Increased recycling of α5β1 integrins by lung endothelial cells in response to tumor necrosis factor

Tumor necrosis factor α (TNF-α) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-α may influence cell-matrix interactions by altering the clustering as well as internalization of the α5β1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-α caused an increase in the intracellular staining of α5β1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37°C after antibody-labeling of their surface α5β1 integrins at 4°C confirmed an increase in the rate of α5β1 integrin internalization which was at least 3 times greater after TNF-α exposure, based on the half-life for antibody-labeled surface integrins to reach equilibrium with non-labeled integrins within the intracellular pool. Interestingly, the total cell surface expression of α5β1 integrins was relatively constant after TNF-α exposure despite the enhanced rate of internalization, suggesting an accelerated recycling of the internalized α5β1 integrins back to the cell surface. This response was confirmed by the measurement of labeled integrin recycling, which showed a significant (P