Sequential migration of granulocytes across monolayers of endothelial and lung epithelial cells: a new model for inflammatory processes in the lung
01 / 2001 - 12 / 2004
In lung inflammatory disorders, circulating leukocytes, primarily neutrophils and eosinophils, migrate into the airway lumen by subsequent transendothelial and transepithelial migration. Whereas the molecular basis of transendothelial migration is relatively well understood, the molecules and mechanisms underlying transepithelial migration are largely unknown. This new proposal represents a follow-up of a currently ongoing project (NAF project 96.43), aimed at developing a transmigration model in which eosinophils or neutrophils are allowed to migrate first across an endothelial monolayer and subsequently across an epithelial monolayer ('bi-layer model'). This is achieved by culturing the endothelial cells on the top and the epithelial cells on the bottom of the same Transwell filter. Recently, we succeeded in establishing this model not only with immortalised endothelial and epithelial cells, but also with primary human endothelial and lung epithelial cells. The model is now established, and the first results have already confirmed its validity. The major conclusion from the most recent work is that paracrine communication exists between the epithelial and endothelial monolayers, resulting .in altered monolayer permeability, expression of adhesion molecules and migration of granulocytes.
This new proposal builds upon these data and proposes to study:
1. The molecular basis of the paracrine communication between the epithelial and endothelial cells: Preliminary experiments have shown that epithelial cells secrete soluble factors [we have already established the release of interleukin (IL)-1, IL-6 and IL-8] that alter adhesion molecule expression and permeability of the endothelial monolayers. The nature of the factors involved, their relative role in the migration of granulocytes across the bi-layer, and the consequences for granulocyte migration will be investigated.
2. The differential use of specific adhesion molecules on the granulocytes as well as on the endothelial and epithelial cells, in the context of migration across the bi-layer: Emphasis will be on the role for CD31, beta1- and beta2-integrins and their ligands, and CD47.
3. The change in molecular 'make-up' of the granulocytes following adhesion to and migration across the bi-layer, and the consequences of these changes for their activation: These analyses will reveal to what extent the expression of relevant adhesion molecules as identified under (2) is altered following transendothelial migration, whether these changes are similar for neutrophils and eosinophils, and whether there are important consequences for subsequent transepithelial migration. In addition, the sensitivity of migrated versus non-migrated cells to activating stimuli will be investigated.
The experiments will be initially performed with human cell lines: primary endothelial and lung epithelial cells as well as with freshly isolated granulocytes from healthy donors. In addition, primary cells (granulocytes) from patients with lung inflammatory disorders will be incorporated in the studies.