[in Japanese]

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Sphingosine 1-phosphate (S1P), a lysophospholipid mediator, is generated from sphingosine by sphingosine kinases and binds 5 types of G protein-coupled S1P receptors. It has been well documented that S1P receptor type 1 (S1P₁) plays an essential role in lymphocyte egress from secondary lymphoid organs (SLO), because lymphocytes are unable to exit from SLO to periphery in mice lacking lymphocytic S1P₁ conditionally. FTY720 (fingolimod) is an orally active first-in-class S1P receptor modulator and is highly effective in various experimental autoimmune disease models including encephalomyelitis, adjuvant- or collagen-induced arthritis, and lupus nephritis. FTY720 is a structural analogue of sphingosine and is effectively converted to an active metabolite, FTY720 phosphate (FTY720-P) by sphingosine kinases. FTY720-P binds to four types of S1P receptors (S1P₁, S1P₃, S1P₄, and S1P₅) except for S1P₂ and acts as an agonist at these receptors. Particularly, FTY720-P strongly internalizes S1P₁ from the cell surface, almost completely inhibits S1P responsiveness of lymphocytes in SLO, and acts as a functional antagonist at lymphocytic S1P1. Consequently, FTY720 inhibits S1P₁-dependent lymphocyte egress from SLO to decrease circulation of lymphocytes including autoreactive T cells and shows immunomodulating effects on autoimmune disease models. Recently, it has been reported that FTY720 has a superior efficacy in relapsing remitting multiple sclerosis patients compared to interferon-β. From these results, it is presumed that S1P₁ is a novel target for the therapy of autoimmune diseases including multiple sclerosis.

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Sjögren's syndrome (SS) is a unique autoimmune disease that shows dry mouth (xerostomia) and dry eye (xerophthalmia). Some SS patients develop visceral involvement, including malignant lymphoma. However, the molecular markers for disease activity or disease progression of SS have not been established. Therefore, the gene expression pattern in SS peripheral blood was examined by DNA microarray analysis. The gene expression patterns of primary and secondary SS were similar, but different from that of rheumatoid arthritis. Interferon (IFN)-inducible genes were upregulated in primary and secondary SS. The interferon gene signature was more prominent in primary SS compared to secondary SS. Primary and secondary SS were distinguishable based on the gene expression pattern of peripheral blood. The most upregulated gene, IFNα-inducible protein 27, showed a significant positive correlation with serum IgG level in SS. IFN-inducible genes could be markers for disease activity. The expression of ribosomal protein S27 and S29 genes was upregulated in peripheral blood from SS patients with marginal zone B cell lymphoma. The levels of ribosomal protein S27 and S29 expression were decreased after chemotherapy against lymphoma. The ribosomal protein S27 and S29 genes could be molecular markers for lymphoma development in SS.

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Human amniotic membrane (AM), a thin intrauterine placental membrane is highly biocompatible, and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We evaluated autologous transplantation of oral mucosal epithelial cells on AM in patients undergoing oral surgeries. Specimens of AM were obtained from women undergoing Caesarean section. Using oral mucosal biopsy specimens obtained from the patients, oral epithelial cells were cultivated on an AM carrier. The resultant sheet was transplanted on the oral mucosal defect. After 2-3 weeks of culture, the cultivated epithelial cells seemed well differentiated and showed stratification into 5-7 layers on AM. Immunohistochemistry demonstrated that the cultivated cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedure, the reconstructed sites did not show infection, bleeding, rejection, or sheet detachment, and the sites achieved a new oral mucous membrane. The cultivated epithelial sheets maintained the properties of a mucous membrane and expressed basement membrane proteins. Autologous transplantation of cultivated oral epithelial sheets was performed, and the transplanted tissue showed adherence to the oral mucosal defect. A long-term follow-up of more than 12 months revealed absence of postoperative recurrence, and the postoperative courses were excellent. These findings showed that this novel epithelial sheet is a useful biomaterial for mucosal reconstruction.

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Objective: A cytokine of the IL-1 family, IL-33, has recently been recognized as one of the key cytokines enhancing Th2-balanced immune regulation through its receptor, ST2. However, the action of IL-33 on allergic effector granulocytes has remained unclear. We thus tested whether IL-33 acts directly on, and affects the functions of, human basophils and eosinophils.
Methods: Basophils and eosinophils were prepared from normal human peripheral blood. Cells were treated with IL-33, and their adhesion, migration, surface CD11b expression, mediator release and survival were assessed in vitro. Expression of ST2 was analyzed at both the mRNA and protein levels.
Results: Analysis by real-time PCR and flow cytometry demonstrated that both basophils and eosinophils expressed mRNA and protein for ST2. Expressions of IL-4 and IL-13 mRNA in basophils were upregulated by IL-33. IL-33 at 1-100 ng/ml potently enhanced the adhesiveness and CD11b expression of basophils and eosinophils. Although IL-33 failed to directly induce degranulation or migration of basophils, it exerted priming effects by enhancing basophil migration toward eotaxin and degranulation in response to IgE-crosslinking stimulus. IL-33 prolonged survival of eosinophils, but not basophils, via suppression of their apoptosis. In addition, blocking of ST2 with neutralizing antibody inhibited IL-33-induced upregulation of basophil and eosinophil functions.
Conclusion: IL-33 is a potent regulator of various functions of basophils and eosinophils. IL-33 may be a key cytokine in the pathogenesis of Th2-dominant allergic diseases, at least partly by acting on effector cells, including basophils and eosinophils.

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Fingolimod (FTY720), a sphingosine 1-phosphate (S1P) receptor modulator, is highly effective in various autoimmune disease models and decreases circulating mature lymphocytes by inhibiting S1P-dependent lymphocyte egress from secondary lymphoid organs (SLO). On the contrary, FTY720 at an oral dose of 1 mg/kg also induces a significant decrease in the number of peripheral blood lymphocytes (PBL), particularly T cells, in alymphoplasia (aly/aly) mice lacking SLO. We demonstrated that there was no contribution of thymus or spleen for the reduction of PBL by FTY720 because FTY720 could also decrease the number of PBL in thymectomized and/or splenectomized aly/aly mice. When mice were given FTY720 at 1 mg/kg orally, the blood concentration of FTY720 is approximately 0.2 μM or less. On the other hand, no apoptosis was seen in aly/aly lymphocytes when they were treated with FTY720 at a concentration of up to 1 μM. Moreover, we found that oral administration of FTY720 at 1 mg/kg to aly/aly mice induced the accumulation of mature lymphocytes, particularly T cells, into the bone marrow but not spleen or thymus. Consequently, it is highly probable that mature lymphocytes depleted from blood by FTY720 were sequestered into the bone marrow in aly/aly mice. The sequestration of T cells into the bone marrow was also induced by S1P lyase inhibitor that disrupts S1P gradients. These results demonstrate that the reduction of PBL in aly/aly mice by FTY720 is due to inhibition of S1P-dependent lymphocyte egress from the bone marrow, but not induction of lymphocyte apoptosis.

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We induced neural cells by treating cynomolgus monkey embryonic stem cells with retinoic acid. The retinoic acid-treated cells had elongated axons and expressed βIII tubulin, neurofilament middle chain (NFM) and Islet1 in vitro, suggesting their differentiation into motoneurons. The monkey ES derived neural cells were transplanted to hemiplegic mice with experimental brain injury. Injured mice with the neural cell graft gradually recovered motor function, whereas injured mice with vehicle (PBS) injection and injured mice with undifferentiated monkey ES cell graft remained hemiplegic. After transplantation into hemiplegic mice, the neural cells that had grafted into the periventricular area migrated and located near the corpus callosum by day 7. The neural cells distributed over the injured cortex at day 21. The cells expressed CXCR4, a receptor for chemokine SDF1. In a microchemotaxis assay, the neural cells responded to SDF1, and AMD3100, an antagonist of CXCR4, abrogated their migration. The injured cortex initially produced SDF1, and the graft expressed CXCR4 in the brain. SDF1 accelerated NCAM mRNA expression in the neural cells in vitro. The neural cells distributed over the cortex expressed L1CAM, NCAM, and N-Cadherin extensively after reaching the injured cortex. Administration of AMD3100 forced the graft to stay at the injection site. Thus, chemokine, chemokine receptor, and neural cell adhesion molecules seem to be involved in the regeneration of neural networks and functional recovery of hemiplegic mice.

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