坂本 明美

Bcl6-deficient (Bcl6(-/-)) mice displayed Th2 type inflammation, which caused by abnormality of non-lymphoid cells. However, initiators for the Th2 type inflammation were not clear. In order to elucidate the initiators, we investigated property and function of mast cells derived from Bcl6(-/-) mice. Mast cells were developed from bone marrow cells cultured with IL-3 (BMMCs). Although the development of BMMCs from Bcl6(-/-) mice was similar to that from wild-type mice, proliferation of Bcl6(-/-) BMMCs stimulated with IL-3 was slightly lower than that of wild-type BMMCs. When these BMMCs were stimulated by Fc epsilon RI/IgE cross-linking, Bcl6(-/-) BMMCs produced Th2 cytokines more than wild-type BMMCs did. Thus, Bcl6(-/-) mast cells are one of the initiators for Th2 type inflammation in Bcl6(-/-) mice, and Bcl6 may be a molecular target for Th2 type allergic diseases. (c) 2005 Elsevier Ltd. All rights reserved.

The role of transforming growth factor (TGF)-beta and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-beta, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null ( null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF-beta caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF-beta in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-beta. TGF-beta increased transcript level of alpha 2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF-beta were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.

The CpG motif in DNA plays a critical role in immunity via modulating the Th1/Th2 balance. In B cells, CpG-containing oligodcoxynucleotides (CpG ODNs) inhibit IL-4-mediated class switch recombination (CSR) to IgG1 and IgE through inhibition of the germline transcription (GLT) of these isotypes. However, the molecular mechanism of this inhibitory effect remains elusive. We showed here that Id2 and Bc16, both of which inhibit IgE GLT and CSR, are not involved in this inhibitory pathway. We demonstrated that there is reduced activity of NFkappaB binding to the IgE promoter and a reduction of Irf4 protein in CpG ODN-treated B cells. These data indicate the critical role of NFkappaB and Irf4 in the regulation of IgE CSR through actions downstream of CpG signaling. (C) 2005 Elsevier Inc. All rights reserved.

Marginal zone (MZ)-B cells participate in very early immune responses and play a pivotal role in the first-line of defense against blood-borne Ags including bacterial LPS. Since splenic B cells from c-fos transgenic (H2-c-fos) mice are hyper-sensitive to LPS stimulation, we examined LPS-sensitivity of MZ-B cells in the spleen of H2-c-fos mice. Here, we show that proliferation of MZ-B cells from H2-c-fos mice stimulated with LPS was larger than that from control mice. Proliferation and IgM production of the H2-c-fos MZ-B cells were also larger than those of splenic follicular (FO)-B cells from the 142-c-fos mice, suggesting that c-fos overexpression augments LPS-sensitivity of MZ-B cells more than that of FO-B cells. Furthermore, the number of MZ-B cells but not that of FO-B cells in the spleen of H2-c-fos mice was two- to three-fold larger than that in control littermates. The number of transitional type II (T2)-B cells in H2-c-fos mice was also larger than that of control littermates. However, the number of transitional type I (T1)-B cells in the spleen of H2-c-fos mice was not larger than that of control mice. Moreover, this c-fos effect on differentiation of MZ-B cells was intrinsic in B cells by the competitive repopulation assay with hematopoietic stem cells of H2-c-fos and control mice. These results suggest that c-fos overexpression in B cells augments differentiation and accumulation of MZ-B cells. (c) 2004 Elsevier Ltd. All rights reserved.

The CpG motif in DNA plays a critical role in immunity via modulating the Th1/Th2 balance. In B cells, CpG-containing oligodcoxynucleotides (CpG ODNs) inhibit IL-4-mediated class switch recombination (CSR) to IgG1 and IgE through inhibition of the germline transcription (GLT) of these isotypes. However, the molecular mechanism of this inhibitory effect remains elusive. We showed here that Id2 and Bc16, both of which inhibit IgE GLT and CSR, are not involved in this inhibitory pathway. We demonstrated that there is reduced activity of NFkappaB binding to the IgE promoter and a reduction of Irf4 protein in CpG ODN-treated B cells. These data indicate the critical role of NFkappaB and Irf4 in the regulation of IgE CSR through actions downstream of CpG signaling. (C) 2005 Elsevier Inc. All rights reserved.

Objective. The bcl-6 proto-oncogene is ubiquitously expressed in various tissues. Since we found out the smaller number of TER119(+) cells in the spleen of neonatal bcl-6-deficient (bcl-6(-/-)) mice compared with that of control (bcl-6(+/+)) littermates, we studied functions of bcl-6 in differentiation of erythroid lineage cells. Materials and Methods. Erythroblasts in the definitive erythropoiesis were separated into four subsets using anti-TER119 and anti-CD71 mAbs. The cell number and property of these four subsets in spleens of neonatal bcl-6(+/+) and bcl-6(-/-) mice were examined using a flow cytometry. Results. bcl-6 mRNA expression was detected in the TER119(high) CD71(high) subset, which is morphologically equivalent to basophilic erythroblasts, by reverse-transcribed polymerase chain reaction. High percentages of cells in the TER119(low)CD71(high) and TER119(high)CD71(high) subsets were in the cell cycle. The cell number of the TER119(high)CD71(high) subset in the spleen and the percentage of reticulocytes in the peripheral blood of neonatal bcl-6(-/-) mice were significantly lower than those of neonatal bcl-6(+/+) mice. However, the percentage of apoptotic cells and that of cells in the cell cycle in the TER119(high)CD71(high) subset of bcl-6(-/-) mice were similar to those of bcl-6(+/+) mice. Conclusion. bcl-6 detected in the TER119(high)CD71(high) subset of erythroblasts in the spleen of neonatal mice may be required to retain the erythroblasts in the cell proliferation stage. (C) 2005 International Society for Experimental Hematology. Published by Elsevier Inc.

We examined effects of c-fos overexpression on the development and property of peritoneal B-1 cells using transgenic (H2-c-fos) mice carrying the c-fos gene under the control of the constitutive H-2K(b) promoter. The number of B-1b cells in the peritoneal cavity of H2-c-fos mice was 4-fold larger than that in control littermates. Although the numbers of total peritoneal B cells and B-1a cells were similar between them, the peritoneal B-2 cell number in H2-c-fos mice was reduced to 50% of control littermates, suggesting the effect of c-fos overexpression on a balance of B-1b and B-2 cells in a peritoneal cavity. Adoptive transfer experiments with hematopoietic stem cells of H2-c-fos and control mice into irradiated H2-c-fos mice demonstrated that the augmentation of B-1b cells is due to the c-fos effect in B cells and the effect on environment of the peritoneal cavity of H2-c-fos mice. When peritoneal B cells were cultured with LPS in the presence or absence of IL-4, cell proliferation of B-1b cells was the highest among these peritoneal B cell subsets, and the proliferation of H2-c-fos B-1b cells was 3-fold higher than that of control B-1b cells. This augmentation is due to the c-fos effect in B cells. IgG1 production of B-1b cells in these cultures was slightly higher than those of B-1a and peritoneal B-2 cells. Thus, the c-fos overexpression augments development of B-1b cells in a peritoneal cavity and proliferation of peritoneal B-1b cells to LPS.

BAZF, a member of the Bcl6 gene family, acts as a sequence-specific transcriptional repressor in association with Bcl6. However, the tissue expression pattern of BAZF differs from that of Bcl6, suggesting a Bcl6-independent function of BAZF. In order to examine the physiological function of BAZF, we generated BAZF-deficient mice and transgenic mice with BAZF-cDNA under the control of the lck proximal promoter (lck-BAZF). These mice were viable and no gross anatomical abnormalities were observed after birth. Since Bcl6 is a key molecule for the generation of memory T cells, we examined the function of T cells of these mice. We show here that cell proliferation of naive CD4 T cells, but not memory ones, of BAZF-deficient mice to anti-CD3 antibody stimulation was impaired. Conversely, cell proliferation of naive CD4 T cells, but not memory ones, of lck-BAZF mice was augmented. Since cell proliferation of naive CD4 T cells of lck-Bcl6 mice to anti-CD3 antibody stimulation was severely impaired, BAZF may attenuate the regulatory effect of Bcl6 on antigenic activation of naive CD4 T cells by Bcl6/BAZF heterodimer formation. These results suggest that BAZF is necessary for activation of naive T cells to antigenic stimulation.

Background. Hematopoietic reconstitution after stem cell transplantation has been analyzed by using stem cells of Ly5 congenic mice. However, the early erythropoiesis has never been analyzed because this marker is not expressed on all of the erythroid lineage cells. The transgenic mouse expressing beta-galactosidase (beta-gal) or green fluorescent protein (GFP) has been reported. Using these markers, we analyzed the early erythropoiesis after stem cell transplantation. Methods. The beta-gal activity and GFP were examined in the hematopoietic cells of ROSA26 and GFP transgenic mice, respectively, by flow cytometry. The primitive hematopoietic stem cell fraction (Lin(-)c-kit(+)Sca-1(+)) in bone marrow (BM) cells of ROSA26 mice was transferred into lethally irradiated mice. The kinetics of hematopoietic reconstitution was analyzed in the BM and spleen after transplantation. Results. The beta-gal activity, but not the GFP and Ly5, was detected in all of the erythroid (TER119(+)) cells. The beta-gal activity was also detected in the donor-derived myeloid (Mac-1(+)), B lymphoid (B220(+)), and T lymphoid (Thy-1(+)) cells in the BM and spleen after stem cell transplantation. The kinetics of the hematopoietic reconstitution demonstrated that early erythroid (TER119(low)-CD71(med)) cells were developed in the BM and spleen within 2 days after transplantation before development of proerythroblasts (TER119(+)CD71(high)), and that massive erythropoiesis and myelopoiesis were observed in the spleen until 2 and 4 weeks after transplantation, respectively. Conclusions. The beta-gal of ROSA26 mice can be a useful marker to identify the donor-derived hematopoietic cells, including early erythroid cells, and the first major wave of erythropoiesis occurring in the spleen after stem cell transplantation.

Clast5/Stra13/DEC1 is a member of the helix-loop-helix family of transcriptional repressors. We have previously shown that Clast5 is rapidly down-regulated upon B cell activation and its overexpression inhibits cell cycle progression in B lymphoma cells. In the present study, we show that Clast5 expression is developmentally regulated during B cell differentiation, being expressed at the progenitor B cells, down-regulated at the precursor B cells, elevated in immature and mature resting B lymphocytes, and down-regulated again in germinal center B ells. To investigate the function of Clast5 in regulating lymphocyte development, we have generated transgenic mice expressing Clast5 in B- and T-lineage cells (Clast5-Tg). Clast5-Tg mice grew and bred normally but their spleen and thymus cellularity was reduced compared with control littermates. The development of B cells in the bone marrow and T cells in the thymus was impaired, with the expansion of progenitor 13 and T cells most strongly affected. The frequency of IL-7-responsive cells in the bone marrow of Clast5-Tg mice was reduced by >80% and their proliferative response to IL-7 was also compromised. Mature B cells from Clast5-Tg mice were hyporesponsive to antigen receptor cross-linking and exhibited mild reduction in the proliferative response to CD40 ligation or lipopolysaccharide stimulation. Moreover, the development of germinal center B cells and antibody production against a T-dependent antigen were reduced in Clast5-Tg mice. These results reveal a critical role for Clast5/Stra13/DEC1 in negatively regulating lymphocyte development and function in vivo.

Clast5/Stra13/DEC1 is a member of the helix-loop-helix family of transcriptional repressors. We have previously shown that Clast5 is rapidly down-regulated upon B cell activation and its overexpression inhibits cell cycle progression in B lymphoma cells. In the present study, we show that Clast5 expression is developmentally regulated during B cell differentiation, being expressed at the progenitor B cells, down-regulated at the precursor B cells, elevated in immature and mature resting B lymphocytes, and down-regulated again in germinal center B ells. To investigate the function of Clast5 in regulating lymphocyte development, we have generated transgenic mice expressing Clast5 in B- and T-lineage cells (Clast5-Tg). Clast5-Tg mice grew and bred normally but their spleen and thymus cellularity was reduced compared with control littermates. The development of B cells in the bone marrow and T cells in the thymus was impaired, with the expansion of progenitor 13 and T cells most strongly affected. The frequency of IL-7-responsive cells in the bone marrow of Clast5-Tg mice was reduced by >80% and their proliferative response to IL-7 was also compromised. Mature B cells from Clast5-Tg mice were hyporesponsive to antigen receptor cross-linking and exhibited mild reduction in the proliferative response to CD40 ligation or lipopolysaccharide stimulation. Moreover, the development of germinal center B cells and antibody production against a T-dependent antigen were reduced in Clast5-Tg mice. These results reveal a critical role for Clast5/Stra13/DEC1 in negatively regulating lymphocyte development and function in vivo.

Bc16 functions as a sequence-specific transcriptional repressor, and Bc16-deficient (Bc16(-/-)) mice have been reported to display Th2-type inflammatory diseases in multiple organs. Since IL-18 is a potent stimulator of Th2 cells, we examined the expression of IL-18 mRNA in bone marrow-derived macrophages from Bc16(-/-) mice after LPS stimulation. Here we show that the expression was strikingly up-regulated after stimulation. The expression was also up-regulated in RAW264 cells, a murine macrophage cell line, by transfection with the dominant negative type of Bc16 gene. We identified a putative Bc16-binding DNA sequence (IL-18BS) upstream of exon 1 of the murine IL-18 gene and three EL-18BSs in the promoter region of human IL-18 gene. Binding of Bc16 in nuclear protein from resting RAW264 cells to murine IL-18BS was detected by gel retardation assay and chromatin immunoprecipitation assay. The binding activity was diminished gradually in RAW264 cells after LPS stimulation. However, the amount of Bc16 protein in these cells was constant over the period examined, suggesting the functional modification of Bc16 protein after stimulation. Furthermore, murine IL-18BS was required for Bc16 to repress the expression of the luciferase reporter gene under control of the IL-18 promoter. Taken together, Bc16 is a key regulator of IL-18 production by macrophages.

Previously we demonstrated that IL-15 and IL-2 control the number of memory CD8+ T cells in mice. IL-15 induces, and IL-2 suppresses the division of these cells. Here we show that CD25+CD4+ regulatory T cells play an important role in the IL-2-mediated control of memory phenotype CD8+ T cell number. in animals, the numbers of CD25+CD4+ T cells were inversely correlated with the numbers of memory phenotype CD8+ T cells with age. Treatment with anti-IL-2 caused CD25+CD4+ T cells to disappear and, concurrently, increased the numbers of memory phenotype CD8+ T cells. This increase in the numbers of CD8+ memory phenotype T cells was not manifest in animals lacking CD4+ cells. Importantly, adoptive transfer of CD25+CD4+ T cells significantly reduced division of memory phenotype CD8+ T cells. Thus, we conclude that CD25+CD4+ T cells are involved in the IL-2-mediated inhibition of memory CD8+ T cell division and that IL-2 controls memory phenotype CD8+ T cell numbers at least in part through maintenance of the CD25+CD4+ T cell population.

Naive T cells proliferate and differentiate into memory cells after antigenic stimulation or in a lymphopenic environment. We showed here transient increases in memory phenotype CD8(+) T cell numbers in the lymphopenic environment of spleens of very young mice. The magnitude of the increase correlated with Bcl-6 expression in the T cells. Bcl-6 controlled the generation and maintenance of antigen-specific memory phenotype CD8(+) T cells in the spleens of immunized mice. These data suggest that Bcl-6, which is essential for memory B cell development in germinal centers, is a key molecule for the establishment not only of memory T cells but also of the peripheral T cell compartment in infancy.

Naive T cells proliferate and differentiate into memory cells after antigenic stimulation or in a lymphopenic environment. We showed here transient increases in memory phenotype CD8(+) T cell numbers in the lymphopenic environment of spleens of very young mice. The magnitude of the increase correlated with Bcl-6 expression in the T cells. Bcl-6 controlled the generation and maintenance of antigen-specific memory phenotype CD8(+) T cells in the spleens of immunized mice. These data suggest that Bcl-6, which is essential for memory B cell development in germinal centers, is a key molecule for the establishment not only of memory T cells but also of the peripheral T cell compartment in infancy.

Memory T cells maintain their numbers for long periods after antigen exposure, Here we show that CD8(+) T cells of memory phenotype divide slowly in animals. This division requires interleukin-15 and is markedly increased by inhibition of interleukin-2 (IL-2), Therefore, the numbers of CD8(+) memory T cells in animals are controlled by a balance between IL-15 and IL-2.