Regulation of β2-integrins by signalling pathways and cytoplasmic interacting partners

摘要

In the immune system, integrin-mediated adhesion is important for leukocyte trafficking, signalling, activation and effector functions, but how integrin-mediated adhesion is regulated in leukocytes is still incompletely understood. The main focus of this thesis was to investigate signalling pathways and mechanisms which regulate lymphocyte adhesion under flow conditions and integrin recycling and integrin-mediated phagocytosis in myeloid cells, respectively.Traditionally, cell adhesion assays have been carried out in static conditions using immobilized ligands. We have now developed shear flow assays to study integrin-mediated cell adhesion and signalling pathways involved in a physiologically-relevant manner. Integrin regulation in naïve T cells has been studied extensively in the past, and therefore the focus of this thesis was to investigate signalling pathways that regulate β2-integrins (particularly LFA-1, Lymphocyte Function-associated Antigen 1) in B cells and effector T cells. It was now shown that B cells do not require Protein Kinase C βeta or Protein Kinase D for integrin-mediated cell adhesion but inhibition of Phosphoinositide 3-kinase and Akt reduced chemokine-(Stromal cell-Derived Factor 1-) induced B cell adhesion to the LFA-1 integrin ligand ICAM-1 (Intracellular Adhesion Molecule 1) under shear stress. In contrast, integrin-mediated adhesion of CD4+ and CD8+ T cells was not affected by Akt inhibition under shear flow conditions, indicating that adhesion in different lymphocyte subtypes is regulated by different signalling pathways. I show here that effector cytotoxic T lymphocytes (CTLs) have high LFA-1 integrin expression and display high spontaneous binding to ICAM-1 under static conditions. Unlike B cells, these cells were able to adhere to ICAM-1 under shear stress in the absence of chemokines. However, cytotoxic T lymphocyte adhesion to ICAM-1 under shear flow was dependent on calcium/calmodulin signalling and an intact actin cytoskeleton. β2-integrins in myeloid cells are continuously recycled and mediate both cell trafficking and phagocytosis of complement (iC3b)-coated particles, but how these processes are regulated remain incompletely understood. I show here that a triple threonine-motif in the β2-integrin cytoplasmic domain is important for in integrin-mediated adhesion in primary mouse macrophages and dendritic cells. This motif also prevents integrin lysosomal degradation in primary myeloid cells, and is required for integrin-mediated phagocytosis of iC3b-coated particles. I also show that an R77H substitution in the extracellular domain of the Mac-1 integrin, which is associated with the inflammatory disorder systemic lupus erythematosus, leads to impaired cell adhesion to ICAM-1 and iC3b as well as impaired phagocytosis. In conclusion, in this thesis I have successfully developed methods to investigate integrin-mediated adhesion in leukocytes. In addition, I have investigated signalling pathways and mechanisms involved in regulation of β2-integrin-mediated functions in primary lymphocytes and myeloid cells. These studies have led to an increased understanding of how integrins in immune cells are regulated both in the presence and absence of shear stress.