Antineutrophil cytoplasmic antibodies stabilize adhesion and promote migration of flowing neutrophils on endothelial cells

137

151

The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCAs) may contribute to the pathogenesis of ANCA-associated vasculitis are not well understood. In this study, both polyclonal ANCAs isolated from patients and chimeric proteinase 3-ANCA induced the release of neutrophil microparticles from primed neutrophils. These microparticles expressed a variety of markers, including the ANCA autoantigens proteinase 3 and myeloperoxidase. They bound endothelial cells via a CD18-mediated mechanism and induced an increase in endothelial intercellular adhesion molecule-1 expression, production of endothelial reactive oxygen species, and release of endothelial IL-6 and IL-8. Removal of the neutrophil microparticles by filtration or inhibition of reactive oxygen species production with antioxidants abolished microparticle-mediated endothelial activation. In addition, these microparticles promoted the generation of thrombin. In vivo, we detected more neutrophil microparticles in the plasma of children with ANCAassociated vasculitis compared with that in healthy controls or those with inactive vasculitis. Taken together, these results support a role for neutrophil microparticles in the pathogenesis of ANCA-associated vasculitis, potentially providing a target for future therapeutics.

Section: Polyclonal Ancas From Patients and Chimeric Ancamentioning

confidence: 99%

Section: Polyclonal Ancas From Patients and Chimeric Ancamentioning

confidence: 99%

Anti-Neutrophil Cytoplasmic Antibodies Stimulate Release of Neutrophil Microparticles

Hong

Eleftheriou

Hussain

et al. 2012

137

151

“…9 -13 They modulate neutrophil adhesion and migration 14,15 and induce F-actin polymerization and cell stiffening, ultimately leading to neutrophil retention in glomerular capillaries. [15][16][17] Although the role of ANCA in vasculitis pathogenesis has been established, the accessibility of ANCA to their antigens in blood is still debated. 18 -20 The absence of ANCA detection on neutrophils in whole blood has been proposed to result from plasma ␣1 anti-trypsin (A1AT) binding to PR3.…”

mentioning

confidence: 99%

Increased Membrane Expression of Proteinase 3 during Neutrophil Adhesion in the Presence of Anti–Proteinase 3 Antibodies

Brachemi¹,

Mambole²,

Fakhouri³

et al. 2007

We investigated membrane proteinase 3 (mPR3) expression during TNF-␣-induced adhesion of neutrophils in the presence of anti-PR3 antibodies, a situation occurring during anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis. Three increasing levels of mPR3 expression were observed on the mPR3 ϩ neutrophil subset after stepwise cell activation. TNF-␣ activation without adhesion, TNF-␣-induced adhesion, and adhesion in the presence of anti-PR3 mAb or human anti-PR3 ANCA resulted, respectively, in a two-, seven-, and 24-fold increase of mPR3 levels. In plasma, anti-PR3 antibodies poorly recognized suspended neutrophils, whereas they bound to mPR3 on adherent cells. mPR3 upregulation was also triggered by IL-8, formyl-methionyl-leucyl-phenylalanine (fMLP), and neutrophil adhesion to activated human umbilical vein endothelial cells. It involved ␤2 integrins and Fc␥ receptor, because it was prevented by anti-CD18 antibodies and was not observed with anti-PR3 F(abЈ) 2 . Furthermore, it was specific to anti-PR3 mAb, and no mPR3 upregulation was observed with antimyeloperoxidase or anti-HLA-ABC mAb. Newly expressed mPR3 molecules, after TNF-induced adhesion, were mobilized from secretory vesicles (CD35 ϩ ) and secondary granules (CD11b ϩ ). The adhesionand antibody-dependent upregulations of mPR3 expression occurred with little azurophilic granule degranulation, no sign of apoptosis, and no further CD177 upregulation. In conclusion, this study describes an amplifying loop in polymorphonuclear neutrophil activation process, whereby ANCA are involved in the membrane expression of their own antigen during cell adhesion. This could explain the restriction of ANCA-associated vasculitis to small vessels, the main site of neutrophil adhesion.

“…Multiple ANCA may bind target antigens on one neutrophil and cross present oligomerized Fc moieties to surrounding neutrophils. However, in a flow assay examining ANCA-induced adhesion of neutrophils to an endothelial monolayer, it was clear that cell-cell contact between neutrophils was not necessary for ANCA-mediated effects (18). Alternatively, ANCA may heterodimerize Fc␥ receptors and target antigens on the surface of individual neutrophils.…”

mentioning

confidence: 99%

Activation of Syk in Neutrophils by Antineutrophil Cytoplasm Antibodies Occurs via Fcγ Receptors and CD18

Hewins¹,

Williams²,

Wakelam³

et al. 2004

Abstract. Antineutrophil cytoplasm antibodies (ANCA) activate TNF-␣-primed neutrophils to undergo a respiratory burst. The intracellular signals that mediate activation have not been studied extensively but could increase the understanding of the pathogenesis small vessel vasculitis. It was demonstrated that ANCA-IgG induced phosphorylation of the tyrosine kinase Syk in TNF-␣-primed neutrophils from healthy donors. Syk was not phosphorylated in response to ANCA F(ab') 2 . Furthermore, Syk phosphorylation was attenuated by blockade of both low-affinity Fc␥ receptors and CD18. Similarly, low-affinity Fc␥ receptor blockade reduced ANCA-induced superoxide production. In patient-derived neutrophils, the high-affinity Fc␥ receptor Fc␥RI was also demonstrated to be involved in ANCA-induced superoxide production. However, Syk phosphorylation was not attenuated by blockade of the Fc␥RI, present on neutrophils from vasculitis patients. The tyrosine kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine inhibited the ANCA-induced respiratory burst and Syk phosphorylation, suggesting that Src kinases lie upstream of Syk activation but downstream of ANCA engagement of Fc␥ receptors. Piceatannol, another tyrosine kinase inhibitor, also inhibited ANCA-induced Syk phosphorylation and the ANCA-stimulated respiratory burst, supporting the proposed functional role for Syk in ANCA signaling. ANCAinduced phosphorylation of Cbl and intracellular calcium transients, potential downstream mediators of Syk activation, were also blocked by tyrosine kinase inhibitors. While it has previously been shown that pertussis toxin diminishes the ANCAinduced respiratory burst, indicating heterotrimeric G protein involvement, Syk phosphorylation and calcium transients were unaffected by pertussis toxin. Collectively, these data show that Syk phosphorylation is induced during ANCA-triggered neutrophil activation.