title: Platelet and red blood cell phagocytosis kinetics are differentially controlled by phosphatase activity within mononuclear cells.
abstract: BACKGROUND
  Anti-D treatment is effective in increasing platelet (PLT) counts in patients with autoimmune thrombocytopenic purpura (AITP); however, the exact mechanism of action is unknown. Previous results have suggested that anti-D-coated red blood cells (RBCs) affect reticuloendothelial system phagocytosis by stimulating agents (e.g., reactive oxygen species) that alter signaling pathways within the phagocyte. To address this, a flow cytometric assay was used to compare the kinetics and signaling pathways responsible for opsonized PLT and RBC phagocytosis.
  
  
  METHODS
  Human RBCs or PLTs were labeled with the fluorescent dye CM-Green, opsonized with Rh immune globulin or anti-MHC, respectively, and incubated with THP-1 monocytes with or without signal transduction inhibitors and intracellular fluorescence was analyzed.
  
  
  RESULTS
  Compared with opsonized PLTs, phagocytosis of opsonized RBCs was significantly slower (p<0.0001) and, within 2 hours, induced a state of phagocytic refractoriness; resting the mononuclear cells (MNCs) for up to 24 hours did not rescue their ability to further mediate PLT phagocytosis. Inhibitors of phosphatidylinositol 3-kinase (wortmannin, LY294002, myricetin, and quercetin), protein kinase C (staurosporine), and Syk kinase (piceatannol) inhibited both opsonized RBC and opsonized PLT phagocytosis. In contrast, opsonized RBC phagocytosis was significantly (p<0.0001) enhanced by the tyrosine phosphatase inhibitor phenyl arsine oxide, whereas PLT phagocytosis was significantly reduced (p<0.0001). Of interest, phosphatase inhibition during opsonized RBC phagocytosis induced a longer (48 hr) phagocytic refractoriness period in the MNCs.
  
  
  CONCLUSIONS
  These results suggest that the early kinetics and signaling events related to phosphatase activity regulate how mononuclear phagocytes engulf opsonized RBCs and induce phagocytic refractoriness for further PLT phagocytosis.
authors.affiliation: Department of Laboratory Medicine, St Michael's Hospital, the Department of Pharmacology, and the Laboratory of Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Rukhsana
authors.initials: R
authors.lastname: Aslam
authors.forename: Michael
authors.initials: M
authors.lastname: Kim
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Speck
authors.forename: Arjuna Contram
authors.initials: AC
authors.lastname: Seetanah
authors.forename: Steven
authors.initials: S
authors.lastname: Molinski
authors.forename: John
authors.initials: J
authors.lastname: Freedman
authors.forename: John W
authors.initials: JW
authors.lastname: Semple
mesh_terms: D001792:Blood Platelets / Q000276:immunology*; D004912:Erythrocytes / Q000276:immunology*; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000201:enzymology* / Q000276:immunology; D009895:Opsonin Proteins / Q000378:metabolism; D010587:Phagocytosis; D010744:Phosphoric Monoester Hydrolases / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D009895:Opsonin Proteins; D010744:Phosphoric Monoester Hydrolases