Platelet P-selectin triggers rapid surface exposure of tissue factor in monocytes.

Tissue factor (TF) plays a central role in haemostasis and thrombosis. Following vascular damage, vessel wall TF initiates the extrinsic coagulation cascade. TF can also be exposed by monocytes. Inflammatory or infectious stimuli trigger synthesis of new TF protein by monocytes over the course of hours. It has also been suggested that monocytes can expose TF within minutes when stimulated by activated platelets. Here, we have confirmed that monocytes rapidly expose TF in whole blood and further demonstrate that platelet P-selectin exposure is necessary and sufficient. Monocyte TF exposure increased within five minutes in response to platelet activation by PAR1-AP, PAR4-AP or CRP-XL. PAR1-AP did not trigger TF exposure on isolated monocytes unless platelets were also present. In whole blood, PAR1-AP-triggered TF exposure required P-selectin and PGSL-1. In isolated monocytes, although soluble recombinant P-selectin had no effect, P-selectin coupled to 2 µm beads triggered TF exposure. Cycloheximide did not affect rapid TF exposure, indicating that de novo protein synthesis was not required. These data show that P-selectin on activated platelets rapidly triggers TF exposure on monocytes. This may represent a mechanism by which platelets and monocytes rapidly contribute to intravascular coagulation.

Using Yoda-1 to mimic laminar flow in vitro: A tool to simplify drug testing.

The endothelium is an attractive drug target and an important site of adverse drug reactions. Endothelial dysfunction is strongly associated with inflammation and contributes to drug-induced cardiovascular toxicity. Endothelial cells in the circulation are exposed to haemodynamic forces including shear stress. Including shear stress may improve future endothelial cell drug discovery or toxicity screening. Piezo-1 is required for endothelial cells to respond to shear stress. In this study, we investigated whether a small molecule activator of Piezo-1, Yoda-1, can mimic the effect of laminar flow-induced shear stress on endothelial cell inflammation, and endothelial cytotoxicity in response to the chemotherapy agent, doxorubicin. First, we tested whether Yoda-1 could mimic the effects of shear stress of expression of the endothelial adhesion molecules, ICAM-1 and VCAM-1. Human umbilical vein endothelial cells (HUVEC) were cultured in static conditions (with or without Yoda-1) or under laminar flow-induced shear stress (5 dyn/cm2). Yoda-1 and laminar flow had similar anti-inflammatory effects, reducing the ability of TNF-α to induce ICAM-1 and VCAM-1 expression. We then tested whether Yoda-1 could mimic the effect of shear stress on doxorubicin-induced cytotoxicity. Both laminar flow and Yoda-1 treatment of static cultures increased the cytotoxicity of doxorubicin. These findings show that Piezo-1 activation with Yoda-1 in static culture leads to an endothelial cell phenotype that mimics endothelial cells under laminar flow. Pharmacological activation of Piezo-1 may be a useful approach to mimic constant shear stress in static cultures, which may improve endothelial drug discovery and toxicity testing.

Cross-reactivity of anti-HMGB1 antibodies for HMGB2.

HMGB1 and HMGB2 are DNA-interacting proteins but can also have extracellular actions during inflammation. Despite their relatively high homology, they may have distinct roles, making it essential to be able to differentiate between the two. Here we examine the specificity of five commercially-available anti-HMGB1 antibodies. By Western blotting of recombinant proteins and HMGB1-/- mouse embryonic fibroblasts, we identified only one HMGB1 antibody that, under our experimental conditions, did not also detect HMGB2. Selecting specific antibodies for HMGB1 and HMGB2 allowed identification of distinct HMGB1 and HMGB2 subcellular pools in primary neutrophils.

Introduction: T Cell Trafficking in Inflammation and Immunity.

T cell migration across vascular endothelium is essential for T cell responses, as through the expression of specific tissue-homing receptors, these cells then access peripheral tissues, with the goal of eliminating invading pathogens and/or tumor cells. However, aberrant trafficking of T cells to peripheral tissues contributes to the development of most chronic inflammatory diseases. Very little is known about the mechanisms by which T cell trafficking is regulated during inflammation, and it is thus difficult to target this aspect of pathology for the development of new therapies. It is therefore important to understand the pathways involved in regulating the recruitment of immune cells.

Endocrine Regulation of Lymphocyte Trafficking In Vitro.

Lymphocyte recruitment in inflammation can be influenced by many molecules including cytokines, chemokines, and adipokines. In our lab, we have examined the effects of the adipokines leptin and adiponectin on lymphocyte migration, and observed modulation of this process. Lymphocyte behavior can be assessed in the lab under static conditions, or can be studied under flow, simulating in vivo conditions. In this chapter, in vitro methods for analyzing adhesion and migration of lymphocytes isolated from blood are described in detail. In static adhesion and migration assays, lymphocytes are allowed to settle on top of endothelial cell monolayers cultured in plates for a desired period of time. In the flow-based assay, lymphocytes are perfused over the endothelium at a continuous rate through microchannels which are commercially available. Depending on the choice of method employed, the efficiency of lymphocytes to adhere to and migrate across the endothelial cell monolayer under different conditions can be evaluated. Static assays are less complex and are of higher throughput. However, these assays provide less detailed information regarding lymphocyte behaviors. On the other hand, the flow-based assays are more difficult to perform, but are more physiologically relevant due to the presence of flow and yield more detailed information about lymphocyte activities such as capture, immobilization, and migration in real-time.