Margaret A. Viss

Antiproteinase 3 Antineutrophil Cytoplasmic Antibodies and Disease Activity in Wegener Granulomatosis

Context Most patients with Wegener granulomatosis have antineutrophil cytoplasmic antibodies (ANCA). The role of ANCA testing in monitoring response to treatment is controversial. Contribution Using data from a large treatment trial, the authors found little association between disease activity and ANCA levels. Decreases in ANCA levels were not associated with remission, and increases were not associated with relapse. Caution Because follow-up duration differed by patient, standard measures of ANCA accuracy, such as sensitivity and specificity for detecting remission and relapse, could not be calculated. Implication Serial ANCA testing should not be used to monitor disease activity or to guide decisions about immunosuppressive treatment in patients with Wegener granulomatosis. The Editors Wegener granulomatosis is characterized by necrotizing granulomatous inflammation and vasculitis, most commonly affecting the respiratory tract and kidneys (1, 2). Remission can be induced in most patients by using glucocorticoids with cyclophosphamide or methotrexate (17), but most patients have relapse when immunosuppression is reduced or withdrawn (1, 2, 6, 8). Consequently, patients experience substantial illness and damage from both the disease and treatment toxicity (1, 9). Accurate assessment of disease activity and prediction of relapse remain the biggest challenges in management of Wegener granulomatosis (10). Most patients with Wegener granulomatosis have antineutrophil cytoplasmic antibodies (ANCA), which produce a cytoplasmic immunofluorescence pattern on ethanol-fixed neutrophils and react with the neutrophil serine protease proteinase 3 (PR3) (1115). Proteinase 3 is synthesized as a proenzyme (pro-PR3) containing an amino-terminal activation dipeptide that preserves PR3 in an inactive state (16). Subsequent cleavage of this dipeptide allows PR3 to assume its active enzyme conformation (mature-PR3) (16). The diagnostic value of ANCA is well established (17, 18); however, the role of serial ANCA measurements during follow-up and their utility in guiding treatment remain controversial (10, 19, 20). A recent study indicated that in individual patients with Wegener granulomatosis, ANCA against pro-PR3 had a stronger correlation with disease activity than did ANCA against mature-PR3 (21). Therefore, we sought to determine whether pro-PR3ANCA levels correlate more strongly with disease activity than do mature-PR3ANCA levels, whether a decrease in pro- or mature-PR3ANCA levels during remission-induction therapy is associated with a shorter time to sustained remission, and whether an increase in pro- or mature-PR3ANCA levels is associated with relapse. Methods This prospective study was done in the context of the WGET (Wegener Granulomatosis Etanercept Trial) (6, 22, 23), a randomized, placebo-controlled trial that evaluated etanercept for maintenance of remission in 180 patients with Wegener granulomatosis at 8 centers across the United States (Appendix 1). All patients met at least 2 of the 5 modified American College of Rheumatology criteria for classification of Wegener granulomatosis and had active disease within 28 days before enrollment and a Birmingham Vasculitis Activity Score for Wegener granulomatosis (BVAS/WG) of at least 3 (22, 24). Follow-up evaluations were done at baseline, after 6 and 12 weeks, and then every 3 months until the end of the trial. Two additional evaluations took place at 3 and 6 months after the end of the trial. During each visit, disease activity was measured by using the BVAS/WG, and serum samples were obtained, frozen, and stored at 80 C. Treatment Patients were treated in a protocol-defined manner with etanercept or placebo in addition to standard therapies. Patients with severe Wegener granulomatosis (life- or organ-threatening disease) received cyclophosphamide and glucocorticoids at enrollment (22, 24). Those with limited (that is, nonsevere) Wegener granulomatosis received methotrexate and glucocorticoids. Medication dosages were tapered according to protocol once disease activity was controlled (6, 22). Assessment of Disease Activity and Definitions of Sustained Remission and Relapse Disease activity was measured by using the BVAS/WG (24). This index considers all manifestations of active disease during the 28 days preceding the date of assessment. A BVAS/WG of 1 or greater is considered active disease, and a BVAS/WG of 0 indicates remission (24). Our analyses focused on first sustained remission and first relapse. Sustained remission was defined as a BVAS/WG of 0 for at least 6 months (6, 22). The PR3-ANCA level at the visit in the middle of this period was considered the PR3-ANCA level at sustained remission. Disease relapse was defined as an increase of at least 1 point in the BVAS/WG in patients who had sustained remission. ANCA Detection Methods A standard immunofluorescence assay and direct enzyme-linked immunosorbent assays (ELISAs) for PR3-ANCA and ANCA against myeloperoxidase were done as described elsewhere (25). Capture ELISA was used to measure PR3-ANCA (2527); levels are expressed as net absorbance, and a level of 0.10 or greater is considered positive (26, 27). The intra-assay and interassay coefficients of variation are 9% and 31%, respectively, for pro-PR3 capture ELISA, and 6% and 28%, respectively, for mature-PR3 capture ELISA. All baseline serum samples were screened at first thaw by using all methods. Patients whose baseline samples tested positive for perinuclear-staining ANCA or ANCA against myeloperoxidase (n= 24) were excluded from further analyses because of substantial differences in disease phenotype between patients positive for ANCA against myeloperoxidase and those positive for PR3-ANCA. In addition, the number of patients who were positive for ANCA against myeloperoxidase was too small for meaningful longitudinal analysis (5, 28, 29). Subsequently, all serum samples were tested for mature- and pro-PR3ANCA in parallel by using capture ELISA at first thaw (except for the baseline samples, which were retested at second thaw). To minimize variability, all serum samples from an individual patient were run at once in the same plate and the same lots of all reagents were used for all assays. Laboratory personnel were blinded to the clinical data. Increase and Decrease of PR3-ANCA Levels We defined an increase in PR3-ANCA levels a priori as an increase of at least 100% in the net absorbance over 6 months. An absolute increase of at least 0.4 was also required to ensure that small elevations were above the intra-assay coefficient of variation. We classified a negative-to-positive conversion of PR3-ANCA status as an increase only if the absolute increase was at least 0.4. Because 6 months corresponded to 3 clinical visits (except for the initial 6 months after enrollment, when it corresponded to 4 clinical visits), we compared the PR3-ANCA levels at each visit with those from the previous 2 to determine whether the criteria for increase were fulfilled. We first looked for an increase in PR3-ANCA 9 months after enrollment (the fourth visit), because that was the first time that a patient could meet the definition of sustained remission. Thus, the PR3-ANCA level at the fourth visit was compared with the levels at the third and second visits after enrollment. No increase in PR3-ANCA before the fourth visit after enrollment was analyzed (Appendix Figure 1). Appendix Figure 1. Diagram of the initial 4 clinical visits. The fourth clinical visit was the first point at which a patient could meet the study definition of sustained remission (SR) (a Birmingham Vasculitis Activity Score for Wegener granulomatosis [BVAS/WG] of 0 for 6 months) and was the first time we looked for an increase in proteinase 3 (PR3) antineutrophil cytoplasmic antibody (ANCA) levels. We compared the net absorbance of PR3-ANCA at this visit with that of the previous 2 visits or the previous 6 months (curved lines). The same comparison was done at every subsequent visit. We defined a decrease in PR3-ANCA levels during the initial 6 months of follow-up as a decline of at least 50% in the net absorbance with an absolute decrease of at least 0.4; for values between 0.1 and 0.4, the capture ELISA needed to yield negative results. Statistical Analysis Descriptive data are summarized as mean (SD), median (interquartile range), or percentages. Groups were compared by using the t test (or the rank-sum test) or the chi-square test (or Fisher exact test), with 95% CIs calculated as appropriate. A P value less than 0.05 was considered statistically significant. We performed unadjusted and adjusted analyses. The adjusting variables were age, sex, disease severity (severe vs. limited Wegener granulomatosis), treatment group (etanercept vs. placebo), disease duration, baseline BVAS/WG, and clinical center (see Appendix 2 for additional details). PR3-ANCA Levels and Disease Activity The cross-sectional and longitudinal associations between the BVAS/WG and the levels of mature- and pro-PR3ANCA were estimated by using random-effect models. We constructed the models with BVAS/WG as the dependent variable and included a random effect for each patient (random intercept) and 2 terms for PR3-ANCA levelsone term for the value of PR3-ANCA level at baseline and the other for the change in PR3-ANCA level from baseline to time t. To assess the magnitude of the association between PR3-ANCA levels and the BVAS/WG, we estimated the relative reduction in the residual variance by comparing the residual variance of the model that included a random effect for each patient and 2 terms for PR3-ANCA levels with the residual variance of a model that only included the random effect for each patient. These analyses included only observations in which both BVAS/WG and PR3-ANCA level were available (9% of the observations had missing PR3-ANCA information). For patients who achieved sustained remission, the mature- and pro-PR3ANCA level

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

ANCA directed against PR3 are highly specific for Wegeners granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3‐ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N‐terminal activation dipeptide of PR3 is required for the binding of PR3‐ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N‐terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3‐S176A and δ‐rPR3‐S176A. rPR3‐S176A contains the N‐propetide Ala‐2‐Glu‐1, δ‐rPR3‐S176A does not. Culture supernatants of rPR3‐S176A and δ‐rPR3‐S176A expressing 293 cells were used as sources of target antigen for PR3‐ANCA testing by capture ELISA. Forty unselected consecutive PR3‐ANCA+ sera were tested. With δ‐rPR3‐S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3‐S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N‐terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3‐ANCA. However, a substantial proportion of PR3‐ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N‐terminal processing. In 15% of sera this PR3‐ANCA subset occurred exclusively. PR3‐ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non‐haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.

Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA

We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC‐1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N‐methoxysuccinyl‐Ala‐Ala‐Ala‐Pro‐Val‐pNA. The enzymatic activity is inhibited by greater than 95% by α1‐PI. The rPR3 and the enzymatically inactive mutant rPR3‐S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3s targeting to granules. This rPR3 is the first to be recognized by most c‐ANCA from WG patients and all anti‐PR3 ANCA that were detected by standard anti‐PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure‐function relationships and interaction with autoantibodies.

Differences between human proteinase 3 and neutrophil elastase and their murine homologues are relevant for murine model experiments

Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3‐related autoimmune processes investigated in wild‐type‐, mNE‐ and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico‐chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc‐AAPV‐pNA and Suc‐AAPV‐pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by α1‐antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom‐designed hNE inhibitor, Val15‐aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species‐specific assessment of neutrophil protease function and inhibition.

Agreement of anti-neutrophil cytoplasmic antibody measurements obtained from serum and plasma.

Serum and plasma are used interchangeably to measure anti‐neutrophil cytoplasmic antibodies (ANCA), even though the release of ANCA target antigens during the preparation of serum could affect ANCA assays and cause discrepancies between the results obtained from serum and plasma. To what extent ANCA test results obtained from serum agree and correlate with results from plasma remains unknown. Therefore, a comprehensive comparison was performed using serum and plasma samples which were collected in 175 patients with active Wegeners granulomatosis at enrolment of a recent randomized trial. These paired serum and plasma samples were subjected to parallel ANCA testing by standard indirect immunofluoresence on ethanol‐fixed neutrophils, a direct enzyme‐linked immunoassay (ELISA) for proteinase 3 (PR3)‐ANCA and myeloperoxidase (MPO)‐ANCA, and two different capture ELISAs for PR3‐ANCA. The concordance of categorical serum and plasma ANCA results was assessed using κ‐coefficients. These were > 0·8 for all assays, indicating a very good concordance between positive and negative serum and plasma results. Spearmans correlation coefficients for serum and plasma PR3‐ANCA values obtained by direct ELISA and both capture ELISAs were ≥ 0·95 (P < 0·0001). Our study shows that serum and plasma samples can be used interchangeably for measuring ANCA.