Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice

Abstract

Antineutrophil cytoplasmic autoantibodies (ANCAs) are identified in the circulation of approximately 80% of patients with pauci-immune necrotizing and crescentic glomerulonephritis and systemic small vessel vasculitis, such as microscopic polyangiitis and Wegener granulomatosis. The most common antigen target for ANCAs is myeloperoxidase (MPO), which is found in neutrophils and monocytes. We report definitive experimental animal evidence that ANCAs are pathogenic. MPO knockout (Mpo(-/-)) mice were immunized with mouse MPO. Splenocytes from these mice or from control mice were injected intravenously into recombinase-activating gene-2-deficient (Rag2(-/-)) mice, which lack functioning B lymphocytes and T lymphocytes. All mice that received splenocytes developed mild to moderate glomerular immune deposits, but only mice that received 1 x 10(8) or 5 x 10(7) anti-MPO splenocytes developed severe necrotizing and crescentic glomerulonephritis, granulomatous inflammation, and systemic necrotizing vasculitis, including necrotizing arteritis and hemorrhagic pulmonary capillaritis. To test the pathogenic potential of antibodies alone, purified anti-MPO IgG or control IgG was injected intravenously into Rag2(-/-) mice and wild-type mice. Mice that received anti-MPO IgG but not mice that received control IgG developed focal necrotizing and crescentic glomerulonephritis with a paucity of glomerular Ig deposition. Thus, anti-MPO IgG alone was able to cause pauci-immune glomerular necrosis and crescent formation in the absence of functional T or B lymphocytes in Rag2(-/-) mice and in the presence of an intact immune system in wild-type C57BL/6J mice. This animal model offers strong support for a direct pathogenic role for ANCA IgG in human glomerulonephritis and vasculitis.

Figure 1

Anti-MPO antibody ELISA titers in Rag2^–/– mice that received 1 × 10⁸ anti-MPO splenocytes (closed diamonds), 5 × 10⁷ anti-MPO splenocytes (filled squares), 1 × 10⁷ anti-MPO splenocytes (filled triangles), 1 × 10⁸ anti-BSA splenocytes (open triangles), or 1 × 10⁸ splenocytes from nonimmunized mice (open circles). Mice that received 5 × 10⁷ or 1 × 10⁷ anti-BSA splenocytes had no values above 15 (data not shown).

Figure 2

Mean BUN and serum creatinine in Rag2^–/– mice 13 days after they received 1 × 10⁸, 5 × 10⁷, or 1 × 10⁷ anti-MPO splenocytes, anti-BSA splenocytes, or nonimmunized control splenocytes. The normal mouse assay reference range was 18–29 mg/dl for BUN and 0.2–0.8 mg/dl for serum creatinine. Samples taken before injection of splenocytes were within the reference ranges (data not shown).

Figure 3

Urinalysis results in Rag2^–/– mice 13 days after they received 1 × 10⁸, 5 × 10⁷, or 1 × 10⁷ anti-MPO splenocytes, anti-BSA splenocytes, or nonimmunized control splenocytes. Samples taken before injection of splenocytes showed mean proteinuria 1.0+, hematuria 0.2+, and leukocyturia 0.0+ (data not shown).

Figure 4

Pathologic findings in Rag2^–/– mice 13 days after they received 1 × 10⁸, 5 × 10⁷, or 1 × 10⁷ anti-MPO splenocytes, anti-BSA splenocytes, or nonimmunized control splenocytes. The extent of glomerular crescent formation is expressed as the mean percent of glomeruli with crescents in each animal. The extent of glomerular necrosis is expressed as the mean percent of glomeruli with necrosis in each animal. The extent of glomerular endocapillary hypercellularity is expressed as the mean on a scale of 0 (none) to 4+ (severe). The extent of glomerular immunostaining for IgG is expressed as the mean on a scale of 0 (none) to 4+ (very intense). Normal Rag2^–/– mice had no crescents, necrosis, endocapillary hypercellularity, or glomerular Ig staining (data not shown).

Figure 5

Glomerular lesions in Rag2^–/– mice 13 days after they received splenocytes. (a) No light microscopic abnormality in a glomerulus from a mouse that received 1 × 10⁷ anti-MPO splenocytes. (b) Moderate (2+) endocapillary hypercellularity in a mouse that received 1 × 10⁸ anti-BSA splenocytes. (c) Segmental fibrinoid necrosis (arrow) in a mouse that received 1 × 10⁸ anti-MPO splenocytes. (d) Cellular crescent (arrow) in a mouse that received 1 × 10⁸ anti-MPO splenocytes. (e) Immunofluorescence staining for fibrin in a crescent in a mouse that received 1 × 10⁸ anti-MPO splenocytes. (f) Predominantly mesangial moderate (2+) immunofluorescence staining for IgG in a mouse that received 1 × 10⁸ anti-BSA splenocytes. Periodic acid Schiff stain for light microscopy is shown.

Figure 6

Systemic vasculitis and granulomatous inflammation in Rag2^–/– mice 13 days after receiving 1 × 10⁸ anti-MPO splenocytes. (a) Necrotizing arteritis in lymph node with transmural fibrinoid necrosis (arrow) (H&E stain). (b) Necrotizing arteritis in spleen with transmural fibrinoid necrosis (arrow) (Masson trichrome stain). (c) Intense acute arteritis in lung with transmural and perivascular infiltration of predominantly neutrophils (Masson trichrome stain). (d) Early focal arteritis in lung with neutrophils and mononuclear leukocytes invading the intima (arrow) (Masson trichrome stain). (e) Pulmonary hemorrhagic capillaritis showing numerous neutrophils marginated within alveolar septal capillaries (arrow) and red blood cells in air spaces. (f) Granulomatous inflammation with multinucleated giant cells in a lymph node.

Figure 7

Glomerular lesions in Rag2^–/– mice 6 days after receiving anti-MPO IgG. (a) Glomerulus with no lesion. (b) Segmental fibrinoid necrosis (arrow). (c) Segmental fibrinoid necrosis with an adjacent small cellular crescent (arrow). (d) Large circumferential cellular crescent (between arrows) completely surrounding a glomerulus. (e) Immunofluorescence microscopy for fibrin showing prominent staining corresponding to segmental necrosis and crescent formation. (f) Immunofluorescence microscopy for IgG showing a paucity of segmental staining corresponding to an area of segmental necrosis. Masson trichrome staining for light microscopy is shown.

Figure 8

Vasculitic lesions in WT B6 mice 6 days after they received anti-MPO IgG. (a) Glomerulus with segmental fibrinoid neurosis (periodic acid Schiff stain). (b) Glomerulus with segmental fibrinoid necrosis and crescent formation (periodic acid Schiff stain). (c) Glomerulus with segmental fibrinoid necrosis and crescent formation (H&E stain). (d) Immunofluorescence microscopy for fibrin showing prominent staining corresponding to segmental necrosis and crescent formation. (e) Necrotizing arteritis with leukocytoclasia in the dermis of the ear (H&E stain). (f) Pulmonary alveolar capillaritis on the left and more normal lung on the right.