Induction of autoimmune gastritis by neonatal thymectomy requires autoantibodies and is prevented by anti-FcγR antibodies.

The autoantibodies (auto-Abs) that are a hallmark of neonatally thymectomized (NTx) mice with autoimmune gastritis (AIG) have been poorly explored. We investigated their immune significance using B cell-deficient (B(-)) mice and found that B(-) mice are totally resistant to AIG but become susceptible to AIG after receiving bone marrow cells from B(+) mice. This susceptibility is most likely caused by the production of auto-Abs by B cells because B(-) pups also became susceptible to AIG when nourished by an AIG dam producing auto-Abs of the IgG class during the suckling period. NTx B(-) mice receiving purified IgG auto-Abs at this developmental stage similarly developed AIG. Auto-Abs probably act on antigen handling for antigen presentation because the treatment of NTx B(+) mice with anti-FcγR Abs prevented the development of AIG. Auto-Abs are indispensable for AIG development but are not sufficient because auto-Ab treatment did not increase AIG incidence in NTx B(+) mice above the baseline.

Population doublings of murine CD4(+) memory T cells during continuous antigen stimulation in vivo.

We investigated the expansion rate of CD4(+) memory T cells using a newly developed in vivo system. Neonatal thymectomy abrogates the subsequent production of T cells and induces autoimmune gastritis (AIG) by the activation of CD4(+) T cells; this disease was transferred into athymic nude mice through the inoculation of splenic CD4(+) memory T cells. The transferred CD4(+) T cells increased logarithmically in number during the first 2months in the spleen of the recipients. The serial transfer of these splenocytes at two-month intervals revealed that the numbers of the AIG-transferable generations were inversely correlated with the age of the first AIG donors. The duration of the AIG-promoting capacity of CD4(+) T cells under continuous antigenic stimulation in vivo was approximately equivalent-one and a half years. These results indicate that there exists an intrinsic population doubling limit in memory CD4(+) T cells similar to that of self-renewing naïve ones.

Dominant trait linked to chromosome 1 in DBA/2 mice for the resistance to autoimmune gastritis appears in bone marrow cells.

Neonatal thymectomy (NTx) induces autoimmune gastritis (AIG) in BALB/c mice, a model for human type A chronic atrophic gastritis, but not in DBA/2 mice and rarely in CDF1 mice (a hybrid of BALB/c and DBA/2 mice). The aim of this study was to clarify the mechanisms of AIG-resistance in mice bearing the dominant trait of DBA/2. Linkage groups associated with, and cells related to AIG resistance were examined with CDF1-BALB/c backcrosses. Intracellular staining and flow-cytometric bead array for several cytokines were performed on NTx BALB/c mice and NTx DBA/2-chimeric BALB/c mice receiving DBA/2-bone marrow cells. In NTx BALB/c mice, IFN-γ-secreting CD4(+) T cells were increased, but not in NTx DBA/2 mice. Because Vß6(+) T cell-bearing mice of half of their backcrosses developed AIG, but the other half of Vß6(+) T cell-negative mice developed scarcely, resistance for AIG generation is associated with the presence of the Mls-1a locus on chromosome 1 in DBA/2 mice, which deletes Vß6(+) T cells. NTx DBA/2-chimera BALB/c mice showed dominant production of IL-10 and resistance for AIG, although the deletion of Vß6(+) T cells was found not to be a cause of AIG-resistance from Mls-1a locus segregation experiments. Although NTx DBA/2-chimeric BALB/c mice did not suffer from AIG, they brought immediate precursors of T cells for AIG. It is concluded that DBA/2 mice generate bone marrow-derived cells that produce anti-inflammatory cytokines to prevent the activation of AIG-T cells.

Limited immune diversity in urodela: chronic transplantation responses occur even with family-disparate xenografts.

Urodele amphibians are thought to have poorer immune responses than evolutionary more ancestral vertebrate classes, such as bony fish. We investigated skin graft rejection and transplantation immunity in Urodele amphibians, Japanese newts, and Asiatic salamanders, and compared these findings to those from transplants in several species of frogs. The skin grafts used in this study were either allogeneic or xenogeneic. The mean survival time of the first set of allografts at 20°C was approximately 60 days for chronic responses in Urodela and 20 days for acute responses in Anura. As the graft survival times of urodeles were significantly longer than those of anurans, even when urodeles were repeatedly grafted from identical donors, there appear to be substantial differences in transplantation immunity between Urodela and Anura. These slow responses in Urodela may not be accompanied by the expansion of cytotoxic T cells, as observed in fish and anuran species, which are known to have functional major histocompatibility complex (MHC)-class I systems. In our study, approximately five histo-incompatible immunogenic components were found to be involved in chronic responses in newts. Similar chronic responses were also observed in xenograft rejection in newts. In contrast, xenografts were rejected in frogs due to an accelerated acute response, possibly involving natural killer cells. Our findings that some anti-allogeneic components appear to be shared with xenogeneic components indicate that the diversification of the acquired immune system is poorly developed in Urodela.

T cells affect thymic involution during puberty by inducing regression of the adrenal reticularis.

The thymus involutes after puberty, although the mechanism by which this process occurs remains poorly understood. The profile of thymic involution, which is inversely correlated with an increase in peripheral T cells, may indicate that the accumulation of T cells in the periphery is related to thymic atrophy. In this study, it was shown that the prevention of T cell generation delayed the initiation of thymic involution. The activation of T cells increased the serum concentration of glucocorticoid (GC) and thymic involution, which was completely prevented by adrenalectomy. In the adrenals of growing mice, the activity of the zona fasciculata, which produces GC, increased and plateaued by the weaning period; however, the zona reticularis (ZR), which produces dehydroepiandrosterone (DHEA) that has anti-GC actions, started to decline just before puberty. Thymic atrophy was preceded by the infiltration of activated T cells into the ZR and by the loss of ZR cells. Thus, T cells are involved in thymic involution, a process which was retarded by DHEA administration, through an increase in GC activity due to ZR cell-killing.

Chronic transplantation immunity in newts: temperature susceptibility of an effector phase in allo-skin graft rejection.

Urodele amphibians are unique due to their greatly reduced immune responsiveness compared to bony fishes, which show acute immune responsiveness. In newts, the mean survival time of allogenic skin grafts in the transplantation immunity was 48.8 ± 8.3 days at 25°C, suggesting that it occurs in a chronic manner. The graft rejection process was categorized into three stages: a latent stage with frequent blood circulation, or the immune induction phase; a vascular stoppage stage with dominant infiltrating cells of T cells; and a rejection stage showing the change of the dominant cells to monocytes/macrophages, probably as effector cells, tetntatively referred to as the immune effector phase. The immune induction phase is susceptible to the cyclophosphamide (CY) mitosis inhibitor, but not to a temperature shift from 18 to 27°C, while the immune effector phase is susceptible to temperature shifts, but not CY-treatment, although the temperature shift failed to shorten the graft survival time to less than 25 days, which nearly equals that of the secondary set of grafts where the lack of complete blood circulation is remarkable and graft rejection is resistant to CY-treatment. In contrast, a very low temperature (5-10°C) completely prevented effector generation in newts; in frogs, however, it is reported that such low temperatures did not prevent the generation of effectors. Taken together, these data suggest that chronic responses in newts are due to effector cells other than cytotoxic T cells; possible effector cells are discussed.

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.

Different pathological phenotypes of autoimmune gastritis induced by neonatal thymectomy between BALB/c and (BALB/c x DBA/2) F1 mice: role of eosinophils in hypertrophic autoimmune gastritis.

BACKGROUND: A unique pathological feature of murine autoimmune gastritis (AIG) is its pronounced mucosal hypertrophy, which is different from human type A chronic atrophic gastritis with pernicious anemia. The aim of this study was to clarify the mechanism of gastric hypertrophy in murine AIG, especially in relation to inflammatory cells infiltrating the gastric mucosa. METHODS: Neonatally thymectomized (NTx) BALB/c and (BALB/c x DBA/2) F1 mice with gastritis were examined histologically and serologically. The T-helper (Th1/Th2) immune balance in the spleen was evaluated by intracellular cytokine staining for interferon-gamma and a flow-cytometric beads array for several cytokines. Additionally, NTx AIG BALB/c mice were orally administered an H(2)-blocker to decrease eosinophils. RESULTS: NTx AIG BALB/c mice exhibited gastritis without stomach hypertrophy at 2 months of age, and developed gastritis with mucous gland hypertrophy accompanied by eosinophil infiltration at 6 months of age. In contrast, NTx AIG (BALB/c x DBA/2) F1 mice displayed gastritis with neither stomach hypertrophy nor eosinophil infiltration even at the age of 6 months. Upregulation of interleukin-4 and granulocyte-macrophage colony-stimulating factor in the spleen was observed in BALB/c mice but not in (BALB/c x DBA/2) F1 mice. Additionally, some NTx AIG BALB/c mice did not show gastric hypertrophy or eosinophil infiltration owing to the administration of an H(2)-blocker. CONCLUSIONS: There are two different pathological phenotypes of murine AIG, chronic gastritis and hypertrophic gastritis, in NTx AIG BALB/c mice. Furthermore, eosinophil infiltration and the Th2 immune response might play a key role in the phenotypic shift from chronic gastritis to hypertrophic gastritis in these mice.

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

The role of chemokines, especially CXCL10/interferon-gamma-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 Crohn's 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.

Adrenal participation in thymocyte death by anti-CD3 antibodies in vivo.

The deletion of CD4- and CD8-double-positive (DP) cells in the thymus after treatment with anti-CD3 antibodies has long been considered as a useful model for clonal deletion during T cell development, although it was reported that DP cell death was not observed in neonates where self-tolerance should be developing. We dealt with the cellular basis of this enigmatic phenomenon in this report. Due to the similar susceptibility to the antibody-treatment in vitro between neonatal and adult thymocytes, critical factors may be outside rather than within the thymus. Indeed, newborn thymus lobes transplanted into recipients of different ages showed an increased susceptibility to the thymo-toxicity as the age of the recipient increased. The thymo-toxicity seems to be based on the adrenal function of glucocorticoid (GC) synthesis, because administration of an inhibitor of GC synthesis significantly reduced the DP cell death by the antibody-treatment. Finally, adrenalectomy completely prevented DP cell death by anti-CD3 antibodies in adult mice. Therefore, the thymocyte death by anti-CD3 antibodies in vivo may not be due to the T cellreceptor mediated selection in the thymus.

Murine serum obtained from bone marrow-transplanted mice promotes the proliferation of hematopoietic stem cells by co-culture with MS-5 murine stromal cells.

To examine whether serum obtained from bone marrow-transplanted mice can selectively expand hematopoietic stem cells (HSCs) among whole bone marrow cells in vitro, whole bone marrow cells were cultured with or without MS-5 murine stromal cells in the presence of serum obtained from transplanted mice on day 3 (day 3 serum) or serum from normal mice for 7 days. When whole bone marrow cells and MS-5 cells were co-cultured in day 3 serum for 7 days, the c-kit-positive, Sca-1-positive, lineage marker-negative cells (KSL cells) expanded approximately 25 times; however, when they were co-cultured in normal serum for 7 days, the KSL cells expanded approximately 1.3 times. Direct contact between the whole bone marrow cells and MS-5 cells was essential for expansion of KSL cells in the co-culture, and it upregulated the expression of some cytokines in MS-5. Above all, the day 3 serum specifically upregulated the expression of SCF, SDF-1 alpha, G-CSF, IL-11 and IL-6 in MS-5. The level of testosterone in the day 3 serum was higher than normal serum and the addition of the testosterone in the culture expanded the KSL cells among whole bone marrow cells on MS-5 cells and also upregulated the expression of SDF-1 alpha, IL-11 and IL-6 in MS-5. These data indicates that the serum of bone marrow-transplanted mice contains a factor(s) that induced changes in the expression levels of various cytokines in MS-5 stromal cells and enabled the MS-5 cells to expand the KSL cells among whole bone marrow cells.

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-gamma (IFN-gamma) 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.