Recruitment of neutrophils into the tissue during inflammation is a crucial component of the immune response. This study aimed to further understand the role of intercellular adhesion molecule-2 (ICAM-2) in this process. Endothelial cell (EC) ICAM-2 has been implicated in neutrophil extravasation however, its precise role in this process is largely unknown. To address this, the current investigation examined the expression and functional role of ICAM-2 in neutrophil-EC interactions in vivo. Analysis of EC ICAM-2 expression was performed in the mouse cremaster muscle using immunofluorescent staining and confocal microscopy. A high EC body expression of ICAM-2 relative to that of EC junctions in post-capillary venules was observed. It was therefore hypothesised that ICAM-2 could potentially be involved in both luminal neutrophil-EC and junctional interactions. This hypothesis was analysed using confocal intravital microscopy (IVM) of cremaster muscles from WT or ICAM-2 KO Lys-eGFP-ki mice (express fluorescent neutrophils) in conjunction with fluorescent labelling of ECs. Neutrophil crawling and transendothelial migration (TEM) dynamics in IL-1β-stimulated post-capillary venules was analysed. A role for ICAM-2 in supporting speed and continuity of crawling and the initiation of TEM was demonstrated. Using functional blocking mAb to MAC-1 in WT and ICAM-2 KOs, the role of ICAM-2 in neutrophil crawling was demonstrated to be governed through a potential interaction with neutrophil MAC-1. It is therefore possible that non-junctional EC ICAM-2 has important roles in regulating neutrophil polarisation during crawling whilst junctional ICAM-2 mediates the opening of EC junctions and/or influencing the site of ‘preferred’ TEM. This study provides the first in vivo evidence for the ability of ICAM-2 to support neutrophil crawling and the initiation of TEM in IL-1β-induced neutrophil extravasation. To extend the above findings in a complex vascular injury model, a cremasteric Shwartzman Reaction, amenable to IVM analysis, was also developed as part of this project.

Leukocyte recruitment to sites of inflammation is a crucial event in host defense against pathogens and tissue injury. Although there is at present much interest in deciphering the mechanisms of leukocyte transendothelial cell migration, little attention has been paid to the subsequent steps, i.e. leukocyte migration through the pericyte layer and the venular basement membrane. In this context, results from this group previously demonstrated that neutrophils preferentially transmigrate through gaps between adjacent pericytes, regions associated with sites of low matrix protein expression within the vascular basement membrane. The aim of this thesis was to extend these findings by investigating the impact of inflammatory mediators on pericyte morphology and vascular basement membrane deposition using both in vitro and in vivo models. Flow cytometry analysis of pericyte-like C3H/10T1/2 cells and primary lung pericytes revealed the expression of key pro-inflammatory molecules on their surface (including cytokine receptors and adhesion molecules) and the regulation of these molecules upon cytokine stimulation. Using the murine cremaster muscle model it was further demonstrated that key neutrophil chemoattractants (i.e. LTB4, KC, C5a and fMLP) induced neutrophil transmigration that was associated with a change in pericyte morphology (as quantified through enlargement of gaps between adjacent pericytes). These changes in pericyte gap size were neutrophil-dependent and mediated by endogenously generated TNF as demonstrated in neutrophil-depleted mice and TNFR-/- mice, respectively. In addition, TNF appeared to mediate post-inflammatory BM deposition in response to LTB4 and was required for chemoattractant-induced vascular permeability. Hence, the results of the present work have demonstrated the ability of pericytes to respond to both cytokines and chemoattractants, suggesting an active role for pericytes in the regulation of inflammatory responses. In addition, findings provide the first evidence for chemoattractant-induced changes in vascular morphology and barrier functions of venular walls in vivo via the release of endogenous TNF as a secondary mediator, effects that may contribute to the pro-inflammatory properties of these stimuli.