Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides are systemic or more limited conditions characterized by necrotizing destruction of small and medium-sized vessels (eg, capillaries, venules, and arterioles). ANCAs are the most predominant autoantibodies in patients affected by vasculitis, but other autoantibodies may also occur, probably reflecting pathogenetic events in affected tissue. These autoantibodies are assumed to play a role in the intiation and propagation of chronic inflammation. ANCAs are valuable for clinical diagnosis, follow-up, and guidance in therapy.
Antineutrophil cytoplasmic autoantibodies (ANCAs) are frequently associated with diseases characterized by the presence of vasculitis, affecting small and medium-sized vessels (eg, arterioles, capillaries, and venules). Although this association was realized already in the mid-1980s, ANCAs were not immediately included in the classification criteria for this type of vasculitis, and no agreed diagnostic criteria for the diagnosis of these conditions exist as yet. Presence of autoantibodies such as ANCAs most likely reflects pathobiologic events in the affected tissue, in this case neutrophils and monocytes attacking the vascular endothelium of small vessels, causing autoantigen release from these cells and presentation to the immune system.
Because endothelial cells (ECs) become activated and damaged by the attack, autoantibodies to EC constituents (anti–endothelial cell antibodies [AECAs]) are also commonly produced in ANCA-associated vasculitis (AAV). During this inflammatory attack on the vessel walls, the basal membrane of some vessels can also be damaged and autoantibodies to the basement membrane α 3 domain of type IV collagen of glomeruli and pulmonary vessels are produced.
The primary events leading to the onset of these necrotizing vasculitides are not known. However, several hypotheses have proposed that infectious agents can trigger and perhaps perpetuate events that, if left untreated, will lead to irreversible tissue damage and organ function failure.
Patients suffering from ANCA-associated small vessel necrotizing vasculitides, such as Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA), and Churg-Strauss syndrome (CSS), may produce autoantibodies to several different autoantigens and structures that are involved in the pathology of these diseases. Although ANCAs have thus lent name to the group of diseases collectively called AAV, they may not be the only interesting autoantibodies in terms of pathophysiology or classification.
Neutrophil-specific autoantibodies
Neutrophil-specific autoantibodies (NSAs) have been known to exist since 1959, when their presence was first described in patients with leukopenia and later in those with ulcerative colitis.
Using indirect immunofluorescence (IIF) technique, the autoantibodies found in leukopenia and ulcerative colitis decorated the nuclei of neutrophils, and therefore later work on NSAs in patients with rheumatoid arthritis and Felty syndrome called them granulocyte-specific antinuclear antibodies. Similar NSAs have been described in chronic active hepatitis, sclerosing cholangitis, Sweet syndrome, and other conditions in which chronic recruitment of neutrophils is an essential part of the inflammatory process. However, most of the NSAs are not directed to the classic cytoplasmic ANCA antigens proteinase 3 (PR3), myeloperoxidase (MPO), or human leukocyte elastase (HLE) but rather to components found in all compartments of neutrophils and monocytes, probably just showing an immune response caused by chronic neutrophil cell death.
When autoantibodies to neutrophils were described in patients with WG, they were termed anticytoplasmic antibodies because of their preferential staining of granular material in the cytoplasm, but this indistinct term was changed to ANCA during the Second International Workshop on ANCA in Noordwijkerhout, The Netherlands. The main reason for this change was the identification of the 2 most important neutrophil granule antigens, PR3 and MPO, as the dominant targets in patients not only with WG but also in patients with MPA and CSS and their limited forms.
ANCA
In 1982, Davies and colleagues first recognized autoantibodies that were specific for neutrophils in small vessel necrotizing glomerulonephritis. It was initially proposed that the antibodies were produced in response to an arbovirus (Ross River virus) infection, although this was never substantiated. Morphologically similar antibodies giving rise to a coarse granular staining of neutrophil cytoplasm ( Fig. 1 ) were subsequently reported in patients with WG, preferentially in those having active disease. This particular pattern of reactivity with neutrophils was then called the classic cytoplasmic ANCA (C-ANCA). In 1988, soon after the first International Workshop on ANCA in Copenhagen, Denmark, the C-ANCA antigen was reported to be directed to the proteolytic enzyme PR3 in azurophilic granules of neutrophils.
ANCAs that caused staining of neutrophil nuclei and their close vicinity were called perinuclear ANCAs (P-ANCAs) ( Fig. 2 ) ; this reactivity is caused by cationic granule antigens, for example, MPO and HLE, that have migrated to the oppositely charged nuclei and was first described for ANCAs directed to MPO. P-ANCAs with specificity for MPO are commonly produced in AAV with kidney or lung manifestations.
Proteinase 3
The conformation of PR3 is essential for its reactivity with PR3-ANCA. PR3 is a linear polypeptide containing 228 amino acids. In 1994, linear epitopes of PR3 were found, and these seemed to occur in regions close to the active site of the enzyme where 4 of 5 epitopes are assumed to be located. The crystal structure of PR3 was resolved in 1996. PR3 is progressively more strongly expressed from the stage of early myelocyte maturation to the fully developed neutrophil. The gene encoding PR3 is located close to the HLE site on chromosome 19.
Myeloperoxidase
MPO is the main antigen for the P-ANCA reactivity in patients with AAV. The enzyme catalyzes the hydrogen peroxide–mediated peroxidation of chloride ions to hypochlorite, and the hypochlorous acid is effective in killing phagocytized bacteria and viruses. However, the hypochlorous acid can also inactivate proteinase inhibitors in blood and tissues, thus indirectly influencing inflammation through induction of highly reactive oxygen radical species among other mechanisms. MPO is a hydrophilic and positively charged molecule located in azurophilic and primary granules. Thus, after fixation of neutrophils and monocytes by ethanol for use as substrate in IIF, the positively charged molecule migrates toward the nucleus and the ensuing staining pattern becomes perinuclear as shown in Fig. 2 .
MPO consists of 2 polypeptides of 467 and 113 amino acids, called the α and β chains, respectively. After glycosylation and final processing, the polypeptides form a 59-kDa α chain and a 13.5-kDa β chain that are linked together in a heterodimeric 140-kDa molecule, in which each half has one α chain and one β chain. The crystal structure has been resolved, and the gene encoding MPO has been found on the long arm of chromosome 17. Reactivity with MPO-ANCA highly depends on conformational preservation of the structure. The MPO molecule is very sensitive to light exposure, whereby it cleaves at position Met 409 of the α chain, a fact that is important for its stability during purification and coating on solid phases.
Human Leukocyte Elastase
HLE is a frequently targeted autoantigen in patients with drug-induced vasculitis or drug-induced lupus and in those with cocaine-induced nasal destruction, which may mimic late-stage WG. HLE-ANCAs are rarely found alone and are mostly accompanied by ANCAs toward other neutrophil antigens.
HLE-ANCAs are rare in idiopathic AAV. If HLE-ANCA is the only ANCA contained in a serum, it gives a P-ANCA pattern.
Human Lysosomal Membrane Protein 2
Autoantibodies to a human lysosomal membrane protein (H-lamp-2) were first described in 78 of 84 individuals with ANCA-associated focal necrotizing glomerulonephritis (FNGN). Kain and colleagues reported that the H-lamp-2 protein is expressed in the plasma membrane of several human cells, such as neutrophils and ECs, but the protein is also integrated into membranes of neutrophil intracellular vesicles. A recent study from the same research group has suggested that the emergence of autoimmunity to H-lamp-2 is caused by molecular mimicry through reactivity with a H-lamp-2 peptide (P41–49) that shows 100% homology to a bacterial adhesin peptide (amino acids 72–80) found on mature FimH protein located on type 1 fimbriae of gram-negative bacteria. Xenogeneic antibodies to H-lamp-2 were found to bind to fixed neutrophils in a C-ANCA–like reaction pattern by IIF as a result of reactivity with the intracellular vesicle membranes mentioned earlier. These autoantibodies are thus not neutrophil-specific because they react with a variety of human cells, most importantly ECs, and it can be discussed whether it is justified to call them ANCA.
The H-lamp-2 protein is assumed to have physiologic roles in cell adhesion, autophagocytosis, and antigen presentation. Autoantibodies to H-lamp-2, however, may be able to induce pauci-immune FNGN per se because a monoclonal antibody to H-lamp-2 was found to induce apoptosis of microvascular endothelium when injected into rats. In addition, rats injected with rabbit antibodies to H-lamp-2 subsequently developed FNGN.
Further studies on the prevalence and levels of anti–H-lamp-2 are needed to judge the potential clinical utility of this autoantibody in monitoring patients with AAV.
Other Antigens
Lactoferrin, bactericidal permeability-increasing protein, azurocidin, and lysozyme, which are also localized in neutrophil granules, may in some cases be targets of NSAs; however, such reactivity is rare in idiopathic small vessel necrotizing vasculitis. In contrast, any of these antigens may be a target in patients suffering from drug-induced vasculitis or drug-induced lupus-like syndromes. In these cases, there are often multiple granule protein targets, a finding so characteristic that a drug-induced syndrome should be suspected whenever multispecific ANCAs are found in serum. Sera from such patients can additionally harbor autoantibodies to histones and β 2 -glycoprotein I, especially if the syndrome has been caused by an antithyroid drug.
ANCA
In 1982, Davies and colleagues first recognized autoantibodies that were specific for neutrophils in small vessel necrotizing glomerulonephritis. It was initially proposed that the antibodies were produced in response to an arbovirus (Ross River virus) infection, although this was never substantiated. Morphologically similar antibodies giving rise to a coarse granular staining of neutrophil cytoplasm ( Fig. 1 ) were subsequently reported in patients with WG, preferentially in those having active disease. This particular pattern of reactivity with neutrophils was then called the classic cytoplasmic ANCA (C-ANCA). In 1988, soon after the first International Workshop on ANCA in Copenhagen, Denmark, the C-ANCA antigen was reported to be directed to the proteolytic enzyme PR3 in azurophilic granules of neutrophils.
ANCAs that caused staining of neutrophil nuclei and their close vicinity were called perinuclear ANCAs (P-ANCAs) ( Fig. 2 ) ; this reactivity is caused by cationic granule antigens, for example, MPO and HLE, that have migrated to the oppositely charged nuclei and was first described for ANCAs directed to MPO. P-ANCAs with specificity for MPO are commonly produced in AAV with kidney or lung manifestations.
Proteinase 3
The conformation of PR3 is essential for its reactivity with PR3-ANCA. PR3 is a linear polypeptide containing 228 amino acids. In 1994, linear epitopes of PR3 were found, and these seemed to occur in regions close to the active site of the enzyme where 4 of 5 epitopes are assumed to be located. The crystal structure of PR3 was resolved in 1996. PR3 is progressively more strongly expressed from the stage of early myelocyte maturation to the fully developed neutrophil. The gene encoding PR3 is located close to the HLE site on chromosome 19.
Myeloperoxidase
MPO is the main antigen for the P-ANCA reactivity in patients with AAV. The enzyme catalyzes the hydrogen peroxide–mediated peroxidation of chloride ions to hypochlorite, and the hypochlorous acid is effective in killing phagocytized bacteria and viruses. However, the hypochlorous acid can also inactivate proteinase inhibitors in blood and tissues, thus indirectly influencing inflammation through induction of highly reactive oxygen radical species among other mechanisms. MPO is a hydrophilic and positively charged molecule located in azurophilic and primary granules. Thus, after fixation of neutrophils and monocytes by ethanol for use as substrate in IIF, the positively charged molecule migrates toward the nucleus and the ensuing staining pattern becomes perinuclear as shown in Fig. 2 .
MPO consists of 2 polypeptides of 467 and 113 amino acids, called the α and β chains, respectively. After glycosylation and final processing, the polypeptides form a 59-kDa α chain and a 13.5-kDa β chain that are linked together in a heterodimeric 140-kDa molecule, in which each half has one α chain and one β chain. The crystal structure has been resolved, and the gene encoding MPO has been found on the long arm of chromosome 17. Reactivity with MPO-ANCA highly depends on conformational preservation of the structure. The MPO molecule is very sensitive to light exposure, whereby it cleaves at position Met 409 of the α chain, a fact that is important for its stability during purification and coating on solid phases.
Human Leukocyte Elastase
HLE is a frequently targeted autoantigen in patients with drug-induced vasculitis or drug-induced lupus and in those with cocaine-induced nasal destruction, which may mimic late-stage WG. HLE-ANCAs are rarely found alone and are mostly accompanied by ANCAs toward other neutrophil antigens.
HLE-ANCAs are rare in idiopathic AAV. If HLE-ANCA is the only ANCA contained in a serum, it gives a P-ANCA pattern.
Human Lysosomal Membrane Protein 2
Autoantibodies to a human lysosomal membrane protein (H-lamp-2) were first described in 78 of 84 individuals with ANCA-associated focal necrotizing glomerulonephritis (FNGN). Kain and colleagues reported that the H-lamp-2 protein is expressed in the plasma membrane of several human cells, such as neutrophils and ECs, but the protein is also integrated into membranes of neutrophil intracellular vesicles. A recent study from the same research group has suggested that the emergence of autoimmunity to H-lamp-2 is caused by molecular mimicry through reactivity with a H-lamp-2 peptide (P41–49) that shows 100% homology to a bacterial adhesin peptide (amino acids 72–80) found on mature FimH protein located on type 1 fimbriae of gram-negative bacteria. Xenogeneic antibodies to H-lamp-2 were found to bind to fixed neutrophils in a C-ANCA–like reaction pattern by IIF as a result of reactivity with the intracellular vesicle membranes mentioned earlier. These autoantibodies are thus not neutrophil-specific because they react with a variety of human cells, most importantly ECs, and it can be discussed whether it is justified to call them ANCA.
The H-lamp-2 protein is assumed to have physiologic roles in cell adhesion, autophagocytosis, and antigen presentation. Autoantibodies to H-lamp-2, however, may be able to induce pauci-immune FNGN per se because a monoclonal antibody to H-lamp-2 was found to induce apoptosis of microvascular endothelium when injected into rats. In addition, rats injected with rabbit antibodies to H-lamp-2 subsequently developed FNGN.
Further studies on the prevalence and levels of anti–H-lamp-2 are needed to judge the potential clinical utility of this autoantibody in monitoring patients with AAV.
Other Antigens
Lactoferrin, bactericidal permeability-increasing protein, azurocidin, and lysozyme, which are also localized in neutrophil granules, may in some cases be targets of NSAs; however, such reactivity is rare in idiopathic small vessel necrotizing vasculitis. In contrast, any of these antigens may be a target in patients suffering from drug-induced vasculitis or drug-induced lupus-like syndromes. In these cases, there are often multiple granule protein targets, a finding so characteristic that a drug-induced syndrome should be suspected whenever multispecific ANCAs are found in serum. Sera from such patients can additionally harbor autoantibodies to histones and β 2 -glycoprotein I, especially if the syndrome has been caused by an antithyroid drug.
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