Satoshi Takaki

Crosstalk between neovessels and mural cells directs the site-specific expression of MT1-MMP to endothelial tip cells

The membrane-anchored matrix metalloproteinase MT1-MMP (also known as Mmp14) plays a key role in the angiogenic process, but the mechanisms underlying its spatiotemporal regulation in the in vivo setting have not been defined. Using whole-mount immunohistochemical analysis and the lacZ gene inserted into the Mmp14 gene, we demonstrate that MT1-MMP vascular expression in vivo is confined largely to the sprouting tip of neocapillary structures where endothelial cell proliferation and collagen degradation are coordinately localized. During angiogenesis in vitro, wherein endothelial cells are stimulated to undergo neovessel formation in the presence or absence of accessory mural cells, site-specific MT1-MMP expression is shown to be controlled by crosstalk between endothelial cells and vascular smooth muscle cells (VSMC). When vessel maturation induced by VSMCs is inhibited by introducing a soluble form of the receptor tyrosine kinase Tek, MT1-MMP distribution is no longer restricted to the endothelial tip cells, but instead distributes throughout the neovessel network in vitro as well as ex vivo. Taken together, these data demonstrate that vascular maturation coordinated by endothelial cell/mural cell interactions redirects MT1-MMP expression to the neovessel tip where the protease regulates matrix remodeling at the leading edge of the developing vasculature.

A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.

Interleukin-5 and Its Receptor System: Implications in the Immune System and Inflammation

Publisher Summary The chapter summarizes advances in interleukin-5 (IL-5) and IL-soluble receptor (IL-SR) research in the study of structure, physiologic functions, and the unique modes of receptor-mediated signaling. The chapter also discusses the pathophysiology of aberrant expression of IL-5 and its receptor. Helper T cells recognize antigenic peptide in the context of class IL1 major histocompatibility complex (MHC) molecules on accessory cells and/or B cells and secrete several soluble factors, including IL-4, IL-5, and IL-6, that can induce B-cell growth and the maturation of B cells. Mouse IL-5 (mIL-5) is a glycoprotein induced in T cells after stimulation with an antigen, such as Mycobacterium tuberculosis or Toxocara canis, and in mast cells upon stimulation with allergen/IgE complex or calcium ionophores. The action of IL-5 in responsive cells is the molecular mechanism of signal transduction cascade after IL-5 binding to the functional IL-5 receptor (IL-5R). The IL-5 signals can be transduced through the high-affinity IL-5R that consists of two different polypeptide subunits: α and β. The cDNAs encoding both α - and β -subunits for mIL-5R have been isolated; the α -subunit was found to be a 60 kDa (p60) protein and the β-subunit (a 130-kDa protein, p130) was identified as the protein identical to the β-subunit for mIL-3R and mGM-CSFR, which can convert the low affinity mILSRα into a high-affinity receptor. The study of mIL-5 originated from the search for the B-cell differentiation factor that induces antigen-primed B cells to differentiate into antigen-specific antibody-producing cells or the proliferation of BCLl B-cell tumor cells.

Lnk negatively regulates self-renewal of hematopoietic stem cells by modifying thrombopoietin-mediated signal transduction

One of the central tasks of stem cell biology is to understand the molecular mechanisms that control self-renewal in stem cells. Several cytokines are implicated as crucial regulators of hematopoietic stem cells (HSCs), but little is known about intracellular signaling for HSC self-renewal. To address this issue, we attempted to clarify how self-renewal potential is enhanced in HSCs without the adaptor molecule Lnk, as in Lnk-deficient mice HSCs are expanded in number >10-fold because of their increased self-renewal potential. We show that Lnk negatively regulates self-renewal of HSCs by modifying thrombopoietin (TPO)-mediated signal transduction. Single-cell cultures showed that Lnk-deficient HSCs are hypersensitive to TPO. Competitive repopulation revealed that long-term repopulating activity increases in Lnk-deficient HSCs, but not in WT HSCs, when these cells are cultured in the presence of TPO with or without stem cell factor. Single-cell transplantation of each of the paired daughter cells indicated that a combination of stem cell factor and TPO efficiently induces symmetrical self-renewal division in Lnk-deficient HSCs but not in WT HSCs. Newly developed single-cell immunostaining demonstrated significant enhancement of both p38 MAPK inactivation and STAT5 and Akt activation in Lnk-deficient HSCs after stimulation with TPO. Our results suggest that a balance in positive and negative signals downstream from the TPO signal plays a role in the regulation of the probability of self-renewal in HSCs. In general, likewise, the fate of stem cells may be determined by combinational changes in multiple signal transduction pathways.

The Role of IL-5 for Mature B-1 Cells in Homeostatic Proliferation, Cell Survival, and Ig Production

B-1 cells, distinguishable from conventional B-2 cells by their cell surface marker, anatomical location, and self-replenishing activity, play an important role in innate immune responses. B-1 cells constitutively express the IL-5R α-chain (IL-5Rα) and give rise to Ab-producing cells in response to various stimuli, including IL-5 and LPS. Here we report that the IL-5/IL-5R system plays an important role in maintaining the number and the cell size as well as the functions of mature B-1 cells. The administration of anti-IL-5 mAb into wild-type mice, T cell-depleted mice, or mast cell-depleted mice resulted in reduction in the total number and cell size of B-1 cells to an extent similar to that of IL-5Rα-deficient (IL-5Rα−/−) mice. Cell transfer experiments have demonstrated that B-1 cell survival in wild-type mice and homeostatic proliferation in recombination-activating gene 2-deficient mice are impaired in the absence of IL-5Rα. IL-5 stimulation of wild-type B-1 cells, but not IL-5Rα−/− B-1 cells, enhances CD40 expression and augments IgM and IgG production after stimulation with anti-CD40 mAb. Enhanced IgA production in feces induced by the oral administration of LPS was not observed in IL-5Rα−/− mice. Our results illuminate the role of IL-5 in the homeostatic proliferation and survival of mature B-1 cells and in IgA production in the mucosal tissues.

A Protein Associated with Toll-Like Receptor 4 (PRAT4A) Regulates Cell Surface Expression of TLR4

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.

Impaired Lymphopoiesis and Altered B Cell Subpopulations in Mice Overexpressing Lnk Adaptor Protein

Lnk is an adaptor protein expressed primarily in lymphocytes and hemopoietic precursor cells. Marked expansion of B lineage cells occurs in lnk−/− mice, indicating that Lnk regulates B cell production by negatively controlling pro-B cell expansion. In addition, lnk−/− hemopoietic precursors have an advantage in repopulating the hemopoietic system of irradiated host animals. In this study, we show that Lnk overexpression results in impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. The representation of both B lineage and T lineage cells was reduced in transgenic mice overexpressing Lnk under the control of a lymphocyte-specific expression vector. Whereas the overall number of B and T cells was correlated with Lnk protein expression levels, marginal zone B cells in spleen and B1 cells in the peritoneal cavity were relatively resistant to Lnk overexpression. The C-terminal tyrosine residue, conserved among Lnk family adaptor proteins, was dispensable for the negative regulatory roles of Lnk in lymphocyte development. Our results illuminate the novel negative regulatory mechanism mediated by the Lnk adaptor protein in controlling lymphocyte production and function.

Mechanism of eosinophil infiltration in the patient with subcutaneous angioblastic lymphoid hyperplasia with eosinophilia (Kimura's disease). Mechanism of eosinophil chemotaxis mediated by candida antigen and IL-5.

Kimuras disease is a chronic granulomatous disease of unknown etiology. Although eosinophilia is one of the characteristic features in this disease, little is known about the mechanism of eosinophilia. In the present study it was demonstrated that interleukin-5 (IL-5) was produced and released from the site of a granuloma and lymph nodes after stimulation with candida antigen. It was also shown that peripheral blood eosinophils from patients with Kimuras disease contained a large proportion of hypodense eosinophils and that their viability was prolonged. These results strongly suggest that locally produced IL-5 induced by candida antigen contributes to the eosinophilia in this disease.

Increased Numbers of B-1 Cells and Enhanced Responses against TI-2 Antigen in Mice Lacking APS, an Adaptor Molecule Containing PH and SH2 Domains

ABSTRACT APS (adaptor molecule containing PH and SH2 domains) is an intracellular adaptor protein that forms an adaptor family along with Lnk and SH2-B. While experiments using cultured cell lines have demonstrated that APS is phosphorylated in response to various stimuli, its in vivo functions remain unclear. We attempted to determine the physiological roles of APS by generating APS-deficient (APS−/− ) mice. APS−/− mice were viable and fertile and showed no abnormalities or growth retardation. Immunologically, APS−/− mice showed normal development and distribution of lymphocytes and myeloid cells, except for increased numbers of B-1 cells in the peritoneal cavity. APS−/− mice exhibited an enhanced humoral immune response against trinitrophenol-Ficoll, a thymus-independent type 2 antigen, while APS−/− B-2 cells exhibited normal proliferative responses and tyrosine phosphorylation of intracellular proteins upon B-cell receptor (BCR) cross-linking. APS colocalized with filamentous actin (F-actin) accumulated during the capping of BCRs in APS-transgenic B cells. After BCR stimulation, F-actin contents were lower in APS−/− B-1 cells than in wild-type B-1 cells. Our results indicate that APS might have a novel regulatory role in actin reorganization and control of B-1 cell compartment size.

Interleukin 5 Plays an Essential Role in Elicitation of Contact Sensitivity through Dual Effects on Eosinophils and B-1 Cells

Background: Elicitation of contact sensitivity (CS) depends on B-1-cell-derived antigen-specific immunoglobulin M (IgM) antibodies that recruit CS effector T cells into the local tissue, which is followed by infiltration of antigen-nonspecific mononuclear cells and polymorphonuclear cells, such as neutrophils and eosinophils. In this study, we investigated the role of interleukin (IL)-5, which has broad effects on both eosinophils and B-1 cells, in elicitation of CS. Methods: IL-5 receptor α-chain-deficient (IL-5Rα–/–) mice and IL-5Rα+/+ mice were contact sensitized with oxazolone hapten. Four days later, mice were challenged with the same hapten, and ear swelling responses were measured at 24 h after challenge. Eosinophil infiltration into the local tissue was determined by examination of skin histology and eosinophil peroxidase activity. To investigate the role of IL-5 in B-1 cell activation, the number of oxazolone-specific IgM-producing cells in the spleen was determined by enzyme-linked immunospot assay. Results: Ear swelling responses in IL-5Rα–/– mice were about half of those in IL-5Rα+/+ mice, and nearly no eosinophil infiltration was observed in IL-5Rα–/– mouse skin. Eosinophil peroxidase activity in the sensitized and challenged IL-5Rα–/– mice was about 11 times less than that in immunized IL-5Rα+/+ mice. Contact sensitization significantly increased in numbers of oxazolne-specific IgM-producing cells in IL-5Rα+/+ mouse spleen, but not in IL-5Rα–/– mouse spleen. Conclusion: We conclude that IL-5-dependent activation of eosinophils and B-1 cells is necessary for induction and elicitation of CS. These findings provide a new insight into complicated mechanisms of CS elicitation and suggest a novel role of IL-5 in the regulation of immune responses.