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Outcome determinants in acute lung injury
P251 Flow cytometric detection of intracellular activation markers of pulmonary cells during acute lung injury
  1. A C Waite,
  2. K P O'Dea,
  3. M R Wilson,
  4. M Takata
  1. Section of Anaesthetics, Pain Medicine and Intensive Care, Imperial College, London, UK

Abstract

Background Activation of intracellular signalling cascades such as mitogen-activated protein (MAP) kinase pathways has been implicated in various animal models of acute lung injury. However, such data rely almost exclusively on measurements within whole lung homogenate samples, so little information is available regarding cell type specific processes. In order to address this, we have developed a flow cytometric technique to identify discrete pulmonary cell populations and examine their early activation in terms of phosphorylation of intracellular MAPkinase pathway intermediates (ERK, p38 and its downstream target MK2).

Methods Anaesthetised C57BL6 mice were given an intratracheal 20 μg dose of lipopolysaccharide (LPS). Mice were sacrificed at various time points, their lungs were removed, immersed in a buffer to fix and permeabilise cells, and a single cell suspension was produced by mechanical disruption. After incubation at 37°C the cell suspension was washed and centrifuged. Samples were stained at room temperature in the dark with antibodies to identify alveolar macrophages (AM) and epithelial cells (AEC), and to measure the intracellular levels of active phosphorylated forms of ERK, p38 and MK2. Finally the cells were washed and resuspended for flow cytometric analysis.

Results AM showed a rapid increase in levels of phosphorylated MK2 (see Abstract P251 Figure 1) as well as p38 and ERK on stimulation by intraalveolar LPS. As MK2 showed the most robust response, we determined its phosphorylation in AEC. Those AEC expressing low levels of surface ICAM-1 (likely type II pneumocytes) showed a pattern of increased MK-2 phosphorylation, while ICAM-1 high expressing AEC (type I pneumocytes) did not show a clear response to LPS stimulation.

Abstract P251 Figure 1

Level of intracellular phosphorylated MK2. A. Alveolar macrophages. B. Epithelial cells.

Conclusion We have developed a method of investigating activation of individual cells within the lung using flow cytometry, in terms of intracellular MAPkinase phosphorylation. Our data show that AM are rapidly activated upon stimulation by intraalveolar LPS, whereas AEC show signs of delayed activation with type II pneumocytes more responsive than type I cells. Applying this technique to other models, in which alternative cell types and/or pathways may be involved, will enhance our understanding of cellular activation and interactions during acute lung injury.

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