Synergistic inflammatory signaling in airway epithelial cells: control of expression levels of protease-activated receptors and interleukin-8 release

5 Abstract Asthma is a chronic inflammatory disease of the airways in which besides migratory cells of the immune system, many resident cells like smooth muscle cells, fibroblasts and epithelial cells play important roles. The airway epithelium acts not only as a physical barrier for inhaled infectious stimuli but also actively participates in acute and chronic inflammatory reactions by releasing proand anti-inflammatory mediators. Exposure of epithelial cells to deleterious factors, like allergens, bacteria, pollutants, and to endogenous proinflammatory factors, triggers defence mechanisms by modulation of expression and secretion of different bioactive molecules such as lipid mediators, cytokines, extracellular matrix proteins. It has been shown that the release of those agents is frequently mediated via activation of protease-activated receptors (PARs). PARs belong to the superfamily of G-protein coupled receptors with a 7-trans-membrane domain structure. They are activated by proteolytic cleavage of their N-terminus. For many potential PAR activators in airways elevated activity has been observed during chronic inflammation. Among those proteases are thrombin, tryptase, human airway trypsin-like protease (HAT) as well as proteases from airborne allergens. However, there is still limited information concerning the question which particular factors are responsible for the alteration of PAR expression and susceptibility in lung epithelial cells. The findings of the present study are as follows: 1. Using RT-PCR, immunocytochemistry and Ca 2+ mobilization measurements, we demonstrated that the airway epithelial cell line A549 expresses PAR-1, PAR-2, and PAR-3. Short-term stimulation of these cells with thrombin, trypsin, and activating peptides of PAR-1, PAR-2, but not PAR-3 and PAR-4, induced a transient rise of [Ca]i. 2. We showed that cationic and anionic trypsin induce Ca mobilization in these cells. Mesotrypsin displays no effect on [Ca]i rise. Furthermore, from desensitization study using PAR-2 AP we conclude that PAR-2 is substrate for cationic and anionic trypsin isoforms in human airway epithelial cells. 3. We evaluated the influence of inflammatory mediators LPS, TNF-α, IL-8 and PGE2 on PAR expression level in A549 cells. We also investigated the influence of continuous PAR activation on PAR expression and release of the proinflammatory chemokine IL-8. We employed three different cells, two airway epithelial cell lines, A549 and HBE cells and primary airway epithelial cells (HSAEC). The bacterial endotoxin LPS after 4 h of stimulation up-regulated PAR-2 expression 2-fold, but the