Role of TNF ligand and receptor family in the lymphoid organogenesis defined by gene targeting

Role of TNF ligand and receptor family in the lymphoid organogenesis defined by gene targeting

Mitsuru Matsumoto

Division of Informative Cytology, Institute for Enzyme Research, University of Tokushima, Tokushima, Japan

Abstract:The molecular basis of lymphoid organogenesis has recently been elucidated using gene-targeted mice. Mice deficient in lymphotoxin-α (LTα) lack lymph nodes and Peyer's patches. The action of LTα in lymphoid organogenesis is mediated mostly by the membrane form of LT by a mechanism independent of TNF receptor I (TNFR-I) or II (TNFR-II). Additionally, follicular dendritic cell (FDC) clusters or germinal centers fail to develop in the spleen of LTα-deficient mice. Mice deficient in either TNFR-I or LTβR also fail to develop splenic FDC clusters and germinal centers, indicating that signaling through both TNFR-I and LTβR is required for the development of these structures. The mechanisms underlying the defective lymphoid organogenesis in LTα-deficient mice, together with a natural mutant strain, alymphoplasia (aly) mice, which manifest a quite similar phenotype to LTα-deficient mice, were investigated by generating aggregation chimeras. These studies demonstrate that LTα and the aly gene product together control lymphoid organogenesis with a close mechanistic relationship in their biochemical pathways through governing distinct cellular compartments;the former acting as a circulating ligand and the latter as a LTβR-signaling molecule expressed by the stroma of the lymphoid organs. J. Med. Invest. 46:141-150, 1999

Keywords:lymphotoxin, TNF, lymph node, spleen, knockout mice

INTRODUCTION
Secondary lymphoid organs such as spleen, lymph node (LN) and Peyer's patches (PPs) are the sites where immune cells interact with each other, and a strong immune response against foreign antigens is initiated. Lymphocytes continuously migrate from the blood to secondary lymphoid organs. The mechanisms for lymphocyte migration have been well documented;homing to LN and PP occurs through the interaction between adhesion molecules expressed on high endothelial venules (HEVs) of the lymphoid tissues and their counter-receptors on lymphocytes (1, 2). Lymphocytes begin to populate LN from the second day after birth, and lympho-cyte diapedesis through the HEV becomes readily apparent from day4 (3). While much is known of the molecular basis for the lymphocyte migration to the developed lymphoid organs, the mechanisms behind the incipient lymphoid organogenesis have been enigmatic. In other word, the molecular basis for the ontogenic development of lymphoid organs is not well understood.
Recent studies with gene-targeted mice manifest-ing abnormal development of the lymphoid organs have provided new insight into these undefined processes (summarized in Table 1). Mice deficient in lymphotoxin-α (LTα) (previously known as TNFβ) were born with a systemic absence of LN and PP(4, 5) with disturbed spleen architecture (6-9), which will be discussed in detail in this article. Cytokine related genes other than LT have been also recog-nized to be involved in the lymphoid organogenesis;mice deficient in either IL-2Rγ chain (10), IL-7Rα or Jak3 (11) have defective PP development. Chemokine receptor CXCR5 (also known as BLR1) has been also demonstrated to be essential for the development of inguinal LN and PP as well as for the formation of B cell follicles in the spleen (12). Recently, it was also demonstrated that mice defi-cient in osteoprotegerin ligand (OPGL) have defec-tive LN genesis (13). In addition, targeted deletion of transcription factors has caused abnormal devel-opment of lymphoid organs and/or lymphoid cells;Hox11-deficient mice lack spleen (14), Id2-deficient mice lack LN and PP (15), and Ikaros-deficient mice lack cells of all lymphoid lineages, LN and PP (16). Mice deficient in both NF-κB1 (p50) and NF-κB2(p52) also lack LN with a disorganized spleen archi-tecture (17). Thus, a broader spectrum of factors than had been anticipated influence the lymphoid organogenesis. Because secondary lymphoid or-gans are essential for the development of immune response, elucidating the genes which control lymphoid organogenesis is critical for a better understanding of the mechanisms for host defence. In this article, I will discuss how the lymphoid organogenesis is controlled at the molecular level focusing on the biology of the LT and tumor necrosis factor (TNF).

I. Ligand and receptor interactions of LT and TNF;a historical perspective
Because both the LTα and the TNF homotrimers were recognized to bind with similar affinity and to activate in similar fashion both of the defined TNF receptors (p55 or TNFR-I, and p75 or TNFR-II)(18), LTα and TNF were for many years thought to be redundant in vivo, differing only in their cel-lular sources and perhaps the signals that induced their expression. Studies by Browning and Ware have, however, demonstrated that LT can exist in two forms, the initially characterized secreted homotrimeric form and a membrane heterotrimer form which in its most prevalent form consists of one LTα subunit associated with two copies of the type II transmembrane protein LTβ (19). The LTβ gene is located adjacent to the TNF locus within the HLA complex (19, 20). The membrane LT protein (LTα1LTβ2) is not a ligand for TNFR-I or TNFR-II, but interacts with another member of the TNF receptor family designated the LTβ receptor (LTβR) (21). The cytoplasmic domain of the LTβR shares little homology with the cytoplasmic domains of TNFR-I or TNFR-II, suggesting that activation of the LTβR should induce cellular responses distinct from those mediated by the defined TNF receptors. These observations suggest that LTα acts to signal two distinct sets of cellular responses, one set deter-mined by its homotrimeric form binding to TNFR-I and/or TNFR-II and one set determined by its membrane form binding to the LTβR or related receptors (20). In this model, we expect that many of the functions of LT in vivo do not overlap with those of TNF.
In addition, a new TNF ligand and receptor family members closely related to LT and TNF have been recently identified. Herpes simplex virus (HSV) 1and 2 infect activated T lymphocytes by attachment of the HSV envelope glycoprotein D to the cellular herpesvirus entry mediator (HVEM) (22). It was shown that HVEM binds two cellular ligands, LTα homotrimer (LTα3) and LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes);LIGHT is a29-kD type II transmembrane protein produced by activated T cells. LIGHT also binds to LTβR as the membrane form of LT does (23). These ligand and receptor interactions are illustrated in Fig. 1.

II. LT as an essential factor for the lymphoid organogenesis
LTα, originally discovered as a pro-inflammatory molecule (24), has turned out to be an essential factor that controls the genesis of the secondary lymphoid organs as well as for the organized spleen architecture (25-27). A spectacular phenotype of mice deficient for LTα was systemic absence of LN and PP (4, 5). Subsequent analyses of mice deficient for other LTα-related molecules have pro-vided more detailed information on the ac-tion of LT in the lymphoid organogenesis (summarized in Table 2). Although mice deficient for LTβ also lacked PP and most LN, they did possess mesenteric LN (28, 29). These results suggested that LTα1LTβ2 plays major roles of LT in the development of LN and PP, and that there also exists an LTα/β heteromer independent pathway re-quired for the development of mesenteric LN. This hypothesis was later proven by the in vivo administration of LTβR-Ig and TNFR-I-Ig fusion protein which block the signals through LTβR and TNFR-I, respec-tively. Normal mice treated in utero with LTβR-Ig alone lacked systemic LN except for mesenteric LN, whereas concomitant administration of LTβR-Ig and TNFR-I-Ig or anti-TNF blocked the development of all LN includ-ing mesenteric LN, indicating that the TNF-TNFR-I axis also contributes to LN genesis (30, 31). Consist-ent with this model, mice deficient for both LTβ and TNFR-I lacked all LN including mesenteric LN (32). Conflicting data, however, also exist. It was demonstrated that mice deficient for LTβR lack all LN including mesenteric LN, suggesting that LTβR is a primary receptor responsible for the develop-ment of all LN (33). Phenotypic difference between LTβ-deficient mice and LTβR-deficient mice also suggested that LTβR binds not only to LTα/β heteromer but to other ligand(s), such as LIGHT (23), which may mediate signals required for the development of mesenteric LN. Thus, the integra-tion of the detailed phenotypic analyses of these gene-targeted mice with current perspectives of LT/TNF biology may illuminate many aspects of the lymphoid organogenesis.

III. LT and TNF as essential factors for the lymphoid organ structure
Studies with knockout mice of LT/TNF-related molecules have also unravelled essential actions of LT and TNF for the organization of lymphoid struc-ture. In mice deficient for either LTα (6, 7), LTβ (28, 29) or TNF (34, 35), organized clusters of follicular dendritic cells (FDCs) and germinal centers (GCs) are absent from the spleen.
GCs are histologically well defined structures that develop in secondary lymphoid organs shortly after challenge with T cell-dependent antigens. B cells within the GC can be clearly identified by their ability to bind to peanut agglutinin (PNA). It has been suggested that it is within GCs that somatic hypermutation and affinity maturation of the antibody response occur (36). Both primary and secondary lymphoid follicles characteristically contain clusters of FDC. FDC trap and can retain antigen-antibody complexes for long periods, apparently by means of receptors for the third complement component (CR) as well as Fcγ receptors (36). We and others have shown that an organized FDC structure, a major morphological characteristic of GCs, is absent in LTα-, LTβ-, TNF-, TNFR-I-, and LTβR-deficient mice (26, 33).

1. Role of LTαand TNFR-I in the development of GC and organized FDC clusters
Interestingly, in LTα-deficient mice, formation of FDC clusters and GCs was restored by transplan-tation of normal bone marrow (BM), indicating that the LTα-expressing cells required to establish these lymphoid structures are derived from BM (6). Sub-sequent analyses have identified B cells as an essen-tial source of LT required for this action (37, 38). In contrast to LTα-deficient mice, when TNFR-I-deficient mice were reconstituted with wild-type BM cells, they showed no detectable FDC clusters or GC formation, suggesting that TNFR-I-expression on non-BM-derived cells is necessary for the establish-ment of these structures (39, 40). An analysis using LTβR-deficient mice has demonstrated similar results;LTβR-deficient mice reconstituted with wild-type BM cells showed no detectable FDC clus-ters or GC formation (41). Thus, both BM-derived (ex-pressing LTα, LTβ and TNF) and non-BM-derived (expressing TNFR-I and LTβR) cells contribute to the organization of the lymphoid structure.
To further demonstrate the role of LTα as a sig-nal required to establish an organized FDC structure and to investigate the cell lineage of FDC, we used BM cells from CR1/2-deficient mice to reconstitute irradiated LTα-deficient mice. In this context, donor CR1/2-deficient BM-derived cells are LTα wild-type, but can be distinguished from the LTα-deficient re-cipient cells by their failure to stain with anti-CR1/2 monoclonal antibody. After reconstitution, spleen sections were stained with anti-CR1/2monoclonal antibody and PNA to assay for the presence of FDC clusters and GC, respectively. Similar to the results ob-tained after transfer of wild-type BM cells, clustered FDCs were identified with anti-CR1/2 monoclonal antibody (39). These results clearly indicate that the clustered FDCs induced in these BM-chimeric mice are derived from the LTα-deficient recipient, and that LTα provides a signal that supports the development of FDC clusters. Thus, LTα is not absolutely required for the production of the FDC lineage, but rather it is necessary for its maturation or for organization of the mature cells.

2. Dysfunctional antibody responses in the absence of GC
When LTα-deficient mice were immunized with low doses of T cell-dependent antigens such as (4-hydroxy-3-nitrophenyl) acetyl-ovalbumin (NP-OVA) in alum, although a strong IgM anti-NP response developed, no IgG anti-NP was observed (7). The development of an IgM anti-NP response indicated that the failure to produce serum IgG anti-NP anti-bodies did not represent failure to deliver antigen to the responding B cells in the absence of LN in LTα-deficient mice. Rather, it appeared to indicate an inability to activate productive isotype switching in the setting of a disturbed primary follicle struc-ture.
LTα-deficient mice also showed an impaired IgG response when immunized with sheep red blood cells (SRBCs) without adjuvant (9). When irradiated wild-type mice received splenocytes from LTα-deficient mice, however, a strong IgG anti-SRBC response was observed, again supporting the idea that LTα-deficient B cells and T cells have no intrinsic defect in ability to generate an IgG response. Rather, the altered microenvironment characteristic of LTα-deficient mice appears to result in an impaired ability to switch to a productive IgG response.

3. Affinity maturation without GC in LTα-deficient mice
Although immunization of LTα-deficient mice with low doses of T cell-dependent antigens elicited an exclusively IgM B cell response as described above, immunization with high doses (100 or 200μg of heavily haptenated NP-OVA) adsorbed to alum resulted in production of serum IgG anti-NP at levels similar to those detected in wild-type mice (7). This ability to form an IgG response permitted us to use these mice to test the role of the splenic GC in the maturation of the immune response. In previous elegant microdissection studies, the GC compart-ment has been shown to contain B lymphocytes with somatic mutations of their rearranged heavy and light chain variable regions, suggesting that it is in the GC that the signals for activation of the somatic mutation process are given (36, 42). We have analyzed the quality of the antibody response in LTα-deficient mice after immunization with high doses of NP-OVA adsorbed to alum. Surprisingly, LTα-deficient mice immunized with high doses of NP-OVA in alum, still without GCs, generate high affinity IgG anti-NP antibodies at levels similar to wild-type mice (7). Furthermore, analysis of the expressed V gene repertoire in splenocytes isolated from the LTα-deficient mice showed activation of a somatic mutation with all of the characteristics of that process in wild-type mice. This included muta-tion of codon 33 in the heavy chain variable region VH186.2 from Trp to Leu in 50%of the amplified VH186.2 sequences. This Trp to Leu mutation has been shown to correlate closely with acquisition of higher affinity for NP and is a reliable marker for the activation of affinity maturation by somatic muta-tion (42). These results demonstrate that somatic mutation and affinity maturation can occur indepen-dent of morphologically defined GCs. They have additional implications regarding the nature of the cells that give signals that regulate the somatic hypermutation response. They suggest that specific cellular elements that are characteristic of GCs are not essential for activation of somatic mutation. For example, clusters of FDCs are absent from the fol-licles of LTα-deficient mice. It remains possible that the splenic white pulp of these animals contains dis-persed FDCs or immature FDC precursors without detectable surface CR1 or immune complex binding activity. Nevertheless, it is clear that the somatic mutation process can occur without the presence of clusters of mature FDCs. The fact that the defect in isotype switching and somatic mutation observed in LTα-deficient mice can be overcome by providing the antigen at high doses together with an adjuvant such as alum (7) or Freund's adjuvant (9) suggests that the physical form and perhaps the density of the antigenic epitopes determines the ability to re-spond in the absence of a proper follicle structure. This is consistent with the role of the natural follicle organization to facilitate focusing of antigen in the environment of the responding B and T lymphocytes.

VI. Analysis of a natural mutant strain resem-bling LTα-deficient mice
In addition to the gene-targeted mice described above, alymphoplasia (aly) mice, an autosomal re-cessive natural mutant strain, have provided a novel and unique model for the abnormal development of lymphoid organs (43). Like LTα-deficient mice, aly mice lack LN and PPs, and the spleen archi-tecture such as the development of GCs and FDC clusters as well as marginal zone formation is disturbed (44, 45). aly mice manifest additional immunodeficiencies including a disorganized thymic architecture, low serum immunoglobulin (Ig) level and impaired allogenic skin rejection, which are not observed in LTα-deficient mice (4, 5, 43). A gene responsible for this mutant strain has just been identified as NF-κB inducing kinase (NIK) (46) by positional cloning (47, 48), but little is known about how the NIK contributes to the lymphoid organo-genesis.
Our studies were undertaken to clarify the mecha-nisms underlying the defective lymphoid organo-genesis in both aly mice and LTα-deficient mice. Two issues were specifically addressed. First, although dominant roles of LTβR as well as supportive roles of TNFR-I in the development of secondary lymphoid organs have been demonstrated as already des-cribed, exactly how LT control the development of lymphoid organs through these receptors was largely unknown. Second, we had no clue as to the role of the aly gene product (i.e. NIK) in the lymph-oid organ development. Consequently, we have generated aggregation chimeras;ex vivo fused morulae were implanted into pseudo-pregnant host females and allowed to develop to term (49). Chi-meric analyses demonstrated that LTα and NIK control lymphoid organogenesis by governing dis-tinct cellular compartments;LTα, expressed by the BM-derived cells, is essential not only for the organization of spleen architecture but also for lymphoid organogenesis. By contrast, the aly gene product NIK from BM-derived cells, if expressed, has no major role in the development of secondary lymphoid organs. Rather, the lack of LN and PPs in aly mice may be caused by a defect of non-BM-derived cells, possibly through a defective development of the incipient stromal cells of the LN and PP (50). Strategies undertaken to clarify these findings are as follows.

1. Complementation of abnormal LN genesis in aly mice with LTα-deficient mice
Although there is some phenotypic difference between LTα-deficient mice and aly mice, these two strains possess an extremely similar phenotype;lack of LN and PPs, and a disorganized spleen architecture. It was still possible that the altered function of NIK perturbs the expression of the membrane form of LT to cause the LTα-deficient phenotype in aly mice. We therefore first examined the expression of the membrane form using LTβR-Ig which binds to membrane-associated LTα1LTβ2. LTβR-Ig bound to activated aly spleen cells, demon-strating that expression of the membrane form of LT is retained in aly mice with this in vitro system.
If the membrane form of LT is present and func-tional per se in aly mice, as suggested by the in vitro studies, cells from aly mice should reverse the phenotype of LTα-deficient mice, and this was the case;a chimeric mouse from LTα-deficient mice and aly mice was generated, in which cells from both strains co-exist and interact with each other throughout development. Upon detailed inspection, mesenteric LN and one lumbar LN were observed in this chimera. Thus, lack of LN genesis in aly mice could not be attributed to the lack of functional membrane-associated LT.

2. A novel approach to define the mechanisms underlying the defective lymphoid organogenesis in LTα-deficient mice and aly mice
1) Defective lymphoid organ development in LTα-deficient mice is almost completely restored by the generation of chimeras with normal animals
Lack of FDC clusters as well as the defective GC formation in the spleen from LTα-deficient mice were restored by the transfer of wild-type BM cells, indicating that the LTα-expressing cells required to establish these lymphoid structures are derived from BM (6, 39). In contrast to in the spleen archi-tecture, the development of LN and PP was not restored in the same animals (51). Given that the lack of LN and PP in LTα-deficient mice is develop-mentally fixed, we speculated that LTα-expressing cells required to generate LN and PP are also BM-derived. This hypothesis, however, has not been formally tested, since BM cells were transferred only into the adult mice in these experiments. We have approached this issue by generating aggre-gation chimeras from LTα-deficient mouse morulae and LTα-sufficient mouse morulae ; both LTα-deficient BM-derived cells and LTα-sufficient BM-derived cells are expected to circulate in the body and to interact with the incipient stromal cells of the lymphoid organs throughout the development. We used GFP-Tg as wild-type mice in order to monitor the chimeric contribution from each strain by the detection of GFP (52). In particular, detection of GFP from the thymocytes and/or splenocytes by flow cytometry enabled us to focus on the chimerism of BM-derived cells which are the major source of membrane-associated LT.
Upon detailed inspection, all 10 chimeric mice generated showed lymphoid organ development indistinguishable from that seen in wild-type mice. Percentages of the LTα-expressing cells evaluated by the detection of GFP from thymocytes and splenocytes varied among the chimeras (ranging from 8 to 66%), and this variation did not appar-ently affect the extent of restoration of lymphoid organogenesis in this range. Development of LN and PP occurred irrespective of the status of GFP ex-pression from the body as long as LTα-expressing cells exist in the spleen and thymus. As expected, spleen architecture was also restored in these mice;organized FDC clusters as well as GCs were pres-ent in the spleens from all chimeric mice. Thus, LTα-expressing BM-derived cells, if present through-out the development, could restore the lymphoid organogenesis in LTα-deficient mice, supporting the idea that LTα-dependent interactions must occur during development in order for LN and PP to develop.

2) Defective lymphoid organ development in aly mice is only partially restored by the generation of chimeras with normal animals
To investigate how NIK contributes to the lymph-oid organogenesis, chimeric mice from aly mice and GFP-Tg were also generated and evaluated for the restoration of lymphoid organ development.
Out of11chimeras generated, two showed no inguinal LN and one had only right inguinal LN. Although 4 mice had more than four PPs, 7mice had either less than three or none at all. Further-more, one mouse which lacked inguinal LN and PPs had only one small mesenteric LN. It is particularly important to note that there were many spleen cells derived from GFP-Tg even in the chimeric animals defective in the development of LN and/or PPs. Because BM-derived cells from GFP-Tg should have the normal NIK, if expressed, a lack of LN and/or PP in these chimeras would be independent of the presence of NIK on the BM-derived cells. This limited restoration of lymphoid organ development sharply contrasted to that seen in the chimeras of LTα-deficient mice and GFP-Tg in which relatively small numbers of LTα-expressing cells were suffi-cient to restore the lymphoid organogenesis, as illustrated in Fig. 2.
Despite the partial restoration of LN and PP devel-opment in these chimeras, histological evaluation showed spleen architecture indistinguishable from that seen in wild-type mice;GC and FDC formation were apparently normal in all 11 chimeras some of which showed abnormal LN and/or PP develop-ment. These results demonstrated that an organized spleen architecture can be formed independent of the defective development of LN and/or PP.

3. Possible involvement of LTβR signaling in the abnormal lymphoid organ development in aly mice
The similar phenotypes of the LTα-deficient and aly mouse strains suggested that there might be a close mechanistic relationship in their affected bio-chemical pathways. The results described above, however, showed no detectable alteration in the expression of the membrane-associated LT ligand in aly mice. We therefore examined downstream elements of the pathway at the level of LTβR signal-ing using embryonic fibroblast (EF) isolated either from aly mice or from C57BL/6 wild-type mice. We found that the up-regulation of VCAM-1 after stimu-lation either with agonistic anti-LTβR monoclonal antibody or with recombinant LTα1LTβ2 was defec-tive in aly EF suggesting that signaling through LTβR is impaired in aly mice. Taken together, we concluded that LTα and NIK together control lymphoid organogenesis, governing distinct cellular compartments but with a close mechanistic relation-ship in their biochemical pathways, ligand-binding and receptor-signaling. Because LTβR is exclusively expressed by non-lymphoid cells (20, 21), a defect in LTβR signaling in aly mice is consistent with the idea that the lack of lymphoid organogenesis in this strain is caused by the defect of non-BM-derived cells as demon-strated by the chimeric analysis.

CONCLUDING REMARKS
The various studies described in this review have established that both LT and TNF provide essential signals for the development of a proper peripheral lymphoid organ structure. Together with experi-ments demonstrating essential roles for CD40 and its ligand (53, 54) and for OX40 and its ligand (55) in the development of a mature antibody response, these studies demonstrate critical roles for multi-ple members of the TNF ligand and receptor family in controlling organized T cell-dependent B cell responses. LT appears to be important both during ontogeny to establish the architecture within which immunocytes interact and later to maintain the full cellular constituency of the lymphoid fol-licles. TNF and TNFR-I appear to act in the es-tablishment of clusters of FDCs within the follicles of the splenic white pulp (6, 34, 39, 40). Signals through CD40 and OX40 participate in the direct interactions between T and B cells during the antigen-driven portion of the immune response. Thus, this family of cytokines acts at multiple levels of lymphoid organ morphogenesis and responsive-ness. Other components of the signaling pathway undoubtedly remain to be defined.
Identification of essential signaling molecules that act to establish a proper peripheral lymphoid tissue structure provides important information that will ultimately lead to the identification of the specific cell types that must interact for the initial establish-ment of this structure. It is now important to define the developmental expression patterns of the dif-ferent members of the TNF ligand and receptor family during ontogeny of the immune system. Once the essential regulatory cells have been iden-tified, then it may become feasible to develop new strategies to interfere with immune responses by changing the lymphoid organ tissue structure.

ACKNOWLEDGEMENTS
I thank David D. Chaplin for encouragement and members of The First Department of Internal Medicine, School of Medicine, Ehime University for support. I wish to thank Editor-in-Chief, Professor Saburo Sone for providing me with the opportunity to write this article.

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Abbreviations:LT, lymphotoxin;LN, lymph node;PP, Peyer's patch;EF, embryonic fibroblast;FDC, follicular dendritic cell;GC, germinal center;HEV, high endothelial venule;TNFR-I, type I TNF receptor;LTβR, lymphotoxin-β receptor;LTβR-Ig, LTβR-IgG1 fusion protein;TNFR-I-Ig, TNFR-I-IgG1 fusion protein;BM, bone marrow;GFP, green fluorescence protein; Tg, transgenic mice; PNA, peanut agglutinin.

Received for publication June 1, 1999;accepted June 16, 1999.
Address correspondence and reprint requests to Mitsuru Matsumoto, M.D., Division of Informative Cytology, Institute for Enzyme Research, University of Tokushima, Kuramoto-cho, Tokushima, 770-8503, Japan and FAX:+81-88-633-7434.