Injection of ANCA-No Neutrophils, No Glomerular Damage: The Role of Neutrophils in the Induction of Glomerulonephritis by Anti-Myeloperoxidase Antibodies. Am J Pathol 167: 39-45, 2005.

Abstract

Since the seminal observations of Davies (1) and later of van der Woude (2), it is well known that antineutrophil cytoplasmic antibodies (ANCA) are found in approximately 80% of patients with pauci-immune necrotizing/crescentic glomerulonephritis and with systemic small vessel vasculitis. The most common epitopes recognized by ANCA are myeloperoxidase (MPO) and proteinase 3 (PR3) (3), the former more frequently, but not uniquely, in Wegener’s granulomatosis, the latter mostly in microscopic polyangiitis (4). ANCA stimulate cytokine-primed neutrophils and monocytes, causing for instance respiratory burst, release of toxic granule constituents such as oxygen metabolites and proteinases, as well as endothelial cell injury (5). However, it had long remained controversial whether ANCA are just a marker of disease or have a causal role in the genesis of necrotizing and crescentic glomerulonephritis. The elegant animal model of Xiao et al. (6) had provided definite proof that ANCA were both necessary and sufficient to cause necrotizing and crescentic glomerulonephritis, as well as systemic necrotizing arteritis and hemorrhagic pulmonary capillaritis. For this purpose the authors used MPO knockout mice for which mouse MPO is a “foreign” substance. They immunized these mice with mouse MPO to obtain mouse MPO antibodies. In a second step, mice which were unable to launch an immune response against such mouse MPO antibodies, i.e., knockout mice with a deletion of the recombinase activating gene 2 and therefore devoid of functional B and T cells, received purified mouse anti-MPO IgG or control IgG by intravenous injection. This maneuver reproduced the features of microscopic polyangiitis, i.e., focal necrotizing and crescentic glomerulonephritis with fibrinoid necrosis, crescent formation, and absence or paucity of glomerular IgG deposits. The same outcome was seen when mouse anti-MPO splenocytes were injected. These experiments with injection of mouse anti-MPO IgG or adoptive transfer of mouse anti-MPO splenocytes left no doubt that MPO antibodies alone were able to cause the disease in the absence of B or T cells. What had remained unclear, however, was the cellular target through which MPO antibodies mediated glomerular and vascular injury. To chase down the culprit and to provide evidence that indeed neutrophils—and not for instance endothelial cells, as postulated by others—were the target of the MPO antibodies and the key effector cells, the authors now recently carried out an additional experiment. They used the model of the neutropenic mouse. After injection of NIMP-R14, a rat monoclonal antibody, the mice were selectively depleted of circulating neutrophils, the number decreasing from 14% to 1%. Neutropenic mice or control mice received a low (50 μg/g body weight) or high (50 μg/g body weight on day 0 and day 3) dose of anti-MPO IgG by intravenous injection. Control mice received an injection of bovine serum albumin. Five days after injection of anti-MPO IgG, mice without neutrophil depletion developed hematuria, proteinuria, and leucocyturia. Renal histology documented focal glomerular necrosis and glomerular crescents. Glomerular neutrophil phenotyping revealed glomerular infiltration by neutrophils in foci of inflammation and necrosis as well as in a few afferent arterioles, while monocytes/macrophages were mainly observed in glomerular crescents. With the higher dose, blood urea nitrogen was increased as well, but interestingly necroses were seen on average only in 17.8 ± 7.8% of glomeruli. All these lesions were attenuated or even absent in neutropenic mice receiving the anti-MPO IgG. The observation of no glomerular injury in the absence of neutrophils points to the neutrophil as the key effector cell in the induction of the acute glomerular injury of MPO-induced glomerulonephritis, although an ancillary role of monocytes is not completely excluded. The smoking gun points to the neutrophil, and this fits in nicely with past observations documenting the ability of human MPO ANCA and PR3 ANCA to activate and degranulate neutrophils causing the release of toxic oxygen metabolites (7), proteases, nitric oxide (8) and inflammatory cytokines (9), facilitating attachment and killing of endothelial cells (10,11). Why is the observation of Xiao et al. important? It points to maneuvers interfering with ANCA-induced neutrophil recruitment and activation as potential therapeutic targets, if this can be achieved safely and without inducing harm.