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Transgenic mice expressing human herpesvirus 8 encoded viral interleukin-6 reveal features of multicentric Castleman’s disease
Abstracts / Cytokine 48 (2009) 46–90 binding to 20 different over-expressed proteins intumor cells were successfully isolated within several weeks. The expression profiles of the identified proteins were then determined by tissue microarray analysis using the scFv-phages. Consequently, we revealed frequent expression of TRAIL-R2 in human breast tumors. Our data demonstrate s the utility of an antibody proteomics system for discovering and validating tumor-related proteins in pharmaceutical proteomics. Currently, we are analyzing the functions of these proteins to use them as diagnostic markers or therapeutic targets.
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taxis, up-regulate the expression of the glucose transporter, Glut-1, and the amino acid transporter 4F2. In addition, using a radio-labelled glucose analog, 2-deoxy-Dglucose, we provide evidence for glucose uptake mediated by CCL5 treatment. Viewed altogether, these findings suggest that CCL5, at concentrations that support optimal chemotaxis, may also influence the metabolic status of activated T cells. doi:10.1016/j.cyto.2009.07.165
doi:10.1016/j.cyto.2009.07.162 PP1-043 Transgenic mice expressing human herpesvirus 8 encoded viral interleukin-6 reveal features of multicentric Castleman’s disease PP1-041 Cancer hazard of carbon nanotubes: Size/shape-dependent induction of DNA damage and inflammation Kohei Yamashita, Yasuo Yoshioka, Hiroyuki Kayamuro, Tokuyuki Yoshida, Kazuma Higashisaka, Yasuhiro Abe, Tomoaki Yoshikawa, Norio Itoh, Shin-ichi Tsunoda, Yasuo Tsutsumi, Poster Presentation I Cancer hazard of carbon nanotubes: Size/shape-dependent induction of DNA damage and inflammation Kohei Yamashita 1,2, Yasuo Yoshioka 1,3, Hiroyuki Kayamuro 1,2, Tokuyuki Yoshida 1,2, Kazuma Higashisaka 1,2, Yasuhiro Abe 1, Tomoaki Yoshikawa 1,2,3, Norio Itoh 1,2, Shinichi Tsunoda 1,3, Yasuo Tsutsumi 1,2,3, 1 Laboratory of Pharmaceutical Proteomics (LPP), National Institute of Biomedical Innovation (NiBio), Osaka, Japan, 2 Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka 565-0871, Japan, 3 The Center for Advanced Medical Engineering and Informatics, Osaka university, Osaka, Japan Recently, promising nanomaterials (NM), such as titanium dioxide and carbon nanotubes (CNTs) are increasingly used for applications in medical science and cosmetics. CNTs are one of the most promissing NM used for a variety of applications in industry and medicine because of distinctive characteristics. However, the safety of CNTs is concerned worldwide because they have needle-like fiber shape looks like asbestos. In this study, we examined the biological impacts, such as cytotoxicity, DNA damage, and inflammation, induced by different-sized multiwalled CNT (MWCNT) and single-walled CNT (SWCNT). At first, we examined cytotoxicity of CNTs on human epithelial cell lines (A549). None of the CNTs had any significant cytotoxicity in A549 cells. DNA damage caused by CNTs was evaluated by Comet assay. As a result, MWCNT induced stronger DNA damage than SWCNT, and the long and thick size/ shape of MWCNT significantly induced DNA damage. We then examined the CNTinduced inflammatory responses. The long and thick size/shape of MWCNT induced IL-1 b production on THP-1 cells. For further investigation of inflammation, C57BL/6 mice were intraperitoneally administered with CNTs and counted infiltrating leukocyte cell numbers in abdominal lavage fluid. A s in the case of Comet assay and cytokine production, the long and thick size/shape of MWCNT-exposed mice exhibited increased total cell numbers. These results indicate that long and thick MWCNT are likely to cause severe biological effects and may lead to be the augmentation of cancer risk. For the future analysis, we will investigate the kinetics of the long and thick MWCNT in the body. doi:10.1016/j.cyto.2009.07.164
PP1-042 The role of CCL5/RANTES in regulating nutrient receptor traffiking, metabolism and protein expression in activated T cells Olivia Chan, Thomas Murooka, Eleanor Fish, Poster Presentation I The role of CCL5/RANTES in regulating nutrient receptor traffiking, metabolism and protein expression in activated T cells Olivia Chan 1, Thomas Murooka 1,2, Eleanor Fish 1,3, 1 Department of Immunology, University of Toronto, Ontario, Canada, 2 The Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, MA, USA, 3 Division of Cellular Molecular Biology, Toronto General Hospital Research Institute, Toronto, Ontario, Canada Recruitment of effector T cells to sites of infection or inflammation is imperative for an effective adaptive immune response. Upon activation, T cells alter their cytokine and chemokine profile, thereby affecting their function and localization. The chemokine receptor, CCR5, is expressed on Th1 cells and binds a number of chemokines including MIP-1a, MIP-1b and CCL5/RANTES. CCL5 activates CCR5 to initiate proliferation, chemotaxis, cytokine production, or apoptosis. We have shown that CCL5/CCR5 signaling activates the mTOR/4E-BP1 pathway to directly modulate mRNA translation. Moreover, CCL5-mediated mTOR activation influences T cell chemotaxis by initiating the translation of chemotaxis-related proteins, including MMP-9 and cyclin D1. Up-regulation of chemotaxis-related proteins may ‘‘prime” T cells for efficient migration. It is now clear that mTOR is a central regulator of cell size, nutrient sensing and glycolysis. In continuing studies, we are investigating the ability of CCL5 to regulate T cell metabolism through the PI-30 K/mTOR pathway. Data generated in ex vivo activated human CD3+ T cells show that CCL5 treatment, at doses that invoke chemo-
Jan Suthaus, Christiane Stuhlman-Laiesz, Wolfram Klapper, Jürgen Scheller, Stefan Rose-John, Poster Presentation I Transgenic mice expressing human herpesvirus 8 encoded viral interleukin-6 reveal features of multicentric Castleman’s disease Jan Suthaus 1, Christiane Stuhlman-Laiesz 2, Wolfram Klapper 2, Jürgen Scheller 1, Stefan Rose-John 1, 1 Department of Biochemistry, Christian-Albrechts-University, Kiel, Germany, 2 Department of Pathology, Haematopathology Section and Lymph Node Registry, Kiel, Germany Human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with the proliferative disorders Kaposi’ssarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. Viral Interleukin-6 (vIL-6), which is encoded by HHV8 shows 25% identity with human Interleukin-6 (IL-6). In contrast to human IL-6, vIL-6 does not require the human IL-6 receptor for gp130 receptor complex formation and initiation of signaling. As a consequence, vIL-6 can stimulate far more targeT cells than human IL-6. Diseases associated with HHV-8 such as Kaposi’s sarcoma, pleural effusion lymphoma and multicentric Castleman’s disease have been associated with dysregulation of IL-6. Therefore we generated transgenic mice, which expressed vIL-6 under the control of MHC class I promotor H2. In these mice, vIL-6 was mainly expressed in lymphoid tissue. Transgenic animals showed vIL-6 levels, which were in the range observed in HHV8 patients. The expression of vIL-6 in spleen, lymph node, thymus and peyer’s patches led to elevated STAT3 phosphorylation and induction of STAT3 target genes such as cmyc or Pim-1. The mice display splenomegaly, multifocal lymphadenopathy and hypergammaglobulinemia. Furthermore, some lymph nodes were characterized by plasmacytosis, follicular hyperplasia and vascular proliferation. All these features are hallmarks of multicentric Castleman’s disease. In Castleman’s disease patients it has been speculated that vIL-6 induced the expression of human IL-6, which in turn is responsible for the development of the disease. With our new animal model this hypothesis is tested by crossing vIL-6 transgenic mice to the background of IL-6 knockout mice. In summary, our vIL-6 transgenic mice suggest that vIL-6 is a decisive viral factor in the pathogenesis HHV-8 related diseases such as multicentric Castleman’s disease. doi:10.1016/j.cyto.2009.07.166
PP1-044 Inhibition by proxy: LPS-driven B cell responses in RP105-deficient mice Jessica L. Allen, Senad Divanovic, David Rawlings, Fred Finkelman, Christopher L. Karp, Poster Presentation I Inhibition by proxy: LPS-driven B cell responses in RP105-deficient mice Jessica L. Allen 1, Senad Divanovic 1, David Rawlings 2, Fred Finkelman 3, Christopher L. Karp 1, 1 Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, and the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA, 2 Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA, 3 Division of Immunobiology, Cincinnati Children’s Hospital Research Foundation, and the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA RP105, a TLR homolog lacking a TIR domain, is an inhibitor of TLR4 signaling in HEK293 cells and primary myeloid cells, and inhibits in vivo LPS-driven proinflammatory responses. On the other hand, splenic B cells from RP105/ mice exhibit impaired LPS-driven proliferation while retaining normal proliferative responses to most other stimuli— something interpreted to indicate that RP105 facilitates TLR4 signaling in B cells. To address this apparent contradiction, we re-examined RP105 modulation of B cell TLR4 signaling. The blunted ex vivo LPS-driven proliferative response of RP105/ splenic B cells cannot be attributed to altered B cell subpopulation frequency. Moreover, purified marginal zone and follicular B cells from RP105/ mice recapitulate the proliferative phenotype. RP105/ mice have a significant increase in splenic leukocyte numbers—with normal leukocyte subpopulation percentages. Of note, BrdU analysis revealed that B cells from RP105/ mice exhibit significantly increased baseline in vivo proliferation (along with decreased in vivo proliferation in response to LPS). The former finding suggested that the proliferative defect observed with RP105/ B cells might not be B cell autonomous. Indeed, after adoptive transfer of RP105/ and RP105+/+ B cells into an RP105+/+ environment (in lMT mice), differences in their LPS-driven ex vivo proliferative responses were
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taxis, up-regulate the expression of the glucose transporter, Glut-1, and the amino acid transporter 4F2. In addition, using a radio-labelled glucose analog, 2-deoxy-Dglucose, we provide evidence for glucose uptake mediated by CCL5 treatment. Viewed altogether, these findings suggest that CCL5, at concentrations that support optimal chemotaxis, may also influence the metabolic status of activated T cells. doi:10.1016/j.cyto.2009.07.165
doi:10.1016/j.cyto.2009.07.162 PP1-043 Transgenic mice expressing human herpesvirus 8 encoded viral interleukin-6 reveal features of multicentric Castleman’s disease PP1-041 Cancer hazard of carbon nanotubes: Size/shape-dependent induction of DNA damage and inflammation Kohei Yamashita, Yasuo Yoshioka, Hiroyuki Kayamuro, Tokuyuki Yoshida, Kazuma Higashisaka, Yasuhiro Abe, Tomoaki Yoshikawa, Norio Itoh, Shin-ichi Tsunoda, Yasuo Tsutsumi, Poster Presentation I Cancer hazard of carbon nanotubes: Size/shape-dependent induction of DNA damage and inflammation Kohei Yamashita 1,2, Yasuo Yoshioka 1,3, Hiroyuki Kayamuro 1,2, Tokuyuki Yoshida 1,2, Kazuma Higashisaka 1,2, Yasuhiro Abe 1, Tomoaki Yoshikawa 1,2,3, Norio Itoh 1,2, Shinichi Tsunoda 1,3, Yasuo Tsutsumi 1,2,3, 1 Laboratory of Pharmaceutical Proteomics (LPP), National Institute of Biomedical Innovation (NiBio), Osaka, Japan, 2 Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka 565-0871, Japan, 3 The Center for Advanced Medical Engineering and Informatics, Osaka university, Osaka, Japan Recently, promising nanomaterials (NM), such as titanium dioxide and carbon nanotubes (CNTs) are increasingly used for applications in medical science and cosmetics. CNTs are one of the most promissing NM used for a variety of applications in industry and medicine because of distinctive characteristics. However, the safety of CNTs is concerned worldwide because they have needle-like fiber shape looks like asbestos. In this study, we examined the biological impacts, such as cytotoxicity, DNA damage, and inflammation, induced by different-sized multiwalled CNT (MWCNT) and single-walled CNT (SWCNT). At first, we examined cytotoxicity of CNTs on human epithelial cell lines (A549). None of the CNTs had any significant cytotoxicity in A549 cells. DNA damage caused by CNTs was evaluated by Comet assay. As a result, MWCNT induced stronger DNA damage than SWCNT, and the long and thick size/ shape of MWCNT significantly induced DNA damage. We then examined the CNTinduced inflammatory responses. The long and thick size/shape of MWCNT induced IL-1 b production on THP-1 cells. For further investigation of inflammation, C57BL/6 mice were intraperitoneally administered with CNTs and counted infiltrating leukocyte cell numbers in abdominal lavage fluid. A s in the case of Comet assay and cytokine production, the long and thick size/shape of MWCNT-exposed mice exhibited increased total cell numbers. These results indicate that long and thick MWCNT are likely to cause severe biological effects and may lead to be the augmentation of cancer risk. For the future analysis, we will investigate the kinetics of the long and thick MWCNT in the body. doi:10.1016/j.cyto.2009.07.164
PP1-042 The role of CCL5/RANTES in regulating nutrient receptor traffiking, metabolism and protein expression in activated T cells Olivia Chan, Thomas Murooka, Eleanor Fish, Poster Presentation I The role of CCL5/RANTES in regulating nutrient receptor traffiking, metabolism and protein expression in activated T cells Olivia Chan 1, Thomas Murooka 1,2, Eleanor Fish 1,3, 1 Department of Immunology, University of Toronto, Ontario, Canada, 2 The Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, MA, USA, 3 Division of Cellular Molecular Biology, Toronto General Hospital Research Institute, Toronto, Ontario, Canada Recruitment of effector T cells to sites of infection or inflammation is imperative for an effective adaptive immune response. Upon activation, T cells alter their cytokine and chemokine profile, thereby affecting their function and localization. The chemokine receptor, CCR5, is expressed on Th1 cells and binds a number of chemokines including MIP-1a, MIP-1b and CCL5/RANTES. CCL5 activates CCR5 to initiate proliferation, chemotaxis, cytokine production, or apoptosis. We have shown that CCL5/CCR5 signaling activates the mTOR/4E-BP1 pathway to directly modulate mRNA translation. Moreover, CCL5-mediated mTOR activation influences T cell chemotaxis by initiating the translation of chemotaxis-related proteins, including MMP-9 and cyclin D1. Up-regulation of chemotaxis-related proteins may ‘‘prime” T cells for efficient migration. It is now clear that mTOR is a central regulator of cell size, nutrient sensing and glycolysis. In continuing studies, we are investigating the ability of CCL5 to regulate T cell metabolism through the PI-30 K/mTOR pathway. Data generated in ex vivo activated human CD3+ T cells show that CCL5 treatment, at doses that invoke chemo-
Jan Suthaus, Christiane Stuhlman-Laiesz, Wolfram Klapper, Jürgen Scheller, Stefan Rose-John, Poster Presentation I Transgenic mice expressing human herpesvirus 8 encoded viral interleukin-6 reveal features of multicentric Castleman’s disease Jan Suthaus 1, Christiane Stuhlman-Laiesz 2, Wolfram Klapper 2, Jürgen Scheller 1, Stefan Rose-John 1, 1 Department of Biochemistry, Christian-Albrechts-University, Kiel, Germany, 2 Department of Pathology, Haematopathology Section and Lymph Node Registry, Kiel, Germany Human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with the proliferative disorders Kaposi’ssarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. Viral Interleukin-6 (vIL-6), which is encoded by HHV8 shows 25% identity with human Interleukin-6 (IL-6). In contrast to human IL-6, vIL-6 does not require the human IL-6 receptor for gp130 receptor complex formation and initiation of signaling. As a consequence, vIL-6 can stimulate far more targeT cells than human IL-6. Diseases associated with HHV-8 such as Kaposi’s sarcoma, pleural effusion lymphoma and multicentric Castleman’s disease have been associated with dysregulation of IL-6. Therefore we generated transgenic mice, which expressed vIL-6 under the control of MHC class I promotor H2. In these mice, vIL-6 was mainly expressed in lymphoid tissue. Transgenic animals showed vIL-6 levels, which were in the range observed in HHV8 patients. The expression of vIL-6 in spleen, lymph node, thymus and peyer’s patches led to elevated STAT3 phosphorylation and induction of STAT3 target genes such as cmyc or Pim-1. The mice display splenomegaly, multifocal lymphadenopathy and hypergammaglobulinemia. Furthermore, some lymph nodes were characterized by plasmacytosis, follicular hyperplasia and vascular proliferation. All these features are hallmarks of multicentric Castleman’s disease. In Castleman’s disease patients it has been speculated that vIL-6 induced the expression of human IL-6, which in turn is responsible for the development of the disease. With our new animal model this hypothesis is tested by crossing vIL-6 transgenic mice to the background of IL-6 knockout mice. In summary, our vIL-6 transgenic mice suggest that vIL-6 is a decisive viral factor in the pathogenesis HHV-8 related diseases such as multicentric Castleman’s disease. doi:10.1016/j.cyto.2009.07.166
PP1-044 Inhibition by proxy: LPS-driven B cell responses in RP105-deficient mice Jessica L. Allen, Senad Divanovic, David Rawlings, Fred Finkelman, Christopher L. Karp, Poster Presentation I Inhibition by proxy: LPS-driven B cell responses in RP105-deficient mice Jessica L. Allen 1, Senad Divanovic 1, David Rawlings 2, Fred Finkelman 3, Christopher L. Karp 1, 1 Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, and the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA, 2 Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA, 3 Division of Immunobiology, Cincinnati Children’s Hospital Research Foundation, and the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA RP105, a TLR homolog lacking a TIR domain, is an inhibitor of TLR4 signaling in HEK293 cells and primary myeloid cells, and inhibits in vivo LPS-driven proinflammatory responses. On the other hand, splenic B cells from RP105/ mice exhibit impaired LPS-driven proliferation while retaining normal proliferative responses to most other stimuli— something interpreted to indicate that RP105 facilitates TLR4 signaling in B cells. To address this apparent contradiction, we re-examined RP105 modulation of B cell TLR4 signaling. The blunted ex vivo LPS-driven proliferative response of RP105/ splenic B cells cannot be attributed to altered B cell subpopulation frequency. Moreover, purified marginal zone and follicular B cells from RP105/ mice recapitulate the proliferative phenotype. RP105/ mice have a significant increase in splenic leukocyte numbers—with normal leukocyte subpopulation percentages. Of note, BrdU analysis revealed that B cells from RP105/ mice exhibit significantly increased baseline in vivo proliferation (along with decreased in vivo proliferation in response to LPS). The former finding suggested that the proliferative defect observed with RP105/ B cells might not be B cell autonomous. Indeed, after adoptive transfer of RP105/ and RP105+/+ B cells into an RP105+/+ environment (in lMT mice), differences in their LPS-driven ex vivo proliferative responses were