Annual Report 2004

Department of Microbiology and Immunology
Division of Immunology

Self-defense against invaded pathogenic microorganisms and foreign antigenic molecules is strictly controlled by the immune system and inflammation. Our major research interests are to elucidate cells and effector molecules in innate and acquired immunity and inflammation. In particular, we are focused on cellular and molecular mechanisms of development and activation of B cells and IgH class switch recombination under the influence of T cells, cytokines and adaptor proteins. We are also interested in elucidating cellular mechanisms of preferential induction of Th1 cells upon immunization with Mycobacterium-derived, Peptide-25 and its derivatives.
In 2003, Professor Dr. Fritz Melchers, Basel University, who were invited by the University of Tokyo as the Eminent Scientist of JSPS, has been joining us for our research projects. Dr. Melchers always encouraged us and suggested important issues for each project. We would like to appreciate his enormous contributions.


Regulatory functions of adapter proteins in the immune system

a. Control of hematopoietic stem/progenitor cell functions by Lnk adaptor protein, which functions as a molecular scaffold linking RTKs with cytoskeletal regulatory components

Satoshi Takaki, Sang-Mo Kwon, Hitoshi Takizawa, Masanori Iseki, Kazuhiro Sudo2, Hideo Ema2, Jun Seita2, Mitsujiro Osawa2, Hiromitsu Nakauchi2 and Kiyoshi Takatsu: 2Laboratory of Stem Cell Therapy, Center for Experimental Medicine, IMSUT

Lnk, a recently identified intracellular adaptor protein, negatively regulates B-lymphopoiesis and early hematopoiesis. The lnk-deficient mice show enhanced B cell production due to the hypersensitivity of B precursors to stem cell factor, SCF. Competitive repopulation assays in irradiated host animals have demonstrated that the ability of hematopoietic progenitors to generate various blood cells is greatly enhanced by the absence of Lnk. We further investigated the effect of lnk-deficiency on the compartment size and ability of hematopoietic stem cells (HSCs), which is strictly regulated in normal conditions. Measurement of competitive repopulation unit (CRU) as well as flow cytometric analysis revealed that there exist nearly 15-fold more functional HSCs in the bone marrow of adult lnk-/- mice compared to normal mice. Clonal analysis by single cell HSC transplantation indicated that a part of lnk-/- HSCs had highly repopulating capability.
Molecular mechanisms underlying Lnk-mediated regulation are not fully understood. We revealed that Lnk could control actin reorganization activated by receptor tyrosine kinases (RTKs), and thereby regulate cell migration. Lnk expressing fibroblasts showed flattened, spreading shapes, prominent actin polymerization accompanied by augmented Rac activation, and impaired migration in a wound-healing assay. Lnk co-immunoprecipitated with Rac, Vav, PAK and filamin A indicating a complex formation of cytoskeletal regulatory proteins supported by Lnk. Adhesion and migration behavior of lnk-/- progenitor cells are now under investigation.

b. Enhancing repopulation ability of hematopoietic stem/progenitor cells by a targeted inhibition of Lnk
Hitoshi Takizawa, Chiyomi Kubo-Akashi, Ikuo Nobuhisa3, Sang-Mo Kwon, Masanori Iseki, Tetsuya Taga3, Kiyoshi Takatsu and Satoshi Takaki: 3Laboratory of Gene Expression and Regulation, Center for Experimental Medicine, IMSUT

Repopulating ability of HSC/Ps in irradiated host animals is greatly enhanced by the absence of Lnk. In lnk-deficient mice, however, neither malignant transformation nor functional defect of blood cells was observed. We identified functional domains of Lnk, generated dominant-negative (DN) Lnk mutants and tested whether DN Lnk mutants could block functions of Lnk endogenously expressed in HSC/Ps and augment repopulation ability of HSC/Ps. Lnk consists of an N-terminal proline-rich region, SH2-, PH- domains and a conserved tyrosine phosphorylation site. Various Lnk mutants were generated and transducted into MC9 mast cells using retroviral vector, and their SCF-dependent growth was evaluated by monitoring eGFP+ cells. While the wild-type Lnk efficiently inhibited growth of MC9 cells, a point mutation in the SH2 domain completely abolished the inhibitory effect. The SH2 mutants acted as DN mutants since they cancelled growth inhibition of MC9 transfectants overexpressing Lnk. The SH2 mutant with deletion of PH domain and C-terminal region was the most effective DN Lnk mutant. We, next, evaluated consequences of the DN Lnk expression in HSC/Ps by competitive repopulation assay. HSC/Ps expressing the DN Lnk was transferred into lethally irradiated host animals and percentage of eGFP+ cells in peripheral blood was monitored. Cells expressing DN Lnk repopulated at much higher percentage in lymphoid and myeloid lineages than cells treated with control vectors. Inhibition of Lnk by the DN mutant could become a potent approach for expansion and regulation of HSC/Ps.

c. Control of actin reorganization and B-1 cell compartment size by adaptor molecule containing PH and SH2 domains, APS
Masanori Iseki, Chiyomi Kubo-Akashi, Sang-Mo Kwon, Nobuaki Yoshida3, Kiyoshi Takatsu and Satoshi Takaki

To understand functions of the Lnk family adaptor proteins further, we tried to identify other members of the family, and isolated mouse APS (adaptor molecule containing PH and SH2 domains). APS is expressed in various tissues including spleen, bone marrow, brain and muscle, and in mature B but not in T or immature B cells. In cell lines, APS is tyrosine phosphorylated upon stimulation with IL-5, IL-3 or anti-IgM. To investigate roles of APS in vivo, we generated APS-/- mice. APS-/- mice were viable, fertile and show no anomalies or growth retardation. Lymphocyte or myeloid cell developments in bone marrow, thymus, spleen and lymph nodes were not perturbed. However, APS-/- mice had more B-1 cells in peritoneal cavity, and showed enhanced humoral immune responses against thymus-independent type-2 (TI-2) antigens, while APS-/- B-2 cells exhibited normal proliferative responses and tyrosine phosphorylation of intracellular proteins upon BCR crosslinking. On the other hand, in transgenic mice overexpressing APS in lymphocytes, the numbers of peritoneal B-1 and splenic B cells were reduced, and proliferation induced by anti-IgM stimulation was impaired. APS colocalized with filamentous actin (F-actin) accumulated during capping of BCR in APS-transgenic B cells. F-actin contents after BCR stimulation was decreased in APS-/- B-1 cells compared to wild-type B-1 cells. Our results indicated that APS may have a novel regulatory role in actin reorganization and control of B-lineage cell compartment size.

d. Roles of Lnk-family adaptor proteins, Lnk, SH2-B and APS in growth and functions of mast cells: APS-deficiency causes augmented degranulation and reduced actin assembly
Chiyomi Kubo-Akashi, Masanori Iseki, Sang-Mo Kwon, Hitoshi Takizawa, Kiyoshi Takatsu and Satoshi Takaki

Lnk, SH2-B and APS form an adaptor protein family conserved from drosophila. SH2-B is originally identified as a protein associated with immunoreceptor tyrosine-based activation motifs (ITAMs) of FcオRI ウ-chain. APS is identified as a potential substrate of c-Kit. Since Lnk, SH2-B and APS are all expressed in bone marrow-derived mast cells (BMMCs), and may play roles in signaling mediated through c-Kit or FcオRI, we investigated consequences of the deficiency either of Lnk, SH2-B or APS in mast cell functions. We established IL-3-dependent BMMCs from lnk-/-, SH2-B-/- and APS-/- mice. IL-3-dependent growth of those cells was comparable. Proliferation or adhesion mediated by c-Kit activation as well as degranulation induced by cross-linking FcオRI were normal in the absence of Lnk or SH2-B. In contrast, APS-/- BMMCs showed augmented degranulation after cross-linking FcオRI compared to wild type cells, while c-Kit-mediated proliferation and adhesion were kept unaffected. The enhanced degranulation from APS-/- BMMCs was due to augmented degranulation from each mast cell but not to increased proportion of cells that underwent degranulation. Calcium influx and tyrosine phosphorylation of various cellular proteins induced by cross-linking FcオRI were normal in the absence of APS, and cell survival mediated by binding of monomeric IgE to FcオRI was also normal. We found, however, that APS-/- BMMCs showed reduced F-actin assembly at steady state. APS-/- cells were resistant to latrunculin, an inhibitor disrupting F-actin microfilaments in FcオRI-mediated degranulation responses. Our results suggest potential roles of APS in controlling actin cytoskeleton and magnitude of degranulation in mast cells.