Barnes, Theresa (2010) Defining the neutrophil phenotype in systemic sclerosis. Doctoral thesis, University of Liverpool.
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Background: Systemic sclerosis (SSc) is a disease characterised by a triad of immunological abnormalities, endothelial cell dysfunction and fibrosis. Neutrophils are the most abundant circulating leukocyte. They contain several mediators which when released can lead to modulation of the inflammatory response, cause endothelial cell activation and injury and eventually lead to fibrosis. The peri-endothelial cell environment in SSc has the potential to lead to neutrophil activation and indeed this has been previously described in the literature in terms of reactive oxygen species (ROS) generation. In this thesis I aim to explore the hypothesis that: “Neutrophils are activated in SSc and contribute to endothelial cell activation and damage”. Methods: The functional phenotype of SSc neutrophils was explored in vitro. Functions investigated included; ROS generation, chemotaxis, integrin expression, apoptosis and CD16 expression. The protein expression of ex vivo SSc neutrophils was compared to healthy control neutrophils. The DIGE technology was used to explore the pan-proteome and iTRAQ was used to focus in on the plasma membrane proteome. The role of neutrophil elastase was explored in SSc by examining the serum neutrophil elastase concentration and activity and correlating these to clinical manifestations of disease. The role of neutrophil: endothelial cell interactions was modelled in vitro using live cell imaging by confocal microscopy looking for evidence of endothelial cell activation (E-selectin expression) and apoptosis. Experiments examined the role of neutrophil derived mediators in these interactions. Results: Functional studies revealed that SSc neutrophils are hypofunctional in terms of spontaneous ROS generation and chemotaxis in vitro. This may reflect in vivo activation. SSc neutrophils are similar in terms of integrin expression and baseline apoptosis to control neutrophils. Pan-proteomic studies reveal neutrophil activation in SSc since changes in protein expression mirror those seen in response to neutrophil activators TNFα and LPS. Proteomic studies also point to neutrophil priming in vivo. Serum neutrophil elastase concentrations and activity were not elevated in SSc, however, discrepancies between concentration and activity suggest a functional deficiency in elastase inhibitors in SSc serum. Serum elastase activity and concentrations were found to be lower in RNP (ribonucleoprotein) positive patients indicating that different mechanisms maybe involved in different SSc subtypes. In vitro models demonstrate that SSc serum causes endothelial cell activation and apoptosis and that neutrophils are essential for this effect. Serine proteases seem to play an important role in inducing apoptosis and IL-6 trans signalling is involved in endothelial cell activation and apoptosis. Neutrophils do not express IL-6 but are dominant sources of the soluble IL-6R which is essential for trans signalling. Conclusions: There is evidence that neutrophils are activated in SSc though; no specific activating signature is identified. In co-cultures, neutrophils are essential for endothelial cell activation and apoptosis in response to SSc serum. Neutrophil mediators including serine proteases and IL6R are likely to play important roles in this effect. Therefore neutrophils may play an important part in the propagation of inflammation and endothelial cell activation which eventually leads to the fibrotic phenotype which is characteristic of this disease.
|Item Type:||Thesis (Doctoral)|
|Subjects:||R Medicine > R Medicine (General)|
|Departments, Research Centres and Related Units:||Academic Faculties, Institutes and Research Centres > Faculty of Medicine > School of Infection & Host Defence|
|Deposited On:||13 Jan 2011 16:53|
|Last Modified:||08 May 2012 12:00|
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