Exogenous dendritic cell homing to draining lymph nodes can be boosted by mast cell degranulation

Cellular Immunology 263 (2010) 204–211

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Exogenous dendritic cell homing to draining lymph nodes can be boosted by mast cell degranulation Shu-Rong Ren a,b, Li-Bin Xu b,c, Zhi-Yuan Wu b, Jun Du b, Mei-Hua Gao a,*, Chun-Feng Qu b,** a

Department of Immunology, Medical College of Qingdao University, Qingdao 266071, China State Key Laboratory of Molecular Oncology, Cancer Institute/Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China c Department of Surgery, Cancer Institute/Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China b

a r t i c l e

i n f o

Article history: Received 22 December 2009 Accepted 30 March 2010 Available online 4 April 2010 Keywords: Dendritic cells Homing to draining lymph nodes Mast cell degranulation

a b s t r a c t Dendritic cells (DCs), as potent antigen presenting cells, are increasingly used for immunotherapeutic approaches, predominantly in oncology. Low efficiency of injected Ag-pulsed DC homing to draining lymph nodes (DLNs) is one of the factors that affect the efficacy of therapy. As Langerhans cell emigration was enhanced after skin mast cell degranulation, we investigated the effect of local mast cell activation on exogenous bone marrow-derived DCs (BM-DCs) homing to DLNs. Product of activated MC/9 mast cells enhanced chemotaxis of BM-DCs to CCL21 in vitro. Intradermal injection of compound 48/80 (c48/80) induced local skin mast cell obvious degranulation and boosted exogenous BM-DC homing to DLNs. Both Ag-specific lymphocyte proliferation and TH1/TH2 cytokine production increased after HBsAg-pulsed BM-DC was injected into c48/80 pretreated mice. These results suggest that transferred DC homing to DLNs promoted by local mast cell degranulation may have potential application to improve DC-based immunotherapy. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Dendritic cells (DCs), the most potent professional antigen presenting cells, play critical roles in triggering immunity to many types of antigens including tumors [1]. DC migration from peripheral tissue to secondary immune organs especially lymph nodes is fundamental prerequisite for initiating an effective immune response. The magnitude of immune response is proportional to the number of antigen-loaded DCs that reached draining lymph nodes (DLNs) [2]. Tumor associated antigen-pulsed DCs have been explored in anti-tumor immunotherapy in clinical practices [3]. However, in clinical trials, the unexpectedly poor mobilization of adoptively transferred DCs to DLNs has limited the efficacy of therapeutic vaccines. Most of the administered DCs stayed in the original site of injection and less than 5% of the cells reached the DLNs [4–6]. Approaches have been sought to facilitate DC homing, which included tissue conditioning with TNF, IL-1 and TLR agonist, or * Correspondence to: M.-H. Gao, Department of Immunology, Medical College of Qingdao University, Room 305, Boya Building, 308 Ningxia Road, Qingdao 266071, China. ** Correspondence to: C.-F. Qu, State Key Laboratory of Molecular Oncology, Cancer Institute/Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 17 Panjiayuan Nanli, Chaoyang District, Beijing 100021, China. Fax: +86 10 6771 3917. E-mail addresses: [email protected] (M.-H. Gao), [email protected] (C.-F. Qu). 0008-8749/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2010.03.017

induction of CCR7 expression on DC [2,7–9]. However, so far no satisfying efficiency of DC homing have been achieved with all the studies published. Techniques facilitating DC homing to DLNs are being required for advancing DC-based immunotherapy. DC homing to DLNs is orchestrated by a complex interplay between chemokines and their receptors, adhesion molecules, as well as matrix metalloproteinases and lipid mediators [10,11]. CCR7 expressed on DCs interacting with its ligands CCL19 and CCL21 is critical for DC homing [12–14]. However, expression of CCR7 alone is not sufficient for DC migration. Signals found at site of inflammation, such as lipid mediators cysteinyl leukotrienes (LTs) and prostaglandin E2 (PGE2), are required to sensitize CCR7-mediated signal transduction and augment DC homing to DLNs [15–17]. In anaphylaxis, a large amount of LTs and PGE2 are produced by activated mast cells [18]. It has been reported that local mast cell activation enhanced Langerhans cell migration from skin to DLNs [19,20]. Recent in vivo studies showed that Bacillus anthracis protective antigen immunization in combination with a mast cell activator compound 48/80 (c48/80) resulted in increased endogenous DC accumulation in DLNs and enhanced protective humoral immune response [21]. But the roles of mast cell activation on modulating exogenous DC homing to DLNs remains unresolved. In the present study, we investigated whether local mast cell degranulation could enhance the migration of transferred bone marrow-derived DCs (BM-DCs) to DLNs, which may have potential therapy application in tumor associated diseases.

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2. Materials and methods 2.1. Mice and reagents Female C57BL/6 mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. Animals were maintained in specific pathogen-free facility and studied at 8–12 weeks of age. All experiments were performed according to the guidelines of Local Animal Experiment Ethics Committee. Recombinant murine granulocyte–macrophage colony stimulating factor (rmGM-CSF) was the supernatant from J588L cells. Recombinant murine TNF-a and CCL21 were purchased from Peprotech. Phycoerythrin (PE) conjugated anti-mouse CD11c, Biotin anti-mouse CD11c, and PE-streptavidin were from eBioscience. FluoresbriteÒ YG Microspheres (1 lm in diameter) was from Polysciences Corporation. Compound 48/80 (c48/80) and LPS were purchased from Sigma. Collagenase D was from Roche. Fetal calf serum (FCS) was obtained from Tissue Culture Biologicals. Purified HBsAg from human serum was purchased from Beijing Hepatitis Research Center. 2.2. Cell line MC/9, a mast cell line derived from mouse fetal liver, was maintained at 37 °C and 5% CO2 in DMEM supplemented with 10% FCS, 100 U/ml penicillin, and 100 lg/ml streptomycin. In addition, 10% of rat splenic cell culture supernatant obtained by Concanavalin A stimulation was added to the culture system. MC/9 degranulation was induced by incubating MC/9 cells (2  106/ml) with c48/80 (final concentration 10 lg/ml) at 37 °C for 30 min and the supernatant was collected and stored at 20 °C for future use. 2.3. Tissue preparation for histological examination C57BL/6 mice were treated with c48/80 and examined for local mast cell degranulation using method described by McLachlan et al. [22] with some modifications. Briefly, mice were given intradermal (i.d.) injection of 25 lg/50 ll c48/80 in scapular area where skin was shaved before injection. After injection, mice were sacrificed at 30 min, 4 h, 8 h, 24 h and 48 h, respectively. The control animals were given i.d. injection of the same volume of normal saline (NS). Skin biopsy samples from the injected area were fixed in 10% formalin and embedded in paraffin. Sections were cut at a thickness of 5 lm and stained with hematoxylin and eosin (H&E) or Toluidine Blue. Intact and degranulated mast cells were counted in continuous 10 high power fields (400) per skin section. Degranulated mast cells were defined as having at least three granules evident outside the cell or having reduced granule density throughout the entire cell. The number of total mast cells equals to the number of intact mast cells plus the number of degranulated mast cells. 2.4. Preparation of BM-DCs BM-DCs were generated from bone marrow progenitor cells using previously described techniques [15,23] with some modifications. Briefly, bone marrow cells were harvested from the femurs and tibias of C57BL/6 mice and depleted of erythrocytes with 1.66% ammonium chloride. Cells were then adjusted to 1  106/ ml with complete medium (RPMI 1640 supplemented with 5% heat-inactivated FCS, 100 U/ml penicillin, 100 lg/ml streptomycin, 50 lM 2-mercaptoethanol and 1:40 rmGM-CSF) and seeded into 6well plates at 4 ml/well. All cultures were incubated at 37 °C in 5% humidified CO2. The culture medium was refreshed every 2 days with half of the old medium being replaced with an equivalent

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volume of complete medium. Non-adherent cells spontaneously released from the clusters were harvested after 6 days of culture and transferred into new 6-well plates. LPS (20 ng/ml) was added on day 7 for cell maturation. The purity of BM-DCs was determined by the expression of CD11c with FACS using PE conjugated antimouse CD11c. 2.5. Chemotaxis assay Chemotaxis of BM-DCs to CCL21 was measured by migration through a polycarbonate filter (5-lm pore size) in 24-well transwell chambers (Corning Costar) with RPMI 1640 supplemented with 0.1% BSA as assay medium. A volume of 600 ll assay medium with or without 100 ng/ml of CCL21 was added to the lower chamber. BM-DCs (5  105 in 100 ll) with or without MC/9 supernatant were added to the upper chamber and incubated for 2 h at 37 °C. The number of migrated cells to the bottom chamber was counted by hemacytometer. Each experiment was performed in triplicate. The number of chemotactic cells equaled to the number of total migrated cells minus the number of spontaneously migrated cells. 2.6. In vivo DC migration assay Propagated BM-DCs were pulsed with YG-microspheres on day 6. On day 7, 25 lg/50 ll of c48/80 was injected into one side of the scapular skin and 50 ll NS into the other side as control. On day 8, 1  106 BM-DCs in four injections were given to each side of the scapular skin. Two days after DC injection, brachial and axillary DLNs were pooled and digested with 2.7 g/L collagenase D for 25 min at 37 °C. EDTA at a final concentration of 10 mM was added during the last 5 min. Single cell suspensions were generated by pressing LN cells through a 70-lM cell strainer. The total cell number was evaluated for each sample by using a hemacytometer. Samples were stained with biotin-conjugated anti-mouse CD11c and PE-streptavidin. After washing twice with PBS, the cells were determined by FACS and analyzed by FlowJo software program. The total number of migrated cells in DLNs equals to the percentage of migrated cells determined by FACS multiplied by the total number of DLNs cells. For fluorescence microscopy, 8 lm cryostat sections of snap-frozen DLNs were prepared as described previously [24] and the migrated BM-DCs were examined with fluorescence microscopy. 2.7. Preparation of HBsAg-pulsed DCs and immunization of mice BM-DCs were generated from bone marrow progenitors using rmGM-CSF as described above. On day 6 of culture, the immature DCs were pulsed with 20 lg/ml purified HBsAg. On day 7, the cells were added LPS (20 ng/ml) overnight for maturation. On day 8, the DCs were washed twice in PBS and resuspended in PBS for injection into the scapular skin of mice (5  105 cells per mice). Mice were divided into four groups with five mice in each group: group I (G1): c48/80 pretreated mice were injected with HBsAg-pulsed BM-DCs; group II (G2): mice were injected with HBsAg-pulsed BM-DCs; group III (G3): mice were injected with non-pulsed BMDCs; group IV (G4): mice did not receive any treatment (naive control). The c48/80 pretreatment and injection of BM-DCs were repeated twice at a 1-week interval in each mouse from the three experiment groups. 2.8. Lymphocyte proliferation assay DLN cells and splenocytes were obtained from each experimental group 3 weeks after the final injection. Single cell suspensions were generated by pressing DLN cells or splenocytes through a

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70-lM cell strainer. Splenic erythrocytes were lysed by 1.66% amino chloride and washed twice with RPMI 1640. Duplicate samples of 2  105 cells in 100 ll RPMI 1640 supplemented with 10% heat-inactivated FCS were seeded in 96-well flat-bottom plates and were stimulated with 2 lg/ml HBsAg. The ability of lymphocyte proliferation was measured by Cell Counting Kit-8 (Dojindo) according to the manufacturer’s instruction. Briefly, the cells were incubated at 37 °C for 72 h. Then 10 ll WST-8 was added to each well. The culture plates were maintained in the same condition for an additional 4 h, and optical density at 450 nm (OD 450) was measured for each well. 2.9. Cytokine secretion To measure the IL-4 and IFN-c secreted by DLN cells and splenocytes from immunized mice, single cell suspensions were generated as describes above. 2.5  106 cells in 0.5 ml medium were seeded in 24-well plates, stimulated with 2 lg/ml HBsAg and supernatant were collected at 72 h. Quantities of secreted IL4 and IFN-c were measured by ELISA using Ready-SET-go sets (eBioscience) as described by the manufacturer. 2.10. Statistical analysis All data are expressed as means ± standard deviations. Statistical analyses of data were performed using GraphPad Prism 5 software. Non-parametric Mann–Whitney U test was performed for

restimulation cytokines. Other data were analyzed with the Student’s two tailed t-test between two groups and ANOVAs for multiple comparisons. Differences were considered significant at p < 0.05.

3. Results 3.1. Compound 48/80 induced skin mast cell degranulation c48/80 is a well known mast cell activator which can cause mast cell degranulation [25]. Treatment with 25 lg of c48/80 was well tolerated by all mice with no apparent ruffling of fur, diarrhea, or other signs of anaphylactic reaction except for local edema and inflammation at later time. Thirty minutes after c48/ 80 injection, there was a striking increase in the number of the degranulated mast cells, which reached the highest level 4 h after c48/80 injection (Fig. 1). However, after the 4th hour, the number of degranulated mast cells counted began to drop gradually, but was still significantly higher than that of the control group at corresponding test time points until 48 h after injection where there was no significant difference in the number of degranulated mast cells between the two groups. Meanwhile, the number of intact mast cells decreased gradually until reaching the lowest level at 24 h after c48/80 treatment (Fig. 1B). At all time points, the number of intact mast cells was significantly lower than that of the control group (p < 0.001). Similarly, the total mast cell number decreased gradually from 4 to 48 h, which was significantly lower

Fig. 1. Histological changes of skin mast cells following intradermal injection of compound 48/80 (c48/80) or normal saline (NS). (A) Toluidine Blue-stained sections are shown at 30 min after injection of NS or c48/80. Intact mast cells (black arrows) are readily detected following injection of NS, but more degranulated mast cells (white arrows) could be detected following injection of c48/80. (B) Quantification of local mast cell numbers in scapular skin after c48/80 injection in 10 high microscopic fields at indicated time (n = 3–5 mice/time point). *p < 0.05; **p < 0.01; ***p < 0.001 vs. NS group.

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than that of the control group (Fig. 1B). In NS treated control groups, there were no obvious changes in mast cell degranulation at all tested time points (data not shown).

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cell number of the lymph nodes dropped significantly compared with that observed at 24 h. 3.3. Identification of BM-DC

3.2. Intradermal injection of c48/80 induced skin inflammation and DLN hypertrophy A variety of stimuli can activate mast cells to release a diverse array of biological active products, many of which including TNFa and IL-1b can mediate proinflammatory effects [26]. We therefore monitored injected site reactions induced by c48/80 activated mast cells. Fig. 2A shows typical skin sections stained with H&E 4, 24 h after c48/80 injection or 30 min after NS injection. No obvious changes were observed in early stages of c48/80 injection between 30 min and 4 h, nor were in control side. Obvious inflammatory cells were present in injected local skin at 8 and 24 h, which could be still observed 48 h after injection. No obvious change was found at distal site of c48/80 injection. To characterize the effect of c48/80 on DLNs, total cell number in the draining brachial and axillary lymph nodes were determined after injection of 25 lg c48/80 in the brachial site at different time. Compared with the control group, lymph node hypertrophy was discernable by 4 h after c48/80 injection with the most significant cell number increase observed at 24 h, which was about twofold greater than that of the control (Fig. 2B). By the time of 48 h, the

After 8 days of culture with rmGM-CSF, BM-DCs were propagated from murine bone marrow progenitor cells and about 1  107 cells were generated per mouse. Moderate expression of CD11c (a characteristic marker of murine DCs) on the cultured bone marrow cells was identified at day 8 (Fig. 3A). In order to distinguish transferred exogenous BM-DC from endogenous DC in DLNs, exogenous BM-DC were cultured with YG-microspheres before transfer so that BM-DC could be labeled with YG-microspheres by uptaking YG-microspheres. In order to determine the proportion of labeled DCs in cultured BM-DCs, CD11c staining was applied to the labeled BM-DC. The results revealed that most of the bead-labeled cells were CD11c+ DCs (Fig. 3B), which ensured most of adoptive infused labeled cells are DCs and can be correctly traced in DLNs in vivo by the labeling method. 3.4. MC/9 degranulation enhanced BM-DC chemotaxis toward CCL21 When incubated with c48/80, MC/9 mast cells were activated to release granules into the supernatant [27]. Therefore, the supernatant was used as product of mast cell degranulation. Transwell

Fig. 2. Local skin inflammation and DLN hypertrophy after c48/80 injection. (A) Typical H&E-stained skin sections are shown from NS-treated mice (control) and c48/80 treated mice at 4 and 24 h after 25 lg of c48/80 injection. Arrow indicates prominent inflammatory infiltrates. (B) Total cell number in the draining brachial and axillary lymph nodes at tested time points following c48/80 injection (n = 3–5 mice/time point). **p < 0.01; ***p < 0.001 vs. control.

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Fig. 3. FACS analysis of BM-DC. (A) Bone marrow cells were cultured with complete medium for 8 days and stained with PE conjugated anti-mouse CD11c. (B) Bone marrow cells were labeled with YG-microspheres on day 6 and stained with PE conjugated hamster IgG (left) or PE conjugated anti-mouse CD11c (right) on day 8.

3.6. Mice pretreated with c48/80 and immunized with HBsAg-pulsed BM-DCs demonstrated enhanced specific lymphocyte proliferation and cytokine secretion

Fig. 4. Chemotaxis assay of BM-DC in response to CCL21. Migration of BM-DC in the absence or presence of different dilution of product from activated MC/9 mast cells (MC) toward CCL21 was assessed by means of transwell chemotaxis assays. **p < 0.01; ***p < 0.001 vs. BM-DC alone.

Exogenous BM-DCs homing to DLNs were increased by local mast cell degranulation (as described in Section 3.5), but whether the increased migration of DC to DLNs actually results in an enhanced immune response was further investigated. HBsAg was used for pulsing BM-DCs before being transferred into c48/80 pretreated mice. The results revealed that DLN cells obtained from mice receiving HBsAg-pulsed BM-DC injection showed stronger lymphocyte proliferation when compared with naïve control (G4). The strongest lymphocyte proliferation was observed in the group treated with c48/80 and HBsAg-pulsed BM-DC (G1), which was significantly greater than that of G2 (p < 0.001) (Fig. 6A). Lymphocyte proliferation from obtained spleen cells was similar to that of DLNs among different groups (Fig. 6A). Both IL-4 and IFN-c were produced in cultured DLNs and spleen cells in all groups. The highest IL-4 and IFN-c levels were observed in G1 (Fig. 6B). Both IL-4 and IFN-c secretion were significantly increased in DLN and spleen cells of G1 group compared with their corresponding G2 group. 4. Discussion

migration assay demonstrated that activated MC/9 product enhanced in vitro chemotaxis of BM-DC to CCL21 (Fig. 4). The number of migrated BM-DC in the presence of MC/9 product was significantly higher compared with BM-DC alone. The number of chemotactic cells was proportional to the concentration of MC/9 activation product and 1:80 dilution of the product could result in significant increase of DC migration. 3.5. c48/80 increased BM-DC homing to DLNs To examine the effect of c48/80 on BM-DC homing to DLNs, microsphere-labeled BM-DCs were injected into the scapular skin of C57BL/6 mice pretreated with or without c48/80. Two days after BM-DC injection, the homing of BM-DCs to DLNs was examined by fluorescent microscope and FACS. The DLNs from c48/80 pretreated and BM-DC injected side demonstrated greater amount of labeled DCs compared with that from BM-DC injected, but without c48/80 pretreated side (Fig. 5A). FACS results showed that most of the bead-labeled cells in DLNs were CD11c+ (Fig. 5B). The number of migrated BM-DCs in DLNs of c48/80 pretreated side was significantly higher than that of the contralateral side (p < 0.001, Fig. 5C). The mean increasing rate was 68.2%.

DC-based immunotherapy for tumors have been focus of many scientific researchers [28]. Most protocols involved the in vitro loading of DC with tumor antigen, followed by induction of maturation and subcutaneous or i.d. injection into host tissues. Although this treatment has been demonstrated to be effective in stimulating immune responses, a variety of factors are being considered to optimize DC vaccination. These include DC lineage, nature of the maturation stimulus, and route, frequency, and dosage of DC injection. The number of DCs injected, and ultimately the number of DCs that ends up in the T cell zone, represent important variables that impact on T cell priming. On one hand, larger number of DCs increases the probability of DC–T cell encounter, and delivers a sustained stimulation through interactions with T cells [29], on the other hand, smaller number of poorly stimulatory DCs induces abortive T cell proliferation and tolerance [30,31]. Mast cells, as highly specialized secretory cells, locate preferentially at the environment interface of the body and play roles both in innate and adaptive immune responses [18]. Mast cells are initiators and effectors of innate immunity, which are essential for the resolution of bacterial infections through neutrophil mobilization to the site of infection [32,33]. Mast cells play critical roles in

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Fig. 5. Adoptively transferred BM-DC in DLNs 48 h after BM-DC injection in the scapular skin. Histological images (A) and FACS analysis (B) of the DLNs of mice 48 h after YGmicrospheres-labeled BM-DCs were injected into c48/80 or NS pretreated scapular skin. (C) The number of total migrated BM-DC in DLNs. ***p < 0.001.

the effector phase of anaphylaxisis such as allergy and asthma. In addition, activated mast cells can secrete a variety of biologically active products which may influence the development, intensity and duration of adaptive immune responses that contribute to host defense against infectious agents or tumors [18]. Studies on animal contact hypersensitivity to FITC showed that mast cell activation and mast cell-derived TNF-a were responsible for efficient DC entry to DLNs [34]. Another report indicated that mast cell-derived histamine was related to DC homing in IgE-mediated anaphylaxis [20]. It was recently reported that mast cell activation induced by c48/80 resulted in the increase of endogenous DCs in DLNs and could be used safely and efficiently in the induction of protective humoral immune responses [21,35]. So we hypothesized that mast cell activation may also enhance transferred DC homing to DLNs, which is a key factor for enhancing the efficacy of antigenloaded DC-based immunotherapy. c48/80, as a well known mast cell activator, can trigger Gi protein-dependent exocytic release of mast cell granules [25,36]. Local injection of 25 lg c48/80 induced significant mast cell degranulation. There was a gradual increase of mast cell degranulation between 30 min and 4 h of c40/80 injection. After 4 h, a drop of the number of mast cell degranulation was found, which may due to the limitation of Toluidine Blue staining in detecting the totally

degranulated cells, which also resulted in a reduction in the total number of mast cells [22]. The number of granule intact mast cells gradually decreased over time, which indicated the increase of activated mast cells. The present study demonstrated exogenous BM-DC homing to DLNs was increased by c48/80 induced mast cell activation. Minimal product of activated MC/9 mast cells significantly enhanced BM-DC chemotaxis to CCL21 (a critical ligand for DC homing to DLNs), which indicated degranulation of mast cells could enhance BM-DC migration to LN-directing cytokine. Intradermal injection of c48/80 induced local skin mast cell obvious degranulation and increased exogenous BM-DC homing to DLNs, which indirectly indicated exogenous DC migration to DLNs was boosted by mast cell degranulation. We chose BM-DC injection at 24 h after c48/80 treatment, when the skin demonstrated the largest number of mast cell activation. Activated mast cells could produce many biological active products such as TNF-a, IL-1, LTs, PGE2, etc., which can facilitate the migration of DCs [18]. It has been reported the lymph node hypertrophy as well as lymphatic vessel expansion induced by proinflammatory substance administration aid in transferred DC homing to the hypertrophied lymph node [37]. In the present study, c48/80 induced local inflammatory response and enlargement of the DLNs, which may be beneficial for exogenous

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Fig. 6. Lymphocyte proliferation assay and cytokine production by DLN and spleen cells. c48/80 pretreated or non-pretreated mice were injected with HBsAg-pulsed BM-DC or BM-DC. The c48/80 pretreatment and injection of BM-DC were repeated twice at a 1-week interval. On day 21 after the final injection, DLN cells (DLN) and spleen cells (SP) were harvested and stimulated with HBsAg in vitro for 72 h. Their ability to proliferate in response to HBsAg was detected by Cell Counting Kit-8 (A). The supernatants of stimulated lymphocytes were harvested and assayed for IL-4 and IFN- by ELISA (B). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

BM-DC homing to DLNs. The detailed mechanism of mast cellmediated BM-DC homing to DLNs need to be investigated. With the demonstration that activated mast cells promoted transferred DC homing to DLN in the present study, we were further interested in the potential association between increased DC homing and possible enhanced immune responses. To our expectation, mice immunized with HBsAg-pulsed BM-DC showed significantly higher levels of both Ag-specific lymphocytes and cytokines, including IFN-c (TH1-type) and IL-4 (TH2-type), if the mice were pretreated with c48/80. In accordance with our results, McGowen et al. demonstrated that C3H/HeN mice have been induced production of TH1,TH2 and TH17 cytokines after intradermal injection of Bacillus anthracis protective antigen in combination with c48/80 [35]. Other researchers have reported Th2-dominance in immune responses induced by mast cell activation [38,39]. The different profile of cytokine production may be due to different immunization protocols used in different research Institutes. In conclusion, local mast cell degranulation boosted exogenous BM-DC homing to DLNs and enhanced HBsAg-pulsed BM-DCs mediated immune responses. These results suggest that transferred DC homing to DLNs promoted by local mast cell degranulation may have potential application to improve DC-based immunotherapy. Acknowledgment This work was supported by basic scientific research fund of Chinese Academy of Medical Sciences (JK2007B06). References [1] J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque, Y.J. Liu, B. Pulendran, K. Palucka, Immunobiology of dendritic cells, Annu. Rev. Immunol. 18 (2000) 767–811.

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