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The granule enzyme myeloperoxidase (MPO) plays an important role in neutrophil antimicrobial responses. However, the severity of immunodeficiency in patients carrying mutations in MPO is variable. Serious microbial infections, especially with Candida species, have been observed in a subset of completely MPO-deficient patients. Here we show that neutrophils from donors who are completely deficient in MPO fail to form neutrophil extracellular traps (NETs), indicating that MPO is required for NET formation. In contrast, neutrophils from partially MPO-deficient donors make NETs, and pharmacological inhibition of MPO only delays and reduces NET formation. Extracellular products of MPO do not rescue NET formation, suggesting that MPO acts cell-autonomously. Finally, NET-dependent inhibition of Candida albicans growth is compromised in MPO-deficient neutrophils. The inability to form NETs may contribute in part to the host defense defects observed in completely MPO-deficient individuals.

Anti-proteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies are capable of activating human neutrophils primed by TNF-alpha in vitro. We described previously the involvement of FcgammaRIIa and beta(2) integrins in this neutrophil activation. In the literature, the requirement of TNF priming has been attributed to an effect of TNF-alpha on the expression of PR3 or MPO on the cell surface. Under our experimental conditions, TNF-alpha (2 ng/ml) increased the binding of the antibody against PR3, whereas binding of the antibody against MPO could hardly be detected, not even after TNF-alpha treatment. The aim of this study was to consider (an)other(s) role(s) for TNF-alpha in facilitating the NADPH-oxidase activation by these antibodies. We demonstrate the early mobilization of the secretory vesicles as a result of TNF-induced increase in intracellular-free calcium ions, the parallel colocalization of gp91(phox), the main component of the NADPH oxidase with beta(2) integrins and FcgammaRIIa on the neutrophil surface, and the FcgammaRIIa clustering upon TNF priming. TNF-alpha also induced redistribution of FcgammaRIIa to the cytoskeleton in a dose- and time-dependent manner. Moreover, blocking CD18 MHM23 antibody, cytochalasin B, and D609 (an inhibitor of phosphatidylcholine phospholipase C) inhibited this redistribution and the respiratory burst in TNF-treated neutrophils exposed to anti-PR3 or anti-MPO antibodies. Our results indicate direct effects of TNF-alpha in facilitating neutrophil activation by these antibodies and further support the importance of cytoskeletal rearrangements in this priming process.

Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase-3 and myeloperoxidase (MPO) activate tumor necrosis factor-alpha-primed neutrophils in vitro. We used neutrophils from one completely and one partially MPO-deficient donor to assess the requirement of MPO expression for neutrophil activation by anti-MPO antibodies. The MPO deficiencies were defined enzymatically, by immunocytochemistry and by immunoblotting. The mutations in the MPO genes of these donors were identified as a combination of a novel splice-site mutation at the 3' end of intron 11 (A-2-->C), a deletion of 14 nucleotides in exon 9 (A1555-C1568), and a novel C1907 --> T (636Thr-->Met) substitution in exon 11 in the completely MPO-deficient donor and as the same splice-site mutation and a novel C995 --> T (332Ala-->Val) substitution in exon 7 in the partially MPO-deficient donor. Monoclonal antibody 4.15 against MPO and MPO-ANCA-immunoglobulin G induced no superoxide anion production in these MPO-deficient neutrophils despite a normal production induced by other stimuli. Thus, the presence of MPO is a conditio sine qua non for neutrophil activation by anti-MPO antibodies. Moreover, we demonstrated that by means of these MPO-deficient cells, hydrogen peroxide may diffuse from neutrophils to surrounding cells, which may contribute to the damage induced by oxygen radicals in the pathology of systemic vasculitides.

Antineutrophil cytoplasmic autoantibodies (ANCA) have been described in sera of patients with several forms of systemic vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. The two main targets of ANCA in vasculitis are proteinase 3 (PR3) and myeloperoxidase (MPO). ANCA are capable of activating neutrophils primed by tumor necrosis factor-alpha (TNF-alpha) in vitro, which may be relevant for the induction of the vascular inflammation observed in vivo. Recently, it has been suggested that engagement of Fc gamma receptor IIa (Fc gamma RIIa) on the neutrophils is involved in the activation by ANCA. In the present study, we show that activation of the neutrophil respiratory burst by anti-PR3 and anti-MPO is strongly enhanced after TNF priming and lost on removal of the Fc parts of the antibodies. Similar results were obtained when the neutrophils were activated with antibodies against known membrane antigens without major changes in the expression of the target antigens. The TNF-induced enhancement of the neutrophil activation was not observed when adherence of the cells was prevented by continuous stirring of the suspension or by the addition of CD18 antibodies before TNF exposure. Hence, our results indicate that engagement of both Fc gamma RIIa and beta 2 integrins is instrumental in neutrophil activation induced by ANCA.

Atypical antineutrophil cytoplasm antibodies (A-ANCA) are defined here as ANCA detected by IIF and not directed against the predominant ANCA antigens, proteinase 3 (PR3) and myeloperoxidase (MPO). A-ANCA are found in a variety of clinical conditions, namely rheumatoid arthritis, inflammatory bowel diseases, chronic hepatic diseases and several infections including HIV infection. They are directed against a variety of still ill-defined neutrophil antigens and most frequently yield a perinuclear pattern (P-ANCA) of binding by indirect immunofluorescence on ethanol fixed neutrophils. This paper reviews the literature on A-ANCA and our recent data suggesting that, among others, cathepsin G is one of the predominant antigen targets of A-ANCA. From a clinical point of view, the distinction between MPO-ANCA and A-ANCA is not possible by indirect immunofluorescence (IIF). The determination of ANCA antigens by specific ELISA is therefore necessary to differentiate P-ANCA with MPO specificity from those with undefined specificity. This is of importance because the clinical value of MPO-ANCA is clearly established while the presence of A-ANCA is difficult to interpret given their occurrence in a large variety of clinical conditions.

Autoantibodies directed against polymorphonuclear neutrophils (PMN) have been observed in serum from patients with ulcerative colitis (UC), Crohn's disease (CD) and primary sclerosing cholangitis (PSC) using indirect immunofluorescence and fixed granulocyte ELISA. Our study demonstrates the presence in the serum of these patients of autoantibodies which bind to an azurophilic granule component distinct from proteinase 3, elastase and myeloperoxidase. These autoantibodies thus belong to the ANCA family, but their antigen specificity differs from the already characterized ANCA antigens. We have found that the same ANCA antigen target, named UC-antigen, was recognized by serum IgG from patients with UC, CD and PSC. It was purified by Matrex Gel Orange A dye affinity chromatography and subsequent immunoabsorption of contaminant proteinase 3 with immobilized anti-proteinase 3 MoAb. The identity between this UC antigen and cathepsin G was demonstrated by their coelution from Matrex Gel Orange A column and the parallel titration of cathepsin G-specific enzymatic activity and UC-ANCA binding, both in partially purified UC antigen and in highly pure cathepsin G. Furthermore, the use of cathepsin G ELISA confirmed that UC, CD and PSC patients' IgG did indeed bind to cathepsin G. Comparison of the results obtained with azurophilic granule- and cathepsin G-ELISA as well as inhibition of ANCA binding by anti-cathepsin G polyclonal antibodies, revealed that in some patients cathepsin G is the main azurophilic granule target of ANCA while others have other ANCA specificities. The fact that UC, CD and PSC are frequently associated with cathepsin G ANCA, while rarely occurring in other types of vasculitis, is intriguing but suggests that these diseases may have a common pathogenetic mechanism.

Antineutrophil cytoplasmic autoantibodies have been recently reported in sera from patients with inflammatory bowel disease. We report our experience based on 90 patients with ulcerative colitis, 148 patients with Crohn's disease, and 60 controls. Determination of antineutrophil cytoplasmic autoantibodies was performed by the indirect immunofluorescence technique on ethanol fixed leucocytes. The specificities for proteinase 3, myeloperoxidase, and lactoferrin were tested by enzyme-linked immunosorbent assay. Forty-three out of 90 (48%) patients with ulcerative colitis, 10 out of 148 (7%) patients with Crohn's disease, and none of controls were positive by indirect immunofluorescence technique. All patients but two with positive immunofluorescence exhibited a perinuclear staining pattern. Among patients with ulcerative colitis, there was no relationship between the presence of perinuclear antineutrophil cytoplasmic autoantibodies and disease location or activity. Seven out of 20 (35%) patients with ulcerative colitis who had a previous colectomy (including 1 with ileoanal anastomosis) had perinuclear antineutrophil cytoplasmic autoantibodies. Antineutrophil cytoplasmic autoantibody specificity was not directed against myeloperoxidase, proteinase 3 or lactoferrin in sera from patients with inflammatory bowel diseases. Inflammatory bowel diseases are associated with a new subset of antineutrophil cytoplasmic autoantibodies. Among patients with Crohn's disease and ulcerative colitis, the sensitivity and specificity of the presence of perinuclear antineutrophil cytoplasmic autoantibodies for ulcerative colitis was 46% and 93%, respectively. Their presence reinforces the likelihood of underlying immunologic dysregulation in ulcerative colitis. Identification of the autoantigen(s) to which these antibodies are directed might facilitate the understanding of inflammatory bowel disease pathophysiology.

We previously described the requirement of tumour necrosis factor-alpha (TNF- α) and the role of β2 integrins in the Fc-gamma receptor IIa (Fc γRIIa)-mediated mechanism of neutrophil activation by antiproteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies. In the present study, we assessed the involvement of Fc γRIIIb by studying the respiratory burst activation of completely Fc γRIIIb-deficient neutrophils primed by TNF- α and exposed to anti-PR3 or anti-MPO. Activation of the NADPH oxidase occurred normally in these neutrophils, which indicates that engagement of Fc γRIIIb is not essential in our model. Experiments performed with neutrophils from severe leucocyte adhesion deficiency (LAD) patients confirmed that β2 integrins play a pivotal role in this activation. We next studied whether adhesion per se, β2-integrin-mediated adhesion, or β2-integrin ligation without adhesion is necessary or sufficient for this activation. Anti-PR3 or anti-MPO induced an Fc γRIIa-dependent burst in TNF-primed neutrophils incubated in wells coated with poly- l-lysine, known to induce β2-integrin-independent adhesion, but this reaction was still inhibited by blocking CD18 antibodies. In a system with granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, which did not enhance adhesion, we measured a similar activation by anti-PR3 or anti-MPO and inhibition by CD18. We also noticed that treatment with the β2-integrin-activating CD18 MoAb KIM185 per se is insufficient for neutrophil activation by anti-PR3 or anti-MPO. We therefore conclude that ligation of β2 integrins rather than adherence per se is essential for this activation, and that TNF- α or GM-CSF is needed for priming but not for adherence. [ABSTRACT FROM AUTHOR]