Biomedical subjects

Harry L Goldsmith

Publications and source records attributed to Harry L Goldsmith.

4 recordsLinked to original sources

PubMed2005

Biomechanics of P-selectin PSGL-1 bonds: shear threshold and integrin-independent cell adhesion.

Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP)-stimulated platelets or P-selectin-bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14 to 3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed using both flow cytometry and high-speed videomicroscopy. We observed that although blocking antibodies against either P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1) alone inhibited platelet-neutrophil adhesion by approximately 60% at 140/s, these reagents completely blocked adhesion at 3000/s. Anti-Mac-1 alone did not alter platelet-neutrophil adhesion rates at any shear rate, though in synergy with selectin antagonists it abrogated cell binding. Unstimulated neutrophils avidly bound Ps-beads and activated platelets in an integrin-independent manner, suggesting that purely selectin-dependent cell adhesion is possible. In support of this, antagonists against P-selectin or PSGL-1 caused dissociation of previously formed platelet-neutrophil and Ps-bead neutrophil aggregates under shear in a variety of experimental systems, including in assays performed with whole blood. In studies where medium viscosity and shear rate were varied, a shear threshold for P-selectin PSGL-1 binding was also noted at shear rates <100/s when Ps-beads collided with isolated neutrophils. Results are discussed in light of biophysical computations that characterize the collision between unequal-size particles in linear shear flow. Overall, our studies reveal an integrin-independent regime for cell adhesion and weak shear threshold for P-selectin PSGL-1 interactions that may be physiologically relevant.

Binding Sites↗

PubMed2004

Cooperativity between selectins and beta2-integrins define neutrophil capture and stable adhesion in shear flow.

A cooperative, sequential process of molecular recognition governs leukocyte capture, rolling, and arrest on inflamed endothelium. Flowing neutrophils are captured via heterotypic adhesive interactions mediated by endothelial E-selectin, whereas homotypic interactions between neutrophils are mediated by L-selectin. To elucidate how each selectin facilitates the transition to CD18-mediated stable adhesion, E-selectin and L-selectin were expressed at defined site density in a murine pre-B-cell line. Direct observation of two-body collisions revealed that 30% of neutrophil interactions with E-selectin transfectants formed doublets at low shear rate G = 14 s(-1) whereas a threshold shear rate 14 s(-1) < or = G < or = 10 s(-1) was necessary for L-selectin adhesion. Adhesion via L-selectin resisted rupture at high shear stress, while E-selectin tethered doublets remained intact longer once formed. Moreover, higher expression of L-selectin (1100 sites/microm2) than that of E-selectin (220 sites/microm2) was required for comparable heterotypic adhesion efficiency. With a threefold rise in active CD18 upregulated on chemotactically stimulated neutrophils, homotypic adhesion efficiency increased 10-fold compared to less than 5-fold for heterotypic adhesion to selectin transfectants. Co-expression of E-selectin and ICAM-1 boosted adhesion efficiency threefold more than either receptor alone over the range of active CD18 expression. These data are the first to quantify adhesion efficiency mediated by selectin tethering and conformational activation of beta2-integrin in neutrophils in shear flow.

Animals↗

PubMed2002

Leukocyte adhesion dynamics in shear flow.

Of the white blood cells that traverse the circulation, the polymorphonuclear leukocytes commonly called neutrophils, are the most numerous, numbering approximately 5000 per microliter of blood. In a sense, these cells are the emergency response unit within the body's circulatory highway since they are the first to be recruited at sites of tissue trauma, infection, or inflammation. The chief function of neutrophils is the capture, phagocytosis, and degradation of foreign invaders. To carry out this critical function, neutrophils sense infection via chemotactic receptors that trigger cellular activation upon ligation of as few as 10-100 bacterial or chemokine peptides. Despite this acute sensitivity to stimuli, neutrophils circulate in healthy individuals largely in a passive state, with a very low efficiency of capture and arrest on quiescent endothelium. Here, we define the efficiency as the fraction of all cells passing by a given length of vessel wall that is captured and achieves firm adhesion. Within seconds of chemotactic signaling, adhesion molecules expressed on the plasma membrane of neutrophils are activated and a rapid boost in the efficiency of stable adhesion is detected. This occurs for both homotypic neutrophil-neutrophil adhesion and neutrophil capture and adhesion on inflamed endothelium. With such large numbers of circulating neutrophils, and with their inherent capability to rapidly adhere to postcapillary venular endothelium, the body has evolved a diverse set of mechanisms to regulate the inflammatory cascade that begins with intracellular signaling and leads to cell arrest and extravasation at sites of tissue insult. In this article we will focus on the interplay between particle-fluid dynamics that involve the shear and normal forces that transport cells transversely and axially within the vessel, and the activation and interaction of adhesion molecules that involve receptor-ligand bond formation that enables the neutrophils to resist wall shear stress and adhere to specific sites of inflammation.

CD18 Antigens↗

PubMed2002

Hydrodynamic shear and tethering through E-selectin signals phosphorylation of p38 MAP kinase and adhesion of human neutrophils.

Recently, we reported that tethering and rolling of neutrophils in shear flow over a substrate of E-selectin signals activation of beta 2-integrins and firm adhesion via an intracellular signaling pathway involving phosphorylation of p38 MAP kinase. In the current study the objective was to examine the molecular mechanisms and shear dependence underlying activation and adhesion of beta 2-integrin during shear-induced collisions between human neutrophils and murine B cells (300.19) transfected to express either E-selectin or L-selectin. Three separate parameters of cell activation were assessed over the time course of application of a defined shear field to heterotypic cell suspensions in a cone-plate viscometer. These were the two-body collision doublet lifetime and capture efficiency, surface upregulation of CD11b/CD18, and tyrosine phosphorylation of p38 MAP kinase. The data indicate that neutrophil adhesion to E-selectin expressing 300.19 cells occurs with a fourfold higher efficiency of firm adhesion than do collisions with L-selectin or parent control cells. Visual analysis of aggregation in a transparent cone-plate rheoscope revealed that the lifetime and efficiency of doublet formation increased fourfold as the applied shear stress increased. Neutrophil tethering via E-selectin was associated with rapid activation as indicated by upregulation of surface CD11b/CD18 and phosphorylation of p38 MAP kinase within seconds of application of shear. Activation greatly exceeded that observed for neutrophils sheared alone or with B cells expressing L-selectin. A distinct dependence of activation on the magnitude of the shear rate suggests a coupling between the fluid mechanical effects of shear and signaling of neutrophil adhesion.

Animals↗