Masahide Asano

Antigen-Specific T Cell Sensitization Is Impaired in IL-17-Deficient Mice, Causing Suppression of Allergic Cellular and Humoral Responses

Interleukin-17 (IL-17) is a proinflammatory cytokine produced by T cells. The involvement of IL-17 in human diseases has been suspected because of its detection in sera from asthmatic patients and synovial fluids from arthritic patients. In this study, we generated IL-17-deficient mice and investigated the role of IL-17 in various disease models. We found that contact, delayed-type, and airway hypersensitivity responses, as well as T-dependent antibody production, were significantly reduced in the mutant mice, while IL-17 deficiency of donor T cells did not affect acute graft-versus-host reaction. The results suggest that impaired responses were caused by the defects of allergen-specific T cell activation. Our findings indicate that IL-17 plays an important role in activating T cells in allergen-specific T cell-mediated immune responses.

Caspase 1-independent IL-1β release and inflammation induced by the apoptosis inducer Fas ligand

Fas ligand is a well-characterized apoptosis inducer. Here we demonstrate that Fas ligand induces the processing and secretion of interleukin-1β (IL-1β) in peritoneal exudate cells. This IL-1β secretion is independent of IL-1β converting enzyme (caspase 1), yet it is inhibited by caspase inhibitors, indicating that a caspase(s) in addition to IL-1β converting enzyme can process IL-1β. Inoculation of tumor cells expressing Fas ligand into wild-type mice induces a massive neutrophil infiltration that is, in contrast, suppressed in IL-1α/β knockout mice. These results demonstrate a newly discovered role for Fas ligand in inflammation, and challenge the dogma that apoptosis does not induce inflammation.

Growth retardation and early death of beta-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells.

Carbohydrate chains on a glycoprotein are important not only for protein conformation, transport and stability, but also for cell–cell and cell–matrix interactions. UDP‐Gal:N‐acetylglucosamine β‐1,4‐galactosyltransferase (GalT) (EC 2.4.1.38) is the enzyme which transfers galactose (Gal) to the terminal N‐acetylglucosamine (GlcNAc) of complex‐type N‐glycans in the Golgi apparatus. In addition, it has also been suggested that this enzyme is involved directly in cell–cell interactions during fertilization and early embryogenesis through a subpopulation of this enzyme distributed on the cell surface. In this study, GalT‐deficient mice were produced by gene targeting in order to examine the pathological effects of the deficiency. GalT‐deficient mice were born normally and were fertile, but they exhibited growth retardation and semi‐lethality. Epithelial cell proliferation of the skin and small intestine was enhanced, and cell differentiation in intestinal villi was abnormal. These observations suggest that GalT plays critical roles in the regulation of proliferation and differentiation of epithelial cells after birth, although this enzyme is dispensable during embryonic development.

Mutually exclusive expression of odorant receptor transgenes.

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, β-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.

Impaired learning with enhanced hippocampal long‐term potentiation in PTPδ‐deficient mice

Protein tyrosine phosphatase δ (PTPδ) is a receptor‐type PTP expressed in the specialized regions of the brain including the hippocampal CA2 and CA3, B lymphocytes and thymic medulla. To elucidate the physiological roles of PTPδ, PTPδ‐deficient mice were produced by gene targeting. It was found that PTPδ‐deficient mice were semi‐lethal due to insufficient food intake. They also exhibited learning impairment in the Morris water maze, reinforced T‐maze and radial arm maze tasks. Interestingly, although the histology of the hippocampus appeared normal, the magnitudes of long‐term potentiation (LTP) induced at hippocampal CA1 and CA3 synapses were significantly enhanced in PTPδ‐deficient mice, with augmented paired‐pulse facilitation in the CA1 region. Thus, it was shown that PTPδ plays important roles in regulating hippocampal LTP and learning processes, and that hippocampal LTP does not necessarily positively correlate with spatial learning ability. To our knowledge, this is the first report of a specific PTP involved in the regulation of synaptic plasticity or in the processes regulating learning and memory.

Phenotypic Analysis of Meltrin α (ADAM12)-Deficient Mice: Involvement of Meltrin α in Adipogenesis and Myogenesis

ABSTRACT Meltrin α (ADAM12) is a metalloprotease-disintegrin whose specific expression patterns during development suggest that it is involved in myogenesis and the development of other organs. To determine the roles Meltrin α plays in vivo, we generated Meltrin α-deficient mice by gene targeting. Although the number of homozygous embryos are close to the expected Mendelian ratio at embryonic days 17 to 18, ca. 30% of the null pups born die before weaning, mostly within 1 week of birth. The viable homozygous mutants appear normal and are fertile. Most of the muscles in the homozygous mutants appear normal, and regeneration in experimentally damaged skeletal muscle is unimpeded. In some Meltrin α-deficient pups, the interscapular brown adipose tissue is reduced, although the penetrance of this phenotype is low. Impaired formation of the neck and interscapular muscles is also seen in some homozygotes. These observations suggest Meltrin α may be involved in regulating adipogenesis and myogenesis through a linked developmental pathway. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a candidate substrate of Meltrin α, and we found that TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ectodomain shedding of HB-EGF is markedly reduced in embryonic fibroblasts prepared from Meltrin α-deficient mice. We also report here the chromosomal locations of Meltrin α in the mouse and rat.

Interleukin-1β, but not interleukin-1α, is required for T-cell-dependent antibody production

Interleukin‐1 (IL‐1) consists of two molecules, IL‐1α and IL‐1β, and IL‐1 receptor antagonist (IL‐1Ra) is a natural inhibitor of these molecules. Although the adjuvant effects of exogenously administered IL‐1 in the humoral immune response are well known, the roles of endogenous IL‐1 and the functional discrimination between IL‐1α and IL‐1β have not been elucidated completely. In this report, we investigated the role of IL‐1 in the humoral immune response using gene‐targeted mice. Both primary and secondary antibody production against T‐dependent antigen, sheep red blood cells (SRBC), was significantly reduced in IL‐1α/β−/− mice, and was enhanced in IL‐1Ra−/− mice. The intrinsic functions of B cells, such as antibody production against type 1 T‐independent antigen, trinitrophenyl–lipopolysaccharide and proliferative responses against mitogenic stimuli, were normal in IL‐1α/β−/− mice. The proliferative response of T cells and cytokine production upon stimulation with anti‐CD3 monoclonal antibody were also normal, as was the phagocytotic ability of antigen‐presenting cells (APCs). However, SRBC‐specific proliferative response and cytokine production of T cells through the interaction with APCs were markedly impaired in IL‐1α/β−/− mice, and enhanced in IL‐1Ra−/− mice. Moreover, we show that SRBC‐specific antibody production was reduced in IL‐1β−/− mice, but not in IL‐1α−/− mice. These results show that endogenous IL‐1β, but not IL‐1α, is involved in T‐cell‐dependent antibody production, and IL‐1 promotes the antigen‐specific T‐cell helper function through the T‐cell–APC interaction.

IL-1 Enhances T Cell-Dependent Antibody Production Through Induction of CD40 Ligand and OX40 on T Cells

IL-1 is a proinflammatory cytokine that plays pleiotropic roles in host defense mechanisms. We investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Ab production against SRBC was significantly reduced in IL-1α/β-deficient (IL-1−/−) mice and enhanced in IL-1R antagonist−/− mice. The intrinsic functions of T, B, and APCs were normal in IL-1−/− mice. However, we showed that IL-1−/− APCs did not fully activate DO11.10 T cells, while IL-1R antagonist −/− APCs enhanced the reaction, indicating that IL-1 promotes T cell priming through T-APC interaction. The function of IL-1 was CD28-CD80/CD86 independent. We found that CD40 ligand and OX40 expression on T cells was affected by the mutation, and the reduced Ag-specific B cell response in IL-1−/− mice was recovered by the treatment with agonistic anti-CD40 mAb both in vitro and in vivo. These observations indicate that IL-1 enhances T cell-dependent Ab production by augmenting CD40 ligand and OX40 expression on T cells.

Ex vivo whole-embryo culture of caspase-8-deficient embryos normalize their aberrant phenotypes in the developing neural tube and heart

Caspase-8 plays the role of initiator in the caspase cascade and is a key molecule in death receptor-induced apoptotic pathways. To investigate the physiological roles of caspase-8 in vivo, we have generated caspase-8-deficient mice by gene targeting. The first signs of abnormality in homozygous mutant embryos were observed in extraembryonic tissue, the yolk sac. By embryonic day (E) 10.5, the yolk sac vasculature had begun to form inappropriately, and subsequently the mutant embryos displayed a variety of defects in the developing heart and neural tube. As a result, all mutant embryos died at E11.5. Importantly, homozygous mutant neural and heart defects were rescued by ex vivo whole-embryo culture during E10.5–E11.5, suggesting that these defects are most likely secondary to a lack of physiological caspase-8 activity. Taken together, these results suggest that caspase-8 is indispensable for embryonic development.

Impairment of skin wound healing in β-1, 4-galactosyltransferase-deficient mice with reduced leukocyte recruitment

Cell-surface carbohydrate chains are known to contribute to cell migration, interactions, and proliferation, but their roles in skin wound healing have not been evaluated. We examined the biological roles of beta4-galactosylated carbohydrate chains in skin wound healing using mutant mice that lack beta-1,4-galactosyltransferase-I (beta4GalT-I), which is responsible for the biosynthesis of the type 2 chain in N-glycans and the core 2 branch in O-glycans. beta4GalT-I-deficient mice showed significantly delayed wound healing with reduced re-epithelialization, collagen synthesis, and angiogenesis, compared with control mice. Neutrophil and macrophage recruitment at wound sites was also impaired in these mice probably because of selectin-ligand deficiency. In accordance with the reduced leukocyte infiltration, the expression levels of macrophage-derived chemokines, transforming growth factor-beta1, and vascular endothelial growth factor were all reduced in beta4GalT-I(-/-) mice. These results demonstrate that beta4-galactosylated carbohydrate chains play a critical role in skin wound healing by mediating leukocyte infiltration and epidermal cell growth, which affects the production of chemokines and growth factors. This study introduces a suitable mouse model for investigating the molecular mechanisms of skin wound healing and is the first report showing that carbohydrate chains have a strong influence on skin wound healing.