A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites.

Barry Mccormick,Julia Y Chu,Jennifer M Felton,Marta Canel,Adriano G Rossi,Laura Norton,Ingeborg Hers,Johanna Ivaska,Helen E Craig,Melina Michael,Ian Dransfield,Leo M Carlin,Takehiko Sasaki,Sonja Vermeren,Matilda Toivakka,Florian Wollweber,Anne L Astier,Johanna Lilja

doi:10.4049/jimmunol.1900443

Abstract

Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation.

Highlights

We demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils
We noted significantly reduced neutrophil numbers in Bronchoalveolar lavage (BAL) from ARAP3deficient mice compared with controls (Fig. 6A)
ARAP3 downstream of PI3K in a negative feedback loop that promotes integrin inactivation (Fig. 7). This mechanism enables rapid switching-off of integrins following ligand binding–induced outside-in signaling. This feedback loop operates in adherent neutrophils, in which ARAP3-dependent neutrophil activities are entirely dependent upon outside-in signaling-induced upstream PI3K activity

Summary

Introduction

The data presented in this study identify that integrin activation triggers a negative feedback loop downstream of PI3K by which ARAP3 promotes integrin inactivation. In keeping with our earlier work, we observed enhanced effector functions, including adhesion, spreading, ROS production, and degranulation, with ARAP3-deficient neutrophils that had been plated onto fibronectin with costimulation by TNF-a (Supplemental Fig. 1A– G) but not upon stimulation with formylated peptides [16]. Not observe any increased surface integrin expression with ARAP3-deficient neutrophils [Supplemental Fig. 1H, 1J, data not shown [16]].

Results

Conclusion