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 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 3

Role for Abl kinases in FcγRIIA-mediated neutrophil functions.

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Role for Abl kinases in FcγRIIA-mediated neutrophil functions. (A) Diffe...
(A) Differentiated HL-60 cells pretreated with vehicle (–), bosutinib (Bos), imatinib (Ima), nilotinib (Nil), or PP2 adherent to ICs or BSA under flow. Fold induction is relative to vehicle (–) on BSA (n = 3). (B) A representative Western blot of cells expressing Abl1 (Abl1 #01 and #00) or control shRNA and analyzed for Abl or β-actin (loading control) (n = 3). (C) ROS in cells in B subjected to FcγRIIA cross-linking (XL) or treated with PMA. The peak level was normalized to control shRNA cells (n = 3). (D) Cells on BSA- or IC-coated plates were stained for actin, and the number of adherent cells (left) and the area of a single spread cell (right) were calculated (n = 3, except in the right panel, n = 2 for treatment with shRNA #00). (E) Cell adhesion to BSA or IC at 0.35 dyn/cm2. Fold induction is relative to control cells on BSA (n = 3). (F) Western blot analysis of indicated proteins with or without FcγRIIA XL for times in seconds. β-Actin is the loading control. One representative of 3 is shown. (G and H) F-actin polymerization analyzed by flow cytometry (mean fluorescence intensity, MFI) at indicated times after XL and staining with NBD-phallacidin. Omission of the primary antibody is the negative control. Shown is 1 representative of 3 experiments for Abl1- or control shRNA–expressing cells (G), and for cells treated with vehicle (–) or indicated inhibitors before XL (H). For A–H, cells were treated with 1 μM of Bcr-Abl inhibitors or PP2, or 3 μM cytochalasin D (cytD). All data are mean ± SEM. *P < 0.05, **P < 0.01, 1-way ANOVA followed by Dunnett’s multiple comparison test for A and C–E.