Objective Syk is a cytoplasmic protein tyrosine kinase that plays a role in signaling via B cell and Fc receptors (FcR). FcR engagement and signaling via Syk is thought to be important in antineutrophil cytoplasm antibody (ANCA) IgG–mediated neutrophil activation. This study was undertaken to investigate the role of Syk in ANCA‐induced myeloid cell activation and vasculitis pathogenesis. Methods Phosphorylation of Syk in myeloid cells from healthy controls and ANCA‐associated vasculitis (AAV) patients was analyzed using flow cytometry. The effect of Syk inhibition on myeloperoxidase (MPO)–ANCA IgG activation of cells was investigated using functional assays (interleukin‐8 and reactive oxygen species production) and targeted gene analysis with NanoString. Total and phosphorylated Syk at sites of tissue inflammation in patients with AAV was assessed using immunohistochemistry and RNAscope in situ hybridization. Results We identified increased phosphorylated Syk at critical activatory tyrosine residues in blood neutrophils and monocytes from patients with active AAV compared to patients with disease in remission or healthy controls. Syk was phosphorylated in vitro following MPO‐ANCA IgG stimulation, and Syk inhibition was able to prevent ANCA‐mediated cellular responses. Using targeted gene expression analysis, we identified up‐regulation of FcR‐ and Syk‐dependent signaling pathways following MPO‐ANCA IgG stimulation. Finally, we showed that Syk is expressed and phosphorylated in tissue leukocytes at sites of organ inflammation in AAV. Conclusion These findings indicate that Syk plays a critical role in MPO‐ANCA IgG–induced myeloid cell responses and that Syk is activated in circulating immune cells and tissue immune cells in AAV; therefore, Syk inhibition may be a potential therapeutic option.
Background/Aims Real Time-Deformability Cytometry (RT-DC) is a novel technique able to identify morpho-rheological characteristics of individual cells such as size, deformability, and elasticity using only 50µl of whole blood. Cells in suspension flow through a microfluidic channel while hydrodynamic force is applied, leading to reversible deformation of individual cells from shear stress and pressure gradients. Cell brightness and area can be used to identify individual cell types. The morpho-rheological characteristics of immune cells in ANCA associated vasculitis (AAV) are unknown. Methods Whole blood from healthy controls (HC), patients with active AAV, or patients with AAV in remission was analysed using RT-DC. The diagnosis of AAV was based upon positive ANCA testing, with either (i) pauci-immune glomerulonephritis, or (ii) typical clinical features of extra-renal AAV. Patients with active disease may have received steroids prior to sampling but those who received other immunosuppression or cytotoxics were excluded. Remission was defined as BVAS of 0 with prednisolone dose ≤7.5mg/day. Blood was collected into K2 EDTA and diluted 1:20 in 1xPBS/0.6% methylcellulose. Cell suspensions were flowed across a Flic20 polydimethylsiloxane microfluidic chip, containing reservoirs connected by a measurement channel with a 20x20 μm2 cross-section. RT-DC measurements were collected at a flow rate of 0.12μL/s using a high-speed CMOS camera to capture images at a rate of 2000 frames/second. ShapeOut software was used to calculate cell size, deformation, and elasticity. Results There was no difference in neutrophil size between patients with active AAV (n = 15), patients with AAV in remission (n = 31), and HC (n = 15). Neutrophils from patients with active AAV were significantly stiffer than patients in remission or HC, displaying decreased deformation (median 0.084, 0.085, and 0.078 au respectively, p = 0.0068) and increased elasticity (median 0.973, 0.968 and 1.006 au respectively, p = 0.0196). In patients with active AAV, there was strong inverse correlation between neutrophil deformation and disease activity measured by BVAS score (r=-0.573, p = 0.032). Neutrophils isolated from healthy donors exhibited a trend towards decreased deformability when primed with TNF and stimulated with MPO-ANCA IgG compared to unstimulated or cells treated with healthy donor IgG (median 0.086, 0.085, and 0.083 for unstimulated, control IgG, and MPO-ANCA IgG respectively, ns). Conclusion Neutrophils from patients display distinct physical properties in active AAV which correlate with disease activity. Similar results are seen when neutrophils are stimulated in vitro with MPO-ANCA IgG. This phenotype of increased cell stiffness may lead to increased neutrophil retention in pulmonary and renal microvasculature, thus increasing the potential for neutrophil-endothelial cell interactions and microvascular damage. Morphorheological parameters can be rapidly measured using a small volume of whole blood; thus, RT-DC may be a useful technique to aid identification of disease activity in AAV and to guide treatment. Disclosure N. Pisacano: None. S.P. McAdoo: None. J. Guck: None. C.D. Pusey: None. E.R. Chilvers: None. A.C. Cowburn: None. K.M. Lodge: None. M.F. Prendecki: None.
BackgroundANCA associated vasculitis (AAV) is characterised by neutrophil-mediated endothelial damage. Neutrophil deformability impacts their ability to traffic through microvasculature. Distinct biophysical properties of neutrophils in patients with active AAV, e.g. in response to ANCA or hypoxia in inflamed tissues, may contribute to endothelial injury.ObjectivesInvestigate how MPO-ANCA IgG (ANCA) treatment and/or hypoxia affect the actin cytoskeleton or biophysical properties of neutrophils, and whether this phenotype is replicated in patients with active AAV.MethodsWhole blood and isolated neutrophils from patients with active AAV (AAV), patients with AAV in remission (rAAV) or healthy controls (HC) was analysed using real time deformability cytometry (RT-DC), a novel microfluidic technique able to identify individual cell biophysical profiles. Deformation was calculated using ShapeOut. Neutrophils were isolated using immunomagnetic separation. Cells were primed with TNF (2 ng/mL, 15 min) and stimulated with healthy donor IgG (CIgG), or ANCA IgG (100 ug/mL, 1 h) under normoxia (21% O2) or hypoxia (5% O2). Cells were stained for F actin (AF488 Phalloidin) and imaged using confocal microscopy. Cell area and total percentage phalloidin staining was analysed (ImageJ).ResultsAAV neutrophils (n=27) were significantly stiffer than rAAV (n=25) or HC (n=15), displaying decreased deformation (median 0.085, 0.086, and 0.076 au for HC, rAAV, and AAV respectively, p=0.0001). There was an inverse correlation between AAV neutrophil deformation and disease activity as measured by BVAS score (r=-0.573, p=0.032). This phenotype of decreased deformation was replicated by ANCA stimulation of HC neutrophils (median 0.086, 0.085 and 0.082 for untreated, CIgG, and ANCA respectively, p=0.05). Hypoxia prevented the effect of ANCA stimulation on HC neutrophils (median 0.078, 0.077, and 0.079 au for untreated, CIgG, and ANCA respectively, ns). ANCA stimulation of HC neutrophils increased cell area (mean 51.56/63.07/88.61 um2, p=0.0008) and total actin signal (mean 27.61/25.86/44.30%, p=<0.0001) (untreated, CIgG, ANCA respectively, Fig 1A-C). At baseline, hypoxic neutrophils were larger with increased actin staining. Under hypoxia the effect of ANCA stimulation was diminished, with a smaller increase in area (mean 60.34/67.48/71.15 um2, p=0.0011) and total actin signal (mean 38.15/46.01/45.49%, p=0.0255) compared to normoxia (Fig 1D-F). AAV neutrophils (n=8) were significantly larger, with more F-actin at baseline and showed a larger increase in actin following ANCA stimulation compared to HC cells: area (mean 75.73/98.68/121.25 um2, p=0.0088) and total actin signal (mean 51.74/74.85/83.44%, p=0.0032) (Fig 1G-I).ConclusionNeutrophils from patients with active AAV display distinct physical properties, which correlate with disease activity. Similar results are seen when neutrophils are stimulatedin vitrowith ANCA. In keeping with this, increased neutrophil actin polymerisation and marked lamellopodia formation is seen following ANCA stimulationin vitro, with the greatest effect seen when cells from patients with active AAV were used. Of interest, incubation under hypoxic conditions diminished the effect of ANCA stimulation suggesting that hypoxia can modulate actin polymerisation. This phenotype of increased cell stiffness in AAV may be due to reorganisation of the actin cytoskeleton and lead to increased neutrophil retention in pulmonary and renal microvasculature, thus increasing the potential for neutrophil-endothelial cell interactions and microvascular damage. Therefore, targeting cytoskeletal changes in AAV may offer a route to improve clinical outcomes.REFERENCESNIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.