Antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis after immunisation with bacterial proteins.

OBJECTIVE: There is circumstantial evidence for a role for infections in the development of the small vessel vasculitides associated with antineutrophil cytoplasmic antibodies (ANCA). The aim of this study was to determine whether the immunisation of rats with bacterial proteins could result in circulating ANCA, T cells with specificity for ANCA antigens, and a systemic vasculitis. METHODS: Adult male Wistar rats were immunised with pasteurised sonicated S. aureus (n = 7), E. coli (n = 8), purified protein derivative (PPD, n = 5), myeloperoxidase (MPO, n = 5) or phosphate-buffered saline (PBS, n = 5), in complete and in incomplete Freund's adjuvant. ANCA were assayed by indirect immunofluorescent (IIF) examination of normal rat neutrophils, and in ELISAs using human proteinase 3 (PR3), MPO and bactericidal/permeability-inreasing protein (BPI). The T cell response to PR3, MPO and BPI was assessed by a whole blood T cell proliferative assay in vitro, and by a delayed type hypersensitivity (DTH) response in vivo. Kidney and bowel were examined histologically for evidence of vasculitis and colitis. RESULTS: One rat from each group immunised with S. aureus or E. coli developed pauciimmune segmental glomerular sclerosis. The rat immunised with E. coli had additionally an arteritis affecting renal interlobular and gut vessels. This rat had circulating C-ANCA, that produced granular cytoplasmic neutrophil fluorescence with central accentuation, but the target antigen could not be determined in ELISAs using human PR3, MPO or BPI. In animals immunised with S. aureus or E. coli, there was no significant T cell proliferative or DTH response specific for human PR3, MPO or BPI. CONCLUSION: The development of ANCA and vasculitis in a rat immunised with bacterial proteins indicates that the relationship between infections and ANCA should be investigated further.

Indirect immunofluorescence (IIF) of normal washed peripheral blood cells to demonstrate antineutrophil cytoplasmic antibodies (ANCA).

BACKGROUND: The "International consensus document on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA)" requires all sera to be examined by indirect immunofluorescence (IIF). However, commercial neutrophil slides are expensive, fluorescence patterns can be difficult to interpret, and coincidental antinuclear antibodies (ANA) cannot be demonstrated; in addition, in house cytospin neutrophil preparations are time consuming to prepare and deteriorate with time. AIMS: To compare the IIF demonstration of ANCA, using washed peripheral blood cell smears, with commercial neutrophil preparations and with ANCA positivity as demonstrated by enzyme linked immunosorbent assay (ELISA). METHODS: Serum fluorescence positivity, pattern, and intensity using washed peripheral blood cell smears were compared with the results obtained using commercial neutrophil slides (INOVA). Fluorescence positivity, pattern, and intensity of 500 sera from consecutive patients with suspected vasculitis tested with washed peripheral blood cells were compared with binding in ELISAs for proteinase 3 (PR3) and myeloperoxidase (MPO). RESULTS: IIF of washed peripheral blood cell smears detected seven of eight sera with cytoplasmic fluorescence (C-ANCA), and 11 of 12 sera with perinuclear fluorescence (P-ANCA) demonstrated using commercial slides. The two sera that were negative by IIF were also negative in the ELISAs for both PR3-ANCA and MPO-ANCA. Of the 500 sera examined, there were 35 (7%) with C-ANCA, 65 (13%) with P-ANCA, and eight (2%) IIF negative sera that were positive by either ELISA. There was a strong correlation between C-ANCA fluorescence and PR3-ANCA values (p < 0.0001), and a moderate to strong correlation between P-ANCA fluorescence and MPO-ANCA values (p < 0.001) when ANCA fluorescence was demonstrated with washed peripheral blood cell smears. CONCLUSIONS: Washed peripheral blood cells are a convenient and useful low cost alternative to commercial or cytospin neutrophil preparations for the IIF demonstration of ANCA.

Clinical associations and characterisation of antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein and azurocidin.

Bactericidal/permeability-increasing protein (BPI) and azurocidin (AZ) are recently described target antigens of antineutrophil cytoplasmic antibodies (ANCA). In this study, BPI-ANCA were demonstrated most often in patients with ulcerative colitis (36/92, 39%), Crohn's disease (17/66, 26%) and cystic fibrosis (11/14, 79%), but also in patients with rheumatoid arthritis (8/40, 20%), systemic lupus erythematosus (SLE) (111/65, 17%) and mixed connective tissue disease (4/18, 22%). BPI-ANCA were also common in sera containing antinuclear (ANA) (9/43, 21%) or antidouble-stranded (ds) DNA (7/28, 25%) antibodies. There was no increased frequency of abnormal alpha1-antitrypsin (alphal1AT) phenotypes in patients with BPI-ANCA, and BPI-ANCA were not more common in individuals with an abnormal phenotype. The predominant IgG subclasses were IgG1 and IgG3; IgA but not IgM was present. Both IgG and IgA BPI-ANCA were high affinity antibodies, and the affinity of IgG antibodies did not change with time in the sera tested. Four of the five sera (80%) containing BPI-ANCA did not bind to denatured, reduced BPI, suggesting that most BPI-ANCA recognised conformational epitopes. AZ-ANCA were demonstrated in 2/11 patients (18%) with Wegener's granulomatosis, 3/12 (25%) with cystic fibrosis and 3/14 (21%) with chronic active hepatitis. AZ-ANCA were present in 5/25 sera (25%) with ANA, but the levels were only marginally elevated. AZ-ANCA were uncommon in patients with inflammatory bowel and rheumatological diseases, and in sera containing other autoantibodies. Again, there was no association with abnormal alpha1-AT phenotypes. BPI represents a major ANCA target antigen in patients with rheumatological as well as inflammatory bowel disease and cystic fibrosis, but AZ-ANCA are uncommon.

Pyoderma gangrenosum with secondary pyarthrosis following propylthiouracil.

The association of pyoderma gangrenosum and arthritic symptoms is well documented. We present a rarely reported variant of this in a 44-year-old woman with pyoderma gangrenosum and bilateral large purulent effusions of her knees. She had no evidence of underlying rheumatoid arthritis or a specific seronegative spondyloarthropathy. Of note she had a history of Graves' disease for which she had been treated with propylthiouracil for 3 years and on investigation at this presentation had a markedly elevated perinuclear antineutrophil cytoplasm antibody (P-ANCA) level with specificities for IgM myeloperoxidase, IgG elastase and IgG lactoferrin. We believe this patient had pyoderma gangrenosum with secondary sterile pyarthrosis and a P-ANCA precipitated by propylthiouracil.

International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA)

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in the primary systemic small vessel vasculitides. ANCA is best demonstrated in these diseases by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 (PR3) or myeloperoxidase (MPO). For ANCA testing in "new" patients, IIF must be performed on all serum samples. Serum samples containing ANCA, any other cytoplasmic fluorescence, or an antinuclear antibody (ANA) that results in homogeneous or peripheral nuclear fluorescence then should be tested in ELISAs for PR3-ANCA and MPO-ANCA. Optimally, ELISAs for PR3-ANCA and MPO-ANCA should be performed on all serum samples. Inclusion of the most recent positive sample in the IIF or ELISA may help demonstrate a change in antibody level. Reports should use recommended terms. Any report of positive neutrophil fluorescence issued before the ELISA results are available should indicate that positive fluorescence alone is not specific for the diagnosis of Wegener granulomatosis or microscopic polyangiitis and that decisions about treatment should not be based solely on the ANCA results.

A review of immunofluorescent patterns associated with antineutrophil cytoplasmic antibodies (ANCA) and their differentiation from other antibodies.

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.

Primary Sjögren's syndrome and gamma heavy chain disease.

We describe a patient who has primary Sjögren's syndrome associated with asymptomatic gamma heavy chain disease and a tubulointerstitial nephritis. Sjögren's syndrome is known to be complicated by lymphoproliferative disorders and tubulointerstitial nephritis but gamma heavy chain disease is rare (approximately 100 cases described). There is one previously reported case of gamma heavy chain disease associated with primary Sjögren's syndrome and 2 cases associated with secondary Sjögren's syndrome. Our patient and the 3 other patients described in the literature did not have evidence of an underlying lymphoproliferative disorder.

Autoantibodies to neutrophil cytoplasmic (ANCA) and endothelial cell surface antigens (AECA) in chronic inflammatory bowel disease.

Sera from 103 patients with chronic inflammatory bowel disease (IBD) were tested prospectively for antibodies against neutrophil cytoplasmic antigens (anti-neutrophil cytoplasm antibodies, ANCA) and endothelial cell surface antigens (anti-endothelial cell antibodies, AECA) by indirect immunofluorescence (IIF) and assays based on whole fixed neutrophils, purified neutrophil enzyme substrates and human umbilical vein endothelial cells. Using IIF, ANCA were found in 26 IBD sera (25%) and in none of 51 controls. Twenty-two positive sera (85%) were from patients with ulcerative colitis (UC). The pattern of distribution of immunofluorescence was always perinuclear (P-ANCA). A majority of UC patients positive for these autoantibodies (68%) had active colitis, but none had evidence of vasculitis. Using a whole neutrophil ELISA, binding was demonstrable in 73% of UC sera compared to 27% of Crohn's (CD) sera and only 4% of controls. Unlike vasculitis sera, UC sera with P-ANCA did not bind strongly to myeloperoxidase (MPO). Forty-five per cent of IBD sera tested positive for IgG AECA in an endothelial cell ELISA, compared to seven of 51 (14%) controls. Binding correlated with both active and extensive colitis. A type of P-ANCA, in most cases distinct from MPO-specific P-ANCA observed in vasculitis, is detected in a significant proportion of patients with UC, but rarely Crohn's colitis and therefore may be of differential diagnostic value. IgG AECA are also frequent in CIBD sera but are less disease specific than ANCA.