Neutrophil FcγRIIA promotes IgG-mediated glomerular neutrophil capture via Abl/Src kinases
Hiroshi Nishi, … , George C. Tsokos, Tanya N. Mayadas
Hiroshi Nishi, … , George C. Tsokos, Tanya N. Mayadas
Published October 2, 2017; First published September 11, 2017
Citation Information: J Clin Invest. 2017;127(10):3810-3826. https://doi.org/10.1172/JCI94039.
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Research Article Inflammation Nephrology

Neutrophil FcγRIIA promotes IgG-mediated glomerular neutrophil capture via Abl/Src kinases

Abstract

The kidney glomerular capillaries are frequent sites of immune complex deposition and subsequent neutrophil accumulation in post-infectious and rapidly progressive glomerulonephritis. However, the mechanisms of neutrophil recruitment remain enigmatic, and there is no targeted therapeutic to avert this proximal event in glomerular inflammation. The uniquely human activating Fc receptor FcγRIIA promotes glomerular neutrophil accumulation and damage in anti–glomerular basement membrane–induced (anti-GBM–induced) glomerulonephritis when expressed on murine neutrophils. Here, we found that neutrophils are directly captured by immobilized IgG antibodies under physiological flow conditions in vitro through FcγRIIA-dependent, Abl/Src tyrosine kinase–mediated F-actin polymerization. Biophysical measurements showed that the lifetime of FcγRIIA-IgG bonds increased under mechanical force in an F-actin–dependent manner, which could enable the capture of neutrophils under physiological flow. Kidney intravital microscopy revealed that circulating neutrophils, which were similar in diameter to glomerular capillaries, abruptly arrested following anti-GBM antibody deposition via neutrophil FcγRIIA and Abl/Src kinases. Accordingly, inhibition of Abl/Src with bosutinib reduced FcγRIIA-mediated glomerular neutrophil accumulation and renal injury in experimental, crescentic anti-GBM nephritis. These data identify a pathway of neutrophil recruitment within glomerular capillaries following IgG deposition that may be targeted by bosutinib to avert glomerular injury.

Authors

Hiroshi Nishi, Kazuhiro Furuhashi, Xavier Cullere, Gurpanna Saggu, Mark J. Miller, Yunfeng Chen, Florencia Rosetti, Samantha L. Hamilton, Lihua Yang, Spencer P. Pittman, Jiexi Liao, Jan M. Herter, Jeffrey C. Berry, Daniel J. DeAngelo, Cheng Zhu, George C. Tsokos, Tanya N. Mayadas

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Figure 1

A small-molecule screen identifies bosutinib as the lead inhibitor of FcγRIIA functions in neutrophils.

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A small-molecule screen identifies bosutinib as the lead inhibitor of Fc...
(A) FcγRIIA+/γ–/– mouse bone marrow neutrophils (BMNs), suspended in fluorescent ROS probes with library compounds (light blue, at 10 μM), were loaded on BSA/anti-BSA IC–coated plates, and fluorescence was read 1 hour later. The Src inhibitor PP2 (red) and the diluent DMSO (vehicle, deep blue) were positive and negative controls, respectively. A profile with representative “hits” (arrows) is shown. (B) Bosutinib (Bos) and PP2 at indicated doses were evaluated and plotted relative to vehicle (0 nM) (n = 3). (C) BMNs were preincubated with mouse anti–human FcγRIIA and then Bos, PP2, or vehicle (Veh). ROS generation was evaluated upon addition of F(ab′)2 anti-mouse IgG in a luminol-based assay. A representative profile is shown (left). The average peak level of ROS was normalized to vehicle (right) (n = 3). (D) Bos-pretreated BMNs were stimulated with PMA, and ROS generation was assessed as in C (n = 3). (E) The number of adherent (left) and spread (right) cells on BSA or IC under static conditions of BMNs pretreated with vehicle (–) or Bos (n = 3). (F) Adhesion under shear flow (0.5 dyn/cm2) to BSA- or IC-coated coverslips, of vehicle- or Bos-pretreated BMNs. The data are presented as the average fold induction normalized to the average value of the vehicle (–)/BSA control (n = 3). Neutrophils were treated with 100 nM Bos or 1 μM PP2 unless indicated otherwise (n = 3). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Dunnett’s multiple comparison test for B and C to assess dose responsiveness of the drugs compared with vehicle, and for E and F.