Lymphocyte Dynamics on Aligned Endothelial Cells

Abstract

Immune cells circulating blood vessels recognize the signatures of tissue inflammation in blood vessels and undergo multiple cascades of interactions with inflamed endothelium to eventually leave out the vessels. Parallel plate flow chamber assay has been extensively used to investigate detailed mechanisms of lymphocyte-endothelial cell (ECs) interactions. However, orientation of ECs, which may play significant roles in lymphocyte dynamics on inflamed endothelium, has not been considered in these assays yet. Indeed, healthy blood vessels are composed of endothelium aligned in parallel with the direction of blood flow. To address the importance of ECs alignment on lymphocyte dynamics, we firstly aligned ECs on the substrates containing nanoscale grooves and ridges. Then, dynamics of T cells on the aligned endothelial layers (either parallel or perpendicular to the direction of flow) and randomly oriented ECs were monitored by video microscopy. Firstly, we quantitatively analyzed the direction of T cell crawling under the conditions stated above. Interestingly, the direction of T cell crawling was not significantly affected by the flow direction, but rather biased toward the direction of EC alignment. Indeed, when junctions and nuclei of ECs were visualized, T cells preferentially migrated along the EC the junctions, while avoided crawling on top of EC nucleus. Based on this observation, we hypothesized that topographical structures and mechanical properties of ECs guided crawling of T cells and performed a number of experiments to test the hypothesis. Secondly, we investigated the effect of EC alignment on transendothelial migration (TEM) of T cells. T cells on poorly-aligned ECs underwent TEM more quickly and frequently than T cells on well-aligned ECs. Further analysis revealed that local alignment of EC junctions surrounded by more than three ECs form preferential sites for TEM of T cells.