UvA-DARE (Digital Academic Repository) In vivo studies on the role of Adhesion-GPCR CD97 in immunity Veninga, H. Publication date 2010 Link to publication Citation for published version (APA): Veninga, H. (2010). In vivo studies on the role of Adhesion-GPCR CD97 in immunity. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:04 Oct 2021 CD55 regulates CD97 expression, granulopoietic activity, and anti- bacterial host defense Henrike Veninga,1 Robert M. Hoek,1 Alex F. de Vos,2 Alex M. de Bruin,1 Dennis Flierman,1 Feng-Qi An,3 Tom van der Poll,2 René A.W. van Lier,1 M. Edward Medof,3 and Jörg Hamann1 1Department of Experimental Immunology and 2Center for Experimental Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam,5 The Netherlands, 3Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA Manuscript in preparation Thesis henrike_HV.indd 77 26-9-2010 22:08:06 Thesis henrike_HV.indd 78 26-9-2010 22:08:06 CD55 regulates CD97 expression and granulopoietic activity, ABSTRACT Next to its well-established activity in regulating complement activation, CD55 is a ligand of the Adhesion class G protein-coupled receptor CD97; however, the relevance of this interaction has remained elusive. We here show that leukocytes fromCd55 -/- mice expressed significantly increased levels of CD97 that normalized after transfer into wild-type mice due to contact with CD55 on both leukocytes and stromal cells. Moreover, mice lacking CD55 displayed granulocytosis with about two-fold more circulating granulocytes in the blood stream, the marginated pool, and the spleen. This granulocytosis was independent of increased complement activity and copied the phenotype previously found in CD97- deficient mice. Augmented numbers of Gr-1-positive cells in cell cycle in the bone marrow indicated a higher granulopoietic activity in mice lacking CD55 or CD97. Concomitant with the increase in blood granulocyte numbers, Cd55-/- mice challenged with the respiratory pathogen Streptococcus pneumoniae developed less bacteremia and died later after infection. Collectively, these data suggest that complement-independent interaction of CD55 with CD97 is functionally relevant and involved in granulocyte homeostasis and host defense. INTRODUCTION (GPCRs), abundantly expressed by virtually Decay-accelerating factor (CD55) is a glycosyl- all immune cells (16-21). Like all Adhesion- phosphatidyl-inositol (GPI)-anchored mole- GPCRs, CD97 is a two-subunit molecule cule on leukocytes, erythrocytes, and serum- consisting of an extracellular α subunit that exposed stromal cells that accelerates the is non-covalently associated with a seven- decay of the complement convertases transmembrane (TM7) β subunit.(18) The C3 and C5 (1). The importance of CD55 two subunits originate from autocatalytic for preventing endogenous cells from processing of the CD97 polypeptide in 5 unwanted complement activation is evident the endoplasmatic reticulum (22). At the from the phenotype of CD55-deficient mice N-terminus, CD97 possesses tandemly that develop exaggerated autoimmune arranged epidermal growth factor (EGF)- reactions in a variety of spontaneous and like domains of which the first two interact induced models (2-7). Furthermore, studies with the N-terminal complement control in Cd55-/- mice showed that complement protein (CCP) domains of CD55 (23,24). activation not only facilitates innate immune When overexpressed in vitro, CD97 can responses but also adaptive immunity facilitate adhesion of CD55-bearing cells (8-12). Next to the well-established (15,19,20,23). However, with 86 μM, the function as regulator of the complement affinity between CD97 and CD55 is low (24), cascade, CD55 is engaged in complement- and investigation of CD97-deficient mice independent processes and is hijacked by revealed no signs of impaired leukocyte several viral and bacterial pathogens to migration (25,26). Moreover, CD97 signaling promote cell adhesion and invasion (13,14). in response to CD55 binding could not be Furthermore, we demonstrated previously demonstrated (21), which was unexpected that CD55 is a binding partner of CD97 (15). for a TM7 receptor. To investigate whether CD97 is a member of the EGF-TM7 family of CD55 and CD97 interact in vivo and to explore Adhesion class G protein-coupled receptors possible physiological consequences of this 79 Thesis henrike_HV.indd 79 26-9-2010 22:08:06 CD55 regulates CD97 expression and granulopoietic activity, interaction, we studied the expression of a polyclonal goat antibody against the α CD97 on leukocytes as well as the size and chain of CD97 (R&D Systems, Wiesbaden, behavior of the granulocyte compartments Germany) and a rabbit polyclonal antibody in CD55-deficient mice. We found that CD97 against the β chain of CD97 (26). expression on leukocytes was constitutively For flow cytometry, CD97 antibodies downregulated by CD55. Moreover, CD55- were biotinylated in house and used in deficient mice, like mice that lack CD97, had combination with streptavidin-conjugated increased levels of circulating granulocytes, APC or PeCy7. In addition, FITC-, PE-, PerCP- which was due to a higher granulopoietic Cy5.5-, PeCy7-, APC-, or biotin-conjugated activity in the bone marrow. Finally, mice mAbs specific for CD3, CD11b, CD45.1, lacking CD55 were better protected against CD45.2, B220, F4/80, Gr-1, Ly6C, NK1.1 (all S. pneumoniae-induced pneumonia. To- eBioscience, San Diego, CA, USA) and Ly6G gether, our data prove cross-talk between (BD Biosciences, San Jose, CA, USA) were CD55 and the Adhesion-GPCR CD97 in vivo used. and indicate a role of this interaction in granulocyte homeostasis and host defense. Flow cytometry Peripheral blood was collected in heparin MATERIALS AND METHODS by heart puncture. Single cell suspensions of spleen were made by mashing the Mice organs through a 70-μm cell strainer. Bone Mice deficient for CD55 Cd55( -/-, synonym: marrow cells were harvested from dissected Daf1-/-) and CD97 (Cd97-/-) have been femurs by flushing the bone marrow plug generated previously by us (27,26). Mice with PBS/0.5% bovine serum albumin lacking the receptors for C3a (C3ar1-/-) and (BSA). Erythrocytes were lysed with a buffer -/- C5a (C5ar1 ) were kind gifts of Prof. Graig containing 155 mM NH4Cl, 10 mM KHCO3, Gerard (Harvard Medical School, Boston, and 1 mM EDTA in all these cell preparations. MA, USA) (28,29). Compound mice were 25 μl whole blood or 5 x 105 splenocytes or created by crossing Cd55-/- mice with Cd97-/-, bone marrow cells were used per staining. C3ar1-/-, and C5ar1-/- mice (10). All genetically Nonspecific binding of mAbs was blocked modified mice were back-crossed to C57BL/6 by adding 10% normal mouse serum and for at least eight generations. C57BL/6 1.25 μg/ml anti-CD16/32 (clone 2.4G2; BD wild-type mice were littermates or were Biosciences) together with the appropriately purchased from Charles River (Maastricht, diluted mAbs in PBS containing 0.5% BSA. The Netherlands). Congenic mice expressed Cells were incubated for 30 min at 4°C, Cd45.1 in the B6.SJL strain. All mice used in followed (where appropriate) by a second this study were matched for age and sex and incubation step with streptavidin-APC kept under specific pathogen-free conditions. or -PeCy7 (eBioscience). Flow cytometric Experiments were approved by the Animal analysis was performed using a FACSCalibur Ethics Committees of our institutions. or FACSCanto (BD Biosciences) and the FlowJo software package (Tree Star, Ashland, Immunological reagents OR, USA). Absolute numbers were calculated Native CD97 was detected with hamster on basis of total cell counts measured on mAbs against EGF domain 1 (clone 1B2), a CASY cell counter (Schärfe, Reutlingen, EGF domain 2 (clone 1A2), and the α chain Germany) or FACSCalibur multiplied by the downstream of EGF domain 3 (clone 1C3) percentage of cells positive for a specific (20,30). For Western blot analysis, we used marker, as measured by flow cytometry. 80 Thesis henrike_HV.indd 80 26-9-2010 22:08:06 CD55 regulates CD97 expression and granulopoietic activity, Western blot analysis Adoptive transfer Immune cells from spleen and blood were Spleens were harvested from congenic wild- isolated as described above and solubilized in type (Cd45.1) or Cd55-/- (Cd45.2) mice. Single a lysis buffer consisting of 2% Triton X-100 in cell suspensions were made as described PBS, supplemented with Complete Protease above under aseptic conditions in sterile Inhibitor Mix (Roche, Mannheim, Germany). PBS, and 25 x 106 congenic wild-type or Lysates were made by agitation on a nutator Cd55-/- cells were injected into the tail vein for 30 min at 4°C, after which insoluble of Cd55-/- or congenic wild-type recipient material was removed by centrifugation in mice, respectively. Blood and spleen from a microfuge at 16,100 x g for 15 min. The recipient mice were collected 24 h after resulting supernatant was used for SDS- the adoptive transfer and analyzed by PAGE. Approximately 3 x 106 cell equivalents flow cytometry for CD97 expression on were loaded on a protein gel and blotted to leukocytes.