Primary human neutrophils and monocytes were recently shown, in static in vitro co-cultures with human endothelial monolayers, to enrich at and actively migrate along endothelial cell-cell boundaries, and a reductionist numerical model indicated that this boundary-following strategy samples more endothelial cells per traveled distance than straight-line motion for tracks shorter than roughly eight cell diameters. Two questions about this phenomenon remain open: the molecular mechanism that confines migrating leukocytes to endothelial boundaries is unidentified, and whether the boundary-following behavior and its search-efficiency advantage operate under physiological conditions (blood flow, three-dimensional vessel geometry, in vivo) is unestablished.

Background

Neutrophils and monocytes are innate immune cells that patrol the vasculature and, during inflammation, leave the bloodstream by adhering to the endothelium (the single cell layer lining blood vessels), crawling over its luminal surface, and crossing it by transendothelial migration. This multistep process is well characterized: after selectin-mediated rolling (selectins are carbohydrate-binding adhesion molecules that slow leukocytes along the vessel wall) and integrin-mediated arrest (integrins are leukocyte adhesion receptors that lock onto immunoglobulin-family ligands on the endothelial surface), leukocytes crawl intraluminally to an exit site and transmigrate, usually between adjacent endothelial cells at their cell-cell junctions (a paracellular route). Intraluminal crawling itself is molecularly defined: neutrophil crawling to emigration sites depends on the beta-2 integrin Mac-1 binding its endothelial ligand ICAM-1 (intercellular adhesion molecule 1), with monocytes and neutrophils showing characteristically different dependencies on LFA-1 (leukocyte function-associated antigen 1, the other major beta-2 integrin on these cells) and Mac-1 for crawling in situ (Phillipson et al. 2006; Sumagin et al. 2010). Exit sites are also spatially organized: leukocytes preferentially bind to endothelial junction regions in venules in vivo (Wojciechowski and Sarelius 2005), monocytes crawl to the nearest junction before transmigrating (Schenkel et al. 2004), and endothelial 'transmigration hotspots' arise through heterogeneous ICAM-1 expression (Grönloh et al. 2023a) while ICAM-2 enables neutrophils to crawl against the direction of flow and find these hotspots (Grönloh et al. 2023b). Soluble inflammatory cues are likewise spatially patterned: chemotactically active chemokines can be sequestered on endothelial heparan sulfate (a sulfated sugar chain on the endothelial cell surface that binds chemokine proteins) and concentrated at junctional regions (Massena et al. 2010), and non-classical monocytes patrol the luminal vascular surface to scan for tissue damage (Auffray et al. 2007). Against this backdrop, Honig et al. (2025) co-cultured primary human neutrophils or monocytes with confluent human umbilical vein endothelial cell (HUVEC) monolayers and, combining immunofluorescence, quantitative single-cell tracking, cross-correlation of cell tracks with endothelial boundaries derived by Voronoi tessellation of endothelial nuclei, and a reductionist Python numerical model comparing migration along endothelial cell-cell boundaries with straight-line migration, found that both leukocyte types are enriched at endothelial cell-cell boundaries and dynamically migrate along them. Their model showed that for walking distances below about eight endothelial cell diameters, boundary-following motion samples more distinct endothelial cells than a straight line, the classical optimal planar search strategy; they argued conceptually that boundary migration should also improve detection of inflammatory chemokines, which are most concentrated near junctions. The work was performed under static in vitro conditions (the published title states this explicitly), and the authors identified the endothelial nucleus as strongly flattened, excluding endothelial surface topology as the main reason for boundary confinement. What remains unresolved is why leukocytes are confined to boundary lines at the molecular level, and whether the boundary-following search strategy is a real physiological adaptation of the vasculature or an artifact of the reductionist static system.

Problem Statement

Identify the molecular mechanism that restricts the intravascular migration of neutrophils and monocytes to endothelial cell-cell boundaries, and determine whether this boundary-restricted migration and its predicted search-efficiency advantage (more endothelial cells sampled per traveled distance, and plausibly enhanced detection of junctional chemokines) operate under physiological conditions. A complete answer must (i) identify the heterotypic leukocyte-endothelial interaction sites or adhesion/signaling molecules that causally confine migrating leukocyte tracks to endothelial boundaries, with perturbation and rescue evidence in an appropriate co-culture or in vivo system, and (ii) establish whether the boundary-following behavior, and the search advantage it confers in the reductionist static model, persists under physiological conditions — fluid shear forces, physiological vessel biophysics and geometry, blood-serum composition, or in vivo.

References

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