Tsunetoshi Itoh

Immunosuppressant FTY720 inhibits thymocyte emigration

One major role of the thymus is to provide the peripheral immune system with mature T cells, but the mechanisms involving the cellular export are not fully understood. In this study, we examined the ability of a novel immunosuppressive reagent, FTY720, to inhibit T cell export from the thymus. Daily administration of FTY720 at a dose of 1 mg / kg resulted in a marked decrease in the number of peripheral blood T lymphocytes. In the thymus, long‐term daily administration of FTY720 caused a three‐ to fourfold increase in the proportion of mature medullary thymocytes (CD4+CD8– and CD4–CD8+) as well as a slight decrease in the double‐positive cell (CD4+CD8+) ratio. Phenotypic analysis (TCRα β, H‐2Kd, CD44, CD69 and CD24) revealed that these increased subsets represent possible peripheral recent thymic emigrants. High level expression of L‐selectin by these subsets further suggests that they were prevented from leaving the thymus. By intrathymic labeling with fluorescein isothiocyanate, only one fourth of labeled cells could be detected in the lymph nodes and in the spleen of FTY720‐treated mice compared to saline‐treated control mice. Taken together, these results suggest that the immunosuppressive action of FTY720, at least in part, could be due to its inhibitory effect on T cell emigration from the thymus to the periphery.

Presence of glucocorticoid receptors in cultured thymic epithelial cells

We investigated the presence of glucocorticoid receptors (GC) in human thymic epithelial cells grown in primary cultures and in a pure epithelial rat cell line. These GR levels were compared to those determined concomitantly in fresh human thymocytes. The average number of sites were 54,457/cell for males (n = 8) and 58,224/cell for females (n = 8) with mean Kd values of 1.5 and 1.7 X 10(-8) M, respectively, in cultured human epithelial cells. These results are comparable to those obtained for rat thymic epithelial cells. Competition experiments showed that the relative affinities of the steroids tested were in decreasing order: dexamethasone greater than progesterone greater than testosterone and estradiol. This observation is compatible with binding to physiological GR. Moreover, the mean GR value appeared to be approximately 10 times higher for human thymic epithelial cells than for thymocytes. Thus, human epithelial cells as well as thymocytes should be considered as a specific target for glucocorticoid hormones.

Effects of sex steroids on the proliferation of thymic epithelial cells in a culture model: A role of protein kinase C

Using a rat thymic epithelial cell line (TEC; IT‐45R1), the present study attempted to elucidate the mechanism of action of sex steroid hormones (SH) on the proliferation of TEC The findings were as follows: (a) the proliferation of TEC in response to SH was mediated through protein kinase C activity introduced as a result of interaction between SH and plasma‐borne inhibitors; (b) the strong inhibitory effect of SH on TEC proliferation might be mediated through the SH receptor pathway because the proliferative response was triggered by progesterone (P) and androgen (A), whereas the inhibitory response was triggered by P, A and oestrogen. These results clearly suggest that the control of TEC proliferation is a ‘shut‐off’ mechanism triggered by high plasma levels of SH. This further refers to the speculation that the development of the normal thymus may be due to a lack of this ‘shut‐off’ mechanism so that development occurs at the adequate plasma SH levels that are often observed before puberty. However, this development is inhibited at the high plasma SH levels after puberty and/or during pregnancy.

Distribution of two types of lymphocytes (intraepithelial and lamina-propria-associated) in the murine small intestine

The intestine, which is exposed to nutrition and to food-derived antigens and microbes including viruses and bacteria, might be an important site for the immune response. Crucial structural and functional differences exist between the small and large intestine, regional differences even having been demonstrated within the small intestine. Accordingly, intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) might be heterogeneous among the different intestinal regions. The aim of this study has been to describe, as accurately as possible, the numbers and T-cell receptor (TCR) phenotypes of IELs and LPLs present in distinct regions of the murine small intestine under physiological conditions. Using an immunohistological technique to differentiate IELs from LPLs, the differential enumeration of IELs and LPLs in distinct regions of the murine small intestine, based upon their definition originally determined by their location, has been performed for the first time and has demonstrated that (1) there are more IELs than LPLs in the duodenum and jejunum, but more LPLs than IELs in the ileum, (2) in the duodenum and jejunum, TCRγδ IELs account for 70%–75% of the total CD3+ IELs, a much greater percentage than previously reported, (3) the ratio of TCRγδ to TCRαβ IELs is inverted in the ileum, with more than 75% IELs being TCRαβ-positive, (4) the lamina propria forms one functional unit throughout the small intestine in terms of the TCR subset components (TCRαβ:TCRγδ=3:1), and (5) the ileum is entirely different from other regions of the small intestine. To deepen our understanding of the functional significance of the small intestine as an immunologically competent organ, the precise distributions of IELs and LPLs, the ratio of their various subsets, and the strict distinction of IELs and LPLs, as described in this study, is indispensable.

A time kinetic study of the effect of aminobisphosphonate on murine haemopoiesis

We previously showed that aminobisphosphonates (aminoBPs), potent inhibitors of bone resorption, increased the number of osteoclasts and granulocytes, and enhanced the cell size of osteoclasts in vivo, indicating that aminoBPs have a profound effect on murine haemopoiesis. The possible effect of an aminoBP (4‐amino‐1‐hydroxybutylidene‐1,1‐bisphosphonate; AHBuBP) on murine haemopoiesis in vivo was examined in more detail. Macroscopically, AHBuBP induced the whitened bone marrow (BM) and splenomegaly. Flow cytometric analysis indicated that in BM, AHBuBP reduced the number of mature monocyte‐macrophage lineage cells and erythroid cells 1 and 2 d after treatment, respectively, whereas it enhanced granulopoiesis on day 4. In the spleen, both erythropoiesis and granulopoiesis were significantly increased. BM haemopoietic progenitors of granulocyte lineage and of monocyte‐macrophage lineage (CFU‐G, CFU‐M and CFU‐GM) were well maintained by the injection of AHBuBP, and even a small increment in these progenitors was observed 2–4 d after treatment. Immunohistochemical examination of BM demonstrated that residential macrophages of erythroblastic islands disappeared. Increased numbers of osteoclasts, as well as enlarged cell size, was confirmed up to 7 d after the treatment, implicating that the inhibition of bone resorption was not due to the reduced generation of osteoclasts by AHBuBP. These results suggest (1) that AHBuBP treatment in vivo rapidly deleted mature residential macrophages from BM, (2) that mature macrophages once deleted did not reappear even when CFU‐M and CFU‐GM increased in number and the number of Mac‐1+/Gr‐1− cells recovered to normal, (3) that BM erythropoiesis was significantly decreased due to the lack of erythroblastic islands, and (4) that compensatory erythropoiesis was evoked in the spleen to induce splenomegaly.

Bacterial superantigens and T cell receptor β-chain-bearing T cells in the immunopathogenesis of ulcerative colitis

Ulcerative colitis (UC) is a chronic relapsing–remitting inflammatory bowel disease (IBD) that affects the colon and the rectum producing debilitating symptoms, which impair ability to function and quality of life. The aetiology of IBD is incompletely understood, but within the lymphocyte population, specific T cell subsets are known to be major factors in the development of intestinal immune pathology while different subsets are essential regulators, controlling IBD. Hence, IBD is thought to reflect dysregulated T cell behaviour. This study was to investigate if the normal molecular configuration of the T cell receptor (TCR) repertoire is compromised in patients with UC. The percentage of T cell‐bearing β‐chain 4 (TCRBV4) was high in patients with UC, and T cells showed polyclonal expansion in the presence of bacterial superantigens (SA) such as streptococcal mitogenic exotoxin Z‐2 (SMEZ‐2), indicating that bacterial SA promote specific TCRBV family expansion. Further, in patients with UC, the duration of UC was significantly longer in patients with skewed TCRBV4 compared with patients without TCRBV4 skewing, suggesting that long‐term exposure to bacterial SA such as SMEZ‐2 might promote systemic immune disorders like the remission‐relapsing cycles seen in patients with UC. In conclusion, our observations in this study support the perception that the systemic activation of T cells by enteric bacterial SA might lead to a dysregulated, but exuberant immune activity causing the remission and flare‐up cycle of mucosal inflammation in patients with UC. Future studies should strengthen our findings and increase understanding on the aetiology of IBD.

Formation of Hassall's corpuscles in vitro by the thymic epithelial cell line IT-26 R 21 of the rat

SummaryIn the monolayer of an established epithelial cell line from the rat thymus, IT-26R21, characteristic cell aggregates quite similar to Hassalls corpuscles were formed. These aggregates were examined by light and electron microscopy, and immunohistochemically. Their interpretation as Hassalls corpuscles is based on the following observations: (1) The aggregates are formed in the monolayer of cells that greatly resemble medullary epithelial cells of the thymus. (2) They consist of flattened epithelial cells in a concentric pattern with one or more degenerating cells in the center. (3) Loss of microvilli suggests that these cells are keratinizing. (4) The aggregates show strongly positive reactions in immunofluorescent staining with antikeratin and antiprekeratin.When Hassalls corpuscles increase in size, cellular proliferation is somewhat suppressed. Both in vivo and in vitro, they may be interpreted as an expression of a changing growth pattern in confined spaces and thus seem to have little immunological function.

Alteration of T-cell receptor repertoires during thymic T-cell development.

The majority of thymocytes die in the thymus, whereas small populations of T cells that are able to specifically recognize an antigen are considered to survive. Although the thymic selection is thought to have a profound effect on T‐cell receptor (TCR) repertoire, little is known how TCR repertoire is formed during the thymocyte developmental process. We examined TCRα‐ and β‐chain variable regions (TCRAV and TCRBV) repertoire in thymic T‐cell subpopulations from mice bearing different major histocompatibility (MHC) haplotypes. In Balb/c mice, but not C57BL/6, remarkable alterations of the TCR repertoire were observed in mature T‐cell subpopulations as previously reported. In contrast, there were no significant differences of TCRBV repertoire between DN3 (CD25+CD44−) and DN4 (CD25−CD44−), and between DN4 and DP. These results suggest that (1) TCR repertoire of mature T cells was formed mainly under the influence of endogenous superantigens, while MHC haplotypes played the least role; (2) the ‘β‐selection’ process during immature stages had little impact on TCRBV repertoire formation; and (3) TCR repertoire in immature T‐cell subpopulations was extremely similar between different strains of mice. We thus consider that pre‐selection TCR repertoire in immature T cells could be determined by some genetic factors conserved among different strains.

Establishment of a myoid cell clone from rat thymus

SummaryIn the course of rat thymus cultures, cloning of myoid cells (myoblasts) was successfully achieved. Myoblasts first appeared in the primary culture of the rat thymus in very limited number, but proliferated rapidly, started to fuse, and formed multinucleated myotubes contracting spontaneously. In the 15th month of culture, myoblasts were detached from the flasks to establish clones by the limiting dilution method. The cloned cells of IT-45R92 were identified as myoid cells; the small spindle-shaped cells proliferated rapidly and fused with each other forming large cross-striated myotubes. Ultrastructurally, thick and thin filaments with A, I, and Z bands as well as H and M bands occupied the cytoplasm of a large myotube. No epithelial origin of IT-45R92 cells was considered, and pluripotent stem cells were regarded as the most likely cells of origin. A difference is suggested between the factor stimulating fusion and the factor stimulating formation of fully differentiated myotubes. The significance of myoid cells is discussed in relation to acetylcholine receptors of IT-45R92 and the possible causes of myasthenia gravis.

Vitamin E enhances T cell differentiation through increased epithelial cell function in rat thymus

This study was designed to elucidate the mechanism on increased T cell differentiation in thymus following vitamin E supplementation and discussed on the changes of epithelial cell (TEC) and macrophage functions in thymus following vitamin E supplementation. Thymic epithelial cells isolated by panning method from thymocytes of rats fed the high vitamin E (500 IU α-tocopherol nicotinate/kg) diet for 7 weeks increased the proportion of CD4+CD8- T cells in thymocytes. In addition, when thymic epithelial cell line IT45-R1 was pretreated with vitamin E for 24 h and then incubated with immature T cells for 48 h, they also enhanced the proportion of CD4+CD8-T cells in thymocytes. On the contrary, the supernatant of IT45-R1 treated in vitro with vitamin E for 24 h did not have any effect on the proportions of T cell subsets in thymocytes. Furthermore, in vitro treatment with vitamin E significantly enhanced the binding capacity of TEC to immature T cells. The expression of adhesion molecule, ICAM-1, on the membrane of TEC was also greatly increased by high vitamin E diet. However, in vitro incubation with macrophages isolated from rats fed the regular or high vitamin E diet did not induce a significant difference in the changes of T cell subsets in immature T cells. These results suggest that vitamin E enhances T cell differentiation through the increase of not macrophage but TEC function in thymus, which is associated with the increased binding capacity of TEC to immature T cells via increased expression of ICAM-1.