Effect of Elastase on the Directional Migration of Lung Fibroblasts within a Three-Dimensional Collagen Matrix

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Abstract
Interactions between airway epithelial cells and bronchial fibroblasts often require close proximity between these cells. Previous studies have demonstrated that airway epithelial cells direct the migration of lung fibroblasts, but the factors that regulate this process during airway injury are not clear. We hypothesized that exposure of culture substrates to proteolytic enzymes, like those present in the inflamed airway, would increase fibroblast recruitment. We also postulated that elastase might affect the epithelium's ability to attract fibroblasts. We used an in vitro model with fibroblasts embedded between two layers of collagen gel to investigate their migration. Embedded fibroblasts exposed to culture medium alone (baseline) had a slight downward migration (migration directed to the upper gel layer expressed as a percentage of total migration was -2.8 +/- 1.4), but medium supplemented with porcine pancreatic elastase (PPE) resulted in a slight upward migration (2.0 +/- 1.4). When airway epithelial cells were cultured on the upper gel surface, the index of directed migration toward them was 15.9 +/- 1.3. Addition of PPE to the culture medium resulted in a significant increase to 22.3 +/- 1.5 (p < .05). Human neutrophil elastase (HNE) produced similar results, and these effects were inhibited by alpha 1-proteinase inhibitor. Similarly, total fibroblasts per 20 high-powered fields were counted in all conditions, suggesting that mitogenic interactions were not important in this system. The percentage of the total fibroblasts migrating at least 5 microns in any direction was also similar in all groups, suggesting chemokinetic mechanisms were not involved. These data suggest that elastase exposure in a model of the human airway increases directed fibroblast migration through the extracellular matrix. This phenomenon may play a role in the development of subepithelial fibrosis seen in inflammatory airway diseases like asthma.