–based direct mechanism, we further focused our efforts on studying the ability of propranolol to block
the E-enhancing effect on Tregs in vitro and provided more direct
evidence to strengthen the in vivo observations. PBMCs from breast
cancer patients were exposed to varying doses of E (0, 1, 5, 10 mM) or
three increasing doses of propranolol (1, 10, 20 mM) administered 30
min prior to E (5 mM) for 3 d and were sequentially cultured for an
additional 4 d. Thereafter, the percentage of CD4+CD25+FOXP3+
Tregs was determined. E (1, 5, 10 mM) significantly increased the
Treg percentage on day 7 with a dose-dependent effect to some extent
(no more than 5 mM). Furthermore, 10 mM propranolol attenuated the
enhancement effect of E (5 mM) on Treg proliferation, which was
completely blocked by 20 mM propranolol, however. The low dose of
propranolol (1 mM) had no obvious effect on the E-induced promotion. Thus, the decreased frequency of Treg observed after
3467
3468
REDUCING REGULATORY T CELL ELEVATION AFTER MASTECTOMY
Table IV. Effects of propranolol on Ag-specific IFN-g secretion by
CD4+ T cells from CD25high-depleted or undepleted PBMCs in
perioperative breast cancer patients using ELISPOT assays
n
+
Pre-OP
POD7
6
Function of total CD4 T cells (SFC/10 PBMC)
Control
15
54 (29, 106)
3 (3, 43)*
Propranolol
14
38 (8, 99)
40 (5, 124)#
Suppressive function of CD25high Tregs (SFC/106 PBMC)
Control
15
43 (29, 63)
51 (9, 106)*
Propranolol
14
26 (10, 42)
13 (218, 53)
Values are expressed as medians (interquartile ranges) of three independent experiments.
*p , 0.05 versus the Pre-OP time point, #p , 0.05 versus the control group.
FIGURE 8. Propranolol treatment attenuated the E-induced increase in
Treg frequency in vitro. PBMCs were plated at 106 cells/ml and stimulated
with anti-CD3/CD28 plus 100 U/ml human rIL-2 in 24-well plates in the
presence of increasing concentrations of E (0, 1, 5, 10 mM) (A), or in the
presence of 5 mM E plus varying doses of propranolol (1, 10, 20 mM) (B) for
3 d. Thereafter, PBMCs were washed and expanded for another 4 d with
anti-CD3/CD28 plus 100 U/ml IL-2. On day 7, PBMCs were harvested to
estimate the proportion of CD4+CD25+FOXP3+ Tregs using flow cytometry.
Data are presented as the mean 6 SD of three independent experiments.
attenuate the immunosuppressive effects of surgical stress in patients who underwent a radical mastectomy. However, a 3- to 5-y
follow-up study to evaluate long-term relapse-free survival rate is
needed to address the clinical significance of perioperative treatment with propranolol in breast cancer patients.
Acknowledgments
We thank Prof. Shuang Liu and Dr. Jingxuan He for assistance with flow
cytometry and IFN-g ELISPOT assays. We are also grateful to Prof. Wei
Tian for critical reading of this manuscript and helpful discussions.
Disclosures
The authors have no financial conflicts of interest.
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