UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No 1166 – ISSN 0346-6612 - ISBN 978-91-7264-526-4 ___________________________________________________________________________ Role of the CD47/SIRPα­interaction in regulation of macrophage phagocytosis Mattias Olsson Umeå 2008 Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, Umeå, Sweden Department of Integrative Medical Biology Section for Histology and Cell biology Umeå University Copyright © Mattias Olsson, 2008 ISSN: 0346-6612 ISBN: 978-91-7264-526-4 Printed in Sweden by Arkitektkopia, Umeå 2008 All previously published papers were reproduced with permission from the publishers. 2 3 Table of contents Abbreviations ............................................................................................................................ 7 Abstract ..................................................................................................................................... 8 List of original papers .............................................................................................................. 9 Background ............................................................................................................................. 10 The innate immune system – overview ............................................................................................. 10 Self vs. non-self discrimination in the innate immune system .......................................................... 10 CD47 ................................................................................................................................................. 11 SIRPα ................................................................................................................................................ 14 Red blood cells .................................................................................................................................. 16 RBC turnover ................................................................................................................................ 16 Platelets ............................................................................................................................................. 18 Macrophages ..................................................................................................................................... 18 Macrophage Subpopulations ......................................................................................................... 19 Phagocytosis ...................................................................................................................................... 19 Pro-phagocytic receptors ................................................................................................................... 20 Fc receptors ................................................................................................................................... 20 Complement receptors ................................................................................................................... 21 Scavenger receptors ....................................................................................................................... 22 Cytoskeleton and signaling in phagocytosis ...................................................................................... 22 Inhibitory regulation through CD47/SIRPα-interaction .................................................................... 24 Materials and methods ........................................................................................................... 27 Animals ............................................................................................................................................. 27 Preparation of bone marrow derived macrophages ........................................................................... 27 Measurement of platelet and red blood cell concentration ................................................................ 27 Platelet and RBC staining .................................................................................................................. 28 PKH26 and PKH67 staining .......................................................................................................... 28 CMFDA staining ........................................................................................................................... 28 RBC in vitro phagocytosis ................................................................................................................ 29 May-Grünewald and Giemsa staining ............................................................................................... 29 Staining of F-actin with phalloidin probes ........................................................................................ 29 Flow cytometric analysis of platelets ................................................................................................ 30 Platelet survival ................................................................................................................................. 30 Thrombin induced CD62P expression ............................................................................................... 30 4 Induction of platelet phosphatidylserine exposure ............................................................................ 31 In Vitro platelet phagocytosis assay .................................................................................................. 31 Experimental ITP .............................................................................................................................. 31 Red blood cell survival ...................................................................................................................... 32 Red blood cell survival in combination with a pathogenic Ab .......................................................... 32 In vitro oxidation of RBCs ................................................................................................................ 32 Liposomes ......................................................................................................................................... 32 Macrophage phagocytosis of oxidized RBCs in vitro ....................................................................... 33 Microscopy of CD47 clustering on RBCs ......................................................................................... 33 Aims ......................................................................................................................................... 34 Results ..................................................................................................................................... 35 Paper I ............................................................................................................................................... 35 CD47-/- mice are thrombocytopenic .............................................................................................. 35 Platelet clearance ........................................................................................................................... 35 In Vitro aging or activation of platelets ......................................................................................... 35 Experimental ITP and FcγR-mediated phagocytosis .................................................................... 36 Gene-dose effect of CD47/SIRPα-signaling ................................................................................. 36 Paper II .............................................................................................................................................. 36 CD47 expression on RBCs ............................................................................................................ 36 In Vitro phagocytosis ..................................................................................................................... 37 RBC survival ................................................................................................................................. 37 Paper III ............................................................................................................................................. 37 Phagocytosis of ox-RBCs is dependent on serum, scavenger receptors and PS expression ......... 37 Oxidized RBCs have reduce mobility of CD47 in the plasma membrane .................................... 38 Effects of CD47 on phagocytosis of ox-RBCs .............................................................................. 38 Paper IV ............................................................................................................................................. 39 Discrimination between wild-type and CD47-/- RBCs during FcγR-mediated phagocytosis in macrophages .................................................................................................................................. 39 Discussion ................................................................................................................................ 40 Paper I: Experimental ITP and macrophage phagocytosis of platelets ............................................. 40 Paper II: Gene-dose effect of CD47 in regulation of FcγR-mediated phagocytosis ......................... 42 Paper III: Role of CD47 in phagocytosis of oxidized RBCs ............................................................