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Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes A.C. Bhingare, T. Ohno, M. Tomura, C. Zhang, O. Aramaki, M. Otsuki, J. Tagami and M. Azuma J DENT RES 2014 93: 288 originally published online 30 December 2013 DOI: 10.1177/0022034513518223 The online version of this article can be found at: http://jdr.sagepub.com/content/93/3/288
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research-article2014
JDR
93310.1177/0022034513518223
RESEARCH REPORTS Biological
A.C. Bhingare1, T. Ohno1, M. Tomura2, C. Zhang1, O. Aramaki3, M. Otsuki3, J. Tagami3, and M. Azuma1*
Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes
1
Department of Molecular Immunology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; 2Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; and 3 Department of Cariology and Operative Dentistry, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; *corresponding author, [email protected] J Dent Res 93(3):288-293, 2014
Abstract
Introduction
Dendritic cell (DC) migration to regional lymph nodes (RLNs) is an essential step in adaptive immunity, and cell-surface antigens on migrating DCs greatly affect the quality and quantity of subsequent immune responses. Although MHC class II+ DC-like cells exist in the dental pulp, the lineage and function of these cells remain unknown. Here, we identified migratory DCs from the dental pulp after cusp trimming and acid etching in KikGR mice, in which the photoconvertible fluorescent protein changed from green to red upon violet light exposure. Two major cell fractions from the dental pulp had migrated to the RLNs at 16 hrs after cusp treatment, which showed the following lineage markers in the main and second fractions: CD11chighCD11b++Ly6Clow Ly6Glow F4/80+ and CD11cmedCD11b+++Ly6C++Ly6 G+++F4/80-, respectively. These lineage markers indicate that the former cells were DCs that had migrated through afferent lymphoid vessels, and the latter were granulocytes recruited via blood circulation. Migratory dental pulp DCs were mature, expressing the highest levels of CD273 (B7-DC) and CD86 co-stimulators and MHC class II. Our results suggest that cariogenic-bacteria-exposed dental pulp DCs migrate to RLNs and there trigger adaptive immune responses.
T
KEY WORDS: co-stimulatory molecules, CD86, CD273, cell trafficking, photoconversion, fluorescence protein.
DOI: 10.1177/0022034513518223 Received October 2, 2013; Last revision November 12, 2013; Accepted December 4, 2013 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
he immune system has evolved to recognize microbial attacks and eliminate infections. Dendritic cells (DCs) exist broadly in the skin and mucosa as the front line of defense against microbial pathogens (Steinman, 2001; Iwasaki, 2007; Steinman and Banchereau, 2007). DCs recognize microbes via pathogen-associated receptors at the periphery, secrete multiple cytokines/ chemokines to initiate innate immunity, and migrate to regional lymph nodes (RLNs) to present antigens to naïve T-cells that trigger adaptive immunity. DCs are heterogeneous cells, and the site-specific microenvironment modulates their phenotypic and functional properties. Odontoblasts, with their cellular processes extending into dentinal tubules, are the first line of defense against cariogenic bacteria in the dental pulp. They express a variety of pathogen-recognition receptors and chemokines, and trigger the initial innate inflammatory responses in cooperation with DC-like cells (Farges et al., 2009). MHC class II-positive DC-like cells with macrophage lineage markers have been found in the dental pulp of the human tooth with caries along with T-cell infiltration (Jontell et al., 1998; Yoshiba et al., 1998; Yoshiba et al., 2003). These observations suggest that dental pulp DCs with captured microbial antigens migrate to the RLNs to present antigens to naïve T-cells, followed by effector T-cell recruitment to the dental pulp. Previously, we demonstrated that 2 distinct cell populations with dendritic morphology exist in the steady-state dental pulp: CD11c+F4/80- and CD11c-F4/80+ cells (Zhang et al., 2006). A few CD11c+F4/80- cells were localized in the pulpdentin border of the central pulp beneath the dental fissure and quickly migrated to the treated cusp side at 2 hrs after cusp trimming. They constitutively express an endocytic receptor CD205 and the pathogen-associated recognition receptors TLR2 and TLR4. In contrast, CD11c-F4/80+ cells were abundantly distributed in the inner pulp and lacked expression of CD205, TLR2, and TLR4. They migrated to the treated side after cusp trimming and exhibited increased cell size and expression of the potent co-stimulator CD86. However, at 24 hrs postcusp treatment, CD86+ cells disappeared from the dental pulp, and the number of CD86+ DCs in the RLN was increased, suggesting migration of dental pulp DCs to RLNs. Migration of DCs from the peripheral tissues to the RLNs can evoke immune responses against pathogens or induce tolerance against tissuespecific self-antigens (Iwasaki and Medzhitov, 2004; Idoyaga et al., 2013). The characterization of migratory DCs from dental pulp will benefit further our understanding of dental pulp immune responses. In this study, we investigated dental pulp DC migration to the RLNs after cusp trimming and determined the antigenic phenotypes.
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J Dent Res 93(3) 2014 289 Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes
Materials & Methods Mice Female five- to six-week-old BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan) and used at 6 to 10 wks of age. ROSA-CAG-loxp-stop-loxp-KikGR knock-in (KI) mice were generated by homologous recombination in a BDF1derived ES cell line with a targeting construct, in which cDNA encoding a loxp-stop-loxp-CAG-KikGR was inserted into the ROSA26 locus. Details will be reported elsewhere (M.T., manuscript in preparation). KikGR mice were generated by mating ROSA-CAG-loxp-stop-loxp-KikGR knock-KI mice with CAGCre mice (Sakai et al., 1995) and back-crossed to C57BL/6 mice more than 12 times. KikGR mice were maintained under specific pathogen-free conditions at Kyoto University. All experimental procedures were reviewed and approved by the Animal Care and Use Committees of Tokyo Medical and Dental University and Kyoto University of Faculty of Medicine.
Cusp Trimming and Acid Etching The mice were anesthetized by intraperitoneal injection of xylazine and ketamine. To expose dentin, the mesial cusps of the lower first molars were trimmed by means of a dental engine with a steel bur (0.6-mm diameter). The trimmed surface was etched with 40% phosphoric acid gel to keep the dentinal tubules open, as described previously (Zhang et al., 2006).
Photoconversion At 2 hrs post-cusp trimming and acid etching in KikGR mice, photoconversion was performed by exposure to violet light for 3 min at 70 mW/cm2 with a 436-nm band-pass filter in Spot UV curing equipment (SP250, USHIO), as described previously (Tomura et al., 2008; Tomura and Kabashima, 2013). To focus as narrowly as possible on the treated molar, an optical fiber (0.75-mm diameter) was fixed at the top of the probe.
Isolation of Lymph Node Cells Submandibular lymph nodes (LNs) from both sides of intact and cusp-treated mice were used as a source of RLNs; inguinal LNs were used as non-RLNs. To isolate a single-cell suspension of LN cells, collected LNs were digested with type I collagenase as described (Chalermsarp and Azuma, 2009; Aramaki et al., 2011). After cells were counted, they were stained for flow cytometry. For the experiments using KikGR mice, pooled RLNs from 2 mice were analyzed.
Monoclonal Antibodies and Flow Cytometry Cells were pre-incubated with anti-CD16/32 (2.4G2, rat IgG2b) mAb to block FcγR and then stained with fluorochromeconjugated or biotinylated monoclonal antibodies (mAbs). mAbs against CD11c (N418, Armenian hamster IgG), CD11b (Mac-1, M1/70, rat IgG2b), F4/80 (BM8, rat IgG2a), Gr-1 (Ly6C/Ly6G, RB6-8C5, rat IgG2b), Ly6G (1A8, rat IgG2a), Ly6C (HK1.4, rat IgG2c), CD205 (NLDC-145, rat IgG2a), CD103 (2E7, Armenian hamster IgG), CD326 (G8.8, rat IgG2a),
CD86 (PO3, rat IgG2a), CD273 (TY25, rat IgG2a), and MHC class II (I-A/I-E, M5/114, rat IgG2b) were used. All FITC-, phycoerythrin (PE)-, allophycocyanin (APC)-, peridininchlorophyll-protein complex-carbocyanin5.5 (PerCP-Cy5.5)-, PE-complex-carbocyanin7 (PE-Cy7)-, APC-eFluor780-, V450-, V500-conjugated, or biotinylated mAbs were obtained from eBioscience (San Diego, CA, USA) or BD-Pharmingen (San Diego, CA, USA). For the biotinylated mAb, APCeFluor780- or V450-streptavidin (BD-Pharmingen) was used. Stained cells were analyzed with a FACSVerse and the FACSuite application (BD Biosciences). LSRFortessa with a FACSDiva application (BD Biosciences) was used for the KikGR mice. All data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
Statistical Analyses Statistical analysis was performed with the Mann-Whitney U test. Values of p < .05 were considered to be significant.
Results Preliminary experiments revealed that the total numbers of RLN cells increased at 12 to 18 hrs post-cusp trimming. Phenotypic changes of RLNs at 16 hrs post-treatment of the lower first molars were compared with those in the submandibular LNs of intact mice. Three cell fractions increased post-cusp treatment: Gr-1+++CD11b+++ (Fr-1), Gr-1++CD11b++ (Fr-2), and Gr-1low CD11b+~++ (Fr-3) (Fig. 1A). Whereas Fr-3 cells were present in the LNs of intact mice, Fr-1 and Fr-2 cells were barely detected; only the Fr-1 and Fr-2 cells were significantly increased posttreatment (Fig. 1B). Because the anti-Gr-1 (RB6-8C5) mAb reacted to both Ly6C and Ly6G, we re-analyzed the phenotype using mAbs specific for either Ly6C or Ly6G (Fig. 1C, left panels). Fr-1 cells (red dots) that expressed CD11b at the highest levels expressed both Ly6C and Ly6G. Fr-2 cells (black dots) expressed the highest levels of Ly6C but low levels of Ly6G. Fr-3 cells (green dots) expressed low levels of both Ly6C and Ly6G. Additional analyses of CD11c and F4/80 revealed that Fr-1 cells were Ly6C++Ly6G+++CD11b+++CD11cmedF4/80-, Fr-2 cells were Ly6C+++Ly6GlowCD11b++CD11clowF4/80+, and Fr-3 cells were Ly6ClowLy6GlowCD11b+~++CD11cmed~highF4/80low~+ (Fig. 1C, right 6 panels). The phenotypes of Fr-1, Fr-2, and Fr-3 cells from intact mice did not show clear differences (data not shown). Monocyte-derived macrophages are discriminated from DCs and granulocytes by the highest expression of Ly6C (Hettinger et al., 2013), and granulocytes are distinguishable by their high-level Ly6G and CD11b, but low-level Ly6C, expression. Thus, we speculated that Fr-1 and Fr-2 cells were granulocytes and macrophages recruited via blood circulation to the inflammatory RLNs, respectively, and the Fr-3 cells were DCs that had migrated via afferent lymphoid vessels. To determine whether the Fr-3 contained migrating DCs from the dental pulp, we used photoconvertible fluorescence protein-KI KikGR mice in which, similar to “Kaede”-transgenic mice (Tomura et al., 2008; Tomura and Kabashima, 2013), intracellular KikGR protein changed from green to red by the exposure of violet light. This enables the monitoring of cell trafficking from special tissue sites to other sites but does not affect
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Appendix Fig. The former cells (black dots) lacked substantial expression of CD11c, F4/80, CD273, and CD86, suggesting a granulocyte phenotype. The latter fraction (red dots) seemed to consist of several populations; however, these were not clearly separated based on the differential expression levels of Gr-1, CD11b, CD11c, and F4/80. Most of them expressed CD11c, and a major subset contained CD11chigh cells, which expressed higher levels of the co-stimulatory molecules CD273 and CD86. These results indicate that CD11chigh cells within the Fr-3 contain migrating DCs from the dental pulp. Next we compared the CD273 and CD86 expression levels within the Fr-3 in intact and cusp-treated mice. Interestingly, CD273+ cells within the Fr-3 expressed high levels of CD11c, and the percentages of CD273+ cells within Fr-3 cells or CD11chigh Fr-3 cells were significantly increased in the cusptreated mice (Fig. 3A). Although most Fr-3 cells expressed CD86 and MHC Figure 1. Three fractions of regional lymph node (RLN) cells increased after cusp treatment. class II in both intact and cusp-treated Submandibular lymph node (LN) cells from intact and cusp-treated mice at 16 hrs after cusp mice, the cusp-treated mice showed trimming were isolated as described in “Materials & Methods”. (A, C) Cells were stained with higher levels of CD86 and MHC class II either FITC-anti-Gr-1 (Ly6C/6G) or anti-Ly6G, V450--anti-Ly6C, APC-anti-CD11b, PerCP-Cy5.5anti-CD11c, and PE-Cy7-anti-F4/80 mAbs or the appropriate fluorochrome-conjugated control expression. CD273+ Fr-3 cells in the Igs. Samples were analyzed with a FACSVerse. An electronic gate was placed to include cusp-treated mice also expressed higher lymphocytes and myeloid cells (hereafter referred to as a large lymphocyte gate) with levels of CD86. These results indicate characteristic forward-scatter (FSC) and side-scatter (SCC) profiles. Representative profiles are CD11chighCD273+ to be a good index of shown. In C, the indicated logical gates for Fr-1 (red), Fr-2 (black), and Fr-3 (green) were migratory dental pulp DCs. placed in the profile of Ly6C and CD11b. The data are displayed as pseudocolor dot plots in CD11chighCD273+ Fr-3 cell analysis A and overlaid contour plots in C. Quadrant markers were positioned to include > 98% of revealed low but substantial levels of cells stained with control Igs in the lower left. (B) The percentages of each fraction within the large lymphocyte gate are shown. Values are the mean ± SD from each group of 7 mice. CD103 (integrin αE) and CD205 *Statistically different from the intact mice (p < .05). (DEC205), and various levels of CD326 (EpCAM) (Fig. 3B). The phenotypes of Fr-1, Fr-2, Fr-3, and CD11chighCD273+ cellular functions. Previously, we showed that the intrapulpal Fr-3 cells are summarized in the Table. cellular movement and CD86 induction occurred at 2 hrs postcusp treatment (Zhang et al., 2006); thus, the treated molars were exposed to violet light after 2 hrs, and then RLN cells were Discussion analyzed. The percentage of Kik-red+ cells increased ten-fold Using photoconvertible fluoroprotein-KI KikGR mice to trace the (from 0.002% to 0.02%) in the RLN of cusp-trimmed mice after trafficking of dental pulp DCs in RLNs, we demonstrated for the photoconversion, and there were clearly fewer in the non-RLNs first time that dental pulp DCs migrated to RLNs at 16 hrs after (Fig. 2A). Only 0.018 ± 0.02% (mean ± SD) RLN cells were cusp trimming. Kik-red+ migrating dental pulp cells contain the Kik-red+ migrating dental pulp cells, which represented approx+ cells with the CD11chighLy6ClowLy6GlowCD11b++F4/80+ phenoimately 1,000 cells per one side of RLN. Kik-red cells from type. In addition, they expressed the highest levels of MHC class cusp-treated and photoconverted mice consisted of 2 fractions at II and potent co-stimulators CD86 and CD273. These results a roughly 1:3 ratio: Gr-1+++ CD11b+++ cells (black dots, that indicate that these cells are definitively DCs rather than macroseemed equivalent to Fr-1) and Gr-1+~++CD11b+~++ cells (red phages. Previously, we demonstrated sentinel CD11c+F4/80dots, that seemed equivalent to a mixture of Fr-2 and Fr-3) (Fig. DC-like cells and interstitial CD11c-F4/80+ macrophage-like 2B). Because different combinations of fluorochrome-conjugated cells by immunohistochemical analyses of dental pulp (Zhang mAbs and flow cytometry were used, the expression levels of et al., 2006). Compared with flow cytometry, the sensitivity of each antigen differed from the data in Fig. 1. However, they are immunohistostaining is clearly inferior and is dependent upon the relatively similar, as shown by the comparative profiles in the
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J Dent Res 93(3) 2014 291 Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes Table. Summary of Migrating Cells in RLNs after Cusp Trimming Fr-3
Phenotype Ly6C Ly6G CD11b CD11c F4/80 CD205 CD326 CD103 CD86 CD273 MHCII
Inflammatory Macrophages
Migrating DCs in the Steady State + Migrating Dental Pulp DCs
Migrating Dental Pulp DCs
+++ low ++ low + low medium
low low +~++ medium - high low~+ low medium - high - / low ~+ medium - high -/+ medium - high
low low ++ high + low medium - high low high + high
Fr-1
Fr-2
Inflammatory Granulocytes ++ +++ +++ medium -
CD11chiCD273+ Fr-3
mAbs and methods used. In fact, the use of a more sensitive immunostaining method revealed that the CD11c-F4/80+ interstitial cells did express CD11c (O.A. and M.A., unpublished observations). Presently, we cannot discriminate whether the migratory Kik-red+ Fr-3 DCs are derived from the sentinel or interstitial DCs, because the properties of DCs in the periphery are modulated by antigen-capture, antigen-processing, and migration to RLNs. Similar to skin and type II mucosal DCs (Iwasaki and Medzhitov, 2004; Iwasaki, 2007), dental pulp DCs might pass through several maturation steps before reaching the RLNs after exposure to antigenic stimuli. Although a specific pathogen was not applied to the trimmed surfaces because of technical difficulties, Figure 2. Trace of cells migrating from dental pulp in KikGR mice. regional lymph node (RLN) salivary dental microbes might have pencells from cusp-treated mice without violet-light exposure (A, left panel) and RLN (A, middle etrated the dentinal tubules after etching, panel and B) and non-RLN (A, right panel) cells from cusp-treated and violet-light exposed mice because the intra-pulpal traffic of dental were isolated at 16 hr. Cells were stained with V450-anti-Gr-1, V500-anti-CD11b, APC-antipulp DCs did not occur without the etchCD11c, APC-efluor780-anti-F4/80, or either biotinylated anti-CD273 or CD86 mAbs, ing treatment (Zhang et al., 2006). followed by APC-efluor780-streptavidin or the appropriate fluorochrome-conjugated control Butcher et al. (2011) demonstrated that Igs. Stained cells were analyzed with LSRFortessa. Photoconverted Kik-red+ cells and unconverted Kik-green+ cells in the large lymphocyte gate are shown in A. In B, an electronic bone-marrow-derived DCs exposed to gate was placed on Kik-red+ cells from cusp-treated and violet light–exposed mice; Streptococcus mutans in vitro processed representative profiles CD11b and Gr-1 are displayed (B, left panel). Further logical gates for bacterial antigens and up-regulated CD11bhighGr-1high (black dots) and CD11bmedGr-1med-low (red dots) cells were placed; expressions expression of MHC class II, CD40, and of the indicated antigens are displayed with CD11c as dotted plots. Quadrant markers were CD86. Our previous studies of migrating positioned to include > 98% of cells stained with control Igs in the lower left. Representative DCs from skin and oral (buccal) mucosa profiles from 3 independent experiments are shown. demonstrated that all hapten antigen (FITC)-captured migrating DCs expressed et al., 2011). Antigen-captured DCs enhanced MHC class II on high levels of MHC class II, despite differential expression of cotheir surfaces and migrated to RLNs. The facts that (1) the stimulatory molecules in the distinct DC subsets, and that CD11chigh cells migrated from dental pulp, (2) they expressed high CD11chigh dermal/interstitial resident DCs expressed high levels of CD273 and CD86 (Chalermsarp and Azuma, 2009; Aramaki levels of MHC class II, and (3) they co-expressed high levels of
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PDCA-1 and CD45R (B220) (data not shown), suggesting that LCs and plasmacytoid DCs are not involved. Although CD273 expression on DCs is up-regulated by various stimuli, its function remains unclear (Tseng et al., 2001; Yamazaki et al., 2002; Geng et al., 2008; Mendoza-Coronel et al., 2011; Inamine et al., 2012). An initial report suggested that this molecule had a costimulatory function (Tseng et al., 2001), but recent reports suggest it to play a regulatory role in DCs in allo-transplantation, bacterial infection, and allergy (Geng et al., 2008; Mendoza-Coronel et al., 2011; Inamine et al., 2012). Migratory dental pulp DCs expressed substantial levels of CD103 and various levels of CD326. Our previous study showed that all migratory DCs from skin and oral mucosa expressed various levels of CD326, but only LCs expressed CD103 (Aramaki et al., 2011). Although particular subsets of intestinal CD103+ DCs play a crucial role in the induction of tolerance to commensal bacteria and food antigens, migratory CD103+ DCs Figure 3. Antigenic properties of migrating dental pulp dendritic cells (DCs). Lymph node (LN) cells from intact and cusp-treated mice were isolated as described in Fig. 1. (A) Cells were from the skin, lung, and intestine effistained with FITC-anti-Gr-1, PE-anti-CD273 or -anti-MHC class II, PerCP-Cy5.5-anti-CD11c, ciently present exogenous antigens to APC-anti-CD11b, and biotinylated-anti-CD86 mAbs, followed by APC-efluor780-streptavidin. induce effector T-cells in RLNs (del Rio Stained cells were analyzed with FACSVerse. A logical gate was placed on Gr-1++CD11b++ et al., 2010; Scott et al., 2011). Therefore, Fr-3 cells as described in Fig. 1A, and expression profiles of the indicated antigens are shown expression of CD103 alone is not closely high + as dotted plots. Values are the mean percentages ± SD of CD11c CD273 cells within the correlated with a regulatory function. CD11chigh Fr-3 cells. *Significantly different from the intact mice (p < .05). (B) RLN cells were Further studies are required to determine stained with FITC-anti-Gr-1, PerCP-Cy5.5-anti-CD11c, APC-anti-CD11b, and either PE-anti-CD326 or biotinylated anti-CD205 and -CD103 mAbs, followed by V450-streptavidin antigenic or tolerogenic function of migraor the appropriate fluorochrome-conjugated control Igs. A logical gate was placed on tory dental pulp DCs. CD11chigh Fr-3 cells, and the indicated antigen expression is shown together with CD11c One-third of the Kik-red+ cells were expression. Representative profiles from 3 independent experiments are shown. Quadrant Ly6G+++CD11b+++ Fr-1 cells, indicating markers were positioned to include > 98% of cells stained with control Igs in the lower left. that these granulocytes existed in the dental pulp at the time of violet light CD273 and CD86 strongly suggest that migrating CD11chigh cells exposure after cusp trimming. Cusp trimming and acid etching are the antigen-captured mature state of DCs. Additionally, the could induce mild pulp injury and cariogenic bacteria exposure. abundance of F4/80+ interstitial DCs in the dental pulp and the These stimuli may induce rapid chemokine secretion from transient induction of CD86 in F4/80+ interstitial DCs at 2 hrs odontoblasts and dental pulp immune cells to recruit granuloafter cusp trimming (Zhang et al., 2006) suggest that CD11chigh cytes into the inflamed dental pulp (Farges et al., 2009), with Fr-3 cells originate from the interstitial dental pulp DCs. further recruitment to RLNs via blood circulation. The migraSubstantial numbers of Fr-3 are cells present even in intact tion of antigen-captured dental pulp DCs into the RLNs might LNs. Ohl et al. (2004) demonstrated that CD11c+MHC class up-regulate levels of LN-targeted chemokines and enhance IIhigh DCs that expressed low levels of co-stimulatory molecules recruitment of granulocytes and macrophages (blood monosuch as CD40, CD80, and CD86 in skin-draining LNs under cytes) in addition to T- and B-cells (von Andrian and Mempel, steady-state conditions were CCR7-dependent migrating DCs. 2003). Fr-3 cells that exhibit relatively low MHC class II and CD86 We demonstrated that dental pulp DCs migrate to the RLNs and expression, but no CD273 expression, in the intact mice might show a mature phenotype. This characterization of migratory dental be migratory DCs under steady-state conditions. Fr-3 cells from pulp DCs contributes to our understanding of the dental pulp cusp-trimmed mice did not express a Langerhans cell (LC) immune responses against cariogenic bacteria and to the developmarker, CD207 (Langerin) and plasmacytoid DC markers, ment of potential intervention for dental caries and pulpitis.
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J Dent Res 93(3) 2014 293 Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes
Acknowledgments This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by grants from the Japan Society for the Promotion of Science. M.A. has received a licensing fee and royalties from eBioscience and BD-Pharmingen. The other authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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Iwasaki A, Medzhitov R (2004). Toll-like receptor control of the adaptive immune responses. Nat Immunol 5:987-995. Jontell M, Okiji T, Dahlgren U, Bergenholtz G (1998). Immune defense mechanisms of the dental pulp. Crit Rev Oral Biol Med 9:179-200. Mendoza-Coronel E, Camacho-Sandoval R, Bonifaz LC, Lopez-Vidal Y (2011). PD-L2 induction on dendritic cells exposed to Mycobacterium avium downregulates BCG-specific T cell response. Tuberculosis (Edinb) 91:36-46. Ohl L, Mohaupt M, Czeloth N, Hintzen G, Kiafard Z, Zwirner J, et al. (2004). CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity 21:279-288. Sakai K, Mitani K, Miyazaki J (1995). Efficient regulation of gene expression by adenovirus vector-mediated delivery of the CRE recombinase. Biochem Biophys Res Commun 217:393-401. Scott CL, Aumeunier AM, Mowat AM (2011). Intestinal CD103+ dendritic cells: master regulators of tolerance? Trends Immunol 32:412-419. Steinman RM (2001). Dendritic cells and the control of immunity: enhancing the efficiency of antigen presentation. Mt Sinai J Med 68:160-166. Steinman RM, Banchereau J (2007). Taking dendritic cells into medicine. Nature 449:419-426. Tomura M, Kabashima K (2013). Analysis of cell movement between skin and other anatomical sites in vivo using photoconvertible fluorescent protein “Kaede”-transgenic mice. Methods Mol Biol 961:279-286. Tomura M, Yoshida N, Tanaka J, Karasawa S, Miwa Y, Miyawaki A, et al. (2008). Monitoring cellular movement in vivo with photoconvertible fluorescence protein “Kaede” transgenic mice. Proc Natl Acad Sci USA 105:10871-10876. Tseng SY, Otsuji M, Gorski K, Huang X, Slansky JE, Pai SI, et al. (2001). B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J Exp Med 193:839-846. von Andrian UH, Mempel TR (2003). Homing and cellular traffic in lymph nodes. Nat Rev Immunol 3:867-878. Yamazaki T, Akiba H, Iwai H, Matsuda H, Aoki M, Tanno Y, et al. (2002). Expression of programmed death 1 ligands by murine T cells and APC. J Immunol 169:5538-5545. Yoshiba K, Yoshiba N, Iwaku M (2003). Class II antigen-presenting dendritic cell and nerve fiber responses to cavities, caries, or caries treatment in human teeth. J Dent Res 82:422-427. Yoshiba N, Yoshiba K, Iwaku M, Ozawa H (1998). Immunohistochemical localizations of class II antigens and nerve fibers in human carious teeth: HLA-DR immunoreactivity in Schwann cells. Arch Histol Cytol 61:343-352. Zhang J, Kawashima N, Suda H, Nakano Y, Takano Y, Azuma M (2006). The existence of CD11c+ sentinel and F4/80+ interstitial dendritic cells in dental pulp and their dynamics and functional properties. Int Immunol 18:1375-1384.
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Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes A.C. Bhingare, T. Ohno, M. Tomura, C. Zhang, O. Aramaki, M. Otsuki, J. Tagami and M. Azuma J DENT RES 2014 93: 288 originally published online 30 December 2013 DOI: 10.1177/0022034513518223 The online version of this article can be found at: http://jdr.sagepub.com/content/93/3/288
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research-article2014
JDR
93310.1177/0022034513518223
RESEARCH REPORTS Biological
A.C. Bhingare1, T. Ohno1, M. Tomura2, C. Zhang1, O. Aramaki3, M. Otsuki3, J. Tagami3, and M. Azuma1*
Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes
1
Department of Molecular Immunology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; 2Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; and 3 Department of Cariology and Operative Dentistry, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; *corresponding author, [email protected] J Dent Res 93(3):288-293, 2014
Abstract
Introduction
Dendritic cell (DC) migration to regional lymph nodes (RLNs) is an essential step in adaptive immunity, and cell-surface antigens on migrating DCs greatly affect the quality and quantity of subsequent immune responses. Although MHC class II+ DC-like cells exist in the dental pulp, the lineage and function of these cells remain unknown. Here, we identified migratory DCs from the dental pulp after cusp trimming and acid etching in KikGR mice, in which the photoconvertible fluorescent protein changed from green to red upon violet light exposure. Two major cell fractions from the dental pulp had migrated to the RLNs at 16 hrs after cusp treatment, which showed the following lineage markers in the main and second fractions: CD11chighCD11b++Ly6Clow Ly6Glow F4/80+ and CD11cmedCD11b+++Ly6C++Ly6 G+++F4/80-, respectively. These lineage markers indicate that the former cells were DCs that had migrated through afferent lymphoid vessels, and the latter were granulocytes recruited via blood circulation. Migratory dental pulp DCs were mature, expressing the highest levels of CD273 (B7-DC) and CD86 co-stimulators and MHC class II. Our results suggest that cariogenic-bacteria-exposed dental pulp DCs migrate to RLNs and there trigger adaptive immune responses.
T
KEY WORDS: co-stimulatory molecules, CD86, CD273, cell trafficking, photoconversion, fluorescence protein.
DOI: 10.1177/0022034513518223 Received October 2, 2013; Last revision November 12, 2013; Accepted December 4, 2013 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
he immune system has evolved to recognize microbial attacks and eliminate infections. Dendritic cells (DCs) exist broadly in the skin and mucosa as the front line of defense against microbial pathogens (Steinman, 2001; Iwasaki, 2007; Steinman and Banchereau, 2007). DCs recognize microbes via pathogen-associated receptors at the periphery, secrete multiple cytokines/ chemokines to initiate innate immunity, and migrate to regional lymph nodes (RLNs) to present antigens to naïve T-cells that trigger adaptive immunity. DCs are heterogeneous cells, and the site-specific microenvironment modulates their phenotypic and functional properties. Odontoblasts, with their cellular processes extending into dentinal tubules, are the first line of defense against cariogenic bacteria in the dental pulp. They express a variety of pathogen-recognition receptors and chemokines, and trigger the initial innate inflammatory responses in cooperation with DC-like cells (Farges et al., 2009). MHC class II-positive DC-like cells with macrophage lineage markers have been found in the dental pulp of the human tooth with caries along with T-cell infiltration (Jontell et al., 1998; Yoshiba et al., 1998; Yoshiba et al., 2003). These observations suggest that dental pulp DCs with captured microbial antigens migrate to the RLNs to present antigens to naïve T-cells, followed by effector T-cell recruitment to the dental pulp. Previously, we demonstrated that 2 distinct cell populations with dendritic morphology exist in the steady-state dental pulp: CD11c+F4/80- and CD11c-F4/80+ cells (Zhang et al., 2006). A few CD11c+F4/80- cells were localized in the pulpdentin border of the central pulp beneath the dental fissure and quickly migrated to the treated cusp side at 2 hrs after cusp trimming. They constitutively express an endocytic receptor CD205 and the pathogen-associated recognition receptors TLR2 and TLR4. In contrast, CD11c-F4/80+ cells were abundantly distributed in the inner pulp and lacked expression of CD205, TLR2, and TLR4. They migrated to the treated side after cusp trimming and exhibited increased cell size and expression of the potent co-stimulator CD86. However, at 24 hrs postcusp treatment, CD86+ cells disappeared from the dental pulp, and the number of CD86+ DCs in the RLN was increased, suggesting migration of dental pulp DCs to RLNs. Migration of DCs from the peripheral tissues to the RLNs can evoke immune responses against pathogens or induce tolerance against tissuespecific self-antigens (Iwasaki and Medzhitov, 2004; Idoyaga et al., 2013). The characterization of migratory DCs from dental pulp will benefit further our understanding of dental pulp immune responses. In this study, we investigated dental pulp DC migration to the RLNs after cusp trimming and determined the antigenic phenotypes.
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J Dent Res 93(3) 2014 289 Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes
Materials & Methods Mice Female five- to six-week-old BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan) and used at 6 to 10 wks of age. ROSA-CAG-loxp-stop-loxp-KikGR knock-in (KI) mice were generated by homologous recombination in a BDF1derived ES cell line with a targeting construct, in which cDNA encoding a loxp-stop-loxp-CAG-KikGR was inserted into the ROSA26 locus. Details will be reported elsewhere (M.T., manuscript in preparation). KikGR mice were generated by mating ROSA-CAG-loxp-stop-loxp-KikGR knock-KI mice with CAGCre mice (Sakai et al., 1995) and back-crossed to C57BL/6 mice more than 12 times. KikGR mice were maintained under specific pathogen-free conditions at Kyoto University. All experimental procedures were reviewed and approved by the Animal Care and Use Committees of Tokyo Medical and Dental University and Kyoto University of Faculty of Medicine.
Cusp Trimming and Acid Etching The mice were anesthetized by intraperitoneal injection of xylazine and ketamine. To expose dentin, the mesial cusps of the lower first molars were trimmed by means of a dental engine with a steel bur (0.6-mm diameter). The trimmed surface was etched with 40% phosphoric acid gel to keep the dentinal tubules open, as described previously (Zhang et al., 2006).
Photoconversion At 2 hrs post-cusp trimming and acid etching in KikGR mice, photoconversion was performed by exposure to violet light for 3 min at 70 mW/cm2 with a 436-nm band-pass filter in Spot UV curing equipment (SP250, USHIO), as described previously (Tomura et al., 2008; Tomura and Kabashima, 2013). To focus as narrowly as possible on the treated molar, an optical fiber (0.75-mm diameter) was fixed at the top of the probe.
Isolation of Lymph Node Cells Submandibular lymph nodes (LNs) from both sides of intact and cusp-treated mice were used as a source of RLNs; inguinal LNs were used as non-RLNs. To isolate a single-cell suspension of LN cells, collected LNs were digested with type I collagenase as described (Chalermsarp and Azuma, 2009; Aramaki et al., 2011). After cells were counted, they were stained for flow cytometry. For the experiments using KikGR mice, pooled RLNs from 2 mice were analyzed.
Monoclonal Antibodies and Flow Cytometry Cells were pre-incubated with anti-CD16/32 (2.4G2, rat IgG2b) mAb to block FcγR and then stained with fluorochromeconjugated or biotinylated monoclonal antibodies (mAbs). mAbs against CD11c (N418, Armenian hamster IgG), CD11b (Mac-1, M1/70, rat IgG2b), F4/80 (BM8, rat IgG2a), Gr-1 (Ly6C/Ly6G, RB6-8C5, rat IgG2b), Ly6G (1A8, rat IgG2a), Ly6C (HK1.4, rat IgG2c), CD205 (NLDC-145, rat IgG2a), CD103 (2E7, Armenian hamster IgG), CD326 (G8.8, rat IgG2a),
CD86 (PO3, rat IgG2a), CD273 (TY25, rat IgG2a), and MHC class II (I-A/I-E, M5/114, rat IgG2b) were used. All FITC-, phycoerythrin (PE)-, allophycocyanin (APC)-, peridininchlorophyll-protein complex-carbocyanin5.5 (PerCP-Cy5.5)-, PE-complex-carbocyanin7 (PE-Cy7)-, APC-eFluor780-, V450-, V500-conjugated, or biotinylated mAbs were obtained from eBioscience (San Diego, CA, USA) or BD-Pharmingen (San Diego, CA, USA). For the biotinylated mAb, APCeFluor780- or V450-streptavidin (BD-Pharmingen) was used. Stained cells were analyzed with a FACSVerse and the FACSuite application (BD Biosciences). LSRFortessa with a FACSDiva application (BD Biosciences) was used for the KikGR mice. All data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
Statistical Analyses Statistical analysis was performed with the Mann-Whitney U test. Values of p < .05 were considered to be significant.
Results Preliminary experiments revealed that the total numbers of RLN cells increased at 12 to 18 hrs post-cusp trimming. Phenotypic changes of RLNs at 16 hrs post-treatment of the lower first molars were compared with those in the submandibular LNs of intact mice. Three cell fractions increased post-cusp treatment: Gr-1+++CD11b+++ (Fr-1), Gr-1++CD11b++ (Fr-2), and Gr-1low CD11b+~++ (Fr-3) (Fig. 1A). Whereas Fr-3 cells were present in the LNs of intact mice, Fr-1 and Fr-2 cells were barely detected; only the Fr-1 and Fr-2 cells were significantly increased posttreatment (Fig. 1B). Because the anti-Gr-1 (RB6-8C5) mAb reacted to both Ly6C and Ly6G, we re-analyzed the phenotype using mAbs specific for either Ly6C or Ly6G (Fig. 1C, left panels). Fr-1 cells (red dots) that expressed CD11b at the highest levels expressed both Ly6C and Ly6G. Fr-2 cells (black dots) expressed the highest levels of Ly6C but low levels of Ly6G. Fr-3 cells (green dots) expressed low levels of both Ly6C and Ly6G. Additional analyses of CD11c and F4/80 revealed that Fr-1 cells were Ly6C++Ly6G+++CD11b+++CD11cmedF4/80-, Fr-2 cells were Ly6C+++Ly6GlowCD11b++CD11clowF4/80+, and Fr-3 cells were Ly6ClowLy6GlowCD11b+~++CD11cmed~highF4/80low~+ (Fig. 1C, right 6 panels). The phenotypes of Fr-1, Fr-2, and Fr-3 cells from intact mice did not show clear differences (data not shown). Monocyte-derived macrophages are discriminated from DCs and granulocytes by the highest expression of Ly6C (Hettinger et al., 2013), and granulocytes are distinguishable by their high-level Ly6G and CD11b, but low-level Ly6C, expression. Thus, we speculated that Fr-1 and Fr-2 cells were granulocytes and macrophages recruited via blood circulation to the inflammatory RLNs, respectively, and the Fr-3 cells were DCs that had migrated via afferent lymphoid vessels. To determine whether the Fr-3 contained migrating DCs from the dental pulp, we used photoconvertible fluorescence protein-KI KikGR mice in which, similar to “Kaede”-transgenic mice (Tomura et al., 2008; Tomura and Kabashima, 2013), intracellular KikGR protein changed from green to red by the exposure of violet light. This enables the monitoring of cell trafficking from special tissue sites to other sites but does not affect
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Appendix Fig. The former cells (black dots) lacked substantial expression of CD11c, F4/80, CD273, and CD86, suggesting a granulocyte phenotype. The latter fraction (red dots) seemed to consist of several populations; however, these were not clearly separated based on the differential expression levels of Gr-1, CD11b, CD11c, and F4/80. Most of them expressed CD11c, and a major subset contained CD11chigh cells, which expressed higher levels of the co-stimulatory molecules CD273 and CD86. These results indicate that CD11chigh cells within the Fr-3 contain migrating DCs from the dental pulp. Next we compared the CD273 and CD86 expression levels within the Fr-3 in intact and cusp-treated mice. Interestingly, CD273+ cells within the Fr-3 expressed high levels of CD11c, and the percentages of CD273+ cells within Fr-3 cells or CD11chigh Fr-3 cells were significantly increased in the cusptreated mice (Fig. 3A). Although most Fr-3 cells expressed CD86 and MHC Figure 1. Three fractions of regional lymph node (RLN) cells increased after cusp treatment. class II in both intact and cusp-treated Submandibular lymph node (LN) cells from intact and cusp-treated mice at 16 hrs after cusp mice, the cusp-treated mice showed trimming were isolated as described in “Materials & Methods”. (A, C) Cells were stained with higher levels of CD86 and MHC class II either FITC-anti-Gr-1 (Ly6C/6G) or anti-Ly6G, V450--anti-Ly6C, APC-anti-CD11b, PerCP-Cy5.5anti-CD11c, and PE-Cy7-anti-F4/80 mAbs or the appropriate fluorochrome-conjugated control expression. CD273+ Fr-3 cells in the Igs. Samples were analyzed with a FACSVerse. An electronic gate was placed to include cusp-treated mice also expressed higher lymphocytes and myeloid cells (hereafter referred to as a large lymphocyte gate) with levels of CD86. These results indicate characteristic forward-scatter (FSC) and side-scatter (SCC) profiles. Representative profiles are CD11chighCD273+ to be a good index of shown. In C, the indicated logical gates for Fr-1 (red), Fr-2 (black), and Fr-3 (green) were migratory dental pulp DCs. placed in the profile of Ly6C and CD11b. The data are displayed as pseudocolor dot plots in CD11chighCD273+ Fr-3 cell analysis A and overlaid contour plots in C. Quadrant markers were positioned to include > 98% of revealed low but substantial levels of cells stained with control Igs in the lower left. (B) The percentages of each fraction within the large lymphocyte gate are shown. Values are the mean ± SD from each group of 7 mice. CD103 (integrin αE) and CD205 *Statistically different from the intact mice (p < .05). (DEC205), and various levels of CD326 (EpCAM) (Fig. 3B). The phenotypes of Fr-1, Fr-2, Fr-3, and CD11chighCD273+ cellular functions. Previously, we showed that the intrapulpal Fr-3 cells are summarized in the Table. cellular movement and CD86 induction occurred at 2 hrs postcusp treatment (Zhang et al., 2006); thus, the treated molars were exposed to violet light after 2 hrs, and then RLN cells were Discussion analyzed. The percentage of Kik-red+ cells increased ten-fold Using photoconvertible fluoroprotein-KI KikGR mice to trace the (from 0.002% to 0.02%) in the RLN of cusp-trimmed mice after trafficking of dental pulp DCs in RLNs, we demonstrated for the photoconversion, and there were clearly fewer in the non-RLNs first time that dental pulp DCs migrated to RLNs at 16 hrs after (Fig. 2A). Only 0.018 ± 0.02% (mean ± SD) RLN cells were cusp trimming. Kik-red+ migrating dental pulp cells contain the Kik-red+ migrating dental pulp cells, which represented approx+ cells with the CD11chighLy6ClowLy6GlowCD11b++F4/80+ phenoimately 1,000 cells per one side of RLN. Kik-red cells from type. In addition, they expressed the highest levels of MHC class cusp-treated and photoconverted mice consisted of 2 fractions at II and potent co-stimulators CD86 and CD273. These results a roughly 1:3 ratio: Gr-1+++ CD11b+++ cells (black dots, that indicate that these cells are definitively DCs rather than macroseemed equivalent to Fr-1) and Gr-1+~++CD11b+~++ cells (red phages. Previously, we demonstrated sentinel CD11c+F4/80dots, that seemed equivalent to a mixture of Fr-2 and Fr-3) (Fig. DC-like cells and interstitial CD11c-F4/80+ macrophage-like 2B). Because different combinations of fluorochrome-conjugated cells by immunohistochemical analyses of dental pulp (Zhang mAbs and flow cytometry were used, the expression levels of et al., 2006). Compared with flow cytometry, the sensitivity of each antigen differed from the data in Fig. 1. However, they are immunohistostaining is clearly inferior and is dependent upon the relatively similar, as shown by the comparative profiles in the
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J Dent Res 93(3) 2014 291 Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes Table. Summary of Migrating Cells in RLNs after Cusp Trimming Fr-3
Phenotype Ly6C Ly6G CD11b CD11c F4/80 CD205 CD326 CD103 CD86 CD273 MHCII
Inflammatory Macrophages
Migrating DCs in the Steady State + Migrating Dental Pulp DCs
Migrating Dental Pulp DCs
+++ low ++ low + low medium
low low +~++ medium - high low~+ low medium - high - / low ~+ medium - high -/+ medium - high
low low ++ high + low medium - high low high + high
Fr-1
Fr-2
Inflammatory Granulocytes ++ +++ +++ medium -
CD11chiCD273+ Fr-3
mAbs and methods used. In fact, the use of a more sensitive immunostaining method revealed that the CD11c-F4/80+ interstitial cells did express CD11c (O.A. and M.A., unpublished observations). Presently, we cannot discriminate whether the migratory Kik-red+ Fr-3 DCs are derived from the sentinel or interstitial DCs, because the properties of DCs in the periphery are modulated by antigen-capture, antigen-processing, and migration to RLNs. Similar to skin and type II mucosal DCs (Iwasaki and Medzhitov, 2004; Iwasaki, 2007), dental pulp DCs might pass through several maturation steps before reaching the RLNs after exposure to antigenic stimuli. Although a specific pathogen was not applied to the trimmed surfaces because of technical difficulties, Figure 2. Trace of cells migrating from dental pulp in KikGR mice. regional lymph node (RLN) salivary dental microbes might have pencells from cusp-treated mice without violet-light exposure (A, left panel) and RLN (A, middle etrated the dentinal tubules after etching, panel and B) and non-RLN (A, right panel) cells from cusp-treated and violet-light exposed mice because the intra-pulpal traffic of dental were isolated at 16 hr. Cells were stained with V450-anti-Gr-1, V500-anti-CD11b, APC-antipulp DCs did not occur without the etchCD11c, APC-efluor780-anti-F4/80, or either biotinylated anti-CD273 or CD86 mAbs, ing treatment (Zhang et al., 2006). followed by APC-efluor780-streptavidin or the appropriate fluorochrome-conjugated control Butcher et al. (2011) demonstrated that Igs. Stained cells were analyzed with LSRFortessa. Photoconverted Kik-red+ cells and unconverted Kik-green+ cells in the large lymphocyte gate are shown in A. In B, an electronic bone-marrow-derived DCs exposed to gate was placed on Kik-red+ cells from cusp-treated and violet light–exposed mice; Streptococcus mutans in vitro processed representative profiles CD11b and Gr-1 are displayed (B, left panel). Further logical gates for bacterial antigens and up-regulated CD11bhighGr-1high (black dots) and CD11bmedGr-1med-low (red dots) cells were placed; expressions expression of MHC class II, CD40, and of the indicated antigens are displayed with CD11c as dotted plots. Quadrant markers were CD86. Our previous studies of migrating positioned to include > 98% of cells stained with control Igs in the lower left. Representative DCs from skin and oral (buccal) mucosa profiles from 3 independent experiments are shown. demonstrated that all hapten antigen (FITC)-captured migrating DCs expressed et al., 2011). Antigen-captured DCs enhanced MHC class II on high levels of MHC class II, despite differential expression of cotheir surfaces and migrated to RLNs. The facts that (1) the stimulatory molecules in the distinct DC subsets, and that CD11chigh cells migrated from dental pulp, (2) they expressed high CD11chigh dermal/interstitial resident DCs expressed high levels of CD273 and CD86 (Chalermsarp and Azuma, 2009; Aramaki levels of MHC class II, and (3) they co-expressed high levels of
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PDCA-1 and CD45R (B220) (data not shown), suggesting that LCs and plasmacytoid DCs are not involved. Although CD273 expression on DCs is up-regulated by various stimuli, its function remains unclear (Tseng et al., 2001; Yamazaki et al., 2002; Geng et al., 2008; Mendoza-Coronel et al., 2011; Inamine et al., 2012). An initial report suggested that this molecule had a costimulatory function (Tseng et al., 2001), but recent reports suggest it to play a regulatory role in DCs in allo-transplantation, bacterial infection, and allergy (Geng et al., 2008; Mendoza-Coronel et al., 2011; Inamine et al., 2012). Migratory dental pulp DCs expressed substantial levels of CD103 and various levels of CD326. Our previous study showed that all migratory DCs from skin and oral mucosa expressed various levels of CD326, but only LCs expressed CD103 (Aramaki et al., 2011). Although particular subsets of intestinal CD103+ DCs play a crucial role in the induction of tolerance to commensal bacteria and food antigens, migratory CD103+ DCs Figure 3. Antigenic properties of migrating dental pulp dendritic cells (DCs). Lymph node (LN) cells from intact and cusp-treated mice were isolated as described in Fig. 1. (A) Cells were from the skin, lung, and intestine effistained with FITC-anti-Gr-1, PE-anti-CD273 or -anti-MHC class II, PerCP-Cy5.5-anti-CD11c, ciently present exogenous antigens to APC-anti-CD11b, and biotinylated-anti-CD86 mAbs, followed by APC-efluor780-streptavidin. induce effector T-cells in RLNs (del Rio Stained cells were analyzed with FACSVerse. A logical gate was placed on Gr-1++CD11b++ et al., 2010; Scott et al., 2011). Therefore, Fr-3 cells as described in Fig. 1A, and expression profiles of the indicated antigens are shown expression of CD103 alone is not closely high + as dotted plots. Values are the mean percentages ± SD of CD11c CD273 cells within the correlated with a regulatory function. CD11chigh Fr-3 cells. *Significantly different from the intact mice (p < .05). (B) RLN cells were Further studies are required to determine stained with FITC-anti-Gr-1, PerCP-Cy5.5-anti-CD11c, APC-anti-CD11b, and either PE-anti-CD326 or biotinylated anti-CD205 and -CD103 mAbs, followed by V450-streptavidin antigenic or tolerogenic function of migraor the appropriate fluorochrome-conjugated control Igs. A logical gate was placed on tory dental pulp DCs. CD11chigh Fr-3 cells, and the indicated antigen expression is shown together with CD11c One-third of the Kik-red+ cells were expression. Representative profiles from 3 independent experiments are shown. Quadrant Ly6G+++CD11b+++ Fr-1 cells, indicating markers were positioned to include > 98% of cells stained with control Igs in the lower left. that these granulocytes existed in the dental pulp at the time of violet light CD273 and CD86 strongly suggest that migrating CD11chigh cells exposure after cusp trimming. Cusp trimming and acid etching are the antigen-captured mature state of DCs. Additionally, the could induce mild pulp injury and cariogenic bacteria exposure. abundance of F4/80+ interstitial DCs in the dental pulp and the These stimuli may induce rapid chemokine secretion from transient induction of CD86 in F4/80+ interstitial DCs at 2 hrs odontoblasts and dental pulp immune cells to recruit granuloafter cusp trimming (Zhang et al., 2006) suggest that CD11chigh cytes into the inflamed dental pulp (Farges et al., 2009), with Fr-3 cells originate from the interstitial dental pulp DCs. further recruitment to RLNs via blood circulation. The migraSubstantial numbers of Fr-3 are cells present even in intact tion of antigen-captured dental pulp DCs into the RLNs might LNs. Ohl et al. (2004) demonstrated that CD11c+MHC class up-regulate levels of LN-targeted chemokines and enhance IIhigh DCs that expressed low levels of co-stimulatory molecules recruitment of granulocytes and macrophages (blood monosuch as CD40, CD80, and CD86 in skin-draining LNs under cytes) in addition to T- and B-cells (von Andrian and Mempel, steady-state conditions were CCR7-dependent migrating DCs. 2003). Fr-3 cells that exhibit relatively low MHC class II and CD86 We demonstrated that dental pulp DCs migrate to the RLNs and expression, but no CD273 expression, in the intact mice might show a mature phenotype. This characterization of migratory dental be migratory DCs under steady-state conditions. Fr-3 cells from pulp DCs contributes to our understanding of the dental pulp cusp-trimmed mice did not express a Langerhans cell (LC) immune responses against cariogenic bacteria and to the developmarker, CD207 (Langerin) and plasmacytoid DC markers, ment of potential intervention for dental caries and pulpitis.
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J Dent Res 93(3) 2014 293 Dental Pulp Dendritic Cells Migrate to Regional Lymph Nodes
Acknowledgments This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by grants from the Japan Society for the Promotion of Science. M.A. has received a licensing fee and royalties from eBioscience and BD-Pharmingen. The other authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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