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CUTTING EDGE |
* Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan;
RIKEN Research Center for Allergy and Immunology, Yokohama, Japan; and
Department of Otolaryngology, Head and Neck Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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The lymphotoxin (LT)
R signaling pathway is essential for the organogenesis of secondary lymphoid tissues, including peripheral lymph nodes (LN) and Peyer’s patches (PP) (2). However, previous reports by our and other groups (3, 4) have demonstrated that NALT organogenesis, unlike that of other secondary lymphoid tissues, can occur independently of the LTR signaling pathway. Inducer cells with phenotypes of CD3–, CD4+, and CD45+ are required for the initiation of the organogenesis of NALT, PP, and LN (1, 2, 3). Lymphoid chemokines, including CXCL13, CCL19, and CCL21, have been shown to be important for the recruitment of CD3–CD4+CD45+ cells to the PP anlagen (5, 6). Not surprisingly then, CXCR5–/– mice and CXCL13–/– mice lack PP and several types of LN such as inguinal and iliac LN (7). Although PP and LN are developed in mice carrying the paucity of LN T cell (plt) mutation, which are known not to produce CCR7 ligands, CCL19 and CCL21, a study using double mutants of CXCL13–/– and plt/plt mice revealed that CCL19, CCL21, and CXCL13 were cooperatively involved in the development of secondary lymphoid organs (7). However, little is known about the involvement of these lymphoid chemokines for the recruitment of CD3–CD4+CD45+ cells to sites of NALT development.
To better understand the varying roles of lymphoid chemokines in the development of NALT and the maintenance of its architecture, we investigated the unique characteristics of NALT development using CXCL13–/– mice and plt/plt mice. Our results provide the first evidence that the initiation of NALT organogenesis is independent of lymphoid chemokines, including CXCL13, CCL19, and CCL21. Lymphoid chemokines have been shown to play an important role in the maturation of the microarchitecture of secondary lymphoid organs (8, 9), and our current findings show that these same chemokines are essential for NALT microarchitecture formation as well.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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BALB/c and C57BL/6 mice were purchased from Japan SLC. The procedure for generating CXCL13–/– mice on a C57BL/6 background was reported previously (10). Plt/plt mice with a BALB/c background were provided from Drs. H. Nakano and T. Kakiuchi (Department of Immunology, Toho University School of Medicine, Tokyo, Japan) (11). CXCL13–/–plt/plt mice were generated by intercrossing CXCL13–/– mice with plt/plt mice. PCR primers D4Mit237 (sense, 5'-TTCAAACTCATGAGTCTATGGGG-3'; antisense, 5'-ATATACACGTAGACTCGCACGC-3') were used to determine the genome type of plt/plt and plt/+ or +/+ (11).
Cell analysis and isolation by flow cytometry
Cells were isolated from the nasal tissues and intestines and then stained with the appropriate fluorescence-conjugated anti-CD3
(145-2C11; BD Pharmingen), anti-CD45 (30-F11; BD Pharmingen), anti-CD4 (L3T4; BD Pharmingen), anti-B220 (RA3-6B2; BD Pharmingen), anti-CD11c (HL3; BD Pharmingen), anti-CXCR5 (2G8; BD Pharmingen), and/or anti-CCR7 (4B12; eBioscience) (3). Cells were then analyzed using a FACSCalibur flow cytometer (BD Biosciences), and data analysis was performed with CellQuest software (BD Biosciences). For the purification of CD3–CD4+CD45+ cells from infant nasal tissues (10-day-old) and embryonic intestines (17-day-old embryos (E17)), we used an AutoMACS (Miltenyi Biotec) combined with a FACSAria cell sorter (BD Biosciences), as described previously (3).
Isolation of NALT anlagen for RT-PCR
Following the manufacturer’s recommendations, we obtained RNA from NALT anlagen by using the laser microdissection (LMD) system (Leica Microsystems). Unfixed nasal tissues isolated from newborn, 7-day-old, 14-day-old, and 6-wk-old mice were frozen in liquid nitrogen. Samples were sectioned into 8-µm thicknesses and immediately fixed in 75% ethanol/diethylpyrocarbonate-treated water for 30 s. Sections were counterstained with toluidine blue (Wako Pure Chemical) for 30 s. After the dehydration with ethanol and xylene, the sections were dissected with a LMD system (Leica Microsystems). The site of NALT formation was captured from each tissue and lysed in TRIzol (Invitrogen Life Technologies) for quantitative RT-PCR using the LightCycler system (Roche Diagnostics) (12).
Primers and hybrid probes for real-time RT-PCR
The primers and hybrid probes used for PCR were as follows: the oligonucleotide primers specific for CXCR5 (sense, 5'-TTCTCCACCCAATGTACC-3'; antisense, 5'-AACCTCTGTCGTCATTCTC-3'), CXCR5 detection FITC-labeled probe (5'-ATTCTACGCACCAATGGGGAAGGAAGCCAACT-3'), and LightCycler Red 640-labeled hybrid probe (5'-GCCTGGGGAAAGCAAGATAGCAAAGTGGTCCTA-3'); the oligonucleotide primers specific for CCR7 (sense, 5'-ATGCTGGCTATGAGTTTC-3'; antisense, 5'-GCTGCTATTGGTGATGTT-3'), CCR7 detection FITC-labeled probe (5'-ATGATCACCTTGATGGCCTTGTTCCGCTCAAAG-3'), and LightCycler Red 640-labeled hybrid probe (5'-TGCGTGCCTGGAGCAAGGTACGGATGATAATGA-3'); the oligonucleotide primers specific for CXCL13 (sense, 5'-GAACAGGCATTTAGTGACAAC-3'; antisense, 5'-TTTTGGAAGCCTGCGTTTT-3'), CXCL13 detection FITC-labeled probe (5'-AATGTGAACTTGTAGCTCGTACTAACAAGAGG-3'), and LightCycler Red 640-labeled hybrid probe (5'-TTGCGAGATGGACTTCAGTTATTTTGCACC-3'); the oligonucleotide primers specific for CCL19 (sense, 5'-GCCAAGAACAAAGGCAACA-3', antisense, 5'-CACACTCACATCGACTCTCTA-3'), CCL19 detection FITC-labeled probe (5'-TGGCCCAGGAAACCAAGGACCA-3'), and LightCycler Red 640-labeled hybrid probe (5'-AAGAGAGGACCAGGCCTCCT-3'); the oligonucleotide primers specific for CCL21a (sense, 5'-ACAGACACAGCCCTCAA-3'; antisense, 5'-CATGAGGTGGCTGCTTT-3'), CCL21a detection FITC-labeled probe (5'-CCAGGAGATCCCCCACGAACTTC-3'), and LightCycler Red 640-labeled hybrid probe (5'-AGCTGGGTGGTTCACGGT-3'); and the oligonucleotide primers specific for GAPDH (sense, 5'-TGAACGGGAAGCTCACTGG-3'; antisense, 5'-TCCACCACCCTGTTGCTGTA-3'), GAPDH detection FITC-labeled probe (5'-CTGAGGACCAGGTTGTCTCCTGCGA-3'), and LightCycler Red 640-labeled hybrid probe (5'-TTCAACAGCAACTCCCACTCTTCCACC-3'). They were designed and produced by Nihon Gene Research Laboratories.
Immunohistochemistry
For confocal microscopic analysis, the nasal tissues and intestines were fixed in 4% paraformaldehyde for the preparation of cryostat sections (5 µm) (3). These tissues were then stained with appropriate fluorescence-conjugated mAb as described above. To assess the formation of the germinal center and follicular dendritic cell (FDC) network in NALT, the previously described nasal immunization protocol was used (3). NALT sections were incubated with biotinylated peanut agglutinin (PNA) (Vector Laboratories) and then stained with FITC-streptavidin (BD Pharmingen). To detect the FDC network, the serial sections were stained with anti-FDC-M1 (BD Pharmingen) and then visualized with FITC-conjugated anti-rat IgG (BD Pharmingen). Histological analysis was performed using a confocal microscope (Leica Microsystems).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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The lymphoid chemokines CXCL13, CCL19, and CCL21 were shown to be involved in the migration of CD3–CD4+CD45+ inducer cells into the PP anlagen (5, 6). Our previous study showed that CD3–CD4+CD45+ inducer cells accumulated at the site of NALT development in mice aged between 7 and 10 days (3). When these lymphoid chemokine-deficient mice were examined, we detected a cluster of the inducer cells at the site of NALT formation in the infant nasal cavity of 10-day-old CXCL13–/– mice, plt/plt mice, and CXCL13–/–plt/plt mice in addition to 10-day-old C57BL/6 and BALB/c mice (Fig. 1A). The size of the CD3–CD4+ inducer cell cluster in the NALT anlagen of CXCL13–/– infant mice and plt/plt infant mice was similar to that observed in control C57BL/6 and BALB/c mice, respectively. When single-cell preparations from nasal tissues of 10-day-old CXCL13–/– mice, 10-day-old plt/plt mice, and 10-day-old CXCL13–/–plt/plt infant mice were examined, CD3–CD4+CD45+ cells were also found (Fig. 2A). The number of CD3–CD4+CD45+ cells isolated from nasal tissues of CXCL13–/– mice, plt/plt mice, and CXCL13–/–plt/plt mice did not differ significantly from that of controls (Fig. 2B). As one might expect based on the previous study (6), several cellular clusters of CD3–CD4+ inducer cells were observed in the intestine of 17-day-old embryos (E17) of C57BL/6 and BALB/c mice (Fig. 1B). In contrast, we could not detect any signs of an accumulation of CD3–CD4+ inducer cells in intestines isolated from E17 CXCL13–/– mice and CXCL13–/–plt/plt mice (Fig. 1B). These results confirm those of a previous study (6) and indicate that the degree to which the initiation of tissue genesis depends on lymphoid chemokines can be used to distinguish NALT inducer cells (NALTi) (independent) from PP inducer cells (PPi) (dependent). Thus, CXCL13 is indispensable for the accumulation of CD3–CD4+CD45+ PPi but not of NALTi in the respective tissue anlagen.
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These data demonstrate that CD3–CD4+CD45+ NALTi can migrate to the site of NALT formation without lymphoid chemokines such as CXCL13, CCL19, and CCL21, which are known to be associated with the other lymphoid tissue genesis programs. Furthermore, the size of the CD3–CD4+CD45+ cell cluster and the number of CD3–CD4+CD45+ cells in infant nasal tissues did not change in lymphoid chemokine-deficient mice. Thus, the PP genesis-associated lymphoid chemokines may not have any involvement in the formation of the NALT anlagen operated by the NALTi. If that is the case, then our efforts should be focused on identifying the molecules that are at work in the migration of NALTi to the NALT anlagen.
The expression of chemokine receptors by NALTi
Inasmuch as the chemokine receptor family of CXCR5 and CCR7 has been shown to play a key role in the migration of PPi to the tissue genesis site (5), it was logical to next examine the use of the chemokines by NALTi. We first performed quantitative RT-PCR to examine the levels of CXCR5 and CCR7 expression by NALTi and PPi. For both chemokine receptors, levels expressed by NALTi were significantly lower than those expressed by PPi (Fig. 3A). Thus, CXCR5 and CCR7 expression by NALTi fell to levels that were barely detectable. The finding was further confirmed by FACS analysis, where PPi expressed CXCR5 and CCR7, especially the CD4high fraction (Fig. 3B). However, NALTi expressed neither CXCR5 nor CCR7 (Fig. 3B).
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signaling, CXCR5 is involved in the induction of LT12 expression on PPi (7). In addition, CXCR5 signaling mediates the activation of 1 integrin expressed on PPi for the interaction of VCAM-1+ICAM-1+ stromal cells at the PP anlagen (6). Although the multipotent function of CXCR5 expressed by PPi is required to initiate the development of PP, NALTi did not express CXCR5. Therefore, NALTi is thought to mediate the initiation of NALT organogenesis without a CXCR5/CXCL13-mediated signal. Furthermore, CCR7 expressed on CD3–CD4+CD45+ cells are cooperatively involved in the organogenesis of PP and other LN (5, 7). Given that neither CCR7 nor CXCR5 is expressed on NALTi (Fig. 3, A and B), it is likely that the initial step of NALT organogenesis is completely independent of the lymphoid chemokine signaling mediated by the corresponding receptors of CXCR5 and CCR7. Uniqueness in the production of lymphoid chemokines by NALT
To further support our findings using immunohistological analysis of the lymphoid tissue genesis in lymphoid chemokine-deficient mice, CXCL13-specific mRNA was rarely produced at the site of NALT formation of newborn and 7-day-old BALB/c and C57BL/6 mice (Fig. 3C). Likewise, the production of CCL19-specific mRNA at NALT anlagen was nil or extremely low in newborn mice. In contrast, we detected constitutive mRNA expression of CCL21 at the site of NALT development in mice newly born up to mice aged 6 wk (Fig. 3C). High levels of mRNA expression for CXCL13, CCL19, and CCL21 were also detected in the NALT of 6-wk-old mice (Fig. 3C). As NALT developed, the expression of lymphoid chemokines, including CXCL13 and CCL19, gradually increased.
CXCL13 and CCL19 have been shown to be produced by VCAM-1+ICAM-1+ stromal cells in the anlagen of PP (5). LTR signaling through the alternative NF-
B pathway by the interaction of stromal cells and PPi is thought to induce CXCL13, CCL19, and CCL21 expression (2). In the case of NALT, neither CXCL13 nor CCL19 was expressed at birth, but the expression of both gradually increased as NALT matured. Thus, it is interesting to postulate that the initial triggering of CXCL13 and CCL19 production is induced by the cluster of NALTi accumulated at the NALT anlagen in the neonatal stage, with lymphoid cells gradually taking over the expression of these two chemokines.
CCL21 is produced by stromal cells in the T cell area, endothelial cells of high endothelial venules, and lymphatic vessels (7). Therefore, since the level of CCL21 expression is high at birth and remains high during the maturation stage related to the other two chemokines, stromal cells and endothelial cells in NALT, including anlagen and adult stages, seem to be a key source for the production of CCL21.
Microarchitecture of NALT in the lymphoid chemokine-null mice
Thus far, our data have demonstrated that the lymphoid chemokines CXCL13, CCL19, and CCL21 are not involved in the induction of NALT organogenesis. The role of lymphoid chemokines for the maintenance of mature NALT in adult mice should be investigated next. The total number of mononuclear cells in NALT of young adult CXCL13–/– mice and plt/plt mice was always lower than in normal mice (Fig. 4A). Of the various lymphoid cell subsets, the population of B220+ B cell saw the greatest decrease, followed by CD3+ cells and CD11c+ cells in the NALT of CXCL13–/– mice (Fig. 4A). Furthermore, we sought to determine whether the migration of B1 and B2 cells into NALT might be altered in CXCL13–/– mice since the most obvious alteration was associated with the B cell subset. The level of CXCR5 expression by B1 cells in NALT was similar to that by B2 cells (our unpublished data). Although it has been established that a major subset of NALT B cells belong to B2 cells (13), both B1 and B2 cells were reduced in the NALT of CXCL13–/– mice (our unpublished data). These data indicate that CXCL13 is required for the migration of both B1 and B2 cells into NALT. Using confocal microscopic analysis, we further showed that the microarchitecture of the B cell area was destroyed in CXCL13–/– mice, leaving the NALT extensively occupied by T cells (Fig. 4B). The formation of a germinal center and FDC network was thus disrupted in the NALT of CXCL13–/– mice (Fig. 4B). In contrast, the T cell area was not observed in the NALT of plt/plt mice (Fig. 4B). The formation of the germinal center and the FDC network was intact in the NALT of plt/plt mice (Fig. 4B). These findings suggest that CXCL13 is involved in the recruitment of lymphocytes into NALT instead of the initiation of NALT tissue genesis. Furthermore, it was previously suggested that CXCL13 contributed to the subsequent microarchitecture formation of the B cell zone in NALT (9). However, it should be noted that B cell themselves are also capable of regulating the microarchitecture formation via the use of LT family-mediated signals (8). LT12-expressing B cells themselves promote the formation of follicles in secondary lymphoid organs (8). Therefore, B cell migration into NALT may also affect the disorganized follicles in the NALT of CXCL13–/– mice. In contrast, CCL19 and CCL21 preferentially promoted T cell migration into NALT but were not involved in the genesis of tissue or the formation of microarchitecture like the germinal center and FDC network.
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R induces the expression of CXCL13 by stromal cells in the B cell area for the recruitment of B cells into the follicular regions and the formation of germinal centers in spleen (7). This evidence provides a logical explanation as to why the microarchitecture of NALT is disorganized in mice lacking LTR signaling (e.g., LT–/– mice, LT–/– mice, and IB kinaseAA mice) (3, 4, 14). Our data further support the findings by Ying et al. (15), which showed that the reduced production of CXCL13, CCL19, and CCL21 in LT and LT deficiency resulted in the disorganization of NALT. Thus, not only do our results confirm the findings that CXCL13 is involved in the maintenance of the microarchitecture of NALT (9) (Fig. 4), they further show that it is not involved in the initiation of the tissue genesis (Figs. 1–3). The analysis of plt/plt mice showed that CCL19 and CCL21 promote T cell migration to NALT. CXCL13 also plays an essential role in the formation of the germinal center and FDC network, whereas CCL19 and CCL21 are not involved. Our findings demonstrate that the lymphoid chemokine family interactions of CXCR5/CXCL13 and CCR7/CCL19 and CCL21 are not essential for the initiation of NALT genesis associated with the NALTi migration into the NALT anlagen. However, as our current study demonstrates, these lymphoid chemokines do play key roles in the creation and maintenance of NALT structure in adult mice. The latter finding is in total agreement with the recent study by Rangel-Monero et al. (9), which showed CXCL13, CCL19, and CCL21 were required for the organization of NALT. Our further examinations suggested that the lymphoid chemokine interactions of CXCR5/CXCL13 and CCR7/CCL19 and CCL21 provide distinct signals for the initiation of tissue genesis, as well as recruitment of lymphoid cells and subsequent microarchitecture formation of different mucosa-associated lymphoid tissues located in the aero-digestive tract.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by the Core Research for Evolutional Science and Technology Program, from Japan Science and Technology Corporation, and a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture and the Ministry of Health and Welfare of Japan. S.F. was supported by research fellowships from the Japan Society for the Promotion of Science for Young Scientists. D.-Y.K. was supported by research fellowships from the Japan Society for the Promotion of Science for Foreign Researchers. 
2 Address correspondence and reprint requests to Dr. Hiroshi Kiyono, Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address: kiyono{at}ims.u-tokyo.ac.jp
3 Abbreviations used in this paper: NALT, nasopharynx-associated lymphoid tissue; PP, Peyer’s patch; LT, lymphotoxin; LN, lymph node; E17, 17-day-old embryo; LDM, laser microdissection; FDC, follicular dendritic cell; PNA, peanut agglutinin; NALTi, NALT inducer cell; PPi, PP inducer cell; MLN, mesenteric LN.
Received for publication May 8, 2006. Accepted for publication August 1, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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R, and NIK signaling pathways but requires the Id2 gene and CD3–CD4+CD45+ cells. Immunity 17: 31-40. [Medline] (LT) and retinoic acid receptor-related orphan receptor-
, but the organization of NALT is LT dependent. J. Immunol. 168: 986-990. 41 integrin activation by CXCR5. Immunity 17: 363-373. [Medline]-chain selectively influences the development of the common mucosal immune system independent IgA-producing B-1 cell in mucosa-associated tissues. J. Immunol. 162: 821-828. B kinase complex kinase activity controls chemokine and high endothelial venule gene expression in lymph nodes and nasal-associated lymphoid tissue. J. Immunol. 173: 6161-6168. This article has been cited by other articles:
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  T. Nagatake, S. Fukuyama, D.-Y. Kim, K. Goda, O. Igarashi, S. Sato, T. Nochi, H. Sagara, Y. Yokota, A. M. Jetten, et al. Id2-, ROR{gamma}t-, and LT{beta}R-independent initiation of lymphoid organogenesis in ocular immunity J. Exp. Med., October 26, 2009; 206(11): 2351 - 2364. [Abstract] [Full Text] [PDF]
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 J. Kunisawa, M. Gohda, Y. Kurashima, I. Ishikawa, M. Higuchi, and H. Kiyono Sphingosine 1-phosphate-dependent trafficking of peritoneal B cells requires functional NF{kappa}B-inducing kinase in stromal cells Blood, May 1, 2008; 111(9): 4646 - 4652. [Abstract] [Full Text] [PDF]
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 M. M. Anis, S. A. Fulton, S. M. Reba, Y. Liu, C. V. Harding, and W. H. Boom Modulation of Pulmonary Dendritic Cell Function during Mycobacterial Infection Infect. Immun., February 1, 2008; 76(2): 671 - 677. [Abstract] [Full Text] [PDF]
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T. Nochi, Y. Yuki, A. Matsumura, M. Mejima, K. Terahara, D.-Y. Kim, S. Fukuyama, K. Iwatsuki-Horimoto, Y. Kawaoka, T. Kohda, et al. A novel M cell specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses J. Exp. Med., November 26, 2007; 204(12): 2789 - 2796. [Abstract] [Full Text] [PDF]
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K. Takamura, S. Fukuyama, T. Nagatake, D.-Y. Kim, A. Kawamura, H. Kawauchi, and H. Kiyono Regulatory Role of Lymphoid Chemokine CCL19 and CCL21 in the Control of Allergic Rhinitis J. Immunol., November 1, 2007; 179(9): 5897 - 5906. [Abstract] [Full Text] [PDF]
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