Two Pathways of Shedding of L-selectin and Cd23 from Human B-lymphocytes
نویسنده
چکیده
...................................................................................................... 1 Acknowledgements ..................................................................................... 4 Declaration .................................................................................................. 5 Abbreviations ...............................................................................................6 Publications .................................................................................................. 8 Chapter 1: General Introduction 1.1 ATP ..................................................................................................9 1.2 Historical Aspects of ATP Receptors................................................10 1.3 Properties of P2X7 Receptor ............................................................14 1.4 Advantages of Using Cells from Chronic Lymphocytic Leukemia (CLL) Patients to Study B-lymphocytes ......................................... 17 1.5 In Vitro Model For Lymphocyte Transendothelial Migration ........... 18 1.6 L-selectin ........................................................................................ 22 1.7 CD23 .............................................................................................. 28 Chapter 2: Materials and Methods 2.1 Materials ......................................................................................... 32 2.2 Solutions ......................................................................................... 33 2.3 Preparation Of HUVEC Culture Flasks And Plates ........................ 36 2.4 Isolation of Human Umbilical Vein Endothelial Cells (HUVEC) ...... 36 2.5 Subculture HUVEC ......................................................................... 37 2.6 Cell Preparation .............................................................................. 37 2.7 Lymphocyte Transmigration Assay By Phase Contrast Microscopy .................................................................................... 38 2.8 Lymphocyte Transmigration Assay by Transmission Electron Microscopy ..................................................................................... 38 2.9 Monitoring L-selectin Expression .................................................. 39 2.10 L-selectin and CD23 Shedding Induced by ATP, BzATP or PMA ........................................................................................... 40 2.11 Measurement of soluble L-selectin using ELISA ........................... 40 2.12 Correlation between L-selectin MESF and Molecules per Lymphocyte ............................................................................. 41 2.13 Measurement of L-selectin and CD23 Changes during Transmigration ................................................................................42 2.14 Ethidium Influx Measurement by Time Resolved Flow Cytometry ............................................................................. 43 2.15 Prepare Cell Lysates .................................................................... 43 2.16 Western Blot for L-Selectin and CD23 ........................................... 44 2.17 Statistics ........................................................................................ 44 Chapter 3 : ATP-induced Shedding of CD23 and L-selectin (CD62L) from Lymphocytes Is Mediated by The Same Receptor but Different Metalloproteases 3.
منابع مشابه
Adenosine triphosphate-induced shedding of CD23 and L-selectin (CD62L) from lymphocytes is mediated by the same receptor but different metalloproteases.
CD23 is a transmembrane protein expressed on the surface of B-lymphocytes that binds IgE, CD21, CD11b, and CD11c. High concentrations of soluble CD23 and L-selectin are found in the serum of patients with B-chronic lymphocytic leukemia (B-CLL). Because extracellular adenosine triphosphate (ATP) causes shedding of L-selectin via activation of P2Z/P2X7 receptors expressed on B-CLL lymphocytes, we...
متن کاملActivation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells.
Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5'-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occ...
متن کاملL-Selectin Shedding Does Not Regulate Constitutive T Cell Trafficking but Controls the Migration Pathways of Antigen-activated T Lymphocytes
L-selectin mediates rolling of lymphocytes in high endothelial venules (HEVs) of peripheral lymph nodes (PLNs). Cross-linking of L-selectin causes proteolytic shedding of its ectodomain, the physiological significance of which is unknown. To determine whether L-selectin shedding regulates lymphocyte migration, a mutant form that resists shedding (LdDeltaP-selectin) was engineered. Transgenic mi...
متن کاملCharacterization of the Rapid Proteolytic Shedding of Murine L-Selectin
The structural requirements for L-selectin shedding were studied in murine leukocytes. Upon activation, L-selectin on both lymphocytes and neutrophils undergoes cleavage by a membrane metalloprotease, resulting in the generation of a soluble ectodomain and a membrane-retained 6 kD fragment. Radiochemical sequencing demonstrated a cleavage site in the membrane-proximal region (MPR) between R321 ...
متن کاملHydroxamate-based metalloprotease inhibitor blocks shedding of L-selectin adhesion molecule from leukocytes: functional consequences for neutrophil aggregation.
L-selectin is an adhesion molecule that mediates the recruitment of neutrophils to inflammatory sites and initiates the migration of lymphocytes into the peripheral lymph nodes. In response to cell activation, L-selectin is shed from the cell surface, and altered levels of functional soluble L-selectin are detected in the plasma of patients suffering from numerous inflammatory diseases as well ...
متن کامل