Antineutrophil cytoplasmic antibodies (ANCAs) were first described 2 decades ago (1, 2). By indirect immunofluorescence (IIF), 2 fluorescence patterns can be distinguished on ethanol-fixed neutrophil cytospin preparations (3, 4). The classic cytoplasmic pattern (cANCA) is caused almost exclusively by antibodies against proteinase 3 (PR3) (5–7). In contrast, the perinuclear fluorescence pattern (pANCA) can be caused by antibodies reacting with a variety of different neutrophil granule constituents, including myeloperoxidase (MPO), lactoferrin, human neutrophil elastase (HNE), and others (8). Classic ANCA reacting with PR3 and pANCA reacting with MPO have become accepted diagnostic tools in the evaluation of patients with Wegener's granulomatosis (WG) and patients with microscopic polyangiitis (MPA) (9). Evidence is mounting that these specific antibodies not only are disease markers but also are pathogenic of small-vessel vasculitis, the characteristic histopathologic hallmark of these disorders (10, 11).
PR3 and HNE belong to the chymotrypsin family of serine proteinases. They share gene localization, amino acid sequence homology, posttranslational intracellular processing, and substantial structural and functional characteristics (12). Despite these similarities and the prominence of PR3 ANCAs in patients with WG and patients with MPA, information about HNE ANCAs remains sparse and inconsistent (13–18). Among patients with autoimmune diseases (including vasculitis), the frequency of HNE ANCAs is thought to be low. However, reported frequencies are widely discrepant, ranging from 0% to 20% in patients with WG or MPA (14, 15, 17). Methodologic issues have been implicated but have not been addressed formally (17). We have expressed recombinant HNE (rHNE) in the human mast cell line HMC-1 (HMC-1/HNE). Using HMC-1/HNE cells as substrate for HNE ANCA detection by IIF, we found a surprisingly high frequency (28%) of HNE ANCAs in a cohort of patients with cocaine-induced midline destructive lesions (CIMDL) (19).
The present study was designed to characterize the presence of HNE ANCAs in patients with CIMDL in comparison with that in a clinically well-characterized cohort of consecutive patients undergoing an evaluation for possible vasculitis (20). HNE ANCA reactivity was determined by 3 different methods, complemented by standard ANCA testing with an IIF method using ethanol-fixed neutrophils as substrate, by PR3 ANCA testing using 3 different assays, and by MPO ANCA testing.
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- PATIENTS AND METHODS
In this report we describe an unexpectedly high frequency (84%) of HNE ANCAs in patients presenting with CIMDL. In contrast, no HNE ANCAs were detected in patients with WG or MPA, and HNE ANCAs were detected only rarely in patients with other autoimmune diseases or vasculitis. Many of the sera obtained from patients with CIMDL also reacted with PR3. Consequently, HNE ANCAs occurring in patients with midline destructive lesions may be discriminatory between CIMDL and WG, whereas PR3 ANCA testing alone may not be relied upon for this purpose. Our findings, which were obtained with 3 different HNE ANCA–detection methods, indicate that differences in the reported frequencies of HNE ANCAs in patients with autoimmune disorders and vasculitis are, in all likelihood, the result of differences in the detection methods applied. In order to achieve optimal analytical sensitivity for the detection of HNE ANCAs, multimodality testing appears preferable.
We previously used HMC-1 cells for the expression of functional human and murine recombinant PR3 (21, 30). Here, we demonstrate that HMC-1 cells can also be used to express functional rHNE with a molecular mass similar to that of PMN HNE (31). Like purified native HNE, the rHNE hydrolyses the substrate N-MeOSucc-AAPV-pNA and is inhibited by α1PI, eglin C, and aprotinin. This indicates that HMC-1 cells process rHNE amino-terminally, allowing the molecule to assume the proper conformation required for enzymatic function. Furthermore, this report represents the first direct documentation of enzymatic activity of rHNE expressed in hematopoietic cells. Other investigators have previously expressed rHNE in the rat basophilic cell line, RBL-1, a cell line also used for the expression of recombinant human cathepsin G and PR3 (32, 33). Our choice of the human HMC-1 cell line over hematopoietic cell lines from other species is of particular importance when the cells expressing recombinant ANCA target antigens are used as substrate for target antigen–specific ANCA testing by IIF. Many patients have antibodies reacting with rodent proteins that generate false-positive IIF results on rodent cells. The use of a human cell line for expression of rHNE circumvents this problem.
HMC-1/HNE cells can be used as target antigen–specific substrate for the detection of HNE ANCAs by IIF, as previously demonstrated for PR3 ANCA detection (22). In a first application of this system we had found HNE ANCAs in 5 sera (28%) obtained from the first 18 patients in this CIMDL cohort, who were part of a clinicopathologic comparison of CIMDL and WG with nasal involvement (19). This initial observation prompted the present systematic and detailed analysis of ANCA reactivity in patients with CIMDL. In contrast to the first study, here we took a substantially different approach to the serologic testing. First, we extended the cohort of CIMDL patients by including 7 additional consecutive CIMDL patients who had presented to the participating centers since January 2000. Second, we based the ANCA frequency analysis solely on the first serum samples obtained at the time of presentation of the CIMDL patients. Third, we included an analysis of all available serial serum samples from this CIMDL cohort in order to identify any shifts in ANCA reactivity over time. Fourth, because of widely discrepant HNE ANCA frequency reports in other disorders, we used 3 different HNE ANCA test methods with 2 different antigen sources in parallel. Fifth, all sera were also tested in parallel by standard IIF on ethanol-fixed neutrophil cytospin preparations, for PR3 ANCAs using 3 different target antigen–specific methods, and for MPA ANCAs by standard direct ELISA. Last, we chose a different control population.
The choice of control populations is always an issue when reports on the diagnostic utility of ANCA testing are interpreted (34). The control population described in the original report included only patients with WG and nasal involvement (19). We retested the serum samples obtained from these 21 WG patients for HNE ANCAs by direct ELISA and capture ELISA and did not detect any HNE ANCAs (data not shown). Because other investigators have previously described HNE ANCAs in patients with a variety of inflammatory conditions (14, 15, 17, 35), we wanted to include a different control population that contained additional WG patients with more heterogeneous organ involvement as well as patients with other autoimmune disorders. Furthermore, we wanted to minimize selection bias. A cohort that fulfilled all of these criteria was readily available from another study (20).
By applying all 3 methods of HNE ANCA testing to the first serum samples obtained at the time of presentation, we found a surprisingly high frequency of HNE ANCAs (84%) in the 25 patients with CIMDL. The capture ELISA was the most sensitive (76%), whereas both the IIF method using HMC-1/HNE cells and the direct ELISA had a sensitivity of 56%. Overall, the correlation between HNE ANCA absorbance readings determined by capture and direct ELISA was poor (Figure 4). However, the analysis of serial samples obtained over the course of several years in some of the CIMDL patients indicates that in some patients these values change in parallel (Figures 5B, C, and E), whereas in others they fluctuate in opposite directions (Figure 5D). The IIF pattern on neutrophils and HMC-1/HNE cells may also vary over time (data not shown). Together, these observations suggest that the HNE ANCA response in CIMDL patients is oligoclonal or polyclonal and variable over time.
The high frequency of HNE ANCAs observed in patients with CIMDL is in stark contrast to the frequency in patients with WG, in whom HNE ANCAs were not detectable by any method. In contrast to only 1 serum sample from the control population (patient 5, who had ITP and UC), sera from many patients with CIMDL showed universal HNE ANCA reactivity across all assays. Another 7 samples from the control population showed positive reactivity for HNE ANCAs in at least 1, but not all, target antigen–specific assays and lacked pANCA reactivity on neutrophils. This might suggest that they represented method-intrinsic false-positive test results. However, this is unlikely, because the reactivity of 4 of the 5 sera that were positive in the direct ELISA could be inhibited specifically by preincubation of the sample with excess HNE.
We could not confirm the previously reported high frequency of HNE ANCAs in patients with MPA, and we did not find any HNE ANCAs in patients with WG (15, 17). It is of interest that in the report by Tervaert et al, 1 of the 2 HNE ANCA–positive patients identified among a cohort of 315 patients with vasculitis and its mimickers had so-called “chronic midline destructive disease” (15).
Several groups of investigators have reported observing HNE ANCAs in patients with systemic lupus erythematosus (SLE) (13, 14, 17, 35). However, this finding could not be confirmed by others (15). Only 6 patients with a firm diagnosis of SLE were included in our cohort, but all of them were HNE ANCA–negative in all assays. Some of the reported HNE ANCAs in patients with SLE may represent drug-induced ANCAs (13, 16, 18). In contrast to patients with WG and MPA, patients with drug-induced vasculitis commonly have multiple simultaneous ANCA specificities (16, 18). Unlike the observations reported in other studies, we did not observe simultaneous reactivity with MPO in any of the HNE ANCA–positive serum samples (14, 16–18). However, sera from many of the patients with CIMDL and 3 of the other HNE ANCA–positive sera also reacted positively with PR3 in at least 1 target antigen–specific assay. The 2 patients with ulcerative colitis were receiving mesalamine, the therapeutically active part of the sulfasalazine molecule.
For some of the CIMDL samples we could detect cross-inhibition of HNE ANCA reactivity by preincubation with PR3, suggesting that some ANCAs cross-react because they recognize antigenic determinants that are conserved on HNE and PR3 (36). Other CIMDL sera seem to contain separate HNE ANCA and PR3 ANCA species. This is also supported by the divergent serial titers (Figures 5D and E). How the exact PR3 epitopes recognized by double-positive sera compare with those recognized by single-positive PR3 ANCA sera from patients with WG, and whether they represent cross-reacting or coexisting antibody species, may be of pathogenic relevance. This is being investigated separately.
The fact that many HNE ANCA–positive CIMDL sera also reacted with PR3, but none of the sera obtained from patients with WG or MPA showed any reactivity with HNE, is of clinical significance. First, it seems that the presence of HNE ANCAs is discriminatory, whereas the presence of PR3 ANCAs is not. Consequently, in the clinical setting of necrotizing inflammation of the upper respiratory tract, additional testing for HNE ANCAs may be useful for differentiating CIMDL from limited WG. Finally, our findings also illustrate the importance of adhering to current consensus guidelines for ANCA testing calling for corroboration of IIF results by target antigen–specific assays or vice versa; only the cANCA/PR3 ANCA and the pANCA/MPO ANCA combinations have a clinically useful positive predictive value for ANCA-associated vasculitis (9, 20, 37). In this context, it is of interest that the only cocaine abuser with nasal septal perforation, who was thought to have WG based on the presence of microabscesses and giant cells in the nasal biopsy specimen (patient 19), had a cANCA pattern on neutrophils by IIF and showed positive reactivity in all 3 PR3 ANCA assays and negative reactivity in all 3 HNE ANCA assays. Most of the patients with CIMDL had a pANCA pattern, but none reacted with MPO.