Anti-neutrophil cytoplasmic antibodies (ANCA) is autoantibodies characteristic of vasculitis diseases. A connection between ANCA and Wegener's granulomatosis was well established. The interaction of both ANCA phenotypes (PR3-ANCA and MPO-ANCA) with leukocytes provoked cell activation, which might be involved in the pathogenesis of ANCA-related Wegener's granulomatosis.
In this study, we examined whether PR3-ANCA sera and purified immunoglobulins from patients with Wegener's granulomatosis prime human monocytic cells for enhanced responses to microbial components in terms of production of proinflammatory cytokines.
Flow cytometry demonstrated that stimulation with antibodies to proteinase 3 enhanced the expression of TLR2, 3, 4, 7, and 9, NOD1, and NOD2 in human mononuclear cells. The sera and purified immunoglobulins significantly primed human mononuclear cells to secrete interleukin-8 in response to microbial components via TLRs and NODs. Priming effects were also observed for the production of interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α. On the other hand, PR3-ANCA-negative sera from patients with polyarteritis nodosa which possibly related to MPO-ANCA and aortitis syndrome as well as control sera from a healthy volunteer did not have any priming effects on PBMCs.
In conclusion, PR3-ANCA prime human mononuclear cells to produce cytokines upon stimulation with various microbial components by up-regulating the TLR and NOD signaling pathway, and these mechanisms may partially participate in the inflammatory process in Wegener's granulomatosis.
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.
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-κB. 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.
Wegener's Granulomatosis and Microscopic Polyangiitis are life-threatening systemic necrotizing vasculitides of unknown aetiology. The appearance of circulating antibodies to neutrophil cytoplasmic antigens (ANCA) is strongly associated with the development of the disease. A link between infection and disease has long been suspected, and the appearance of ANCA antibodies has been reported following bacterial and viral infections. The depletion of circulating B cells with monoclonal antibody therapy can induce remission, and this observation suggests a pathogenic role for B cells in this disease. As bacterial DNA is known to induce B cell proliferation and antibody production via TLR-9 stimulation, we have explored the possibility that unmethylated CpG oligodeoxynucleotide, as found in bacterial and viral DNA, may play a role in stimulating circulating autoreactive B cells to produce ANCA in patients with vasculitis.
We have confirmed that unmethylated CpG oligonucleotide is a potent stimulator of antibody production by PBMC in vitro. The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls. In spite of this, PR3 ANCA+ patients synthesised significantly higher amount of IgG ANCA than normal controls. In MPO ANCA+ patients, there was a tendency for patients to produce higher amount of ANCA than controls, however, the difference did not reach significance. Furthermore, we were able to detect circulating MPO-reactive B cells by ELISpot assay from the peripheral blood of 2 MPO+ ANCA vasculitis patients. Together, this indicates that circulating anti-neutrophil autoreactive B cells are present in ANCA+ vasculitis patients, and they are capable of producing antibodies in response to CpG stimulation. Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases.
Circulating ANCA autoreactive B cells are present in patients with ANCA+ vasculitis. The production of ANCA from these cells in response to unmethylated CpG stimulation lead us to propose that stimulation of these cells by immunostimulatory DNA sequences such as CpG oligodeoxynucleotide during infection may provide a link between infection and ANCA associated vasculitis. This phenomenon may also apply to other antibody mediated autoimmune diseases.
Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis.
A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria.
We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti–proteinase 3 ANCA was associated with HLA-DP and the genes encoding α1-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P = 6.2×10−89, P = 5.6×10−12, and P = 2.6×10−7, respectively). Anti–myeloperoxidase ANCA was associated with HLA-DQ (P = 2.1×10−8).
This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA–associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA–associated vasculitis and myeloperoxidase ANCA–associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.)
Anti-neutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of small vessel vasculitis, so called ANCA-associated vasculitis. The international consensus requires testing by indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils (ethN) as screening followed by confirmation with enzyme-linked immunosorbent assays (ELISAs). This study evaluates the combination of cell- and microbead-based digital IIF analysis of ANCA in one reaction environment by the novel multiplexing CytoBead technology for simultaneous screening and confirmatory ANCA testing. Sera of 592 individuals including 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 77 with inflammatory bowel syndrome, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis and 125 blood donors were tested for cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA) by classical IIF and ANCA to proteinase 3 (PR3) and myeloperoxidase (MPO) by ELISA. These findings were compared to respective ANCA results determined by automated multiplex CytoBead technology using ethN and antigen-coated microbeads for microbead immunoassays. There was a good agreement for PR3- and MPO-ANCA and a very good one for P-ANCA and C-ANCA by classical and multiplex analysis (Cohen's kappa [κ] = 0.775, 0.720, 0.876, 0.820, respectively). The differences between classical testing and CytoBead analysis were not significant for PR3-ANCA, P-ANCA, and C-ANCA (p<0.05, respectively). The prevalence of confirmed positive ANCA findings by classical testing (IIF and ELISA) compared with multiplex CytoBead analysis (IIF and microbead immunoassay positive) resulted in a very good agreement (κ = 0.831) with no significant difference of both methods (p = 0.735). Automated endpoint-ANCA titer detection in one dilution demonstrated a very good agreement with classical analysis requiring dilution of samples (κ = 0.985). Multiplexing by CytoBead technology can be employed for simultaneous screening and quantitative confirmation of ANCA. This novel technique provides fast and cost-effective ANCA analysis by automated digital IIF for the first time.
AIM: To describe the neutrophil fluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) with different antigen specificities, and by other auto- and alloantibodies. BACKGROUND: Most sera from patients with active generalised Wegener's granulomatosis result in diffusely granular cytoplasmic neutrophil fluorescence with internuclear accentuation (cANCA) and proteinase 3 (PR3) specificity. About 80% of the sera from patients with microscopic polyangiitis result in perinuclear neutrophil fluorescence with nuclear extension (pANCA) and myeloperoxidase (MPO) specificity, or a cANCA pattern with PR3 specificity. However, many different neutrophil fluorescence patterns are noted on testing for ANCA in routine immunodiagnostic laboratories. METHODS: Sera sent for ANCA testing, or containing a variety of auto- and alloantibodies, were studied. They were examined by indirect immunofluorescence according to the recommendations of the first international ANCA workshop, and for PR3 and MPO specificity in commercial and in-house enzyme linked immunosorbent assays (ELISA). RESULTS: Sera with typical cANCA accounted for only half of all neutrophil cytoplasmic fluorescence. Other sera had "flatter" fluorescence without internuclear accentuation, and the corresponding antigens included MPO and bactericidal/permeability increasing protein (BPI), but were usually unknown. Peripheral nuclear fluorescence without nuclear extension occurred typically when the antigens were BPI, lactoferrin, lysozyme, elastase, or cathepsin G. Most types of ANA were evident on ethanol fixed neutrophil nuclei. AntidsDNA, antiRo, and antilamin antibodies resembled pANCA. Antimicrobial and antiribosomal antibodies produced cytoplasmic fluorescence, and antiGolgi antibodies, a pANCA. Sera from patients with anti-smooth muscle antibodies were associated with cytoplasmic fluorescence. There was no neutrophil fluorescence with anti-skeletal muscle and anti-heart muscle antibodies, anti-liver/kidney microsomal, antithyroid microsomal, or antiadrenal antibodies. Alloantibodies such as antiNB1 typically resulted in cytoplasmic fluorescence of only a subpopulation of the neutrophils. CONCLUSIONS: The ability to distinguish between different neutrophil fluorescence patterns, and the patterns seen with other auto- and alloantibodies is helpful diagnostically. However, the demonstration of MPO or PR3 specificity by ELISA will indicate that the neutrophil fluorescence is probably clinically significant, and that the diagnosis is likely to be Wegener's granulomatosis or microscopic polyangiitis.
OBJECTIVE—To calculate the positive predictive value (ppv) of cytoplasmic anti-neutrophil cytoplasmic antibodies (c-ANCAs) and anti-proteinase 3 (PR 3) antibodies for Wegener's granulomatosis (WG) and to evaluate their association with other diseases.
METHODS—The clinical files of all 94 patients who had a positive c- or perinuclear (p)-ANCA test, or both, in the laboratory of the University Hospital, Leuven between April 1995 and March 1996 and who attended the Internal Medicine Department of the hospital were retrospectively studied.
RESULTS—Of the 94 patients with ANCAs (fluorescence titre ⩾ 1/40), 57 were c-ANCA positive and 45 p-ANCA positive (eight were simultaneously c- and p-ANCA positive). Of the 57 c-ANCA positive patients, 23 had WG. The ppv for WG thus was 40%. This value did not increase by defining a higher threshold for a positive ANCA. There was not a good relation between ANCA titres and disease activity in the WG patients, nor was there a relation between anti-PR 3 antibody levels and WG disease activity. The ppv of anti-PR 3 antibodies for WG however was very high (85%). There was a positive correlation between the level of (hyper) gammaglobulinaemia and c-ANCA titres in those patients with final diagnoses not known to be associated with c-ANCA. Forty five patients had positive p-ANCAs. The largest group were those with inflammatory bowel disease (n = 20, of whom the majority had colitis ulcerosa or primary sclerosing cholangitis, or both); the great majority of these patients had no anti-myeloperoxidase antibodies. Vasculitis was present in eight patients, of whom two had WG (both were also c-ANCA positive).
CONCLUSION—There is a low ppv of c-ANCAs for WG, caused by a high percentage of PR 3 negative, positive c-ANCA determinations, possibly related to hypergammaglobulinaemia. Anti-PR 3 antibodies have a high ppv for WG. However, neither c-ANCA titre, nor the level of anti-PR 3 antibodies correlated with the activity of the disease.
Keywords: antineutrophil cytoplasmic antibodies; vasculitis; Wegener's granulomatosis; hypergammaglobulinaemia
Cytoplasmic antineutrophil cytoplasmic antibodies (cANCA) that accompany the neutrophilic vasculitis seen in Wegener's granulomatosis (WG), are directed against proteinase-3 (PR-3), a serine proteinase which is located in azurophilic granules of neutrophils and monocytes. PR-3, when expressed on the surface of TNFalpha-primed neutrophils, can directly activate neutrophils by complexing cANCA and promoting concomitant Fcgamma receptor (FcgammaR) cross-linking. Although the neutrophil's pathogenic role in WG has been studied, the role of the monocyte has not been explored. The monocyte, with its ability to release cytokines and regulate neutrophil influx, also expresses PR-3. Therefore, the monocyte may play a significant role in WG via the interaction of surface PR-3 with cANCA, inducing cytokine release by the monocyte. To test this hypothesis, monocytes were studied for PR-3 expression and for IL-8 release in response to cANCA IgG. PBMC obtained from healthy donors displayed dramatic surface PR-3 expression as detected by immunohistochemistry and flow cytometry in response to 0. 5-h pulse with TNFalpha (2 ng/ml). Purified monoclonal anti-PR-3 IgG added to TNFalpha-primed PBMC induced 45-fold more IL-8 release than an isotype control antibody. Furthermore, alpha 1-antitrypsin (alpha1-AT), the primary PR-3 antiprotease, inhibited the anti-PR-3 induced IL-8 release by 80%. Importantly, Fab and F(ab')2 fragments of anti-PR-3 IgG, which do not result in Fcgamma receptor cross-linking, do not induce IL-8 release. As a correlate, IgG isolated from cANCA positive patients with WG induced six times as much PBMC IL-8 release as compared to IgG isolated from normal healthy volunteers. Consistent with PR-3 associated IL-8 induction, alpha1-AT significantly inhibited this effect. These observations suggest that cANCA may recruit and target neutrophils through promoting monocyte IL-8 release. This induction is mediated via Fcgamma receptor cross-linking and is regulated in part by alpha1-AT.
Purpose of Review
Anti-neutrophil cytoplasmic autoantibodies (ANCA) are associated with vasculitis. Current therapy involves administration of toxic therapy that is not optimally effective. The review will summarize evidence for the pathogenicity of ANCA, which will suggest possible strategies for improving treatment.
Pauci-immune small vessel vasculitis is associated with antibodies against myeloperoxidase (MPO-ANCA) and proteinase 3 (PR3-ANCA). One research group has reported a high frequency of autoantibodies against lysosomal-associated membrane protein 2 (LAMP-2) in patients with MPO-ANCA or PR3-ANCA. Epigenetic dysregulation appears to be the basis for increased MPO and PR3 neutrophil gene expression in ANCA disease. Release of neutrophil extracellular traps (NETS) may be involved in initiating the ANCA autoimmune response and causing vessel injury. Generation of C5a by alternative pathway activation is involved in pathogenesis in mouse models. Intervention strategies in mice that target antigens, antibodies and inflammatory signaling pathways may translate into novel therapies. Animal models of LAMP-ANCA and PR3-ANCA disease have been proposed. Molecular mimicry and responses to complementary peptides may be initiating events for ANCA. T cells, including regulatory T cells, have been implicated in the origin and modulation of the ANCA, as well as in the induction of tissue injury.
Our basic understanding of the origins and pathogenesis of ANCA disease is advancing. This deeper understanding already has spawned novel therapies that are being investigated in clinical trials. This brief review shows that there are more questions than answers, and new questions are emerging faster than existing questions are being answered.
Vasculitis; Pathogenesis; Antineutrophil Cytoplasmic Autoantibodies; ANCA
Proteinase 3 (PR3)-specific antineutrophil cytoplasmic antibodies (ANCA) are highly specific for the autoimmune small vessel vasculitis, Wegener’s granulomatosis (WG). PR3-ANCA have proven diagnostic value but their pathogenic potential and utility as a biomarker for disease activity remain unclear. PR3-ANCA recognize conformational epitopes, and epitope-specific PR3-ANCA subsets with variable impact on biological functions of PR3 have been postulated. The aims of this study were to identify specific PR3 surface epitopes recognized by monoclonal antibodies (moAbs) and to determine whether the findings can be used to measure the functional impact of epitope-specific PR3-ANCA and their potential relationship to disease activity. We used a novel flow cytometry assay based on TALON-beads coated with recombinant human (H) and murine (M) PR3 and 10 custom-designed chimeric human/mouse rPR3-variants (Hm1–5/Mh1–5) identifying 5 separate non-conserved PR3 surface epitopes. Anti-PR3 moAbs recognize 4 major surface epitopes, and we identified the specific surface location of 3 of these with the chimeric rPR3-variants. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3 was measured indirectly using a capture-ELISA system based on the different epitopes recognized by capturing moAbs. Epitope-specific PR3-ANCA capture-ELISA results obtained from patient plasma (n=27) correlated with the inhibition of enzymatic activity of PR3 by paired IgG preparations (r=0.7, P<0.01). The capture-ELISA results also seem to reflect disease activity. In conclusion, insights about epitopes recognized by anti-PR3 moAbs can be applied to separate PR3-ANCA subsets with predictable functional qualities. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3, a property linked to disease activity, can now be gauged using a simple epitope-based capture-ELISA system.
ANCA; proteinase 3; Wegener’s granulomatosis; vasculitis
Antineutrophil cytoplasmic autoantibodies (ANCAs) directed to proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA) are strongly associated with the ANCA-associated vasculitides—Wegener’s granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome. Clinical observations, including the efficacy of B-cell depletion via rituximab treatment, support—but do not prove—a pathogenic role for ANCA in the ANCA-associated vasculitides. In vitro experimental studies show that the interplay of ANCA, neutrophils, the alternative pathway of the complement system, and endothelial cells could result in lysis of the endothelium. A pathogenic role for MPO-ANCA is strongly supported by in vivo experimental studies in mice and rats, which also elucidate the pathogenic mechanisms involved in lesion development. Unfortunately, an animal model for PR3-ANCA–associated Wegener’s granulomatosis is not yet available. Here, cellular immunity appears to play a major role as well, particularly via interleukin-17–producing T cells, in line with granulomatous inflammation in the lesions. Finally, microbial factors, in particular Staphylococcus aureus and gram-negative bacteria, seem to be involved in disease induction and expression, but further studies are needed to define their precise role in disease development.
Wegener’s granulomatosis; MPO-ANCA; Microscopic polyangiitis; hLAMP-2 autoantibodies; Churg-Strauss syndrome; Staphylococcus aureus; Necrotizing crescentic glomerulonephritis; FimH; ANCA-associated vasculitis; Animal models; Antineutrophil cytoplasmic autoantibodies; Th17 cells; ANCA; T-regulatory cells; Proteinase 3; PR3-ANCA; Myeloperoxidase
Clinical and serological profiles of idiopathic and drug-induced autoimmune diseases can be very similar. We compared data from idiopathic and antithyroid drug (ATD)-induced antineutrophil cytoplasmic antibody (ANCA)-positive patients. From 1993 to 2003, 2474 patients were tested for ANCA in the Laboratory for Allergy and Clinical Immunology in Belgrade. Out of 2474 patients, 72 (2.9%) were anti-proteinase 3 (PR3)- or anti-myeloperoxidase (MPO)-positive and their clinical and serological data were analyzed. The first group consisted of ANCA-associated idiopathic systemic vasculitis (ISV) diagnosed in 56/72 patients: 29 Wegener's granulomatosis (WG), 23 microscopic polyangiitis (MPA) and four Churg-Strauss syndrome. The second group consisted of 16/72 patients who became ANCA-positive during ATD therapy (12 receiving propylthiouracil and four receiving methimazole). We determined ANCA and antinuclear (ANA) antibodies by indirect immunofluorescence; PR3-ANCA, MPO-ANCA, anticardiolipin (aCL) and antihistone antibodies (AHA) by ELISA; and cryoglobulins by precipitation. Complement components C3 and C4, alpha-1 antitrypsin (α1 AT) and C reactive protein (CR-P) were measured by nephelometry. Renal lesions were present in 3/16 (18.8%) ATD-treated patients and in 42/56 (75%) ISV patients (p <0.001). Skin lesions occurred in 10/16 (62.5%) ATD-treated patients and 14/56 (25%) ISV patients (p <0.01). ATD-treated patients more frequently had MPO-ANCA, ANA, AHA, aCL, cryoglobulins and low C4 (p <0.01). ISV patients more frequently had low α1 AT (p = 0.059) and high CR-P (p <0.001). Of 16 ATD-treated patients, four had drug-induced ANCA vasculitis (three MPA and one WG), while 12 had lupus-like disease (LLD). Of 56 ISV patients, 13 died and eight developed terminal renal failure (TRF). There was no lethality in the ATD-treated group, but 1/16 with methimazole-induced MPA developed pulmonary-renal syndrome with progression to TRF. ANCA-positive ISV had a more severe course in comparison with ATD-induced ANCA-positive diseases. Clinically and serologically ANCA-positive ATD-treated patients can be divided into two groups: the first consisting of patients with drug-induced WG or MPA which resemble ISV and the second consisting of patients with LLD. Different serological profiles could help in the differential diagnosis and adequate therapeutic approach to ANCA-positive ATD-treated patients with symptoms of systemic disease.
Anti‐neutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) are highly specific for Wegener's granulomatosis (WG). Evidence for a pivotal role of PR3‐ANCA in the induction of vasculitis has been demonstrated. B cell clusters have been observed within endonasal biopsy specimens.
To determine whether B cell selection and maturation take place in granulomatous lesions of WG.
Granulomatous lesions and the immunoglobulin (VH) gene repertoire from nasal tissue of six WG patients—two active and two smouldering localised WG (ANCA negative, restricted to respiratory tract), plus one active and one smouldering PR3‐ANCA positive generalised WG—were characterised by immunohistochemistry, polymerase chain reaction, cloning, DNA sequencing and database comparison.
B lymphocyte‐rich, follicle‐like areas were observed proximal to PR3 positive cells and plasma cells in granulomatous lesions; 184 VH genes from these granulomatous lesions were compared with 84 VH genes from peripheral blood of a healthy donor. The mutational pattern of VH genes from active WG resembled memory B cells. Structural homologies of VH genes from granulomatous lesions to PR3‐ANCA encoding genes were detected. Significantly more genes (55%, 45%, and 53%, respectively) from active WG compared with the healthy repertoire carried mutations to negatively charged amino acids within the binding site coding regions, favouring affinity to the positively charged PR3.
Selection and affinity maturation of potentially PR3‐ANCA producing autoreactive B cells may start in granulomatous lesions, thereby contributing to disease progression from ANCA negative localised to PR3‐ANCA positive generalised WG.
B lymphocyte; Wegener's granulomatosis; PR3; PR3‐ANCA; VH genes
Before the mid-1980s the only autoantibody widely used to assist in diagnosing vasculitic disease was IgG antibody to the α3 domain of the noncollagenous part of type IV collagen (anti-glomerular basement membrane). Since that time, antineutrophil cytoplasmic antibodies (ANCAs) directed at the azurophilic granule proteins proteinase-3 and myeloperoxidase have been established as clinically useful autoantibodies to support a diagnosis of Wegener's granulomatosis, microscopic polyangiitis, Churg–Strauss syndrome and limited forms of these primary, small vessel necrotizing and often granulomatous vasculitides. The establishment of standardized methods for identifying those antibodies was needed before they could be used in clinical practice. The levels of both types of ANCAs tend to increase in parallel with the degree of clinical disease activity, and they decrease with successful immunosuppressive therapy. More than one assay may have to be used to discover imminent exacerbations in proteinase-3-ANCA associated syndromes. Although autoantibodies to endothelial cells may be important players in the pathogenesis of several vasculitic conditions, they have not gained clinical popularity because of lack of standardized detection methods.
antiendothelial antibodies; antineutrophil cytoplasm antibodies; prognostics; vasculitis
The prevalence of antineutrophil cytoplasmic antibodies (ANCA)
was studied in 12 children with Wegener's granulomatosis. The serum
samples were taken in the active phase of disease and were screened for
ANCA by indirect immunofluorescence with normal neutrophils and enzyme
linked immunosorbent assay (ELISA) using crude neutrophil extract,
proteinase 3, myeloperoxidase, cathepsin G, lactoferrin, and elastase
as antigens. Of these 12 patients, 10 were positive for ANCA in the
active phase of their illness, and they showed a predominantly
cytoplasmic ANCA staining pattern on indirect immunofluorescence. There
were high titres of ANCA directed against crude neutrophil extract,
proteinase 3, myeloperoxidase, and cathepsin G. IgM isotypes occurred
as commonly as IgG isotypes. Therefore, screening for ANCA is usually
but not invariably positive in children with Wegener's granulomatosis.
Specific diagnosis still relies on clinical and pathological features,
and the value of ANCA in the diagnosis of paediatric Wegener's
granulomatosis requires further study.
The glycosylation status of autoantigens appears to be crucial for the pathogenesis of some autoimmune diseases, since carbohydrates play a crucial role in the distinction of self from non-self. Proteinase 3 (PR3), the main target antigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener’s granulomatosis (WG), contains two Asn-linked glycosylation sites. The present study explores the influence of the glycosylation status of PR3 on the PR3 recognition by ANCA in a well characterized population of patients with WG.
Forty-four patients with WG (459 serum samples) who participated in a multicenter randomized trial, were tested by capture ELISA for ANCA against PR3 and deglycosylated recombinant variants of PR3.
The patients were followed for a median of 27 months, and the median number of serum samples per patient was 10. At baseline, the correlation between the levels of ANCA against PR3 and against all the deglycosylated recombinant variants of PR3 were greater than 0.94 (ρ<0.001 for all the comparisons). Longitudinal analyses comparing the levels of ANCA against PR3 versus all the deglycosylated recombinant variants of PR3, using linear mixed models, showed no significant statistical differences (ρ≥0.90 in all cases).
The glycosylation status of PR3 has no impact on its recognition by ANCA in WG.
ANCA; biological markers; glycosylation; proteinase 3; Wegener’s granulomatosis
The complement system is crucial for the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In particular, C5a and its receptor on neutrophils, CD88, play a central role. The functional role of the second receptor of C5a, C5L2, remains unclear. In the current study, we investigated the role of C5L2 in C5a-primed neutrophils for ANCA-induced activation.
The effect of blocking C5L2 by anti-human C5L2 blocking antibody were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on membrane-bound proteinase 3 (mPR3) and concentration of myeloperoxidase (MPO) in supernatant of C5a-primed neutrophils. An antagonist for CD88 was also employed.
Blocking C5L2 resulted in a significantly decreased MPO concentration in the supernatant of C5a-primed neutrophils. mPR3 expression increased from 209.0±43.0 in untreated cells to 444.3±60.8 after C5a treatment (P<0.001), and decreased to 375.8±65.44, 342.2±54.3 and 313.7±43.6 by pre-incubating blocking C5L2 antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-priming group, P<0.001, P<0.001, and P<0.001), respectively. In C5a-primed neutrophils, subsequently activating with MPO-ANCA-positive IgG, the MFI value was 425.8±160.6, which decreased to 292.8±141.2, 289.7±130.0 and 280.3±136.4 upon pre-incubation with mouse anti-human C5L2 blocking antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-primed neutrophils, for MPO-ANCA-positive IgG-induced activation, P<0.05, P<0.05, and P<0.05), respectively. Blocking C5L2 also resulted in significantly decreased C5a-primed neutrophils for PR3-ANCA-positive IgG-induced activation. Moreover, the lactoferrin concentration in the supernant significantly decreased in pre-incubation with anti-human C5L2 blocking antibody, compared with C5a-primed neutrophils induced by PR3- or MPO-ANCA-positive IgG.
C5L2 may be implicated in the pro-inflammatory role in C5a-primed neutrophils for ANCA-induced activation.
The neutrophil azurophil granule constituent proteinase 3 (PR3) is the principal antigen for anti-neutrophil cytoplasmic antibodies (ANCA) in Wegener's granulomatosis. The conformation of the mature PR3 enzyme results from intracellular post-translational processing. The nascent molecule undergoes proteolytic cleavage of the amino-terminal signal peptide and activation dipeptide and of a carboxy-terminal peptide extension. The conformation of PR3 is stabilized by four disulfide bonds and, to a lesser extent, by asparagine-linked glycosylation. Most anti-neutrophil cytoplasmic antibodies directed against proteinase 3 (PR3-ANCA) recognize conformational epitopes. The expression of recombinant PR3 has provided a better understanding of the significance of the various intracellular processing steps for enzymatic activity and recognition by PR3-ANCA.
ANCA; proteinase 3; recombinant proteins
Background: Previous studies have shown considerable variation in diagnostic performance of enzyme linked immunosorbent assays (ELISAs) for measuring antineutrophil cytoplasmic antibodies (ANCA) specific for proteinase 3 (PR3) and myeloperoxidase (MPO).
Objective: To analyse the performance characteristics of different commercially available direct ANCA ELISA kits.
Methods: ELISA kits for detecting PR3-ANCA and MPO-ANCA from 11 manufacturers were evaluated. Serum samples were taken from patients with Wegener's granulomatosis (15), microscopic polyangiitis (15), other vasculitides (10), and controls (40). Results were compared with data obtained by indirect immunofluorescence (IFT). The diagnostic performance of the tests was analysed and compared by receiver operating characteristic (ROC) curve analysis.
Results: Applying the manufacturers' cut off resulted in great variation in sensitivity of the commercial PR3-ANCA kits for diagnosing Wegener's granulomatosis (ranging from 13.3% to 66.7%), and of the MPO-ANCA kits for diagnosing microscopic polyangiitis (ranging from 26.7% to 66.7%). Specificities were relatively constant (from 96.0% to 100%). IFT was superior to all ELISAs (C-ANCA for Wegener's granulomatosis: sensitivity 73.3%, specificity 98%; P-ANCA for microscopic polyangiitis: sensitivity 86.7%, specificity 98%). The sensitivities of PR3-ANCA and MPO-ANCA ELISA kits were increased by lowering the cut off values. This reduced specificity but increased overall diagnostic performance.
Conclusions: The low sensitivity of some commercial kits reflects the high cut off levels recommended rather than methodological problems with the assays. Comparative analyses using sera from well characterised patients may help identify optimum cut off levels of commercial ANCA ELISA tests, resulting in better comparability of results among assays from different manufacturers.
Granulomatosis with polyangiitis, also known as Wegener’s granulomatosis, is a chronic systemic inflammatory disease that can also involve the eyes. We report a case of massive retinal and preretinal hemorrhages with perivascular changes as the initial signs in granulomatosis with polyangiitis (Wegener’s granulomatosis).
A 39-year-old Caucasian male presented with blurred vision in his right eye, myalgia and arthralgia, recurrent nose bleeds and anosmia. Fundus image of his right eye showed massive retinal hemorrhages and vasculitis-like angiopathy, although no fluorescein extravasation was present in fluorescein angiography. Laboratory investigations revealed an inflammation with increased C-reactive protein, elevated erythrocyte sedimentation rate and neutrophil count. Tests for antineutrophil cytoplasmic antibodies (ANCA) were positive for c-ANCA (cytoplasmatic ANCA) and PR3-ANCA (proteinase 3-ANCA). Renal biopsy demonstrated a focal segmental necrotizing glomerulonephritis. Granulomatosis with polyangiitis (Wegener’s granulomatosis) was diagnosed and a combined systemic therapy of cyclophosphamide and corticosteroids was initiated. During 3 months of follow-up, complete resorption of retinal hemorrhages was seen and general complaints as well as visual acuity improved during therapy.
Vasculitis-like retinal changes can occur in Wegener’s granulomatosis. Despite massive retinal and preretinal hemorrhages that cause visual impairment, immunosuppressive therapy can improve ocular symptoms.
Granulomatosis with polyangiitis; Wegener’s granulomatosis; Retinal vasculitis; Hemorrhages; Cyclophosphamide
Proteinase-3 (PR-3) is a neutral serine proteinase present in azurophil granules of human polymorphonuclear leukocytes and serves as the major target antigen of antineutrophil cytoplasmic antibodies with a cytoplasmic staining pattern (c-ANCA) in Wegener's granulomatosis (WG). The WG disease appears as severe vasculitis in different organs (e.g. kidney, nose and lung). Little is known about the expression and distribution of PR-3 in the lung. We found that PR-3 is expressed in normal lung tissue and is upregulated in lung tissue of patients with WG. Interestingly, the parenchymal cells (pneumocytes type I and II) and macrophages, and not the neutrophils, express PR-3 most strongly and may contribute to lung damage in patients with WG via direct interaction with antineutrophil cytoplasmic antobodies (ANCA). These findings suggest that the PR-3 expression in parenchymal cells of lung tissue could be at least one missing link in the etiopathogenesis of pulmonary pathology in ANCA-associated disease.
granuloma; in situ hybridization; pneumocytes; proteinase-3; Wegener's granulomatosis
The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. There is a growing body of evidence that T cells may contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Predominantly, T cells and monocytes are found in inflammatory infiltrates in patients with Wegener's granulomatosis (WG). The production of ANCA appears to be T-cell-dependent. T lymphocytes from the peripheral blood of patients with ANCA-associated vasculitis have been shown to proliferate in response to proteinase 3 (PR3). These and other findings outlined in this review indicate T-cell involvement, although further studies are still needed to elucidate the exact contribution of T cells to the pathogenesis of systemic vasculitis.
proteinase 3; systemic vasculitis; T cells; Wegener's granulomatosis
Subacute bacterial endocarditis (SBE) occasionally exhibits positive cytoplasmic anti-neutrophil cytoplasmic antibody (c-ANCA) of the anti-proteinase-3 (PR-3) type. Clinically, it mimics ANCA-associated vasculitis, such as Wegener's disease with glomerulonephritis. Lung abscesses are the most common manifestation of lung involvement. We herein report a case of culture-negative SBE strongly c-ANCA/PR3-positive accompanied by pulmonary involvement and glomerulonephritis. In this case, we took biopsies of both the lung and kidney, although renal biopsy is usually preferred over lung biopsy. The lung biopsy showed severe alveolar capillaritis, suggesting vasculitis consistent with polyangiitis. The renal biopsy revealed glomerulonephritis with a membranoproliferative pattern. To our knowledge, this is the first such reported case.
A 68-year-old Chinese male patient presented to our hospital with a fever, cough, chest pain, and recurrent peripheral edema. He had a past medical history significant for treated schistosomiasis 20 years previously. Physical examination revealed palpable purpura, mild hypertension, hepatosplenomegaly, and a holosystolic cardiac murmur (Levine 2/6). Echocardiography showed tricuspid valve vegetations with moderate to severe regurgitation. Serum c-ANCA/PR3 and cryoglobulin were strongly positive. Renal biopsy results indicated membranoproliferative glomerulonephritis with several crescents. Chest CT revealed multiple intraparenchymal and subpleural nodules, and lung biopsy showed polyangiitis. The patient’s ANCA titers, glomerulonephritis, and pulmonary injury all resolved after antibiotic therapy.
SBE may present with positive c-ANCA/PR3, multiple pulmonary nodules, pulmonary polyangiitis, and glomerulonephritis clinically mimicking granulomatosis with polyangiitis (Wegener's granulomatosis).
Subacute bacterial endocarditis; PR3/c-ANCA; Granulomatosis with polyangiitis (Wegener's granulomatosis); Glomerulonephritis
Anti-neutrophil cytoplasmic antibody (ANCA) vasculitis is a complex disease, with much debate about the utility of systems for classification and diagnosis. We compared three currently used classification systems in predicting disease prognosis.
Three classification systems were applied to 502 patients with biopsy proven ANCA vasculitis: the Chapel Hill Consensus Conference (CHCC) definition with categories for granulomatosis with polyangiitis (GPA, Wegener’s granulomatosis), microscopic polyangiitis (MPA) and the Kidney Limited Disease (KLD); European Medicines Agency (EMA) system with categories for GPA and MPA, and classification based on ANCA specificity (PR3 versus MPO). Outcomes included treatment resistance, relapse, end stage kidney disease (ESKD) and death. Proportional hazards models were compared between systems using an information-theoretic approach to rank models by predictive fit. Hazard Ratios (HR) with 95% confidence intervals (CI) and p-values are reported.
ANCA specificity was predictive of relapse, with PR3-ANCA patients almost twice as likely as those with MPO-ANCA to relapse (HR=1.89, 95% CI=1.33–2.69, p=0.0004), and ANCA specificity had the best predictive model fit (Model Rank=1) compared to CHCC and EMA. CHCC and EMA systems did not predict relapse. By ANCA specificity, categories of GPA, MPA and KLD did not distinguish differences in probability of relapse-free survival. None of the systems predicted treatment resistance, ESKD or death.
ANCA specificity independently predicts relapse among patients with ANCA vasculitis with renal disease. Classification and diagnostic systems that incorporate ANCA specificity, such as PR3-ANCA-MPA and MPO-ANCA-MPA, provide a more useful tool for predicting relapse than the clinic pathologic category alone.