Masamichi Hosono

A novel murine model of aging, Senescence-Accelerated Mouse (SAM)

Senescence-Accelerated Mouse (SAM) has been under development by our research team at Kyoto University since 1970, based on the AKR/J strain donated by the Jackson Laboratory in 1968. The SAM mouse has an accelerated senescence and age-associated pathologies such as senile amyloidosis, senile osteoporosis, degenerative joint disease, cataract, deficits in learning and memory, brain atrophy, hyperinflation of lungs, hearing impairment and so on. SAM research is advancing world-wide and attempts are being made to clarify fundamental mechanisms involved in primary aging processes, pathogenesis of age-associated pathologies and effective methods to modulate or ameliorate the advance of senescence and disease processes involved in age-associated pathologies.

A conserved epitope on H+,K+-adenosine triphosphatase of parietal cells discerned by a murine gastritogenic T-cell clone

BACKGROUND/AIMS H+,K(+)-adenosine triphosphatase (H+,K(+)-ATPase) of parietal cells is an organ-specific enzyme recognized by autoantibodies found in human and murine autoimmune gastritis (AIG). Murine AIG can be induced in BALB/c mice by thymectomy 3 days after birth and is a T cell-mediated disease. This study examined the specificity of T cells that cause AIG and the role of H+,K(+)-ATPase in this disease. METHODS From an AIG mouse, a gastritogenic T-cell clone (II-6) was established, and its reactivity to synthetic peptides of H+,K(+)-ATPase was tested. RESULTS II-6 cells are CD4+, V beta 14+, and interferon gamma producers. Adoptive transfer of II-6 cells to syngeneic nude mice resulted in AIG without the production of autoantibodies to parietal cells. The II-6 cells were responsive not only to murine but also to human and porcine parietal cells. Their proliferation was also induced by amino acids 891-905 (alpha 891) and 892-906 (alpha 892) of the alpha subunit of porcine and human H+,K(+)-ATPase, respectively. CONCLUSIONS The T-cell response to a single epitope of H+,K(+)-ATPase, the amino acid sequence of which is conserved among at least three mammals tested, is sufficient to cause AIG. Autoantibodies to parietal cells are not detected in these AIG mice.

Involvement of pro-inflammatory cytokines and microglia in an age-associated neurodegeneration model, the SAMP10 mouse

The SAMP10 mouse strain is a model of brain aging in which senescence is characterized by cerebral atrophy and neurodegeneration phenotypes. To investigate the role of neuroinflammation in the age-associated neurodegeneration of SAMP10 mice, we assessed the expression of several cytokines and chemokines in the atrophy-prone brain region of SAMP10, and control, SAMR1 mice, which show a normal aging process. We also studied morphological changes in microglia with advancing age in atrophied regions. The expression of IL-1beta and IFN-gamma mRNA was about 2-fold greater in SAMP10 mice as compared to SAMR1 mice throughout their life span. The expression of IL-6 mRNA was 2.0-fold greater in SAMP10 mice as compared to SAMR1 mice at 14 months of age, although there was no difference at 3 months of age. Fourteen-month-old mice had a 2.1-fold greater expression of TNF-alpha mRNA than 3-month-old mice in both strains. The expression of MCP-1 mRNA was greater in SAMP10 mice than SAMR1 mice, and tended to increase with advancing age. Activated microglia were rarely observed in both strains at 3 months of age. At 14 months of age, however, SAMP10 mice had a 5.6-fold greater number of activated microglia than SAMR1 mice. The aforementioned results suggest the presence of a higher pro-inflammatory status in the atrophy-prone brain region of SAMP10 mice as compared to SAMR1 mice. Neuroinflammation is a possible mechanism of age-associated neurodegeneration in SAMP10 mice.

Blockade of interferon-γ-inducible protein-10 attenuates chronic experimental colitis by blocking cellular trafficking and protecting intestinal epithelial cells

The role of chemokines, especially CXCL10/interferon‐γ‐inducible protein 10 kDa (IP‐10), a chemokine to attract CXCR3+ T‐helper 1‐type CD4+ T cells, is largely unknown in the pathophysiology of inflammatory bowel disease; ulcerative colitis and Crohns disease. The authors have earlier shown that IP‐10 neutralization protected mice from acute colitis by protecting crypt epithelial cells of the colon. To investigate the therapeutic effect of neutralization of IP‐10 on chronic colitis, an anti‐IP‐10 antibody was injected into mice with newly established murine AIDS (MAIDS) colitis. Anti‐IP‐10 antibody treatment reduced the number of colon infiltrating cells when compared to those mice given a control antibody. The treatment made the length of the crypt of the colon greater than control antibody. The number of Ki67+ proliferating epithelial cells was increased by the anti‐IP‐10 antibody treatment. Terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL)+ apoptotic cells were observed in the epithelial cells of the luminal tops of crypts in control MAIDS colitis, whereas TUNEL+ apoptotic epithelial cells were rarely observed with anti‐IP‐10 antibody treatment. In conclusion, blockade of IP‐10 attenuated MAIDS colitis through blocking cellular trafficking and protecting intestinal epithelial cells, suggesting that IP‐10 plays a key role in the development of inflammatory bowel disease as well as in chronic experimental colitis.

Age-related decline in humoral immunity caused by the selective loss of TH cells and decline in cellular immunity caused by the impaired migration of inflammatory cells without a loss of TDTH cells in SAMP1 mice

We investigated the cellular basis of the age-related decline in antibody (Ab) and delayed-type hypersensitivity (DTH) responses to sheep red blood cells (SRBC) in vivo in short-lived senescence-accelerated mouse (SAM) P1. In SAMP1 mice, age-related decreases in CD4+ T cells in the peripheral blood occurred earlier than in control mice and occurred in parallel with the age-related decline in Ab and DTH responses. In addition, the involution of the thymus was faster. The injection of thymic T cells from young mice before sensitization completely restored the Ab responses in aged SAMP1 mice. These data suggest that the age-related decline in Ab response is due to the age-related early loss of helper-T (TH) cells. On the other hand, the local transfer of spleen cells from sensitized aged donors into the footpads of naive syngeneic recipients evoked strong DTH responses, demonstrating the existence of DTH-mediating T (TDTH) cells in the spleens of aged SAMP1 mice. Moreover, the local injection of naive spleen cells from young donors, together with the antigen, into the footpads caused DTH responses in sensitized aged recipients. These findings indicate that TDTH cells were induced and were able to migrate and function as effector cells in aged mice. When naive spleen cells from aged donors were injected locally into the footpad, they restored the DTH response in aged mice, but this effect did not work if the cells were injected intravenously. This demonstrates that the inflammatory cells of the aged mice were able to work at the local site, but could not migrate there. The intravenous injection of naive spleen cells from young donors restored the DTH response in aged mice, suggesting that the endothelial cells of aged mice were not impaired and permitted the inflammatory cells to migrate into the extravascular tissues. Thus, although the age-related decline of the Ab and DTH responses occur in parallel, we found different effects of aging on TH and TDTH cells in SAMP1 mice. Furthermore, our data suggest that the reason for the low DTH response in aged SAMP1 mice is not the loss of TDTH cells, but rather the impaired migration of inflammatory cells into the local site.

Immune abnormality in relation to nonimmune diseases in sam mice

A series of related strains of senescence-accelerated mouse (SAM) shows strain-unique age-related diseases, such as amyloidosis, deficit in learning and memory, osteoporosis, and brain atrophy, while many of these disease-prone mouse (SAMP) strains have impaired immune activity as young adults, and have a short life span, probably not due directly to the diseases. Because the mean life span was prolonged and the time of the disease onset was delayed by a low-calorie dietary condition or a specific pathogen-free environment, both of which ameliorate the impaired immune activity, the enhancement of immune activity may help decrease the deteriorative process of aging, to that seen in ordinary strains of mice. Studies using the SAMP model may help elucidate the role of immunity in the aging process. Herein, we review the cellular and genetic basis of the immune abnormality in SAMP mice, then discuss the relationship between immune abnormality and development of the age-related disease, senile amyloidosis, findings obtained on SAMP hybrid mice and congenic mice for disease-related genes. Activation of the gene(s) for senile amyloid per se shortened the life span, and the early development of the immune dysfunction primarily seems to be both genetically and physiologically independent of amyloidosis, although the disease may be indirectly modified in the aged with depressed immune activity.

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. I. Preferential induction of tolerance to mls antigens and resistance to allo-MHC antigens

Neonatal tolerance inducibility of self-major histocompatibility complex (MHC)-class II-associated antigens was compared with that of allo-class II antigens. BALB/c (H-2d, Mlsb) mice, less than 24 hr after birth, were intravenously injected with bone marrow cells of either (BALB/c X DBA/2)F1 (H-2d, Mlsb/a, semiallogeneic at the Mls locus) or (BALB/c X B10.BR)F1 (H-2d/k, Mlsb; semiallogeneic at the MHC), as antigens. The mice were tested for in vivo immune activity of class II-reactive T cells by means of the popliteal lymph node-swelling assay. They developed tolerance, irrespective of type of antigens, showing profoundly suppressed host-versus-graft reaction, and those tolerized to the allo-MHC antigens accepted skin grafts of the corresponding allogeneic mice. In the thymus and spleen of the Mls-tolerant mice, antigen-specific class II-reactive T-cell activity was completely abolished, without the apparent involvement of suppressor cells. In contrast, the activity in allo-MHC-tolerant mice was not reduced in either thymus or peripheral lymphoid organs, suggesting that systemic hyporesponsiveness is attributable to reversible suppression of immune competent cells. The resistance for cell-level tolerance induction to allo-class II antigens may not be ascribed to the active participation of allo-MHC antigens in prevention of or in escape from tolerance induction or both, since an injection of bone marrow cells of both Mls and H-2-semiallogeneic (DBA/2 X B10.BR)F1 (H-2d/k, Mlsa/b) mice could induce tolerance to Mlsa-H-2d antigens in newborn thymus cells.

Thymic Dendritic Cells and B Cells: Isolation and Function

The thymus is the primary organ in which T cells undergo rearrangement of T cell receptor alpha and beta genes, positive selection for affinity to self MHC products, and elimination (negative selection) of reactivity to self antigens. These events require an interaction of the developing T cell with other cell types in the thymus. The latter include epithelial cells, macrophages, dendritic cells, and the recently described thymic B cells the majority of which are CD5+. Here we review the identification and isolation of thymic dendritic cells and CD5+ B cells. We consider phenotype, ontogeny, and function, including possible contributions to the induction of self tolerance. Thymic dendritic cells are similar to spleen dendritic cells, but are larger and exhibit a few differences in phenotype. Dendritic cells from both organs are equally potent accessory cells for the MLR and lectin-induced, T cell proliferation. Thymic dendritic cells have higher levels of Fc receptors and support anti-CD3 dependent mitogenesis. Thymic CD5+ B cells share phenotypic features with peritoneal CD5+ B cells. However thymic B cells neither proliferate nor form antibody producing cells in response to the stimulation with LPS or anti-IgM plus IL-4, but do respond to stimulation with MHC class II-restricted helper T cells. Thymic dendritic cells and CD5+ B cells both appear at a similar time in ontogeny, about 14 d of gestation, which is the time T cell differentiation begins to take place. Dendritic cells from spleen, which are potent activators for peripheral T cells, are also potent inactivators for thymic-derived cytotoxic T cells. A correlation between reactivity to MIs products and the expression of TCR-V beta genes is well documented, and B cells are the primary APC for this antigen. Therefore, thymic CD5+ B cells may be a good tool for the investigation of tolerance to M1s products.

Studies on B‐Cell Memory

Effects of LPS on primary and secondary antibody responses to typical TI‐2 antigens were investigated in mice. Simultaneous injection of LPS with a TI‐2 antigen showed only little adjuvant effect on the following primary antibody response to the antigen. In contrast, either a single or multiple injections of LPS, prior to the immunization with a TI‐2 antigen, significantly augmented the following primary antibody response to the antigen. LPS, however, inhibited the development of B‐cell memory to a TI‐2 antigen when administered together with the antigen. Moreover, an injection of LPS in mice, which had strong IgM and IgG B‐cell memories to a TI‐2 antigen, caused disappearance or profound reduction of the memories. The results suggest that LPS produced by gram‐negative bacteria exerts inhibitory effects on the development and continuation of B‐cell memory to bacterial infections.

Insufficient interleukin‐2 production from splenic CD4+ T cells causes impaired cell proliferation and early apoptosis in SAMP1, a strain of senescence‐accelerated mouse

We examined the proliferative and cytokine‐producing activities of CD4+ T cells from young mice of the senescence‐accelerated mouse strain SAMP1, which had shown markedly low T‐dependent antibody‐producing responses. When splenic T cells were cultured with concanavalin A (Con A), the percentage of CD4+ cells decreased earlier in SAMP1 than in C3H/He mice. At 40 hr of culture, the percentage of BrdU‐labelled proliferating CD4+ cells increased strongly in C3H/He, but only slightly in SAMP1. When purified CD4+ T cells were cultured with Con A, the percentage of 5‐bromo‐2′‐deoxyuridine (BrdU)‐labelled cells peaked at around 48 hr of culture in both strains, but decreased significantly at 64 hr in SAMP1. The production of interleukin (IL)‐2 but not IL‐4 or interferon‐γ (IFN‐γ) was significantly lower in SAMP1 than in C3H/He at 48 hr of culture. IL‐2 production was also markedly low in SAMP1, even under the stimulation of anti‐CD3 with anti‐CD28 antibodies. The frequency of cells producing IL‐2 was significantly lower in SAMP1 than in C3H/He at 6–24 hr of culture with Con A. The percentage of annexin‐positive and propidium iodide (PI)‐negative apoptotic cells was significantly higher in SAMP1 than in C3H/He at 96 hr of culture. Exogenous IL‐2 prevented the decrease in BrdU‐labelled cells and the increase in apoptotic cells in the SAMP1 cell culture. These results indicate that SAMP1 CD4+ T cells cannot produce IL‐2 at levels sufficient to support cell proliferation and survival. This may account for the weak T‐dependent antibody response in SAMP1 mice.