FUNCTIONAL AND MOLECULAR ASPECTS OF ION CHANNELS IN MACROPHAGES by MIN RU QIU This thesis is submitted to the University of New South Wales in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Medicine Sydney, Australia, 2003 ii PUBLICATIONS AND ABSTRACTS RESULTING FROM THIS PROJECT Publications 1: Qiu MR, Campbell TJ, Breit SN. A potassium ion channel is involved in cytokine production by activated human macrophages. Clin Exp Immunol. 2002 Oct;130(1):67- 74. 2: Warton K, Tonini R, Fairlie WD, Matthews JM, Valenzuela SM, Qiu MR, Wu WM, Pankhurst S, Bauskin AR, Harrop SJ, Campbell TJ, Curmi PM, Breit SN, Mazzanti M. Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1. J Biol Chem. 2002 Jul 19;277 (29):26003-11. 3: Harrop SJ, Demaere MZ, Fairlie WD, Reztsova T, Valenzuela SM, Mazzanti M, Tonini R, Qiu MR, Jankova L, Warton K, Bauskin AR, Wu WM, Pankhurst S, Campbell TJ, Breit SN, Curmi PM. Crystal structure of the soluble form of the intracellular chloride Ion channel CLIC1 (NCC27) at 1.4A resolution. J Biol Chem. 2001 Sep; 10: 10 4: Valenzuela SM, Mazzanti M, Tonini R, Qiu MR, Warton K, Musgrove EA, Campbell TJ, Breit SN. The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle. J Physiol. 2000 Dec 15;529 Pt 3:541-52. 5: Walker BD, Singleton CB, Bursill JA, Wyse KR, Valenzuela SM, Qiu MR, Breit SN, Campbell TJ. Inhibition of the human ether-a-go-go-related gene (HERG) potassium channel by cisapride: affinity for open and inactivated states. Br J Pharmacol. 1999 Sep; 128 (2):444-50. iii 6: Singleton CB, Valenzuela SM, Walker BD, Tie H, Wyse KR, Bursill JA, Qiu MR, Breit SN, Campbell TJ. Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells. Br J Pharmacol. 1999 Jun;127 (4):941-8. 7: Walker BD, Valenzuela SM, Singleton CB, Tie H, Bursill JA, Wyse KR, Qiu MR, Breit SN, Campbell TJ. Inhibition of HERG channels stably expressed in a mammalian cell line by the antianginal agent perhexiline maleate. Br J Pharmacol. 1999 May;127 (1):243-51. 8: Martin D, Bursill J, Qiu MR, Breit SN, Campbell T. Alternative hypothesis for efficacy of macrolides in acute coronary syndromes. Lancet. 1998 Jun 20;351 (9119):1858-9. Abstracts and posters. 1. Min Ru Qiu, Klaus Matthaei, Stella M. Valenzuela, Terence J. Campbell, and Samuel N. Breit. Initial phenotyping and generation of the NCC27 Cl- channel KO mice. ComBio, Darling Harbour, Sydney, Australia, 2002 2. Min Ru Qiu, Klaus Matthaei, W. Douglas Fairlie, Peter K. Brown, Stella M. Valenzuela, Terence J. Campbell, and Samuel N. Breit. Generation of mice with modification and conditional deletion of the NCC27 Cl- channel gene. Structure and function of ion channel, Leura, Australia, 2001 and The 11th St. Vincent’s Campus Research Symposium, Sydney, 2001 3. Min Ru Qiu, Anthony G. Moore, Terence J. Campbell, and Samuel N. Breit. A potassium ion channel is involved in cytokine production by activated human macrophages. Structure and function of ion channel, Leura, Australia, 2001 and The 10th St. Vincent’s Campus Research Symposium, Sydney, 2000 iv 4. Min Ru Qiu, Klaus Matthaei, Stella M. Valenzuela, Terence J. Campbell, and Samuel N. Breit. Cloning and characterization of the mouse NCC27 gene. The 9th St. Vincent’s Campus Research Symposium, Sydney, 1999. v DEDICATION THIS THESIS IS DEDICATED TO MY FATHER, QIU SHI LIANG, AND MY MOTHER, DING BING QING FOR THEIR LIFE TIME SUPPORT. vi ACKNOWLEDGEMENTS I would like express my special gratitude to my supervisor Professor Samuel N. Breit for all his great direction, support, and encouragement during the whole course of this project. I would also like to thank Professor Ronald Penny, the former director of the Centre for Immunology, for accepting me to undertake my PhD at the Centre for Immunology, St Vincent’s Hospital, Sydney, Australia. In addition, I am grateful for the financial support from Austalian Postgraduate Award during my study. Special thanks go to Dr Klaus Matthaei, head of the gene targeting group, JCSMR, ANU, Canberra. Without his great help, my project would not go smoothly as it did. Furthermore, my thanks also go to our collaborators, Professor Terence J. Campebell from Department of Clinical Pharmacology and Cardiology, St Vincent’s Hospital and Dr Paul M.G. Curmi from Initiative for Biomolecular Structure, School of Physics, University of New South Wales. I also like to extend my thanks to my colleagues at the Centre for Immunology for their great support, assistance, and advice over the years. In particular, Stella Valenzuela, Peter Brown, Tony Moore, Douglas Fairlie, Asne Bauskin, Kristina Warton, Ning Xu, Greg Pankhurst, John Zaunders, and Marker Hunter for their assistance and those helpful discussions. I am very grateful to Bill Sewell, Kristina Warton, Natasha Foster, Asne Bauskin, Tamara Kuffner, Greg Pankhurst, and Wendy Gold for their proof reading of my thesis manuscripts and their valuable comments and suggestions. Finally, I would like to express my sincere thanks to my parents and family for their support and patience over the years. Most importantly, I really thank my husband Andrew Ning Han for his support throughout the course of my studies. I can not forget to thank my little boy, James Han for his good behavior and support over the years. vii ABSTRACT Monocytes/macrophages play very important roles in innate and adaptive immunity. Ion channels are small molecules embedded in the cell membrane and they play fundamental roles in cell function. Both topics have been extensively studied in isolation, however the role of ion channels in macrophage function is far less understood. In this thesis, the functional and molecular aspects of two ion channels expressed in macrophages, Kor, a potassium channel, and CLIC1, a chloride channel were studied. The biological function of Kor and CLIC1 in activated human macrophages was examined using ion channel blockers. In addition, the role of CLIC1 in the cell cycling of CHO-K1 cells was also investigated. The in vitro studies showed that Kor and CLIC1 are involved in cytokine production by PMA-activated human macrophages and that CLIC1 is also involved in the cell cycling of CHO-K1 cells. Despite providing interesting data, the results of the in vitro studies were difficult to interpret due to the uncertain specificities of the Cl- channel blockers. Therefore, to understand the biological role of CLIC1 in vivo, a gene targeting experiment was performed to create a CLIC1 knock-out (KO) mouse. This involved cloning the mouse CLIC1 gene, making a targeting vector, producing targeted ES cells, and generating a CLIC1 knock-in (KI) mouse which carries a flag tag at the N-terminal and three loxP sites in the targeted locus. Crossing the CLIC1 KI mouse with the TNAP-Cre mouse, a strain over expressing Cre recombinase under a TNAP promoter, a CLIC1 KO mouse was generated. The initial phenotype analysis showed no major development or growth abnormality in the CLIC1 KO mouse. Instead, hyperplasia of megakaryocytes and possible erythroid cells in the spleen and bone marrow was observed suggesting some degree of abnormality in the haematopoeitic system. Furthermore, a comparison of wild type mice with the CLIC1 KO mouse showed that CLIC1 protein expresses at high levels in monocytes, lymphocytes, platelets, and tissue macrophages of normal animals tissues, such as spleen, kidney (mesangial cell), and liver (kupffer cells). This further indicates that CLIC1 may play a significant role in regulating functions of platelets, lymphocytes, and specially tissue macrophages. More extensive studies can now be performed on the CLIC1 KO mouse to clarify the biological function of CLIC1. In viii summary, the generation of the CLIC1 KO mouse provides a valuable model to study the biological function of CLIC1 both in vivo and in vitro. ix TABLE OF CONTENTS CHAPTER 1 LITERATURE REVIEW ................................................................ 1 1.1 Introduction ......................................................................................................................................... 1 1.2 Macrophages ........................................................................................................................................ 1 1.2.1 Introduction.................................................................................................................................... 1 1.2.2 Origin of mononuclear phagocytes. ............................................................................................... 2 1.2.3 Tissue distribution and heterogeneity of mononuclear phagocytes. .............................................. 3 1.2.4 Gene expression of macrophages................................................................................................... 7 1.2.5 Function of macrophages............................................................................................................... 8 1.2.5.1 Endocytosis and phagocytosis................................................................................................ 8 1.2.5.2 Antigen processing and presentation.................................................................................... 11 1.2.5.3 Secretion of cytokines and other factors .............................................................................. 12 1.2.5.4