ABSTRACT
The diagnosis of systemic vasculitis (SV) is a major clinical challenge due to the very different forms of presentation and requires an interdisciplinary approach. Targeted laboratory diagnostics support making the diagnosis, differential diagnosis and classification and are also a key component in the detection of active organ manifestations and treatment complications. The basic laboratory tests include the erythrocyte sedimentation rate (ESR), Creactive protein (CRP), blood count, serum creatinine, urinalysis, specific autoantibodies, complement, immunoglobulins, cryoglobulins and hepatitis B and C serology. Antineutrophil cytoplasmic autoantibodies (ANCA), antiglomerular basement membrane antibodies (anti-GBM antibodies) and anti-C1q antibodies are valuable laboratory markers for the diagnosis of the various forms of small vessel vasculitis. There are no specific laboratory tests for the diagnosis of medium and large vessel vasculitis. Despite advances in our understanding of the pathogenesis of vasculitis, no biomarkers have yet been identified that can be reliably used to guide treatment or that are useful in distinguishing vasculitis from other inflammatory diseases such as infections or treatment complications.
Subject(s)
Antibodies, Antineutrophil Cytoplasmic , Biomarkers , Vasculitis , Humans , Biomarkers/blood , Vasculitis/diagnosis , Vasculitis/blood , Vasculitis/immunology , Antibodies, Antineutrophil Cytoplasmic/blood , Antibodies, Antineutrophil Cytoplasmic/immunology , Autoantibodies/blood , Clinical Laboratory Techniques/methods , Diagnosis, DifferentialABSTRACT
OBJECTIVES: To determine the spectrum of anti-neutrophil cytoplasmic antibody (ANCA) antigen-specificities in eosinophilic granulomatosis with polyangiitis (EGPA), an ANCA-associated vasculitis (AAV) entity. METHODS: We conducted a retrospective analysis of 73 EGPA patients from three German tertiary referral centres for vasculitis. In addition to in-house ANCA testing, pentraxin 3 (PTX3)- and olfactomedin 4 (OLM4)-ANCA were determined using a prototype cell-based assay for research (EUROIMMUN, Lübeck, Germany). Patient characteristics and clinical manifestations were evaluated and compared based on ANCA status. RESULTS: Myeloperoxidase (MPO)-ANCA positive patients (n=8; 11%) significantly more frequently displayed peripheral nervous system (PNS) and pulmonary involvement and less frequently heart involvement compared to MPO-ANCA negative patients. PTX3-ANCA positive patients (n=5; 6.8%) had a significantly higher prevalence of ear, nose and throat, pulmonary, gastrointestinal and PNS involvement, and a lower prevalence of renal and central nervous system involvement compared to PTX3-ANCA negative patients. Proteinase 3 (PR3)-ANCA and OLM4-ANCA were detected in 2 patients (2.7%) each with multiorgan involvement. One PR3-ANCA positive patient was also positive for bactericidal permeability increasing protein (BPI)-ANCA. CONCLUSIONS: In addition to MPO, the spectrum of ANCA antigen specificities includes various other target antigens such as PR3, BPI, PTX3, and OLM4, potentially segregating further EGPA subgroups. A lower prevalence of MPO-ANCA was detected in this study compared with other studies. OLM4 is reported as novel ANCA antigen-specificity in EGPA, and thus AAV.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis , Churg-Strauss Syndrome , Granulomatosis with Polyangiitis , Humans , Antibodies, Antineutrophil Cytoplasmic , Retrospective Studies , Granulomatosis with Polyangiitis/diagnosis , Churg-Strauss Syndrome/diagnosis , Myeloblastin , PeroxidaseABSTRACT
OBJECTIVE: This study was performed to evaluate the diagnostic accuracy of novel line and dot immunoassays for detection of MPO and PR3 ANCA. METHODS: Sera from 50 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 45 disease controls were tested by IIF and for the presence of PR3-ANCA and MPO-ANCA by four different line or dot immunoassays, as well as by a chemiluminescence immunoassay. RESULTS: The area under the curve of the receiver operating characteristic curve to discriminate AAV from controls was 0.858 (95% CI 0.785-0.931) for the IIF method. For the antigen-specific immunoassays, the area under the curve varied between 0.869 (95% CI 0.797-0.941) and 0.936 (95% 0.886-0.985). CONCLUSIONS: Our comparison of various ANCA detection methods showed a high degree of diagnostic precision for all of the PR3- and MPO-ANCA line and dot immunoassays investigated. The performance was equal to or better than the performance of IIF. These results indicate that novel line and dot immunoassays can serve as a first-line test method in patients with the suspected diagnosis of AAV.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/diagnosis , Antibodies, Antineutrophil Cytoplasmic/immunology , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Case-Control Studies , Female , Humans , Immunoassay/methods , Male , Sensitivity and SpecificityABSTRACT
OBJECTIVES: To evaluate the autoantibody profile in eosinophilic granulomatosis and polyangiitis (EGPA) patients. METHODS: 33 EGPA patients were tested for anti-neutrophil cytoplasmic antibodies (ANCA), antinuclear antibodies (ANA), rheumatoid factor (RF), anti-alpha-enolase antibodies, and anti-eosinophil peroxidase (EPO) antibodies. Granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), hypereosinophilic syndrome (HES), rheumatoid arthritis (RA), primary biliary cirrhosis (PBC) patients and healthy subjects were tested as a control group. RESULTS: Anti-alpha-enolase antibodies were positive in 82% of EGPA patients at high titers. Although a high sensitivity was shown for an anti-alpha-enolase antibody titer above 1/100 (82%), the specificity for EGPA remained low (44%) (AUC=0.653, p=0.008). Anti-alpha-enolase antibodies predominated in males with EGPA (p=0.048) and were associated with skin involvement (p=0.040). Most of the EGPA patients positive for anti-alpha enolase antibodies (20 out of 27) had a negative indirect immunofluorescence test (IFT) for ANCA. ANCA were positive in 8 EGPA patients (24%) with a perinuclear pattern in all but one patient. The ANCA-target antigen was myeloperoxidase (MPO) and/or alpha-enolase. A usually fine-speckled ANA pattern was observed in 42% of the EGPA patients. RF was positive in 1 (6%) of the 18 EGPA patients tested. There was no association between the presence and levels of autoantibodies and EGPA disease activity. None of the patients and controls was positive for anti-EPO antibodies. CONCLUSIONS: Alpha-enolase may be a target of autoimmunity in EGPA patients and shows usually negative ANCA IFT results.
Subject(s)
Granulomatosis with Polyangiitis , Microscopic Polyangiitis , Antibodies, Antineutrophil Cytoplasmic , Humans , Male , Peroxidase , Phosphopyruvate HydrataseABSTRACT
An international consensus on anti-neutrophil cytoplasm antibodies (ANCA) testing in eosinophilic granulomatosis with polyangiitis (EGPA) is presented. ANCA, specific for myeloperoxidase (MPO), can be detected in 30-35% of EGPA patients. MPO-ANCA should be tested with antigen-specific immunoassays in any patient with eosinophilic asthma and clinical features suggesting EGPA, including constitutional symptoms, purpura, polyneuropathy, unexplained heart, gastrointestinal or kidney disease, and/or pulmonary infiltrates or hemorrhage. A positive MPO-ANCA result contributes to the diagnostic workup for EGPA. Patients with MPO-ANCA associated EGPA have more frequently vasculitis features, such as glomerulonephritis, neuropathy, and skin manifestations than patients with ANCA negative EGPA. However, the presence of MPO-ANCA is neither sensitive nor specific enough to identify whether a patient should be subclassified as having "vasculitic" or "eosinophilic" EGPA. At present, ANCA status cannot guide treatment decisions, that is, whether cyclophosphamide, rituximab or mepolizumab should be added to conventional glucocorticoid treatment. In EGPA, monitoring of ANCA is only useful when MPO-ANCA was tested positive at disease onset.
ABSTRACT
OBJECTIVE: This multicentre study was performed to evaluate the diagnostic accuracy of a wide spectrum of novel technologies nowadays available for detection of myeloperoxidase (MPO) and proteinase 3 (PR3)-antineutrophil cytoplasmic antibodies (ANCAs). METHODS: Sera (obtained at the time of diagnosis) from 251 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 924 disease controls were tested for the presence of cytoplasmic pattern/perinuclear pattern and atypical ANCA (A-ANCA) by indirect immunofluorescence (IIF) (at two sites) and for the presence of PR3-ANCA and MPO-ANCA by eight different immunoassays. RESULTS: The area under the curve (AUC) of the receiver operating characteristic curve to discriminate AAV from controls was 0.923 (95% CI 0.902 to 0.944) and 0.843 (95% CI 0.814 to 0.871) for the two IIF methods. For the antigen-specific immunoassays, the AUC varied between 0.936 (95% CI 0.912 to 0.960) and 0.959 (95% CI 0.941 to 0.976), except for one immunoassay for which the AUC was 0.919 (95% CI 0.892 to 0.945). CONCLUSIONS: Our comparison of various ANCA detection methods showed (i) large variability between the two IIF methods tested and (ii) a high diagnostic performance of PR3-ANCA and MPO-ANCA by immunoassay to discriminate AAV from disease controls. Consequently, dual IIF/antigen-specific immunoassay testing of each sample is not necessary for maximal diagnostic accuracy. These results indicate that the current international consensus on ANCA testing for AAV needs revision.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/blood , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/diagnosis , Antibodies, Antineutrophil Cytoplasmic/blood , Myeloblastin/immunology , Peroxidase/immunology , Area Under Curve , Case-Control Studies , Fluorescent Antibody Technique, Indirect , Immunoassay/methods , ROC CurveABSTRACT
Objective: The objective of this multicentre study was to improve the clinical interpretation of PR3- and MPO-ANCAs as an adjunct for the diagnosis of ANCA-associated vasculitis (AAV) by defining thresholds and test result intervals based on predefined specificities and by calculating test result interval-specific likelihood ratios (LRs). Methods: Eight different PR3- and MPO-ANCA immunoassays from seven companies were evaluated using 251 diagnostic samples from AAV patients and 924 diseased controls. Results: Thresholds for antibody levels were determined based on predefined specificities (95, 97.5, 99 and 100%) and used to delimit test result intervals. Test result interval-specific LRs were determined. For all assays, the LR for AAV increased with increasing antibody level. For all but one immunoassay, high antibodies levels (associated with LR >55) were found in a substantial fraction (>65%) of patients. The area under the curve (AUC) of receiver operating characteristics analysis of a diagnostic approach in which positive results were confirmed by IIF or another immunoassay was not substantially higher than the AUC of performing immunoassay only. The highest AUC was found when immunoassay was combined with another immunoassay or with IIF. Conclusion: To diagnose AAV based on PR3- and MPO-ANCA, it is useful to define thresholds for antibody levels and to assign test result interval-specific LRs. Higher antibody levels are associated with a higher likelihood for disease. Such information improves clinical interpretation.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/diagnosis , Antibodies, Antineutrophil Cytoplasmic/blood , Myeloblastin/immunology , Peroxidase/immunology , Biomarkers/blood , Case-Control Studies , Humans , Immunoassay/methods , Likelihood Functions , Sensitivity and SpecificityABSTRACT
OBJECTIVES: The aim of this study was to analyse the role of netting neutrophils in the pathogenesis of granulomatosis with polyangiitis (GPA), especially their interplay with peripheral blood mononuclear cells (PBMCs). METHODS: The amount of cell-free DNA (cfDNA) was determined in sera from GPA patients (pairs active/inactive state of disease, n=18) and from healthy controls (HCs, n=10). Furthermore, we performed in vitro incubation experiments using PBMCs and NETs from patients and HCs for accessing the effect of NETs on PBMC behaviour. We determined proliferation of T- and B-cells (CSFE assay), B-cell maturation (CD38 staining and flow cytometry), production of IgG (ELISpot, ELISA), and secretion of the cytokines IFN-γ, IL-4, IL-10, IL-17A (ELISA). RESULTS: We detected a significant increase in serum cfDNA levels of GPA patients compared to HCs. The concentration of cfDNA was associated with disease activity. NETs of patients and HCs induced proliferation of CD4+ T- cells and CD19+ B-cells and maturation of B-cells. Furthermore, we detected an increase in IL-17A secretion after stimulating PBMCs with NETs. A significant difference between PBMCs from GPA patients and HCs was not detectable. CONCLUSIONS: NETs activate PBMCs of HCs and GPA patients. Our findings give supportive evidence that NETosis plays a role in the pathogenesis of GPA.
Subject(s)
B-Lymphocytes/immunology , Extracellular Traps/immunology , Granulomatosis with Polyangiitis/immunology , Lymphocyte Activation , Neutrophils/immunology , Paracrine Communication , T-Lymphocytes/immunology , B-Lymphocytes/metabolism , Biomarkers/blood , Case-Control Studies , Cell Proliferation , Cells, Cultured , DNA/blood , Extracellular Traps/metabolism , Female , Granulomatosis with Polyangiitis/blood , Granulomatosis with Polyangiitis/diagnosis , Humans , Interleukin-17/blood , Male , Neutrophils/metabolism , Phenotype , T-Lymphocytes/metabolism , Th17 Cells/immunology , Th17 Cells/metabolismABSTRACT
Autoantibodies to nuclear antigens arise in human autoimmune diseases, but a unifying pathogenetic mechanism remains elusive. Recently we reported that exposure of neutrophils to inflammatory conditions induces the citrullination of core histones by peptidylarginine deiminase 4 (PAD4) and that patients with autoimmune disorders produce autoantibodies that recognize such citrullinated histones. Here we identify histone H1 as an additional substrate of PAD4, localize H1 within neutrophil extracellular traps, and detect autoantibodies to citrullinated H1 in 6% of sera from patients with systemic lupus erythematosus and Sjögren's syndrome. No preference for deiminated H1 was observed in healthy control sera and sera from patients with scleroderma or rheumatoid arthritis. We map binding to the winged helix of H1 and determine that citrulline 53 represents a key determinant of the autoantibody epitope. In addition, we quantitate RNA for H1 histone subtypes in mature human neutrophils and identify citrulline residues by liquid chromatography and tandem mass spectrometry. Our results indicate that deimination of linker histones generates new autoantibody epitopes with enhanced potential for stimulating autoreactive human B cells.-Dwivedi, N., Neeli, I., Schall, N., Wan, H., Desiderio, D. M., Csernok, E., Thompson, P. R., Dali, H., Briand, J.-P., Muller, S., Radic, M. Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity.
Subject(s)
Autoantibodies/immunology , Autoimmunity/immunology , Histones/immunology , Neutrophils/immunology , Amino Acid Sequence , Autoantigens/immunology , Autoimmune Diseases/immunology , Epitopes/immunology , Humans , Immunoglobulin G/immunology , Molecular Sequence Data , Sequence AlignmentABSTRACT
Anti-neutrophil cytoplasmic autoantibodies (ANCA) with specificity for proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA) are a defining feature of ANCA-associated vasculitides (AAV). They play a pivotal role in disease pathophysiology and have strongly improved early diagnosis and treatment of these infrequent, but potentially fatal diseases. Neutrophils and their products are major players in initiating the autoimmune response and tissue destruction in vasculitic as well as granulomatous inflammation. This review highlights recent findings on old and novel players (ANCA, neutrophils, neutrophil extracellular traps, fibroblasts, immune cells and complement) and puts them into context with the current understanding of disease mechanisms in AAV.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/etiology , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Autoimmunity/immunology , Complement System Proteins/immunology , Humans , Neutrophils/immunologySubject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/diagnosis , Antibodies, Antineutrophil Cytoplasmic/blood , Immunoassay/standards , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/blood , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Calibration , Data Interpretation, Statistical , Diagnosis, Differential , Humans , Immunoassay/methods , Likelihood Functions , Myeloblastin/immunology , Peroxidase/immunology , Reference Standards , Sensitivity and SpecificityABSTRACT
Autoimmune diseases are rare, but their incidence has increased over the past decades. Interestingly, the co-occurrence of autoimmune diseases is well documented; however, data on the presence of more than one specific autoantibody in healthy individuals are not available. Here, we investigated the prevalence of several autoantibodies in a cohort of over 6000 healthy persons. While individual autoantibodies were rarely detected (i.e. ranging from 0.3% for ANCA to 4.6% for anti-TPO), the cumulative prevalence of the tested autoantibodies was as high as 10%. Furthermore, our results demonstrate co-occurrence of ANA with specific autoantibodies that target TPO, CCP and Dsg1/3, while ANCA and autoantibodies to PCA and BP180/BP230 were not more frequent in ANA-positive compared to ANA-negative samples. This indicates that shared and independent mechanisms influence loss of tolerance to distinct sets of self-antigens.
Subject(s)
Autoantibodies/blood , Autoimmune Diseases/immunology , Blood Donors , Adolescent , Adult , Aged , Antibodies, Antineutrophil Cytoplasmic/immunology , Antibodies, Antinuclear/immunology , Cohort Studies , Female , Humans , Male , Middle Aged , Prevalence , Young AdultABSTRACT
A key feature of granulomatosis with polyangiitis (GPA; or Wegener's granulomatosis) is the granulomatous inflammation of the upper respiratory tract, which leads to the subsequent destruction of adjacent tissues. The aim of our work was to study the histopathological and cellular components of tissue destruction of human GPA tissue transplanted into immunodeficient mice. Biopsy specimens from patients with active GPA (n = 10) or sinusitis (controls, n = 6) were s.c. co-implanted with healthy allogeneic human nasal cartilage into immunodeficient pfp/rag2(-/-) mice. Transplants were examined for their destructive capability of the allografted human cartilage. In addition, nasal fibroblasts from patients with GPA (n = 8) and control healthy nasal fibroblasts (n = 5) were cultured, and cell proliferation and apoptosis were quantified. mRNA and protein levels of matrix metalloproteinases and cytokines were evaluated at baseline and after proinflammatory stimulation. GPA implants showed massive destruction of the co-implanted human cartilage, whereas cartilage destruction was only marginal in control samples. Destruction was mediated by human fibroblasts and could be inhibited by corticoid treatment. The up-regulated production of matrix metalloproteinases 1, 3, and 13 and cytokines IL-6 and IL-8 was found in vivo and in vitro. Although proliferation of isolated fibroblasts was comparable between GPA and controls, GPA samples showed a significant delay of apoptosis. The destruction of nasal cartilage in GPA is mainly mediated by fibroblasts that can be blocked by corticosteroids, and this tissue destruction is not dependent on the influx of leukocytes.
Subject(s)
Fibroblasts/physiology , Granulomatosis with Polyangiitis/pathology , Nasal Cartilages/pathology , Adult , Aged , Animals , Apoptosis/physiology , Cell Proliferation , Cells, Cultured , Cytokines/biosynthesis , Dexamethasone/pharmacology , Dexamethasone/therapeutic use , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Profiling/methods , Glucocorticoids/pharmacology , Glucocorticoids/therapeutic use , Granulomatosis with Polyangiitis/complications , Granulomatosis with Polyangiitis/drug therapy , Humans , Immune Tolerance , Male , Matrix Metalloproteinases/biosynthesis , Mice , Mice, Knockout , Middle Aged , Nasal Cartilages/transplantation , Nasal Mucosa/pathology , Nasal Mucosa/transplantation , Nose Deformities, Acquired/etiology , Nose Deformities, Acquired/pathology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction/methods , Young AdultABSTRACT
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.
Subject(s)
Arthritis, Rheumatoid/immunology , Autoantibodies/immunology , Lupus Erythematosus, Systemic/immunology , Systemic Vasculitis/immunology , Arthritis, Rheumatoid/diagnosis , Autoantibodies/blood , Autoantibodies/isolation & purification , Biomarkers/blood , Disease Progression , Humans , Intercellular Signaling Peptides and Proteins/blood , Intercellular Signaling Peptides and Proteins/immunology , Lupus Erythematosus, Systemic/diagnosis , Progranulins , Protein Array Analysis , Systemic Vasculitis/diagnosisABSTRACT
OBJECTIVE: The aim of the study was to characterize the expression of TLR2, TLR4 and TLR9 in PMNs of patients with granulomatosis with polyangiitis (GPA) and to elucidate the role of these receptors in GPA with respect to neutrophil activation. METHODS: The expression of TLR2, TLR4 and TLR9 was determined on ex vivo PMNs in whole blood samples of GPA patients (n = 35) and healthy controls (HCs) (n = 24). Isolated PMNs were stimulated in vitro with TLR agonists and assessed for degranulation, membrane proteinase 3 (mPR3) expression, soluble l-selectin shedding and cytokine production (IL-8) in five GPA patients and five HCs. The priming effects of TLR2 and TLR9 ligation were assessed by measurement of serine protease activity after stimulation with PR3-ANCA. RESULTS: There were no significant differences in the ex vivo expression of TLRs on PMNs in HCs and GPA patients. Stimulation of TLR4 and TLR9 induced MPO release, stimulation with TLR2, TLR4 and TLR9 ligands elicited IL-8 production and stimulation of TLR2 and TLR9 led to an upregulation in mPR3 expression on PMNs with no significant differences between GPA and HC after 1 or 24 h stimulation. Priming of PMNs with TLR2 and TLR9 ligands induced degranulation after subsequent stimulation with PR3-ANCA, which was comparable to priming with TNF-α. CONCLUSION: Expression of TLR2, TLR4 and TLR9 in PMNs and the TLR-induced activation of PMNs was comparable in GPA and HC. mPR3 upregulation by TLR2 and TLR9 stimulation and the priming effect of TLR ligands on PMNs may have a potential implication for triggering disease activity during infection in GPA.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/metabolism , Neutrophil Activation/physiology , Neutrophils/metabolism , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 9/metabolism , Adult , Aged , Aged, 80 and over , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/physiopathology , Case-Control Studies , Cohort Studies , Female , Humans , Interleukin-8/metabolism , Male , Matrix Metalloproteinase 16/metabolism , Middle Aged , Peroxidase/metabolism , Toll-Like Receptor 4/metabolism , Up-Regulation , Young AdultABSTRACT
OBJECTIVE: To test the hypothesis that autoantigen modifications by peptidylarginine deiminase type 4 (PAD-4) increase immunoreactivity. METHODS: We assembled sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Felty's syndrome (FS), and antineutrophil cytoplasmic antibody-associated vasculitides (AAVs), as well as sera from control subjects without autoimmune diseases. The sera were tested for binding to activated neutrophils, deiminated histones, and neutrophil extracellular chromatin traps (NETs). IgG binding to lipopolysaccharide-activated neutrophils was assessed with confocal microscopy, and binding to in vitro-deiminated histones was measured using enzyme-linked immunosorbent assay (ELISA) and Western blotting. In addition, we quantitated histone deimination in freshly isolated neutrophils from the blood of patients and control subjects. RESULTS: Increased IgG reactivity with activated neutrophils, particularly binding to NETs, was paralleled by preferential binding to deiminated histones over nondeiminated histones by ELISA in a majority of sera from FS patients but only in a minority of sera from SLE and RA patients. Immunoblotting revealed autoantibody preference for deiminated histones H3, H4, and H2A in most FS patients and in a subset of SLE and RA patients. In patients with AAVs, serum IgG preferentially bound nondeiminated histones over deiminated histones. Increased levels of deiminated histones were detected in neutrophils from RA patients. CONCLUSION: Circulating autoantibodies in FS are preferentially directed against PAD-4-deiminated histones and bind to activated neutrophils and NETs. Thus, increased reactivity with modified autoantigens in FS implies a direct contribution of neutrophil activation and the production of NET-associated nuclear autoantigens in the initiation or progression of FS.
Subject(s)
Autoantibodies/immunology , Felty Syndrome/immunology , Histones/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Arthritis, Rheumatoid/immunology , Autoantigens/immunology , Female , Humans , Lupus Erythematosus, Systemic/immunology , Male , Middle Aged , Neutrophils/immunologyABSTRACT
During the past 30 years, remarkable progress has been achieved in understanding the role of the antineutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3) and myeloperoxidase (MPO) in so-called "ANCA-associated vasculitides". In the right clinical context, ANCAs are a good seromarker for these diseases, have improved nomenclature of systemic vasculitides and have contributed to new pathogenic concepts. However, problems with the clinical applications of ANCA testing in daily practice remain. They can be summarised as follows: assay standardisation and performance; the use of ANCA testing in a clinical setting with a low pretest probability; the relationship between ANCA titres and disease activity remains unclear. The solution to problems regarding the ANCA diagnosis is focussed on the fundamental methods, i.e., correct implementation of IFT and ELISA, the cautious use of commercial assays and restricting the use of the tests to clinical situations with a rather high pretest probability of AAV.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/diagnosis , Antibodies, Antineutrophil Cytoplasmic/blood , Enzyme-Linked Immunosorbent Assay , Myeloblastin/immunology , Peroxidase/immunology , Serologic Tests , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/blood , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Biomarkers/blood , Enzyme-Linked Immunosorbent Assay/standards , Humans , Practice Guidelines as Topic , Predictive Value of Tests , Prognosis , Serologic Tests/standards , Severity of Illness IndexABSTRACT
The detection of circulating autoantibodies against a variety of structural and functional molecules present in ubiquitous or tissue-specific cells is critical to the diagnosis of many autoimmune diseases (systemic - such as systemic rheumatic diseases - and organ-specific diseases). In particular, the determination of autoantibodies is one of the classification and/or diagnostic criteria for some autoimmune diseases and has a relevant predictive value, since many autoantibodies can be detected years before the clinical manifestation of a disease.The identification of these antigen-antibody systems and the simultaneous development of laboratory methods for detecting and measuring autoantibodies are considered one of the milestones in the history of clinical immunology over the last 60 years. Many different immunoassay methods have been developed and used in laboratory practice, from the early conventional (or monoplex) analytical methods that can detect single autoantibodies to the newer multiplex platforms that can quantify dozens of molecules. Various diagnostic immunoassays commonly used in current laboratory practice for the detection of autoantibodies are presented in this review.