One of the most characteristic features of the immune system is the migration of neutrophils during inflammation. Their movement inside the vasculature lined with endothelial cells is governed by a process known as the leukocyte adhesion cascade. Neutrophils crawl with the help of their surface integrins on the apical endothelium and adhere to the cell adhesion molecules before transmigrating to the basal endothelium and reaching the site of transmigration. This unique capability to migrate against fluid shear flow is called upstream migration. This distinctive behavior plays a critical role in immune responses and inflammatory processes. The speed and location at which neutrophils can transmigrate through the endothelium to reach the inflammatory site is vital. The molecular mechanisms guiding this phenomenon are not yet fully understood despite its significance. Mac-1, also known as macrophage-1 antigen and leukocyte function-associated antigen-1 (LFA-1) serves as the chemokine-activated integrin molecule on the neutrophils which adhere to cell adhesion molecules like intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelium before they transmigrate into the basal lamina and reach the infection site [1]. Previous research from our lab demonstrated that blocking Mac-1 on HL-60 cells induces upstream migration against shear flow [2]. Similarly, in T-cell migration, blocking LFA-1 has been observed to facilitate upstream migration [1]. Notably, T cells moving on vascular endothelial cell surfaces exhibit a twofold increase in transmigration speed when crawling upstream compared to downstream, suggesting that upstream migration may expedite extravasation by allowing cells to tunnel beneath the endothelial monolayer, reducing transit time to inflammatory sites. Various literature studies have indicated that this directional migratory behavior is probably linked to several signal-transducing pathways. Here in this work, we perform microfluidic assays to analyze the directionality of neutrophil movement under shear flow in the presence of various inhibitors against the key players of those pathways. We hypothesize to identify the molecular signals or cues downstream of Mac-1 which will enable neutrophils a faster transmigration against shear flow to reach the injury site. We will also analyze the different transmigration patterns among the various neutrophils with integrin gene knockouts (Mac-1/LFA-1) through surface material characterization to delve into the biomechanics of upstream migration. We believe that through a comprehensive approach integrating foundational investigations with in-vivo and in-vitro models that mimic physiological conditions, we aim to identify the pivotal signaling pathways governing upstream migration in neutrophils. The long-term objective of studying these pathways will be to provide our understanding of upstream migration during inflammatory responses and lead to better patient outcomes in various inflammatory diseases.
