The role of neutrophils in the induction of glomerulonephritis by anti-myeloperoxidase antibodies

Abstract

In humans, circulating anti-neutrophil cytoplasm autoantibodies (ANCAs) with specificity for myeloperoxidase (MPO) are strongly associated with the development of pauci-immune necrotizing and crescentic glomerulonephritis (NCGN). In mice, we have demonstrated that intravenous injection of mouse antibodies specific for mouse MPO induces NCGN that closely mimics the human disease. We now report that the development of NCGN in this experimental model is accompanied by glomerular accumulation of neutrophils and macrophages. Neutrophil infiltration was most conspicuous at sites of glomerular necrosis and crescent formation, with macrophages also most numerous in crescents. Lymphocytes, however, were sparse in acute lesions. Importantly, mice that were depleted of circulating neutrophils with NIMP-R14 rat monoclonal antibodies were completely protected from anti-MPO IgG-induced NCGN. These findings provide direct evidence that neutrophils play a major role in the pathogenesis of anti-MPO-induced NCGN in this animal model and implicate neutrophils in the induction of human ANCA disease. This raises the possibility that therapeutic strategies to reduce circulating neutrophils could be beneficial to patients with ANCA-induced NCGN.

Figure 1

Neutrophil depletion by NIMP-R14. B6 mice were injected either with 1 mg of NIMP-R14 rat anti-murine neutrophil monoclonal antibody (n = 7) (open circles) or control rat IgG (n = 6) (filled diamonds). Circulating neutrophils were quantified at different time points by cell counting of blood smears stained with Diff-Quik. Data are expressed as percentage of neutrophils in the blood. A single dose of the NIMP-R14 caused severe neutropenia in mice for more than 5 days.

Figure 2

Neutrophil depletion prevents anti-MPO antibody-induced necrotizing and crescentic glomerulonephritis. B6 mice were pretreated with the neutrophil-depleting antibody NIMP-R14 (n = 6) or control rat IgG (n = 6). Sixteen hours later, the mice received the pathogenic anti-MPO IgG (50 μg/g body weight). Mice were sacrificed for pathological examination 6 days after receiving anti-MPO IgG. All mice that received neutrophil-depleting antibodies and anti-MPO IgG had normal glomeruli by light microscopy (left). In contrast, all mice that received control rat IgG and anti-MPO IgG developed segmental fibrinoid necrosis (long arrow, middle, and arrow, right) and cellular crescents (short arrow, middle). Periodic acid-Schiff stain.

Figure 3

Glomerular leukocytes 6 days after injection of a single dose of anti-MPO IgG. The panels show infiltration of an inflamed glomerulus by neutrophils that are scattered throughout the tuft (top left marked with anti-Gr-1), macrophages that are concentrated in the crescent (top right marked with anti-CD68 and bottom right marked with anti-F4/80), and a few lymphocytes (bottom left).

Figure 4

Glomerular neutrophil accumulation in B6 mice that received two doses of anti-MPO IgG on day 0 and day 3 followed by sacrifice on day 6. Mice that received anti-BSA IgG had only rare neutrophils in a few glomeruli (not shown). Immunoenzyme microscopy using an antibody specific for mouse neutrophils demonstrated that mice that received anti-MPO IgG had a marked increase in glomerular neutrophils especially at sites of necrosis or crescent formation. a: The glomerulus has segmental fibrinoid necrosis (arrow) with brown-staining neutrophils most numerous at the margins of the necrosis. b: The glomerular tuft on the left has scattered clusters of neutrophils and the hilar arteriole on the right (arrow) contains aggregated neutrophils. c: At the plane of section examined, the glomerular tuft has a few scattered neutrophils and Bowman’s space contains numerous neutrophils (arrow). d: The glomerulus has extensive fibrinoid necrosis (between arrows) with some residual neutrophils at the periphery in a crescent. Within the necrosis, there are scattered brown granules that probably are neutrophil fragments.