Studying Inflammatory Responses of Endothelial Cells and Leukocytes in Perfused Microchannels

Circulating leukocytes must adhere to the endothelial cells (EC) that form the lining of blood vessels, and migrate through them to carry out their protective immune functions. During inflammation this recruitment is typically controlled by cytokines released from tissue that act on the EC. The endothelial cells respond by increasing the expression of adhesion molecules on their surface (to capture flowing leukocytes), and also by presenting chemotactic agents (to induce the captured cells to migrate). This recruitment process is influenced by the local haemodynamic milieu in several ways: interactions with red cells modify the distribution of leukocytes in the blood stream; flow velocity and shear stress influence the formation and breakage of adhesive bonds; flow forces act on EC and modify their responses to inflammmatory cytokines. Microchannels have been widely used to study these processes, especially the specific receptors required for capture of isolated flowing leukocytes and their ability to support adhesion as a function of fluid shear stress. We developed a versatile system based on pre-fabricated glass capillaries with rectangular cross-section (microslides) in which we cultured EC, and which could also be coated with purified adhesion receptors for reductive studies. We also developed fluoresence-microscope-based systems for using these microslides to observe adhesion in flowing whole blood, and multiple parallel cultures for studying the effects of conditioning the EC by growth at different levels of shear stress before investigations. The microslides are available in various dimensions, and smaller versions can be used to generate high circulatory stresses when small volumes of materials (such as blood from genetically modified mice) are available. With these systems, we have for instance, been able to show how varying the concentration and aggregability of red blood cells alters leukocyte adhesion, and how expression levels of endothelial genes which underly inflammatory responses are modified by culture at a range of shear stresses mimicking different regions of the circulation.