Anti-proteinase 3 antibodies (c-ANCA) prime CD14-dependent leukocyte activation

In Wegener's granulomatosis (WG), a pathogenetic role has been proposed for circulating anti-neutrophil-cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3). Disease activation in WG appears to be triggered by bacterial infections. In the present study, we characterized the effect of anti-PR3 antibodies on in vitro activation of isolated monocytes and neutrophils by the bacterial cell-wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Although sole incubation of monocytes and neutrophils with monoclonal anti-PR3 antibodies induced the release of minor quantities of the chemokine interleukin-8 (IL-8), preincubation with anti-PR3 antibodies, but not with isotype-matched control immunogloblin G (IgG), resulted in a markedly enhanced IL-8 liberation upon LPS challenge. The priming response was evident after 2 h of preincubation with anti-PR3 and peaked after 6 h. The anti-PR3-related priming was also observed for tumor necrosis factor alpha (TNF-alpha) and IL-6 synthesis. Comparable priming occurred when leukocytes were preincubated with ANCA-IgG derived from WG serum but not with normal IgG. The priming effect of the anti-PR3 antibody pretreatment was reproduced for LTA challenge of monocytes and neutrophils but not for leukocyte stimulation with TNF-alpha. Flow cytometric analysis revealed an increase in monocyte and neutrophil membrane CD14 expression during the anti-PR3 priming. We conclude that cytoplasmic ANCA specifically prime CD14-dependent monocytes and neutrophils for activation. The resulting enhanced responsiveness to bacterial pathogens may contribute to the development and maintenance of inflammatory lesions during active WG.     

Antibodies to proteinase 3 prime human oral, lung, and kidney epithelial cells to secrete proinflammatory cytokines upon stimulation with agonists to various Toll-like receptors, NOD1, and NOD2

Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies, the detection of which in serum can be used in the diagnosis of Wegener's granulomatosis (WG). Proteinase 3 (PR3) is a major target antigen of ANCA in WG patients, and the interaction of PR3 ANCA with leukocytes causes a debilitating autoimmune disease. The first signs and symptoms in WG patients are observed in the oral cavity, lungs, and kidneys. Human epithelial cells generally do not secrete proinflammatory cytokines upon stimulation with pathogen-associated molecular patterns (PAMPs). In this study, anti-PR3 antibodies (Abs) and PR3 ANCA-containing sera from WG patients endowed human oral, lung, and kidney epithelial cells with responsiveness to PAMPs in terms of the production of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1, and tumor necrosis factor alpha. Protease-activated receptor-2 (PAR-2) agonist peptides mimicked the priming effects of PR3 ANCA against PAMPs. Furthermore, the anti-PR3 Ab-mediated cell activation was significantly abolished by RNA interference targeting PAR-2 and NF-kappaB. This is the first report of priming effects of anti-PR3 Abs (PR3 ANCA) on epithelial cells. The results suggest that anti-PR3 Abs (PR3 ANCA) prime human epithelial cells to produce cytokines upon stimulation with various PAMPs, and these mechanisms may be involved in severe chronic inflammation in WG.     

Anti-neutrophil cytoplasmic antibodies target sequential functional proteinase 3 epitopes in the sera of patients with Wegener’s granulomatosis

Many patients with Wegener’s granulomatosis (WG) have anti-neutrophil cytoplasmic antibodies (c-ANCA). Aside from being a diagnostic marker, these autoantibodies may play roles in disease pathogenesis. Proteinase 3 (PR3) is the primary target of c-ANCA in WG patient sera. Of 60 c-ANCA-positive patients, 10 patients were selected for detailed humoral epitope analysis, contingent upon serum availability, using samples with positive levels of anti-PR3 by enzyme-linked immunosorbent assay (ELISA). Sequential epitope specificities of anti-PR3 antibodies detected by screening the maximally overlapping solid-phase octapeptides of PR3 showed seven major common antigenic targets bound by WG patient sera. These include novel and previously identified sequential PR3 epitopes bound by c-ANCA. B cell epitope prediction algorithms identified all or part of the seven defined epitopes. Several epitopes share sequence and structural proximity with functional sites, including the catalytic triad and proposed binding sites of other potential proteins [PR3 complementary peptide and soluble endothelial protein C receptor (sEPCR)]. Epitope 4 (VVLGAHNVRTQ) had the highest binding prevalence (90%) and epitope 2 (AQPHSRPYMAS) has the highest average reactivity of the antigenic regions. Epitope 4 includes the interaction site between sEPCR and PR3 which may serve as an important interaction to down-regulate inflammation. Epitopes 3, 5 and 7 are in direct proximity to amino acids that form the catalytic triad of the protein. c-ANCA targets both unique and previously known sequential PR3 peptides. This information may prove useful in understanding anti-PR3-mediated disease pathogenesis in systemic vasculitides.     

Anti-proteinase 3 antibody activation of neutrophils can be inhibited by alpha1-antitrypsin.

Wegener's granulomatosis (WG) is classically associated with the presence of cytoplasmic antineutrophil cytoplasmic autoantibodies (c-ANCA). Proteinase 3 (PR3), the target antigen for c-ANCA, is inhibited by the antiprotease alpha1-antitrypsin (A1AT), and recent studies have demonstrated that WG patients who are A1AT-deficient have a worse clinical course, suggesting that a protease-antiprotease imbalance may play a role in WG. We evaluated the effect of A1AT on anti-PR3 antibody-induced activation of neutrophils. The neutrophil was chosen because of its central role in the pathogenesis of WG. Isolated neutrophils from healthy controls were incubated with tumor necrosis factor (TNF)-alpha to induce surface expression of PR3. Subsequently, they were stimulated with a monoclonal antibody to PR3, resulting in a significant increase in respiratory burst. Addition of A1AT (1 mg/ml) to the TNF-alpha- primed cells before the addition of the anti-PR3 antibody resulted in a 47% reduction in anti-PR3 antibody-induced activation. A1AT mediated this inhibitory action by preventing anti-PR3 antibody binding to PR3 on the cell, thereby preventing the PR3-FcgammaR11a cross-linkage required for cell activation. Further, anti-PR3 antibody-induced activation of neutrophils from WG patients can be reduced by 56% with A1AT. These data suggest that protease-antiprotease interactions may play a pivotal role in neutrophil activation in WG.     

Antineutrophil cytoplasmic antibodies induce decreased CD62L expression and enhanced metabolic activity in monocytes

Monocyte in vitro activation by antimyeloperoxidase (anti-MPO)- and antiproteinase-3 (anti-PR3)-positive sera, corresponding immunoglobulin G (IgG) fractions and monoclonal antibodies against MPO and PR3 was evaluated. The expression of adhesion molecules, l-selectin (CD62L) and CR3 (CD11b), involved in leucocyte endothelial adhesion, and metabolic activity, measured as the production of hydrogen peroxide, were analysed. Decreased expression of CD62L was demonstrated in monocytes after incubation with antineutrophil cytoplasmic antibody (ANCA)-positive sera. This finding was not accompanied by changes in CD11b expression. Metabolic activity was increased in monocytes after incubation with ANCA-positive IgG fractions as well as after incubation with monoclonal anti-MPO and anti-PR3. These findings support the concept that the pathophysiological effect of ANCA is partly mediated through the action on crucial events in monocyte activation, such as CD62L downregulation and oxygen radical production.     

Subthreshold concentrations of anti-proteinase 3 antibodies (c-ANCA) specifically prime human neutrophils for fMLP-induced leukotriene synthesis and chemotaxis

Anti-neutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3) possess a high sensitivity and specificity for Wegener's granulomatosis. Due to their capacity of directly activating neutrophils, a pathogenetic role for these autoantibodies has been proposed. We investigated the impact of subthreshold concentrations of monoclonal anti-PR3 antibodies (anti-PR3; 0.1 microg/mL) on neutrophil activation elicited by a secondary agent. Preincubation with anti-PR3 resulted in a massive amplification of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukotriene (LT) generation, with a marked increase in the liberation of LTB4, LTA4, and 5-hydroxyeicosatetraenoic acid (5-HETE). This priming commenced within 2.5 min, with a maximum after 5-7.5 min. Moreover, anti-PR3 pretreatment markedly enhanced PMN movement toward fMLP. The priming effect of anti-PR3 toward fMLP challenge was reproduced by c-ANCA, but not by F(ab)2 fragments of the antibodies and isotype-matched control IgG. Generation of superoxide anion and release of elastase were suppressed in anti-PR3-pretreated neutrophils undergoing fMLP challenge. In contrast, neutrophil activation by platelet-activating factor (PAF) or the calcium ionophore A23187 remained unaffected. We conclude that subthreshold concentrations of anti-PR3 antibodies selectively modify neutrophil responses to fMLP, with enhancement of leukotriene generation and chemotaxis, but suppression of respiratory burst and degranulation. Such priming might contribute to localized neutrophil accumulation together with blunted host defense in Wegener's granulomatosis.     

Constitutive membrane expression of proteinase 3 (PR3) and neutrophil activation by anti-PR3 antibodies

Antineutrophil cytoplasm autoantibodies with specificity for proteinase 3 (PR3) are thought to play a major role in the pathogenesis of Wegener's granulomatosis (WG), presumably by their potential to activate neutrophils. In patients with WG, high expression of PR3 on the surface of nonprimed neutrophils is associated with an increased incidence and rate of relapse. In this study, we analyzed the functional significance of constitutive PR3 expression for neutrophil activation as induced by anti-PR3 antibody. Therefore, primed and nonprimed neutrophils were stimulated with the monoclonal anti-PR3 antibody PR3G-3. Activation was measured as actin polymerization by the phalloidin assay as an early, detectable activation event and oxidative burst by the dihydrorhodamine assay, as a late, detectable activation event. In contrast to the oxidative burst, we found that anti-PR3 antibody-induced actin polymerization could be triggered in neutrophils without priming with tumor necrosis factor alpha (TNF-alpha). In addition, a correlation was found between the level of PR3 expression on the surface of these nonprimed neutrophils and the degree of actin polymerization. However, after priming with TNF-alpha, no correlation was found between membrane expression of PR3 and the level of actin polymerization or respiratory burst as induced by anti-PR3 antibody. These data suggest that the presence of PR3 on the surface of nonprimed neutrophils has consequences for their susceptibility to the initial activation step by anti-PR3 antibodies. These data may be relevant in view of the observed relation between membrane expression of PR3 on nonprimed neutrophils of patients with WG and their susceptibility for relapses.     

TNF-alpha is secreted by monocytes in transit to become macrophages, but not by peripheral blood monocytes, following OK-432 (lyophilized S. pyogenes) stimulation

OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied proinflammatory interleukin (IL) secretion following OK-432 stimulation of total blood, peripheral blood mononuclear cell (PBMC) and purified monocytes in vitro. OK-432 stimulation of purified monocytes gave IL-1beta, IL-1RA, IL-6, IL-12p40 and tumour necrosis factor (TNF)-alpha response. OK-432 stimulation of cells within blood did, however, not yield TNF-alpha secretion. When PBMC or monocytes were cultured in low-attachment wells a decreased IL secretion was observed compared to adherent cells. Inhibition of Syk kinase with piceatannol, only at high, non-specific doses, but not PI3 kinase inhibition with LY294002 or Wortmannin, decreased monocyte IL response to OK-432. This shows that beta(1-3)-integrin receptor function is not necessary for monocyte OK-432-stimulated TNF-alpha secretion. Direct blockage of the beta(2)-integrin (CD18) receptor by anti-CD18 antibody was also unable to prevent the stimulating effects of OK-432 in human monocytes. On the other hand, Syk phosphorylation is elevated upon adherence of monocytes and this is further increased by OK-432 stimulation, as shown by Western blot. The Fc-receptor was also ruled out as a main receptor of the OK-432 monocyte response. In conclusion, TNF-alpha secretion is only found in monocytes removed from blood. This TNF-alpha secretion is not mediated through the beta(1-3)-integrin receptors. OK-432 may act as a target-seeking substance whereby only monocytes adhered, e.g. to a tumour cell, become cytotoxic in part explaining why OK-432 is well suited as a cancer treatment drug.     

Priming by tumor necrosis factor-alpha of human neutrophil NADPH-oxidase activity induced by anti-proteinase-3 or anti-myeloperoxidase antibodies

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.     

Membrane molecules which trigger the production of interleukin-1 and tumor necrosis factor-alpha by lipopolysaccharide-stimulated human monocytes.

We have investigated the role of the membrane molecules CD11/CD18 and CD14 which may mediate the binding of lipopolysaccharide (LPS) to human monocytes, in the induction of the production and release of interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-alpha) by LPS-stimulated cells. Blockade of CD11a, CD11b and CD18 with saturating concentrations of specific mAb did not inhibit the release of cytokines from LPS-stimulated monocytes. In contrast, inhibition of the release of IL-1 beta and TNF-alpha occurred in monocytes cultures that had been pretreated with either of two monoclonal antibodies (mAb) recognizing different epitopes on the CD14 molecule. The binding of LPS to CD14 has been previously shown to require serum factors. In the present study, we found that serum had an enhancing effect on the release of IL-1 and TNF-alpha from LPS-stimulated cultures of normal human monocytes. The inhibitory effect of anti-CD14 mAb was, however, observed in cultures performed in the presence or in the absence of serum, suggesting that triggering of IL-1/TNF-alpha release by CD14 is independent of LPS-binding proteins or other serum proteins. IL-1 beta and TNF-alpha were also released from LPS-stimulated cultures of monocytes from patients with paroxysmal nocturnal hemoglobinuria lacking expression of CD14. Thus, CD14 but not CD11/CD18 can trigger serum-dependent and independent cytokine release from endotoxin-stimulated normal human monocytes; CD14 is not, however, the only LPS receptor that is involved in the secretory response of endotoxin-stimulated cells.     

Human mast cells expressing recombinant proteinase 3 (PR3) as substrate for clinical testing for anti-neutrophil cytoplasmic antibodies (ANCA)

We have expressed conformationally intact, enzymatically active recombinant PR3 in HMC-1 cells (HMC-1/PR3 cells) that is recognized by C-ANCA. Here we directly compared the clinical utility of C-ANCA testing by indirect immunofluorescence (IIF) using HMC-1/PR3 cell cytospin versus polymorphonuclear neutrophil (PMN) cytospin preparations and commercially available anti-PR3 ELISA kits. Two hundred sera were tested independently by three investigators: 101 previously determined to be C-ANCA-positive by routine clinical laboratory testing using standard IIF on PMN cytospins, and 99 control samples chosen primarily because they contained antibodies against other cytoplasmic target antigens. Discrepant test results between the two cellular substrates were found in seven samples: 2/7 were PMN-positive and HMC-1/PR3 cell-negative (one Sjögren's syndrome, one hand injury); 5/7 were PMN-negative and HMC-1/PR3-positive (all Wegener's granulomatosis (WG)). All C-ANCA-positive WG patients were also positive on HMC-1/PR3 cells. IIF using HMC-1/PR3 cells was as sensitive as the most sensitive anti-PR3 ELISA (79.8% versus 80.7%, P= 0.739), and more sensitive than standard IIF C-ANCA testing using PMN cytospins (79.8% versus 75.2%, P= 0.025) or the anti-PR3 ELISA with the least false-positive test results (79.8% versus 63%, P< 0.01). These findings indicate that HMC-1/PR3 cells are a very sensitive antigen-specific substrate for clinical anti-PR3 ANCA testing which appears superior to standard C-ANCA testing using PMN cytospin substrates and anti-PR3 ELISA. Our results also suggest that in WG the C-ANCA fluorescence pattern is not caused by antibodies against target antigens other than PR3.     

Antibodies to proteinase 3 mediate expression of vascular cell adhesion molecule-1 (VCAM-1)

VCAM-1 was first identified as an adhesion molecule induced on human endothelial cells (HEC) by inflammatory cytokines such as IL-1, tumour necrosis factor (TNF), and lipopolysaccharide (LPS). The molecule binds to a variety of leucocytes, including B cells, T cells, basophils, eosinophils and monocytes. Vascular expression of VCAM-1 has been associated with a number of disease states, including rheumatoid arthritis and vasculitis. The detection of antineutrophil cytoplasmic antibodies (ANCA), especially to proteinase 3 (PR3), has become important in the diagnosis of Wegener’s granulomatosis (WG) and related vasculitides. Recently we were able to demonstrate a direct effect of anti-PR3 antibodies on neutrophil–endothelial interactions (Blood 1993; 82:1221). Binding of anti-PR3 antibodies to their antigen translocated into the membrane of HEC leads to an enhanced adhesion of neutrophils via induction of E-selectin (Clin Exp Immunol 1993; 94:440). The aim of this study was to investigate the effect of anti-PR3 antibodies on the expression of VCAM-1. HEC was isolated from umbilical vein and cultured on microtitre plates. After preincubatiion with purified anti-PR3 antibody, purified control antibodies (SS-A, SS-B, RNP) (IgG and F(ab′)₂ fragments) or different cytokines (controls), VCAM-1 was detected on the surface of unfixed HEC by cyto-ELISA and polymerase chain reaction analysis. Incubation of HEC with anti-PR3 antibodies led to a marked increase of endothelial VCAM-1 expression with a peak after 8 h. Incubation with TNF-α also led to maximal VCAM-1 expression after 4–6 h (control). Increased adhesion of T lymphocytes to HEC after binding of anti-PR3 antibodies to their antigen could be confirmed by performing adherence assays. This effect could be inhibited by antibodies to VLA-4. In conclusion, we have been able to show that cytokine-like effects of anti-PR3 antibodies on HEC are not limited to induction of neutrophil adhesion. Anti-PR3 antibodies may thus contribute to the regulation of T lymphocyte migration from the blood by HEC in ANCA-related vasculitides.     

Antibodies to proteinase 3 prime human monocytic cells via protease-activated receptor-2 and NF-kappaB for Toll-like receptor- and NOD-dependent activation

Anti-neutrophil cytoplasmic Abs against proteinase 3 (PR3) have been detected in relation to a wide range of inflammatory conditions, and the interaction of anti-PR3 Abs with leukocytes provokes cell activation, although how is not clear. Flow cytometric analysis revealed an increase in cell-surface CD14, Toll-like receptor (TLR)2, TLR4 and intracellular TLR3, TLR7, TLR8, TLR9, NOD1 and NOD2 expression during anti-PR3 priming in human monocytic THP-1 cells. Anti-RP3 Abs markedly promoted the release of IL-8 induced by chemically synthesized TLR and NOD ligands mimicking bacterial components: TLR2-agonistic lipopeptide (FSL-1), TLR3-agonistic poly I:C, TLR4-agonistic lipid A (LA-15-PP), TLR7/8-agonistic single stranded RNA (ssPolyU), TLR9-agonistic bacterial CpG DNA, NOD1-agonistic FK156/565 and NOD2-agonistic muramyldipeptide (MDP) in THP-1 cells and human peripheral blood mononuclear cells, although sole incubation with anti-PR3 Abs induced only a low level of IL-8. The priming response was evident after 2h of preincubation with anti-PR3 Abs and peaked after 6h. Priming was also observed for the production of TNF-alpha and monocyte chemoattractant protein-1. An RNA interference assay revealed that anti-PR3 Abs activated THP-1 cells in a PR3- and protease-activated receptor-2-dependent manner. Furthermore, the anti-PR3 Ab-mediated cell activation was significantly abolished by RNA interference targeted at PR3 mRNA and by inhibition of phospholipase C and NF-kappaB. These results suggest that anti-PR3 Abs prime human monocytic cells to produce cytokines upon stimulation with various bacterial components by up-regulating the TLR and NOD signaling pathway, and that these mechanisms may actively participate in the inflammatory process.     

Monocyte responses to sulfatide from Mycobacterium tuberculosis: inhibition of priming for enhanced release of superoxide, associated with increased secretion of interleukin-1 and tumor necrosis factor alpha, and altered protein phosphorylation.

In monocytes, sulfatide, a lipid from Mycobacterium tuberculosis, blocked priming for enhanced release of superoxide (O2-) by the macrophage activating factors lipopolysaccharide, gamma interferon, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and muramyl dipeptide. Sulfatide, in the presence of lipopolysaccharide, also caused increased secretion of IL-1 beta and TNF-alpha into monocyte culture medium. Sulfatide altered the pattern of phosphorylation of monocyte proteins. Cell lysates prepared from monocytes treated with sulfatide showed decreased activity of protein kinase C, but sulfatide did not directly inhibit protein kinase C activity when added to lysates. A known inhibitor of protein kinase C, staurosporine, also inhibited O2- release and caused increased secretion of IL-1 beta. Thus, sulfatide appeared to indirectly affect protein kinase C, implicating protein kinase C as part of the mechanism of priming. Because sulfatide blocked priming for enhanced release of O2-, which could interfere with monocyte bactericidal activity, while causing enhanced secretion of IL-1 beta and TNF-alpha, which could promote formation of granulomata, sulfatide might be an important factor in the pathogenesis of M. tuberculosis.     

Actinobacillus actinomycetemcomitans serotype b-specific polysaccharide antigen stimulates production of chemotactic factors and inflammatory cytokines by human monocytes.

Serotype b-specific polysaccharide antigen (SPA) was extracted from whole cells of Actinobacillus actinomycetemcomitans Y4 by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300. SPA induced the release of monocyte and leukocyte chemotactic factors by human monocytes. Polymyxin B had almost no effect on the release of monocyte chemotactic factor, but a monoclonal antibody against SPA markedly inhibited it. Human monocytes stimulated with SPA exhibited the increased mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and a neutrophil chemotactic factor, interleukin-8 (IL-8). On the other hand, SPA induced the release of IL-1, IL-6, and tumor necrosis factor (TNF) and enhanced the expression of IL-1alpha, IL-1beta, IL-6, and TNF alpha (TNF-alpha) mRNAs. Human monocytes expressed MCP-1 and IL-8 mRNAs when stimulated by human recombinant IL-1alpha, I1-1beta, IL-6, and TNF-alpha, suggesting that these inflammatory cytokines induced by SPA might participate in the production of chemotactic factors in human monocytes.     

Human proteinase 3 can inhibits LPS-mediated TNF-alpha production through CD14 degradation: lack of influence of antineutrophil cytoplasmic antibodies

The present study was conducted to investigate if proteinase-3 (PR3) is able to influence lipopolysaccharide (LPS) responses of monocytes via degradation of CD14 and if antineutrophil cytoplasmic antibodies (ANCA) may modify this process. Recombinant (r) CD14 and CD14 expressed on monocytes were investigated for PR3 mediated degradation by SDS-PAGE and FACS analysis, respectively. TNF-alpha production in whole blood was used to determine functional consequences of CD14 degradation. PR3 degraded rCD14 in a dose- and time-dependent fashion. Major degradation products were found with apparent molecular weight of 45, 25 and 10 kDa. Treatment of PR3 with PMSF completely abolished CD14 degradation. ANCA IgG did not inhibit CD14 degradation. In whole blood, addition of PR3 resulted in diminished CD14 expression on monocytes. In contrast, CD14 was increased in a subpopulation of cells that expressed major histocompatibility (MHC) class II and PR3, but lacked expression of CD64 and CD16. LPS mediated TNF-alpha production in whole blood was significantly inhibited when preincubated with PR3. This study demonstrates that PR3 can degrade rCD14 and that PR3 differentially affects CD14 expression in subsets of monocytes. ANCA IgG does not play a significant role herein.     

Alpha 1-antitrypsin deficiency and anti-proteinase 3 antibodies in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis.

alpha 1-antitrypsin (alpha 1-AT) is a naturally occurring inhibitor of proteinase 3 (PR3) and elastase, two of the target antigens of anti-neutrophil cytoplasmic antibodies (ANCA). An increased incidence of alpha 1-AT phenotypes associated with dysfunctional alpha 1-AT or low serum levels has been reported in patients with anti-PR3 antibodies. We have studied the relationship between ANCA, and phenotypes and serum levels of alpha 1-AT. Phenotypes usually associated with a moderate or severe reduction in alpha 1-AT serum levels or in dysfunctional activity were found more often in individuals with anti-PR3 antibodies than in the general population: four of the 31 patients (13%) with anti-PR3 antibodies had phenotypes MZ (n = 2), S (n = 1) or Z (n = 1) (P < 0.05). However, the corresponding alpha 1-AT serum levels were normal (n = 3) or elevated (n = 1). None of the 31 sera with anti-PR3 antibodies had low levels of alpha 1-AT. No abnormal alpha 1-AT phenotype was demonstrated in seven patients with anti-elastase antibodies, despite a low level of alpha 1-AT in one serum. Anti-myeloperoxidase antibodies are common in patients with ANCA, but no abnormal phenotype or low serum alpha 1-AT level was demonstrated in any of 29 sera containing these antibodies. Finally anti-glomerular basement membrane (GBM) antibodies occur occasionally in patients with ANCA-associated diseases, but again none of 10 sera had an abnormal alpha 1-AT phenotype or low serum level. ANCA were not demonstrated by indirect immunofluorescence in any serum from 73 patients with abnormal alpha 1-AT phenotypes. These results confirm that patients with anti-PR3 antibodies often have alpha 1-AT phenotypes that are usually associated with low serum levels of alpha 1-AT or with dysfunctional protein. Nevertheless, the incidence of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is very low. This probably reflects the rarity of Wegener's granulomatosis, the major disease associated with anti-PR3 antibodies, and the relative frequency of abnormal alpha 1-AT phenotypes. The mechanism for the development of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is not clear, but may relate to the increased propensity of unbound and uninhibited PR3 to stimulate autoantibody production.     

Opsonization of apoptotic neutrophils by anti-neutrophil cytoplasmic antibodies (ANCA) leads to enhanced uptake by macrophages and increased release of tumour necrosis factor-alpha (TNF-alpha)

Since proteinase 3 (PR3)-ANCA interact with PR3 on the surface of apoptotic polymorphonuclear neutrophils (PMN) and ingestion of apoptotic PMN is known to modulate macrophage inflammatory reactions, we raised the question whether PR3-ANCA-opsonized apoptotic PMN influence the uptake by macrophages and their state of activation. We therefore analysed the effects of PR3-ANCA-opsonized apoptotic PMN on the uptake process by enzymatic assay. We further investigated the production of TNF-alpha, IL-10, IL-12 and the secretion of lipid inflammatory mediators (TxB2, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2)) by human monocyte-derived macrophages using FACS and ELISA methods. We show that PMN-opsonization by PR3-ANCA substantially enhances phagocytosis by macrophages and thereby triggers the production of TNF-alpha and TxB2. These in vitro findings indicate that PR3-ANCA opsonization of apoptotic PMN might be an important mechanism in the pathogenesis of Wegener's granulomatosis (WG), prompting macrophages to produce proinflammatory mediators. These mediators, mainly TNF-alpha, might prime further PMN leading to perpetuation of the known priming-dependent mechanisms of ANCA action.     

Antigen specificities and clinical distribution of ANCA in kidney diseases

Summary The antigenic specificity and clinical distribution of the antineutrophil cytoplasmic antibodies (ANCA) in kidney diseases have recently been extensively studied. In patients with systemic vasculitis, the great predominance of two major ANCA antigens, proteinase 3 (PR3) and myeloperoxidase (MPO), is now established. PR3 and MPO are colocalized in the azurophilic granules of neutrophils and translocated to the cell surface during activation, and thus are able to interact with autoantibodies after neutrophil preactivation. Furthermore, by comparison of amino acid and DNA sequences, it has been shown that PR3 is identical to myeloblastin, which has been described independently and is involved in the control of growth and differentiation of leukemic cells. Aside from the two major ANCA antigens, a number of neutrophil cytoplasmic antigens recognized by ANCA have been identified, including human leukocyte elastase, lactoferrin, CAP57, and cathepsin G. These rare ANCA specificities occur in a limited number of patients. The variety of ANCA antigen specificities contrasts, however, with the fact that the vast majority of ANCA-positive sera are monospecific for one single ANCA antigen.With regard to clinical distribution, ANCA have major diagnostic significance in the four conditions in which they are frequently detected: Wegener's granulomatosis (WG), Churg and Strauss Syndrome (CSS), microscopic periarteritis (MPA), and necrotic and crescentic glomerulonephritis (NCGN). However, the initial dichotomy between MPO-associated vasculitis (NCGN, MPA) and that associated with anti-PR3 antibodies (WG) appears far from absolute.Abbreviations ANCA antineutrophil cytoplasm antibodies - PR3 proteinase 3 - MPO myeloperoxidase - CAP57 cationic antimicrobial protein 57 kDa - WG Wegener's granulomatosis - CSS Churg and Strauss syndrome - MPA microscopic periarteritis - NCGN necrotic and crescentic glomerulonephritis - IIF indirect immunofluorescence - HLE human leukocyte elastase - GBM glomerular basement membrane - IgAN IgA nephropathy - HSP Henoch-Schönlein purpura Preprint of a lecture to be read at the 22nd Congress of the Gesellschaft für Nephrologie, Heidelberg, September 15–18, 1991 (Editor: Prof. Dr. E. Ritz, Heidelberg)     

ANCA and associated diseases: immunodiagnostic and pathogenetic aspects

The past decade has seen an explosion of data on the new group of autoantibodies known collectively as ANCA (anti-neutrophil cytoplasmic antibodies). ANCA are specific for granule proteins of granulocytes and monocytes and induce distinct fluorescence patterns, e.g. the cytoplasmic (classic) cANCA and the perinuclear pANCA. cANCA is induced by antibodies directed against Proteinase 3 (PR3; PR3-ANCA) in about 90% of all ANCA-positive sera, and pANCA is induced by antibodies against myeloperoxidase (MPO; MPO-ANCA) in about 40%. A further staining pattern, which does not have a clear cut association with a distinct granule protein, is sometimes seen in chronic inflammatory bowel diseases. PR3-ANCA are serological markers for Wegener's granulomatosis (WG) and MPO-ANCA are associated with certain subtypes of primary vasculitides. Evidence exists that both the autoantigen and ANCA participate in the pathogenesis of at least the group of‘ANCA-associated vasculitides'.