Direct Digital Engagement of Patients and Democratizing Health Care

Direct Digital Engagement of Patients and Democratizing Health Car

Influence of stromal cells on lymphocyte adhesion and migration on endothelial cells

Methods are described for analysing adhesion and migration of isolated lymphocytes on endothelial cell monolayers which have been co-cultured with different stromal cells, with or without additional cytokine treatment. The different cells types are grown on opposite sides of 3.0 or 0.4μm pore filters, depending on whether migration through the whole construct is to be analysed, or adhesion to the endothelial cells alone. Assays may be ‘static’ or filters can be incorporated into flow chambers so that cell behaviour can be directly observed under conditions simulating those in vivo. In general, by choice of method, one can evaluate efficiency of attachment, and ability of cells to migrate across the endothelial monolayer, through the filter and through the stromal cell layer. Fluorescence microscopic examination of fixed filters can be used e.g., to ascertain whether lymphocytes are retained by stromal cells. In general, static assays have the higher throughput and greatest ease of use, while the flow-based assays are more physiologically-relevant and allow detailed recording of cell behaviour in real time

Duffy antigen receptor for chemokines and CXCL5 are essential for the recruitment of neutrophils in a multicellular model of rheumatoid arthritis synovium

OBJECTIVE: The role of chemokines and their transporters are poorly described in rheumatoid arthritis (RA). Evidence suggests that CXCL5 plays an important role as it is abundant in RA tissue and its neutralization moderates joint damage in animal models of arthritis. The chemokine transporter, Duffy Antigen Receptor for Chemokines (DARC), is also upregulated in early RA. Here we investigate the role of CXCL5 and DARC in regulating neutrophil recruitment using an in vitro model of the RA synovium. METHODS: To model the RA synovium, rheumatoid fibroblasts (RAF) were cocultured with endothelial cells (EC) for 24h. Gene expression in cocultured cells was investigated using TaqMan gene arrays. Roles of CXCL5 and DARC were determined by incorporating cocultures into a flow-based adhesion assay, where their function was demonstrated by blocking neutrophil recruitment with neutralizing reagents. RESULTS: EC-RAF coculture induced chemokine expression in both cell types. While CXC chemokines were modestly upregulated in EC, CXCL1, CXCL5 and CXCL8 expression were greatly increased in RAF. RAF also promoted the recruitment of flowing neutrophils to EC. Anti-CXCL5 antibody abolished neutrophil recruitment by neutralizing CXCL5 expressed on EC, or when used to immuno-deplete coculture conditioned medium. DARC was also induced on EC by coculture and an anti-Fy6 antibody or siRNA targeting of DARC expression effectively abolished neutrophil recruitment. CONCLUSION: For the first time in a model of human disease, the function of DARC has been demonstrated as essential for editing the chemokine signals presented by EC and for promoting unwanted leukocyte recruitment

A novel mechanism of neutrophil recruitment in a co-culture model of the rheumatoid synovium

OBJECTIVE: Rheumatoid arthritis (RA) is classically thought of as a Th1, T lymphocyte–driven disease of the adaptive immune system. However, cells of the innate immune system, including neutrophils, are prevalent within the diseased joint, and accumulate in large numbers. This study was undertaken to determine whether cells of the rheumatoid stromal microenvironment could establish an inflammatory environment in which endothelial cells are conditioned in a disease-specific manner to support neutrophil recruitment. METHODS: Human umbilical vein endothelial cells (ECs) and fibroblasts isolated from the synovium or skin of RA patients were established in coculture on opposite sides of porous transwell filters. After 24 hours of EC conditioning, the membranes were incorporated into a parallel-plate, flow-based adhesion assay and levels of neutrophil adhesion to ECs were measured. RESULTS: ECs cocultured with synovial, but not skin, fibroblasts could recruit neutrophils in a manner that was dependent on the number of fibroblasts. Antibody blockade of P-selectin or E-selectin reduced neutrophil adhesion, and an antibody against CD18 (the β2 integrin) abolished adhesion. Blockade of CXCR2, but not CXCR1, also greatly inhibited neutrophil recruitment. Interleukin-6 (IL-6) was detectable in coculture supernatants, and both IL-6 and neutrophil adhesion were reduced in a dose-dependent manner by hydrocortisone added to cocultures. Antibody blockade of IL-6 also effectively abolished neutrophil adhesion. CONCLUSION: Synovial fibroblasts from the rheumatoid joint play an important role in regulating the recruitment of inflammatory leukocytes during active disease. This process may depend on a previously unsuspected route of IL-6–mediated crosstalk between fibroblasts and endothelial cells

ADAM10-interacting tetraspanins Tspan5 and Tspan17 regulate VE-cadherin expression and promote T lymphocyte transmigration

Abstract The recruitment of blood leukocytes across the endothelium to sites of tissue infection is central to inflammation, but also promotes chronic inflammatory diseases. A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane molecular scissor that is implicated in leukocyte transmigration by proteolytically cleaving its endothelial substrates. These include VE-cadherin, a homotypic adhesion molecule that regulates endothelial barrier function, and transmembrane chemokines CX3CL1 and CXCL16, which have receptors on leukocytes. However, a definitive role for endothelial ADAM10 in transmigration of freshly isolated primary leukocytes under flow has not been demonstrated, and the relative importance of distinct ADAM10 substrates is unknown. Emerging evidence suggests that ADAM10 can be regarded as six different molecular scissors with different substrate specificities, depending on which of six TspanC8 tetraspanins it is associated with, but TspanC8s remain unstudied in leukocyte transmigration. In the current study, ADAM10 knockdown on primary HUVECs was found to impair transmigration of freshly isolated human peripheral blood T lymphocytes, but not neutrophils or B lymphocytes, in an in vitro flow assay. This impairment was due to delayed transmigration rather than a complete block, and was overcome in the presence of neutrophils. Transmigration of purified lymphocytes was dependent on ADAM10 regulation of VE-cadherin, but not CX3CL1 and CXCL16. Tspan5 and Tspan17, the two most closely related TspanC8s by sequence, were the only TspanC8s that regulated VE-cadherin expression and were required for lymphocyte transmigration. Therefore endothelial Tspan5- and Tspan17-ADAM10 complexes may regulate inflammation by maintaining normal VE-cadherin expression and promoting T lymphocyte transmigration.</jats:p

Monocyte subsets coregulate inflammatory responses by integrated signaling through TNF and IL-6 at the endothelial cell interface.

Two major monocyte subsets, CD14(+)CD16(−) (classical) and CD14(+/dim)CD16(+) (nonclassical/intermediate), have been described. Each has different functions ascribed in its interactions with vascular endothelial cells (EC), including migration and promoting inflammation. Although monocyte subpopulations have been studied in isolated systems, their influence on EC and on the course of inflammation has been ignored. In this study, using unstimulated or cytokine-activated EC, we observed significant differences in the recruitment, migration, and reverse migration of human monocyte subsets. Associated with this, and based on their patterns of cytokine secretion, there was a difference in their capacity to activate EC and support the secondary recruitment of flowing neutrophils. High levels of TNF were detected in cocultures with nonclassical/intermediate monocytes, the blockade of which significantly reduced neutrophil recruitment. In contrast, classical monocytes secreted high levels of IL-6, the blockade of which resulted in increased neutrophil recruitment. When cocultures contained both monocyte subsets, or when conditioned supernatant from classical monocytes cocultures (IL-6(hi)) was added to nonclassical/intermediate monocyte cocultures (TNF(hi)), the activating effects of TNF were dramatically reduced, implying that when present, the anti-inflammatory activities of IL-6 were dominant over the proinflammatory activities of TNF. These changes in neutrophil recruitment could be explained by regulation of E-selectin on the cocultured EC. This study suggests that recruited human monocyte subsets trigger a regulatory pathway of cytokine-mediated signaling at the EC interface, and we propose that this is a mechanism for limiting the phlogistic activity of newly recruited monocytes