Biomimetic post-capillary venule expansions for leukocyte adhesion studies

Bryan L. Benson, Lucy Li, Jay T. Myers, R. Dixon Dorand, Umut A. Gurkan, Alex Y. Huang, Richard M. Ransohoff
2018 Scientific Reports  
Leukocyte adhesion and extravasation are maximal near the transition from capillary to post-capillary venule, and are strongly influenced by a confluence of scale-dependent physical effects. Mimicking the scale of physiological vessels using in vitro microfluidic systems allows the capture of these effects on leukocyte adhesion assays, but imposes practical limits on reproducibility and reliable quantification. Here we present a microfluidic platform that provides multiple (54-512) technical
more » ... licates within a 15-minute sample collection time, coupled with an automated computer vision analysis pipeline that captures leukocyte adhesion probabilities as a function of shear and extensional stresses. We report that in post-capillary channels of physiological scale, efficient leukocyte adhesion requires erythrocytes forcing leukocytes against the wall, a phenomenon that is promoted by the transitional flow in postcapillary venule expansions and dependent on the adhesion molecule ICAM-1. The leukocyte adhesion cascade, by which peripheral blood immune cells exit from flowing blood and migrate into tissues, is highly constrained by biological regulation, statistics, and biophysics. First, molecular regulations on the cellular level constrains the efficiency of leukocyte adhesion. Defects in integrin expression 1-3 , selectin ligand sialylation 4 , or integrin affinity upregulation 5,6 all lead to severe functional immunodeficiencies, while excessive leukocyte adhesion and accumulation is a contributor to numerous disease states, classically autoimmunity, but also including atherosclerosis 7 , acute lung injury 8 , and sickle cell disease 9 . This regulation occurs with high statistical efficiency. In healthy physiology, 25 billion leukocytes traverse the 100,000 kilometers of vasculature in the body, one circuit per minute, with significant adhesion occurring only in homeostatic sites such as bone marrow, high endothelial venules of secondary lymphoid sites, thymus, spleen, and liver 10-14 . The biophysical context in which these processes are regulated is also challenging. More than half of the vasculature length is made up of capillaries -vessels that are physically smaller than the 8-15 micrometer diameter of leukocytes -where adhesion would result in plugging of the capillary blood flow and local ischemia. Yet, given the speed of blood flow, adhesive interactions between leukocytes and endothelium must occur within fractions of a second 15 for any adhesion to be spatially relevant. Despite these challenges, leukocyte adhesion is largely restricted to post-capillary venules (PCVs) 16, 17 , vessels of about 20-50 micrometers in diameter that immediately follow capillaries. Such efficient regulation on a single-cell basis in a stochastic biological system requires multiple overlapping factors to achieve. For example, while shear stress has a significant influence on leukocyte adhesion, it alone is not sufficient to predict the locations where such adhesion occurs 18 . Likewise, while endothelial cell adhesion molecule expression is maximal in PCVs of 25 micrometer diameter 19 , it is not constrained to zero in capillaries. Consequently, multiple levels of regulation may act in a coordinated fashion to control leukocyte adhesion. In vivo, Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 Scientific REPORTS | (2018) 8:9328 |
doi:10.1038/s41598-018-27566-z pmid:29921896 pmcid:PMC6008471 fatcat:lz4t7oet7zckdflop3iope64be