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TuJ1 (class III β-tubulin) expression suggests dynamic redistribution of follicular dendritic cells in lymphoid tissue
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European Journal of Cell Biology 84 (2005) 453–459 www.elsevier.de/ejcb
TuJ1 (class III b-tubulin) expression suggests dynamic redistribution of follicular dendritic cells in lymphoid tissue Seungkoo Leea, Kyungho Choia, Hanjong Ahnb, Kyuyoung Songc, Jongseon Choed, Inchul Leee, a
Asan Institute for Life Sciences, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea Department of Urology, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea c Department of Biochemistry and Molecular Biology, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea d Department of Microbiology and Immunology, Kangwon National University College of Medicine, Chunchon, Kangwon 200-701, Republic of Korea e Department of Pathology, University of Ulsan, College of Medicine, 388-1 Poongnap-Dong, Seoul 138-736, Republic of Korea b
Abstract Follicular dendritic cells (FDCs) play central roles in the B cell survival, proliferation, and differentiation into memory cells. Here, we show that TuJ1 (class III b-tubulin) is expressed strongly in FDCs of human lymphoid tissue. TuJ1 has been a marker of neurons in the central and peripheral nervous systems from the early stage of neural differentiation. FDCs expressed TuJ1 protein diffusely in both light and dark zones of germinal centers in all human lymphoid tissues. In contrast, CD21 expression was relatively concentrated to the light zone, suggesting that TuJ1 was a marker for FDCs with broader spectrum than CD21. In addition to the germinal center, there were single TuJ1expressing cells scattered in the mantle zone, blurring the border of the FDC network. In human tonsils, single scattered TuJ1-positive cells were also present in the crypt epithelium, suggesting a dynamic redistribution of FDCs among the antigen-rich epithelium, mantle zone, and germinal center. Such migration of FDCs could reflect a way of direct transport of various antigens carried on their surface to the germinal center, and a basis for the polarity of lymphoid follicles toward the epithelium in mucosa-associated lymphoid tissues. HK cells, cultured FDCs, also expressed TuJ1. The expression of TuJ1 by FDCs suggests that they may share certain biological characteristics of the neural system. r 2004 Elsevier GmbH. All rights reserved. Keywords: TuJ1; Follicular dendritic cell (FDC); HK cell; Germinal center; Polarity
Introduction Follicular dendritic cells (FDCs) are found in lymphoid follicles of lymph nodes, spleen, and tonsils (MacLennan, 1994; Cyster et al., 2000; van Nierop and de Groot, 2002; Li and Choi, 2002). They display elaborate cytoplasmic processes and form a fine reticular Corresponding author. Tel.: +82 2 3010 4551; fax: +82 2 472 7898.
E-mail address: [email protected] (I. Lee). 0171-9335/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2004.11.001
network (Klaus et al., 1980). Although FDCs constitute a minor cellular component within lymphoid tissue, they play central roles in B cell survival, proliferation, and differentiation into memory cells (MacLennan, 1994; Choe et al., 1997; Tew et al., 2001; van Nierop and de Groot, 2002; Li and Choi, 2002). FDCs are also engaged in cross-talks with T cells. FDCs express a receptor for IL-4, which is expressed by T cells in the germinal center (Butch et al., 1993). T cell stimulation results in an enhanced proliferation of FDC-like cells (Kim et al.,
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1994). FDCs mediate up-regulation of chemokine receptors on CD4 T cells (Estes et al., 2002). FDCs present native antigens in the form of immunecomplexes on their surface through Fc receptors, such as FcgRIIb (CD32) and FceRII (CD23), and complement receptors, such as CR2 (CD21) and CR1 (CD35) (reviewed in Kosco-Vilbois, 2003). Interactions between FDCs and germinal center B cells are facilitated by pairs of adhesion molecules; lymphocyte function-associated antigen 1 (LFA) with intercellular adhesion molecule 1 (ICAM1) and very late antigen 4 (VLA4) with vascular–cell adhesion molecule 1 (VCAM1) (Koopman et al., 1994). Recently, FDCs have been shown to be a portal of prion neuroinvasion (Prinz et al., 2003). FDCs produce B-lymphocyte chemoattractants (BLC) for B cell chemotaxis (Cyster et al., 2000). Many other antigens have been shown to be expressed in FDCs including CD44, acetylcholine esterase (Lampert and Van Noorden, 1996), epidermal growth factor receptor (Sun et al., 2003), CD137 (Lindstedt et al., 2003), estrogen receptor (Sapino et al., 2003), and FDCSP (Marshall et al., 2002). Numerous monoclonal antibodies have been developed for the characterization and purification of human FDCs (Naiem et al., 1983; Parwaresch et al., 1983; Farace et al., 1986; Butch et al., 1994; Raymond et al., 1997; Liu et al., 1997; Li et al., 2000; Lee et al., 2003; Bofill et al., 2000). TuJ1 (class III b-tubulin) is expressed in neurons of the central and peripheral nervous systems (Alexander et al., 1991; Katsetos et al., 1998), and has been used widely as a marker of neural differentiation (Jung et al., 2004; Shim et al., 2004; Chang et al., 2004; Williams et al., 2004). Here, we show that FDCs also express TuJ1 protein strongly. Possible biological implications are discussed.
antigen pre-absorbed antibody was applied. Monoclonal anti-TuJ1 (Covance, Richmond, CA), anti-CD21 (Novocastra, Newcastle, UK), anti-CD68 (DAKO, Glostrup, Denmark), anti-synaptophysin (DakoCytomation, Carpinteria, CA), anti-neurofilament (NF-L) (DakoCytomation), anti-S-100 protein (Zymed, South San Francisco, USA), anti-chromogranin A(DakoCytomation), anti-cytokeratin antibody AE1/AE3 (Zymed) were applied for 2 h at room temperature. Monoclonal antibody 3C8 (Lee et al., 2003) and affinitypurified rabbit anti-TRF2 antibody (Song et al., 2000) were described previously. After washing, the immunostaining was done using the avidin-biotinylated horseradish peroxidase complex (ABC) method. The ABC complex was developed by immersing slides in diaminobenzidine as chromogen and was counterstained with hematoxylin.
Materials and methods
Western blot
Tissue samples and antibodies
Whole-protein lysates from tonsils and HK cells were analyzed. HK cells were clarified once in cold PBS by centrifugation and solubilized for 30 min at 2 107 cells/ ml in ice-cold Nonidet P-40 lysis buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P-40) containing 1 mM PMSF, 10 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin A (Calbiochem, San Diego, CA). The lysate was subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was blocked for 30 min in 3% BSA in TTBS (Tris-buffered saline supplemented with 0.1% Tween 20), incubated for 1 h with anti-TuJ1 antibody, and thereafter washed three times for 10 min in TTBS. The membrane was subsequently incubated for 1 h with alkaline phosphatase-conjugated anti-mouse IgG1 in
Paraffin-embedded human lymphoid tissues were obtained from Autopsy and Surgical Pathology files at Asan Medical Center, Seoul, Korea. Fresh tonsil samples were obtained from tonsillectomy specimens, with the patients’ written consent after pathologic examination. Two FDC sarcomas were similarly examined. They were from a palatine tonsil of a 48-year-old woman and an intra-abdominal lymph node from a 52year-old woman, measuring 1.5 and 5.0 cm in diameter, respectively. For the immunohistochemical staining, sections of 4 mm thickness were prepared from paraffin blocks. Microwave antigen retrieval was applied in 0.01 M sodium citrate buffer (pH 6.0). For negative control,
HK cells and immunofluorescence staining HK cells were prepared and maintained as described previously (Kim et al., 1994; Lee et al., 2003). HK cells were grown in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FBS, 2 mM Lglutamine (Life Technologies), and 80 mg/ml gentamicin (SoloPak Laboratories, Elk Grove Village, IL). For immunofluorescence microscopy, HK cells grown on coverslips were fixed in cold methanol for 5 min. Anti-TuJ1 antibody was applied for 2 h at room temperature. As a control, normal mouse serum was used. FITC-conjugated anti-mouse IgG secondary antibody (Zymed, South San Francisco, CA) was applied as second antibodies for 30 min. After Washing with PBS 3 times, nuclei were stained with DAPI (Sigma, St. Louis, MO), and mounted. Cells were viewed using Olympus Vanox-S fluorescence microscope.
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3% BSA in TTBS and washed 5 times. The antibody binding was visualized using NBT/BCIP (Calbiochem) developing solution.
Results Upon the immunohistochemical screening, TuJ1 was extensively positive in neural tissues, as described
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previously (Katsetos et al., 1998; Jung et al., 2004). Unexpectedly, we found that germinal centers were immunostained strongly for TuJ1 in human lymphoid follicles of all lymphoid tissues. TuJ1 immunostaining was most prominent in the secondary follicles in both light and dark zones of germinal centers (Fig. 1a, b). The number of TuJ1-positive cells and the staining intensity varied considerably among the tissues and individual lymphoid follicles. The TuJ1-positive cells had large nuclei and dendritic processes, being compatible with
Fig. 1. (a) Typical secondary follicle in tonsil. Crypt epithelium (Ep), mantle zone (MZ), light zone (LZ), and dark zone (DZ) are designated (H & E staining, 50). (b) Diffuse TuJ1 immunostaining in the germinal center (ABC staining, 50). (c) On higher magnification, TuJ1-immunostaining is in the cytoplasm of large cells with dendritic configuration. Scattered single cells are also present in the mantle zone near the light zone blurring the border (arrows) ( 300). (d) Single TuJi-positive cells in the crypt epithelium (Ep), mantle zone (MZ) and germinal center (LZ) ( 120). (e). CD21 immunostaining of FDCs concentrated to the light zone (LZ) whereas the dark zone (DZ) is relatively spared. B cells in the mantle are also positive ( 50). The immunostaining is mostly on the plasma membrane. (f). Crypt epithelium shown by cytokeratin immunostaining (arrowheads) ( 50). (g) CD68immunostaining showing irregularly scattered macrophage/histiocytes ( 50). (h) S-100 protein-positive dendritic cells in the crypt epithelium (arrowheads) and interfollicular compartment (arrows), but not in the germinal center ( 50).
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Fig. 2. (a) HK cells consisting of FDCs with large ovoid nuclei (arrows) and reticular fibroblasts with slender nuclei (DAPI staining). (b) Fibrillar cytoplasmic staining for TuJ1 is found in FDCs by immunofluorescence microscopic analysis (microtubules; arrows).
FDCs (Fig. 1c, d). The immunostaining pattern partly overlapped with that of CD21, another marker of FDCs, which was mostly in the light zone of germinal center (Fig. 1e). The cytoplasmic staining pattern of TuJ1 was distinct from the cytoplasmic membranestaining pattern of CD21 (Fig. 1e). Neuroendocrine markers such as synaptophysin, chromogranin, or neurofilament were negative in the germinal center (data not shown). TRF2, which was recently found in neuroglial processes (Jung et al., 2004), was also negative. Intriguingly, FDC sarcomas were positive for CD21 but not for TuJ1 (data not shown). In tonsils, lymphoid follicles keep the polarity having the mantle zone toward the crypt epithelium (Fig. 1a). In addition to the germinal center, single TuJ1-expressing cells were also scattered in the mantle zone, rendering the border of the FDC network irregular or moth-eaten (Fig. 1c, d). On the other hand, the dark zone side of the FDC network was clearly outlined. Single TuJ1-positive cells were also scattered in the crypt epithelium occasionally (Fig. 1d). In the mantle zone and epithelium, they were either round or dendritic shape. Even in the latter, the cytoplasmic processes were not as prominent as the ones in the germinal center. Such distribution of TuJ1-positive cells strongly suggested a dynamic redistribution of FDCs among the antigen-rich epithelium, mantle zone, and germinal center (Fig. 1d). The number of single positive cells varied considerably among the tonsils. The distribution of TuJ1 cells was distinct from CD68-positive macrophage/histiocytes or S-100 proteinpositive dendritic cells, which could have a dendritic appearance similar to FDCs. The former distributed irregularly throughout the lymphoid tissue (Fig. 1g), and the latter localized in the tonsillar crypt epithelium and interfollicular area (Fig. 1f, h). The distribution of S-100-positive cells was compatible with their function of antigen-presentation to the T cell population, and mutually exclusive with that of TuJ1-positive cells in the lymphoid follicle. In the crypt epithelium, the S-100 protein-positive dendritic cells were much more scattered than TuJ1-positive cells, and increased considerably in the inflammatory environment.
Fig. 3. Western blots probed for TuJ1. lane 1: whole tonsil sample, lane 2: HK cells.
To confirm the TuJ1 expression by FDCs, we also analyzed the cultured FDC cells. HK cells had elongated cytoplasm and appeared to consist of two subpopulations according to the nuclear configuration (Fig. 2a, b): one with large nuclei and the other with small and slender nuclei with dense DNA staining, which were compatible with FDCs and reticular fibroblasts (reticulum cells), respectively (Cyster et al., 2000). By immunofluorescence microscopy, the large cells, but not the small ones, showed fibrillar staining for TuJ1 in the cytoplasm compatible with microtubules (Fig. 2b). Upon Western blotting, TuJ1 was detected as a major band at around 50 kDa in both tonsillar tissues and HK cells similarly (Fig. 3). It was the only immunoreactive band on the blot without any other cross-reactive minor bands.
Discussion We have shown that TuJ1 protein was expressed by FDCs in human lymphoid tissues and cultured HK cells. The expression of TuJ1 was partly overlapping with another FDC marker, CD21, in lymphoid follicles. The FDC network was diffusely immunostained in the light and dark zones altogether whereas the CD21 expression was relatively concentrated in the light zone
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of germinal centers. Thus, it was suggested that TuJ1 was a marker for FDCs with broader spectrum than CD21, and that TuJ1-positive FDCs consisted of CD21-positive and -negative subpopulations. Further studies are necessary for the biological analysis of FDC subpopulations. The biological significance of TuJ1 expression by FDCs is not clear. It has been suggested that dendritic configuration of FDCs is quite reminiscent of neurons with fine dendrites (Kosco-Vilbois, 2003). On the other hand, other neural markers such as synaptophysin, chromogranin, and neurofilament are negative, excluding FDCs from the neural system. We have observed that TuJ1 may be expressed in certain epithelial and connective tissue cells that may not be related to neural differentiation (manuscript in preparation). However, the possibility remains that FDCs may share certain biological characteristics with the neural system partly. The single scattered TuJ1-positive cells in the mantle suggest that a subpopulation of FDCs is undergoing dynamic redistribution. The fact that the FDC network abutting on the mantle zone is not clearly bordered favors this notion. It seems that the light zone border of germinal centers is ‘open (irregular)’ whereas the opposite side, the dark zone border, is ‘closed (linear)’ for the direct communication to the extra-follicular environment. The ‘migrating’ subpopulation may reflect active transportation of various antigens on their surface and/or new FDC recruitment to the germinal center. The presence of TuJ1-positive cells in the crypt epithelium seems to favor the former. It has been shown that the tonsillar crypt epithelium presents various antigens and plays a key role in the initiation of immune responses (reviewed by Nave et al., 2001). The origin of FDCs has been a controversial issue (MacLennan, 1994; Cyster et al., 2000; van Nierop and de Groot, 2002; Li and Choi, 2002), and is beyond the scope of this short report. FDCs are known to be radioresistant, stable cells that may not be easily replaced (Kinet-Denoel et al., 1982; Balogh et al., 2001). On the other hand, it was shown that they could be replaced partly by donor cells in bone marrow-transplanted animal models (Kapasi et al., 1998). The presence of ‘‘antigen transporting cells with dendritic configuration’’ has been suggested (Szakal et al., 1983). It was reported that certain mononuclear cells in lymph or blood were positive for anti-FDC monoclonal antibodies (Parwaresch et al., 1983; Kosco et al, 1992). It was also shown that FDCs moved toward nerves to ‘‘deliver’’ prion proteins (Prinz et al., 2003). Taken together, our data supports the concept that the FDC network is a dynamic structure that evolves depending on the active migration of FDCs carrying various antigens. Such redistribution of FDCs appears to be quite reminiscent of the shuttling of antigen-presenting cells (APCs) between the antigen-rich area and the T cell compart-
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ment: it could be one of the antigen sources necessary for the education of the B cell compartment. The TuJ1 expression pattern also points out an interesting but not-so-well-investigated aspect of lymphoid follicle development, i.e. the polarity. In mucosaassociated lymphoid tissues (MALT), the mantle zone of a follicle is directed to the major source of outside antigens, which is the specialized epithelium. Similarly, the follicles in the cortex of lymph nodes tend to be directed to the marginal sinus, which is the entry site of antigens. However, it has not been explained satisfactorily why and how such a pattern is developed and/or maintained. Our data strongly suggests that the migrating FDCs could not only transport the antigens to the germinal center but also control the ongoing dynamic restructuring of lymphoid follicles. In this process, B cells may also participate actively in collaboration with FDCs (Ansel et al., 2000). Further studies are necessary regarding the characterization of FDC subpopulations as well as dynamic redistribution and restructuring of lymphoid follicles.
Acknowledgements This work was supported by a National Research Laboratory Grant from the Ministry of Science and Technology (MOST) of Korea.
References Alexander, J.E., Hunt, D.F., Lee, M.K., Shabanowitz, J., Michel, H., Berlin, S.C., MacDonald, T.L., Sundberg, R.J., Rebhun, L.I., Frankfurter, A., 1991. Characterization of posttranslational modifications in neuron-specific class III beta-tubulin by mass spectrometry. Proc. Natl. Acad. Sci. USA 88, 4685–4689. Ansel, K.M., Ngo, V.N., Hyman, P.L., Luther, S.A., Forster, R., Sedgwick, J.D., Browning, J.L., Lipp, M., Cyster, J.G., 2000. A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 406, 309–314. Balogh, P., Aydar, Y., Tew, J.G., Szakal, A.K., 2001. Ontogeny of the follicular dendritic cell phenotype and function in the postnatal murine spleen. Cell. Immunol. 25, 45–53. Bofill, M., Akbar, A., Amlot, P., 2000. Follicular dendritic cells share a membrane-bound protein with fibroblasts. J. Pathol. 191, 217–226. Butch, A.W., Chung, G.-H., Hoffman, J.W., Nahm, M.H., 1993. Cytokine expression by germinal center cells. J. Immunol. 150, 39–47. Butch, A.W., Hug, B.A., Nahm, M.H., 1994. Properties of human follicular dendritic cells purified with HJ2, a new monoclonal antibody. Cell Immunol. 155, 27–41. Chang, C.W., Tsai, C.W., Wang, H.F., Tsai, H.C., Chen, H.Y., Tsai, T.F., Takahashi, H., Li, H.Y., Fann, M.J., Yang, C.W., Hayashizaki, Y., Saito, T., Liu, F.C., 2004.
ARTICLE IN PRESS 458
S. Lee et al. / European Journal of Cell Biology 84 (2005) 453–459
Identification of a developmentally regulated striatumenriched zinc-finger gene, Nolz-1, in the mammalian brain. Proc. Natl. Acad. Sci. USA 101, 2613–2618. Choe, J., Kim, H.S., Armitage, R.J., Choi, Y.S., 1997. The functional role of BCR stimulation and IL-4 in the generation of human memory B cells from germinal center B cells. J. Immunol. 159, 3757–3766. Cyster, J.G., Ansel, K.M., Reif, K., Ekland, E.H., Hyman, P.L., Tang, H.L., Luther, S.A., Ngo, V.N., 2000. Follicular stromal cells and lymphocyte homing to follicles. Immunol. Rev. 176, 181–193. Estes, J.D., Keele, B.F., Tenner-Racz, K., Racz, P., Redd, M.A., Thacker, T.C., Jiang, Y., Lloyd, M.J., Gartner, S., Burton, G.F., 2002. Follicular dendritic cell-mediated upregulation of CXCR4 expression on CD4 T cells and HIV pathogenesis. J. Immunol. 169, 2313–2322. Farace, F., Kieffer, N., Caillou, S., Vainchenker, W., Tursz, T., Dokhelar, M.C., 1986. A 93-kDa glycoprotein expressed on human cultured monocytes and dendritic reticulum cells defined by an anti-K562 monoclonal antibody. Eur. J. Immunol. 16, 1521–1526. Jung, Y., Lee, S., Bang, S., Kim, S., Choi, K., Lee, C., Lee, S.G., Kim, C.J., Song, K., Lee, I., 2004. TRF2 is in neuroglial cytoplasm and induces neurite-like processes. FEBS Lett. 557, 129–132. Kapasi, Z.F., Qin, D., Kerr, W.G., Kosco-Vilbois, M.H., Shultz, L.D., Tew, J.G., Szakal, A.K., 1998. Follicular dendritic cell (FDC) precursors in primary lymphoid tissues. J. Immunol. 160, 1078–1084. Katsetos, C.D., Karkavelas, G., Herman, M.M., Vinores, S.A., Provencio, J., Spano, A.J., Frankfurter, A., 1998. Class III beta-tubulin isotype (beta III) in the adrenal medulla: I. Localization in the developing human adrenal medulla. Anat. Rec. 250, 335–343. Kim, H.-S., Zhang, X., Choi, Y.S., 1994. Activation and proliferation of follicular dendritic cell-like cells by activated T lymphocytes. J. Immunol. 153, 2951–2961. Kinet-Denoel, C., Heinen, E., Radoux, D., Simar, L.J., 1982. Follicular dendritic cells in lymph nodes after X-irradiation. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. 42, 121–130. Klaus, G.G., Humphery, J.H., Kunkl, A., Dongworth, D.W., 1980. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53, 3–28. Koopman, G., Keehnen, R.M., Lindhout, E., Newman, W., Shimizu, Y., van Seventer, G.A., de Groot, C., Pals, S.T., 1994. Adhesion through the LFA-1 (CD11a/CD18)-ICAM1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells. J. Immunol. 152, 3760–3777. Kosco, M.H., Pflugfelder, E., Gray, D., 1992. Follicular dendritic cell-dependent adhesion and proliferation of B cells in vitro. J. Immunol. 148, 2331–2339. Kosco-Vilbois, M.H., 2003. Are follicular dendritic cells really good for nothing? Nat. Nat. Rev. Immunol. 3, 764–769. Lampert, I.A., Van Noorden, S., 1996. Acetyl cholinesterase is expressed in the follicular dendritic cells of germinal centres: differences between normal and neoplastic follicles. J. Pathol. 180, 169–174.
Lee, I.Y., Ha, K.S., Choe, J., 2003. 3C8 antigen is a novel protein expressed by human follicular dendritic cells. Biochem. Biophys. Res. Commun. 303, 624–630. Li, L., Choi, Y.S., 2002. Follicular dendritic cell-signaling molecules required for proliferation and differentiation of GC-B cells. Semin. Immunol. 14, 259–266. Li, L., Zhang, X., Kovacic, S., Long, A.J., Bourque, K., Wood, C.R., Choi, Y.S., 2000. Identification of a human follicular dendritic cell molecule that stimulates germinal center B cell growth. J. Exp. Med. 191, 1077–1083. Lindstedt, M., Johansson-Lindbom, B., Borrebaeck, C.A., 2003. Expression of CD137 (4-1BB) on human follicular dendritic cells. Scand. J. Immunol. 57, 305–310. Liu, Y.-J., Xu, J., de Bouteiller, O., Parham, C.L., Grouard, G., Djossou, O., de Saint-Vis, B., Lebecque, S., Banchereau, J., Moore, K.W., 1997. Follicular dendritic cells specifically express the long CR2/CD21 isoform. J. Exp. Med. 185, 165–170. MacLennan, I.C.M., 1994. Germinal centers. Annu. Rev. Immunol. 12, 117–139. Marshall, A.J., Du, Q., Draves, K.E., Shikishima, Y., Hay Glass, K.T., Clark, E.A., 2002. FDC-SP, a novel secreted protein expressed by follicular dendritic cells. J. Immunol. 169, 2381–2389. Naiem, M., Gerdes, J., Abdulaziz, Z., Stein, H., Mason, D.Y., 1983. Production of a monoclonal antibody reactive with human dendritic reticulum cells and its use in the immunohistological analysis of lymphoid tissue. J. Clin. Pathol. 36, 167–175. Nave, H., Gebert, A., Pabst, R., 2001. Morphology and immunology of the human palatine tonsil. Anat. Embryol. (Berl). 204, 367–373. Parwaresch, M.R., Radzun, H.J., Hansmann, M.-L., Peters, K.P., 1983. Monoclonal antibody Ki-M4 specifically recognizes human dendritic reticulum cells (follicular dendritic cells) and their possible precursor in blood. Blood 62, 585–590. Prinz, M., Heikenwalder, M., Junt, T., Schwarz, P., Glatzel, M., Heppner, F.L., Fu, Y.X., Lipp, M., Aguzzi, A., 2003. Positioning of follicular dendritic cells within the spleen controls prion neuroinvasion. Nature 425, 957–962. Raymond, I., Saati, T.A., Tkaczuk, J., Chittal, S., Delsol, G., 1997. CNA.42, a new monoclonal antibody directed against a fixative-resistant antigen of follicular dendritic reticulum cells. Am. J. Pathol. 151, 1577–1585. Sapino, A., Cassoni, P., Ferrero, E., Bongiovanni, M., Righi, L., Fortunati, N., Crafa, P., Chiarle, R., Bussolati, G., 2003. Estrogen receptor alpha is a novel marker expressed by follicular dendritic cells in lymph nodes and tumorassociated lymphoid infiltrates. Am. J. Pathol. 163, 1313–1320. Shim, J.W., Koh, H.C., Chang, M.Y., Roh, E., Choi, C.Y., Oh, Y.J., Son, H., Lee, Y.S., Studer, L., Lee, S.H., 2004. Enhanced in vitro midbrain dopamine neuron differentiation, dopaminergic function, neurite outgrowth, and 1methyl-4-phenylpyridium resistance in mouse embryonic stem cells overexpressing Bcl-XL. J. Neurosci. 24, 843–852. Song, K., Jung, D., Jung, Y., Lee, S.G., Lee, I., 2000. Interaction of human Ku70 and TRF2. FEBS Lett. 481, 81–85.
ARTICLE IN PRESS S. Lee et al. / European Journal of Cell Biology 84 (2005) 453–459
Sun, X., Chang, K.C., Abruzzo, L.V., Lai, R., Younes, A., Jones, D., 2003. Epidermal growth factor receptor expression in follicular dendritic cells: a shared feature of follicular dendritic cell sarcoma and Castleman’s disease. Hum. Pathol. 34, 835–840. Szakal, A.K., Holmes, K.L., Tew, J.G., 1983. Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. J. Immunol. 131, 1714–1727.
459
Tew, J.G., Wu, J., Fakher, M., Szakal, A.K., Qin, D., 2001. Follicular dendritic cells: beyond the necessity of T-cell help. Trends Immunol. 22, 361–367. van Nierop, K., de Groot, C., 2002. Human follicular dendritic cells: function, origin and development. Semin. Immunol. 14, 251–257. Williams, B.P., Milligan, C.J., Street, M., Hornby, F.M., Deuchars, J., Buckley, N.J., 2004. Transcription of the M1 muscarinic receptor gene in neurons and neuronal progenitors of the embryonic rat forebrain. J. Neurochem. 88, 70–77.
European Journal of Cell Biology 84 (2005) 453–459 www.elsevier.de/ejcb
TuJ1 (class III b-tubulin) expression suggests dynamic redistribution of follicular dendritic cells in lymphoid tissue Seungkoo Leea, Kyungho Choia, Hanjong Ahnb, Kyuyoung Songc, Jongseon Choed, Inchul Leee, a
Asan Institute for Life Sciences, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea Department of Urology, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea c Department of Biochemistry and Molecular Biology, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea d Department of Microbiology and Immunology, Kangwon National University College of Medicine, Chunchon, Kangwon 200-701, Republic of Korea e Department of Pathology, University of Ulsan, College of Medicine, 388-1 Poongnap-Dong, Seoul 138-736, Republic of Korea b
Abstract Follicular dendritic cells (FDCs) play central roles in the B cell survival, proliferation, and differentiation into memory cells. Here, we show that TuJ1 (class III b-tubulin) is expressed strongly in FDCs of human lymphoid tissue. TuJ1 has been a marker of neurons in the central and peripheral nervous systems from the early stage of neural differentiation. FDCs expressed TuJ1 protein diffusely in both light and dark zones of germinal centers in all human lymphoid tissues. In contrast, CD21 expression was relatively concentrated to the light zone, suggesting that TuJ1 was a marker for FDCs with broader spectrum than CD21. In addition to the germinal center, there were single TuJ1expressing cells scattered in the mantle zone, blurring the border of the FDC network. In human tonsils, single scattered TuJ1-positive cells were also present in the crypt epithelium, suggesting a dynamic redistribution of FDCs among the antigen-rich epithelium, mantle zone, and germinal center. Such migration of FDCs could reflect a way of direct transport of various antigens carried on their surface to the germinal center, and a basis for the polarity of lymphoid follicles toward the epithelium in mucosa-associated lymphoid tissues. HK cells, cultured FDCs, also expressed TuJ1. The expression of TuJ1 by FDCs suggests that they may share certain biological characteristics of the neural system. r 2004 Elsevier GmbH. All rights reserved. Keywords: TuJ1; Follicular dendritic cell (FDC); HK cell; Germinal center; Polarity
Introduction Follicular dendritic cells (FDCs) are found in lymphoid follicles of lymph nodes, spleen, and tonsils (MacLennan, 1994; Cyster et al., 2000; van Nierop and de Groot, 2002; Li and Choi, 2002). They display elaborate cytoplasmic processes and form a fine reticular Corresponding author. Tel.: +82 2 3010 4551; fax: +82 2 472 7898.
E-mail address: [email protected] (I. Lee). 0171-9335/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2004.11.001
network (Klaus et al., 1980). Although FDCs constitute a minor cellular component within lymphoid tissue, they play central roles in B cell survival, proliferation, and differentiation into memory cells (MacLennan, 1994; Choe et al., 1997; Tew et al., 2001; van Nierop and de Groot, 2002; Li and Choi, 2002). FDCs are also engaged in cross-talks with T cells. FDCs express a receptor for IL-4, which is expressed by T cells in the germinal center (Butch et al., 1993). T cell stimulation results in an enhanced proliferation of FDC-like cells (Kim et al.,
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1994). FDCs mediate up-regulation of chemokine receptors on CD4 T cells (Estes et al., 2002). FDCs present native antigens in the form of immunecomplexes on their surface through Fc receptors, such as FcgRIIb (CD32) and FceRII (CD23), and complement receptors, such as CR2 (CD21) and CR1 (CD35) (reviewed in Kosco-Vilbois, 2003). Interactions between FDCs and germinal center B cells are facilitated by pairs of adhesion molecules; lymphocyte function-associated antigen 1 (LFA) with intercellular adhesion molecule 1 (ICAM1) and very late antigen 4 (VLA4) with vascular–cell adhesion molecule 1 (VCAM1) (Koopman et al., 1994). Recently, FDCs have been shown to be a portal of prion neuroinvasion (Prinz et al., 2003). FDCs produce B-lymphocyte chemoattractants (BLC) for B cell chemotaxis (Cyster et al., 2000). Many other antigens have been shown to be expressed in FDCs including CD44, acetylcholine esterase (Lampert and Van Noorden, 1996), epidermal growth factor receptor (Sun et al., 2003), CD137 (Lindstedt et al., 2003), estrogen receptor (Sapino et al., 2003), and FDCSP (Marshall et al., 2002). Numerous monoclonal antibodies have been developed for the characterization and purification of human FDCs (Naiem et al., 1983; Parwaresch et al., 1983; Farace et al., 1986; Butch et al., 1994; Raymond et al., 1997; Liu et al., 1997; Li et al., 2000; Lee et al., 2003; Bofill et al., 2000). TuJ1 (class III b-tubulin) is expressed in neurons of the central and peripheral nervous systems (Alexander et al., 1991; Katsetos et al., 1998), and has been used widely as a marker of neural differentiation (Jung et al., 2004; Shim et al., 2004; Chang et al., 2004; Williams et al., 2004). Here, we show that FDCs also express TuJ1 protein strongly. Possible biological implications are discussed.
antigen pre-absorbed antibody was applied. Monoclonal anti-TuJ1 (Covance, Richmond, CA), anti-CD21 (Novocastra, Newcastle, UK), anti-CD68 (DAKO, Glostrup, Denmark), anti-synaptophysin (DakoCytomation, Carpinteria, CA), anti-neurofilament (NF-L) (DakoCytomation), anti-S-100 protein (Zymed, South San Francisco, USA), anti-chromogranin A(DakoCytomation), anti-cytokeratin antibody AE1/AE3 (Zymed) were applied for 2 h at room temperature. Monoclonal antibody 3C8 (Lee et al., 2003) and affinitypurified rabbit anti-TRF2 antibody (Song et al., 2000) were described previously. After washing, the immunostaining was done using the avidin-biotinylated horseradish peroxidase complex (ABC) method. The ABC complex was developed by immersing slides in diaminobenzidine as chromogen and was counterstained with hematoxylin.
Materials and methods
Western blot
Tissue samples and antibodies
Whole-protein lysates from tonsils and HK cells were analyzed. HK cells were clarified once in cold PBS by centrifugation and solubilized for 30 min at 2 107 cells/ ml in ice-cold Nonidet P-40 lysis buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P-40) containing 1 mM PMSF, 10 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin A (Calbiochem, San Diego, CA). The lysate was subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was blocked for 30 min in 3% BSA in TTBS (Tris-buffered saline supplemented with 0.1% Tween 20), incubated for 1 h with anti-TuJ1 antibody, and thereafter washed three times for 10 min in TTBS. The membrane was subsequently incubated for 1 h with alkaline phosphatase-conjugated anti-mouse IgG1 in
Paraffin-embedded human lymphoid tissues were obtained from Autopsy and Surgical Pathology files at Asan Medical Center, Seoul, Korea. Fresh tonsil samples were obtained from tonsillectomy specimens, with the patients’ written consent after pathologic examination. Two FDC sarcomas were similarly examined. They were from a palatine tonsil of a 48-year-old woman and an intra-abdominal lymph node from a 52year-old woman, measuring 1.5 and 5.0 cm in diameter, respectively. For the immunohistochemical staining, sections of 4 mm thickness were prepared from paraffin blocks. Microwave antigen retrieval was applied in 0.01 M sodium citrate buffer (pH 6.0). For negative control,
HK cells and immunofluorescence staining HK cells were prepared and maintained as described previously (Kim et al., 1994; Lee et al., 2003). HK cells were grown in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FBS, 2 mM Lglutamine (Life Technologies), and 80 mg/ml gentamicin (SoloPak Laboratories, Elk Grove Village, IL). For immunofluorescence microscopy, HK cells grown on coverslips were fixed in cold methanol for 5 min. Anti-TuJ1 antibody was applied for 2 h at room temperature. As a control, normal mouse serum was used. FITC-conjugated anti-mouse IgG secondary antibody (Zymed, South San Francisco, CA) was applied as second antibodies for 30 min. After Washing with PBS 3 times, nuclei were stained with DAPI (Sigma, St. Louis, MO), and mounted. Cells were viewed using Olympus Vanox-S fluorescence microscope.
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3% BSA in TTBS and washed 5 times. The antibody binding was visualized using NBT/BCIP (Calbiochem) developing solution.
Results Upon the immunohistochemical screening, TuJ1 was extensively positive in neural tissues, as described
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previously (Katsetos et al., 1998; Jung et al., 2004). Unexpectedly, we found that germinal centers were immunostained strongly for TuJ1 in human lymphoid follicles of all lymphoid tissues. TuJ1 immunostaining was most prominent in the secondary follicles in both light and dark zones of germinal centers (Fig. 1a, b). The number of TuJ1-positive cells and the staining intensity varied considerably among the tissues and individual lymphoid follicles. The TuJ1-positive cells had large nuclei and dendritic processes, being compatible with
Fig. 1. (a) Typical secondary follicle in tonsil. Crypt epithelium (Ep), mantle zone (MZ), light zone (LZ), and dark zone (DZ) are designated (H & E staining, 50). (b) Diffuse TuJ1 immunostaining in the germinal center (ABC staining, 50). (c) On higher magnification, TuJ1-immunostaining is in the cytoplasm of large cells with dendritic configuration. Scattered single cells are also present in the mantle zone near the light zone blurring the border (arrows) ( 300). (d) Single TuJi-positive cells in the crypt epithelium (Ep), mantle zone (MZ) and germinal center (LZ) ( 120). (e). CD21 immunostaining of FDCs concentrated to the light zone (LZ) whereas the dark zone (DZ) is relatively spared. B cells in the mantle are also positive ( 50). The immunostaining is mostly on the plasma membrane. (f). Crypt epithelium shown by cytokeratin immunostaining (arrowheads) ( 50). (g) CD68immunostaining showing irregularly scattered macrophage/histiocytes ( 50). (h) S-100 protein-positive dendritic cells in the crypt epithelium (arrowheads) and interfollicular compartment (arrows), but not in the germinal center ( 50).
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Fig. 2. (a) HK cells consisting of FDCs with large ovoid nuclei (arrows) and reticular fibroblasts with slender nuclei (DAPI staining). (b) Fibrillar cytoplasmic staining for TuJ1 is found in FDCs by immunofluorescence microscopic analysis (microtubules; arrows).
FDCs (Fig. 1c, d). The immunostaining pattern partly overlapped with that of CD21, another marker of FDCs, which was mostly in the light zone of germinal center (Fig. 1e). The cytoplasmic staining pattern of TuJ1 was distinct from the cytoplasmic membranestaining pattern of CD21 (Fig. 1e). Neuroendocrine markers such as synaptophysin, chromogranin, or neurofilament were negative in the germinal center (data not shown). TRF2, which was recently found in neuroglial processes (Jung et al., 2004), was also negative. Intriguingly, FDC sarcomas were positive for CD21 but not for TuJ1 (data not shown). In tonsils, lymphoid follicles keep the polarity having the mantle zone toward the crypt epithelium (Fig. 1a). In addition to the germinal center, single TuJ1-expressing cells were also scattered in the mantle zone, rendering the border of the FDC network irregular or moth-eaten (Fig. 1c, d). On the other hand, the dark zone side of the FDC network was clearly outlined. Single TuJ1-positive cells were also scattered in the crypt epithelium occasionally (Fig. 1d). In the mantle zone and epithelium, they were either round or dendritic shape. Even in the latter, the cytoplasmic processes were not as prominent as the ones in the germinal center. Such distribution of TuJ1-positive cells strongly suggested a dynamic redistribution of FDCs among the antigen-rich epithelium, mantle zone, and germinal center (Fig. 1d). The number of single positive cells varied considerably among the tonsils. The distribution of TuJ1 cells was distinct from CD68-positive macrophage/histiocytes or S-100 proteinpositive dendritic cells, which could have a dendritic appearance similar to FDCs. The former distributed irregularly throughout the lymphoid tissue (Fig. 1g), and the latter localized in the tonsillar crypt epithelium and interfollicular area (Fig. 1f, h). The distribution of S-100-positive cells was compatible with their function of antigen-presentation to the T cell population, and mutually exclusive with that of TuJ1-positive cells in the lymphoid follicle. In the crypt epithelium, the S-100 protein-positive dendritic cells were much more scattered than TuJ1-positive cells, and increased considerably in the inflammatory environment.
Fig. 3. Western blots probed for TuJ1. lane 1: whole tonsil sample, lane 2: HK cells.
To confirm the TuJ1 expression by FDCs, we also analyzed the cultured FDC cells. HK cells had elongated cytoplasm and appeared to consist of two subpopulations according to the nuclear configuration (Fig. 2a, b): one with large nuclei and the other with small and slender nuclei with dense DNA staining, which were compatible with FDCs and reticular fibroblasts (reticulum cells), respectively (Cyster et al., 2000). By immunofluorescence microscopy, the large cells, but not the small ones, showed fibrillar staining for TuJ1 in the cytoplasm compatible with microtubules (Fig. 2b). Upon Western blotting, TuJ1 was detected as a major band at around 50 kDa in both tonsillar tissues and HK cells similarly (Fig. 3). It was the only immunoreactive band on the blot without any other cross-reactive minor bands.
Discussion We have shown that TuJ1 protein was expressed by FDCs in human lymphoid tissues and cultured HK cells. The expression of TuJ1 was partly overlapping with another FDC marker, CD21, in lymphoid follicles. The FDC network was diffusely immunostained in the light and dark zones altogether whereas the CD21 expression was relatively concentrated in the light zone
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of germinal centers. Thus, it was suggested that TuJ1 was a marker for FDCs with broader spectrum than CD21, and that TuJ1-positive FDCs consisted of CD21-positive and -negative subpopulations. Further studies are necessary for the biological analysis of FDC subpopulations. The biological significance of TuJ1 expression by FDCs is not clear. It has been suggested that dendritic configuration of FDCs is quite reminiscent of neurons with fine dendrites (Kosco-Vilbois, 2003). On the other hand, other neural markers such as synaptophysin, chromogranin, and neurofilament are negative, excluding FDCs from the neural system. We have observed that TuJ1 may be expressed in certain epithelial and connective tissue cells that may not be related to neural differentiation (manuscript in preparation). However, the possibility remains that FDCs may share certain biological characteristics with the neural system partly. The single scattered TuJ1-positive cells in the mantle suggest that a subpopulation of FDCs is undergoing dynamic redistribution. The fact that the FDC network abutting on the mantle zone is not clearly bordered favors this notion. It seems that the light zone border of germinal centers is ‘open (irregular)’ whereas the opposite side, the dark zone border, is ‘closed (linear)’ for the direct communication to the extra-follicular environment. The ‘migrating’ subpopulation may reflect active transportation of various antigens on their surface and/or new FDC recruitment to the germinal center. The presence of TuJ1-positive cells in the crypt epithelium seems to favor the former. It has been shown that the tonsillar crypt epithelium presents various antigens and plays a key role in the initiation of immune responses (reviewed by Nave et al., 2001). The origin of FDCs has been a controversial issue (MacLennan, 1994; Cyster et al., 2000; van Nierop and de Groot, 2002; Li and Choi, 2002), and is beyond the scope of this short report. FDCs are known to be radioresistant, stable cells that may not be easily replaced (Kinet-Denoel et al., 1982; Balogh et al., 2001). On the other hand, it was shown that they could be replaced partly by donor cells in bone marrow-transplanted animal models (Kapasi et al., 1998). The presence of ‘‘antigen transporting cells with dendritic configuration’’ has been suggested (Szakal et al., 1983). It was reported that certain mononuclear cells in lymph or blood were positive for anti-FDC monoclonal antibodies (Parwaresch et al., 1983; Kosco et al, 1992). It was also shown that FDCs moved toward nerves to ‘‘deliver’’ prion proteins (Prinz et al., 2003). Taken together, our data supports the concept that the FDC network is a dynamic structure that evolves depending on the active migration of FDCs carrying various antigens. Such redistribution of FDCs appears to be quite reminiscent of the shuttling of antigen-presenting cells (APCs) between the antigen-rich area and the T cell compart-
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ment: it could be one of the antigen sources necessary for the education of the B cell compartment. The TuJ1 expression pattern also points out an interesting but not-so-well-investigated aspect of lymphoid follicle development, i.e. the polarity. In mucosaassociated lymphoid tissues (MALT), the mantle zone of a follicle is directed to the major source of outside antigens, which is the specialized epithelium. Similarly, the follicles in the cortex of lymph nodes tend to be directed to the marginal sinus, which is the entry site of antigens. However, it has not been explained satisfactorily why and how such a pattern is developed and/or maintained. Our data strongly suggests that the migrating FDCs could not only transport the antigens to the germinal center but also control the ongoing dynamic restructuring of lymphoid follicles. In this process, B cells may also participate actively in collaboration with FDCs (Ansel et al., 2000). Further studies are necessary regarding the characterization of FDC subpopulations as well as dynamic redistribution and restructuring of lymphoid follicles.
Acknowledgements This work was supported by a National Research Laboratory Grant from the Ministry of Science and Technology (MOST) of Korea.
References Alexander, J.E., Hunt, D.F., Lee, M.K., Shabanowitz, J., Michel, H., Berlin, S.C., MacDonald, T.L., Sundberg, R.J., Rebhun, L.I., Frankfurter, A., 1991. Characterization of posttranslational modifications in neuron-specific class III beta-tubulin by mass spectrometry. Proc. Natl. Acad. Sci. USA 88, 4685–4689. Ansel, K.M., Ngo, V.N., Hyman, P.L., Luther, S.A., Forster, R., Sedgwick, J.D., Browning, J.L., Lipp, M., Cyster, J.G., 2000. A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 406, 309–314. Balogh, P., Aydar, Y., Tew, J.G., Szakal, A.K., 2001. Ontogeny of the follicular dendritic cell phenotype and function in the postnatal murine spleen. Cell. Immunol. 25, 45–53. Bofill, M., Akbar, A., Amlot, P., 2000. Follicular dendritic cells share a membrane-bound protein with fibroblasts. J. Pathol. 191, 217–226. Butch, A.W., Chung, G.-H., Hoffman, J.W., Nahm, M.H., 1993. Cytokine expression by germinal center cells. J. Immunol. 150, 39–47. Butch, A.W., Hug, B.A., Nahm, M.H., 1994. Properties of human follicular dendritic cells purified with HJ2, a new monoclonal antibody. Cell Immunol. 155, 27–41. Chang, C.W., Tsai, C.W., Wang, H.F., Tsai, H.C., Chen, H.Y., Tsai, T.F., Takahashi, H., Li, H.Y., Fann, M.J., Yang, C.W., Hayashizaki, Y., Saito, T., Liu, F.C., 2004.
ARTICLE IN PRESS 458
S. Lee et al. / European Journal of Cell Biology 84 (2005) 453–459
Identification of a developmentally regulated striatumenriched zinc-finger gene, Nolz-1, in the mammalian brain. Proc. Natl. Acad. Sci. USA 101, 2613–2618. Choe, J., Kim, H.S., Armitage, R.J., Choi, Y.S., 1997. The functional role of BCR stimulation and IL-4 in the generation of human memory B cells from germinal center B cells. J. Immunol. 159, 3757–3766. Cyster, J.G., Ansel, K.M., Reif, K., Ekland, E.H., Hyman, P.L., Tang, H.L., Luther, S.A., Ngo, V.N., 2000. Follicular stromal cells and lymphocyte homing to follicles. Immunol. Rev. 176, 181–193. Estes, J.D., Keele, B.F., Tenner-Racz, K., Racz, P., Redd, M.A., Thacker, T.C., Jiang, Y., Lloyd, M.J., Gartner, S., Burton, G.F., 2002. Follicular dendritic cell-mediated upregulation of CXCR4 expression on CD4 T cells and HIV pathogenesis. J. Immunol. 169, 2313–2322. Farace, F., Kieffer, N., Caillou, S., Vainchenker, W., Tursz, T., Dokhelar, M.C., 1986. A 93-kDa glycoprotein expressed on human cultured monocytes and dendritic reticulum cells defined by an anti-K562 monoclonal antibody. Eur. J. Immunol. 16, 1521–1526. Jung, Y., Lee, S., Bang, S., Kim, S., Choi, K., Lee, C., Lee, S.G., Kim, C.J., Song, K., Lee, I., 2004. TRF2 is in neuroglial cytoplasm and induces neurite-like processes. FEBS Lett. 557, 129–132. Kapasi, Z.F., Qin, D., Kerr, W.G., Kosco-Vilbois, M.H., Shultz, L.D., Tew, J.G., Szakal, A.K., 1998. Follicular dendritic cell (FDC) precursors in primary lymphoid tissues. J. Immunol. 160, 1078–1084. Katsetos, C.D., Karkavelas, G., Herman, M.M., Vinores, S.A., Provencio, J., Spano, A.J., Frankfurter, A., 1998. Class III beta-tubulin isotype (beta III) in the adrenal medulla: I. Localization in the developing human adrenal medulla. Anat. Rec. 250, 335–343. Kim, H.-S., Zhang, X., Choi, Y.S., 1994. Activation and proliferation of follicular dendritic cell-like cells by activated T lymphocytes. J. Immunol. 153, 2951–2961. Kinet-Denoel, C., Heinen, E., Radoux, D., Simar, L.J., 1982. Follicular dendritic cells in lymph nodes after X-irradiation. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. 42, 121–130. Klaus, G.G., Humphery, J.H., Kunkl, A., Dongworth, D.W., 1980. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53, 3–28. Koopman, G., Keehnen, R.M., Lindhout, E., Newman, W., Shimizu, Y., van Seventer, G.A., de Groot, C., Pals, S.T., 1994. Adhesion through the LFA-1 (CD11a/CD18)-ICAM1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells. J. Immunol. 152, 3760–3777. Kosco, M.H., Pflugfelder, E., Gray, D., 1992. Follicular dendritic cell-dependent adhesion and proliferation of B cells in vitro. J. Immunol. 148, 2331–2339. Kosco-Vilbois, M.H., 2003. Are follicular dendritic cells really good for nothing? Nat. Nat. Rev. Immunol. 3, 764–769. Lampert, I.A., Van Noorden, S., 1996. Acetyl cholinesterase is expressed in the follicular dendritic cells of germinal centres: differences between normal and neoplastic follicles. J. Pathol. 180, 169–174.
Lee, I.Y., Ha, K.S., Choe, J., 2003. 3C8 antigen is a novel protein expressed by human follicular dendritic cells. Biochem. Biophys. Res. Commun. 303, 624–630. Li, L., Choi, Y.S., 2002. Follicular dendritic cell-signaling molecules required for proliferation and differentiation of GC-B cells. Semin. Immunol. 14, 259–266. Li, L., Zhang, X., Kovacic, S., Long, A.J., Bourque, K., Wood, C.R., Choi, Y.S., 2000. Identification of a human follicular dendritic cell molecule that stimulates germinal center B cell growth. J. Exp. Med. 191, 1077–1083. Lindstedt, M., Johansson-Lindbom, B., Borrebaeck, C.A., 2003. Expression of CD137 (4-1BB) on human follicular dendritic cells. Scand. J. Immunol. 57, 305–310. Liu, Y.-J., Xu, J., de Bouteiller, O., Parham, C.L., Grouard, G., Djossou, O., de Saint-Vis, B., Lebecque, S., Banchereau, J., Moore, K.W., 1997. Follicular dendritic cells specifically express the long CR2/CD21 isoform. J. Exp. Med. 185, 165–170. MacLennan, I.C.M., 1994. Germinal centers. Annu. Rev. Immunol. 12, 117–139. Marshall, A.J., Du, Q., Draves, K.E., Shikishima, Y., Hay Glass, K.T., Clark, E.A., 2002. FDC-SP, a novel secreted protein expressed by follicular dendritic cells. J. Immunol. 169, 2381–2389. Naiem, M., Gerdes, J., Abdulaziz, Z., Stein, H., Mason, D.Y., 1983. Production of a monoclonal antibody reactive with human dendritic reticulum cells and its use in the immunohistological analysis of lymphoid tissue. J. Clin. Pathol. 36, 167–175. Nave, H., Gebert, A., Pabst, R., 2001. Morphology and immunology of the human palatine tonsil. Anat. Embryol. (Berl). 204, 367–373. Parwaresch, M.R., Radzun, H.J., Hansmann, M.-L., Peters, K.P., 1983. Monoclonal antibody Ki-M4 specifically recognizes human dendritic reticulum cells (follicular dendritic cells) and their possible precursor in blood. Blood 62, 585–590. Prinz, M., Heikenwalder, M., Junt, T., Schwarz, P., Glatzel, M., Heppner, F.L., Fu, Y.X., Lipp, M., Aguzzi, A., 2003. Positioning of follicular dendritic cells within the spleen controls prion neuroinvasion. Nature 425, 957–962. Raymond, I., Saati, T.A., Tkaczuk, J., Chittal, S., Delsol, G., 1997. CNA.42, a new monoclonal antibody directed against a fixative-resistant antigen of follicular dendritic reticulum cells. Am. J. Pathol. 151, 1577–1585. Sapino, A., Cassoni, P., Ferrero, E., Bongiovanni, M., Righi, L., Fortunati, N., Crafa, P., Chiarle, R., Bussolati, G., 2003. Estrogen receptor alpha is a novel marker expressed by follicular dendritic cells in lymph nodes and tumorassociated lymphoid infiltrates. Am. J. Pathol. 163, 1313–1320. Shim, J.W., Koh, H.C., Chang, M.Y., Roh, E., Choi, C.Y., Oh, Y.J., Son, H., Lee, Y.S., Studer, L., Lee, S.H., 2004. Enhanced in vitro midbrain dopamine neuron differentiation, dopaminergic function, neurite outgrowth, and 1methyl-4-phenylpyridium resistance in mouse embryonic stem cells overexpressing Bcl-XL. J. Neurosci. 24, 843–852. Song, K., Jung, D., Jung, Y., Lee, S.G., Lee, I., 2000. Interaction of human Ku70 and TRF2. FEBS Lett. 481, 81–85.
ARTICLE IN PRESS S. Lee et al. / European Journal of Cell Biology 84 (2005) 453–459
Sun, X., Chang, K.C., Abruzzo, L.V., Lai, R., Younes, A., Jones, D., 2003. Epidermal growth factor receptor expression in follicular dendritic cells: a shared feature of follicular dendritic cell sarcoma and Castleman’s disease. Hum. Pathol. 34, 835–840. Szakal, A.K., Holmes, K.L., Tew, J.G., 1983. Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. J. Immunol. 131, 1714–1727.
459
Tew, J.G., Wu, J., Fakher, M., Szakal, A.K., Qin, D., 2001. Follicular dendritic cells: beyond the necessity of T-cell help. Trends Immunol. 22, 361–367. van Nierop, K., de Groot, C., 2002. Human follicular dendritic cells: function, origin and development. Semin. Immunol. 14, 251–257. Williams, B.P., Milligan, C.J., Street, M., Hornby, F.M., Deuchars, J., Buckley, N.J., 2004. Transcription of the M1 muscarinic receptor gene in neurons and neuronal progenitors of the embryonic rat forebrain. J. Neurochem. 88, 70–77.