Probing dynamic cell–substrate interactions using photochemically generated surface-immobilized gradients: application to selectin-mediated leukocyte rolling

Abstract

Model substrates presenting biochemical cues immobilized in a controlled and well-defined manner are of great interest for their applications in biointerface studies that elucidate the molecular basis of cell receptor-ligand interactions. Herein, we describe a direct, photochemical method to generate one-component surface-immobilized biomolecular gradients that are applied to the study of selectin-mediated leukocyte rolling. The technique employs benzophenone-modified glass substrates, which upon controlled exposure to UV light (350 – 365 nm) in the presence of protein-containing solutions facilitate the generation of covalently immobilized protein gradients. Conditions were optimized to generate gradient substrates presenting P-selectin and PSGL-1 (P-selectin Glycoprotein Ligand-1) immobilized at site densities over a 5- to 10-fold range (from as low as ~200 molecules/μm2 to as high as 6000 molecules/μm2). The resulting substrates were quantitatively characterized via fluorescence analysis and radioimmunoassays before their use in the leukocyte rolling assays. HL-60 promyelocytes and Jurkat T lymphocytes were assessed for their ability to tether to and roll on substrates presenting immobilized P-selectin and PSGL-1 under conditions of physiologically relevant shear stress. The results of these flow assays reveal the combined effect of immobilized protein site density and applied wall shear stress on cell rolling behavior. Two-component substrates presenting P-selectin and ICAM-1 (intercellular adhesion molecule-1) were also generated to assess the interplay between these two proteins and their effect on cell rolling and adhesion. These proof-of-principle studies verify that the described gradient generation approach yields well-defined gradient substrates that present immobilized proteins over a large range of site densities that are applicable for investigation of cell-materials interactions, including multi-parameter leukocyte flow studies. Future applications of this enabling methodology may lead to new insights into the biophysical phenomena and molecular mechanism underlying complex biological processes such as leukocyte recruitment and the inflammatory response.

Bromelain Decreases Neutrophil Interactions with P-Selectin, but Not E-Selectin, In Vitro by Proteolytic Cleavage of P-Selectin Glycoprotein Ligand-1

“…Using a molecularly general method previously developed in our lab for the covalent and controllable photochemical immobilization of biomolecules on planar glass substrates [14] , we generated and characterized substrates presenting defined “high” and “low” levels of P- or E-selectin ( Figures 1 – 2 and Figure S1 in File S1 ). Substrates were characterized with fluorescence imaging and average fluorescence intensity values were converted into biomolecule site densities using an established fluorescence-radioactivity correlation for P-selectin [15] and E-selectin (Figure S2 in File S1 ).…”

“…Given these temporal selectin expression dynamics, we felt it would be beneficial to independently probe bromelain’s effects on neutrophil interactions with substrates presenting each of these glycoproteins. To achieve this, we utilized a photochemical surface modification strategy [14] developed in our lab to generate substrates presenting controlled densities of P-selectin or E-selectin [15] , and then used these substrates to investigate the effect of bromelain treatment on the ability of human neutrophils to tether and roll in flow assays ( Figure 1 ).…”

The Interaction of Leukocytes and Adhesion Molecules in Mesenteric Microvessel Endothelial Cells after Internal Capsule Hemorrhage

“…The molecular basis of the adhesion was the expression change of CAMs on the surface of leukocytes and microvessel endothelial cells [15]. CAMs participate in cell–cell adhesion and are of great importance in binding and interactions of cells with components of the extracellular matrix.…”

CHAPTER 7. Interfaces in Composite Materials

“…65 The interface could also affect the recruitment of leukocytes and the inflammatory response. 66 This is particularly important for implantable Figure 7.1 (I) Diagrammatic representation of the interface between a filler particle and a polymer matrix in a composite; the interface composed of adsorbed polymer chain that has point contact (anchor or trains) with the filler particle, unadsorbed polymer segment (e.g., loops and tails) that are entangled with other chains adsorbed on filler surface. Modified from Ciprari et al 51 (II) Diagrammatic representation of cell-biomaterial surface interaction: once the biomaterial is implanted into the body, blood contact occurs; water molecules and proteins from the blood become adsorbed onto the surface of the biomaterial (a), then a specific and complex protein layer forms over the surface providing binding sites for cell surface integrins (b), immune cells e.g., macrophages and neutrophils reach the implanted site to phagocytose the implanted material otherwise they fuse together forming foreign-body giant cells that release cytokines to recruit fibroblasts (c); fibroblasts infiltrate the surface and secrete collagen and other proteins of the extracellular matrix (ECM) (d) that subsequently form a fibrous capsules around the implanted biomaterial (e).…”