Analysis of anti-neutrophil cytoplasmic antibodies (ANCA): frequency and specificity in a sample of 191 homozygous (PiZZ) alpha1-antitrypsin-deficient subjects

Marie A. P. Audrain1, Richard Sesboüé2, Thierry A. R. Baranger3, Jane Elliott4, Angelo Testa3, Jean-Pierre Martin2, C. Martin Lockwood4 and Vincent L. M. Esnault,1,3

1 Immunology Department 3 Nephrology and Clinical Immunology Department, Hôtel Dieu, Nantes, France 2 INSERM Unité 295, Faculté de Médecine-Pharmacie Rouen, France 4 University of Cambridge, School of Clinical Medicine, Cambridge, UK



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. ANCA are autoantibodies directed against polymorphonuclear cell antigens, mainly proteinase 3 (PR3) and myeloperoxidase (MPO), which are implicated in the pathogenesis of small-vessel necrotizing vasculitis. Alpha1-antitrypsin is the main inhibitor of neutral serine proteinase [i.e. human leukocyte elastase (HLE) and PR3] present in PMN alpha-granules ({alpha}Gr). An association first reported by us between PR3 ANCA and the deficient PiZZ phenotype in ANCA-positive systemic vasculitis, now widely confirmed by others, led us to study the incidence and specificity of ANCA among PiZZ subjects.

Methods. We tested a population of 191 PiZZ (273 sera) for ANCA activity versus 272 PiMM matched control subjects using {alpha}Gr or antigen-specific ELISA [PR3, HLE, MPO, lactoferin (LF) and bactericidal/ permeability increasing protein (BPI)].

Results. The incidence of antibodies directed against {alpha}Gr and HLE but not PR3, MPO, LF or BPI was increased in the PiZZ as compared to the PiMM group (Fisher probability respectively P<0.0001 and P<0.05).

Conclusions. ANCA not directed against classical antigens (MPO and PR3) may be found in PiZZ patients. However, these patients do not develop systemic vasculitis features. Therefore, alpha1-antitrypsin deficiency is not sufficient to induce ANCA positive vasculitides, and may only act as a second hit amplifying factor.

Keywords: alpha1-antitrypsin; ANCA; elastase; pro-tease inhibitor; proteinase 3; systemic vasculitis



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Anti-neutrophil cytoplasmic antibodies (ANCA) are autoantibodies implicated in the pathogenesis of necrotizing vasculitis affecting small vessels (arterioles, capillaries and venules), particularly in Wegener's granulomatosis [1] and microscopic polyangiitis [2]. They are considered to be good markers of disease activity [3]. The main antigenic targets for ANCA are proteinase 3 (PR3) [4], myeloperoxidase (MPO) [5] and more rarely lactoferrin (LF) [6], human leukocyte elastase (HLE) [7] and bactericidal/permeability-increasing protein (BPI) [8].

Alpha1-antitrypsin ({alpha}1AT) is the main inhibitor of neutral serine proteinase (HLE and PR3) [9] present in alpha-granules ({alpha}Gr) of polymorphonuclear leukocytes. {alpha}1AT is encoded by a highly polymorphic gene, with at least 75 alleles at the so-called protease inhibitor (Pi) locus [10,11]. Pi alleles may be classified as either normal (M) or deficient (Z) according to their circulating {alpha}1AT function and levels. Because the two parental alleles are codominantly expressed, homozygous PiZZ is a severely deficient, heterozygous PiMZ a moderately deficient and PiMM a normal {alpha}1AT phenotype. Severe {alpha}1AT deficiency is recognized as a risk factor for the development of panlobular emphysema and various hepatic disorders in children and adults [1214] as well as autoimmune disorders such as anterior uveitis [15], systemic lupus erythematosus [16] and neutrophilic ulcerative panniculitis [17,18]. In adults with {alpha}1AT deficiency, some isolated case reports have indicated the possible occurrence of systemic vasculitis [1922] and necrotizing glomerulonephritis [2022].

Our observation that PR3 ANCA is associated with the deficient PiZZ phenotype in ANCA-positive systemic vasculitis [23] has been widely confirmed by others [2431], and led us to investigate the incidence of ANCA in a large series of 191 homozygous PiZZ-deficient patients versus 272 normal (PiMM) matched subjects. All sera were tested for ANCA activity by ELISAs specific for {alpha}Gr, MPO, LF and HLE. Sera with anti-{alpha}Gr antibodies were tested for anti-PR3 and anti-BPI activity. Indirect immunofluorescence was performed for all positive sera.



   Subjects and methods
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 Subjects and methods
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Patients
Two hundred and seventy-three serum samples from 191 PiZZ subjects diagnosed during the period 1988 to 1993 were studied versus 272 matched sera obtained from 272 PiMM patients during the same period. These sera were collected among a large series of patients routinely referred by clinicians for Pi ({alpha}1AT) phenotyping. PiZZ homozygotes corresponded to patients or relatives detected by systematic familial inquiry. PiMM patients were matched for age, sex, referring geographical area and their clinical status of patient or healthy relative. No clinical data were available before ANCA determination. Sera were stored at -20°C until use. All sera were tested for ANCA activity on antigen-specific ELISAs (anti-{alpha}Gr, anti-MPO, anti-LF and anti-HLE). Only sera with anti-{alpha}Gr antibodies were tested for anti-PR3 and anti-BPI activity, since in the coating condition used in this anti-{alpha}Gr assay only these autoreactivities are detected, despite the presence of significant amount of MPO and HLE in the preparation. Indirect immunofluorescence (IIF) was performed for all positive sera. Clinical data were requested retrospectively by questionnaire for all ANCA-positive patients after ANCA testing.

Alpha-granule purification and anti-{alpha}Gr ELISA
Briefly, polymorphonuclear leukocytes were separated on a methylcellulose-hypaque density gradient. After hypotonic lysis of erythrocytes and washing in phosphate-buffered saline (PBS) without Ca2+ and Mg2+, the pellet was diluted in buffer containing KCl, NaCl, MgCl2, PIPES and protease inhibitors, according to Borregaard et al. [32]. Primary and secondary granules were then separated by nitrogen cavitation of the neutrophils (350 psi, 20 min). The lysate was centrifuged, and the postnuclear supernatant collected and recentrifuged on a discontinuous Percoll gradient. The azurophilic fraction was collected and cryopreserved until use. For ELISA, azurophilic granules were lysed with 1% Triton X-100 and centrifuged. The supernatant was diluted in phosphate buffer, pH 11.9, 0.025 M, and used for microplate coating. After non-specific sites were blocked with PBS containing 2% bovine serum albumin (BSA, Sigma, St Louis, MO, USA), sera diluted 1:50 were incubated for 1 h at 37°C before incubation of an alkaline phosphatase-conjugated anti-human IgG (Sigma A3150, St Louis, MO, USA). Binding was detected by addition of pNPP, with reading of absorbance at 405 nm. The average optical density (OD) of tested sera was compared with that obtained using a reference panel of normal human sera (30 blood donors). Positivity was defined by an OD value higher than the reference mean plus 2 standard deviation (SD).

Antigen-specific assays
IgG anti-MPO and anti-LF ELISA were performed as previously described [6,33]. IgG anti-PR3 autoantibodies were detected using a commercially available ELISA kit (SHIELD, Dundee, UK).

IgG anti-HLE activity was searched for by ELISA as follows: Commercially purified HLE (Calbiochem, La Jolla, CA, USA) was diluted in carbonate buffer, pH 9.6, 0.02 M and coated at 0.5 µg/ml on microtiter plates overnight at +4°C. Serum samples (diluted 1:50) were applied to the plates in duplicate after non-specific binding sites were blocked with PBS containing 2% BSA. The detection system was an alkaline phosphatase-conjugated goat polyclonal anti-human immunoglobulin G reagent (Sigma A-3150, St Louis, MO, USA) diluted 1:1000 before addition of the substrate. The positivity level was defined by an OD value higher than 2 SD over the mean binding of 30 normal human sera.

IgG anti-BPI activity was searched for by ELISA as previously described [8].

ANCA indirect immunofluorescence (IIF)
IIF was performed as described [34]. Positive IIF were classified as either cytoplasmic (C-ANCA), perinuclear (P-ANCA) or atypical (X-ANCA). P-ANCA and X-ANCA were also tested for anti-nuclear activity (ANA) on HEp2 cells (Kallestad Diagnostics, Chaska, MN, USA).

Alpha1-antitrypsin ({alpha}1AT) assays
Pi phenotypes were determined by isoelectric focusing as described [35], except that Pharmalytes 4.2-4.9 (Pharmacia, St Quentin en Yvelines, France) were used.

Statistical analysis
Fisher's exact test was used to compare the distribution of specific autoantibodies in the two groups.



   Results
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 Subjects and methods
 Results
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 References
 
ELISA determination (Table 1Go and 3Go)
Forty-five PiZZ patients and 18 PiMM patients exhibited at least one ANCA antigen specific ELISA positivity (Table 1Go).


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Table 1. Antigen-specific ELISA in 191 PiZZ and 272 PiMM subjects

 

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Table 3. Clinical features of ANCA positive patients

 
Anti-{alpha}Gr ELISA was positive for 35/191 subjects in the PiZZ group versus 8/272 in the PiMM control group, a difference highly significant (P<0.0001) with Fisher's exact test.

Anti-MPO ELISA showed that 2/191 PiZZ and 3/272 PiMM subjects had anti-MPO antibodies (difference not statistically significant). Anti-MPO-positive PiZZ patients also exhibited anti-{alpha}Gr and anti-HLE activity, and one had anti-BPI activity. No other autoantibody activity was found in the three anti-MPO-positive PiMM patients.

Anti-LF ELISA detected 4/191 PiZZ and 2/272 PiMM with anti-LF antibodies (difference not statistically significant). One of the four anti-LF-positive PiZZ patients also exhibited anti-{alpha}Gr and anti-BPI activity. No other autoantibody activity was found in anti-LF-positive PiMM patients.

Anti-HLE ELISA showed that 11/191 PiZZ and 5/272 PiMM subjects had anti-HLE antibodies, a statistically significant difference (P<0.05). Five anti-HLE-positive PiZZ patients also had anti-{alpha}Gr antibodies, including one with anti-{alpha}Gr and anti-MPO activity, and one with anti-{alpha}Gr, anti-MPO and anti-BPI activity. No other autoantibody activity was found in the 5 anti-HLE-positive PiMM patients.

Anti-PR3 ELISA was used to test anti-{alpha}Gr-positive sera for anti-PR3 activity. No PiZZ (0/35) and only one PiMM serum exhibited anti-PR3 activity (difference not statistically significant).

Anti-BPI ELISA was used to test anti-{alpha}Gr-positive sera for anti-BPI activity. Fourteen out of 35 PiZZ subjects and 4/8 PiMM patients had anti-BPI antibodies (difference not statistically significant). One anti-BPI PiZZ patient also had anti-HLE activity, one had anti-LF activity, and one had anti-MPO and anti-HLE activity. No other specific autoantibody activity was found in the 4 anti-BPI-positive PiMM patients.

ANCA IIF (Table 2Go)
ANCA detected by IIF in 21/191 PiZZ patients showed a cytoplasmic pattern in 15 cases and perinuclear fixation in 6 (Table 2Go). The incidence of C-ANCA (P<0.005) and P-ANCA (P<0.05) was significantly higher in the PiZZ group than PiMM controls.


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Table 2. ANCA indirect immunofluorescence in 191 PiZZ and 272 PiMM subjects

 

Clinical features
The main clinical features are summarized in Table 3Go. Diagnoses were obtained in 27/45 ANCA-positive PiZZ patients and 3/18 ANCA-positive PiMM subjects.

Two PiZZ patients had systemic involvements. One with anti-{alpha}Gr, anti-LF and anti-BPI autoantibodies presented with rapidly progressive glomerulonephritis as well as emphysema, and the other with anti-{alpha}Gr had an unclassified vasculitis.

One PiMM patient with anti-PR3 antibodies presented with Wegener's granulomatosis.

The male/female ratio was 24/21 in ANCA-positive PiZZ and 14/4 in ANCA-positive PiMM (P=0.092). There was no difference in mean age between ANCA- positive PiZZ and PiMM groups (mean±SD: 48.2±18 and 47.8±22 respectively).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We first reported a higher incidence of deficient Pi phenotype in patients presenting PR3-ANCA-associated vasculitis, which suggested a possible involvement of protease/protease inhibitor imbalance in the pathogenesis of some PR3-ANCA-positive systemic vasculitis [23,31,36]. The present study demonstrates that ANCA positivity is a rare but significant event in subjects with severely deficient Pi phenotype. These ANCA are directed against {alpha}Gr antigens but not against classical ANCA antigens, except HLE which is also a serine protease specifically inhibited by {alpha}1AT. Although four PiZZ sera exhibited multiple ANCA specificities (one MPO/HLE, one LF/BPI, one HLE/ BPI, and one MPO/HLE/BPI), this cannot be an artefact due to non specific binding to plastic since all sera were at least negative in one ELISA assay. However, these ANCA ELISA proved to be highly sensitive since only 21 out of the 45 PiZZ subjects with ANCA ELISA positivity exhibited positive ANCA IIF. The clinical significance of these ANCA remains unclear. Several autoantibody specificities have been described in the normal immune repertoire. After in vitro stimulation of normal B lymphocytes, ‘natural’ autoantibodies may be produced, including rheumatoid factor [37] and anti-nuclear [38] and anti-histone antibodies [39]. Sera of patients with monoclonal gammopathies may also exhibit ANCA activity without vasculitis features [40]. The ANCA-positive PiZZ subjects had a mean age similar to that of ANCA-positive PiMM matched controls and patients presenting with ANCA-positive systemic vasculitis [41]. This is in contrast with the situation for ‘natural’ antinuclear autoantibodies that are found mainly in elderly subjects [42]. There was an even distribution by gender in ANCA-positive PiZZ subjects as observed in systemic vasculitis patients [41].

However, ANCA positivity was relatively rare (23.6%) in our PiZZ subjects. ANCA activity directed against classical antigens was found in only 2/191 PiZZ patients, with a specificity for MPO and not PR3. Clinical features of systemic vasculitis were found in only 2/191 PiZZ subjects. The only PR3-ANCA-positive patient with Wegener's granulomatosis was in the PiMM group. These patients with classical ANCA positive systemic vasculitis had probably been referred for Pi phenotyping because of pulmonary systemic vasculitis involvement. Therefore, protease inhibitor deficiency is not sufficient to significantly increase the risk for developing ANCA positive vasculitides.

Chronic infection often precedes overt vasculitis, and anti-protease deficiency may prevent normal limitation of proteolytic activity after neutrophil activation, involving sustained ANCA antigen presentation that may lead to ANCA production [36]. The PiZZ patients with pulmonary emphysema may be exposed to more chronic inflammation of the lung than their PiMM controls. {alpha}1AT deficiency may also worsen the clinical presentation of systemic vasculitis, as suggested by the poor outcome of PR3-ANCA-positive patients with the PiZZ phenotype [43]. Therefore, alpha1-antitrypsin deficiency may only act as a second hit amplifying factor.



   Acknowledgments
 
This work was supported by the Délégation à la Recherche Clinique of the Nantes University Hospital.



   Notes
 
Pr VLM Esnault, Service de Néphrologie-Immunologie Clinique, Hôtel Dieu, 30 bd Jean Monnet, F-44093 Nantes, France. Back



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