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.
| Gevrey, Jean-Claude; Isaac, Beth M; Cox, Dianne (2005) Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine). J Immunol 175:3737-45 |
| Kheir, Wassim Abou; Gevrey, Jean-Claude; Yamaguchi, Hideki et al. (2005) A WAVE2-Abi1 complex mediates CSF-1-induced F-actin-rich membrane protrusions and migration in macrophages. J Cell Sci 118:5369-79 |
| Cox, Dianne; Berg, Jonathan S; Cammer, Michael et al. (2002) Myosin X is a downstream effector of PI(3)K during phagocytosis. Nat Cell Biol 4:469-77 |
| Cox, D; Dale, B M; Kashiwada, M et al. (2001) A regulatory role for Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) in phagocytosis mediated by Fc gamma receptors and complement receptor 3 (alpha(M)beta(2); CD11b/CD18). J Exp Med 193:61-71 |
