Signaling by Cytoplasmic Tyrosine Kinases in Leukocytes

Summary

Principal Investigator: Clifford A Lowell
Abstract: DESCRIPTION (provided by applicant): The Src-family tyrosine kinases play pivotal roles in initiating intracellular signaling from a diverse repertoire of receptors on innate immune leukocytes. The Src-family kinases initiate intracellular signaling from these receptors by phosphorylating specific tyrosine residues within ITAM (Immunoreceptor Tyrosine-based Activation Motifs) domains that are either part of the cytoplasmic domain of the receptor or are located on receptor-associated signaling adapters, such as the FcR? or DAP-12 molecules. Phosphorylation of these ITAM domains leads to recruitment of Syk/Zap70 kinases which in turn initiate downstream signaling, leading to cellular activation. Src-family kinases also phosphorylate inhibitory receptors on specific tyrosines within ITIM (Immunoreceptor Tyrosine-based Inhibitory Motifs) domains that are located within the cytoplasmic regions of each receptor. ITIM phosphorylation leads to recruitment of the tyrosine phosphatase SHP-1, which in turn dephosphorylates both the Src kinases as well as downstream substrates in the signaling cascade. The overall balance between activating and inhibitory signaling determines the cellular response. As a result, the functions of the Src-family kinases are opposed in large part by SHP-1, which serves as a major brake to intracellular signaling in innate cells. Mutation of the SHP-1 gene (Ptpn6) results in severely hyperactive lymphocytes, myeloid cells and platelets. As a result, SHP-1 deficient animals (motheaten (me/me) or motheaten viable (mev/mev)) develop dramatic autoimmunity, inflammation and early mortality due to lung inflammation (~4 weeks for complete SHP-1 loss in me/me mice and ~8-10 weeks in the hypomorphic mev/mev mice). Though these SHP-1 deficient animals have been available for years, the complex interactions and indirect effects between the many hyperactive leukocyte subsets has limited their utility. Thus it remains unclear which specific SHP-1 regulated signaling pathways in each of the different hyperactive leukocyte types is responsible for the different aspects of the autoimmune/inflammatory disease. To address this problem, we have bred the Ptpn6flx/flx (SHP-1flx) mice to a series of Cre expressing animals to achieve lineage-specific deletion of SHP-1. Deletion of SHP-1 specifically in neutrophils (using MRP8-Cre) recapitulates much of the inflammatory disease in present in mev/mev mice but the animals don't get autoimmunity. Deletion of SHP-1 in dendritic cells (using CD11c-Cre) results in autoimmunity, but no inflammation. Deletion of SHP-1 in both neutrophils and macrophages (using LysM-Cre) results in no phenotype at all (despite the presence of activated neutrophils). Finally, deletion of SHP-1 in platelets (using PF4-Cre) results in early mortality due to lung inflammation, without evidence of other disease. By subdividing the overall disease phenotype caused by global SHP-1 deficiency, this set of lineage-specific mutant mice will allow us to characterize the in vivo physiologic functions of SHP-1 regulated signaling in defined leukocytes which has been impossible to do with previous animal models.. We hypothesize that dysregulation of integrin and Toll-like receptor (TLR) signaling are the major drivers to the inflammatory versus autoimmune phenotypes in these different conditional SHP-1 mutant mice. We will examine this using both biochemical and genetic approaches, the latter of which will depend on the generation of double floxed mice that delete specific signaling pathways in each conditional murine line (for example SHP-1flxMyD88flxCD11c-Cre). We will also carry out unbiased screens of signaling pathways in the cells from the various conditional mutant mice. Finally, we will carry out platelet studies and use a novel two-photon imaging method to examine platelet/neutrophil interactions in the inflamed lungs of SHP-1flxPF4-Cre mice, to determine the mechanisms by which dysregulation of platelet function can lead to lethal pulmonary inflammation. Overall, these studies will help define how hyperactivation of specific tyrosine kinase based signaling pathways leads to inflammation and autoimmunity. Since it is well appreciated that alterations in these signaling pathways play a major role in human autoimmune, inflammatory and malignant disease, a more complete in vivo dissection of SHP-1 regulated signaling will help guide therapeutic development. PUBLIC HEALTH RELEVANCE: Immune cells sense pathogens such as bacteria, fungi and viruses through a complex array of cellular receptors. Binding of the pathogen molecules to these receptors activates intracellular enzymes that initiate intracellular signaling pathways that lead to immune cell activation to eliminate the pathogen. Appropriate control of these intracellular signaling pathways is critical to prevent immune cell hyperactivation that can cause damage to host tissues during responses to pathogen invasion. One of the major molecules that downregulates immune cells are the tyrosine phosphatase SHP-1. We have generated a series of mutant mice that lack the SHP-1 protein in defined immune cell types. These conditional SHP-1 mutant animals develop a spectrum of diseases, including severe skin inflammation, autoimmunity or lethal pulmonary inflammation. By studying the specific intracellular signaling pathways that are affected by these different SHP-1 conditional mutations, we will gain a new insight into the regulation of immune cell reactivity. This will lead to better design of therapeutcs to target immune diseases, as well as leukemia and lymphoma.
Funding Period: 2000-08-01 - 2017-01-31
more information: NIH RePORT

Top Publications

  1. ncbi Mac-1 signaling via Src-family and Syk kinases results in elastase-dependent thrombohemorrhagic vasculopathy
    Junichi Hirahashi
    Department of Pathology, Center for Excellence in Vascular Biology, Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
    Immunity 25:271-83. 2006
  2. pmc Multimolecular signaling complexes enable Syk-mediated signaling of CD36 internalization
    Bryan Heit
    Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
    Dev Cell 24:372-83. 2013
  3. pmc Tracing conidial fate and measuring host cell antifungal activity using a reporter of microbial viability in the lung
    Anupam Jhingran
    Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
    Cell Rep 2:1762-73. 2012
  4. pmc Macrophages require Skap2 and Sirpα for integrin-stimulated cytoskeletal rearrangement
    Francis J Alenghat
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
    J Cell Sci 125:5535-45. 2012
  5. pmc Dectin-1-triggered recruitment of light chain 3 protein to phagosomes facilitates major histocompatibility complex class II presentation of fungal-derived antigens
    Jun Ma
    Division of Immunology, Cedars Sinai Medical Center, Los Angeles, California 90048, USA
    J Biol Chem 287:34149-56. 2012
  6. ncbi Distinct and overlapping functional roles of Src family kinases in mouse platelets
    S Severin
    Centre for Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
    J Thromb Haemost 10:1631-45. 2012
  7. pmc Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury
    Helena Block
    Department of Anesthesiology and Critical Care Medicine, University of Munster, 48149 Munster, Germany
    J Exp Med 209:407-21. 2012
  8. pmc B-cell adaptor for PI3K (BCAP) negatively regulates Toll-like receptor signaling through activation of PI3K
    Minjian Ni
    Immunology Program, Benaroya Research Institute, Seattle, WA 98101, USA
    Proc Natl Acad Sci U S A 109:267-72. 2012
  9. pmc Receptor-like tyrosine phosphatases CD45 and CD148 have distinct functions in chemoattractant-mediated neutrophil migration and response to S. aureus
    Jing W Zhu
    Department of Medicine, Howard Hughes Medical Institute, Rosalind Russell Medical Research Center for Arthritis, University of California, San Francisco, San Francisco, CA 94143, USA
    Immunity 35:757-69. 2011
  10. ncbi Class I phosphoinositide-3-kinases and SRC kinases play a nonredundant role in regulation of adhesion-independent and -dependent neutrophil reactive oxygen species generation
    Laura Fumagalli
    Section of General Pathology, Department of Pathology and Diagnostics, University of Verona, Verona 37134, Italy
    J Immunol 190:3648-60. 2013

Research Grants

Detail Information

Publications40

  1. ncbi Mac-1 signaling via Src-family and Syk kinases results in elastase-dependent thrombohemorrhagic vasculopathy
    Junichi Hirahashi
    Department of Pathology, Center for Excellence in Vascular Biology, Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
    Immunity 25:271-83. 2006
    ..These data suggest that Mac-1 engagement of complement activates tyrosine kinases to promote elastase-dependent blood vessel injury in vivo...
  2. pmc Multimolecular signaling complexes enable Syk-mediated signaling of CD36 internalization
    Bryan Heit
    Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
    Dev Cell 24:372-83. 2013
    ..By coupling to FcRγ, CD36 is able to engage Src-family kinases and Syk, which in turn drives the internalization of CD36 and its bound ligands...
  3. pmc Tracing conidial fate and measuring host cell antifungal activity using a reporter of microbial viability in the lung
    Anupam Jhingran
    Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
    Cell Rep 2:1762-73. 2012
    ....
  4. pmc Macrophages require Skap2 and Sirpα for integrin-stimulated cytoskeletal rearrangement
    Francis J Alenghat
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
    J Cell Sci 125:5535-45. 2012
    ....
  5. pmc Dectin-1-triggered recruitment of light chain 3 protein to phagosomes facilitates major histocompatibility complex class II presentation of fungal-derived antigens
    Jun Ma
    Division of Immunology, Cedars Sinai Medical Center, Los Angeles, California 90048, USA
    J Biol Chem 287:34149-56. 2012
    ....
  6. ncbi Distinct and overlapping functional roles of Src family kinases in mouse platelets
    S Severin
    Centre for Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
    J Thromb Haemost 10:1631-45. 2012
    ..The aims of this study were to quantitate SFK members present in platelets and to analyze their contribution to platelet regulation using glycoprotein VI (GPVI) and intregrin αIIbβ3, and in vivo...
  7. pmc Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury
    Helena Block
    Department of Anesthesiology and Critical Care Medicine, University of Munster, 48149 Munster, Germany
    J Exp Med 209:407-21. 2012
    ..Thus, SLP-76 and ADAP are involved in E-selectin-mediated integrin activation and neutrophil recruitment to inflamed kidneys, which may underlie the development of life-threatening ischemia-reperfusion-induced AKI in humans...
  8. pmc B-cell adaptor for PI3K (BCAP) negatively regulates Toll-like receptor signaling through activation of PI3K
    Minjian Ni
    Immunology Program, Benaroya Research Institute, Seattle, WA 98101, USA
    Proc Natl Acad Sci U S A 109:267-72. 2012
    ..Therefore, BCAP is an essential activator of the PI3K pathway downstream of TLR signaling, providing a brake to limit potentially pathogenic excessive TLR responses...
  9. pmc Receptor-like tyrosine phosphatases CD45 and CD148 have distinct functions in chemoattractant-mediated neutrophil migration and response to S. aureus
    Jing W Zhu
    Department of Medicine, Howard Hughes Medical Institute, Rosalind Russell Medical Research Center for Arthritis, University of California, San Francisco, San Francisco, CA 94143, USA
    Immunity 35:757-69. 2011
    ..Moreover, our results suggest that CD45 and CD148 preferentially target different SFK members (Hck and Fgr versus Lyn, respectively) to positively and negatively regulate GPCR pathways...
  10. ncbi Class I phosphoinositide-3-kinases and SRC kinases play a nonredundant role in regulation of adhesion-independent and -dependent neutrophil reactive oxygen species generation
    Laura Fumagalli
    Section of General Pathology, Department of Pathology and Diagnostics, University of Verona, Verona 37134, Italy
    J Immunol 190:3648-60. 2013
    ....
  11. pmc Distinct roles for neutrophils and dendritic cells in inflammation and autoimmunity in motheaten mice
    Clare L Abram
    Department of Laboratory Medicine and the Program in Immunology, University of California, San Francisco 94143, USA
    Immunity 38:489-501. 2013
    ..Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease...
  12. pmc STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense
    Hong Zhang
    Department of Laboratory Medicine
    Blood 123:2238-49. 2014
    ..These results demonstrate the critical role of STIM1-mediated SOCE and define major protein targets of calcium signaling in neutrophil activation during inflammatory disease. ..
  13. pmc Requirement for MyD88 signaling in B cells and dendritic cells for germinal center anti-nuclear antibody production in Lyn-deficient mice
    Zhaolin Hua
    Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
    J Immunol 192:875-85. 2014
    ....
  14. pmc B cell-specific loss of Lyn kinase leads to autoimmunity
    Chrystelle Lamagna
    Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143
    J Immunol 192:919-28. 2014
    ..Our results demonstrate that B cell-intrinsic Lyn-dependent signaling pathways regulate B cell homeostasis and activation, which in concert with B cell-specific MyD88 signaling pathways can drive the development of autoimmune disease. ..
  15. pmc Expression of the TEL-Syk fusion protein in hematopoietic stem cells leads to rapidly fatal myelofibrosis in mice
    Michelle T Graham
    Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, United States of America
    PLoS ONE 8:e77542. 2013
    ..These data indicate that expression of TEL-Syk in fetal liver hematopoietic cells results in JAK-independent STAT5 phosphorylation ultimately leading to a uniquely aggressive and lethal form of myelofibrosis. ..
  16. ncbi The multifaceted functions of neutrophils
    Tanya N Mayadas
    Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women s Hospital and Harvard Medical School, Boston, Massachusetts 20115 email
    Annu Rev Pathol 9:181-218. 2014
    ..These newly appreciated contributions of neutrophils are described in the setting of several inflammatory and autoimmune diseases. ..
  17. pmc Neutrophils mediate antibody-induced antitumor effects in mice
    Marcello Albanesi
    Departement d Immunologie, Laboratoire Anticorps en Thérapie et Pathologie, Institut Pasteur, Paris, France
    Blood 122:3160-4. 2013
    ....
  18. pmc Hyperactivated MyD88 signaling in dendritic cells, through specific deletion of Lyn kinase, causes severe autoimmunity and inflammation
    Chrystelle Lamagna
    Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA
    Proc Natl Acad Sci U S A 110:E3311-20. 2013
    ..Thus, we demonstrate that hyperactivation of MyD88-dependent signaling in DCs is sufficient to drive pathogenesis of lupus-like disease, illuminating the fact that dysregulation in innate immune cells alone can lead to autoimmunity. ..
  19. pmc Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors
    Christel Verollet
    Centre National de la Recherche Scientifique CNRS, Unité Mixte de Recherche UMR 5089, Institut de Pharmacologie et de Biologie Structurale IPBS, Toulouse, France
    FASEB J 27:3608-18. 2013
    ..This phenotype was still detectable in adults. In summmary, Hck is one of the very few effectors of preosteoclast recruitment described to date and thereby plays a critical role in bone remodeling...
  20. pmc Strain-dependent induction of neutrophil histamine production and cell death by Pseudomonas aeruginosa
    Xiang Xu
    Department of Medicine, University of California at San Francisco, San Francisco, California, USA
    J Leukoc Biol 91:275-84. 2012
    ..These findings raise the possibility that Pseudomonas-stimulated neutrophils can enhance airway inflammation by producing histamine...
  21. pmc Deletion of Syk in neutrophils prevents immune complex arthritis
    Emily R Elliott
    Biomedical Sciences Program, University of California, San Francisco, San Francisco, CA 94143, USA
    J Immunol 187:4319-30. 2011
    ..Basophil-deficient mice also responded normally to K/BxN serum transfer. These results demonstrate that Syk-dependent signaling in neutrophils alone is critically required for arthritis development in the serum transfer model...
  22. pmc Generation of a novel system for studying spleen tyrosine kinase function in macrophages and B cells
    Allison L Miller
    Department of Laboratory Medicine, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143, USA
    J Immunol 182:988-98. 2009
    ....
  23. ncbi Transmigration of neutrophils across inflamed endothelium is signaled through LFA-1 and Src family kinase
    Melissa R Sarantos
    Department of Biomedical Engineering, University of California, Davis, CA 95616, USA
    J Immunol 181:8660-9. 2008
    ..We conclude that dimeric bond clusters of LFA-1/ICAM-1 provide a key outside-in signal for orienting cytoskeletal dynamics that direct PMN extravasation at sites of inflammation...
  24. pmc Myeloid Src kinases regulate phagocytosis and oxidative burst in pneumococcal meningitis by activating NADPH oxidase
    Robert Paul
    Department of Neurology, Klinikum Grosshadern, Ludwig Maximilians University, Marchioninistr 15, D 81377 Munich, Germany
    J Leukoc Biol 84:1141-50. 2008
    ..These data support the role of SFKs as critical mediators of CR3 signal transduction in host defense...
  25. ncbi The diverse functions of Src family kinases in macrophages
    Clare L Abram
    Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA
    Front Biosci 13:4426-50. 2008
    ..In general, SFKs may function more like rheostats, influencing the amplitude of many pathways...
  26. pmc Increased TLR responses in dendritic cells lacking the ITAM-containing adapters DAP12 and FcRgamma
    Ching Liang Chu
    Immunology Research Center, National Health Research Institutes, Taiwan
    Eur J Immunol 38:166-73. 2008
    ....
  27. ncbi Immunoreceptor-like signaling by beta 2 and beta 3 integrins
    Zoltan Jakus
    Department of Physiology, Semmelweis University School of Medicine, 1088 Budapest, Hungary
    Trends Cell Biol 17:493-501. 2007
    ..These and other reports reveal an unexpected similarity between the signal-transduction mechanisms used by integrins and immune recognition receptors...
  28. ncbi Convergence of immunoreceptor and integrin signaling
    Clare L Abram
    Department of Laboratory Medicine, University of California, San Francisco, CA 94143 0451, USA
    Immunol Rev 218:29-44. 2007
    ..In this review, we discuss the convergence of immunoreceptor and integrin signaling, focusing on how these pathways modulate leukocyte activation...
  29. pmc Neutrophil histamine contributes to inflammation in mycoplasma pneumonia
    Xiang Xu
    Cardiovascular Research Institute, Department of Microbiology Immunology, University of California at San Francisco, and Veterans Affairs Medical Center, San Francisco 94121, USA
    J Exp Med 203:2907-17. 2006
    ..These findings suggest that neutrophils, provoked by mycoplasma, greatly expand their capacity to synthesize histamine, thereby contributing to lung and airway inflammation...
  30. pmc Multiple roles of Lyn kinase in myeloid cell signaling and function
    Patrizia Scapini
    Department of Laboratory Medicine, University of California, San Francisco, CA 94143 0451, USA
    Immunol Rev 228:23-40. 2009
    ..In this review, we discuss the current knowledge of the duplicitous nature of Lyn in the modulation of myeloid cell signaling and function...
  31. pmc The ins and outs of leukocyte integrin signaling
    Clare L Abram
    Program in Immunology, Department of Laboratory Medicine, University of California, San Francisco, California 94143 0451, USA
    Annu Rev Immunol 27:339-62. 2009
    ....
  32. pmc B cell-derived IL-10 suppresses inflammatory disease in Lyn-deficient mice
    Patrizia Scapini
    Department of Laboratory Medicine and Microbiology Immunology, University of California, San Francisco, CA 94143, USA
    Proc Natl Acad Sci U S A 108:E823-32. 2011
    ....
  33. pmc Overview: studying integrins in vivo
    Clifford A Lowell
    Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA
    Methods Mol Biol 757:369-97. 2012
    ....
  34. pmc Neutrophils give us a shock
    Clifford A Lowell
    Department of Laboratory Medicine, UCSF, San Francisco, California 94143 0451, USA
    J Clin Invest 121:1260-3. 2011
    ..These exciting results suggest that we have to reevaluate our models for anaphylaxis in humans, which will have a direct impact on our therapeutic approaches for prevention of this potential deadly hypersensitivity reaction...
  35. pmc Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection
    Lynn A Kamen
    Program in Immunology, Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
    J Immunol 186:1656-65. 2011
    ..These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents...
  36. pmc Myeloid cells, BAFF, and IFN-gamma establish an inflammatory loop that exacerbates autoimmunity in Lyn-deficient mice
    Patrizia Scapini
    Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
    J Exp Med 207:1757-73. 2010
    ..Our findings uncover an important pathological role of BAFF in autoimmune disorders...
  37. pmc The expanding roles of ITAM adapters FcRgamma and DAP12 in myeloid cells
    Jessica A Hamerman
    Immunology Program, Benaroya Research Institute at Virginia Mason, Seattle, WA, USA
    Immunol Rev 232:42-58. 2009
    ..In this review, we discuss the newly described receptors that utilize DAP12 and/or FcRgamma adapters to modulate innate immune responses...
  38. pmc Neutrophil-specific deletion of Syk kinase results in reduced host defense to bacterial infection
    Jessica A Van Ziffle
    Immunology Program and the Department of Laboratory Medicine, University of California San Francisco, 513 Parnassus Ave, San Francisco, CA 94143 0451, USA
    Blood 114:4871-82. 2009
    ..These results indicate that loss of Syk kinase-mediated integrin signaling impairs leukocyte activation, leading to reduced host defense responses...
  39. pmc Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice
    Clare L Abram
    Department of Laboratory Medicine and the Program in Immunology, University of California, San Francisco, CA 94143, USA
    J Immunol Methods 408:89-100. 2014
    ..By focusing on myeloid subsets, we directly compare the relative efficiency and specificity of myeloid deletion in these strains under steady-state conditions. ..

Research Grants30

  1. Mental Stress Ischemia: Prognosis and Genetic Influences
    Arshed A Quyyumi; Fiscal Year: 2013
    ....
  2. Analyses of neutrophil interaction with immune complexes
    Tanya N Mayadas; Fiscal Year: 2013
    ..abstract_text> ..