Increased neutrophil membrane expression and plasma level of proteinase 3 in systemic vasculitis are not a consequence of the - 564 A/G promotor polymorphism

Abstract

Several findings link proteinase 3 (PR3) to small vessel vasculitis. Besides being a major target of anti-neutrophil cytoplasm antibodies (ANCA), previous findings have shown increased circulating levels of PR3 in vasculitis patients, increased levels of neutrophil membrane-PR3 (mPR3) expression and a skewed distribution of the - 564 A/G polymorphism in the promotor region of the PR3 gene. In this study we elucidate how these three findings relate to each other. The plasma concentration of PR3 was measured by enzyme-linked immunosorbent assay (ELISA), mPR3 expression by fluorescence activated cell sorter (FACS) and the gene polymorphism by real-time polymerase chain reaction (PCR). We compared results from 63 patients with ANCA-associated systemic vasculitis (AASV) with 107 healthy blood donors. In accordance with previous reports, AASV patients had increased plasma concentrations of PR3 compared to healthy controls (mean 224 microg/l versus 155 microg/l, P < 0.0001). They also showed an increased number of mPR3-positive neutrophils (60%versus 42%, P < 0.001). However, contrary to a previous report, we found no skewed distribution of the polymorphism in PR3 gene. There was a weak correlation between mPR3 mean fluorescence intensity (MFI) and plasma PR3 among healthy controls and myeloperoxidase-ANCA (MPO-ANCA)-positive patients (r = 0.24, P = 0.015 and r = 0.52, P = 0.011, respectively). In conclusion, increased plasma PR3 and high expression of mPR3 are associated with small vessel vasculitis, but neither of them is a consequence of the - 564 A/G polymorphism of the PR3 gene promotor.

Fig. 1

Distribution of the −564 A/G polymorphism in the promotor region of the proteinase 3 gene. Black colour represents individuals homozygous for the A allele, white colour represents individuals homozygous for the G allele and grey colour represents heterozygotes. The results of 189 healthy blood donors are shown in (a) and 136 anti-neutrophil cytoplasm antibodies-associated systemic vasculitis patients are shown in (b).

Fig. 2

Membrane expression of proteinase 3 (PR3) on neutrophils. Neutrophils stained with anti-PR3 murine monoclonal antibodies and followed by fluorescein isothiocyanate-conjugated anti-mouse antibodies were analysed by flow cytometry. (a) Shows the percentage of membrane-PR3⁺ neutrophils in 107 healthy blood donors, 58 patients with anti-neutrophil cytoplasm antibodies (ANCA)-associated systemic vasculitis (AASV) and two subsets of the same patients (23 having myeloperoxidase–ANCA and 34 having PR3–ANCA). (b) Shows the expression index of mPR3 on neutrophils measured by arbitrary units (AU) for the same groups as in (a). All results are given as mean values ± s.d.

Fig. 3

Phenotype distribution of membrane-proteinase 3 (mPR3) on neutrophils. Individuals divided into three predefined groups, high (black)/intermediate (grey)/low (white), according to their percentage of mPR3⁺ neutrophils [6]. The distribution of these three groups is shown in 58 patients with anti-neutrophil cytoplasm antibodies-associated systemic vasculitis compared to 107 healthy blood donors.

Fig. 4

Concentration of proteinase 3 in the plasma measured by enzyme-linked immunosorbent assay; 130 healthy blood donors are compared with 63 patients with anti-neutrophil cytoplasm antibodies-associated systemic vasculitis and two subsets of the same patients (26 with myeloperoxidase and 37 with Wegener’s granulomatosis.

Fig. 5

Scatter diagrams showing the correlation between the mean fluorescence intensity of membrane proteinase 3 (mPR3) on neutrophils and plasma PR3 concentrations. (a) Shows the results of 102 healthy blood donors. (b) Shows the results of 58 patients with anti-neutrophil cytoplasm antibodies (ANCA)-associated systemic vasculitis. Circles indicate 23 myeloperoxidase–ANCA- positive patients and triangles 34 PR3–ANCA-positive patients.