Evidence for a remote lung response in endotoxin-mediated direct lung injury : a strategic application of microarray technology

Endotoxin, a lipopolysaccharide component of the membrane of gram-negative bacteria, is responsible for rapid triggering of innate defence mechanisms in the lung. Such mechanisms, featuring local accumulation of neutrophil leucocytes, concomitant modulation of local immunoregulatory cytokine expression and the development of interstitial oedema are the hallmarks of an intense inflammatory response and comprise the clinical definition of acute lung injury (ALI). Although a proportion of individuals with ALI will progress towards a more severe form of lung injury referred to as the acute respiratory distress syndrome (ARDS) the mechanisms underlying such susceptibility are unknown. One potential avenue through which individuals may exhibit variable susceptibility to progression is in the nature and extent of the whole organ response to a local challenge. However, there is relatively little known in relation to whether the lung will mount such a 'remote' response to local challenge. The central hypothesis underlying this thesis is that the whole lung will respond to local challenge with endotoxin in the absence of any overt phenotypic change throughout the organ. The work presented here is directed towards characterising the molecular aspects of this response using an ovine model. Quantitative RT-PCR analysis of the cytokine expression in directly challenged and contra lateral 'remote' lung segments showed that the lung produced the anti-inflammatory cytokine IL-10 remotely whilst the pro¬ inflammatory cytokines IL-1 (3, IL-6 and TNFa, which are associated with acute inflammation, were expressed in the directly challenged segment and correlated with the observed neutrophil influx to the challenged lung segment. To further this characterisation a novel microarray platform was designed to specifically probe the genomic profile of the sheep lung. This process included the construction of a subtracted lung library and the identification of ovine homologs within a commercially available bovine genomic library. The selected elements were combined to make a cDNA microarray which was used to probe the nature of the remote response after LPS instillation. A remote response was observed. The response was highly complex, involving mediators from diverse pathways and functional groups including cytoskeletal genes, ribosomal genes, mitochondrial products, prostaglandins and immune mediators. The characteristics of the transcriptional response in the remote segment were considered broadly anti¬ inflammatory in nature. This study has shown for the first time that the lung responds as a whole organ to local inflammatory insult. Variation in the nature and extent of this remote response amongst individuals is proposed as a factor underlying susceptibility to progressive lung injury and ARDS. The study has also generated and utilised a novel ovine microarray, and produced a subtracted library of ovine lung sequences. Both of these represent considerable new resources for the study of pulmonary responses in both natural and experimental models of disease in sheep.