Human monocytes comprise three distinct subsets, defined by their relative expression of CD14 and CD16. These subsets appear to have different functional roles, which are only recently becoming delineated. Functional differences include the manner in which different monocytes interact with blood endothelial cells. Interaction with endothelial cells in the micro-vasculature controls access of monocytes to tissues. This interaction is of particular interest in inflammatory diseases such as Rheumatoid Arthritis (RA), in which inflammatory cell infiltration into target tissues causes the majority of the associated damage. The studies in this thesis aim to examine the molecular interactions occurring between monocytes of different subsets and the endothelium during different kinds of locomotion. A multi-step, systems-based approach was taken. First, analysis of macro- and micro-vascular endothelium by flow cytometry revealed differences in the cell surface expression of a range of well-described adhesion/function-associated molecules in both resting and activated states. Second, these differences were further investigated by gene array analysis of micro-vascular endothelium under resting and inflammatory conditions. In a limited number of clinical cases, endothelial cells were isolated from RA synovium and characterised by gene expression microarray. These data showed that similar cellular pathways are up-regulated by RA synovial endothelium and micro-vascular endothelium under in vitro inflammatory conditions. In the third step, micro-vascular endothelium gene expression was analyzed in parallel with that of the three major monocyte subsets in order to identify potential receptor-ligand binding pairs. Six putative receptor-ligand pairs were taken forward for functional analysis. In order to facilitate this, a novel in vitro migration assay was developed to enable simultaneous monitoring of the locomotory behaviour of all three monocyte types over endothelial monolayers by live-cell, fluorescent time-lapse imaging, with the whole system subjected to physiologically relevant shear flow. In this final fourth step, each monocyte subset was shown to preferentially perform different types of locomotory behaviour in a resting state. The roles of the six putative receptor-ligand pairs in each type of behaviour, for each monocyte subset, were then investigated using blocking reagents. As a result, the work in this thesis contributes to the functional delineation of human monocyte subsets and provides insight into the molecular basis for monocyte-endothelial interaction behaviour.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628457 |
| Date | January 2014 |
| Creators | Collison, Joanna |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://kclpure.kcl.ac.uk/portal/en/theses/a-systemsbased-approach-to-examine-the-molecular-basis-of-interactions-between-human-monocytes-and-human-microvascular-endothelium(2949c6e1-fc2c-4bcc-a884-780fac689c42).html |
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