A novel 4-dimensional live-cell imaging system to study leukocyte-endothelial dynamics in ANCA-associated vasculitis

Abstract

Neutrophils, monocytes and the endothelium are critical to ANCA-associated vasculitis (AAV) pathogenesis. This study aimed to develop a 4-dimensional (4D) live-cell imaging system that would enable investigation of spatial and temporal dynamics of these cells in health and disease. We further aimed to validate this system using autologous donor serum from AAV patients and polyclonal ANCA IgG, as well as exploring its potential in the pre-clinical testing of putative therapeutic compounds. Neutrophils and monocytes were isolated from peripheral venous blood of AAV patients or healthy controls and co-incubated on an endothelial monolayer in the presence of autologous serum. Alternatively, polyclonal ANCA IgG was used, following TNF-α priming, and imaged in 4-dimensions for 3 hours using a spinning disc confocal microscope. Volocity 6.3® analysis software was used for quantification of leukocyte dynamics. The use of autologous serum resulted in increased neutrophil degranulation (P = 0.002), transmigration (P = 0.0096) and monocyte transcellular transmigration (P = 0.0013) in AAV patients. Polyclonal MPO-ANCA IgG induced neutrophil degranulation (P < 0.001) in this system. C5aR1 antagonism reduced neutrophil degranulation (P < 0.0002). We have developed a novel 4D in vitro system that allows accurate quantification of multiple neutrophiland monocyte-endothelial interactions in AAV in a single assay. This system has the potential to highlight dynamics key to pathophysiology of disease, as well investigating the impact of potential therapeutics on these functions.

Immune cells fold and damage fungal hyphae

“…For some experiments, BMDMs were prepared from knockout mice lacking CARD9, Dectin-1, or MyD88 (43,49,76). BMDMs were treated at the following final concentrations in the specified carriers: 5 μM cytochalasin D in dimethyl sulfoxide (DMSO), 1 μM latruncilin A in DMSO, 20 μM blebbistatin in DMSO, 10 μM colchicine in EtOH, 100 μg/mL laminarin in RPMI, anti-Cd11b or isotype (abcam 8878) added at 33 μg/mL, anti-CD18 or isotype (abcam 119830) added at 33 μg/mL, Human monocyte-derived macrophages were prepared from the blood of healthy volunteers using density centrifugation followed by magneticactivated cell sorting of CD14+ populations (Miltenyi Biotech) and cultured in DMEM containing 10% autologous human serum (77). For experiments with differentially activated monocytes, cells were left untreated until day 7 to generate M0 cells.…”

Autoantibodies as Biomarker and Therapeutic Target in Systemic Sclerosis

“…In AAV, ANCAs lead to an activation of neutrophils primed by complement fragment C5a or cytokines (TNF-α, IL-1β), resulting in a translocation of PR3 or MPO to the cellular surface. After interaction between ANCAs with the target antigens PR3 or MPO and Fcγ receptors IIa or IIIb, intravascular near-wall degranulation of neutrophils and subsequent endothelial cell damage occurs [ 96 , 97 , 98 ]. ANCA-induced monocyte activation is mediated by a similar mechanism [ 99 , 100 ].…”