Project: Research project

Project Details


DESCRIPTION (Taken from the applicant's abstract): Arthritis, whether due to
bacterial infections or autoimmune diseases, is characterized by the presence
of phagocytic leukocytes in the joint spaces. Phagocytes are capable of
ingesting pathogenic microbes, apoptotic cells and immune complexes by multiple
cell surface receptors. They have found that inhibition of phosphatidylinositol
3-kinase (PI 3-kinase) activity using pharmacologic agents inhibits FcgR- and
complement receptor (CR3)-mediated phagocytosis. Manipulation of
phosphatidylinositol (3,4,5) trisphosphate (PIP3) content by expression of WT
and catalytically-inactive alleles of a PIP3 phosphates (the SH2 domain
containing inositol 5' phosphates, SHIP) also regulates phagocytosis. Finally,
macrophages derived from SHIP knock-out mice display enhanced For FcgR and
CR3-mediated phagocytosis, implicating PIP3 in promoting phagocytosis. This
research proposal will address the mechanisms by which these enzymes regulate
phagocytosis. In Specific Aim 1, they will determine whether the SH2 domain of
SHIP is required for its anti-phagocytic function. They will subclone
Myc-tagged alleles of SHIP into a vector (pSFFV) that gives high expression
levels in macrophage cell lines. In Specific Aim 2, they will determine whether
known SHIP interacting proteins, such as Shc, participate in SHIP
down-modulation of FcR- and CR3-mediated phagocytosis. They will use
co-immunoprecipitation assays to identify in vivo SHIP-binding proteins
following ligation of FcgR or CR3. For in vitro assays they will employ
affinity chromatography using a GST fusion of the SHIP SH2 domain. In addition,
they will also use affinity chromatography to identify potentially novel
SHIP-interacting proteins. In Specific Aim 3, they will determine whether PIP3
production is required for CR3 "inside-out" signaling, "outside-in" signaling,
or both using primary macrophages and transfected cells lines. They will study
the role that PIP3 plays in "inside-out" signaling of CR3 using the following
assays: surface expression of CR3, presence of an activation-dependent epitope,
receptor clustering, cytoskeletal association, and CR3mediated binding to ICAM
or fibrinogen. To study "outside-in" signaling they will use cell lines that
express constitutively active CR3 and measure PI 3-kinase activity, PIP3
production, spreading on ICAM or fibrinogen, actin assembly and phagocytosis
following CR3 ligation.
Effective start/end date4/1/003/31/05


  • Cell Biology
  • Immunology


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