Signal transduction pathways regulating granulocyte differentiation and function

Abstract

Human granulocytes play an important role in host defense by mediating inflammatory responses to invading microorganisms. Defects in granulocyte differentiation or improper functioning of these cells can lead to disease pathogenesis. Granulocyte maturation, priming and activation are under the control of cytokines and chemoattractants. This study focuses on the intracellular signaling mechanism regulating granulocyte differentiation and functioning. To address this, several points of research or questions were investigated. 1) What are the roles of intracellular pathways in the cytokine induced maturation of hematopoietic progenitors towards granulocytes ? To address this question, an in ex-vivo differentiation model was developed, in which isolated umbilical cord blood CD34+ progenitors were differentiated towards neutrophils. In Chapter 2 we have investigated the differences in the maturation program induced by G-CSF or a combination of IL-3 and G-CSF. By using pharmacological inhibitors we demonstrate the importance of PI3K and MEK in the maturation process of hematopoietic progenitors towards neutrophils. 2) Identification of novel kinases present in human granulocytes. Since granulocytes have specific effector functions we hypothesized that specific signaling molecules could be present in these cell. To this purpose we describe in Chapter 3 polymerase chain reaction analysis using degenerate primers against conserved regions of kinases, leading to the identification of CKLiK. We characterize CKLiK as a Ca2+ and calmodulin dependent kinase, whose expression is restricted to granulocytes. For analysis of the role of CKLiK in granulocyte functioning, we describe in Chapter 4 the role of Ca2+ and calmodulin regulated pathways by using a Ca2+ ionophore and a calmodulin antagonist. In Chapter 5 we describe the regulation and functional characterization of CKLiK in human granulocytes, by using an antibody against CKLiK and an cell permeable inhibitory peptide. 3) What genes are regulated by cytokines in human granulocytes? Exposure of inflammatory mediators, such as GM-CSF, to mature granulocytes leads to a pre-activated or primed phenotype. In Chapter 6 we identified GM-CSF regulated genes, by a differential display technology, in which the gene encoding 5-lipoxygenase activating protein was identified as a cytokine regulated gene in vitro as well as in vivo (Chapter 6). Taken together this thesis provides novel insights into granulocyte signal transduction pathways and identifies new signals that can control granulocyte maturation, priming, activation and functioning.