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: firstname.lastname@example.org or C.P. Zhong, Department of Anatomy, Histology and Embryology, Shanghai Medical College of Fudan University, 138 Yixueyuan Road, Shanghai 200032, China. E-mail: email@example.com 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. LITERATURE CITED Forster R, Davalos-Misslitz AC, Rot A. 2008. CCR7 and its ligands: balancing immunity and tolerance. Nat Rev Immunol 8:362–371. 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