European Journal of Pharmacology 796 (2017) 110114

MARK
Effect of Dezocine on IL-12 and IL-10 secretion and lymphocyte activation by
culturing dendritic cells from human umbilical cord blood
Chang Fenga, Man Fengb, Ran Jiaoc, Dongyi Liua, Yanwu Jina, Xin Zhaoa,, Ruixue Xiaob
a
Department of Anesthesiology, The Second Hospital of Shandong University, 247 Bei Yuan Street, Jinan 250033, China b Department of Pathology, Affiliated Hospital of
Shandong Academy of Medical Sciences, Shandong Academy of Medical Sciences, 38 Wu Yingshan Road, Jinan 250031, China c Department of Operation Room,
Ophthalmologic Hospital of Shandong Province, 372 Jing Si Road, Jinan 250021, China

ARTICLEINFO ABSTRACT
Keywords: Dezocine has been generally utilized for pain therapy and auxiliary anesthesia. Although it has some advantages on the
Dezocine prevention of some anesthesia related complications, its effect on immune responses remains unclear. Our study investigated
Dendritic cells the effects of Dezocine on IL-10 and IL-12 secretion and lymphocytes activation by culturing dendritic cells (DCs), and revealed
Immunity the underlying mechanism. Mononuclear cells were divided into negative control group (GN), positive control group (GP),
IL-12 experimental group (GD; GD5, D7, D9). DCs morphological structure was performed by microscope and its phenotypes were
IL-10 evaluated by flow cytometry. IL12 and IL-10 levels were determined by ELISA and lymphocyte proliferation capacity was
performed by MTT assay. Results showed that typical morphological characters of DCs were observed in GP and GD. The
positive cell percentages of CD83, HLA-DR, CD80, CD86 and CD40 in GD were lower than those in GP, but higher than the GN
group (P < 0.01). IL-12 level in GD was higher than in GP, however, IL-10 was opposite (P < 0.01). The optical density in GD was
lower than in GP (P < 0.05). There were no dose-dependent relationships correlated with DCs phenotypes, IL-12 and IL-10
secretion and lymphocytes activation (P > 0.05). Our conclusion was that Dezocine might play a role in immunity by regulating
IL-12 and IL-10 secretion, and affecting lymphocyte activity in process of DCs maturation. Our findings reveal an unexpected
immuno-regulatory function of Dezocine in DCs and provide an important insight for investigating the effect of opioid drugs
in immunologic responses.

1. Introduction
Various researches showed that surgery trauma might suppress the
immune responses of patients with operation, and also led to several
complications, such as infection, inflammation, multiple organ dysfunction
syndrome (MODS), tumor metastasis (Bezerra et al., 2015; Jung et al., 2016;
Ng and Lau, 2015; Norberg et al., 2015). Except of these, anesthesia methods
and medicines had been proved to affect the immunologic function of patients
with tumor (Cakmakkaya et al., 2014; Chen et al., 2015; Zhao and Mo, 2015).
In addition, more research were focus on the effect of opioid drugs on the
immune responses recently.

Corresponding author.
E-mail address: 15953107775@163.com (X. Zhao).

http://dx.doi.org/10.1016/j.ejphar.2016.12.035
Received 11 July 2016; Received in revised form 20 December 2016; Accepted 21 December 2016
Available online 22 December 2016
0014-2999/ 2016 Elsevier B.V. All rights reserved.
C. Feng et al.
Dezocine is one of opioids drugs as partial -receptor agonist, a receptor
antagonist (O'Brien and Benfield, 1989). Previous research revealed that
Dezocine performed various advantages on clinical implications and novel
molecular targets. Current clinical settings also indicated that it had benefit on
the prevention of multiple anesthesia related complications (He et al., 2015;
Liu et al., 2015; Zhou et al., 2015). Thus, it has been widely used in analgesia
and auxiliary anesthesia recently. OBrien group found that Dezocine had
analgesic effect with 59 ng/ml concentration (O'Brien and Benfield, 1989).
Some researchers reported that its analgesic effect was equal to morphine, in
additionally, other researches implied that its effect was even 2 times stronger
than morphine. At present, Dezocine was widely used in clinical anesthesia
combined with other analgesic medicines, but its effects on immune function
remained unknown. The data from Declue AE's group indicated that morphine
and other opioids could alter immune and apoptotic pathways in dogs (Declue
et al., 2014). Boland JW and his partners firstly proposed that opioids have
different effects on the immune function by acting on natural killer (NK), T
cells, neutrophils and monocytes. They also systematically evaluate these
effect of opioids in vitro. These results demonstrated that none of the opioids
affect immune parameters, but some therapeutic opioids suppress IL-6
production (Boland et al., 2014). However, more clinical and studies in vitro
will be needed to confirm their findings.
Dendritic cells (DCs) are important drivers and regulators of innate and
adaptive immune responses. They are involved in tissue injuries, exogenous
antigens uptake, promoting T lymphocytes activation. Of note, they can active
T and B lymphocytes, especially priming naive T cell response by multiple
classical pathways (Merad et al., 2013). With the acknowledge to biological
characters of DCs, previous research demonstrated that mature DCs can be
induced in vitro, and markers of mature DCs were identified, for example,
CD1, CD11c, CD40, CD83, CD80, CD86, HLA-DR, MHCand etc. All of them
played crucial roles in anti-tumor immunity, autoimmune diseases and anti-
pathogens infection. Meanwhile, some studies indicated that DCs also express
functional kappa-opioid receptors, and can be activated by some specific
agonists (Kirst et al., 2002). However, research on the effects of opioid drugs
especial Dezocine on DCs remains unclear.
In our study, we collected DCs by separating mononuclear cells from
human umbilical cord blood, and investigate its effect of Dezocine on IL-10 and
IL-12 secretion and lymphocyte activation. We also explore its immuno-
regulation mechanism, which may provide a novel theoretical basis for
researching on potential effect of opioid drugs in immune responses.
2. Materials and methods
2.1. Isolation and preparation of human umbilical cord blood mononuclear
cells
Under sterile conditions, human umbilical cord blood (HUCB) was collected
from 20 healthy full-term pregnancies after informed written consent
according to guidelines approved by the Shandong University Ethics
Committee. The mononuclear fraction was separated from HUCB by density
gradient centrifugation. Experimental procedures were as following
descriptions. Cord blood was diluted with normal saline, and subsequently
subjected to 1.077 kg/l Ficoll solution (Shanghai Bioleaf Biotech Co., China).
The mixture was centrifuged at 700g for 20 min at room temperature,
mononuclear cells (MNC) were isolated from the interphase and centrifuged
at 700g 10 min at room temperature, the liquid at the top of sample-medium
interface containing mononuclear cells was discarded. The process was
repeated twice and all cells were washed with saline. The monocytes were
cultured in 10% fetal calf serum medium at 2.0106 cells/ml/well in 24-well
tissue culture plates (Shanghai Hengfei Biotech Co., China) and incubated in a
humidified 5% CO2 incubator at 37 C. The cells were maintained for 2 h
without changing the medium, discarded suspension cell and left anchorage-
independent cell, following the steps, cells was cultured at 1.0106
European Journal of Pharmacology 796 (2017) 110114
cells/ml/well in each group. All culturing procedures were performed under
sterile conditions.
2.2. Group allocation and cell morphology observation
Group N: Negative control group, the cells were cultured at rhGMCSF 50
ng/ml, rhIL-4 10 ng/ml and equal volume of physiological saline as D9 group
described as following for 10 days;.
Group P: Positive control group, the cells were cultured at rhGMCSF 50
ng/ml, rhIL-4 10 ng/ml and rhTNF- (Peprotech, Inc. USA) 50 ng/ml for 10
days;.
Group D (D5, D7, D9): experimental group, the cells were cultured at rhGM-
CSF 50 ng/ml, rhIL-4 10 ng/ml and Dezocine (Yangtze River Pharmaceutical
Group Co., China) for 10 days. The concentration was 5 ng/ml, 7 ng/ml, 9 ng/ml
(D5, D7, D9) respectively.
The cells in each group were cultured by changing culture plates once each
two days till to the endpoint, each group was made in triplicate and was
observed under an inverted light microscope.
2.3. Cell surface molecules phenotypes analysis of DCs
The DCs cultured for 10 days were collected from each group and washed
with phosphate buffered saline (PBS) twice, the suspension cells were
adjusted to 1106/ml and incubated with antibody CD40, CD83, CD80, CD86,
HLA-DR (BD Pharmingen, American), in the dark for 45 min at 4 C, after
washed twice with PBS, the immune phenotypes of DCs were detected by flow
cytometer (Epics elite, Beckman Coulter, USA).
2.4. The IL-12 and IL-10 level detection
10 days after cell culturing, DCs supernatants were collected from each
group, the levels of IL-12 and IL-10 in supernatants were measured by enzyme
linked immunosorbent assay (Elisa Kit, Shanghai Yanyu Biotech Co., China) as
following the manufacturer's protocol.
2.5. The mixed lymphocyte culture (MLC) and allogenic T-cell proliferation
activity assay
After culturing 10 day, the DCs in each group were treated with 25 ng/ml
mitomycin C (Sangon Biotech Co., Ltd., Shanghai, China) for 45 min in
incubator. The DCs were as stimulator cells for the MLC after being inhibited
proliferation capacity. On the other hand, 20 ml peripheral blood was
collected from a healthy adult volunteer, and peripheral blood mononuclear
cells were separated by density gradient centrifugation, cells were washed
three times with RPMI 1640 (Yuanlong Biotech Co., Shanghai, China) and were
incubated for 2 h at 37 C in a humidified atmosphere with 5% CO2. Responder
cells for MLC were suspended at 2.0106/ml, and were cultured for 7 days in
RPMI 1640 medium with 10% FBS. 100 l stimulator cells (S) (1.0103, 1.0104,
1.0105) and 100 l responder cells (R) (2.0105) were mixed and co-cultured
in 96-well plate for 6 days, each group was made in triplicate. and 5 mg/ml 20
l 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide
(Shanghai Hao Yang Biotechnology limited company, China) per well were
added and cultured for 4 h. After centrifuged, the supernatant in the culture
was dropped, subsequently, 150 l/well dimethyl sulfoxide was added. The
optical density (OD) values were measured under 570 nm wavelength by an
automatic enzyme linked immunosorbent assay (BIOB-EL10A, China), the
results were recorded by the mean OD values of three well (Huang et al.,
2013).
2.6. Statistical analysis
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Fig. 1. DCs morphology observation. a: Cultured for 6 days in GN. b: Cultured for 6 days in GP. c: Cultured for 6 days in GD; A: Cultured for 10 days in GN. B: Cultured for 10 days in GP. Cultured for 10
days in GD. (Under an inverted microscope, original magnification 400).

The quantitative data were recorded as mean S.D and analyzed by one-
way ANOVA and t-test or t-test method using the SPSS 17.0 software. A P
value of < 0.05 was considered significant.

3. Results

3.1. DCs morphology observation

After cultured for one day, most of cells were adherent growing in clusters
in three groups, only few suspended cells were observed under an invert light
microscope. But in group P and D, the percentage of suspended cells was
increased with mini protrusions on the 6th days. Frequently, on the 10th days,
these cells had typical spherical shape with characteristic short hairy
Fig. 2. Expression of DCs surface markers CD40, CD83, CD80, CD86, HLA-DR by FCM. Their
protrusions on their surface (Fig. 1). Also, the FCAS data indicated that
percentages of positive cells in GP were higher than in GN, but in GD (GD5, GD7, GD9) were lower
Dezocine did not significantly induce DCs apoptosis or necrosis than in GP. Compared to GN, P < 0.01; compared to GP, P < 0.01.
(Supplementary Fig. S1). However, the protrusions in group D were not Fig. 3. The levels of IL-12, IL-10 were measured by ELISA. Their level in GP and GD were higher
obviously gradually with the Dezocine concentration increasing. than in GN, and the IL-12 level in GD (GD5, GD7, GD9) was higher than in GP, but IL-10 levels was
lower than in GP. Compared to GN, P < 0.05; compared to GP, P < 0.01.
3.2. Effects of Dezocine on the DCs immune phenotypes by FCM The positive cell percentage of DC surface markers CD40, CD83, CD80,
CD86, HLA-DR in GP were higher than in GN (P < 0.01), but their expressions in

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GD (GD5, GD7, GD9) were lower than in GP (P < 0.01), their expression hadnt
dose dependent among GD (GD5, GD7, GD9) (P > 0.05) (Fig. 2).
3.3. Effects of Dezocine on the IL-12, IL-10 level by ELISA
The levels of IL-12, IL-10 both in GP and GD were higher than in GN (P <
0.05), and the IL-12 level in GD (GD5, GD7, GD9) was higher than in GP, but IL-
10 level was lower than in GP (P < 0.01), there wasnt dose dependent on IL-
12 and IL-10 secretion (P > 0.05) (Fig. 3).
3.4. Effects of Dezocine on the mixed lymphocyte proliferation by
MTT assay
The OD absorbance in GP and GD (GD5, GD7, GD9) was higher than in GN (P
< 0.05). The immature DCs in GN had little effects on T lymphocyte
proliferation. The mature DCs in GP and GD had a stronger capacity to induce
T lymphocyte proliferation, while GD (GD5, GD7, GD9) groups performed lower
level compared to GP group
(Fig. 4).
4. Discussion
As professional classical antigen presenting cells, DCs was firstly
discovered in 1973 by Ralph Steinman (Steinman and Cohn, 1973), which
induces cytotoxic T cell response predominately and plays central roles in
mediating immune function of anti-neoplasma and
Fig. 4. Mixed lymphocytes proliferation assay (OD value) by MTT. Different numbers of
stimulating cells were cultured with T cells as indicated in X-axis. The OD values in GP and GD
(GD5, GD7, GD9) was higher than in GN, but in GD was lower than in GP with different stimulating
cells numbers. Compared to GN, P < 0.05; compared to GP, P <
0.05.
anti-inflammation (Klechevsky, 2015). Normally, the percentage of DCs in
human peripheral blood mononuclear cells was less than 1.0%. The number of
DCs is also impaired obviously under tumor environment or after
chemotherapy (Vakkila et al., 2004). Therefore, it is very difficult to isolate DCs
from human or animal due to its limited numbers. Recently, it was found that
GM-CSF, IL-4 can promote the DCs transformation from mononuclear cells and
inhibit the occurrence of granulocytes and macrophage. In additionally, GM-
CSF-driven DCs also develop into mature status and perform strongly ability to
activate T lymphocyte (Morse et al., 1997).
Opioid receptors take an important role in regulating immune responses
induced by opioid drugs. The surface of DCs contains various opioid receptors,
and the expression levels of these receptors are inducible in the process of DCs
maturing (Li et al., 2009; McCarthy et al., 2001). Dezocine as an opioid drugs
with partial -receptor agonist, a -receptor antagonist, had an analgesia
effects when its steady state plasma concentration comes to 59 ng/ml, which
was only one to fifth of morphine (Hoskin and Hanks, 1991). In our study, the
results showed that Dezocine could drive the DCs into mature stage. Dezocine-
induced mature DCs had the typical spherical shape with characteristic short
European Journal of Pharmacology 796 (2017) 110114
hairy protrusions on their surface. We concluded that the interaction of
Dezocine with opioid -receptor on the of DCs surface activate the DCs, and
also promote the expression of DCs markers.
By phenotype analysis, our data identified that the expression of CD83,
HLA-DR, CD80, CD86, CD40 in GD and GP were higher than in GN (P < 0.01).
Rouard et al. found that CD83 was a specific marker on DCs surface and
indicated that DCs had strong capacity to activate T lymphocyte (Rouard et al.,
2000). In additionally, CD40 is also essential in activation of dendritic cells, it
enhances immunity depending on effective APC licensing through T cell
activation. Some research also found that some cancer patients showed
increased expression of CD40. However, further studies of the role of CD40 in
cancer treatment were needed (Lee et al., 2014; Murray et al., 2015). Previous
research found that CD80, CD86 were important for mature DCs activation.
They took part in T cell activation by binding to co-stimulators on adaptive
immune cell surface. Besides, HLA-DR as one of MHCmolecules, it can
process antigen to CD4+ T cells (Chen et al., 1998). Therefore, we speculated
that Dezocine promote the DCs mature and also played a crucial role in T cell
immune response. In our study, the expression of DCs surface markers didnt
show a correlation with increasing concentration of Dezocine. Esche et al.
(1999) demonstrated that both of low and high concentration (1.0 and 10,000
ng/ml) morphine inhibits the expression of DCs surface receptor, but the
middle concentration 10100 ng/ml) increases their expressions. Lin et al.
(2009) also reported that fentanyl had a dosedependent effect on dendritic
spines. Fentanyl at low concentration (0.010.1 M) led to the collapse of
dendritic spines, but had opposite effects at the high concentration (110
mM). As described above, whether the expression of DCs surface maker had
relationship with Dezocine concentration would be investigated in future.
IL-12 is a key cytokine in mediating cellular immunity, especially in inducing
Th1 differentiation both in vivo and in vitro (Jahn et al., 2010). Compared to
the role of IL-12, IL-10 is an suppressive factor by acting on antigen-presenting
cells to inhibit cytokines secretion by Th1 cells (Couper et al., 2008; Steinbrink
et al., 1997). Some researchers also found that the process of maturation of
DCs could increase the IL12 secretion and the expression of co-stimulatory
molecules, which could activate T cell differentiation (Napolitani et al., 2005;
St John et al., 2007). Our data revealed that Dezocine upregulated IL-12 level,
but decreased IL-10 expression. The results also revealed that Dezocine group
promoted T cells proliferation. With regard to these results described above,
we speculated that Dezocine might affect immunity by enhancing T cell activity
and regulating proinflammatory cytokines secretion. Moreover, Messmer et
al. (2006) found that morphine enhanced the T cell activation, and increased
the IL-12 secretion by targeting P38 MAPK pathway which was consistent with
our results. Immunological effects of Dezocine remains poorly understood, so
we still need collect more samples to elucidate the effects and potential
mechanism of Dezocine on immunity in future.
5. Conclusions
Dezocine as a widely used analgesic drug, its role in immunoregulation
remains unclear. Our results demonstrate that Dezocine would mediate
immune function by affecting inflammatory factors and T cell activity. Our
finding provide theoretical basis for guiding clinical anesthetist to make a
reasonable choice, especially for patients with immune compromised.
Disclosure of conflict of interest
The authors declare no competing financial interests.
Acknowledgements
This study was supported by Youth Fund of the Second Hospital of
Shandong University, Shandong Province, China (No. Y2013010044).
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