The pathogenesis of small-vessel vasculitis is critically associated with antineutrophil cytoplasmic autoantibodies (ANCAs) (1). The two major antigens are myeloperoxidase (MPO) and proteinase 3 (PR3). MPO ANCA is mainly detected in the sera of patients with microscopic polyangiitis (MPA), whereas serum PR3 ANCA is a marker of granulomatosis with polyangiitis (Wegener's). Patients with MPO ANCA–associated vasculitis (MPO AAV), especially those with MPA, frequently have rapidly progressive renal failure caused by crescentic glomerulonephritis and sometimes develop pulmonary hemorrhage due to alveolar capillaritis (2). MPO ANCA can activate neutrophils primed by proinflammatory cytokines, such as tumor necrosis factor α, to release reactive oxygen species and lytic enzymes, and consequently injure small-vessel endothelial cells (3, 4). Transfer of splenocytes from MPO-deficient mice immunized with mouse MPO into wild-type mice results in the development of pauci-immune systemic small-vessel vasculitis (5). The results of these in vitro and in vivo experiments clearly indicate that MPO ANCA plays a pathogenic role in the development of the small-vessel vasculitis subset MPO AAV. However, the mechanism of MPO ANCA production remains unknown.
A unique mode of neutrophil cell death, which is characterized by the release of chromatin fibers and intracytoplasmic proteins, including MPO, PR3, lactoferrin, and bactericidal/permeability increasing protein (BPI), to outside of the cells, has recently been discovered. This type of cell death, caused by neutrophil extracellular traps (NETs), is a defense mechanism used by the host to trap and kill invading microbes, which functions even after the neutrophils are dead (6, 7). Interestingly, NETs have been detected in the lesions of crescentic glomerulonephritis in patients with MPO AAV, and circulating MPO–DNA complexes, which might be derived from the in situ NETs, are elevated in the serum (8). In addition, digestion of NETs has been shown to be poor in a subgroup of patients with systemic lupus erythematosus (SLE) because of the presence of DNase I inhibitors or anti-NET antibodies in the serum (9). These findings suggest that impaired regulation of NETs triggers an autoimmune response against components of NETs and induces autoimmune diseases, including MPO AAV and SLE.
Propylthiouracil (PTU) is an antithyroid drug used for the treatment of hyperthyroidism. Approximately 30% of the patients who receive PTU produce MPO ANCA, and some of them develop MPO AAV (10). The majority of PTU is metabolized in the liver, but a portion is modified by MPO in neutrophils (11). The metabolites of PTU generated by MPO easily bind to diverse proteins and exhibit cytotoxicity; therefore, this is considered to be a possible pathogenesis of agranulocytosis, which is sometimes an adverse effect of PTU (12).
Based on these findings, we hypothesized that PTU can influence the regulation of NETs and induce the production of MPO ANCA. In the present study, we examined whether the addition of PTU influences the formation of NETs induced by phorbol myristate acetate (PMA) and the degradation of NETs by DNase I in vitro. Furthermore, we examined whether the NETs generated by PMA and PTU together induce MPO ANCA and MPO AAV in vivo.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
In the present study, we first demonstrated that the addition of PTU to NET induction resulted in abnormal conformation of NETs in vitro. We also demonstrated that the abnormally constituted NETs were not easily released into the liquid phase. Furthermore, very little of the abnormal NETs were digested by DNase I, which functions as a regulator of NETs in vivo. These findings suggest that NETs induced under the condition of PTU treatment undesirably remain in the tissue. Purified DNA was digested by DNase I even in the presence of PTU, indicating that PTU did not inhibit DNase I activity directly (data not shown). Although further investigations are needed to clarify the precise mechanism, the metabolites of PTU generated by MPO may bind to DNA and interfere with the extension of chromatin fibers. Since DNase I recognizes and cleaves the phosphodiester linkage of DNA, the metabolites of PTU may mask the recognition sites of DNase I. Consequently, the abnormal NETs containing intracellular granules such as MPO exist for a long time outside of the cells.
The immunization of rats with the abnormal NETs resulted in the production of MPO ANCA in vivo. These findings clearly indicate that MPO involved in the abnormal NETs can be a target of the immune system. NETs involve not only MPO, but also other intracytoplasmic proteins, including PR3, lactoferrin, and BPI; however, MPO ANCA is specifically induced by PTU treatment. This may indicate a conformational alteration of MPO. It has been reported that the conformation of heme proteins in MPO was altered when PTU was metabolized by MPO (18). Although the conformational change in MPO induced by PTU should be clarified in future studies, the exposure of modified MPO proteins can induce a breakdown of tolerance to self MPO.
MPO is expressed exclusively in neutrophils and monocytes in the peripheral blood and their precursors in the bone marrow. In addition, formation of NETs is a characteristic of these cells. Although the contribution of neutrophils to the mechanism of MPO ANCA production was demonstrated in the present study, it remains unknown whether monocytes and bone marrow myeloid cells are also involved in the mechanism. This should be elucidated in future studies.
Interestingly, WKY rats immunized with the abnormal NETs (PTU/NET-immunized rats) developed pulmonary hemorrhage with MPO ANCA production, which could be regarded as a phenotype of MPO AAV. These findings suggested the potential pathogenicity of MPO ANCA driven by the modified MPO involved in the abnormal NETs. However, glomerulonephritis, which is a major characteristic of MPO AAV, did not develop in these rats. Therefore, we attempted to establish a more adequate model of MPO AAV through endogenous generation of abnormal NETs.
For this purpose, WKY rats were given oral PTU and injected intraperitoneally with PMA. It has been reported that lipopolysaccharide pretreatment of the recipient mice causes an increase in the disease frequency and severity in MPO ANCA transfer experiments (19). These findings are consistent with the ANCA cytokine sequence theory that activation of neutrophils by cytokines is critical for the development of vasculitis (20).
It has also been reported that intradermal injection of PMA induced acute neutrophil infiltration (17). In this study, intraperitoneal injection of PMA was used to stimulate neutrophils in vivo. Consequently, in addition to MPO ANCA production and pulmonary hemorrhage, pauci-immune glomerulonephritis, which is a hallmark of MPO AAV, developed in PTU/PMA-treated rats. These findings suggested that the systemic activation of neutrophils by intraperitoneal injection of PMA might crucially contribute to the development of glomerulonephritis. However, severe glomerular lesions with crescent formation were not observed, even in PTU/PMA-treated rats. This may be related to the fact that only a small percentage of patients with MPO ANCA caused by PTU administration develop rapidly progressing renal failure. It has been shown that the disease severity of MPO AAV is associated with the epitope specificity and affinity of MPO ANCA (21, 22). It is possible that MPO ANCA induced by a combination of PMA and PTU may be a low-risk autoantibody.
Nevertheless, the important issue demonstrated in this study is that impaired regulation of NETs can trigger an autoimmune response to intracytoplasmic proteins, which are antigens of ANCA. It has been reported that excessive formation of NETs was induced by influenza virus infection (23). Undetermined environmental factors that take the place of PTU, e.g., infectious agents, can induce NET disorder and trigger MPO ANCA production, resulting in the development of MPO AAV.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Ishizu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Tomaru, Ishizu.
Acquisition of data. Nakazawa, Suzuki, Hasegawa, Kobayashi.
Analysis and interpretation of data. Nakazawa, Tomaru, Masuda, Nishio, Kasahara, Ishizu.