Granulocyte-specific Antinuclear Antibodies Research Articles

Antineutrophil cytoplasmic antibodies: Major autoantigens, pathophysiology, and disease associations

Antineutrophil cytoplasmic antibodies (ANCA) are important serological markersfor the primary systemic vasculitides, including microscopic polyarteritis and necrotizing crescentic glomerulonephritis. Numerous reports have established the clinical utility of ANCA titer in monitoring disease activity, relapses, and response to treatment. ANCA, detected by indirect immunofluorescence (IIF) assays using patient's serum and ethanol-fixed human neutrophils, produce two common fluorescent staining patterns: cytoplasmic (C-ANCA), involving a 29-kD neutral serine protease termed proteinase 3 (PR3), and perinuclear (P-ANCA), the result mainly of myeloperoxidase (MPO), but occasionally by other components of the azurophilic granules including lysozyme, elastase, cathepsins, and lactoferrin. Some sera contain granulocyte-specific antinuclear antibodies (GS-ANA), which require formaldehyde fixation of neutrophils to cross link cytoplasmic antigens for distinguishing between ANCA and the GS-ANA by IIF. Positive IIF is confirmed by Western blot analysis or specific enzyme-linked immunosorbent assay for PR3, MPO, and other neutrophil granule antigens. The C-ANCA pattern is highly specific for Wegener's granulomatosis, a disease characterized by granulomatous inflammation, necrotizing and crescentic glomerulonephritis, and vasculitis; P-ANCA is found in sera of individuals with vasculitis, glomerulonephritis, and several other diseases. ANCA are predominantly immunoglobulin (Ig)G isotype, but may be IgM and IgA. Various pathophysiologic mechanisms have been proposed involving ANCA-mediated neutrophil activation in a hypothetical model of vasculitic diseases: positive signals via the FcγRII (CD32) receptor after IgG-ANCA binding to membrane-associated PR3, relevant cytokines, production of adhesion molecules on both activated neutrophils and endothelial cells, and the release of neutrophil reactive oxygen species and degranulation causing endothelial cell damage. Interference of C-ANCA with PR3 proteolysis and PR3 inhibition physiologically by the α 1-proteinase inhibitor may have a pathogenic role. No convincing data have been reported for the existence of autoreactive T lymphocytes reactive to any degree with the neutrophil azurophilic enzymes. Studies of various drug- and infectious agent-related diseases and ANCA may contribute to understanding the mechanism(s) involved in some vasculitides.

Effect of proctocolectomy on serum antineutrophil cytoplasmic antibodies in patients with chronic ulcerative colitis.

To study the effect of proctocolectomy on the antineutrophil cytoplasmic antibody (ANCA) titres in association with ulcerative colitis. Serum samples were taken from 15 patients with ulcerative colitis immediately before and at a mean of 24 months after proctocolectomy. Indirect immunofluorescence for ANCA and enzyme immunoassays for myeloperoxidase and proteinase-3 antibodies were employed. A liver biopsy was taken from every patient during the proctocolectomy, and serum liver enzyme activities were also determined. Before proctocolectomy, 13 of the 15 patients had perinuclear antineutrophil cytoplasmic antibodies (p-ANCA). Additionally, one patient had a low tire of classical cytoplasmic ANCA and one had granulocyte specific antinuclear antibodies. After proctocolectomy, the ANCA titres decreased in 10 patients, in two of whom they became negative. The titres remained the same in four patients with positive ANCA and increased twofold in one patient. Only one patient was proteinase-3 antibody positive and all 15 patients were myeloperoxidase antibody negative. The clinical condition improved in all patients, irrespective of the ANCA status after proctocolectomy. Seven patients, all of whom were positive for p-ANCA before proctocolectomy, had histological liver abnormalities. No correlation was observed between serum liver enzyme levels and ANCA staining patterns or titres. Proctocolectomy decreased the ANCA titres in the majority of our patients, suggesting that reduction of the inflammation or the available antigenic material modifies the immune disturbance related to ulcerative colitis.

Antineutrophil cytoplasmic antibodies in inflammatory arthritis--potential for misdiagnosis?

Antineutrophil cytoplasmic antibodies (ANCA) are well described in Wegener's granulomatosis and some forms of vasculitis. They have also been described in patients with arthritis, but the specificity of these ANCA and their relationship to the presence of vasculitis, antinuclear antibodies (ANA) and granulocyte-specific ANA (GS-ANA), and to disease activity are uncertain. We studied 101 patients with forms of inflammatory arthritis and detected four cytoplasmic ANCA, eight perinuclear ANCA and 16 atypical ANCA. There was no association between the presence of ANCA and ANA or rheumatoid factor. No anti-PR3 antibodies were found and no strong anti-myeloperoxidase antibodies were detected. Four GS-ANA were detected and were distinct from ANCA. There was no association between rheumatoid arthritis disease activity or disability and ANCA status. ANCA did not predict vasculitis over a 3 yr follow-up. These ANCA appear to be epiphenomena. Their importance lies in their potential to mislead physicians towards a misdiagnosis of vasculitis.

Anti-granulocyte perinuclear antibodies but not anti-neutrophil cytoplasmic antibodies (ANCA) in rheumatoid arthritis.

We studied 45 patients with rheumatoid arthritis for the presence of ANCA. These antibodies were determined by indirect immunofluorescence (IIF) and by enzyme-linked immunosorbent assays (ELISAs) using as a substrate purified myeloperoxidase and purified extract of azurophilic granules. By IIF, we found a characteristic perinuclear immunostaining pattern in 21 cases (47%). However, no patient had a positive result by the two ELISAs performed. Patients with a positive IIF result had significantly higher levels of anti-nuclear and anti-ds DNA antibodies than those with a negative IIF result. Therefore, these antibodies must correspond to the previously reported as granulocyte specific antinuclear antibodies (GS-ANA).

Anti-neutrophil antibodies in chronic hepatitis and the effect of α-interferon therapy

In 34 patients with non-A non-B, 28 with type B and 11 with autoimmune chronic hepatitis, anti-neutrophil antibodies were investigated using indirect immunofluorescence and anti-myeloperoxidase antibodies by enzyme-linked immunosorbent assay. Granulocyte-specific antinuclear antibodies, were detected in 14 patients with advanced stages of non-A, non-B hepatitis (41%). Their presence correlated with histological features of disease activity but not with response to interferon therapy. Within 24 h after the first dose of interferon, 9 of these became negative and 3 more became negative after 1, 3 and 5 months. Myeloperoxidase-positive perinuclear neutrophil cytoplasmic antibodies were detected in a single patient and increased reaching a peak level after 8 weeks of interferon, decreasing thereafter. In type B, all were negative before and during the 6 months of therapy. In 6 patients with autoimmune hepatitis (55%), myeloperoxidase-negative perinuclear neutrophil cytoplasmic antibodies were detected in high titers. The association of granulocyte-specific anti-nuclear antibodies with non-A, non-B hepatitis support the hypothesis that hepatitis C virus infection might trigger humoral autoimmune response. In chronic autoimmune hepatitis, perinuclear neutrophil cytoplasmic antibodies appear as another marker of autoimmunity.

Anti-granulocyte antibodies (C-ANCA, P-ANCA, GS-ANA) studied by confocal scanning laser fluorescence microscopy, ELISA, and chemiluminescence techniques.

Fifty-nine patient sera with antibodies against human polymorphonuclear neutrophil granulocyte (PMN) antigens, as determined primarily by indirect immunofluorescence microscopy (IIF) screening, were further analysed by enzyme-linked immunosorbent assays (ELISA). The antibodies were primarily characterized by their immunomorphological staining patterns on ethanol-fixed PMN as judged by conventional IIF microscopy, i.e. anti-neutrophil cytoplasmic antibodies (ANCA) giving a pancytoplasmic granular staining pattern (C-ANCA) or a diffuse perinuclear cytoplasmic pattern (P-ANCA), or granulocyte-specific anti-nuclear antibodies (GS-ANA) producing a homogeneous or peripheral nuclear staining pattern. The three distinct patterns were confirmed by confocal scanning laser IIF microscopy. As antigen substrates in the ELISA tests we used an extract from azurophil PMN granules, myeloperoxidase (MPO), and lactoferrin. As expected, most (but not all) of the C-ANCA positive sera turned out positive in the alpha-ELISA assay. Both P-ANCA and GS-ANA positive sera had high frequencies of antibodies against MPO. Occasional P-ANCA positive sera contained antilactoferrin antibodies. Although P-ANCA and GS-ANA in general probably represent the same type of auto-antibodies, we regard it appropriate to make a distinction between the two patterns, until the existence of 'true' granulocyte-specific ANAs has been ruled out. All sera were analysed for their ability to activate PMN in vitro as judged by the generation of a chemiluminescence (CL) response. Sera containing C-ANCA, as well as sera containing P-ANCA or GS-ANA, showed high frequencies of positive CL tests using 'resting' isolated PMN. The reactions were diminished, but not always abolished, by heat-treatment of the sera.

Antineutrophil Cytoplasmic Autoantibodies Antigen Specificity

The results presented during the Third International ANCA Workshop, Washington, DC, 1990, allowed a better definition of the antigenic specificity of the antineutrophil cytoplasmic autoantibodies (ANCA). The large predominance of two major antigen specificities for proteinase 3 (PR3) and myeloperoxidase (MPO), in the group of vasculitic patients, was confirmed. PR3 and MPO are colocalized in the azurophilic granules of neutrophils and translocated to the cell surface during activation and thus are able to interact with ANCA after neutrophil preactivation. Furthermore, by comparison of amino acid and DNA sequences, the agreement was reached that PR3 was identical to AGP7, p29, and myeloblastin, described independently and involved in the control of growth and differentiation of leukemic cells. In addition to the two major ANCA antigens, a number of neutrophil cytoplasmic antigens recognized by ANCA have been previously identified (human leukocyte elastase [HLE], lactoferrin). It was established during the Third Workshop that these rare ANCA specificities, occurring in a limited number of patients, include a cationic antimicrobial protein (CAP57) and cathepsin G. However, the variety of ANCA antigen specificities contrasts with the fact that the vast majority of ANCA-positive sera are monospecific for a single ANCA antigen. Finally, the fine specificity of granulocyte-specific antinuclear antibodies (GS-ANA) occurring in rheumatoid arthritis and ulcerative colitis is still unknown, but clearly a substantial proportion of GS-ANA belongs to the ANCA family.

An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA)

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of circulating anti-neutrophil cytoplasm antibodies (ANCA), which are defined by a diffuse, granular staining of the cytoplasm of alcohol-fixed human neutrophils by indirect immunofluorescence (IIF). Detection of anti-neutrophil cytoplasm antibodies has a high sensitivity and specificity for active Wegener's granulomatosis (WG) and reflects the effect of treatment. In the present enzyme-linked assay, immunoplates were coated with the cytoplasmic α fraction of neutrophils obtained from apparently healthy human donors by nitrogen bomb cavitation and subsequent Percoll gradient centrifugation. Alkaline phosphatase-labelled anti-human IgG was used as a secondary antibody. Diluted sera from 70 patients with WG and 16 patients with other diseases with anti-myeloperoxidase antibodies (anti-MPO) were examined. It is concluded that the ELISA accurately detects IIF ANCA positive patients with WG, is helpful in detecting WG patients in remission, is not influenced by the presence of anti-MPO and may help in detecting ANCA in cases with granulocyte-specific anti-nuclear antibodies since this IIF pattern obscures the IIF ANCA patterns. The ELISA with titration can be carried out in 3.5 h whereas a rapid test just to detect ANCA can be performed in 30 min.

Absence of Circulating Antineutrophil Cytoplasm Antibodies (ANCA) in Severe Vasculitis Associated with Rheumatoid Arthritis

Sixteen patients with classical rheumatoid arthritis (RA) complicated by severe vasculitis were studied and compared with a matched control group of 17 RA patients without vasculitis. A control group of 24 patients with unrelated disease was also included. Neither antineutrophil cytoplasmic antibodies (C-ANCA) nor anti-myeloperoxidase (anti-MPO) were found. Granulocyte specific antinuclear antibodies ("GS-ANA") were found in a higher frequency in the vasculitis group (75%) than in the RA control group (41%), but the difference was not statistically significant. No patient in the control group without RA had "GS-ANA".

Circulating anti‐neutrophil cytoplasm antibodies in patients with rapidly progressive glomerulonephritis and extracapillary proliferation

We investigated 39 patients with rapidly progressive glomerulonephritis and extracapillary proliferation for the presence of circulating anti-neutrophil cytoplasm antibodies (ANCA) by indirect immunofluorescence. Patients with antibodies against the glomerular basement membrane, systemic lupus erythematosus or with glomerulonephritis following infection and where renal disease could be ascribed to treatment with drugs were not included. IgG class ANCA producing diffuse cytoplasmic immunostaining were detected in 54% of the patients. With reservation for the difficulties involved in distinguishing between different forms of systemic vasculitis, these autoantibodies were present in patients with Wegener's granulomatosis (12 out of 17 patients), polyarteritis nodosa (4/6), idiopathic extracapillary glomerulonephritis (1/4), and with less distinct syndromes (4/10), but not in Henoch-Schönlein's purpura (0/2). Another 18% of the patients showed granulocyte-specific anti-nuclear antibodies' with perinuclear immunostaining. This pattern was recently reported to indicate the presence of antibody against myeloperoxidase of alcohol-fixed neutrophils. Autoimmune mechanisms, particularly those involving components of neutrophil granulocytes, could play a significant role in development of extracapillary glomerulonephritis.

Radiographic progression of rheumatoid arthritis related to some clinical and laboratory parameters.

In 188 patients followed for 3 to 12 years, the radiologic course of rheumatoid arthritis was assessed in 20 joint groups. A severe course in most joints was related to the presence of rheumatoid factor and to high values of the ESR. Granulocyte-specific antinuclear antibodies were related to a severe course in most joints. The presence of nodules, the Rose-Waaler titre and the presence of organ non-specific antinuclear antibodies were generally unrelated to the course of the disease.

Granulocyte-specific antinuclear antibodies. Possible significance for the pathogenesis, clinical features and diagnosis of rheumatoid arthritis.

Publisher Summary This chapter focuses on granulocyte-specific antinuclear antibodies (GS-ANA). GS-ANA in rheumatoid arthritis and Felty's syndrome are mainly type G immunoglobulin antibodies with a tendency to engage in circulating and synovial fluid immune complexes––these are produced locally in the rheumatoid synovium indicating a relationship to pathogenesis. Some evidences suggest role for GS-ANA in pathogenesis, such as the higher prevalence and levels found in active disease and the presence of GS-ANA in local and circulating immune complexes.

Antinuclear antibodies in ankylosing spondylitis, psoriatic arthritis, and psoriasis.

Granulocyte-specific antinuclear antibodies (GS-ANA) were detected in the sera of 5 of 88 patients with ankylosing spondylitis (AS) and in 7 of 52 cases of psoriatic arthritis (PsA), but were not found in 91 patients with malignant or non-malignant chest disease nor in 25 cases of psoriasis. Organ non-specific ANA were present in serum from 6 cases of AS and 1 of PsA. None of the sera gave significant levels for soluble immune complexes as detected by a C1q-binding assay. The presence of antinuclear antibodies was not associated with clinical features or drug therapy in either AS or PsA.

THE PREVALENCE AND POSSIBLE SIGNIFICANCE OF IgD GRANULOCYTE‐SPECIFIC ANTINUCLEAR ANTIBODIES IN NEUTROPENIC AND NON‐NEUTROPENIC CASES OF RHEUMATOID ARTHRITIS

Sera from 21 rheumatoid arthritis patients with accompanying neutropenia (less than or equal to 2000 neutrophils/microliter) and 45 rheumatoid arthritis patients without neutropenia were studied for the occurrence of IgD granulocyte-specific antinuclear antibodies. Such antibodies were found in 67 per cent of the neutropenic and 18 per cent of the non-neutropenic cases (p less than 0.001). The titres of IgD granulocyte-specific antinuclear antibodies varied independently of the titres of IgD and complement-fixing granulocyte-specific antinuclear antibodies, but showed some covariation with granulocyte-specific antinuclear antibodies of the IgA and IgM classes most probably reflecting a broad polyclonal antibody response to phagocyte nuclear antigens in the serologically highly active neutropenic cases. Gel filtration studies on selected sera containing IgD granulocyte-specific antinuclear antibodies indicated participation of these antibodies in immune complexes. It is thus possible that IgD granulocyte-specific antinuclear antibodies may have some significance for the rheumatoid inflammatory processes.

Eosinophil-specific and other granulocyte-specific antinuclear antibodies in juvenile chronic polyarthritis and adult rheumatoid arthritis.

Sera from 151 children of whom 112 had juvenile chronic polyarthritis (JCP), and from adults with rheumatoid arthritis (RA), and from healthy pregnant females were tested for the presence of granulocyte-specific antinuclear antibodies (GS-ANA). These were detected in 20% of sera from cases of JCP, in 68% of adult RA, but in none of the controls. Eosinophil-specific ANA were the only ANA present in 18% of positive children and 54% of the positive adults. GS-ANA in children were predominantly IgG and of low titre. Heat-stable GS-ANA were detected in sera from eight children but none bound complement. The presence of GS-ANA was not significantly associated with sex, age of onset, duration of disease, mean active joint count, mean ESR, nor with the presence of fever, rash, splenomegaly, amyloidosis, pericarditis, or rheumatoid factor.

Immunologic and clinical observations of granulocyte-specific antinuclear antibodies.

AbstractThe presence of granulocyte‐specific antinucleoprotein antibodies was established by absorption tests in 31 sera and unmasked in three additional sera. The majority of patients demonstrated rheumatoid arthritis, but 2 patients had lupoid hepatitis. Differences in fluorescent patterns of staining and degrees of heterologous reactivity delineated two separate granulocyte‐specific antinuclear antibodies, one of which showed a significant association with granulocytopenia.