Systemic neutrophil activation in COPD : Analysis of gene expression profiles and intracellular signaling

Abstract

Granulocytes are thought to play an important role in the maintenance and progression of chronic diseases, such as COPD, allergic asthma, and rheumatoid arthritis. Despite the importance of granulocytes in these diseases surprisingly little is known about the processes occurring in vivo that regulate inappropriate granulocyte functioning. In this thesis, gene expression of neutrophils has been analyzed in vitro and in vivo, to characterize chronic systemic inflammation and identify mediators that are playing a role in the pathogenesis of COPD. To understand the finely tuned process of neutrophil functioning in vivo, it is also necessary to define components of the signaling pathways responsible for the priming and activation of effector functions in granulocytes. In this thesis, research questions were addressed, concerning granulocyte functioning in vitro and in vivo.

Are changes in gene expression of peripheral blood neutrophils a result of the pathogenetic mechanisms underlying the development of COPD and can these gene profiles be used to define penotypes and severity of the disease? Exposure of neutrophils to cytokines or chemokines, results in their activation and changes in gene expression profiles. In Chapter 2, we utilise gene array technology and real time reverse trancriptase polymerase chain reaction to define the gene expression profiles of peripheral blood neutrophils of COPD patients compared with in vitro stimulated neutrophils. In Chapter 3, we use the same techniques, to investigate differences between peripheral blood neutrophils of COPD patients during a stable phase of their disease and during an exacerbation. Furthermore, we investigated the kinetics of activation of peripheral blood neutrophils in COPD patients in a longitudial setting.

What is the role of Src kinases and small GTPase Rac in granulocyte functioning? To address this question, we use pharmacological inhibitors and a recently developed technique of protein transduction. In Chapter 5 we investigate the role of Src kinases in neutrophil functioning, whereas in Chapter 6 the role of Rac is investigated in peripheral blood eosinophil functioning by introducing a constitutively active mutant into these cells. In Chapter 7, we describe an ELISA technique developed for non-radioactive semi-quantitative analysis of the activation state of several kinases in adherent and suspension cells using phosphospecific antibodies