Yasuo Amoh

Induction of donor-specific T cell anergy by portal venous injection of allogeneic cells

The mechanisms behind tolerance induction by portal venous (pv) injection of allogeneic cells are investigated. When a hematopoietic stem cell (HSC)-enriched population of BALB/c bone marrow was pv injected into C57BL/6 mice, the response of the T cells in the B6 mice to BALB/c alloantigens in mixed lymphocyte reaction (MLR) decreased until day 4 after the injection. Neither clonal deletion of V beta 11+ T cell nor donor-specific suppressor activity was observed. When recipient T cells were separated into CD4+ and CD8+ cells, only the CD8+ cell population showed donor-specific tolerance. The donor cells were trapped and retained in the host liver. MHC class I antigens were highly expressed on the trapped cells whereas class II antigens or B7 costimulatory molecules were not. The tolerance to BALB/c alloantigens in MLR was obtained also by the pv injection of Meth A, a BALB/c-derived sarcoma cell line. However, tolerance was not induced by the pv injection of B7-transfected Meth A cells. In addition to MLR, tolerance was also observed in DTH responses, and this was also due to the unresponsiveness of CD8+ cells to the donor alloantigens. However, the BALB/c-specific DTH responses were not suppressed after the pv injection of B7-transfected Meth A cells. These results strongly suggest that the tolerance induced by pv injection of allogeneic cells is due to clonal anergy generated by the absence of costimulatory signals in the interaction between donor-specific CD8+ T cells and donor hematopoietic cells trapped in the host liver.

EFFECT OF BONE MARROW TRANSPLANTATION ON ANTI-PHOSPHOLIPID ANTIBODY SYNDROME IN MURINE LUPUS MICE

The (NZW x BXSB)F1 (W/BF1) mouse is known to be an animal model of systemic lupus erythematosus (SLE) and immune thrombocytopenic purpura (ITP). These mice produce not only anti-DNA antibodies but also anti-platelet antibodies, resulting in decreased platelet counts. They show a high level of proteinuria, increased white blood cell (WBC) counts, hypertension, and myocardial infarction due to the high levels of anti-cardiolipin antibodies. When W/BF1 mice (4-5 months) were lethally irradiated and then reconstituted with T cell-depleted bone marrow cells of normal BALB/c mice (8 weeks), 60% of the mice survived more than one year. The WBC and platelet counts in the mice were normalized, and the levels of anti-DNA and anti-platelet antibodies decreased. The renal dysfunction was also ameliorated as indicated by a lower level of proteinuria, lower levels of serum creatinine (S-CRTN) and blood urea nitrogen (BUN), and by improved histology. The blood pressure (BP) of the treated W/BF1 mice decreased due to the improved renal functions. In contrast to the non-treated W/BF1 mice which died of myocardial infarction or renal failure by the age of 7 months, the treated W/BF1 mice showed no evidence of myocardial infarction even one year after BMT. This was due to the lower cardiolipin levels.

Effects of Administration of Monoclonal Antibodies (anti-CD4 or anti-CD8) on the Development of Autoimmune Diseases in (NZW × BXSB)F1 Mice

(NZW x BXSB)F1 (W/BF1) mice spontaneously develop autoimmune diseases, characterized by lymphadenopathy, lupus nephritis, and immune thrombocytopenia associated with various autoantibodies such as anti-DNA, anti-platelet and anti-cardiolipin antibodies (Abs). In the present study, we investigate the effects of administration of monoclonal Abs (anti-CD4 or anti-CD8 mAb) on the development of autoimmune diseases in W/BF1 mice. MAb was administered from the age of 7 weeks. Prolongation of survival rate and reduction of severity of autoimmune diseases were observed after treatment with anti-CD4 mAb. However, anti-CD8 mAb treatment accelerated the diseases. Serum levels of IFN-gamma and IL-10 in old W/BF1 mice were significantly high, whereas IL-4 levels were low in comparison with those of young W/BF1 mice; the expression of mRNA of IFN-gamma, IL-4 or IL-10 in CD4+ T cells of old W/BF1 mice was parallel to the serum levels of each cytokine. These observations suggest that CD4+ cells are involved in the development of autoimmune diseases in W/BF1 mice, and that CD8+ cells have a suppressive effect on the development of autoimmune diseases in W/BF1 mice.

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.

Carcinoma of the colon in children: Case report and review of the Japanese literature

A 15-year-old female presenting with anemia and positive for occult blood was diagnosed as having an adenomatous polyp with mild atypia in the cecum by colonoscopy. Microscopically, the majority of the surface of the tubulovillous adenoma was occupied by a well-differentiated adenocarcinoma, indicating that the adenoma-carcinoma sequence is involved in the development of colon cancers, even in children. Forty-three cases of proven carcinoma of the colon in Japanese children aged under 15 years are also reviewed. The majority of the patients were aged over 10. Although an emergency laparotomy was undertaken in 42.5% of these patients, the signs and symptoms observed in these children did not markedly differ from those of adults. Colon cancer should not be excluded in children only on the basis of age, and barium enema and colonoscopy should therefore be applied to pediatric patients with unexplainable bleeding and abdominal pain, especially those over 10 years of age.

Increased neutrophil chemotaxis in obstructive jaundice: an in vitro experiment in rats.

Background and Aims: Changes in neutrophil functions in obstructive jaundice have been poorly understood. An in vitro experimental study was performed to evaluate the effect of obstructive jaundice on the functions of macrophages (secretion of neutrophil chemoattractants) and neutrophils (chemotaxis and superoxide anion generation).

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.

Influence of biliary obstruction on neutrophil chemotaxis

Abstract: The effect of obstructive jaundice on neutrophil chemotactic function was investigated, with a potent chemotactic factor, IL-8 (recombinant rat GRO-β), in rats that received 7-day bile duct ligation. Carrageenin or IL-8 was injected into a preformed air pouch, and exudate was collected 4 h later for measurement of myeloperoxidase activity. In vitro chemotaxis of peripheral neutrophils to IL-8 was evaluated by a modified Boyden chamber method. Both carrageenin and IL-8 induced significantly pronounced intra-air pouch neutrophil recruitment in the bile duct-ligated group compared with a sham-ligated group. In vitro neutrophil chemotaxis was significantly increased in the bile duct-ligated group compared with the sham-ligated group. The present experimental model suggests enhanced neutrophil chemotaxis to IL-8 in obstructive jaundice.

The appearance of unusual phenotypic cells (CD4+ Mac-1+ class II+) in the liver of (NZW × BXSB)F1 mice is possibly an animal model for autoimmune hepatitis

The male (NZW x BXSB)F1 (W/BF1) mouse, a murine model for autoimmune diseases, shows hepatosplenomegaly with lymphoid cell infiltration in the liver by 20 weeks of age. The majority of infiltrating cells are T cells, B cells and plasma cells, as seen in autoimmune hepatitis. Together with the increase in serum glutamate pyruvate transaminase (GPT) levels, anti-dsDNA antibody (Ab) and circulating immune complex (CIC) levels increase with age. These findings are compatible with those of autoimmune hepatitis in humans. In addition, a unique finding in this mouse is the accumulation of CD4+ Mac-1+ Class II+ cells in the sinusoidal space. The cells have the capacity to proliferate and differentiate into macrophages in vitro, indicating that they are the precursors of macrophages. This W/BF1 mouse provides a useful tool for not only analyzing the pathogenesis of autoimmune hepatitis but also establishing a new therapeutic strategy for it. In addition, we discuss the significance of the appearance of abnormal cells in autoimmune-prone mice.

Age-Dependent Abnormalities of Hematopoietic Stem Cells in (NZW × BXSB)F1 Mice

The (NZW × BXSB)F1 (W/BF1) mouse is known as an autoimmune‐prone strain which develops lupus nephritis, thrombocytopenia due to platelet‐specific autoantibodies, leukocytosis, and myocardial infarction. In this experiment, we investigated the age‐dependent abnormalities of the hematopoietic stem cells (HSCs) and hematopoiesis in this mouse. White blood cell counts (especially Mac‐1‐ or Gr‐1‐positive cells) in the peripheral blood of 12‐week‐old W/BF1 mice increased in comparison with those of four‐week‐old W/BF1 or normal mice. To investigate whether the abnormal hematopoiesis can be attributed to the HSCs of W/BF1 mice, colony‐forming unit in spleen (CFU‐S) and colony‐forming unit in culture (CFU‐C) assays were performed. Day 12 CFU‐S counts of 12‐week‐old W/BF1 mice significantly increased in comparison with those of four‐week‐old W/BF1 mice or normal mice. In the CFU‐C assay, CFU‐GEMM and CFU‐GM counts in 12‐week‐old W/BF1 mice increased in comparison with those of four‐week‐old W/BF1 or control mice. The bone marrow cells (BMCs) from 12‐week‐old W/BF1 mice showed a high level of G‐CSF and a low level of GM‐CSF in mRNA expression. To examine the effect of HSCs from 12‐week‐old W/BF1 mice on the onset of autoimmune diseases and the abnormal hematopoiesis, T‐ and B‐cell‐depleted BMCs of four‐week‐old or 12‐week‐old W/BF1 mice were transplanted to C3H mice. Recipient C3H mice that had received the BMCs from 12‐week‐old W/BF1 mice showed an earlier onset of autoimmune diseases and a shorter survival rate than those that had received the BMCs from four‐week‐old W/BF1 mice. These data suggest that the HSCs from 12‐week‐old W/BF1 mice showing the symptoms of autoimmune diseases have the capacity to induce autoimmune diseases earlier than the HSCs from four‐week‐old W/BF1 mice.