Ryoji Yasumizu

Experimental autoimmune thyroiditis induced by thyroglobulin-pulsed dendritic cells

Dendritic cells (DCs), which are the most effective professional antigen-presenting cells (APCs), initiate and regulate immune responses. In this report, we examine the role of DCs in the induction of autoimmune thyroiditis. Experimental autoimmune thyroiditis (EAT) induced by immunization with thyroglobulin (Tg) plus adjuvant is considered to be an animal model of autoimmune thyroiditis, and is categorized as a T cell-mediated autoimmune disease. To examine the contribution of DCs to EAT, naive DCs were purified from high responder B10BR mice and pulsed with various concentrations of porcine Tg (pTg). These pTg-pulsed DCs were transferred without adjuvant to syngenic BIOBR mice to induce EAT. Mice that had received pTg-pulsed DCs showed thyroiditis, and the degree of thyroiditis induced was positively correlated to the amounts of pTg used for the incubation (pulsing) of DCs. The severity of thyroiditis was also correlated to the amounts of anti-pTg IgG2a antibodies and IFN-gamma in the recipient sera, but not to IL-4 or IL-10, indicating that Th1 cells are mainly activated by pTg-pulsed DCs and attributable to the pathogenesis of EAT.

Comparison of bone marrow cells harvested from various bones of cynomolgus monkeys at various ages by perfusion or aspiration methods: a preclinical study for human BMT.

Using cynomolgus monkeys, we have previously established a new method for harvesting bone marrow cells (BMCs) with minimal contamination of the BMCs with T cells from the peripheral blood. We originally conducted this new “perfusion method” in the long bones (the humerus, femur, and tibia) of cynomolgus monkeys.

Treatment and transfer of emphysema by a new bone marrow transplantation method from normal mice to Tsk mice and vice versa.

We have recently established a new bone marrow transplantation (BMT) method in which bone marrow cells are injected into the intrabone marrow (IBM). In the present study, we used an animal model for emphysema (tight‐skin [Tsk] mouse) to examine whether IBM‐BMT could be used to treat emphysema in Tsk mice. IBM‐BMT was carried out from C3H mice into Tsk mice (8–10 weeks old) that had already shown emphysema. Six months after transplantation, the lungs of all the Tsk mice treated with IBM‐BMT [C3H→Tsk] showed similar structures to those of normal mice, whereas the [Tsk→Tsk] mice showed emphysema, as seen in age‐matched Tsk mice. Next, we attempted to transfer emphysema from Tsk mice to C3H mice by IBM‐BMT. Six months after IBM‐BMT, the [Tsk→C3H] mice showed emphysema. These results strongly suggest that emphysema in Tsk mice originates from defects of stem cells in the bone marrow.

Analyses of very early hemopoietic regeneration after bone marrow transplantation: comparison of intravenous and intrabone marrow routes.

In bone marrow transplantation (BMT), bone marrow cells (BMCs) have traditionally been injected intravenously. However, remarkable advantages of BMT via the intra‐bone‐marrow (IBM) route (IBM‐BMT) over the intravenous route (IV‐BMT) have been recently documented by several laboratories. To clarify the mechanisms underlying these advantages, we analyzed the kinetics of hemopoietic regeneration after IBM‐BMT or IV‐BMT in normal strains of mice. At the site of the direct injection of BMCs, significantly higher numbers of donor‐derived cells in total and of c‐kit+ cells were observed at 2 through 6 days after IBM‐BMT. In parallel, significantly higher numbers of colony‐forming units in spleen were obtained from the site of BMC injection. During this early period, higher accumulations of both hemopoietic cells and stromal cells were observed at the site of BMC injection by the IBM‐BMT route. The production of chemotactic factors, which can promote the migration of a BM stromal cell line, was observed in BMCs obtained from irradiated mice as early as 4 hours after irradiation, and the production lasted for at least 4 days. In contrast, sera collected from the irradiated mice showed no chemotactic activity, indicating that donor BM stromal cells that entered systemic circulation cannot home effectively into recipient bone cavity. These results strongly suggest that the concomitant regeneration of microenvironmental and hemopoietic compartments in the marrow (direct interaction between them at the site of injection) contributes to the advantages of IBM‐BMT over IV‐BMT.

Isolation of murine pluripotent hemopoietic stem cells in the Go phase

A method to purify pluripotent hemopoietic stem cells in the Go phase from mouse bone marrow was established. Bone marrow cells from 5-fluorouracil (5-FU)-treated mice were fractionated by Percoll density gradient. The cells with density between 1.063 and 1.075 were further separated into wheat germ agglutinin (WGA)-positive and -negative cells using fluorescent-activated cell sorter (FACS) after staining with fluorescein isothiocyanate-conjugated WGA (FITC-WGA). An assay for spleen colony-forming units (CFU-S) revealed that the WGA-positive cells (1 X 10(6)) produced 1380 CFU-S (about 150 times of the number in the original bone marrow cells) on day 12 (but no CFU-S on day 8), whereas the WGA-negative cells produced no CFU-S. Thus, the stem cells in the Go phase are found to be enriched 150 times in 5-FU-treated WGA-positive cells.

Marked Increase in Number of Dendritic Cells in Autoimmune-Prone (NZW × BXSB)F1 Mice with Age

Here, we report that the number of CD11c+CD3− B220− cells increases in autoimmune‐prone male (NZW × BXSB)F1 (W/BF1) mice with age. The CD11c+CD3−B220− cells from W/BF1 mice show a typical stellate shape and induce the proliferation of T cells. In the CD11c+CD3−B220− cells from W/BF1 mice, CD11b (Mac‐1α), NK 1.1, and CD95 (Fas) are upregulated in comparison with normal mice, while the expression of CD8α, CD117 (c‐kit), CD135 (Flk‐2/Flt‐3), and Sca‐1 decreases. There is a significant increase in Flt‐3L (FL) mRNA in the bone marrow of W/BF1 mice with age. Moreover, activated hemopoietic cells express high levels of FL. The injection of CD11c+CD3−B220− cells from old W/BF1 mice to young W/BF1 mice transiently induces autoimmune disease (thrombocytopenia). These results suggest that hyperproduction of FL from activated hemopoietic cells induces a dramatic increase in the number of dendritic cells in aged W/BF1 mice, followed by the acceleration of autoimmunity.

c‐kit<low Pluripotent Hemopoietic Stem Cells Form CFU‐S on Day 16

Using Ly5 congenic mice, we characterized the early differentiation step of pluripotent hemopoietic stem cells. Lineage− (Lin−)/CD71− cells in the bone marrow cells were separated into major histocompatibility complex (MHC) class Ihigh/c‐kitlow and MHC class Ihigh/c‐kit

Intrathymically Injected Hemopoietic Stem Cells Can Differentiate into All Lineage Cells in the Thymus : Differences between c-kit[+] Cells and c-kit[<low] Cells

To investigate whether hemopoietic stem cells (HSCs) can differentiate into all lineage cells even in the thymus, we injected two types of HSCs (c‐kit+ and c‐kit

CHANGES IN MARKERS, RECEPTORS AND ADHESION MOLECULES EXPRESSED ON MURINE HEMOPOIETIC STEM CELLS AFTER A SINGLE INJECTION OF 5-FLUOROURACIL

Cytokines play a crucial role in the differentiation and proliferation of hemopoietic cells, and it has recently been found that adhesion molecules play crucial roles not only in differentiation and proliferation, but also in the homing and other functions of hemopoietic cells. We have very recently established a new method for purifying pluripotent hemopoietic stem cells (P‐HSC) in mice by injecting 5‐fluorouracil (5‐FU). The P‐HSC were found to be low‐density, lineage marker‐negative (Lin−), CD71− and major histocompatibility complex class Ihigh. In the present study, we analyze changes in the expression of various HSC markers (Sca‐1 and CD34), receptors (c‐kit and interleukin‐6 receptor [IL‐6R]) and adhesion molecules (very late activation antigen‐4 [VLA‐4], lymphocyte function‐associated antigen‐1 [LFA‐1], and CD44) after 5‐FU injection. The percentage of Sca‐1+ cells increases after 5‐FU treatment, reaching a maximum on day 3, whereas the percentage of IL‐6R+ cells decreases, reaching a minimum on day 3. The percentage of CD34+ cells does not change after 5‐FU treatment. The percentages of both c‐kitlow and c‐kithigh cells decrease, reaching a minimum on day 3 after 5‐FU treatment, whereas the percentage of c‐kit− cells reciprocally increases, reaching a maximum on day 3. However, there is no change in the expression of adhesion molecules (VLA‐4, LFA‐1 and CD44) on the P‐HSC.

Development of insulin-dependent diabetes mellitus in [(NOD + BALB/c) --> NOD] mixed allogeneic bone marrow chimeras.

To examine the possibility that the bone marrow cells of BALB/c genotype interfere with the development of insulitis and diabetes in NOD mice, we transplanted BALB/c bone marrow cells mixed with NOD bone marrow cells into NOD mice. The [(NOD + BALB/c) --> NOD] chimeras developed insulitis and diabetes, indicating that BALB/c bone marrow cells do not interfere with the development of the disease in NOD mice. Surprisingly, these mice have been reconstituted with only NOD hematolymphoid cells. When the pancreatic tissues from newborn NOD and BALB/c mice were grafted into [(NOD + BALB/c) --> NOD] chimeras, the BALB/c pancreatic tissues were rejected, whereas the NOD graft showed insulitis. Furthermore, the spleen cells of the chimeras showed responsiveness to BALB/c spleen cells in mixed lymphocyte reaction and generated cytotoxic T lymphocytes specific for the H-2d and third party targets. These findings indicate that the hematolymphoid cells (including hemopoietic stem cells) of NOD mice are more resilient than those of normal BALB/c mice, and that insulin-dependent diabetes mellitus will recur after bone marrow transplantation unless the hematolymphoid cells of NOD mice are completely destroyed by irradiation.