Neutrophil Serine Proteases Promote IL-1β Generation and Injury in Necrotizing Crescentic Glomerulonephritis

Abstract

The pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated necrotizing crescentic GN (NCGN) is incompletely understood. Dipeptidyl peptidase I (DPPI) is a cysteine protease required for the activation of neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase, and proteinase 3, which are enzymes that modulate inflammation. We used a mouse model of anti-myeloperoxidase (MPO) antibodyinduced NCGN to determine whether active NSPs contribute to its pathogenesis. MPO-deficient animals immunized with murine MPO, irradiated, and transplanted with wild-type bone marrow developed NCGN. In contrast, transplantation with bone marrow that lacked DPPI or lacked both neutrophil elastase and proteinase 3 protected mice from NCGN induced by anti-MPO antibody. The kidneys of mice reconstituted with DPPI-deficient bone marrow generated significantly less IL-1b than did those of mice reconstituted with wild-type bone marrow; similarly, in vitro, DPPI-deficient monocytes produced significantly less IL-1b in response to anti-MPO antibody than did wild-type monocytes. This reduction in IL-1b was NSP dependent; exogenous addition of PR3 restored IL-b production in DPPI-deficient monocytes. Last, the IL-1 receptor antagonist anakinra protected animals against anti-MPO antibody-induced NCGN (16.7%66.0% versus 2.4%61.7% crescents), suggesting that IL-1b is a critical inflammatory mediator in this model. These data suggest that the development of anti-MPO antibody-induced NCGN requires NSP-dependent IL-1b generation and that these processes may provide therapeutic targets for ANCAmediated diseases in humans.

Phagocyte NADPH Oxidase Restrains the Inflammasome in ANCA-Induced GN

“…Bringing our data and the previous NSP study 14 together, we postulate that two independently acting ANCA-mediated pathways induce IL-1b generation in monocytes and thereby, cause NCGN, namely an NSP-dependent, inflammasome/caspase-1-independent pathway and a parallel inflammasome/caspase-1-dependent pathway (Supplemental Figure 1). The latter seems to be restrained by ANCA-induced Phox-generated ROS providing a pivotal control loop limiting and terminating ANCA-induced inflammation.…”

“…In fact, active NSPs were more important than caspase-1 in ANCA-induced IL-1b generation by monocytes. 14 In our experiments, the accelerated disease phenotype of Phoxdeficient BM showed strongly increased macrophage influx and IL-1b in kidneys from mice transplanted with gp91 phox2/2 and p47 phox2/2 BM compared with WT BM. The IL-1b values were 757.06120.3 pg/mg with WT BM versus 1708.76360.3 pg/mg in the two combined Phox-deficient BM-transplanted animals (P,0.01).…”

“…Numerous in vitro studies established an ANCA-mediated robust Phox-dependent respiratory burst in neutrophils and although less well studied, monocytes. 2,[11][12][13][14]38,39 Together with the fact that ROS modify and damage proteins and lipids, ANCA-induced ROS release is considered a key pathogenic component for necrotizing small vessel vasculitis. However, our data underscore the need to study candidate mechanisms in complex in vivo conditions.…”

“…2,[11][12][13] We used a murine ANCA NCGN model and recently showed that IL-1b provides an important disease mediator and that proteolytically active NSPs are essential for processing pro-IL-1b to IL1b. 14 The in vivo role of phagocyte Nox (Phox) in NCGN is, however, not yet established. Activated Phox consists of a heterodimeric heme protein from gp91 phox (Nox2) and p22 phox complemented by p47 phox , p67 phox , p40 phox , and Rac2.…”

Localization and Functionality of the Inflammasome in Neutrophils

“…Previous studies supported the notion that serine proteases are the key component in generating mature IL-1␤ protein in neutrophils (6,11,12,16). However, this view has been challenged by a recent elegant study demonstrating that murine neutrophils are also capable of generating mature IL-1␤ protein independent from serine proteases (18,19).…”