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SLCCCR7 Stimulates the Proliferation of BMDCs by the pNF-╬║B p65 Pathway.

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THE ANATOMICAL RECORD 293:48–54 (2010)
SLC/CCR7 Stimulates the Proliferation
of BMDCs by the pNF-jB p65 Pathway
SHUANG ZHOU, RILUN LI, JIE QIN, CUIPING ZHONG,* AND CHUNMIN LIANG*
Department of Anatomy, Histology and Embryology, Shanghai Medical College of Fudan
University, Shanghai 200032, China
ABSTRACT
The chemokine receptor CCR7 is highly expressed in dendritic cells
(DCs), T cells, and other immune effector cells. One of the high-affinity
ligand that can bind to CCR7 is the secondary lymphoid tissue chemokine
(SLC). The SLC/CCR7 axis plays an important role in the immune system
by inducing the chemotaxis and migration of immune effector cells. In
this study, we examined the effect of SLC at different concentrations (0,
50, 100, 200, 300, and 400 ng/mL) on the proliferation of bone-marrowderived dendritic cells (BMDCs). ELC (CCL19), another high-affinity
ligand for CCR7, was used as the control at the same time. We found that
SLC directly stimulated the proliferation of BMDCs and enhanced the
antigen-presenting function and CCR7 expression. Western blot analysis
showed that pNF-jBp65 was involved in this mechanism. We also found
that the NF-jB inhibitor PDTC could specifically block the proliferation
and CCR7 expression of BMDCs induced by SLC or ELC (200 ng/mL).
The results suggested that there were cross-talk signals between the chemotaxis and proliferation of BMDCs involving the SLC/CCR7 axis. Anat
C 2009 Wiley-Liss, Inc.
Rec, 293:48–54, 2010. V
Key words: secondary lymphoid
BMDCs; pNF-Bp65
Chemokines are a superfamily of small, basic, and
chemotactic proteins that mediate the trafficking activity
of immune cells by interacting with specific transmembrane G-protein coupled receptors (von Andrian and
Mackay, 2000; Zlotnik and Yoshie, 2000; Rot and von
Andrian, 2004). Secondary lymphoid tissue chemokine
(SLC), also known as CCL21/Exodus-2, is an important
member of the CC chemokine sub-family. It is constitutively expressed in high levels by stromal cells in the
T-cell area of secondary lymphoid tissue organs (Yoshida
et al., 1998). Chemokine receptor 7 (CCR7), the receptor
for SLC, is expressed on all naı̈ve T cells, some memory
T cells, B cells, and dendritic cells (DCs). Therefore, the
SLC/CCR7 signal axis plays a central role in lymphocyte
trafficking and homing to secondary lymphoid tissues.
In vivo and in vitro functional studies have demonstrated the highly significant role of SLC/CCR7 in the
control of DCs migration to and from sites of antigen
challenge and in the development of peripheral lymphoid
organs (Nagira et al., 1998; Tangemann et al., 1998; Willimann et al., 1998; Forster et al., 1999, 2008; Gunn
et al., 1999; Ueno et al., 2002).
C 2009 WILEY-LISS, INC.
V
tissue
chemokine;
CCR7;
Despite the well-characterized roles of the SLC/CCR7
axis in chemotaxis, very few studies have investigated the
effect of the SLC/CCR7 axis on the proliferation or maturation of bone-marrow-derived dendritic cells (BMDCs).
Recently, we found that SLC contributed to the maturation of BMDCs, strengthened their antigen-presenting
functions, and induced secretion of IL-12 and IFN-c
(Liang et al., 2007). These findings support and extend
Grant sponsor: National Natural Science Foundation of China
(NSFC); Grant numbers: 30500280, 30871312.
*Correspondence to: C.M. Liang, Department of Anatomy,
Histology and Embryology, Shanghai Medical College of Fudan
University, 138 Yixueyuan Road, Shanghai 200032, China. Fax:
8621-54237027. E-mail: cmliang@fudan.edu.cn or C.P. Zhong,
Department of Anatomy, Histology and Embryology, Shanghai
Medical College of Fudan University, 138 Yixueyuan Road,
Shanghai 200032, China. E-mail: cpzhong@shmu.edu.cn
Received 12 May 2009; Accepted 10 July 2009
DOI 10.1002/ar.21015
Published online in Wiley InterScience (www.interscience.wiley.
com).
PROLIFERATION OF BMDCs
49
Fig. 1. Proliferation assay of BMDCs treated with different concentrations of SLC. (A) Incubation of BMDCs with different concentrations
of SLC in vitro for 12 hr. The proliferation of BMDCs was stimulated
by SLC in a concentration-dependent manner. a, 0; b, 50; c, 100; d,
200; e, 300; f, 400 ng/mL SLC. Bar ¼ 100 lm. (B) CCK-8 assay of
BMDCs proliferation treated with different concentrations of SLC for
12 hr. The optimal concentration of SLC for BMDCs proliferation was
200 ng/mL. Results represent three independent experiments. *P <
0.05, **P < 0.01, as compared with cultures with 0 ng/mL SLC.
the results of previous studies by demonstrating that the
observed SLC-mediated antitumor effect is T-cell dependent. SLC treatment resulted in systemic immune
responses, which were accompanied by a significant
increase in the expression of IFN-c, GM-CSF, and IL-12
(Yang et al., 2004). However, the mechanism of the SLCstimulated proliferation of BMDCs remains unknown. In
this study, we examined the effect of SLC at different concentrations (0, 50, 100, 200, 300, and 400 ng/mL) on the
proliferation of semi-mature BMDCs and observed the
direct stimulating effect of SLC on BMDCs. We also dem-
onstrated that pNF-jB p65 was involved in the stimulation of proliferation involving the SLC/CCR7 signals.
MATERIALS AND METHODS
Mice and Reagents
C57BL/6J (H-2b) female mice, 6–8 weeks of age, were
purchased from the Chinese Academy of Science and
housed at the Animal Maintenance Facility of Shanghai
Medical College, Fudan University. RPMI 1640 medium
and heat-inactivated fetal calf serum were obtained from
50
ZHOU ET AL.
GIBCO-BRL (Gaithersburg, MD). Recombinant mouse
SLC and ELC (99% purity, <1 endotoxin unit/lg), GMCSF, and IL-4 were from PeproTech (Rockey Hill, NJ).
Monoclonal rat anti-mouse CCR7 antibody was obtained
from eBioScience (San Diego, CA). Monoclonal antibodies
for NF-jB p65, phospho-NF-jB p65, b-actin, and E2F-1
were purchased from Cell Signaling Technology (Beverly,
MA). E2F-2 mAb was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Corresponding HRP-conjugated
secondary antibodies were from KPL (Gaithersburg, MD).
The CCR7 inhibitor pertussis toxin (PTX) and the NF-jB
inhibitor pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma-Aldrich (St. Louis, MO).
Generation of BMDCs
BMDCs were generated as described previously with
minor modifications (Liang et al., 2007). Briefly, bone
marrow cells flushed from tibias and femurs were
depleted of erythrocytes by incubating in 0.9% ammonium chloride for 3 min at 37 C. The cells were washed
in HBSS and cultured in complete culturing medium
(CM) containing 10% FCS with 10 ng/mL GM-CSF and
TABLE 1. SLC/CCR7 enhanced the APC
function of BMDCs
DC:T ¼ 1:10
Groups
Control BMDCs
BMDCs þ PTX þ SLC
BMDCs þ SLC
BMDCs þ PTX
PI (X s)
Sample (n)
12
12
12
12
2.95
7.411
10.562
6.883
0.17
1.042*
1.84**
1.29*
20 ng/mL IL-4 at 2 106 cells/mL. BMDCs were induced
with GM-CSF plus TNF-a for additional 3 days from
day 6, then nonadherent cells were harvested by gentle
pipetting and stained with anti-CD11c antibody-conjugated microbeads (Miltenyi Biotec, Auburn, CA) to magnetically sort CD11cþ BMDCs. The purity of the sorted
BMDCs (semi-mature BMDCs) was consistently greater
than 90% as analyzed by immunofluorescence staining.
Cell Proliferation Assay
Aliquots of 2 106 BMDCs isolated by CD11cþ
microbeads were incubated in RPMI 1640 supplemented
with 10% FCS and 20 mmol/L HEPES buffer for 30 min
at 37 C and then were respectively stimulated with 0,
50, 100, 200, 300, and 400 ng/mL SLC for 12 hr. A cell
count kit-8 (CCK-8, Beyotime, China) was used in this
experiment to quantitatively evaluate the cell proliferation and to determine the optimal concentration of SLC.
The absorbances at 450 nm were determined using a
microplate reader (Model 680, Bio-Rad, Hercules, CA).
RPMI 1640 medium containing 10% CCK-8 was used as
a control. Cell proliferation index (PI) was calculated by
the formula: PI ¼ OD of experimental group/ OD of control group. In another experiment, BMDCs (2 106/mL)
were pretreated at 37 C with or without PDTC (100 lM)
for 1 hr, then incubated with SLC or ELC (200 ng/mL)
for 0, 1, 2, 4, 6, 12, and 24 hr, followed by visual observation under a microscope and by CCK-8 assay for the
statistic analysis of BMDCs proliferation.
Proliferation Assay of Cocultured Lymphocytes
Compared with control, *P < 0.05, **P < 0.01.
Methyl thiazolyl tetrazolium (MTT) assay was used to
measure proliferation. Briefly, control BMDCs (1 105/
200 lL) and BMDCs treated with SLC (200 ng/mL) were
Fig. 2. Expressions of CCR7, NF-jB p65, p-NF-jB p65, and E2F in
BMDCs treated with SLC. (A) CCR7, NF-jB p65, p-NF-jB p65, and
E2F were detected by Western blot at 1hr and 12 hr after SLC (200
ng/mL) was added into the medium. BMDCs treated with SLC at 0 hr
were used as an experimental control. b-actin was used as a quantitative control. CCR7 expression was increased at 1 hr and 12 hr after
stimulation with SLC. SLC signaling activated the NF-jB p65 pathway
and initiated phosphorylation of NF-jB p65. E2F expression did not
change following SLC stimulation. (B) Histogram for the densitometric
analysis of each protein level. Expression of each protein is shown as
relative density. Average of three independent experiments was used.
*P < 0.05, **P < 0.01, as comparison with the control (0 hr).
PROLIFERATION OF BMDCs
51
Fig. 3. Proliferation assay of BMDCs at different times of incubation
with SLC or ELC (200 ng/mL). (A) Observation of BMDC proliferation
under a microscope. BMDCs were pretreated with or without 100 lM
PDTC (NF-jB inhibitor) at 37 C for 1 hr, and then incubated with SLC
or ELC (200 ng/mL) for 0, 6, 12, and 24 hr. Bar ¼ 100 lm. (B) CCK-8
assay of BMDCs proliferation at 0, 1, 2, 4, 6, 12, and 24 hr after incubation with SLC (200 ng/mL). BMDCs were pretreated with or without
PDTC (100 lM) at 37 C for 1 hr. Results represent three independent
experiments. *P < 0.05, **P < 0.01, as compared with cultures in the
presence of PDTC.
cocultured with T cells at a 1:10 ratio for 24 hr, which
were plated in 96-well round-bottom microtiter plates.
BMDCs pretreated with PTX (100 ng/mL) were used as
an experimental control. It was followed by addition of
20 lL of MTT stock solution to each well. Then the
plates were incubated in a 5% CO2 incubator at 37 C for
5 hr, and the medium was removed and collected for further use. Dimethyl sulfoxide (DMSO) (200 lL) was
added to each well. Absorbance at 450 nm was measured
within 1 hr.
Western Blot Analysis
BMDCs treated with SLC (200 ng/mL) were washed
twice with cold PBS, lysed with lysis buffer (RIPA) (50
mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1%
52
ZHOU ET AL.
Differences were considered statistically significant with
P < 0.05 and highly significant with P < 0.01.
RESULTS
Proliferation of BMDCs Stimulated
with SLC In Vitro
Fig. 3. (Continued)
SDS, 1% Triton X-100, 1% sodium deoxycholate, 0.1 mM
Na3VO4, 25 mM NaF, 1 mM PMSF, 10 mg/mL aprotinin,
10 mg/mL leupeptin) and incubated for 30 min on ice. After centrifugation at 14,000 rpm at 4 C for 30 min,
supernatants were collected and protein concentrations
were determined by BCA assay (Pierce, Rockford, IL).
For detection of the nuclear translocation of NF-jB p65,
cytoplasmic and nuclear extracts were prepared using
NE-PER nuclear and cytoplasmic extraction kit (Pierce,
Rockford, IL). Equal amounts of denatured proteins were
separated on a 10% SDS-PAGE gel and transferred onto
a PVDF membrane (Millipore, Bedford, MA). Membranes
were blocked with 5% nonfat milk in TBST (1 TBS containing 0.1% Tween 20), and then incubated with primary antibodies overnight. After washing with TBST for
three times, the membranes were incubated with HRPconjugated secondary antibodies and developed with
SuperSignal West Pico chemiluminescence substrate
(Pierce, Rockford, IL). Band intensities were quantified
using Band Leader software (Magnitec, Tel Aviv, Israel).
Immunofluorescence Analysis
BMDCs isolated by CD11cþ microbeads were plated
into 24-well culture plates, incubated for 30 min in
RPMI 1640 with 10% FCS and 20 mmol/L HEPES buffer
at 37 C in 5% CO2, and pretreated at 37 C with or without PDTC (100 lM) for 1 hr before incubation for 24 hr
with SLC or ELC (200 ng/mL). Cells were rinsed with
PBS and fixed with ice-cold acetone for 15 min. Nonspecific binding was minimized by blocking with 2.5% BSA
in PBS for 1 hr at room temperature. The samples were
incubated overnight with rat anti-mouse CCR7 mAb (1/
200 dilution in PBS; CST, Beverly, MA) at 4 C, washed
three times with PBS for 5 min each, and stained with
FITC-conjugated goat anti-rat immunoglobulin G secondary antibody (1/100 dilution in PBS; KPL, Gaithersburg, MD) for 30 min at 4 C. Isotype-matched antibodies
were used as a control.
Statistical Analysis
For comparisons of the various treatment groups, oneway ANOVA was performed. All statistical analyses
were performed using the SPSS statistical software
package (SPSS 12.0 for Windows; SPSS, Chicago, IL).
To assess whether SLC directly induces the proliferation of BMDCs, BMDCs were incubated in the presence
of SLC at different concentrations (0, 50, 100, 200, 300,
and 400 ng/mL). We observed under a microscope that
SLC stimulated the proliferation of clones of BMDCs as
the concentrations of SLC increased from 0 to 400 ng/
mL (Fig. 1A). Further quantitative evaluation was determined by CCK-8 analysis, and the data showed that the
proliferation was significantly different under different
concentrations of SLC (*P < 0.05, **P < 0.01, Fig. 1B).
The proliferation rate got to the highest level from the
SLC concentration of 200 ng/mL.
Upregulation of the Antigen-presenting
Function of BMDCs by SLC
The mixed lymphocyte reaction (MLR) assay revealed
that BMDCs treated with SLC (200 ng/mL) had significantly enhanced antigen-presenting function, which was
demonstrated by the pronounced T cell stimulation.
Moreover, the CCR7 inhibitor PTX downregulated the
antigen-presenting function of BMDCs (*P < 0.05, **P <
0.01, Table 1).
pNF-jB p65 was Involved in the SLC-Mediated
Proliferation of BMDCs
To gain a better understanding of the mechanism of
SLC/CCR7 axis-mediated proliferation of semi-mature
BMDCs, we analyzed the phosphorylation of NF-jB p65
and the nuclear transcriptors E2F-1 and E2F-2 by western blot at 1 hr and 12 hr after stimulation with SLC
(200 ng/mL). The data indicated that NF-jB p65 in the
cytoplasm of SLC-treated BMDCs was downregulated,
whereas the level of pNF-jB p65 was markedly up regulated in the nucleus. We found that the levels of E2F-1
and E2F-2 did not change significantly in BMDCs (*P <
0.05, **P < 0.01, Fig. 2).
Specific Inhibition of the Proliferation
of BMDCs by PDTC
To demonstrate that the proliferation of BMDCs is
related with SLC/CCR7 axis, we incubated BMDCs with
ELC as a positive control, which is another ligand for
CCR7. We observed that both SLC and ELC (200 ng/mL)
stimulated the proliferation of BMDCs at 6, 12, and
24 hr much more than the control (0 hr). At the same
time, we found that the specific inhibitor for NF-jB,
PDTC, blocked the proliferation of BMDCs (Fig. 3A).
CCK-8 assay was used and demonstrated that the SLCmediated proliferation of BMDCs was specifically inhibited by PDTC (*P < 0.05, **P < 0.01, Fig. 3B).
Inhibition of the CCR7 Expression
in BMDCs by PDTC
The CCR7 expression in BMDCs was analyzed by
western blot analysis at 1 hr and 12 hr after stimulation
with SLC (200 ng/mL). The result showed that the
PROLIFERATION OF BMDCs
53
Fig. 4. Immunofluorescence analysis of the CCR7 expression in BMDCs. BMDCs were stimulated with
SLC or ELC (200 ng/mL) for 24 hr after being pretreated with or without 100 lM PDTC at 37 C for 1 hr.
Bar ¼ 50 lm.
CCR7 expression was increased (*P < 0.05, Fig. 2). Immunofluorescence analysis of the CCR7 expression in
BMDCs stimulated with SLC or ELC (200 ng/mL) for
24 hr also showed a strong positive staining, whereas
PDTC could specifically inhibit the expression of CCR7
(Fig. 4).
DISCUSSIONS
The chemokine receptor CCR7 contains transmembrane domains and transduces its signals through heterotrimeric G proteins, mainly of the Gai subclass, and
their downstream effectors (Mellado et al., 2001). CCR7
is highly expressed in semi-mature and mature DCs. It
has been clearly demonstrated that SLC (CCL21) can
vigorously promote migration of DCs (Gunn et al., 1999;
Nakano and Gunn, 2001). Based on its role in the trafficking of lymphocytes and DCs to LNs, CCR7 is
regarded as a central organizer of the primary protective
immune response. Despite the critical importance of
SLC (CCL21)/CCR7 in immune response, little is known
about the signals induced by this axis. CCR7, like other
chemokine receptors, is capable of initiating a complex
cascade of intracellular signal transduction that may be
relevant to cellular activation in addition to the migration event. It has been found that in human DCs the
54
ZHOU ET AL.
SLC/CCR7 axis activates two independent signaling
modules: one involving Gai and a hierarchy of MAPK
family member that regulates chemotaxis, and the other
involving Rho/Pyk2/cofilin that regulates the migratory
speed of DCs (Riol-Blanco et al., 2005). Apart from the
aforementioned reports, a number of recent studies have
indicated that CCR7 controls the cytoarchitecture, rate
of endocytosis, survival, migratory speed, and maturation of the DCs (Sánchez-Sánchez et al., 2006). Moreover, it has been found that CCR7 uses the transcription
factor NF-jB to regulate CCR7-dependent survival in
the DCs (Sánchez-Sánchez et al., 2004). Therefore,
CCR7 may use independent signaling modules to regulate its multiple functions in DCs.
In this study, we found that the SLC/CCR7 directly
stimulated the proliferation of BMDCs together with elevated antigen-presenting function and CCR7 expression.
Our findings are consistent with those of a previous
study, which indicated that SLC (CCL21) and ELC
(CCL19), another ligand for CCR7, are potent natural
adjuvant for terminal activation of DCs (Marsland et al.,
2005). Our previous preliminary studies using the
Superarray gene chip and qPCR assays revealed that
several signals, such as the PKC pathway, PI-3 kinase
pathway, NF-jB pathway, and cyclins pathway, were
functionally activated on stimulation of the SLC/CCR7
axis (data not shown). We explored the potential mechanisms involved in these signals and deduced that pNFjB p65 was involved not only in the survival of DCs, as
previously reported (Sánchez-Sánchez et al., 2004), but
also in the proliferation of BMDCs. It was evident that
the NF-jB inhibitor PDTC specifically blocked the proliferation and CCR7 expression of BMDCs treated with
SLC or ELC. The results suggested that there were
cross-talk signals between the chemotaxis and proliferation of BMDCs involving the SLC/CCR7 axis.
The transcription factor E2F plays a central role in
the cell cycle through its ability to activate genes
involved in cell division. Therefore, we investigated the
potential role of E2F and its effect on cyclins in the development of semi-mature BMDCs. We did not find
obvious changes in the expression levels of E2F-1 and
E2F-2 after stimulation with SLC (CCL21). While the
data may indicate that E2F is not involved in the proliferation of semi-mature BMDCs, it can also be inferred
that there may be other pathways that affect the cyclins
of BMDCs in the SLC/CCR7 axis.
In summary, our studies on the functional capabilities
of the SLC/CCR7 axis and mechanisms by which it regulates the different functions of BMDCs or other cells will
contribute to the better understanding of the molecular
and cellular processes of the immune system.
ACKNOWLEDGMENTS
The authors thank Professor Zhang Su-chun from the
University of Wisconsin-Madison for his critical readings
and comments.
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