Nano-to-Microscale Mechanical Switches and Fuses Mediate Adhesive Contacts between Leukocytes and the Endothelium

As a vital part of the inflammatory response, the leukocyte adhesion cascade involves the capture of white blood cells at the vascular endothelium, a progressively slowing, “rolling” motion of cells along the vessel walls, firm cell adhesion at target sites, and eventually, the transmigration of activated cells into the inflamed tissue1-4 (for an instructive overview see also http://bme.virginia.edu/ley/). Studies aiming to reveal the biophysical mechanisms underlying these processes face the difficult challenge of covering a large range of relevant scales: nanoscale interactions between adhesive molecules, local deformations of subcellular structures under point loads, and whole-cell activation and motion. Yet multiscale approaches also offer particularly rewarding insights, for example, by providing an understanding of single-molecule interactions within their natural, larger-scale biological context and, conversely, by tracing macroscopically observed cellular phenomena directly to their molecular origins. The present work focuses on nano-to-microscale regulatory aspects that mediate the initial stages of the adhesion cascade. Early events, such as the capture of leukocytes and their initial rolling exploration at the endothelium, occur usually too fast to be regulated biologically, i.e., through the expression of proteins. We thus expected that most mechanisms governing initial leukocyte adhesion under a broad range of blood-flow conditions had to be “preprogrammed” into molecular-to-subcellular structures and that they had to be primarily mechanical. Accordingly, our experimental approach was to study in vitro the nano-to-micromechanical response of individual leukocytes that were loaded at a point with a rapidly increasing force. The adhesion bond between P-selectin (expressed in vivo on the endothelium and on activated platelets), and its ligand PSGL-1 (resident in the leukocyte membrane) is a key player in the initial adhesion stages.4,5 We exploited this bond to form point attachments between polymorphonuclear leukocytes (PMNs) and functionalized microspheres that had been coated with P-selectin. To distinguish the dynamic properties of an individual P-selectin:PSGL-1 bond from mechanisms governed by other cellular structures, we also tested this bond on its own using isolated PSGL-1 that had been immobilized on a second batch of microspheres. Both types of interaction, i.e., P-selectin:PSGL-1 as well as P-selectin:PMN, were characterized by dynamic force spectroscopy6,7 over exceptionally wide ranges of forceloading rates. Complementary test surfaces were first brought into soft, feedback-controlled contact to allow for the formation of bonds between immobilized, reactive molecules. Then, while moving the surfaces apart at preset velocities, we recorded the force experienced by attachments between them. Piconewton forces were reported by the biomembrane force probe,8,9 a prototypical biophysical tool that is uniquely suited to study the dynamics of biologically relevant, ultraweak interactions. This approach enabled us to inspect in detail two possible candidates for principal regulatory mechanisms in early leukocyte adhesion. One is a serial molecular attachment consisting of (i) the extracellular P-selectin:PSGL-1 bond and (ii) a putative, weak biochemical link that anchors the intracellular tail domain of the transmembrane PSGL-1 to the cortical cytostructure.10 The other is a hierarchy of rheological cell responses to point loads: from slow viscoelastic deformation of the whole cell, and a soft-elastic displacement of the cell cortex, to the extrusion of thin membrane tubes (“tethers”) that can easily growsat quasiconstant pulling forcessto several micrometers in length. The formation of tethers from fluid membranes under point loads is a ubiquitous phenomenon, as evidenced by a large body of work on lipid vesicles11-14 and on various types of biological cells.15-18 A number of pioneering studies also investigated the mechanical properties of PMN tethers19-21 and their importance in leukocyte rolling.22,23 Perhaps even more interesting than these regulatory mechanisms by themselves is the intriguing interplay between them. For example, the lifetime of weak attachments under stress depends critically on the history of force application. In the present case where the leukocyte itself links adhesive molecules to rigid surfaces, any subcellular soft structures will act to dampen the sudden impact of force and thus modulate the force history experienced by the actual adhesion bonds. Therefore, the lifetime, or strength, of adhesive attachments is directly affected by the rheology of cell deformation under point loads. On the other hand, the extrusion of tethers from PMNs appears to commence only * Corresponding author e-mail: [email protected]. † Boston University. # Current address: Department of Biomedical Engineering, University of California, Davis, 451 East Health Sciences Drive, Davis, CA 95616. ‡ Department of Pathology, University of British Columbia. § Department of Physics and Astronomy, University of British Columbia. 1482 J. Chem. Inf. Model. 2005, 45, 1482-1490