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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.)

Objectives: Polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) are relatively common inflammatory disorders. Establishing the diagnosis however may be difficult, since so far no specific biomarkers of the disorders are available., Methods: As a screening procedure, the authors used protein arrays for the detection of new autoantigens in GCA and PMR. The results of the protein array were confirmed by different ELISAs detecting IgG antibodies against the human ferritin heavy chain, N-terminal 27 amino acids of the human ferritin heavy chain or the homologous peptide of Staphylococcus epidermidis. Sera of patients with only GCA (n=64), only PMR (n=47) and both PMR and GCA (n=31) were used., Results: In the ELISA using the human ferritin peptide, the sensitivity of IgG antibodies against ferritin was 92% in 36 GCA and/or PMR patients before initiation of treatment, 22/32 (69%) in patients with disease flares and 64/117 (55%) in the total cohort including treated and inactive patients. In controls, the false positive rate was 11/38 (29%) in systemic lupus erythematosus, 1/36 (3%) in rheumatoid arthritis, 0/31 (0%) in late onset rheumatoid arthritis, 3/46 (6.5%) in B-non-Hodgkin's lymphoma and 1/100 (1%) in blood donors. In the ELISA using the ferritin peptide of S epidermidis, 89% of 27 patients with untreated GCA and PMR were positive., Conclusion: Antibodies against the ferritin peptide were present in up to 92% of untreated, active GCA and PMR patients. They can be useful as a diagnostic marker of PMR and GCA.

Wegener's granulomatosis (WG) is a severe autoimmune disorder ranging from localized granulomatous disease to generalised anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. A previous analysis of immunoglobulin heavy chain genes derived from tissue, i.e. Wegener's granuloma indicated selection and affinity maturation towards local antigen(s). The current study focused on determining the specificity of immunoglobulins from distinct B lymphocytes out of Wegener's granuloma. Four pairs of variable region immunoglobulin light and heavy chain genes, isolated before, were recombinantly expressed using the baculovirus/insect cell system. These immunoglobulins were then analysed for their antigenic target employing a protein macroarray based upon a human fetal brain tissue cDNA expression library. The lysosomal transmembrane protein 9B, a key regulator for TNFα activation, was identified as the putative antigenic target of two immunoglobulins and a tetraspanin, which might play a role in leukocyte activation and motility, was identified as the putative antigenic target of another one. Recombinant monoclonal antibodies out of Wegener's granuloma represent a new tool aiding in elucidation of its and WG immunopathogenesis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

History: A 27-year-old woman from Cameroon was admitted because of arthralgia, myalgia and severe thrombocytopenia (20,000/ micro l). She had been suffering from weakness, recurrent febrile episodes, generalized lymphadenopathy and pancytopenia for 2 years. Having a typical autoantibody constellation and fulfilling four (pleurisy, autoimmune-hemolytic anemia, antinuclear antibodies (ANA), anti-Sm antibodies) of the American College of Rheumatology (ACR) classification criteria, systemic lupus erythematosus (SLE) had been diagnosed at another hospital. Treatment with corticosteroids and azathioprine did not improve her condition., Investigations: Abnormal laboratory findings were pancytopenia, elevated markers of inflammation and extreme hypergammaglobulinemia (70 %) with polyclonal IgM (73 g/l). Antinuclear antibodies (ANA), anti-Sm-, anti-Scl 70-, anti-U1-RNP-, anti-histo-, anti-leukocyte- and IgM anticardiolipin antibodies were detected. A bone marrow biopsy showed polyclonal B-cell and plasma cell infiltrates. Examination of peripheral blood smears disclosed trypanosoma brucei infection., Diagnosis, Treatment and Course: After the diagnosis of stage 2 West African trypanosomiasis (sleeping sickness) specific treatment was initiated leading to subsequent remission of the disease., Conclusion: This case report underlines the importance of a thorough differential diagnosis in cases of suspected autoimmune disease. Induction of autoantibodies during infectious diseases may be misleading. The use of the ACR criteria for SLE must be restricted to the classification of proven connective tissue diseases.

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.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a system of autoantibodies which are strongly associated with the primary systemic vasculitides (PSV). cANCA, as detected by indirect immunofluorescence, are mostly reactive to proteinase 3 (PR3) and bear high sensitivity and specificity for Wegener's granulomatosis. pANCA have varied subspecificities and clinical associations. The most important subspecificity of pANCA is anti-myeloperoxidase (MPO), which is strongly associated with microscopic polyaniitis and pauci-immune crescentic glomerulonephritis. pANCA also occur at low to moderate frequency in other PSV, collagen vascular disease, inflammatory bowel disease and autoimmune liver disease. In systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), pANCA of varied subspecificity have been found, including anti-MPO at low rate and low titer, while cANCA and anti-PR3 were generally absent. Consequently, anti-PR3 (PR3-ANCA) and anti-MPO (MPO-ANCA) at moderate and high titer are distinguishing features of the PSV and, in an appropriate clinical setting, argue strongly against the presence of SLE or RA. Since no significant clinical association has been found for other pANCA subspecificities, cANCA, PR3-ANCA and MPO-ANCA remain the critical elements of ANCA testing in the clinical diagnosis of generalized autoimmune diseases.

This article summarizes the most recent findings concerning the clinical relevance of antineutrophil cytoplasmic autoantibody (ANCA) testing for patients with idiopathic vasculitis and with diseases known to be associated with secondary vasculitis. The clinical value of granular cytoplasmic pattern (c)ANCA (proteinase 3 [PR3]-ANCA) and perinuclear fluorescence pattern (p)ANCA (myeloperoxidase [MPO]-ANCA) testing in Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA), respectively, is now well established; however, the various subspecificities beside myeloperoxidase (MPO), which also include the perinuclear staining pattern, are detectable not only in vasculitis, but equally in a heterogeneous patient population with a spectrum of autoimmune diseases and idiopathic chronic inflammatory diseases of the bowel, liver, and so forth. Future studies must establish the specificity, sensitivity, and role of these pANCA subspecificities usually measured by enzyme-linked immunosorbent assays for distinct disease entities in clinical medicine. In summary, despite the relatively poor understanding of the immunopathogenesis of ANCA-associated disease, cANCA (PR3-ANCA) and pANCA (MPO-ANCA) continue to be important clinical markers of the so-called "ANCA-associated vasculitides" (i.e., WG, MPA, and Churg-Strauss syndrome).

ANCA-associated vasculitides (AAV) include primary vasculitides that affect predominantly small vessels such as Wegener's Granulomatosis (WG), Microscopic Polyangiitis (MPA) and Churg-Strauss Syndrome (CSS). The former is closely associated with cANCA, induced by proteinase 3, MPA is characterized by pANCA with MPO specificity and the latter, CSS, can--to a far lesser extent--be associated with, either c- or pANCA. All AAV have in common (as opposed to immune-complex-vasculitides) that they occur without complement consumption and show histologically no deposition of immune complexes (pauci-immune vasculitis). Clinically, the AAV show quite variable courses. The mostly clinically rather inapparent initial phase, which is typically granulomatous in WG and CSS, is mostly followed by a potentially life-threatening phase of full blown generalized systemic vasculitis. Frequently, rheumatic complaints are the initial symptom. The characteristic clinical features in the full blown generalized vasculitis are mostly found in the upper respiratory tract, the lung and/or the kidney. Subsequently, the clinical pictures of AAV will be described, especially taking into consideration the rheumatic manifestations as early symptoms. Furthermore, pathodynamics and differential diagnoses of AAV will be regarded.

Recent studies on the long-term clinical course of ANCA-associated vasculitides have revealed considerable variation in the clinical severity of the disease. The most effective standard therapy (i.e., daily cyclophosphamide + prednisolone, "FAUCI-scheme") is associated with high treatment-related morbidity and mortality as well as a high incidence of relapse. On the other hand, less toxic therapeutic strategies (e.g., monthly bolus of cyclophosphamide or weekly low-dose methotrexate) are being pursued with remarkable success in non-life threatening disease. Intractable cases (under daily cyclophosphamide + prednisolone) may profit from additional high dose IVIG therapy or from monoclonal antibodies against CD4 and CDw52 molecules. Plasmapheresis can be effective in combination with the standard therapy in dialysis patients with necrotizing glomerulonephritis (in Wegener's granulomatosis or microscopic polyarteritis). On the other hand, Wegener's granulomatosis restricted to the upper and/or lower respiratory tract ("Initial WG") responds to trimethoprimsulfamethoxazole. In future, therefore, an individualized strategy adapted to extension and activity has to be developed for the treatment of each patient with ANCA-associated vasculitis.

The ANCA-associated vasculitides (Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis) are characterized by a broad spectrum of clinical manifestations and a highly variable clinical course. Due to greatly improved diagnostic techniques the number of newly diagnosed cases has increased rapidly within the past few years. This facilitated more individualized treatment and led to the concept of stage-adapted treatment. Immunodiagnostic parameters are being utilized increasingly to supplement clinical and radiological findings. Inflammatory lesions are visualized by, for example, e.g. rhinoscopy, sinoscopy, and bronchoscopy or by radiological techniques including cranial and skeletal muscle magnetic resonance imaging, pulmonary high-resolution computed tomography, and digital subtraction angiography. These techniques not only serve to detect the extent of tissue and organ involvement, but also to direct bioptic procedures. Conventional histopathology must be supplemented by immunohistology in order to distinguish between the "immune complex vasculitides" and the ANCA-associated "pauci-immune 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.

Objective: To assess the effectiveness of pulse cyclophosphamide (CYC) in the treatment of Wegener's granulomatosis (WG) and to identify the patients who are responsive to the treatment., Methods: The prospective study included 43 patients with biopsy-proven WG. Clinical, radiographic, laboratory, and immunologic data were evaluated for predictive values regarding the outcome of pulse CYC therapy., Results: Only 42% of the patients showed complete or partial remission that lasted at least 6 months after cessation of pulse CYC therapy. These responders had a higher frequency of disease activity limited to the upper and lower respiratory tract (39%, versus 8% in the nonresponder group; P < 0.05) and had lower titers of classic antineutrophil cytoplasmic antibody (cANCA) prior to treatment (< 1:64 42%, versus 6% in the nonresponder group; P < 0.05). In the 58% of patients who did not respond to pulse CYC treatment, there was both systemic disease involving more than 4 organ systems (mainly, the heart, nervous system, eye, and skin) and constitutional symptoms. Serious side effects induced by pulse CYC occurred in only 1 patient., Conclusion: Based on these findings, pulse CYC therapy appears to be effective in WG patients with moderate disease activity and low titers of cANCA, but of little benefit in patients with severe WG. Pulse CYC should therefore not be used as first-line therapy in patients with severe and rapidly progressing forms of WG associated with high titers of cANCA.

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.

Sera of 30 patients with clinically well established Thromboangiitis obliterans were tested for the presence of ANCA by means of immunofluorescence and ELISA. ANCA did occur neither in active nor in inactive disease.

Relapsing polychondritis is a systemic disease associated with a destruction of cartilage in various parts of the body. Sera from six patients with relapsing polychondritis and one patient with microscopic polyarteritis nodosa as well as from six controls were analyzed by immunoblotting and ELISA. All patients had autoantibodies against native collagens II and IX. The serum from one patient showed a strong reaction with all three collagen chains of the high molecular weight fraction of collagen IX after denaturation; sera from four patients showed autoantibodies against alpha 2 (XI) and sera from three patients showed autoantibodies against the covalently cross-linked gamma component of collagen XI. The presence of autoantibodies against collagens II, IX, and XI, which form the major fibrillar scaffold in cartilage and mediate the interaction of collagen fibrils and proteoglycan, suggests that autoantibodies against cartilaginous collagen may play a crucial role in the pathogenesis of relapsing polychondritis and microscopic polyarteritis nodosa.

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.

The antigen specificity of autoantibodies causing perinuclear staining of granulocytes and monocytes (pANCA) was evaluated by analyzing 3000 sera, which were sent to us for screening of anticytoplasmic antibodies (ACPA, synonym: cANCA, anti-proteinase 3). In 620 sera, perinuclear staining was found. Antigen specificity was investigated by a myeloperoxidase ELISA and indirect immunofluorescence with Hep2 cells specific for antinuclear antibodies (ANA). Only 9.8% of the 620 sera showed reactivity with myeloperoxidase (AMPO), while 85.6% contained ANA which induced a pANCA-like staining. A further 4.6% of the 620 sera were neither ANA nor AMPO positive. Therefore, pANCA in general is only an indication that one should look for AMPO or other antilysosomal autoantibodies, when ANA have been excluded. To investigate the disease specificity of AMPO, we examined sera from patients with several well-defined autoimmune diseases. There were only very few positive results in collagen vascular diseases (3/114) (positive/total), primary systemic vasculitis (1/116) and clinically and histologically proven Wegener's granulomatosis (2/213). On the other hand, AMPO were present in patients with different forms of glomerulonephritis (45/192), especially crescentic glomerulonephritis (CGN) (34/79) without immune deposits in their biopsy specimen (3/30 showed trace deposits of IgM). There were, however, additionally 11 patients with symptoms resembling WG (who were cANCA negative, w/o characteristic WG biopsy), who had no obvious renal symptoms. These findings indicate that AMPO are primarily associated with idiopathic GN, especially CGN. Together with anti-proteinase-3 antibodies and anti-glomerular basement membrane antibodies they are an essential serologic parameter in the diagnosis of unclear systemic diseases with renal involvement.

Sera of 108 patients with chronic inflammatory bowel disease (IBD) and 13 control sera were screened for antibodies against neutrophil cytoplasmic antigens (ANCA) by an indirect immunofluorescence test. 37 out of 64 sera (58%) from patients with ulcerative colitis (UC) produced a fine granular and perinuclear ANCA staining pattern (p-ANCA) clearly different from the typically diffuse and granular cytoplasmic ANCA fluorescence (c-ANCA) seen in active Wegener's granulomatosis (WG). Only 1 of the 44 sera from patients with Crohn's disease and none of the control sera showed positive p-ANCA reactions. Only 1 of the 64 CU sera was positive for antinuclear antibodies, and another one showed a positive reaction in the anti-proteinase-3 ELISA which is specific for WG. Antibodies against myeloperoxidase were negative in the CU sera. 31 out of the 37 p-ANCA-positive sera (84%) were obtained from patients with high disease activity. p-ANCA titers became negative after long-term steroid therapy and after complete colectomy. These preliminary data suggest that ANCA screening may be of value in differentiating IBD and in monitoring disease activity and drug effects in UC patients.

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.