The role of monocytes in sepsis-associated pulmonary microvascular injury

Although monocyte margination to the lungs is enhanced during endotoxemia, little is known of their role in sepsis-associated acute lung injury (ALI). Moreover, monocytes exists as two functionally distinct subsets ‘inflammatory’ Gr-1high and ‘resident’ Gr-1low, which exhibit heterogeneous inflammatory responses upon LPS stimulation both in vivo and in vitro but, the signalling mechanisms regulating their differential responses are unknown. The objective of this research was to define the role of monocyte-endothelial interactions during pulmonary microvascular injury, in response to LPS challenge, using an in vitro monocyte-endothelial coculture model. The specific aims were to: establish a primary lung endothelial cell line culture; investigate priming and enhancement of monocyte pro-inflammatory response; investigate the role of the p38 MAPK pathway in the differential monocyte subset cytokine response, and investigate the role of LPS-mediated TLR4 signalling for monocyte-endothelial reciprocal activation. Primary mouse lung endothelial cells (MLEC), that were successfully cultured, displayed typical morphological and phenotypic properties of the vascular endothelium. During coculture with MLEC, monocytes produced significantly higher levels of TNF when treated with a single high dose of LPS when compared with monocytes cultured alone. Also, upon LPS stimulation, Gr-1high monocytes expressed significantly higher levels of TNF, IL-6, COX-2, and iNOS in comparison to Gr-1low monocytes. Furthermore, significantly higher levels of p38, a critical regulator of inflammatory cytokines, were induced in Gr-1high monocytes in comparison to Gr-1low monocytes. Finally, using a chimeric system of TLR4+/+ (LPS responsive) and TLR4-/- (LPS unresponsive) monocytes and endothelial cells, it was demonstrated that monocytes could activate endothelial cells through TNF-dependent signalling, while endothelial cells could induce TNF production in monocytes. These data indicate an important role for monocytes, especially the Gr-1high subset in the pathogenesis of ALI, and suggest that the p38 MAPK pathway could play a pivotal role in regulating monocyte-induced pulmonary inflammation during sepsis-associated ALI.EThOS - Electronic Theses Online ServiceBiotechnology and Biological Sciences Research Council (BBSRC)GBUnited Kingdo