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

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 (alphaGr). 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 alphaGr or antigen-specific ELISA [PR3, HLE, MPO, lactoferin (LF) and bactericidal/ permeability increasing protein (BPI)]. RESULTS: The incidence of antibodies directed against alphaGr 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.

Treatment of graft-versus-host disease by extracorporeal photochemotherapy: a pilot study.

BACKGROUND: Graft-versus-host disease (GVHD) is a major complication after bone marrow transplantation, which may be refractory to immunosuppressive drugs. As preliminary case reports suggested that extracorporeal photochemotherapy (ECP) using a Therakos device might be beneficial, we conducted a pilot study to assess the efficacy and safety of a new ECP method that does not require administration of 8-methoxypsoralen (8-MOP) to the patient. METHODS: ECP was performed three times a week for 3 weeks and then tapered according to the patient's course. Soluble 8-MOP was added ex vivo to an enriched mononuclear cell suspension obtained by a cell separator. This cellular suspension was then ultraviolet A irradiated and reinfused into the patient. Evaluation was performed using specific objective tests depending on clinical conditions. RESULTS: The two patients in the study with acute GVHD and severe liver dysfunction resistant to steroid pulse showed no improvement with ECP treatment. The five patients with chronic GVHD (c-GVHD) had the following clinical features: three patients had myositis and two patients had severe cutaneous c-GVHD, including one patient with sclerodermoid lesions, one with bronchiolitis obliterans, one with bronchitis, and one with liver involvement. Immunosuppressive drugs were either prohibited or ineffective. The number of procedures for each patient ranged from 13 to 30. Cytapheresis required the use of a double-lumen catheter (4/5) or an arteriovenous fistula (1/5). No side effects were related to 8-MOP or ultraviolet A irradiation. Four of five patients improved after ECP; one patient with bronchiolitis obliterans, a fibrotic condition, remained stable. CONCLUSIONS: ECP treatment may be helpful for the treatment of severe c-GVHD and the avoidance of increased immunosuppression.

Anti-native and recombinant myeloperoxidase monoclonals and human autoantibodies.

Myeloperoxidase (MPO) is one of the main antigen targets of anti-neutrophil cytoplasmic antibodies (ANCA) in systemic vasculitides. It has been suggested that anti-MPO antibodies may recognize a single epitope on recombinant MPO. If confirmed on native MPO, this might allow specific therapeutic intervention with anti-idiotypic MoAbs to prevent antibody antigen interaction which is thought to cause activation of neutrophils and vasculitis. We searched for restriction in the epitope recognition profile in 50 patients with anti-MPO autoantibodies, using both native and recombinant MPO. Mouse monoclonals were purified and tested in competition assays. At least four epitopes were identified on native MPO using these monoclonals and only two were conserved on recombinant MPO. We found that human MPO autoantibody response was not restricted to a single epitope on native MPO, as all sera tested did not show the same profile in competitive studies with monoclonals. Furthermore, 30% of human anti-native MPO sera failed to recognize rMPO.

Anti-neutrophil cytoplasm antibodies in patients with ACR criteria for polyarteritis nodosa: help for systemic vasculitis classification?

The american college of rheumatology (ACR) proposed in 1990 revised clinical criteria for systemic vasculitis classification to define homogeneous group of patients for clinical trials. However, microscopic polyarteritis (MPA) was not clearly identified from polyarteritis nodosa (PAN). Since anti-neutrophil cytoplasm antibodies (ANCA) are markers of disease activity of small vessel vasculitides including MPA, we tested the clinical significance of ANCA in 24 patients with PAN according to the ACR 1990 criteria. Two of 24 patients had ANCA, as defined by indirect immunofluorescence on normal human neutrophils, antigen-specific ELISA and Western blot analysis. However, they exhibited histologically proven small vessel but not medium vessel vasculitis. Furthermore, they had neither artery microaneurysms nor large organ injury consequent upon large vessel occlusion. Although they satisfied ACR criteria for PAN, they probably were misclassified and should be considered as MPA. We conclude that: (i) ANCA are not found in patients with classical PAN in the absence of MPA features; (ii) caution should be exercised when defining PAN according to the ACR 1990 criteria; (iii) ANCA may help systemic vasculitis classification.

High immunoreactivity of lactoferrin contaminating commercially purified myeloperoxidase.

Three sets of experiments were performed to investigate the quality of myeloperoxidase (MPO) preparations and anti-MPO reagents. In the first experiment, two groups of three and four mice were immunized with commercially purified MPO (Calbiochem). Immunization was performed in PBS in the first group and in acetate buffer in the second. From the first group, five monoclonals were raised, and their specificities examined by ELISA and immunoblotting. Surprisingly, these antibodies reacted with lactoferrin (LF) and not MPO. In the second group, 13 monoclonals were raised; six of these reacted with MPO and seven reacted with LF. In a second set of experiments, MPO and LF reactivity were tested in different buffer conditions in the ELISA procedure. Slight variations in the detection of contaminating LF were found. In a third experiment, polyclonal reagents directed against MPO and LF were tested in MPO immunoblotting studies. A polyclonal anti-MPO reagent reacted not only with MPO but also with contaminating material including LF. The anti-MPO polyclonal reagent also reacted with LF on immunoblotting. We conclude that: (i) caution should be exercised when defining anti-neutrophil cytoplasm specificities of human sera and monoclonals by ELISA, (ii) the low concentration of contaminating LF in the commercially purified reference MPO preparation should be taken into consideration since it appears to have high immunoreactivity, (iii) changes in MPO immunoreactivity may occur under different buffer and pH conditions.

Absence of antineutrophil cytoplasmic antibodies in giant cell arteritis.

OBJECTIVE: To determine the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in patients with active giant cell arteritis (GCA). METHODS: 23 patients with GCA were selected according to ACR 1990 criteria. Sera were harvested in all patients at an active stage of the disease and during followup (1 to 3 sera/patient for a total of 50 sera). ANCA positivity was searched for by indirect immunofluorescence (IIF) and enzyme linked immunosorbent assay (ELISA) using a neutrophil extract, and antigen specificity was determined by proteinase 3 (PR3), lactoferrin (LF) and myeloperoxidase (MPO) ELISA: RESULTS: Only 1/23 patients exhibited reactivity in IgG ANCA ELISA and IIF, with borderline anti-MPO reactivity in ELISA which was not inhibited by preincubation with MPO in the liquid phase, and no reactivity in Western blot analysis. Specificity could not be demonstrated in another patient who had positive IgG ANCA ELISA but negative ANCA IIF and negative antigen specific ELISA: All other patients were ANCA negative. CONCLUSION: As our patients with GCA did not exhibit typical ANCA when validated antigen specific assays were used, careful laboratory controls and clinical evaluation would seem essential in cases of apparent ANCA positivity.