The Role of NMDA-Receptors (NMDARs) and NMDAR Antagonists in Murine T- and B-Lymphocyte Function Dissertation Zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität Magdeburg von M.Sc. Narasimhulu Simma geb. am 16.08.1981 in Srikakulam, India Gutacher: Prof. Dr. rer. nat. Ursula Bommhardt Prof. Dr. Dr. Sven Meuth eingereicht am: 23.06.2015 verteidigt am: 10.12.2015 Acknowledgements Acknowledgements It gives me immense pleasure to thank my mentor Prof. Dr. rer. nat. Ursula Bommhardt for her precious scientific suggestions and professional guidance that helped me a lot throughout the whole project. She was more than a modest supervisor of my PhD thesis; she really took care of my professional life and fostered my knowledge through scientific discussions. Finally, I would like to thank her for her never-ending motivation and encouragement throughout my research work. I am also very grateful to Prof. Dr. med. Burkhart Schraven, not only for giving me the opportunity to work in the Institute, but also for his continuous and critical support which helped me to develop my scientific work in a fruitful way. I would like to thank Dr. Martin Heine for enriching my scientific knowledge and valuable support throughout my research as collaboration partner. I would like to thank my colleagues Tanima Bose, Theresa Lowinus, Judith Mankiewicz, Sascha Kahlfuss, and my colleague and friend A. Varma for their support and help in correcting the thesis. I also would like to thank Gabi Weitz for her technical support during my project and all IMKI colleagues, technicians and scientists for their helpful scientific discussions and advice. I would like to thank my friends Aditya, Koteswara Rao, Sai Srinivas and Pavan for their continuous support during my Magdeburg life. Finally, I would like to thank my family and my beloved parents for their ever-lasting support throughout of my life. 2 Table of Contents Table of Contents Acknowledgements ................................................................................................................... 2 Table of Contents ..................................................................................................................... 3 Summary ................................................................................................................................... 6 Zusammenfassung .................................................................................................................... 7 1. Introduction .......................................................................................................................... 9 1.1 The adaptive immune system ........................................................................................... 9 1.2 Development and function of T cells ............................................................................... 9 1.2.1 T-cell signalling ....................................................................................................... 12 1.2.2 The Ca2+ - calcineurin - NFAT pathway ................................................................. 14 1.2.3 The Ras-Erk-AP-1 pathway .................................................................................... 15 1.2.4 Activation of the PI3-K-Akt-mTOR pathway ......................................................... 15 1.3 Development and function of B cells ............................................................................. 16 1.3.1 B- cell signalling ..................................................................................................... 17 1.4 TLR signalling ................................................................................................................ 19 1.5 Neuronal glutamate receptors ......................................................................................... 20 1.6 NMDARs and their antagonists ..................................................................................... 21 1.7 GluRs in immune cells ................................................................................................... 23 1.8 Aims of the study ........................................................................................................... 24 2. Material and Methods ........................................................................................................ 25 2.1 Material .......................................................................................................................... 25 2.1.1 Cell culture material and reagents ........................................................................... 25 2.1.2 Antibodies (Abs) ..................................................................................................... 26 2.1.2.1 Abs for cell stimulation ................................................................................................. 26 2.1.2.2 Abs for Western blot ..................................................................................................... 26 2.1.2.3 Abs for flow cytometry ................................................................................................. 27 2.1.3 Radioactive material ................................................................................................ 27 2.1.4 Inhibitors ................................................................................................................. 27 2.1.5 Chemicals and kits................................................................................................... 27 2.1.6 Miscellaneous material ............................................................................................ 29 2.1.7 Instruments .............................................................................................................. 30 2.1.8 Software................................................................................................................... 31 2.1.9 Mouse lines.............................................................................................................. 32 3 Table of Contents 2.2 Methods .......................................................................................................................... 32 2.2.1 Isolation of immune cells ........................................................................................ 32 2.2.1.1 Isolation of thymocytes, spleen and lymph node cells .................................................. 32 2.2.1.2 Isolation of CD4+ T cells .............................................................................................. 32 2.2.1.3 Isolation of B cells ......................................................................................................... 33 2.2.2 Isolation of RNA and RT-PCR ............................................................................... 34 2.2.2.1 PCR primers and RT-PCR reaction ............................................................................... 35 2.2.3 Isolation of DNA ..................................................................................................... 37 2.2.3.1 PCR of genomic DNA ................................................................................................... 38 2.2.4 Agarose gel electrophoresis..................................................................................... 39 2.2.5 Proliferation assay ................................................................................................... 40 2.2.5.1 T-cell proliferation ........................................................................................................ 40 2.2.5.2 B-cell proliferation ........................................................................................................ 40 2.2.6 Western blot analysis ............................................................................................... 40 2.2.6.1 Preparation of total protein cell extracts ........................................................................ 42 2.2.6.2 Preparation of cytoplasmic and nuclear protein extracts ............................................... 43 2.2.6.3 SDS PAGE and Western blot ........................................................................................ 44 2.2.7 Flow cytometry........................................................................................................ 44 2.2.8 Intracellular cytokine staining and IL-10-GFP expression assay ............................ 45 2.2.9 ELISA ...................................................................................................................... 45 2.2.10 Statistical analysis ................................................................................................. 46 3. Results ................................................................................................................................. 47 3.1. The function of NMDARs in murine T cells ................................................................ 47 3.1.1 Analysis of NMDAR expression in thymocytes and CD4+ T cells ........................ 47 3.1.2. NMDAR antagonists inhibit T-cell proliferation ................................................... 48 3.1.3 GluN1 protein expression in murine T cells is elusive ........................................... 49 3.1.4 NMDAR antagonist ifenprodil attenuates TCR-induced signalling ....................... 54 3.2 The effects of NMDAR antagonists on B cells .............................................................