Investigating the reverse transmigration of neutrophils in human and murine in vitro models of inflammation

Diapouli, Frantzeska-Maria (2011). Investigating the reverse transmigration of neutrophils in human and murine in vitro models of inflammation. University of Birmingham. Ph.D.

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Abstract

The aim of this project was the study of neutrophil recruitment and reverse transmigration using murine and human in vitro models of inflammation. Murine in vitro models of inflammation were developed using an immortalised microvascular cell line (MCEC-1) and primary murine vascular endothelial cells (mEC) isolated from heart and lung. We found that MCEC-1 could recruit murine neutrophils without the requirement of cytokine stimulation, although efficient transmigration did require such a stimulus. Primary cells required cytokine stimulation to recruit mEC. Interestingly, and in contrast to human EC, mEC were relatively insensitive to TNF-α stimulation, although IL-1β was a good stimulus for adhesion and migration. Using the IL-1β driven system we generated reverse migrated murine neutrophils and their phenotype and prolonged survival were assessed. The effect of shear stress and nitric oxide on the regulation of the process of reverse migration was examined. Using adoptive transfer strategies we investigated the fate of mRPMNs in vivo. A significant part of this work involved the study of human reverse migrated neutrophils at a proteomic level using two-Dimensional Fluorescence Gel Electrophoresis methodology to identify changes in neutrophils associated with reverse migration process. We found that murine reverse migrated neutrophils had a very similar surface phenotype to human reverse migrated cells. They also showed prolonged survival. However, our preliminary data on trafficking in vivo did not give a clear indication about their fate upon adoptive transfer into recipient mice. In vitro studies showed that flow generated shear stress and nitric oxide delayed, but did not inhibit, the process of reverse migration. Finally, the proteomics study revealed a number of metabolic, cytoskeletal and regulatory proteins that were differentially expressed in human reverse migrated neutrophils although the functional significance of these changes is yet to be explored.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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