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The detection of circulating autoantibodies against a variety of structural and functional molecules present in ubiquitous or tissue-specific cells is critical to the diagnosis of many autoimmune diseases (systemic - such as systemic rheumatic diseases - and organ-specific diseases). In particular, the determination of autoantibodies is one of the classification and/or diagnostic criteria for some autoimmune diseases and has a relevant predictive value, since many autoantibodies can be detected years before the clinical manifestation of a disease.The identification of these antigen-antibody systems and the simultaneous development of laboratory methods for detecting and measuring autoantibodies are considered one of the milestones in the history of clinical immunology over the last 60 years. Many different immunoassay methods have been developed and used in laboratory practice, from the early conventional (or monoplex) analytical methods that can detect single autoantibodies to the newer multiplex platforms that can quantify dozens of molecules. Various diagnostic immunoassays commonly used in current laboratory practice for the detection of autoantibodies are presented in this review., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Autoantibodies to nuclear antigens arise in human autoimmune diseases, but a unifying pathogenetic mechanism remains elusive. Recently we reported that exposure of neutrophils to inflammatory conditions induces the citrullination of core histones by peptidylarginine deiminase 4 (PAD4) and that patients with autoimmune disorders produce autoantibodies that recognize such citrullinated histones. Here we identify histone H1 as an additional substrate of PAD4, localize H1 within neutrophil extracellular traps, and detect autoantibodies to citrullinated H1 in 6% of sera from patients with systemic lupus erythematosus and Sjögren's syndrome. No preference for deiminated H1 was observed in healthy control sera and sera from patients with scleroderma or rheumatoid arthritis. We map binding to the winged helix of H1 and determine that citrulline 53 represents a key determinant of the autoantibody epitope. In addition, we quantitate RNA for H1 histone subtypes in mature human neutrophils and identify citrulline residues by liquid chromatography and tandem mass spectrometry. Our results indicate that deimination of linker histones generates new autoantibody epitopes with enhanced potential for stimulating autoreactive human B cells.-Dwivedi, N., Neeli, I., Schall, N., Wan, H., Desiderio, D. M., Csernok, E., Thompson, P. R., Dali, H., Briand, J.-P., Muller, S., Radic, M. Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity., (© FASEB.)

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To test the hypothesis that autoantigen modifications by peptidylarginine deiminase type 4 (PAD-4) increase immunoreactivity., Methods: We assembled sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Felty's syndrome (FS), and antineutrophil cytoplasmic antibody-associated vasculitides (AAVs), as well as sera from control subjects without autoimmune diseases. The sera were tested for binding to activated neutrophils, deiminated histones, and neutrophil extracellular chromatin traps (NETs). IgG binding to lipopolysaccharide-activated neutrophils was assessed with confocal microscopy, and binding to in vitro-deiminated histones was measured using enzyme-linked immunosorbent assay (ELISA) and Western blotting. In addition, we quantitated histone deimination in freshly isolated neutrophils from the blood of patients and control subjects., Results: Increased IgG reactivity with activated neutrophils, particularly binding to NETs, was paralleled by preferential binding to deiminated histones over nondeiminated histones by ELISA in a majority of sera from FS patients but only in a minority of sera from SLE and RA patients. Immunoblotting revealed autoantibody preference for deiminated histones H3, H4, and H2A in most FS patients and in a subset of SLE and RA patients. In patients with AAVs, serum IgG preferentially bound nondeiminated histones over deiminated histones. Increased levels of deiminated histones were detected in neutrophils from RA patients., Conclusion: Circulating autoantibodies in FS are preferentially directed against PAD-4-deiminated histones and bind to activated neutrophils and NETs. Thus, increased reactivity with modified autoantigens in FS implies a direct contribution of neutrophil activation and the production of NET-associated nuclear autoantigens in the initiation or progression of FS., (Copyright © 2012 by the American College of Rheumatology.)

The detection of autoantibodies is an important element in the diagnosis and monitoring of disease progression in patients with autoimmune diseases. In laboratory diagnostic tests for connective tissue and autoimmune liver diseases, indirect immunofluorescence on HEp-2 cells plays a central role in a multistage diagnostic process. Despite the high quality of diagnostics, findings at different laboratories can differ considerably due to a lack of standardization, as well as subjective factors. The present paper formulates recommendations for the standardized processing and interpretation of the HEp-2 cell test for the detection of non-organ-specific (especially antinuclear) antibodies. It provides requirements regarding the diagnostic tests used, instructions for laboratory procedure and evaluation, and recommendations for interpretation. For an optimal laboratory diagnostic process, it is useful to have an informative, tentative clinical diagnosis and an experienced laboratory diagnostician. In addition, the following key elements are recommended: initial screening using indirect immunofluorescence on carefully chosen HEp-2 cells beginning with a serum dilution of 1:80 and evaluation under a microscope with powerful illumination; results from a titer of 1:160 upwards being considered positive; internal laboratory quality control; and standardized interpretation. The aim is to improve diagnostic tests and care of patients with autoimmune diseases as a central concern of the European Autoimmunity Standardization Initiative (EASI).

Systemic autoimmune diseases are characterized by the presence of antinuclear autoantibodies (ANA). Diluted patient sera are typically used to screen for the presence of ANA by immunfluorescence microscopy with fixed HEp-2 cells. Despite high-quality test kits, reports of different laboratories frequently present controversial results. This article recommends unified processing and interpretation of HEp-2 based screening for autoantibodies. Suggestions are made for the selection of high-quality test kits, optimized processing and diagnostic procedures. In addition to a relevant clinical diagnosis and an experienced laboratory specialist, the following procedure is highly recommended to achieve good laboratory practice: Initial HEp-2 based screening by indirect immunofluorescence, starting with a 1:80 serum dilution, and evaluation in a bright fluorescence microscope, pathological values from a titer of 1:160 upwards, internal quality checks and unified interpretation. We aim to improve diagnosis and care of patients with autoimmune diseases as a central focus of the European Autoimmunity Standardization Initiative (EASI).

Objective: Cytokines and growth factors can be a target of autoantibodies in systemic inflammatory diseases. We examined whether patients with neutropenia and either Felty's syndrome (FS) or systemic lupus erythematosus (SLE) have autoantibodies against granulocyte colony-stimulating factor (G-CSF) and whether these autoantibodies are functionally relevant., Methods: Fifteen patients with neutropenia due to FS were matched for age, sex, and disease activity with 16 normocytic rheumatoid arthritis (RA) control patients. Sixteen patients with SLE and neutropenia were matched with 16 normocytic SLE control patients. Antibodies against G-CSF were measured by enzyme-linked immunosorbent assay and Western blotting. Antibody specificity was verified by competitive inhibition using recombinant human G-CSF. The effect of anti-G-CSF antibodies on the functional activity of their target molecule was measured in a bioassay using G-CSF-sensitive murine 32D cells., Results: IgG anti-G-CSF was found in 11 FS patients, 6 SLE patients with neutropenia, 6 SLE control patients, and none of the RA control patients. IgM anti-G-CSF was found in 6 neutropenic and 3 normocytic SLE patients. Anti-G-CSF antibodies were associated with an exaggerated serum level of G-CSF and a low neutrophil count. A neutralizing effect of anti-G-CSF antibodies on its target molecule was found in 3 of the 9 patients tested. Irrespective of the presence or absence of anti-G-CSF antibodies, neutropenic patients with FS and SLE had exaggerated serum levels of G-CSF., Conclusion: Anti-G-CSF autoantibodies are common in neutropenia due to FS and SLE. In individual patients, these autoantibodies have a neutralizing capacity. In patients without neutralizing antibodies, hyposensitivity of the myeloid cells to G-CSF appears to be central to the pathogenesis of the neutropenia in FS and SLE.

Background: Bactericidal permeability increasing protein (BPI) is an antibacterial product of neutrophilic granulocytes that can serve as target antigen for antineutrophil cytoplasmic antibodies (ANCA). The clinical associations of autoantibodies against BPI (BPI-ANCA) are essentially unclear., Patients and Methods: 587 sera from patients with chronic inflammatory bowel diseases, inflammatory hepatobiliary diseases, primary systemic vasculitides and other rheumatological diseases were examined for BPI-ANCA by mono-specific ELISA and a standard indirect immunofluorescence test for ANCA. (ACD-CPR versus S-CPR). The treatment groups were similar with respect to age, sex, time interval from collapse to CPR, defibrillation and first epinephrine medication. There was no difference between the ACD group and the standard CPR group in terms of survival rates and neurologic outcome. No differences occurred concerning complications of CPR., Conclusion: In our two-tiered EMS system with physician-staffed ambulances ACD-CPR neither improved nor impaired the survival rates and the neurological prognosis in patients with out-of-hospital cardiac arrest. Our results are in accordance with other studies carried out in EMS systems, with first tier call-response intervals between 4 and 6 min., Results: The prevalence of BPI-ANCA was 43% in ulcerative colitis, 23% in Crohn's disease, 35% in primary sclerosing cholangitis, 25% in primary biliary cirrhosis and 29% in autoimmune hepatitides. In a spectrum of systemic vasculitides, inflammatory joint diseases and collagen vascular diseases the prevalence was only 3 to 11%. In contrast to PR3-ANCA and MPO-ANCA, BPI-ANCA was not associated with a particular pattern of fluorescence in the immuno-fluorescence test on ethanol- and formalin-fixed neutrophils., Conclusion: This study shows that BPI-ANCA is the third ANCA specificity, besides PR3-ANCA and MPO-ANCA, with a limited spectrum of clinical associations. The diagnostic and prognostic relevance of BPI-ANCA in the above clinical conditions is being examined prospectively.

Among the anti-neutrophil cytoplasmic antibodies (ANCA), those targeting proteinase 3 (PR3) have a high specificity for Wegener's granulomatosis (WG). It is known that a preceding priming of neutrophils with cytokines is a prerequisite for membrane surface expression of PR3, which is then accessible to autoantibody binding. Employing a monoclonal antibody directed against human PR3 and ANCA-positive serum from WG patients with specificity for PR3, we now investigated the role of free arachidonic acid (AA) in autoantibody-related human neutrophil activation. Priming of neutrophils with tumor necrosis factor (TNF-alpha) for 15 min or exposure to anti-PR3 antibodies or incubation with free AA (10 microM) as sole events did not provoke superoxide generation, elastase secretion or generation of 5-lipoxygenase products of AA. Similarly, the combination of TNF-alpha-priming and AA incubation was ineffective. When TNF-alpha-primed neutrophils were stimulated by anti-PR3 antibodies, superoxide and elastase secretion was provoked in the absence of lipid mediator generation. However, when free AA was additionally provided, a strong activation of the 5-lipoxygenase pathway was demasked, with the appearance of excessive quantities of leukotriene (LT)B4, LTA4, and 5-hydroxyeicosatetraenoic acid. Moreover, superoxide and elastase secretion were markedly amplified, and studies with 5-lipoxygenase inhibitors and a LTB4-antagonist demonstrated this was due to an LTB4-related autocrine loop of cell activation. In contrast, the increased synthesis of platelet-activating factor in response to TNF-alpha-priming and anti-PR3 stimulation did not contribute to the amplification loop of neutrophil activation under the given conditions. We conclude that anti-PR3 antibodies are potent inductors of the 5-lipoxygenase pathway in primed human neutrophils, and extracellular free AA, as provided at an inflammatory focus, synergizes with the autoantibodies to evoke full-blown lipid mediator generation, granule secretion and respiratory burst. Such events may be enrolled in the pathogenesis of focal necrotizing vascular injury in Wegener's granulomatosis.

TGF-beta is a multifunctional cytokine modulating the onset and course of autoimmune diseases as shown in experimental models. The aim of this study was to investigate TGF-beta expression in ANCA-associated vasculitis (AAV), and the possible interactions of this cytokine with lysosomal enzymes identified as ANCA autoantigens (e.g. PR3). This included TGF-beta effects on the translocation of the lysosomal enzymes to the cell surface of polymorphonuclear neutrophils (PMN), and the presumed activation of non-bioactive, latent TGF-beta by these enzymes. Patients with various types of systemic vasculitis (SV) were studied, including three different types of AAV (Wegener's granulomatosis (WG), Churg-Strauss syndrome (CSS) and microscopic polyangiitis (MPA)). Regardless of the type of assay applied, the TGF-beta 1 isoform was found to be over-expressed in SV, including AAV, and to correlate with disease activity as shown for WG. Mean TGF-beta 1 plasma levels in AAV patients ranged from 8.9 ng/ml (WG) to 13.3 ng/ml (CSS) (control 4.2 ng/ml; P < 0.01), while TGF-beta 2 levels were not elevated. Flow cytometry analysis showed TGF-beta 1 to be a potent translocation factor for PR3 comparable to other neutrophil-activating factors such as IL-8. PR3 membrane expression on primed PMN increased by up to 51% after incubation with TGF-beta 1. PR3 itself was revealed as a potent activator of latent TGF-beta, thus mediating bioeffects of this cytokine. These findings, together with other features of TGF-beta such as induction of angiogenesis and its strong chemotactic capacity, indicate that TGF-beta might serve as a proinflammatory factor in SV, especially in AAV.

Objective: To determine the incidence of antineutrophil cytoplasmic antibodies (ANCA) in rheumatoid arthritis (RA), to detect the target antigens of ANCA, and to compare clinical and laboratory data of ANCA+ with ANCA- patients with RA., Methods: 385 sera of patients with RA were screened for ANCA by indirect immunofluorescence. ANCA+ sera were further analyzed for target antigens by ELISA: The ANCA+ patients were compared to randomly selected ANCA- patients with RA with respect to serological and radiological variables and extraarticular involvement., Results: ANCA were found in 16% of patients with RA (61/385 sera). All sera showed a perinuclear (pANCA) pattern. Antibodies directed against proteinase 3 were not observed. The analysis of ANCA+ and ANCA- patients revealed that the pANCA+ group exhibited significantly higher serological markers of inflammation (p < 0.005) and a higher incidence of rheumatoid factor (p < 0.005). Furthermore, vasculitic involvement was found at a higher frequency (p < 0.05) in the pANCA+ group. Five patients in the pANCA+ group had pulmonary involvement, but none in the pANCA- group., Conclusion: pANCA in RA may be a marker for a more aggressive course of disease in respect to serological variables and extraarticular manifestations including rheumatoid vasculitis and lung involvement.

It was our purpose to determine the immunodiagnostic value of ANCA directed against BPI in diseases known to be associated with ANCA, such as ANCA-associated vasculitides, inflammatory bowel disease (IBD) and the associated condition primary sclerosing cholangitis. The immunoreactivity of recombinant PBI (rBPI) was established in order to develop an ELISA specific for rBPI. By means of this assay, BPI-ANCA were assessed in sera of 178 patients with IBD or the associated disorder primary sclerosing cholangitis, 112 patients with ANCA-associated vasculitides, and in sera of 182 disease and 140 health controls. BPI-ANCA were found to be closely associated with IBD and primary sclerosing cholangitis (34% and 44% of ANCA-positive sera, respectively). By contrast, BPI-ANCA positivity was low (<10%) in the double-negative sera of patients with ANCA-associated vasculitides and in disease and health controls. BPI-ANCA appear to constitute an important marker for IBD and primary sclerosing cholangitis, but not for the ANCA-associated vasculitides.

In this uncontrolled study 15 patients with ANCA-associated systemic vasculitis, who were poor responders to conventional therapy, were treated with single or multiple courses of intravenous immunoglobulin (IVIG), 30 g/day over 5 days. Clinical and serological evaluation was performed before and 4 weeks after IVIG. Six of the 15 patients experienced clinically significant benefit from IVIG. Improvement was confined to single organ manifestations (skin, ENT findings), no improvement was seen with conjunctivitis and scleritis, pericarditis or nephritis. No patient experienced complete remission after IVIG. Repeated courses of IVIG at 4-week intervals were no more effective than single courses. In six anti-proteinase 3 (PR3)-positive patients pretreatment sera were incubated with F(ab')2 fragments of the IVIG preparation in vitro to measure the inhibitory effect of IVIG on anti-PR3 activity. An inhibition of anti-PR3 activity by 25-70% was observed; this did not correlate with clinical effects. Approximately 40% of patients benefited from IVIG treatment, though complete remission of disease activity did not occur. Neither clinical characteristics nor the inhibitory effect of the IVIG preparation on serum anti-PR3 activity in vitro predicted clinical response to this treatment modality.

Objective: To examine the prevalence, subspecificities, and clinical associations of antineutrophil cytoplasmic antibodies (ANCA) in patients with systemic lupus erythematosus (SLE)., Methods: One hundred fifty-seven sera from 120 patients with SLE were examined for classic (c) and perinuclear (p) pattern ANCA by indirect immunofluorescence. Antibody subspecificities were determined by enzyme-linked immunosorbent assay (ELISA). Serologic results were correlated with clinical manifestations as categorized by the BILAG (British Isles Lupus Assessment Group) index., Results: ANCA were found in 40 of the 157 sera (25%). Only a pANCA, not a cANCA, pattern of fluorescence was seen. By ELISA testing, 16 sera reacted to lactoferrin, 8 to elastase, and 4 to lysozyme. There was no reactivity to proteinase 3 (PR3) or myeloperoxidase (MPO). No correlation of pANCA, or any of the ANCA subspecificities, with organ system involvement, as categorized by the BILAG index, was found. Notably, there was no correlation of ANCA results with lupus vasculitis., Conclusion: The absence of cANCA, anti-PR3, and anti-MPO shows that with appropriate assay conditions, ANCA testing assists in the differentiation between SLE and the ANCA-associated vasculitides. The lack of a correlation between pANCA or any ANCA subspecificity and clinical manifestations suggests that ANCA do not identify particular clinical subsets among SLE patients, including those with lupus vasculitis.

Antineutrophil cytoplasmic antibodies (ANCA) encompass a heterogeneous group of autoantibodies targeting antigens in neutrophils (PMN), monocytes, and endothelial cells. ANCA are routinely detected by the indirect immunofluorescence technique (IFT) and at least three different patterns of fluorescence can be distinguished which have been assigned the acronyms cANCA, pANCA and aANCA. cANCA is mostly induced by proteinase 3 (PR3) antibodies (PR3-ANCA), and pANCA by myeloperoxidase (MPO) antibodies (MPO-ANCA), while aANCA has unidentified subspecificity. Over the past decade, ANCA have been the subject of extensive investigation. They have proved to be of significant value both as diagnostic tools and for follow-up in several forms of systemic vasculitis (e.g. Wegener's granulomatosis, WG; microscopic polyarteritis, MPA; Churg-Strauss syndrome, CSS) which are now termed 'ANCA-associated vasculitides'. Furthermore, it is suspected that the presence of ANCA is an important factor in the pathogenesis of these disease groups. Data regarding the detection of ANCA and their diagnostic value and role in the pathogenesis of vasculitic disorders will be discussed in this review. Growing evidence points to a pathophysiological and diagnostic relevance of the distribution of the ANCA target antigens PR3 and MPO (presence in the circulation, on cell membranes, and in tissue extracellularly). An autoimmune process has been implicated in the pathogenesis of ANCA-associated vasculitis, but it is uncertain which mechanism underlies the induction of the ANCA-related immunoresponse. In this paper mechanisms such as antigenic cross-reactivity between human PMN proteins and extrinsic antigens by molecular mimicry, idiotypic immunoglobulin regulation, and T-cell reactivity to PR3 and MPO will be discussed.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a heterogeneous group of autoantibodies with a wide and diverse range of clinical associations. Over the past decade ANCA have been the subject of extensive investigations. In vasculitis the diagnostic utility of proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA for Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA), respectively, is now well established. Because of their significance as tools for diagnosis and prognosis of "ANCA-associated vasculitides," these autoantibodies have been analyzed extensively as markers for underlying immunopathogenic disturbances. Data regarding the detection of ANCA and their diagnostic value and role in the pathogenesis of rheumatic disorders will be discussed in this review. Growing evidence points to a pathophysiologic and diagnostic relevance of the ANCA target antigens PR3 and MPO. To date, there is mounting evidence that ANCA can have pathophysiologic effects on neutrophils, and may play a direct role in ANCA-associated vasculitides. A pathogenic model for ANCA-mediated vasculitis will be presented in this paper.

Antineutrophil cytoplasmic antibodies (ANCA) are a heterogeneous group of autoantibodies with a wide and diverse range of clinical associations. In vasculitis, the diagnostic utility of proteinase 3 (PR3)-ANCA and myeloperoxidase-ANCA for Wegener's granulomatosis and microscopic polyangiitis, respectively, is now well established. Because of their significance as tools for diagnosis and prognosis, these autoantibodies have been analyzed extensively as markers for underlying immunopathogenic disturbances. In this review, we consider recent advances in the understanding of ANCA, focusing on their detection, diagnostic value, and role in the pathogenesis of vasculitis. In addition, promising new ways have been developed to elucidate the pathophysiologic and diagnostic relevance of the ANCA target antigens PR3 and myeloperoxidase. A great deal of attention and controversy has focused on the possible mechanisms underlying the ANCA-related immune response, such as antigenic cross-reactivity between human polymorphonuclear leukocyte proteins and extrinsic antigens by molecular mimicry, idiotype network regulation, and T cell reactivity to PR3 and myeloperoxidase.

The presence of anti-neutrophil cytoplasmic Abs (ANCA) in many patients with systemic vasculitis suggests that ANCA may play a role in disease pathogenesis. Neutrophils from patients with Wegener's granulomatosis often express ANCA target Ags (myeloperoxidase (MPO) and proteinase 3 (PR3)) on their surface, making these intracellular primary granule enzymes accessible to these autoantibodies. Similarly, normal neutrophils can be induced to translocate MPO and PR3 to the cell surface in vitro, and we demonstrate that murine mAb ANCA IgG, but not IgM, binds to the ANCA target and engages the Fc gamma RIIa ligand-binding site on the surface of human neutrophils. In contrast to ANCA IgM, ANCA IgG also induces an oxidative burst in neutrophils (oxidation of dihydrorhodamine = 91 +/- 15 fluorescence units with anti-PR3 IgG vs 17 +/- 2 with anti-PR3 IgM, p < 0.001). Blockade of the ligand-binding site of Fc gamma RIIa with an antibinding site mAb Fab significantly reduces this ANCA IgG-triggered production of reactive oxygen species (p < 0.01). Similarly, human ANCA bind the ANCA target, engage Fc gamma RIIa, and induce an oxidative burst in neutrophils. The allelic phenotype of Fc gamma RIIa strongly influences the Fc gamma receptor engagement by ligand, and Fc gamma RIIa homozygous donors differ by more than threefold in the quantitative production of reactive oxygen intermediates (ROI) (p < 0.01). Thus, engagement of Fc gamma RIIa by the Fc region of ANCA is one mechanism by which these autoantibodies activate receptor-mediated signal transduction systems in human neutrophils to initiate programs of inflammation and tissue injury. Fc gamma receptor alleles may represent heritable disease risk factors influencing the magnitude of such a process.

Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (PR-3), have proved to be a useful clinical tool confirming the diagnosis or monitoring disease activity of Wegener's granulomatosis (WG). Although several concepts concerning the pathophysiologic potentials of ANCA have been discussed, only sparse data about ANCA-endothelium interactions have been available. In this study, we have investigated the expression of PR-3 in cytokine-treated human endothelial cells using purified anti-PR-3 antibodies of patients with WG, murine and human monoclonal anti-PR-3 antibodies as probes. We were able to show that tumor necrosis factor-alpha, interleukin-1 alpha/beta, and interferon-gamma led to an increased PR-3 expression in the cytoplasm of endothelial cells by performing polymerase chain reaction analysis, Western blot, cyto-enzyme-linked immunosorbent assays, and confocal laser scanning microscopy. Moreover, PR-3 was also translocated into the cell membrane, becoming accessible to ANCA. Our data suggest a possible direct pathogenic effect of anti-PR-3 antibodies in WG and other vasculitides. Anti-PR-3 antibodies represent an important missing link in ANCA-endothelial interactions.

A real explosion of data is seen concerning the detection of autoantibodies collectively known as antineutrophil cytoplasmic antibodies (ANCA) recognizing granule proteins of granulocytes and monocytes. Distinct fluorescence staining patterns result from their diverse substrate specificities, and can even identify several disease entities mainly in the etiopathogenetically unclear group of primary systemic vasculitides (PSV). The well-documented disease specificity of ANCA recognizing proteinase 3 (PR3), and recent in vitro findings concerning the effect of cytokines on immunocompetent cells including the endothelium are the rationale for the presentation of a new model explaining the development of systemic vasculitis.

Wegener's autoantigen (WA), a 29 kD multifunctional protein, is the principal target antigen of autoantibodies associated with Wegener's granulomatosis (WG). WA was first identified as proteinase 3 (PR3), which is now known to be identical with myeloblastin and AGP7. Like other lysosomal proteins, WA/PR3 displays enzymatic activity, differentiation factor activity for myeloid precursor cells, and antimicrobial functions. Neutrophilic polymorphonuclear leukocytes (PMN) and a subpopulation of monocytes contain high levels of WA/PR3 in their myeloperoxidase-positive granules. The autoantibodies from WG sera produce a finely granular, centrally accentuated fluorescence pattern on PMN and monocytes and have been designated 'classic' pattern antineutrophil cytoplasmic autoantibodies (cANCA). However, PMN/monocyte activation (in vitro/ex vivo) is associated with the translocation of WA/PR3 on the cytoplasm membrane. WA/PR3 is accessible to the WG-associated autoantibody: cANCA stimulate cytokine-preactivated PMN to produce oxygen radicals and to degranulate. Furthermore, cANCA interfere with the biological functions of WA/PR3 (e.g. inhibition of elastinolytic activity). Hence, cANCA represents not only the best seromarker for WG so far available, but several lines of evidence indicate that the autoantibodies against WA/PR3 play a major role in the pathogenesis of this enigmatic disease.

It was the aim of this study to reevaluate the diagnostic significance and clinical implication of ANCA after testing sera from 13,606 patients for the presence of ANCA. Our data confirm the high specificity (97%) and sensitivity (80%) of cANCA for Wegener's granulomatosis. pANCA were found in renal vasculitides (60%), collagen vascular diseases (SLE 20%, Sjögren's syndrome 26%, polymyositis 16%) and rheumatic disorders (Felty's syndrome 50%, rheumatoid arthritis 20%). A third fluorescence pattern in sera of patients with inflammatory bowel disease (ulcerative colitis 28/72, Crohn's disease 6/84), here called xANCA, was seen. Target antigens of granule proteins from PMN and monocytes (proteinase 3, myeloperoxidase, elastase, cathepsin G, lactoferrin, lysozyme) were identified.

We studied the presence of proteinase 3 (PR3), myeloperoxidase (MPO) and elastase (HLE) on the plasma membrane of neutrophils in patients with biopsy-proven Wegener's disease (WG), pANCA-positive vasculitis, control patients (SLE, rheumatoid arthritis, ankylosing spondylitis), sepsis patients and healthy donors. We found an overexpression of PR3 on the cell surface of neutrophils in WG, ANCA-associated vasculitis and during infection (sepsis). Thus PR3 becomes accessible to ANCA. Furthermore we detected intracytoplasmic IgG antibodies in PMN from patients with WG by immunoelectron microscopy and direct immunofluorescence. Our findings support the pathophysiological role of ANCA.

Several concepts concerning the pathogenicity of antineutrophil cytoplasmic antibodies (ANCA) exist, but till now only sparse data about ANCA-endothelial interactions are available. In this study we have investigated the expression of proteinase 3 (PR-3) in human umbilical endothelial cells (HEC) using purified anti-PR-3 antibodies (C-ANCA) of patients with Wegener's granulomatosis (WG) and monoclonal antibodies to PR-3 (human and murine) as probes. Performing cyto-ELISAs, laser scanning microscopy and Western blot we were able to show that treatment of HEC with IL-1-alpha led to an increased PR-3 expression in the cytoplasm and to a transient translocation into the EC-membrane. Representing an important missing link of ANCA-endothelial interactions, our data give a hint at a possible direct pathogenicity of anti-PR-3 antibodies in WG and other vasculitides.

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'.

ANCA-positive sera from 1138 patients and ANCA-negative sera from 90 patients were screened for autoantibodies directed against lysozyme (LZ) by ELISA. Sera from 120 patients did react with LZ. 99 sera bound to LZ only, whereas 56 sera bound to further granule proteins, especially cathepsin G and lactoferrin. In the routine ANCA screening, most of the anti-LZ-positive sera showed a pANCA fluorescence. In total, 8% of 674 pANCA-positive sera did react with LZ. Clinically, anti-LZ antibodies were associated inflammatory rheumatologic, -renal and -bowel diseases.

As other vasculitic-syndromes Takayasu's-Arteritis needs a long-term immunosuppressive therapy in order to control disease-activity. As steroids create many problems in the long run other immunosuppressives should be considered. We report 3 cases treated with low-dose methotrexate. In all patients the arteritis was controlled, steroids reduced or stopped. In the effort to find autoantibodies 16 patients were screened for c-/p-ANCA and neutrophil granular enzymes. In no patient a positive result could be obtained.

Antineutrophil cytoplasmic autoantibodies (ANCA) specific for constituents of neutrophil primary granules and monocyte lysosomes have been demonstrated in various vasculitic disorders. The staining pattern in indirect immunofluorescence microscopy using alcohol-fixed neutrophils as substrate allows distinction among 3 types of ANCA: 1) classic anti-neutrophil cytoplasmic antibody (cANCA, formerly known as ACPA); 2) a type with a perinuclear/nuclear staining pattern produced when alcohol-fixed neutrophils are used as substrate (pANCA); and 3) a mixture of both of the above types (xANCA, also described recently as pANCA). Most cANCA are directed against proteinase 3 ("Wegener's autoantigen"). Some pANCA have specificity for myeloperoxidase and are associated with idiopathic crescentic glomerulonephritis ("renal vasculitis") and other systemic vasculitides exhibiting a paucity of immune deposits in blood vessels. In addition to being a useful serological marker, ANCA appear to be directly involved in the pathogenesis of systemic vasculitis. ANCA can activate cytokine-primed granulocytes and monocytes to undergo a respiratory burst and degranulation. This effect leads to vasculitis through the attachment of these cells to the vascular endothelium primed by cytokine-induced expression of adhesion molecules (E-LAM 1) on the endothelium. Thus, the release of toxic oxygen radicals and lytic enzymes is capable of causing vascular damage. In the present paper we report on the main target antigens and on the history, nomenclature, laboratory methods, and etiopathological implication of ANCA. Additional pathophysiological aspects of ANCA and/or autoreactive T cells and immunoregulatory events are also discussed.

Autoantibodies against neutrophil cytoplasmic antigens (ANCA) produce two major immunofluorescence (IF) patterns on ethanol-fixed granulocytes: the "classical" (centrally accentuated) C-ANCA, associated with Wegener's granulomatosis (WG), and P-ANCA (perinuclear), which mainly occur in renal vasculitis. Rheumatic manifestations are an important clinical finding in systemic vasculitis, often preceding a fulminant course and sometimes imitating various rheumatic disorders. We analyzed the incidence of ANCA in rheumatic patients and looked for the frequency of rheumatic symptoms in systemic vasculitis. In WG (n = 186), we found rheumatic symptoms in 55% (myalgia, 45%; arthritis, 21%); in 90%, rheumatic complaints were associated with active vasculitis. In 730 patients with various rheumatic conditions (eg, 268 rheumatoid arthritis, 130 systemic lupus erythematosis [SLE], 32 sharp-S, 50 ankylosing spondylitis, 43 systemic sclerosis) no C-ANCA were found. On the contrary, the P-ANCA pattern was seen in seven of 62 giant cell arteritis, five of 27 Felty's/Still's syndrome, and four of 130 SLE patients in addition to renal vasculitis (21/74). We demonstrated that 95% of C-ANCA-positive sera react with proteinase 3 (PR3 or myeloblastin). Using monoclonal antibodies, we showed that PR3 is expressed on the plasma membrane of neutrophil granulocytes and monocytes; thus, PR3 autoantigens are accessible for circulating antibodies. The detection of ANCA in sera from vasculitis and other rheumatic diseases is of immunodiagnostic value and provides new insight in the pathogenesis of systemic vasculitides.

Objective: To study immunological markers and compare these markers with standard measures for the clinical and immunological follow up of vasculitis activity in hepatitis C virus (HCV) associated cryoglobulinaemic vasculitis (CV).Methods: Serial serum samples from eight patients with newly diagnosed HCV associated CV were followed during interferon alpha treatment induced remission of the CV. Vasculitis activity and disease extent were evaluated with the Birmingham vasculitis activity score (BVAS) and disease extent index (DEI). Cryoglobulinaemia, complement levels (C3c, C4, and CH50), rheumatoid factor (RF), autoantibodies such as antinuclear antibodies, soluble interleukin 2 receptor (sIL2r), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble CD30 (sCD30) were determined.Results: All patients achieved either complete or partial remission of their CV during interferon alpha treatment. There was a significant reduction in vasculitis activity and disease extent (BVAS, DEI), cryoglobulinaemia, RF, sIL2r, sICAM-1, and sCD30. Complement C3c levels increased significantly during this period. Erythrocyte sedimentation rate and levels of complement C4 and CH50 did not change significantly. Both clinical measures (BVAS and DEI) correlated significantly only with C3c and sCD30.Conclusions: Although this study was of only a small group of patients, it shows that BVAS and DEI as clinical measures and C3c and sCD30 as immunological markers may be useful in the follow up of disease activity of HCV associated CV. The data indicate that activity of the humoral (cryoglobulinaemia, RF, autoantibodies) and cellular (sIL2r, sICAM-1, sCD30) immune response and endothelial damage (sICAM-1) are found in HCV associated CV. [ABSTRACT FROM AUTHOR]

Objective: To examine the prevalence, binding sites and functional interactions of antineutrophil cytoplasmic autoantibodies (ANCA) against the bactericidal/permeability increasing protein (BPI) in reactive arthritis (ReA).Methods: Sera were analysed for the occurrence of ANCA by indirect immunofluorescence microscopy (IIF) and ELISA. Binding sites were determined using BPI, lipopolysaccharid binding protein (LBP), and fusion proteins of both proteins in ELISA. In addition, the effect of antibodies on the antibiotic activity of BPI was examined.Results: BPI-ANCA was found in patients with Yersinia- and Salmonella-triggered ReA and directed against the C-terminal portion of BPI. Goat anti BPI antibodies recognising this part inhibited the antibiotic activity of BPI in vitro.Conclusion: BPI-ANCA was associated with ReA triggered by Salmonella and Yersinia infection. Directed against the C-terminal part of BPI, it can potentially inhibit its antibiotic activity and might be useful to identify patients with infectious bowel disease prone to extraintestinal sequelae. [ABSTRACT FROM AUTHOR]