Abstract
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 replicates 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 post-capillary venule expansions and dependent on the adhesion molecule ICAM-1.
Highlights
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
Our results indicate that interactions with other flowing cells within the microchannel, which are governed by rheological stresses, rather than rheological stresses per se, most strongly determine leukocyte adhesion probability
Initial experiments with leukocytes in media alone without erythrocytes suggested that extensional stress itself could modulate leukocyte adhesion, independent of margination
Summary
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. Defects in integrin expression[1,2,3], 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, and including atherosclerosis[7], acute lung injury[8], and sickle cell disease[9] This regulation occurs with high statistical efficiency. 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. Inflammation triggers multiple coordinated changes, such as increasing adhesion molecule[20], selectin[21], and arrest chemokine expression[22], the endothelial barrier becoming more permeable to inflammatory signals from the parenchyma[23], vascular geometry changing from more capillary-like to more PCV-like[24,25], and erythrocyte aggregation promoting margination of leukocytes to the vascular wall[26,27]
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