Allosteric modulation of proteinase 3 activity by anti-neutrophil cytoplasmic antibodies in granulomatosis with polyangiitis
Introduction
Anti-neutrophil cytoplasmic autoantibodies (ANCA) reacting with proteinase 3 (PR3) have a high positive predictive value for the diagnosis of granulomatosis with polyangiitis (GPA), formerly known as Wegener's granulomatosis (WG), but their role in the pathogenesis of the disease and as a biomarker of disease activity and relapse risk remains a controversial issue [1], [2]. When ANCA disappear during therapy, the likelihood of a relapse within the subsequent five-year period is reduced [3]. However, the persistence or increase of ANCA levels is not reliably associated with relapses [2], [4]. Only less than 10% variation in disease activity was estimated to be attributable to changes in ANCA levels [2].
PR3-ANCA are directed to different non-overlapping epitopes on the surface of the native molecule and may vary between patients and over the course of the disease. Variable epitope recognition, binding affinities and association/dissociation rate constants of PR3-ANCA, all contribute to their heterogeneity and may result in functional differences. The functional effects of different PR3-ANCA are difficult to quantify in small plasma samples, and consequently their pathogenic potential and clinical associations are poorly understood. A few small studies have focused on a subset of PR3-ANCA which reduced the catalytic activity of PR3 and impaired its inhibition by the major plasma protease inhibitor, alpha-1-proteinase inhibitor (α1PI). Their results have suggested that disease activity of GPA may be more closely related to the appearance and rise of PR3-inhibiting ANCA than to total PR3-ANCA levels measured conventionally [4], [5], [6]. As PR3 is cleared by complex formation with α1PI and this complexation is dependent on the activity of PR3, inhibitory PR3-ANCA may delay the clearance of PR3 and contribute to the pathogenesis via this mechanism.
Genetic studies [7], [8] and a recently published genome wide association study [9] further support this hypothesis. PR3- and MPO-ANCA-associated autoimmune vasculitis were found to be genetically distinct, and only the PR3-ANCA associated clinical condition was associated with certain risk alleles of the α1PI (serpinA1) and PR3 (PRTN3) loci. In particular, deficiency alleles of serpinA1, the S- and Z-variants, were identified as risk factors for ANCA-positive GPA, suggesting that an imbalance between PR3 activity and the α1PI inhibitor facilitates the development of GPA. Inhibitory PR3-ANCA may thus reduce the clearance of PR3 by α1PI, and consequently inhibitory PR3-ANCA and low levels of α1PI could promote the disease process in the same direction.
The question as to whether specific PR3-ANCA subsets and particularly those inhibiting PR3 activity are associated with relapses, specific organ involvement and overall disease activity, has remained unanswered, largely because the quantification of functional effects of PR3-ANCA could not be conducted efficiently on small plasma samples from large cohorts of patients.
Recent progress in the development of highly sensitive extended peptide substrates for PR3 and of optimized fluorophore-quencher pairs permitted us to establish a new ultrasensitive assay to quantify the inhibitory capacity of PR3-ANCA in small plasma volumes. Contamination of IgG preparations by α1PI is excluded by our protocol, and the inhibitory effect of PR3-ANCA is determined at very low levels of the active target antigen. Availability of plasma samples collected from a well characterized GPA cohort during the conduct of the Wegener Granulomatosis Etanercept Trial (WGET) [10], allowed us to explore clinical associations with inhibitory PR3-ANCA.
Section snippets
Patients
This study used plasma from patients with GPA obtained during the conduct of the WGET. 180 patients were enrolled into this trial [10]. All patients were diagnosed with GPA, fulfilling at least 2 of 5 modified criteria for the classification of GPA from the American College of Rheumatology. At enrollment all patients further had a Birmingham Vasculitis Activity Score for Wegener's Granulomatosis (BVAS/WG) of ≥3. 128 of these patients were diagnosed with severe disease and 52 patients with
Detection of activity modifying PR3-ANCA in plasma samples
The low proportion of PR3-ANCA within the total IgG preparations from patients, the low affinity of most PR3-ANCA to PR3, the high levels of natural PR3 inhibitors in plasma samples, and the limited volume of plasma available from patients, all represent significant challenges that have precluded a systematic screening and characterization of functional effects of PR3-ANCA from large, well characterized patient cohorts to date. To overcome these challenges, we established a novel sensitive
Discussion
The evidence that PR3-ANCA play a major disease-causing role at the onset of local or systemic disease has been a matter of debate since the discovery of ANCA. Whether changes of total ANCA levels can predict relapses or disease progression is even more controversial. Increasing ANCA levels are not considered as a reliable guide for pre-emptive treatments [2]. A distinct subset of so-called inhibitory ANCA has been proposed as a more reliable biomarker of imminent clinical deterioration. The
Disclosure statement
The authors declare that they have no conflict of interests.
Acknowledgment
The authors thank Heike Kittel for her excellent technical assistance, Oliver Eickelberg for his continuous interest in the project and Karl-Heinz Wiesmüller, EMC Microcollections, for the design and synthesis of FRET-substrates. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 261382 (INTRICATE). The WGET trial was supported by the National Institute of Arthritis and Musculoskeletal and Skin
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