Objective: Periodontitis is a chronic inflammatory disease in which chemokines have been shown to have an important role. Chemokines are inflammatory mediators and attract leukocytes to sites of inflammation. This is achieved by chemokine binding at the luminal endothelial cell (EC) surface and presentation to leukocyte chemokine receptors. The present study aimed at understanding the role of endothelial microstructures in presenting chemokines to leukocytes resulting in transendothelial migration.
Methods: ECs were cultured in the presence of chemokines and analysed for microstructure formation by phase contrast and electron microscopy. Transwell experiments were used to study neutrophil transendothelial migration. iTRAQ mass spectrometry was employed to examine intracellular changes in proteins following chemokine stimulation.
Results: CXCL8, an inflammatory chemokine, was shown to induce microstructures formation, namely filopodia and microvilli up to 10 micrometres long, on ECs. The same effects were observed with chemokines CXCL10 (IP-10) and CCL5 (RANTES). In vitro studies revealed colocalisation of CXCL8 and heparan sulphate (HS) on the endothelial microstructures. This suggested that the HS-CXCL8 interaction had an important role in leukocyte transendothelial migration, since removing HS caused a significant decrease in the number of leukocytes undergoing transendothelial migration. Transmission electron microscopy imaging showed that CXCL8-stimulated microstructures interacted with leukocytes prior to transendothelial migration occurring, suggesting that the microvilli were involved in leukocyte transendothelial migration. Mass spectrometry analysis of CXCL8-stimulated ECs showed significant changes in cytoskeletal proteins, adhesion and extracellular matrix proteins, and signal transduction proteins. This suggested that CXCL8 binding to receptors on the EC surface caused an alteration in signalling pathways, a reorganisation of the cytoskeleton and a disruption of the basement membrane, in order for the microstructures to form.
Conclusions: Overall, there appears to be an important function for EC microstructures in inflammation. Chemokines stimulate the formation of EC microstructures leading to their presentation to leukocytes.