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Developmental Stage-Dependent Collaboration between the TNF Receptor-Associated Factor 6 and Lymphotoxin Pathways for B Cell Follicle Organization in Secondary Lymphoid Organs¹

Junwen Qin^*, Hiroyasu Konno^*, Daisuke Ohshima^*, Hiromi Yanai^*, Hidehiko Motegi^*, Yusuke Shimo^*, Fumiko Hirota, Mitsuru Matsumoto, Satoshi Takaki, Jun-ichiro Inoue^2,* and Taishin Akiyama^2,*

^* Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, Tokushima, Japan; and Department of Community Health and Medicine, Research Institute International Medical Center of Japan, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Signal transduction pathways regulating NF-B activation essential for microenvironment formation in secondary lymphoid organs remain to be determined. We investigated the effect of a deficiency of TNFR-associated factor 6 (TRAF6), which activates the classical NF-B pathway, in splenic microenvironment formation. Two-week-old TRAF6-deficient mice showed severe defects in B cell follicle and marginal zone formation, similar to mutant mice defective in lymphotoxin (Lt) β receptor (LtβR) signal induction of nonclassical NF-B activation. However, analysis revealed a TRAF6 role in architecture formation distinct from its role in the early neonatal Lt signaling pathway. LtβR signal was essential for primary B cell cluster formation with initial differentiation of follicular dendritic cells (FDCs) in neonatal mice. In contrast, TRAF6 was dispensable for progression to this stage but was required for converting B cell clusters to B cell follicles and maintaining FDCs through to later stages. Fetal liver transfer experiments suggested that TRAF6 in radiation-resistant cells is responsible for follicle formation. Despite FDC-specific surface marker expression, FDCs in neonatal TRAF6-deficient mice had lost the capability to express CXCL13. These data suggest that developmentally regulated activation of TRAF6 in FDCs is required for inducing CXCL13 expression to maintain B cell follicles.

	Introduction

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Secondary lymphoid organs provide an environment that facilitates interactions among lymphocytes and between lymphocytes and APCs, interactions that are essential for initiation of an effective immune response. Although the structures and the development of the secondary lymphoid organs during ontogeny have been studied in detail, the molecular mechanisms involved in their development and organization remain unclear (1, 2, 3, 4). Studies with gene-targeting techniques revealed that lymphotoxin (Lt)³ β receptor-mediated signaling is required for the development of the secondary lymphoid organs including lymph nodes and Peyer’s patches (2, 5, 6). Intracellular signaling from Lt β receptor (LtβR) induces transcriptional activation by members of the NF-B family through both the canonical and noncanonical pathways (7, 8). Deficiency in NF-B-inducing kinase (NIK) or RelB, which are essential for the noncanonical pathway, results in the absence of lymph nodes and Peyer’s patches in mice (7, 8). These results suggest that the noncanonical NF-B activation pathway plays a critical role in the development of these secondary lymphoid organs (8, 9).

The microarchitecture of the splenic white pulp, in which the immune response against blood-borne Ags initiates, requires LtβR signaling for development (6, 10). The white pulp in naive adult mice is divided into the B cell follicles, the T cell zone, and the marginal zone. The regulated interaction between lymphocytes and stromal cells is essential for development of these organized structures in the white pulp. For development of B cell follicles, the interaction between B cells and FDCs, a subset of stromal cells in lymphoid follicles, is crucial (11). Stimulation of LtβR on FDCs by B cell surface-anchored Lt1β2 complex induces FDC expression of CXCL13, a chemokine that attracts B cells. Interaction of CXCL13 with its receptor, CXCR5, on B cells increases cell surface expression of Lt1β2, which in turn stimulates the LtβR on FDCs to express CXCL13. This process provides a positive feedback loop that is essential for the development of primary B cell follicles (12).

TNFR-associated factor 6 (TRAF6) is an adaptor protein that transduces signals from cell surface receptors such as the Toll/IL-1R family, CD40, and receptor activator of NF-B (RANK)/TNF-related activation-induced cytokine (TRANCE) ligand to induce the activation of the canonical NF-B pathway and mitogen-activated kinase cascades (13, 14). Analyses of TRAF6-deficient (TRAF6^–/–) mice indicated that TRAF6 is essential for the development and organization of the various primary and secondary lymphoid organs (15, 16, 17, 18). The bone marrow cavity of TRAF6^–/– mice is size-limited due to severe osteopetrosis resulting from a defect in osteoclast formation (15) (16). Furthermore, we recently reported that TRAF6 is essential for the formation of the thymic microenvironment, which directs the induction of self-tolerance (17). TRAF6^–/– mice as well as RANK and RANK ligand (RANKL)-deficient mice lack lymph nodes but not Peyer’s patches, suggesting that signal transduction through a RANKL-RANK-TRAF6 pathway is required for lymph node development (18).

In this study, we investigate the role of TRAF6 in the organization of the microarchitecture of the splenic white pulp. Our data indicate that B cell follicle development within the spleen during the neonatal period is regulated by at least two distinct signal transduction pathways with respect to the requirement for TRAF6. The signal pathway consisting of the Lt1β2-LtβR-NIK-RelB axis, which is independent of TRAF6, is necessary for B cell cluster formation during early neonatal development. In contrast, the TRAF6-dependent pathway is not required for organization during the early neonatal period, but instead contributes to the development and maintenance of B cell follicles during the late neonatal period.

	Materials and Methods

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Mice

TRAF6^–/– mice (C57BL/6 and BALB/c background) were generated in our laboratory as previously described (15, 17). The phenotypes observed in the present work are common between C57BL/6 and BALB/c backgrounds. Aly/+ and aly/aly mice were purchased from CLEA Japan. Male aly/aly mice were crossed with aly/+ female, and the genotypes of pups was determined if the lymph nodes were present. RelB^–/– mice were purchased from The Jackson Laboratory. RAG2^–/– mice were provided from Dr. F. W. Alt (Harvard University Medical School, Boston, MA), and LT^–/– mice were obtained from Dr. D. D. Chaplin (University of Alabama, Birmingham, AL). All mice were maintained under specific pathogen-free conditions and were handled in accordance with the Guidelines for Animal Experiments of the Institute of Medical Science (University of Tokyo, Tokyo, Japan).

Reagents

The following reagents were used in the present studies: biotin-conjugated anti-mouse B220 (Biolegend), biotin-conjugated anti-mouse IgM (clone R6-60.2; BD Pharmingen), biotin-conjugated rabbit anti-mouse IgM (Pierce), biotin-conjugated anti-mouse CD3 (clone 145-2C11; BD Pharmingen), anti-mouse follicular dendritic cell (FDC)-M1 (BD Pharmingen), anti-mouse mucosal addressin cell adhesion molecule-1 (MAdCAM-1; BD Pharmingen), anti-mouse monocytes/macrophases (MOMA)-1 (BMA Biomedical), anti-mouse SIGN-R1 (clone ER-TR9) Hycult Biotechnology), biotin-conjugated anti-mouse CD35 (CR1, clone 8C12; BD Pharmingen), biotinylated anti-mouse FDC-M2 (clone FDC-M2; ImmunoKontact), anti-mouse CXCL13 Ab (R&D Systems), alkaline phosphatase-conjugated streptavidin (Zymed Laboratories), HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology), Alexa Fluor 488 donkey anti-goat IgG (H+L; Molecular Probes), Alexa Fluor 594 donkey anti-rat IgG (Molecular Probes), Alexa Fluor 488-conjugated streptavidin (Molecular Probes), Alexa Fluor 546-conjugated streptavidin (Molecular Probes), Alexa Fluor 488-conjugated anti-rabbit IgG (Molecular Probes), Alexa Fluor 546-conjugated anti-rabbit IgG (Molecular Probes), Alexa Fluor 546-conjugated anti-rat IgG (Molecular Probes), anti-FcIII/IIR Ab (clone 2.4G2; BD Pharmingen), PE-conjugated anti-mouse B220 (clone RA3-6B2; BD Pharmingen), FITC-conjugated anti-mouse IgM (clone 11/41; eBioscience), FITC-conjugated anti-mouse IgD (clone 11-26; eBioscience), PE-conjugated anti-mouse CXCR5 (BD Pharmingen), streptavidin-CyChrome (BD Pharmingen), monoclonal anti-mouse LTβR Ab (clone 4H8 WH2; Alexis), anti-phospho-IB Ab (Ser^32/36) (clone 5A5; Cell Signaling), anti-mouse NF-B p52 Ab (clone C-5; Santa Cruz Biotechnology), anti-phospho-Jun-N-terminal nucleotide kinase (JNK) Ab (Cell Signaling Technologies), and anti-JNK Ab (clone C-17; Santa Cruz Biotechnology).

Histological and immunohistochemical analyses

After wild-type or mutant mice were killed at indicated ages, spleens and Peyer’s patches were removed and frozen quickly in OCT compound (Sakura Finetek), or fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. Paraffin sections (5-µm thick) were stained with H&E and examined by light microscopy. For immunohistochemistry, frozen sections (5-µm thick) were cut and fixed in acetone for 20 min at 4°C. For color detection, endogenous peroxidase was quenched with 0.2% H₂O₂/methanol. After blocking with 10% normal goat serum (Zymed Laboratories) or 10% normal donkey serum (Sigma-Aldrich) for 30 min at room temperature, sections were incubated with primary Abs containing blocking solution for 60 min at room temperature or overnight at 4°C. Sections were then washed with PBS (pH 7.4) and incubated with the proper secondary Ab for 50 min at room temperature. Confocal color images were obtained with a Radiance 2000 (Zeiss). Color development was conducted with an AP Reaction kit (Vector Laboratories) or a diaminobenzidine-peroxidase reaction kit (KPL).

Western blot analysis

Total spleen from wild-type or mutant mice was homogenized in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40) containing protease inhibitor (1 mM PMSF). The lysates were boiled and separated by polyacrylamide/SDS gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane (Millipore). After blocking with 5% skim milk in TBS, the membrane was immunoblotted with specific Abs in 5% skim milk. After treating with HRP-conjugated secondary Ab, the bands were visualized by ECL or ECL plus Western Blotting System (GE Healthcare).

Flow cytometric analysis

Mice were killed at the indicated neonatal ages, and single-cell suspensions were prepared from spleen. Erythrocytes were lysed in ammonium chloride buffer. For analysis, 1 x 10⁶ cells/sample were suspended in 100 µl of ice-cold PBS containing 4% FBS and 10 mM HEPES, and labeled with Abs. Labeled cells were analyzed with a fluorescence-activated cell sorter. (Epics XL; Beckmann Coulter). Dead cells were excluded for analysis by staining with 7-aminoactinomycin D (Wako).

Semiquantitative RT-PCR analysis

Total RNA was extracted with TRIzol (Invitrogen Life Technologies), and reverse transcription was performed with the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies) according to the manufacturer’s instructions. Primer sets used in the present studies were described elsewhere (19).

Isolation of FDC-enriched cells, IgM-positive B cells, and total B cells from spleen

For isolation of FDC-enriched cells, spleen was digested in RPMI 1640 (Invitrogen Life Technologies) medium containing 0.25 mg/ml collagenase/disperse (Roche) and 1% DNase I (Takara). For isolation of IgM-positive B cells, cell suspension of spleen was prepared by teasing the tissues between two frosted microscope slides (Matsunami). After removing the erythrocytes by ACK buffer, a single-cell suspension was labeled with FDC-M1 Ab, biotin-conjugated FDC-M2 Ab, or IgM Ab. FDC-enriched cells and IgM-positive B cells were positively selected by MACS system with anti-rat microbeads or anti-biotin microbeads (Miltenyi Biotec). For isolation of total B cells from spleen, B cells were negatively selected from single-cell suspension by the B Cell Isolation kit of the MACS system. Purity of B cells was 85–95%.

Fetal liver cell transfer analysis

Fetal liver cells were obtained from embryonic day 14 TRAF6^–/– and wild-type mice and injected i.v. to the lethally irradiated (950 rad) RAG2^–/– mice. Six to 8 wk after the transplantation, the transferred mice were sacrificed and the spleen was used for the immunostaining analysis.

B cell transfer to RAG2^–/– mice

Splenic B cells from TRAF6^–/– and wild-type mice at PN 11.5 were transferred to RAG2^–/– mice i.v. Two weeks after the transfer, the transferred mice were sacrificed, and the spleen was used for the immunostaining analysis.

Restoration of B cells into TRAF6^–/– spleen

B cells were negatively selected by MACS system (Miltenyi Biotec) from spleens of adult (8 wk old) CD45.1-congenic mice. The selected B cells (1.5 x 10⁸ cell) were injected in neonatal TRAF6^–/– mice (postnatal (PN) 7.5). The injected mice were analyzed at PN 14.5. The restored B cell number was determined as 2.0 x 10⁷ cells by counting CD45.1-labeled cells.

Treatment of mouse embryonic fibroblast cells (MEFs) with agonistic anti-LtβR Ab

Embryos (E14.5) from wild-type, TRAF6^–/–, or aly/aly mice were used to isolate MEFs. MEFs were expanded for two passages before assay. MEFs were cultured in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine (Wako Chemicals), 100 U/ml penicillin, and 100 µg/ml streptomycin. MEFs were treated with agonistic LtβR mAb (2 µg/ml) for the indicated time. After treatment of the agonistic Ab, the cell were lysed by a buffer containing 3% SDS, 5% 2-ME, and 10% glycerol. Then, the whole cell lysates were analyzed by Western blot.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
TRAF6^–/– mice display defective architecture in spleen

We first examined architecture in paraffin-embedded sections of spleen obtained from postnatal (PN) 14.5 TRAF6^–/– mice and wild-type mice by H&E staining. Defective splenic architecture was observed in the TRAF6^–/– mice, with a reduction in the number of lymphocytes in the white pulp (Fig. 1A). The structure of the red pulp region was not significantly affected by TRAF6 deficiency. Immunohistological analysis of spleen cryosections indicated that B cell follicles (as detected with anti-IgM Ab) in the white pulp were almost completely absent in spleen from TRAF6^–/– mice (Fig. 1A). The T cell area (detected with anti-CD3 Ab) was present, but the accumulation of T cells appeared to be less efficient compared with that in wild-type mice (Fig. 1B, upper panels). In addition, a scattered distribution of IgM-positive cells was observed in the T cell area. It is known that B cell follicular formation is dependent on the interaction between B cells and FDCs (2, 11). To analyze the development of FDCs in the spleen of TRAF6^–/– mice, we performed immunostaining of cryosections of spleen with FDC-M1 Ab. As shown in Fig. 1B (lower panels), FDC-M1-positive cells were not detected in TRAF6^–/– spleen at PN 14.5. These data indicate that TRAF6 is essential for the development of primary B cell follicles.

View larger version (74K): [in this window] [in a new window]   FIGURE 1. Disorganization of splenic architecture in TRAF6–/– mice. A, Disruption of splenic architecture in TRAF6–/– mice. Paraffin-embedded spleen sections were stained with H&E staining (upper panels; x40 magnification, error bars 400 µm, lower panels; x200 magnification, error bars 200 µm). B, Disruption of B cell follicles in the spleen of TRAF6–/– mice. Spleen sections were immunostained with the combination of anti-IgM Ab (green) and anti-CD3 Ab (red) (upper panels) or the combination of anti-IgM Ab (green) and FDC-M1 Ab (red) (lower panels). Scale bars, 200 µm. C, Disorganization of the marginal zone in TRAF6-deficient spleen. Cryosections of spleen were double-stained with anti-IgM (green) and anti-MAdCAM-1 Abs (red) (upper panels), anti-IgM (green), and MOMA-1 Abs (red) (middle panels), or stained with ER-TR9 Ab (red) (lower panels). Scale bars, 200 µm.

Development of B cell follicles in the spleen is divided into two stages differing in their requirement for the TRAF6 or Lt signaling pathway

The phenotype of TRAF6^–/– mice with respect to the disorganization of B cell follicles and the marginal zone was similar to the phenotypes of mutant mice defective in molecules involved in LtβR signaling such as Lt, NIK, and RelB (19, 23, 24, 25, 26, 27, 28, 29, 30). We then compared the process of B cell follicle formation from PN 3.5 to PN 11.5 between TRAF6^–/– mice, Lt-deficient (Lt^–/–) mice, aly/aly mice, which carry a point mutation in the NIK gene, and RelB-deficient (RelB^–/–) mice by double-staining spleen sections with anti-IgM and FDC-M1 Abs. In wild-type mice, the separation of IgM⁺ cells from the perivascular area due to the influx of T cells was detected at PN 5.5 after the accumulation of IgM⁺ cells in the perivascular area (Fig. 2 and data not shown). At PN 5.5, the accumulated IgM⁺ cells do not form follicle-like structures, but instead form cluster-like structures (31, 32). FDC-M1-positive cells were observed within and near the cluster of IgM-positive cells. The cross-sectional area of IgM-positive cell clusters enlarged gradually and appeared to develop into follicle-like structures with FDC-M1-positive cells at PN 8.5. This observation is consistent with previous reports (31, 32, 33). In Lt^–/– mice, aly/aly mice and RelB^–/– mice, no enlargement of the B cell clusters was observed and a subset of IgM-positive cells remained around the perivascular area until PN 5.5 (Fig. 2). During the neonatal period investigated, no follicle-like structures or B cell clusters were observed nor were FDC-M1-positive cells detected in spleens of Lt^–/–, aly/aly, or RelB^–/– mice. In contrast, the separation of B cell clusters from the perivascular area, B cell cluster enlargement, and early development of FDC-M1-positive cells were observed in spleens of TRAF6^–/– mice from PN 3.5 to PN 5.5 and were indistinguishable from that observed in wild-type spleens (Fig. 2). Strikingly, at PN 8.5, the B cell cluster was disturbed, and follicle-like structure formation did not occur in TRAF6^–/– mice. By PN 11.5, the B cell clusters had almost completely disappeared. It should be noted that although the number of FDC-M1-positive cells was diminished, such cells were still present in TRAF6^–/– spleen through PN 11.5. However, these FDC-M1-positive cells disappeared by PN 14.5 (Fig. 1A). Therefore, the B cell follicles were abolished before FDCs disappeared. These data indicate that Lt, NIK, and RelB, which are components of the noncanonical pathway of LtβR signaling, are essential for primary B cell cluster formation during early neonatal development. TRAF6 is necessary for development and maintenance of B cell follicles only after PN 8.5 but is not essential for the organization of B cell clusters during the early neonatal period. Therefore, B cell follicle formation is regulated by a combination of the early TRAF6-independent, Lt-dependent pathway, and late TRAF6-dependent pathway.

View larger version (51K): [in this window] [in a new window]   FIGURE 2. B cell follicle development in the spleen of wild-type, TRAF6–/–, Lt–/–, aly/aly, or RelB–/– mice during the neonatal period. Cryosections of the spleen from wild-type, TRAF6–/–, Lt–/–, aly/aly, or RelB–/– mice at PN 3.5, 5.5, 8.5, or 11.5 were double-stained with anti-IgM (green) and FDC-M1 (red) Abs. Scale bars, 100 µm.

Directed movement of lymphocytes within lymphoid organs is dependent on the expression of several chemoattractants by stromal cells (2, 4, 11). The interaction between CXCL13 and its receptor, CXCR5, is critical for the development of B cell follicles in spleen (12), whereas the migration of T cells in the white pulp is regulated by the interaction between CCL19 or CCL21 and CCR7 (2). We performed semiquantitative RT-PCR analysis to investigate the expression level of these chemokines and their receptors in spleen from TRAF6^–/– mice (Fig. 3A). The expression of CXCL13 was 100 times lower in TRAF6-deficient spleen than in wild-type spleen. Levels of expression of CCL19, CCL21, CXCR5, and CCR7 were also decreased.

View larger version (46K): [in this window] [in a new window]   FIGURE 3. Reduced expression of chemokines and chemokine receptors in the spleen of TRAF6–/– mice. A, Semiquantitative RT-PCR analyses (10-fold serially diluted cDNA template) of chemokine (CXCL13, CCL19, and CCL21), chemokine receptor (CXCR5, CCR7), and β-actin transcripts (loading control) in TRAF6-deficient and wild-type spleen. B, Western blot analysis of total spleen lysates from TRAF6–/– mice (knockout (KO)) and wild-type control (WT) with anti-CXCL13 Ab. C, Immunohistological analysis of CXCL13 expression in the splenic white pulp of the TRAF6–/– mice and wild-type littermate. Consecutive cryosections of spleen were stained with H&E (upper panels) or with the combination of anti-IgM (blue) and anti-CXCL13 (brown) Abs (lower panels). Scale bars, 200 µm. D, Kinetics of the CXCL13 expression in spleens of wild-type and TRAF6–/– mice during the neonatal period. Expression of CXCL13 at the indicated ages was determined by RT-PCR. Upper panel, The expression profile of CXCL13; lower panel, expression of β-actin as a loading control.

The expression of CXCL13 in FDC requires TRAF6

The expression of CXCL13 by FDCs plays a crucial role in B cell follicle formation (2, 11, 12). The reduction of CXCL13 expression in spleens of TRAF6^–/– mice at PN 8.5, when FDCs are still present, might be ascribed to the inability of FDCs to express CXCL13. We performed immunostaining of spleens with anti-CXCL13 Ab at PN 5.5, 8.5, and 11.5 (Fig. 4A). At PN 5.5, expression of CXCL13 was detected across the entire spleen in both TRAF6^–/– mice and wild-type littermate. At PN 8.5 and 11.5, expression of CXCL13 was observed around FDC-M1-positive cells in wild-type spleen, whereas the level of CXCL13 expression around FDC-M1-positive cells was significantly lower in spleens of TRAF6^–/– mice.

View larger version (47K): [in this window] [in a new window]   FIGURE 4. TRAF6 is required for expression of CXCL13 by FDC. A, FDCs in the spleen of TRAF6–/– mice express the low level of CXCL13 expression. Cryosections of spleen from wild-type or TRAF6–/– mice at the indicated ages were stained with a combination of anti-CXCL13 (green) and FDC-M1 (red) Abs. Scale bars, 100 µm. B, Semiquantitative RT-PCR analysis (10-fold serially diluted cDNA template) of the expression of CXCL13 in the FDC-enriched cells magnetically sorted from wild-type or TRAF6–/– spleen on day 5.5, 8.5, or 11.5.

Disorganized spleen microarchitecture is independent of the TRAF6 deficiency in lymphocytes

Because follicle formation depends on the interaction between B cells and stromal cells, a defect in B cell function, such as reduced CXCR5 expression due to TRAF6 deficiency, may cause the disruption of the B cell follicles. We analyzed the B cells in spleens from neonatal TRAF6^–/– mice by flow cytometry over the neonatal period. In neonatal mice (from PN 3.5 to PN 14.5), the spleen contains a considerable number of B220⁺IgM⁺IgD^– immature B cells and B220⁺IgM^–IgD^– cells in addition to the B220⁺IgM^lowIgD⁺ mature B cells, which compose the major cell fraction in adult B cell follicles (32). We focused on IgM⁺ B cells in the spleen due to their involvement in B cell follicle formation through expression of high levels of CXCR5 (34). From PN 5.5 to 8.5, the number of IgM⁺ B cells among total splenocytes in TRAF6^–/– mice was slightly lower than that in wild-type mice, however, the difference was not significant (Fig. 5A and Table I). At PN 11.5, the number of IgM-positive B cells in TRAF6^–/– mice was 10 times lower than that in wild-type mice. Importantly, the reduction in the number of IgM-positive cells in TRAF6^–/– mice was not statistically significant on PN 8.5 (p > 0.05) when the B cell follicle structure of TRAF6-deficient spleen was already disturbed. These data suggest that the reduced number of IgM-positive cells underlies the reduction of CXCR5 expression in the whole TRAF6-deficient spleen on PN 14.5 (Fig. 3A). To confirm that the level of CXCR5 on IgM-positive B cells and the ratio of CXCR5-positive cells to total IgM-positive B cells are comparable between wild-type mice and TRAF6^–/– mice, IgM-positive cells were sorted from the spleen of TRAF6^–/– mice and control littermates on PN 11.5, and expression of CXCR5 was analyzed by semiquantitative RT-PCR (Fig. 5B) and flow cytometric analysis (Fig. 5C). As expected, the level of CXCR5 expression and the ratio of CXCR5-expressing cells in IgM-positive cells obtained from TRAF6^–/– mice spleen were similar to those of wild-type mice.

View larger version (65K): [in this window] [in a new window]   FIGURE 5. Disorganization of B cell follicles in TRAF6–/– mice is not the result of B cell dysfunction due to TRAF6 deficiency. A, Number of IgM-positive cells in spleens of wild-type and TRAF6–/– mice at the different neonatal ages. Data are mean ± SD. B, Semiquantitative RT-PCR analysis (10-fold serially diluted cDNA template) of CXCR5, Lt (Lt), Lt β (Ltβ), and β-actin (loading control) in the IgM-positive B cells sorted magnetically from spleens of TRAF6–/– and wild-type mice at PN 14.5. C, Flow cytometric analysis of CXCR5 expression on the cell surface in IgM-positive B cells sorted magnetically from the spleen of TRAF6–/– and wild-type mice. D, Splenic structure in B cell restored TRAF6–/– mice. Sorted B cells were i.p. injected into neonatal TRAF6–/– mice (PN 7.5). The sections of spleen from the B cell restored TRAF6–/– mice (PN 14) were stained with the combination of anti-IgM (green) and FDC-M1 (red) Abs. Scale bars, 200 µm. E, Splenic B cells transfer to RAG2–/– mice. B cells sorted from spleens of wild-type and TRAF6–/– mice were i.v. transferred to RAG2–/– mice. Fourteen days after transfer, spleens from the recipient RAG2–/– mice were stained with a combination of anti-IgM (green) and FDC-M1 (red) Abs (upper panels), anti-IgM (green), and MAdCAM-1 (red) Abs (middle panels) or anti-IgM (green) and MOMA-1 (red) Abs (lower panels). Scale bars, 200 µm. F, Flow cytometric analysis of CD4+CD3–CD11c– cells in the spleen of TRAF6–/– and wild-type mice. Ratio of CD4+CD3– (rectangle) in CD11c-negative cells is indicated. Figure shows a result of two independent experiments. G, Splenic structure in fetal liver transplantation chimeric mice. Irradiated RAG2–/– mice were reconstituted with fetal liver cells from TRAF6–/– mice or wild-type control. Eight weeks after the transfer, the sections of spleen from the chimeric mice was stained with the combination of anti-IgM (green) and FDC-M1 (red) Abs (upper panels), anti-IgM (green) and CD3 (red) Abs (upper middle panels), anti-IgM (green) and MadCAM-1 (red) Abs (lower middle panels), or anti-IgM (green) and MOMA-1 (red) Abs (lower panels). Scale bars, 200 µm.

Another possible argument is that IgM-positive cells that have moved from the impaired TRAF6^–/– bone marrow environment are somehow dysfunctional and incapable of forming the B cell follicles. To rule out this possibility, B cells were sorted from TRAF6^–/– mice and wild-type spleens at PN 11.5, the time point at which B cell follicles of TRAF6^–/– mice have become disrupted, and then transferred into RAG2^–/– mice. B cells derived from TRAF6^–/– splenocytes as well as from wild-type splenocytes restored the B cell follicles, FDCs, and marginal zone in the spleens of RAG2-deficient mice (Fig. 5E). These data suggest that the disorganization of TRAF6-deficient spleen is not ascribed to dysfunction of B cells or low number of mature B cells.

It was recently reported that CD4⁺CD3^– accessory cells express high levels of Lt, Ltβ, and TNF- (35). We addressed whether the deficiency of TRAF6 caused the developmental defect of CD4⁺CD3^– accessory cells. Flow cytometric analysis revealed that the ratio of CD4⁺CD3^–CD11c^– in splenocytes is comparable between wild-type and TRAF6^–/– mice (Fig. 5F) or rather higher in TRAF6^–/– mice, suggesting that the development of CD4⁺CD3^– accessory cells is not significantly impaired by deficiency of TRAF6.

To determine whether TRAF6 deficiency in hemopoietic cells disturbs splenic microarchitecture, we performed transplantations of TRAF6-deficient and wild-type fetal liver cells into lethally x-ray-irradiated RAG2-deficient (RAG2^–/–) mice. Immunohistochemical analysis revealed that the splenic architecture of chimeric mice reconstituted with TRAF6-deficient fetal liver cells did not differ from that of chimeras reconstituted with wild-type fetal liver cells (Fig. 5G). These data suggest that the disorganized splenic microarchitecture in the TRAF6^–/– mice is not due to TRAF6 deficiency in the hemopoietic cells.

TRAF6 is not involved in the noncanonical NF-B pathway triggered by ligation of LtβR

TRAF6 was reported to be involved in the canonical pathway of NF-B, activated by signaling from RANK, CD40 or Toll/IL-1Rs but not by signaling from TNFR type I (13). We examined the role of TRAF6 in NF-B activation triggered by LtβR ligation, which is essential for the formation of B cell follicles. Primary fibroblasts obtained from TRAF6-deficient or wild-type embryos were treated with agonistic anti-LtβR Ab. We examined processing of p100 to p52, an essential step in the activation of the noncanonical pathway (7, 8), by Western blotting of whole cell lysates. As shown in Fig. 6A, induction of processing from p100 to p52 occurred normally in TRAF6-deficient cells as well as wild-type cells in response to LtβR ligation. In contrast, as described previously (36, 37), processing of p100 in embryonic fibroblasts prepared from aly/aly mice was severely impaired (Fig. 6A). The LtβR signaling also activates the canonical NF-B activation pathway as well as the noncanonical pathway (7, 8, 9). We investigated whether TRAF6 is involved in activation of the canonical NF-B pathway in response to the LtβR ligation. Phosphorylation of IB induced by ligation of LtβR was observed in TRAF6-deficient cells as well as wild-type cells (Fig. 6B). Phosphorylation of JNK was also observed in TRAF6-deficient cells (Fig. 6C). These data indicate that TRAF6 is dispensable for NF-B activation and JNK activation triggered by LtβR ligation and is consistent with the observation that TRAF6 is not required for early B cell cluster formation, which requires the Lt pathway. These data strongly suggest that TRAF6 regulates the development of B cell follicles and FDCs through the expression of CXCL13 independent of the Lt pathway.

View larger version (70K): [in this window] [in a new window]   FIGURE 6. TRAF6 is dispensable in the processing of NF-B activation by ligation of LtβR. Embryonic fibroblasts from wild-type, TRAF6–/–, and aly/aly mice were treated with an agonistic LtβR Ab (2 µg/ml) for the indicated time. Whole cell extracts were analyzed by Western blotting with a p52/p100-specific Ab (A), a phospho-IB-specific Ab (B), or a phospho-JNK specific Ab (C).

We next examined whether a similar defect in follicle formation occurs during the development of other secondary lymphoid organs. TRAF6^–/– mice have no lymph nodes but have Peyer’s patches (18). Cryosections of the Peyer’s patches were analyzed on PN 8.5, 11.5, and 14.5. B cell follicles and FDCs were observed in Peyer’s patches in both wild-type and TRAF6^–/– mice at PN 8.5. However, at PN 11.5, the B cell follicles showed signs of disruption, and the number of FDC-M1-positive cells was decreased but still present in the Peyer’s patches in TRAF6^–/– mice at PN 14.5 (Fig. 7A). Furthermore, CXCL13 expression (Fig. 7, B and C) was also diminished in TRAF6-deficient Peyer’s patches. Importantly, comparable development of FDCs and IgM-positive follicles during the early neonatal period and the subsequent disorganization of the B cell follicles were observed in Peyer’s patches as well as in the splenic white pulp of TRAF6^–/– mice, suggesting that the requirement for TRAF6 in B cell follicle formation is common in the secondary lymphoid organs.

View larger version (77K): [in this window] [in a new window]   FIGURE 7. Development of B cell follicles and CXCL13 expression in Peyer’s patches of TRAF6–/– mice. A, The process of development of the B cell follicles in Peyer’s patches in wild-type (upper panels) and TRAF6–/– mice (lower panels) are shown. Scale bars, 200 µm. Each panel is divided into the H&E staining image (upper) and double immunostaining with anti-IgM (green) and FDC-M1 (red) Abs (lower) of consecutive cryosections from the Peyer’s patches. B, Semiquantitative RT-PCR analyses (10-fold serially diluted cDNA templates) of CXCL13 and β-actin (loading control) in the Peyer’s patches of TRAF6–/– mice and wild-type mice. C, Expression and distribution of CXCL13 in Peyer’s patches of wild-type and TRAF6–/– The upper part of each panel shows H&E staining, and the lower part shows immunostaining with anti-CXCL13 Ab (brown) of consecutive cryosections. Scale bars, 200 µm.

	Discussion

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B cell follicle formation is dependent on the secretion of CXCL13 from stroma cells, especially, FDCs (11, 12). Previous studies indicated that the LtβR signaling pathway is essential for the expression of CXCL13 (19, 23, 24, 25, 26, 27, 28, 29, 30). Fetal liver transfer experiments provided evidence that TRAF6 is required in stroma cells for B cell follicle formation. Immunostaining and semiquantitative RT-PCR analyses strongly suggested that FDCs in TRAF6-deficient spleen at PN 8.5 are incapable of secreting CXCL13. These data suggest that the TRAF6-mediated pathway directs CXCL13 expression in FDCs, enabling B cell follicle development. However, it is possible that TRAF6-dependent signaling may not directly induce expression of CXCL13 but instead play roles in promoting B cells to remain adjacent to FDCs by regulating the expression of cell adhesion molecules or B cell survival factor. Such molecules would indirectly affect the expression of CXCL13 through the regulation of the positive feedback loop between B cells and FDCs. Thus, it still remains to be determined whether CXCL13 is a direct target molecule of the TRAF6-dependent signal pathways.

Another interesting observation is that B cell follicles are completely collapsed in the spleen of TRAF6^–/– mice. Previous study indicated that B cell area of CXCL13^–/– mice failed to organize in polarized follicle structure (12). However, the B cell area forms a ring of cells around T cell zone (12). The phenotype of TRAF6^–/– spleen appears to be different from that of CXCL13^–/– spleen. These data suggested that the TRAF6-signal is required for maintaining B cell area in addition to polarized follicle formation and FDC development by inducing CXCL13. The fact that the number of IgM⁺ B cells was largely reduced in TRAF6^–/– spleen implicates that the TRAF6 signal is also required for B cell survival. Detailed analysis is necessary to address this hypothesis.

It was recently reported that CD4⁺CD3^– accessory cells expresses high levels of Lt, Ltβ, and TNF- (35). Even if these cells seem not to be involved in the expression of CXCL13 and FDC development, they are sufficient for the segregation between B and T cell zones in Lt^–/– mice (35). As seen in Fig. 5F, development of CD4⁺CD3^– accessory cells appears not to be impaired in the spleen of TRAF6^–/– mice. However, it is still possible that, in addition to the CXCL13 expression and FDC development, the TRAF6 signal regulates the function of these cells to induce the segregation between B and T cell zones.

It remains unknown what ligand and receptor TRAF6 uses for signal transduction to develop and maintain the B cell follicles and FDCs at PN 8.5. Around PN 8.5, B cells from the bone marrow start entering the spleen. It is possible that in TRAF6^–/– mice, stimulation of FDCs with mature or transitional B cells derived from the bone marrow is impaired. Thus, the TRAF6-dependent signal in FDCs may be stimulated only by the ligand expressed by bone marrow-derived B cells. In contrast, the TRAF6-independent Lt1β2-dependent signal might be required for stromal cells, which may be a precursor of FDCs, to express CXCL13 in response to activation by immature B cells or other type of lymphocytes present in the early neonatal period. Members of the Toll/IL-1R family, which use TRAF6 as a signal transducer, have not been reported to be involved in the organization of the lymphoid organs. CD40- and CD40L-deficient mice were reported to be defective in germinal center formation (38). However, this defect is most likely due to the absence of the interaction between CD40 on B cells and CD40L on T cells, which are activated by the immunization of thymus-dependent Ags, because the splenic architecture in naive CD40-deficient mice was not impaired.

RANKL/TRANCE-deficient mice show altered development of B cell follicles, in which some of the B cell areas in the spleen lack the follicle-like structure (39). However, this phenotype was observed in only some of the mutant mice and was considerably milder than that of TRAF6, suggesting that the RANKL/TRANCE-RANK-TRAF6 axis could contribute to, but is not essential for, B cell follicle formation.

Previous studies revealed that NF-B activation is necessary for the development of organization in the secondary lymphoid organs. It is known that TRAF6 induces activation of the canonical NF-B activation pathway and the MAPK cascades (13). In this study, we have shown that TRAF6 is not essential for the noncanonical NF-B activation induced by LtβR ligation. Recently, it was reported that FDC-specific deletion of IKKβ did not affect the architecture of the splenic white pulp (40). These data suggest that the signal downstream of TRAF6 required for the B cell follicle development may not be the canonical NF-B activation pathway. However, as the authors pointed out, it is possible that residual IKKβ due to a delay in the deletion of IKKβ compared with the timing at its requirement functions to develop B cell follicles in FDCs (40). The authors used transgenic mice in which Cre was under the regulation of the CD21/CR2 promoter. Previous analysis revealed that a considerable number of FDCs double-labeled with FDC-M1 and CR1/2 (35% of total FDC-M1-positive cells) are present at PN 10 (33).

At this time point, the effect of TRAF6 deficiency on B cell follicle development is already evident. Thus, in the study of FDC-specific IKKβ-deficient mice, IKKβ protein could persist in FDCs to the stage in which the TRAF6-dependent signal is required.

Our present study revealed that the TRAF6-dependent signal regulating B cell follicle and FDC development is independent of, but collaborates with, the Lt pathway. Further detailed studies of the TRAF6-dependent signaling pathway may clarify the molecular basis for the formation of the splenic microenvironment, which is essential for an efficient immune response. Such a fuller understanding may lead to exploitation of novel targeting molecules for controlling the immune response or immune system diseases.

	Acknowledgments

 
We thank F. W. Alt for RAG2-deficient mice, D. D. Chaplin for Lt-deficient mice, Dr. R. Whittier for critical reading of the manuscript, and other laboratory members for technical help and useful discussion.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants-in-Aid for Special Coordination Funds for Promoting Science and Technology (to J.-i.I.), a Grant in-Aid for Priority Area Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.A. and J.-i.I.) and by The Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T.A.).

² Address correspondence and reprint requests to Dr. Taishin Akiyama, Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan; E-mail address: taishin{at}ims.u-tokyo.ac.jp or Dr. Jun-ichiro Inoue, Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan; E-mail address: jun-i{at}ims.u-tokyo.ac.jp

³ Abbreviations used in this paper: Lt, lymphotoxin; LtβR, Lt β receptor; NIK, NF-B-inducing kinase; TRAF6, TNFR-associated factor 6; RANK, receptor activator of NF-B; RANKL, RANK ligand; TRANCE, TNF-related activation-induced cytokine; FDC, follicular dendritic cell; MAdCAM-1, mucosal addressin cell adhesion molecule-1; PN, postnatal; MEF, mouse embryonic fibroblast; MOMA, anti-monocytes/macrophases; JNK, Jun-N-terminal nucleotide kinase; ER-TR9, anti-mouse SIGN-R1 (clone ER-TR9).

Received for publication May 31, 2007. Accepted for publication September 6, 2007.

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