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Anti-neutrophil cytoplasmic autoantibodies (ANCAs) are directed against lysosomal components of neutrophils. ANCAs directed to proteinase 3 and myeloperoxidase (MPO) in particular are associated with distinct forms of small vessel vasculitides. MPO is an abundant neutrophil-derived heme protein that is part of the antimicrobial defense system. The protein is typically present in the azurophilic granules of neutrophils, but a large portion may also enter the extracellular space. It remains unclear why MPO is frequently the target of antibody-mediated autoimmune responses. MPO is a homodimeric glycoprotein, posttranslationally modified with complex sugars at specific sites. Glycosylation can strongly influence protein function, affecting its folding, receptor interaction, and backbone accessibility. MPO potentially can be heavily modified as it harbors 5 putative N -glycosylation sites (10 in the mature dimer). Although considered important for MPO structure and function, the full scope and relative abundance of the glycans attached to MPO is unknown. Here, combining bottom-up glycoproteomics and native MS approaches, we structurally characterized MPO from neutrophils of healthy human donors. We quantified the relative occupancy levels of the glycans at each of the five sites and observed complex heterogeneity and site-specific glycosylation. In particular, we detected glycosylation phenotypes uncommon for glycoproteins in the extracellular space, such as a high abundance of phosphorylated high-mannose species and severely truncated small glycans having the size of paucimannose or smaller. We hypothesize that the atypical glycosylation pattern found on MPO might contribute to its specific processing and presentation as a self-antigen by antigen-presenting cells., (© 2019 Reiding et al.)

Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO 409-428 ), can induce anti-MPO autoimmunity. The peptide (6PGD 391-410 ) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD 391-410 , or with S. aureus containing a plasmid expressing 6PGD 391-410 , develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD 391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD 391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.

Current therapies for treating antineutrophil cytoplasm autoantibody (ANCA)-associated vasculitis include cyclophosphamide and corticosteroids. Unfortunately, these agents are associated with severe adverse effects, despite inducing remission in most patients. Histone deacetylase inhibitors are effective in rodent models of inflammation and act synergistically with many pharmacological agents, including alkylating agents like cyclophosphamide. EDO-S101 is an alkylating fusion histone deacetylase inhibitor molecule combining the DNA alkylating effect of Bendamustine with a pan-histone deacetylase inhibitor, Vorinostat. Here we studied the effects of EDO-S101 in two established rodent models of ANCA-associated vasculitis: a passive mouse model of anti-myeloperoxidase IgG-induced glomerulonephritis and an active rat model of myeloperoxidase-ANCA microscopic polyangiitis. Although pretreatment with EDO-S101 reduced circulating leukocytes, it did not prevent the development of passive IgG-induced glomerulonephritis in mice. On the other hand, treatment in rats significantly reduced glomerulonephritis and lung hemorrhage. EDO-S101 also significantly depleted rat B and T cells, and induced DNA damage and apoptosis in proliferating human B cells, suggesting a selective effect on the adaptive immune response. Thus, EDO-S101 may have a role in treatment of ANCA-associated vasculitis, operating primarily through its effects on the adaptive immune response to the autoantigen myeloperoxidase., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) is a typical disease of the elderly. In AAV, there is an age-specific increase in disease incidence with age being a predictor of disease outcome. In this study, we aimed to determine the contribution of age to the development of AAV employing a mouse model of anti-myeloperoxidase (MPO) antibody-mediated glomerulonephritis., Methods: Anti-MPO IgG and lipopolysaccharide (LPS)-mediated glomerulonephritis was induced in 3- and 18-month-old C57Bl6 mice. Clinical and pathological parameters of disease severity, alterations in the immune system and kidney specific changes in these mice were evaluated., Results: Eighteen-month-old mice developed increased disease severity upon injection of anti-MPO IgG/LPS compared with 3-month-old mice. This was evidenced by increased albuminuria, more extensive glomerular capillary necrosis and increased glomerular neutrophil accumulation. Glomerular crescent formation was mild in both young and old mice. Old mice displayed higher plasma interleukin-6 levels as well as higher proportions of circulating neutrophils and activated monocytes compared with young mice. In addition, renal mRNA levels of inflammatory genes and endothelial adhesion molecules were higher in 18-month-old mice compared with 3-month-old mice., Conclusion: In conclusion, our results indicate that aged mice develop more severe clinical and pathological disease upon induction of anti-MPO IgG/LPS-mediated glomerulonephritis. These findings may be attributed to age-related changes in the immune system as well as in the kidney itself.

ANCA vasculitis encompasses several autoimmune conditions characterised by destruction of small vessels, inflammation of the respiratory tract and glomerulonephritis. Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3). Clinical and experimental data suggest that pathogenesis is driven by ANCA-mediated activation of neutrophils and monocytes. We investigated a potential role for distinct monocyte subsets. We found that the relative proportion of intermediate monocytes is increased in patients versus control individuals, and both MPO and PR3 are preferentially expressed on these cells. We demonstrate that MPO and PR3 are expressed independently of each other on monocytes and that PR3 is not associated with CD177. MPO expression correlates with that of Fc receptor CD16 on intermediate monocytes. Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1β, IL-6 and IL-8 production. In concordance with the observed higher surface expression of MPO on intermediate monocytes, this subset produces the highest quantity of IL-1β in response to anti-MPO stimulation. These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) comprise granulomatosis with polyangiitis (GPA), primarily associated with antibodies to proteinase 3 (PR3-ANCA); microscopic polyangiitis (MPA); and eosinophilic granulomatosis with polyangiitis (EGPA), both principally associated with antibodies to myeloperoxidase (MPO-ANCA). Genetic and environmental factors are involved in their etiopathogenesis, with a possible role for silica exposure in AAVs and Staphylococcus aureus infection in GPA. The distinct associations of PR3-ANCA and MPO-ANCA with different HLA class II antigens, which are stronger than those with the associated diseases, suggest a pathogenic role for those ANCAs and indicate that GPA and MPA are different diseases. Both in vitro and in vivo experimental data have shown that MPO-ANCA can induce necrotizing small-vessel vasculitis and glomerulonephritis. The additional role of the alternative pathway of complement activation has been demonstrated in human and experimental pathology. Also, T cells seem to be involved in lesion development, particularly in the kidney. Granuloma formation, as seen in PR3-ANCA-associated GPA, is not well explained by the presence of autoantibodies in experimental models. Here, T cells seem crucial. Recently obtained insights into the pathogenesis of AAVs have led to more targeted treatment of these life-threatening diseases., (Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocyte's surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPO's affinity to both the endothelial and the leukocyte's surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.

Antimyeloperoxidase antibodies can cause crescentic glomerulonephritis and pulmonary hemorrhage. Toll-like receptors (TLRs) respond to infectious agents activating host defenses, whereas infections potentially initiate disease and provoke relapses. Neutrophils were found to be key effector cells of injury in experimental models, as disease does not occur in their absence and injury is enhanced by lipopolysaccharide (LPS). In this study, highly purified LPS (a pure TLR4 ligand) acted with antimyeloperoxidase antibodies to synergistically increase kidney and lung neutrophil recruitment and functional injury; effects abrogated in TLR4-deficient mice. Increased kidney TLR4 expression after stimulation predominantly occurred in glomerular endothelial cells. Enhanced glomerular neutrophil recruitment correlated with increased kidney mRNA expression of CXCL1 and CXCL2 (homologs of human CXCL8), whereas their preemptive neutralization decreased neutrophil recruitment. Disease induction in bone marrow chimeric mice showed that TLR4 in both bone marrow and renal parenchymal cells is required for maximal neutrophil recruitment and glomerular injury. Further studies in human glomerular cell lines stimulated with LPS found that glomerular endothelial cells were the prominent sources of CXCL8. Thus, our results define a role for TLR4 expression in bone marrow-derived and glomerular endothelial cells in neutrophil recruitment and subsequent functional and histological renal injury in experimental antimyeloperoxidase glomerulonephritis.

Atherosclerosis is linked to inflammation. HDL protects against atherosclerotic cardiovascular disease, mainly by mediating cholesterol efflux and reverse cholesterol transport (RCT). The present study aimed to test the impact of acute inflammation as well as selected acute phase proteins on RCT with a macrophage-to-feces in vivo RCT assay using intraperitoneal administration of [(3)H]cholesterol-labeled macrophage foam cells. In patients with acute sepsis, cholesterol efflux toward plasma and HDL were significantly decreased (P < 0.001). In mice, acute inflammation (75 microg/mouse lipopolysaccharide) decreased [(3)H]cholesterol appearance in plasma (P < 0.05) and tracer excretion into feces both within bile acids (-84%) and neutral sterols (-79%, each P < 0.001). In the absence of systemic inflammation, overexpression of serum amyloid A (SAA, adenovirus) reduced overall RCT (P < 0.05), whereas secretory phospholipase A(2) (sPLA(2), transgenic mice) had no effect. Myeloperoxidase injection reduced tracer appearance in plasma (P < 0.05) as well as RCT (-36%, P < 0.05). Hepatic expression of bile acid synthesis genes (P < 0.01) and transporters mediating biliary sterol excretion (P < 0.01) was decreased by inflammation. In conclusion, our data demonstrate that acute inflammation impairs cholesterol efflux in patients and macrophage-to-feces RCT in vivo in mice. Myeloperoxidase and SAA contribute to a certain extent to reduced RCT during inflammation but not sPLA(2). However, reduced bile acid formation and decreased biliary sterol excretion might represent major contributing factors to decreased RCT in inflammation.

Myeloperoxidase (MPO) is a heme-containing peroxidase abundantly expressed in neutrophils and to a lesser extent in monocytes. Enzymatically active MPO, together with hydrogen peroxide and chloride, produces the powerful oxidant hypochlorous acid and is a key contributor to the oxygen-dependent microbicidal activity of phagocytes. In addition, excessive generation of MPO-derived oxidants has been linked to tissue damage in many diseases, especially those characterized by acute or chronic inflammation. It has become increasingly clear that MPO exerts effects that are beyond its oxidative properties. These properties of MPO are, in many cases, independent of its catalytic activity and affect various processes involved in cell signaling and cell-cell interactions and are, as such, capable of modulating inflammatory responses. Given these diverse effects, an increased interest has emerged in the role of MPO and its downstream products in a wide range of inflammatory diseases. In this article, our knowledge pertaining to the biologic role of MPO and its downstream effects and mechanisms of action in health and disease is reviewed and discussed.

Lung neutrophilia is common to a variety of lung diseases. The production of reactive oxygen and nitrogen species during neutrophil oxidative burst has been associated with protein and DNA damage. Myeloperoxidase (MPO) is an enzyme stored in the azurophilic granula of neutrophils. It is important in host defense because it generates the reactive oxidant hypochlorous acid and has been described to play a role in the activation of neutrophils during extravasation. We hypothesized that MPO contributes directly to the development of acute lung neutrophilia via stimulation of neutrophil extravasation and indirectly to the subsequent production of cytokines and chemokines in the lung. To test this hypothesis, wild-type (WT) and Mpo(-/-) mice were given a single LPS instillation, after which the development of neutrophil-dominated lung inflammation, oxidative stress, and cytokine and chemokine levels were examined. Mpo(-/-) mice demonstrated a decreased lung neutrophilia that peaked earlier than neutrophilia in WT mice, which can be explained by decreased neutrophil chemoattractant levels in LPS-exposed Mpo(-/-) compared with WT mice. However, oxidative stress levels were not different in LPS-exposed WT and Mpo(-/-) mice. Furthermore, in vivo findings were confirmed by in vitro studies, using isolated neutrophils. These results indicate that MPO promotes the development of lung neutrophilia and indirectly influences subsequent chemokine and cytokine production by other cell types in the lung.

During extensive inflammation, neutrophils undergo secondary necrosis causing myeloperoxidase (MPO) release that may damage resident lung cells. Recent observations suggest that MPO has pro-inflammatory properties, independent of its enzymatic activity. The aims of the present study were to characterise MPO internalisation by lung epithelial cells and to investigate the effect of MPO on oxidative stress, DNA damage and cytokine production by lung epithelial cells. Human alveolar and bronchial epithelial cells were stimulated with MPO, with or without priming the cells with pro-inflammatory stimuli. MPO protein was detected in the cell cytoplasm. Expression of haemoxygenase (HO)-1 and DNA strand breakage were determined. The production of interleukin (IL)-8 and -6 were measured. Analyses of MPO-stimulated cells demonstrated MPO presence in the cells. HO-1 expression was increased after MPO stimulation and increased further when cells were primed before MPO stimulation. MPO exposure also induced DNA strand breakage. Interestingly, MPO inhibited IL-8 production in bronchial, but not alveolar epithelium. In conclusion, alveolar and bronchial epithelial cells can internalise myeloperoxidase. Stimulation with myeloperoxidase increases haemoxygenase-1 expression and DNA strand breakage, suggesting cell damaging capacity of myeloperoxidase. In addition, myeloperoxidase inhibited interleukin-8 production by bronchial epithelial cells, indicating a negative feedback loop for neutrophil recruitment.

Objective: Atherosclerosis is a chronic inflammatory disease in which the immune system plays an important role. Neutrophils have not been thoroughly studied in the context of atherogenesis. Here, we investigated neutrophils in the development of murine atherosclerotic lesions., Methods and Results: LDLR-/- mice were given a high-fat diet for different time periods and subsequently atherosclerotic lesions were studied by immunohistochemistry. Staining with anti-Ly-6G monoclonal antibody, a specific marker for neutrophils, revealed a marked accumulation of neutrophils during atherosclerosis development. Neutrophils were observed in the lesion, attached to the cap, and in the arterial adventitia. In addition, at some sites, neutrophil accumulation colocalized with endothelial E-selectin expression. Immunofluorescence double staining with anti-myeloperoxidase and anti-Ly-6G antibodies demonstrated the presence of myeloperoxidase in atherosclerotic lesions and its colocalization with neutrophils. After introducing the high-fat diet, levels of circulating myeloperoxidase in plasma strongly increased, with a peak at 6 weeks and a subsequent decrease to almost normal levels after 16 weeks of diet., Conclusions: We here demonstrate for the first time the presence of neutrophils and myeloperoxidase in murine atherosclerotic lesions. As a major cell type in inflammatory responses the neutrophil may also be an important mediator in the development of atherosclerosis.

In this study the role of myeloperoxidase (MPO) in a murine (C57BL/6) model of ischemia and reperfusion (I/R)-induced renal failure was investigated. The renal function after I/R was analyzed in MPO-deficient (Mpo(-/-)) mice and compared with wild-type (WT) controls. A significant reduction in renal function loss (blood urea nitrogen) was observed after 24 hours of reperfusion of ischemically damaged kidneys in Mpo(-/-) mice compared with I/R WT controls (I/R Mpo(-/-) = 31.3 +/- 1.7 mmol/L versus I/R WT = 42.8 +/- 2.1 mmol/L, sham = 7.0 +/- 0.5 mmol/L; P = 0.003). The early reperfusion phase (2 hours of reperfusion) was characterized by a substantial increase in apoptosis and early complement activation, surprisingly similar in Mpo(-/-) and WT mice. Improved renal function in Mpo(-/-) mice after extended reperfusion was accompanied by a reduced neutrophil influx (P = 0.017) compared with WT controls. Activation and deposition of complement was not significantly reduced in Mpo(-/-) mice compared with WT controls after 24 hours of reperfusion, indicating no specific in vivo role for MPO in activating complement after renal I/R. Taken together, these results demonstrated an important contribution of MPO in the induction of organ damage after renal I/R by influencing critical factors such as neutrophil extravasation but not complement activation.

In mice, administration of murine anti-myeloperoxidase (MPO) IgG induces pauci-immune necrotizing crescentic glomerulonephritis. Recent studies in this model indicate a crucial role for complement activation in disease induction. Here, we investigated the effect of pretreatment or intervention with a C5-inhibiting monoclonal antibody (BB5.1) in the mouse model of anti-MPO IgG-induced glomerulonephritis. Mice received BB5.1 8 h before or 1 day after disease induction with anti-MPO IgG and lipopolysaccharide. Mice were killed after 1 or 7 days. Control antibody-pretreated mice developed hematuria, leukocyturia and albuminuria, and glomerulonephritis with a mean of 21.0+/-8.8% glomerular crescents and 12.8+/-5.5% glomerular capillary necrosis. BB5.1 pretreatment prevented disease development, as evidenced by the absence of urinary abnormalities, a marked reduction in glomerular neutrophil influx at day 1 and normal renal morphology at day 7. Importantly, BB5.1 administration 1 day after disease induction also resulted in a marked attenuation of urinary abnormalities and a more than 80% reduction in glomerular crescent formation. In conclusion, inhibition of C5 activation attenuates disease development in a mouse model of anti-MPO IgG-induced glomerulonephritis. These results favor further investigations into the role of complement activation in human MPO-anti-neutrophil cytoplasmic autoantibody-mediated glomerulonephritis, and indicate that inhibition of C5 activation is a potential therapeutic approach in this disease.

Clinical and experimental data indicate that anti-neutrophil cytoplasmic autoantibodies (ANCAs) cause glomerulonephritis and vasculitis. Here we report the first evidence that complement is an important mediator of ANCA disease. Transfer of anti-myeloperoxidase (MPO) IgG into wild-type mice or anti-MPO splenocytes into immune-deficient mice caused crescentic glomerulonephritis that could be completely blocked by complement depletion. The role of specific complement activation pathways was investigated using mice with knockout of the common pathway component C5, classic and lectin binding pathway component C4, and alternative pathway component factor B. After injection of anti-MPO IgG, C4-/- mice developed disease comparable with wild-type disease; however, C5-/- and factor B-/- mice developed no disease. To substantiate a role for complement in human ANCA disease, IgG was isolated from patients with myeloperoxidase ANCA (MPO-ANCA) or proteinase 3 ANCA (PR3-ANCA) and from controls. Incubation of MPO-ANCA or PR3-ANCA IgG with human neutrophils caused release of factors that activated complement. IgG from healthy controls did not produce this effect. The findings suggest that stimulation of neutrophils by ANCA causes release of factors that activate complement via the alternative pathway, thus initiating an inflammatory amplification loop that mediates the severe necrotizing inflammation of ANCA disease.

Background: In a substantial proportion of patients with crescentic glomerulonephritis (CGN), both anti-glomerular basement membrane (GBM) antibodies and antineutrophil cytoplasmic antibodies (ANCAs) with specificity for myeloperoxidase (MPO-ANCA) are detected. In the present study, we questioned whether histological and clinical features of patients with both ANCA and anti-GBM antibodies differ from those of patients with either ANCA or anti-GBM alone., Methods: We reviewed the Limburg renal biopsy registry (1978 to 2003; n = 1,373) for cases of CGN. The presence of linear fluorescence on renal biopsy and the presence of ANCA and/or anti-GBM antibodies were measured. Subsequently, we assessed patient characteristics and follow-up and compared histological findings among the different groups., Results: We identified 46 MPO-ANCA-positive, 10 double-positive, and 13 anti-GBM-positive patients. Mean ages were 63, 64, and 52 years (P = 0.04), and serum creatinine levels were 5.0, 10.3, and 9.6 mg/dL (445, 910, and 850 micromol/L), respectively (P = 0.01). Granulomatous periglomerular inflammation was found in either MPO-ANCA- or double-positive patients, but not in anti-GBM-positive patients with CGN without MPO-ANCAs. Patient survival among the 3 groups was different, although not statistically significant (log rank P = 0.17, with 75%, 79%, and 100% alive at 1 year, respectively). Renal survival analysis showed significant differences among the 3 groups (P = 0.04, with 65%, 10%, and 15% off dialysis therapy at 1 year, respectively)., Conclusion: In patients with both anti-GBM antibodies and MPO-ANCAs, histological findings differ from those of patients with anti-GBM antibodies only. However, renal survival in these patients is not better than that in anti-GBM-positive patients and is worse compared with patients with MPO-ANCAs only.

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.

Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and idiopathic pauci-immune necrotizing crescentic glomerulonephritis are associated with myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibodies (ANCAs). Clinical and experimental evidence indicates that ANCA and proinflammatory stimuli of infectious origin act synergistically to cause vasculitis. We tested this hypothesis in a recently developed mouse model of anti-MPO IgG-induced glomerulonephritis by using bacterial lipopolysaccharide (LPS) as the proinflammatory stimulus. Systemic administration of LPS dose dependently increased renal injury induced by anti-MPO IgG as demonstrated by increased glomerular crescent formation and glomerular necrosis. In the early phase, LPS enhanced anti-MPO IgG-induced glomerular neutrophil accumulation. Furthermore, a transient induction of circulating tumor necrosis factor (TNF)-alpha levels, followed by a marked increase in circulating MPO levels, was observed on administration of LPS. In vitro, anti-MPO IgG induced a respiratory burst in murine neutrophils only after priming with TNF-alpha. Finally, anti-TNF-alpha treatment attenuated, but did not prevent, the LPS-mediated aggravation of anti-MPO IgG-induced glomerulonephritis. In conclusion, our study demonstrates that ANCA and proinflammatory stimuli act synergistically to induce vasculitic disease and suggests potential benefits of inhibiting TNF-alpha bioactivity in treating human ANCA-associated necrotizing crescentic glomerulonephritis.

Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome and idiopathic pauci-immune necrotizing crescentic glomerulonephritis are strongly associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). These ANCA-associated vasculitides can serologically be separated into myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA positive patients. The unique properties of the antigen targeted by the anti-MPO antibodies could help to explain the specific characteristics of MPO-ANCA associated disease. Recently, an animal model has been developed that proves that anti-mouse MPO immunoglobulins alone are capable of causing disease similar to that in humans. Also, the in vitro pathologic effects of binding of MPO-ANCA to MPO are better understood. MPO-ANCA can activate (primed) neutrophils directly causing extensive reactive oxygen species formation and degranulation of neutrophil constituents, including MPO, resulting in a destructive inflammatory response towards the vessel wall. MPO-ANCA can prevent the clearing and inactivation of MPO by ceruloplasmin as well, resulting in increased myeloperoxidase activity. Myeloperoxidase produces not only the strong oxidant bleach (hypochlorous acid) out of hydrogen peroxide and chloride ions but also oxidizes LDL into a macrophage high-uptake form, inactivates protease inhibitors, and consumes nitric oxide. These may contribute to endothelial dysfunction and add to the chronic renal lesions observed in patients with MPO-ANCA. MPO levels are influenced by genetic factors including two, MPO463 and MPO129, single nucleotide polymorphisms. The MPO 463 polymorphism has been associated with an increased risk of development of MPO-ANCA associated disease.

Myeloperoxidase (MPO) catalyses the formation of hypochlorous acid and is involved in many (patho)physiological processes. The present study was designed to determine the effect of two MPO promoter polymorphisms (463G/A and 129G/A) on enzyme activity. In 243 healthy controls, genotypes were determined and MPO activity was measured on a single-cell level using a haematological analyser. The 129G/A polymorphism reduces MPO activity in neutrophils, whereas for the 463G/A polymorphism, only gender-dependent differences in MPO activity in older age groups could be found. When studying these polymorphisms in disease, therefore, age and gender should be included in the analysis.

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.

The important issue addressed by the studies presented here is the mechanism of neutrophil-mediated damage to endothelial and epithelial cells during inflammation. Binding of neutrophil-released granule proteins to endothelial cells may be involved in vascular damage in patients with inflammatory vascular diseases. We have determined whether granule proteins proteinase 3(PR3) and/or myeloperoxidase (MPO) are internalized into endothelial cells, as examined by UV light, confocal, and electron microscopy. Coincident induction of apoptosis and/or the generation of intracellular oxidants were monitored. The results indicate that human endothelial cells (human umbilical vein endothelial cells, human umbilical arterial endothelial cells, human lung microvascular endothelial cells) internalize both PR3 and MPO, which are detected on the cell surface, in the cytoplasm, and possibly nuclear. Epithelial cells (small airway epithelial cells) internalized MPO but not PR3, implying that the mechanism of PR3 internalization may be cell-type specific and different from that of MPO. Internalization of PR3, but not MPO, correlated with activation of apoptosis. Internalization of MPO correlated with an increase in intracellular oxidant radicals. The requirement for the proteolytic activity of PR3 for the induction of apoptosis was examined by generating PR3-truncated fragments that did not contain the components of the catalytic triad. An apoptotic function was localized to the C-terminal portion of PR3. These studies reveal novel mechanisms by which the neutrophil granule proteins PR3 and MPO contribute to tissue injury at sites of inflammation.

The lung is a common target in systemic vasculitides associated with antineutrophil cytoplasmic antibodies (ANCA). In the present study, we tested the hypothesis that the presence of antibodies directed against myeloperoxidase (MPO) induces pulmonary (vasculitic) lesions when neutrophils release lysosomal enzymes. Brown Norway (BN) rats were immunized with human MPO in complete Freund's adjuvant (CFA) or with CFA alone. Two weeks after immunization, rats had developed antibodies to human and rat MPO. Next, isolated single left lung perfusion was performed with human neutrophil lysosomal extract containing MPO and proteolytic enzymes. Rats were killed at 15 min, 4 h, and 10 d after perfusion. Tissue samples from the left and right lung were examined for vasculitic lesions and inflammatory cell infiltrates. At 15 min and 4 h, left lungs from control and MPO-immunized rats showed a mild influx of polymorphonuclear cells. At 10 d, patchy inflammatory cell infiltrates, consisting predominantly of polymorphonuclear leukocytes (PMNs) and monocytes, were observed throughout the parenchyma of the left lung in MPO-immunized rats. Occasionally, granuloma-like lesions, giant cells, and foci of alveolar hemorrhage were observed as well. Far less severe lesions were seen in control immunized rats. Strikingly, at 10 d after perfusion, severe pulmonary tissue injury was observed also in right lungs from MPO-immunized rats whereas right lungs from control immunized rats appeared normal. The lesions were characterized by influx of PMNs and monocytes and, in some rats, foci of alveolar hemorrhage. These studies suggest that the presence of an anti-MPO directed autoimmune response contributes to generalized pulmonary tissue injury after local release of products of activated neutrophils, which supports a pathogenic role of MPO-ANCA.

Nitric oxide radicals are recognized as important mediators in various physiological and pathophysiological processes. During inflammation, increased amounts of nitric oxide (NO) are produced, but it is unclear whether NO radicals are either protective or harmful. To obtain more insight into the role of NO in glomerular inflammation, we studied the temporal expression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) in conjunction with platelet aggregation, inflammatory cell influx, superoxide anion production cells, and nitrotyrosine formation in an experimental model of anti-myeloperoxidase (MPO) associated necrotizing crescentic glomerulonephritis (NCGN). Brown Norway rats were immunized with MPO in complete Freund's adjuvant (CFA) or CFA alone. After two weeks, the left kidney was perfused with a neutrophil lysosomal extract and H2O2. Rats were sacrificed at 24 hours, four days, and 10 days after perfusion. Kidney sections were stained by immunohistochemistry for eNOS, iNOS, platelets, nitrotyrosines, polymorphonuclear cells (PMN), monocytes, and T-cells. Superoxide anion producing cells were identified by enzyme cytochemistry using diaminobenzidine. Strong staining for eNOS was found in glomerular capillaries and interstitial tubular capillaries and larger vessels from non-perfused kidneys. At 24 hours after perfusion, glomerular and interstitial eNOS staining was greatly reduced, which was associated with massive platelet aggregation. At later time points, eNOS expression was absent in severely damaged glomeruli. Inducible NOS expression was found at all time points in infiltrating inflammatory cells, which by double labeling studies were identified as PMNs and monocytes. The peak in iNOS expression was observed at four days after perfusion but declined thereafter. Superoxide anion and nitrotyrosine generating cells were also found at all time points, but were most abundantly present at four days after perfusion, coinciding with the peak in iNOS expression. Double labeling experiments revealed that most nitrotyrosine generating cells also produced superoxide anions and expressed iNOS. In conclusion, these studies suggest that during the course of anti-MPO associated NCGN, loss of NO production by eNOS in conjunction with NO radical production by iNOS contribute to tissue injury. This is compatible with a protective role for eNOS contrasting with the possibly harmful effects of iNOS in anti-MPO associated NCGN.

The strong association of anti-neutrophil cytoplasmic antibodies with various forms of systemic vasculitis suggests a role for these autoantibodies in the pathophysiology of systemic vasculitis. In the present study, we tested the hypothesis that release of neutrophil lysosomal enzymes in the presence of an anti-myeloperoxidase (anti-MPO) immune response may underlie the development of systemic vasculitis. Brown Norway rats were immunized with MPO in complete Freund's adjuvant or complete Freund's adjuvant alone. Two weeks after immunization, rats bad developed antibodies to human and rat MPO as measured by enzyme-linked immunosorbent assay. Next, rats were intravenously infused with 400 micrograms of a human neutrophil lysosomal extract containing 200 micrograms of MPO followed by 0.5 ml of a 1 mmol/L solution of H2O2 through a cannula inserted into the right jugular vein. Rats were sacrificed at 4 hours, 24 hours, 7 days, or 14 days, and several organs (lungs, heart, liver, spleen, gut, and kidneys) were examined for vasculitic lesions and inflammatory cell infiltrates. Macroscopically, patchy hemorrhagic spots were observed in the lungs and gut of MPO-immunized rats at days 7 and 14 after systemic infection of the neutrophil lysosomal extract and H2O2. Such changes were not observed at earlier time points or in control immunized rats. Histologically, the lungs of MPO-immunized rats sacrificed at days 7 and 14 showed patchy inflammatory cell infiltrates associated with vasculitis, granuloma formation, giant cells, and foci of hemorrhage. At 14 days, early signs of fibrosis were found with deposition of collagen and proliferation of fibroblasts. Furthermore, a prominent leukocytoclastic vasculitis was found in the small intestine of these rats characterized by fibrinoid necrosis and an extensive neutrophilic infiltrate. No inflammatory changes were found in the other organs studied (heart, liver, spleen, and kidneys). Control immunized rats, sacrificed at days 7 and 14 showed only some small foci of inflammatory infiltrates in the lungs whereas no inflammatory changes were found in the gastrointestinal tract. These studies show that release of products from activated neutrophils in the presence of anti-MPO autoantibodies may be relevant to the pathogenesis of anti-MPO-associated vasculitides.

Autoantibodies to myeloperoxidase (MPO) are present in sera from patients with various forms of vasculitis-associated glomerulonephritis. Evidence for a pathogenic role of anti-MPO antibodies has been provided mainly by in vitro studies. We studied the pathogenic role of autoantibodies to MPO in a rat model of mild immune-mediated glomerular injury. Brown Norway rats were immunized with human MPO in complete Freund's adjuvant or with complete Freund's adjuvant alone. At 2 weeks after immunization, rats had developed antibodies to human and rat MPO as detected by indirect immunofluorescence, enzyme-linked immunosorbent assay, and immunoprecipitation. At this time point, rats were intravenously injected with a subnephritogenic dose of 150 micrograms of rabbit anti-rat GBM. Rats were sacrificed at 4 hours, 24 hours, 4 days, and 10 days after antibody administration. Control immunized rats developed mild glomerulonephritis characterized by slight proteinuria at day 10 (14.8 +/- 8.1 mg/24 hours) and moderate intraglomerular accumulation of ED1+ macrophages. Crescent formation, tuft necrosis, and tubular atrophy were not observed in those rats. In contrast, rats immunized with MPO developed severe glomerulonephritis characterized by the early occurrence of severe hematuria, marked proteinuria at day 10 (76.2 +/- 18.2 mg/24 hours), and massive glomerular deposition of fibrin. Complement and rat IgG were present in insudative lesions, but no linear pattern along the glomerular capillary wall was observed. By light microscopy, severe glomerular lesions were found at day 10 consisting of crescent formation and fibrinoid necrosis of capillary loops. In the interstitium, tubular necrosis and atrophy and marked interstitial mononuclear infiltration were found in conclusion, autoantibodies to MPO severely aggravate subclinical anti-GBM disease demonstrating their in vivo pathogenic potential.