Autoantibodies against Linear Epitopes of Myeloperoxidase in Anti ...

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Autoantibodies against Linear Epitopes of Myeloperoxidase in Anti–Glomerular Basement Membrane Disease Jian-nan Li,*†‡§ Zhao Cui,*†‡§ Jia Wang,*†‡§ Shui-yi Hu,*†‡§ Xiao-yu Jia,*†‡§ Zhe Guan,| Min Chen,*†‡§ Can Xie,| and Ming-hui Zhao*†‡§¶

Abstract Background and objectives Approximately 20%–30% of patients with anti–glomerular basement membrane disease present coexisting anti-myeloperoxidase (MPO) autoantibodies. We previously showed the recognition of a linear fragment of the MPO heavy chain N-terminus (1H, MPO279–409) in plasma from most double-positive patients. Herein, we investigated the frequency of autoantibodies against overlapping 1H-derived linear peptides in plasma from patients with anti–glomerular basement membrane disease. Design, setting, participants, & measurements We synthesized 13 overlapping linear peptides (1H–1 to 1H–13) covering MPO279–409. We retrospectively collected plasma samples from 67 patients with anti–glomerular basement membrane disease from 1996 to 2012, and we screened them for IgG autoantibodies by ELISA using intact human MPO and the overlapping peptides as antigens, and we further investigated the clinical significance. Autoantibody binding to the linear MPO structure was confirmed by Western blotting. Results We followed up the 67 patients until 2015, with a median follow-up time of 10.0 (2.3–36.0) months, and 56 ESRD events occurred among the 67 patients with follow-up data. Plasma from 23.9% (16) of the patients recognized intact human MPO, whereas 62.7% (42) plasma samples recognized MPO279–409 linear peptides. Of the 13 linear peptides, 1H–4 (44.8%, 30 patients) and 1H–12 (40.3%, 27 patients) exhibited the highest recognition frequencies. Patients with autoantibodies against 1H–11 or 1H–12 (MPO371–400) were older (46.1618.8 versus 34.1616.6 years; P,0.01), had higher serum creatinine upon diagnosis (median 7.8 mg/dl, interquartile range 4.9–12.6 mg/dl versus median 5.4 mg/dl, interquartile range 2.4–7.3 mg/dl; P=0.02), and had a higher probability of progressing to ESRD; however, multivariate Cox regression analysis showed that 1H–11 or 12 reaction was not an independent risk factor for renal failure (hazard ratio, 1.2; 95% confidence interval, 0.8 to 2.8; P=0.19). Conclusions Autoantibodies against linear peptides of MPO can be detected in the majority of patients with anti– glomerular basement membrane disease, and several are associated with disease severity. The potential common pathogenic mechanism between anti–glomerular basement membrane antibodies and anti-MPO autoantibodies in anti–glomerular basement membrane disease requires further investigation. Clin J Am Soc Nephrol 11: 568–575, 2016. doi: 10.2215/CJN.05270515

Introduction Goodpasture syndrome is an autoimmune disorder characterized by the presence of anti–glomerular basement membrane (GBM) autoantibodies, resulting in rapidly progressive glomerulonephritis and severe lung hemorrhage, with a high frequency of ESRD and death (1–3). The target antigen of the anti-GBM antibodies is noncollagenous domain 1 of the a3 chain of type IV collagen [a3(IV)NC1], which has two major epitopes: EA and EB (4,5). Approximately 20%–30% of patients with antiGBM disease present serum ANCAs, which are mainly directed against myeloperoxidase (MPO) (6). These patients are defined as “double positive” and have severe clinical manifestations and poor 568

Copyright © 2016 by the American Society of Nephrology

outcomes (7–9). The considerable overlap of the two rare diseases might indicate an underlying common pathogenic mechanism. Several studies have suggested that MPO-ANCA could cause glomerular injury, leading to the exposure of cryptic epitopes on a3(IV) NC1 and thereby inducing anti-GBM autoantibody production (10). However, only 5%–10% of patients with ANCA-associated vasculitis have coexisting anti-GBM disease (6,7,11), which casts doubt on this hypothesis. As confirmed by previous studies, no cross-reaction occurs between autoantibodies against a3(IV)NC1 and MPO (12). These autoantibodies are detected on the basis of recognition of the conformational epitopes on intact antigen molecules. In a recent study

*Renal Division, Department of Medicine, Peking University First Hospital, Beijing, China; †Institute of Nephrology, Peking University, Beijing, China; ‡Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China; §Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education of China, Beijing, China; ║State Key Laboratory of Membrane Biology, Laboratory of Molecular Biophysics, School of Life Sciences, Peking University, Beijing, China; and ¶PekingTsinghua Center for Life Sciences, Beijing, People’s Republic of China Correspondence: Dr. Ming-hui Zhao, Renal Division, Department of Medicine, Peking University First Hospital, Beijing 100034 China. Email: mhzhao@bjmu. edu.cn

www.cjasn.org Vol 11 April, 2016

Clin J Am Soc Nephrol 11: 568–575, April, 2016

on ANCA-associated vasculitis, several MPO linear epitopes were identified as pathogenic, even in ANCAnegative disease (13). These findings implicate diverse autoantibodies against different MPO epitopes in the pathogenesis of ANCA-associated vasculitis. We recently screened autoantibodies against six fragments of the MPO molecule, covering both the light and the heavy chains, in plasma from patients with MPO-ANCA– associated vasculitis; a linear fragment of the N-terminal portion of the MPO heavy chain, 1H279–409, was recognized in the majority of samples (61.5%, eight out of 13) from double-positive patients (14). We hypothesized that double positivity in anti-GBM disease might be largely underestimated. Therefore, in this study, we designed a panel of overlapping synthetic linear peptides covering the N-terminus of the MPO heavy chain. Serum autoantibodies against these linear peptides were detected in a large cohort of patients with anti-GBM disease. Clinical and pathologic associations were also analyzed, with the aim of exploring potential mechanisms for MPO-ANCA generation in anti-GBM disease.

Materials and Methods Patients and Plasma This study included 67 patients with anti-GBM disease who visited Peking University First Hospital from 1996 through 2012. Clinical data were collected at the time of diagnosis and during follow-up. The follow-up was started in 2001, with visits every 1–3 months after the patients were discharged. The end date of follow-up in this study was 2015. All patients were positive for circulating antiGBM autoantibodies, as detected by ELISA using purified bovine a(IV)NC1 (Euroimmun, Lubeck, Germany) and recombinant human a3(IV)NC1 as solid-phase antigens. Sixteen of the 67 patients were positive for MPO-ANCA, as detected by an indirect immunofluorescence assay and antigen-specific ELISA using purified MPO (Euroimmun). Plasma was collected before immunosuppressive therapy or plasma exchange was initiated. Plasma from 44 patients with MPO-ANCA–associated vasculitis without coexisting anti-GBM antibodies served as a disease control, and plasma from 55 healthy blood donors served as a normal control. Plasma samples were stored at 220°C until further use. Multiple freeze-thaws were avoided, and none of the plasma samples was heat treated before use. The research was in compliance with the Declaration of Helsinki and was approved by the ethics committee of Peking University First Hospital. Written informed consent was also obtained from each participant. Preparation of Linear Peptides A panel of 13 sequential overlapping peptides was synthesized, covering amino acids (aa) 279–410 of MPO (14). Each peptide was 20 aa in length, with ten overlapping aa. The peptides were synthesized using an automatic peptide synthesizer with 9-fluorenylmethyloxycarbonyl chemistry (Beijing Scilight Biotechnology Ltd Co., Beijing, China), and purified on a reverse-phase CIS column by preparative HPLC. Purified peptides were analyzed by HPLC for purity and by mass spectrometry to verify that they had the correct sequence. Peptides with

The coexistence of anti-GBM antibodies and MPO-ANCA, Li et al.

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purity .98% were used for further tests. The sequence of each peptide is listed in Table 1. Twenty peptides unrelated to MPO or GBM were also synthesized as peptide controls. In addition, a linear fragment of the MPO C-terminus (H4; aa 622–745) was produced in Escherichia coli, as previously described (14). Determination of Antigen Specificity by ELISA The synthetic peptides were diluted to 10 mg/ml with coating buffer (0.05 M bicarbonate buffer, pH 9.6), used to coat the wells of half of a polystyrene microtiter plate (Nunc-Immuno plate; Nunc, Roskilde, Denmark) and incubated overnight at 4°C. The other half of the plate was coated with coating buffer alone to establish antigen-free wells. Plasma was diluted to 1:100 in PBS containing 0.1% Tween-20 and 1% BSA, added in duplicate, and incubated at 37°C for 60 minutes. Binding was detected with alkaline phosphatase–conjugated goat anti–human IgG (Fc specific; Sigma-Aldrich, St. Louis, MO) at a dilution of 1:5000. P-nitrophenyl phosphate (1 mg/ml; Sigma) was used in substrate buffer (1 M diethanolamine and 0.5 mM magnesium chloride [pH 9.8]), and color development was measured spectrophotometrically at 405 nm (BioRad, Tokyo, Japan) after 30 minutes. Each plate contained positive, negative, and blank controls. The plasma from the 55 healthy blood donors was used to generate cutoff values (mean+2SDs) for each peptide. The samples were re-examined when a SEM.10% was found. The results were normalized to the positive control and are expressed as a percentage of the OD value of the positive control. Cross-Reaction between Autoantibodies against a3(IV)NC1 and MPO Peptides Cross-reactions between autoantibodies against the linear peptides of MPO and a3(IV)NC1 were investigated using an inhibition ELISA. Briefly, polystyrene microtiter plates were coated with soluble linear peptides (1H–1 to 1H–13) at a concentration of 10 mg/ml. Meanwhile, diluted plasma was preincubated with the soluble linear peptides (1H–1 to 1H–13) or recombinant human a3(IV)NC1 at concentrations ranging from 0.5 to 2 mg/ml at 37°C for 60 minutes. The mixtures were then transferred to the linear peptide–coated microtiter plates, and the bound autoantibodies were detected with alkaline phosphatase–conjugated secondary antibodies, as described above. Detection of Circulating anti-MPO Autoantibodies by Western Blot Analysis Intact human MPO (Merck Millipore, Billerica, MA) was boiled in sample buffer and subjected to 12.5% SDS-PAGE at 80 V under reducing conditions with 5% b-mercaptoethanol. The protein was then transferred to a nitrocellulose membrane (Merck Millipore) and blocked in Tris-buffered saline containing Tween-20 and 20 g/l skimmed milk (TBSTM; 0.01 M Tris hydrochloride [pH 7.2], 0.15 M sodium chloride, 0.1% Tween-20, and 20 g/l skimmed milk) for 60 minutes at room temperature. Strips were then incubated with plasma diluted to 1:50 in TBSTM at 4°C overnight, followed by incubation with peroxidase-conjugated secondary antibodies. The proteins were then revealed on autoradiographic film using the Enhanced Chemiluminescence Plus Western blotting detection system (GE Healthcare,

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Table 1. Amino acid sequences of the linear peptides on the MPO 1H fragment and the frequencies and levels of serum autoantibodies from patients with anti-GBM disease

Peptide 1

H–1 H–2 1 H–3 1 H–4 1 H–5 1 H–6 1 H–7 1 H–8 1 H–9 1 H–10 1 H–11 1 H–12 1 H–13 1

Sequence from the N-Terminus of the MPO Heavy Chain

Position from the N-Terminus

Recognition by Anti-GBM Plasma, % (no. out of 67)

Median Autoantibody Levels

VNCETSCVQQPPCFPLKIPP PPCFPLKIPPNDPRIKNQAD NDPRIKNQADCIPFFRSCPA CIPFFRSCPACPGSNITIRN CPGSNITIRNQINAL ITIRNQINALTSFVD QINALTSFVDASMVYGSEEPLA ASMVYGSEEPLARNLRNMSNQL RNLRNMSNQLGLLAVNQRFQ GLLAVNQRFQDNGRALLPFD DNGRALLPFDNLHDDPCLLT NLHDDPCLLTNRSARIPCFL NRSARIPCFLAGDTRSSEMP

279–298 289–308 299–318 309–328 319–333 324–338 329–350 339–360 351–370 361–380 371–390 381–400 391–410

25.4% (17) 13.4% (9) 10.4% (7) 44.8% (30) 16.4% (11) 17.9% (12) 19.4% (13) 20.9% (14) 16.4% (11) 19.4% (13) 35.8% (24) 40.3% (27) 10.4% (7)

0.63 0.65 0.43 0.63 0.56 0.39 0.31 0.37 0.48 0.44 0.45 0.66 0.52

GBM, glomerular basement membrane; MPO, myeloperoxidase.

Waukesha, WI). Plasma from a patient with MPO-ANCA– associated vasculitis and rabbit anti-MPO polyclonal antibodies (Merck Millipore) were used as positive controls. Complement C3 Deposition Assay A complement C3 deposition assay was performed according to a previously described method (15,16). Briefly, microtiter plates were coated with 2 mg/ml recombinant human a3(IV)NC1 or MPO linear peptides, and respective affinitypurified autoantibodies against 1H–4, 1H–11, and 1H–12 from plasma from six patients with anti-GBM disease were added to form immune complexes. Then, 1% fresh normal human plasma was added to provide complement components, and complement activation was evaluated by C3c deposition ELISA (see Supplemental Material). Statistical Analysis The normally distributed quantitative parameters were expressed as the mean6SD, and the quantitative parameters that were not normally distributed were expressed as the median and interquartile range. Differences in quantitative parameters were assessed using the t test or nonparametric tests. Differences in qualitative data were analyzed using the chi-squared test or Fisher exact test. Moreover, Pearson and Spearman rank correlations were performed to analyze the association between the levels of autoantibodies against linear peptides and the clinical data, as appropriate. Peptides with ,5% recognition were excluded from further statistical analyses. Kaplan–Meier analysis was used to investigate patient survival, and renal survival was assessed using the logrank test. A P value ,0.05 was considered significant. The analyses were performed using SPSS statistical software (version 10.0; SPSS Inc., Chicago, IL).

Results Patient Data Sixteen (23.9%) of the 67 patients had coexisting MPOANCA. These patients were significantly older than those

without ANCAs (57.7614.8 versus 34.5616.1 years; P,0.001). The median duration of follow-up was 10.0 (2.3–36.0) months, and 56 (n=67; 83.6%) ESRD events occurred. At the end of 1 year of follow-up, 86.6% (58 out of 67) of patients survived, and 19.4% (13 out of 67) of patients had normal renal function. The outcomes of the double-positive patients were similar to those of the patients with anti-GBM autoantibodies alone. In total, 32 of the 67 patients underwent a renal biopsy, and direct immunofluorescence showed linear IgG deposition along the GBM, with or without complement C3. Patients with ESRD and very old individuals (.75 years old) did not undergo kidney biopsy. The presence of antiGBM antibodies was confirmed by ELISA and Western blot analysis using recombinant human a3(IV)NC1 as a solid-phase antigen. Frequency of Serum Autoantibodies Recognizing Linear Peptides of MPO We previously reported that MPO peptide recognition was restricted to the 1H fragment in double-positive patients (14). Therefore, we synthesized 13 sequential linear peptides covering the entire length of MPO 1H279–409, or 1H–1 to 1H–13, with each overlapping by ten aa (Figure 1, Table 1). Plasma from 42 (62.7%) of the 67 patients with anti-GBM disease demonstrated reactivity with at least one peptide. Among the 16 patients with coexisting MPO-ANCA, 13 (81.3%) showed plasma reactivity with at least one linear peptide; for the 51 patients without ANCA, 29 (56.9%) had plasma reactivity with at least one peptide. Autoantibodies against linear peptides were more common in classic double-positive patients (3, 1–9 versus 1, 0–4; P=0.04). In total, 44 patients with MPO-ANCA–associated vasculitis served as disease controls. Plasma from 39 of these patients (88.6%) demonstrated reactivity with at least one linear peptide, which was significantly higher reactivity than that observed for anti-GBM plasma (P,0.001) (Figure 1). Circulating autoantibodies binding to the linear structure of MPO in patients with anti-GBM disease were further



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Figure 1. | Recognition frequency of linear peptides of the MPO 1H fragment in patients with anti-GBM disease and ANCA-associated vasculitis. (A) Detection rates in patients with anti-GBM disease (n=67). (B) Comparison of the detection rates among patients with anti-GBM autoantibodies alone (n=51), double-positive patients (n=16), and patients with ANCA-associated vasculitis (n=44). GBM, glomerular basement membrane; H1-n, 1H–n; MPO, myeloperoxidase.

confirmed by Western blot analysis using intact human MPO as the ligand under reducing conditions (Figure 2). Plasma from patients with anti-GBM disease recognizing the linear peptides of MPO could also bind to a band representing the MPO heavy chain. The specificity of circulating autoantibodies against the linear peptides of MPO in all 67 patients with anti-GBM disease was further confirmed by ELISA against 20 linear peptides unrelated to MPO or a3(IV)NC1. Only two of the 20 peptides were recognized, by two of the 67 (3.0%) plasma samples. In

addition, the specificity of the autoantibodies against the N-terminus of the MPO heavy chain was confirmed by coating antigen-free wells with H4 (MPO622–745). The difference in net OD values between H4 (MPO622–745)–coated wells and antigen-free wells was not significant (P=0.99). Frequencies of Serum Autoantibodies Recognizing Each Linear Peptide of MPO Among the 13 peptides, 1H–4, 1H–11, and 1H–12 were most commonly recognized by plasma from patients with

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Figure 2. | Western blot analysis of binding to the linear structure of MPO under reducing conditions. Lane 1 was blotted with rabbit anti– human MPO; lanes 2–8 were blotted with plasma from patients with anti-GBM disease without MPO-ANCA, as assessed by routine ELISA screening; lane 9 was blotted with the serum of a patient with MPOANCA–associated vasculitis; and lane 10 was blotted with serum from a healthy control. Lanes 2–9 indicate antibody binding to the MPO heavy chain (above 55 kD). GBM, glomerular basement membrane; MPO, myeloperoxidase.

anti-GBM disease, with prevalence of 44.8% (30 out of 67 patients), 35.8% (24 of 67) and 40.3% (27 of 67), respectively (Figure 1). The binding levels for these peptides are shown in Figure 3. Compared with patients with anti-GBM autoantibodies alone, classic double-positive patients showed a significantly higher frequency of 1H–5 (43.8% versus 9.8%; P=0.001) and 1 H–6 (50.0% versus 7.8%; P,0.001) recognition (Figure 1). Clinical Association of Autoantibodies Recognizing Linear Peptides of MPO In general, patients with autoantibodies against any linear peptide were older than patients without the autoantibodies (45.2619.4 versus 31.3613.6 years; P,0.01). The clinical association of autoantibodies recognizing each linear peptide was also analyzed. Because 1H–11 and 1 H–12 are two adjacent peptides, both on the MPO surface, we combined the two peptides as 1H–11 to 12 (defined as the detection of autoantibodies against 1H–11 or 1H–12). Compared with patients lacking autoantibodies against 1 H–11 to 12 (Table 2), patients with autoantibodies against 1 H–11 to 12 showed significantly higher serum creatinine

at the time of diagnosis (7.8 mg/dl, 4.9–12.6 mg/dl, versus 5.4 mg/dl, 2.4–7.3 mg/dl; P=0.02) and more linear peptides were recognized by their sera (4, 3–9 versus 0, 0–1; P,0.001). In comparison with patients lacking antibodies against 1 H–11 to 12, patients with these autoantibodies showed a significantly higher probability of progression to ESRD (hazard ratio [HR], 1.8; 95% confidence interval [95% CI], 1.1 to 3.0; P=0.03) (Figure 4); however, multivariate Cox regression analysis showed that 1H–11 to 12 reaction was not an independent risk factor for renal failure (HR, 1.2; 95% CI, 0.8 to 2.8, P=0.19). Cross-Reaction between Autoantibodies against a3(IV)NC1 and MPO Peptides In the inhibition ELISA, binding to MPO peptides could be inhibited by the corresponding MPO peptides in a dosedependent manner, but not by soluble a3(IV)NC1, indicating that the autoantibodies against the linear peptides of MPO and a3(IV)NC1 were distinct populations (see Supplemental Figure 1). Measurement of Complement Activation by Anti-1H–4, Anti-1H–11, and Anti-1H–12 Autoantibodies Autoantibodies against 1H–4, 1H–11, and 1H–12 from the plasma of six patients with anti-GBM disease were affinity purified in microtiter plates, with an average concentration of 0.0360.02 mg/ml. C3c deposition activated by the 1H–4, 1H–11, and 1H–12 immune complexes was significantly higher than deposition in the healthy controls (P,0.001) (see Supplemental Figure 2).

Discussion In our previous study, plasma from eight of 13 (61.5%) patients with anti-GBM disease with coexisting MPOANCA could recognize MPO279–409 (14). In this study, we further investigated the recognition of linear peptides covering MPO279–410 by plasma from patients with antiGBM disease. Plasma from 42 patients (62.7%) with antiGBM disease recognized linear peptides of MPO279–410,

Figure 3. | Levels of autoantibodies against 1H–4, 1H–11, and 1H–12 in patients with anti-GBM autoantibodies alone (anti-GBM), doublepositive patients (DP), patients with ANCA-associated vasculitis (AASV) and healthy controls (HC). GBM, glomerular basement membrane; H1-n, 1H–n.



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Table 2. Clinical and histopathological data for patients with anti–glomerular basement membrane disease with or without autoantibodies against 1H–11 to 12

H–11 To 12 Negative

P Value

33 46.1618.8 18/15 7 (21.2) 8.361.9 16 (30.3) 1.7 (0.9–3.5) 7 (21.2) 7.8 (4.9–12.6)

34 34.1616.6 21/11 13 (38.2) 9.262.1 13 (42.7) 2.8 (1.7–5.2) 13 (38.2) 5.4 (2.4–7.3)

0.01 0.36 0.11 0.23 0.42 0.07 0.09 0.02

90.2615.9 4 (3–9)

74.6626.9 0 (0–1)

0.05 ,0.001

1

Parameter Number Age (years) Sex (male/female) Hemoptysis, n (%) Hemoglobin (g/dl) Oliguria/anuria, n (%) Urinary protein (g/24 h) Gross hematuria, n (%) Serum creatinine upon diagnosis (mg/dl) Crescents in glomeruli (%) Number of linear peptides recognized Renal survival at 1 year, n (%) Patient survival at 1 year, n (%) 1

H–11 To 12 Positive

7 (21.2) 28 (84.8)

1

4 (12.1) 30 (90.1)

0.03 0.01

H–11 to 12, combination of 1H–11 and 1H–12.

which was a much higher degree of recognition than for native MPO (23.9%, 16 of 67 patients). No cross-reaction was found between autoantibodies against a3(IV)NC1 and MPO peptides; thus, the autoantibodies against linear peptides of MPO279–410 were distinct from those against a3(IV) NC1. The large proportion of patients with anti-GBM disease with coexisting autoantibodies against linear peptides of MPO279–410 indicates that “double positivity” should not be regarded as a coincidence. In general, autoreactive T cells, recognizing linear peptides presented by antigenpresenting cells, initially activate B cells to produce autoantibodies against the same linear peptide. Subsequently,

intra- and interepitope spreading occurs (17,18) through somatic recombination to produce autoantibodies with high affinity to various conformational epitopes (19,20). The discovery of autoantibodies specific to linear peptides of MPO indicates that autoreactive T cells against MPO might appear in the majority of patients with anti-GBM disease, inducing autoreactive B cell activation against linear MPO. During the development and progression of anti-GBM disease, epitope spreading from linear peptides to conformational a3(IV)NC1 has been shown in vivo in rats and confirmed in patients (21–23). We speculated that intramolecular epitope spreading might also occur from autoreactive B cells against the linear peptides of MPO

Figure 4. | Renal outcomes of patients with anti-GBM disease with or without autoantibodies against MPO 1H–11 to 12. Cum., cumulative; GBM, glomerular basement membrane; 1H–11 to 12, combination of 1H–11 and 1H–12; H1-11 to 12, 1H–11 to 12; MPO, myeloperoxidase.

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to autoreactive B cells against conformational MPO. In fact, autoantibodies against the linear peptides of MPO were detected more commonly in “double-positive” patients who possessed autoantibodies against conformational MPO; thus, the appearance of MPO-ANCA might be associated with autoantibodies reactive to linear MPO peptides through the epitope-spreading process. Another attractive hypothesis is that anti-GBM autoantibodies and MPO-ANCA, at least in anti-GBM disease, may arise from the same unknown origin. An initial linear epitope in either MPO or the a3(IV)NC1 molecule might be responsible for the production of both autoantibodies through intra- and intermolecular epitope–spreading processes. According to our previous study on anti-GBM disease, P14 (a3129–150) is a major linear B cell epitope on a3(IV)NC1, which is the mutual B and T cell epitope in the development of anti-GBM disease (24). Interestingly, autoantibodies against P14 were also found more frequently in double-positive patients. In this study, autoantibodies against 1H–4 to 6 (MPO 1 309–338) and H–11 to 12 (MPO371–400) were also more commonly detected in double-positive patients. In addition, most of the peptides in 1H279–409, and particularly 1H–4 and 1H–12, are on the surface of MPO (see Supplemental Figure 3). Although we failed to identify any aa sequence similarity among the three linear peptides, the homology of the critical aa of the T cell epitope could be within the linear peptide but without continuity. Exploring the critical motif of the T cell epitope in anti-GBM disease might be helpful in searching for cross-reactivity between anti-GBM antibodies and MPO-ANCA at the T cell level. Interestingly, in this study, the presence of serum autoantibodies against the linear peptide 1H–11 to 12 (MPO371–400) was associated with the severity of antiGBM disease. In particular, patients with autoantibodies against 1H–11 to 12 presented higher serum creatinine upon diagnosis and worse renal outcome during followup. Thus, 1H–11 to 12 (MPO381–400) might constitute a risk epitope. This possibility was supported by a recent study, in which a sole linear epitope on the MPO molecule (MPO447–459) recognized by plasma from ANCA-negative vasculitis patients correlated with disease severity. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice (13). In this study, autoantibodies against 1H–11 and 1H–12 resulted in complement fixation, which might be a mechanism by which the autoantibodies induce kidney injury. The frequent recognition of the linear peptide 1H–11 to 12 (MPO371–400) might suggest that extensive epitope spreading occurred in these patients. The patients with positive anti-1H–11 to 12 autoantibodies also showed more autoantibodies against other linear peptides of MPO. Moreover, the close relationship between extensive epitope spreading and severe kidney injury has been shown in human anti-GBM disease (13). A limitation of our study is that we did not synthesize overlapping linear peptides covering the entire length of MPO. Because the 1H fragment was detected with the highest frequency among the light and heavy chains of MPO and was more detectable in double-positive patients, we focused on this fragment. As a result, we might have

missed several potential linear epitopes on the MPO molecule; this possibility deserves further investigation. In conclusion, autoantibodies against linear peptides of MPO can be detected in the majority of patients with antiGBM disease, and several are associated with disease severity, suggesting a potential common pathogenic mechanism for anti-GBM antibodies and MPO-ANCA in antiGBM disease. Acknowledgments This work was supported by a grant from the Chinese 973 Project (no. 2012CB517702), a grant from the Natural Science Fund of China to the Innovation Research Group (81321064), grants from the National Natural Science Fund of China (81370801, 81370740, 81330020) and the Seeding Grant for Medicine and Life Sciences of Peking University (2014-MB-23). Disclosures None. References 1. Goodpasture EW: The significance of certain pulmonary lesions in relation to the etiology of influenza. Am J Med Sci 158: 863– 870, 1919 2. Wilson CB, Dixon FJ: Anti-glomerular basement membrane antibody-induced glomerulonephritis. Kidney Int 3: 74–89, 1973 3. Salama AD, Levy JB, Lightstone L, Pusey CD: Goodpasture’s disease. Lancet 358: 917–920, 2001 4. Pedchenko V, Bondar O, Fogo AB, Vanacore R, Voziyan P, Kitching AR, Wieslander J, Kashtan C, Borza DB, Neilson EG, Wilson CB, Hudson BG: Molecular architecture of the Goodpasture autoantigen in anti-GBM nephritis. N Engl J Med 363(4): 343–354, 2010 5. Vanacore R, Ham AJ, Voehler M, Sanders CR, Conrads TP, Veenstra TD, Sharpless KB, Dawson PE, Hudson BG: A sulfilimine bond identified in collagen IV. Science 325: 1230–1234, 2009 6. Hellmark T, Niles JL, Collins AB, McCluskey RT, Brunmark C: Comparison of anti-GBM antibodies in sera with or without ANCA. J Am Soc Nephrol 8: 376–385, 1997 7. Levy JB, Hammad T, Coulthart A, Dougan T, Pusey CD: Clinical features and outcome of patients with both ANCA and anti-GBM antibodies. Kidney Int 66: 1535–1540, 2004 8. Zhao J, Yang R, Cui Z, Chen M, Zhao MH, Wang HY: Characteristics and outcome of Chinese patients with both antineutrophil cytoplasmic antibody and antiglomerular basement membrane antibodies. Nephron Clin Pract 107: c56–c62, 2007 9. Yang R, Hellmark T, Zhao J, Cui Z, Segelmark M, Zhao MH, Wang HY: Antigen and epitope specificity of anti-glomerular basement membrane antibodies in patients with goodpasture disease with or without anti-neutrophil cytoplasmic antibodies. J Am Soc Nephrol 18: 1338–1343, 2007 10. Serratrice J, Chiche L, Dussol B, Granel B, Daniel L, Jego-Desplat S, Disdier P, Swiader L, Berland Y, Weiller PJ: Sequential development of perinuclear ANCA-associated vasculitis and antiglomerular basement membrane glomerulonephritis. Am J Kidney Dis 43: e26–e30, 2004 11. Jayne DR, Marshall PD, Jones SJ, Lockwood CM: Autoantibodies to GBM and neutrophil cytoplasm in rapidly progressive glomerulonephritis. Kidney Int 37: 965–970, 1990 12. Short AK, Esnault VL, Lockwood CM: Anti-neutrophil cytoplasm antibodies and anti-glomerular basement membrane antibodies: two coexisting distinct autoreactivities detectable in patients with rapidly progressive glomerulonephritis. Am J Kidney Dis 26: 439–445, 1995 13. Roth AJ, Ooi JD, Hess JJ, van Timmeren MM, Berg EA, Poulton CE, McGregor J, Burkart M, Hogan SL, Hu Y, Winnik W, Nachman PH, Stegeman CA, Niles J, Heeringa P, Kitching AR, Holdsworth S, Jennette JC, Preston GA, Falk RJ: Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis. J Clin Invest 123: 1773–1783, 2013


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Received: May 13, 2015 Accepted: January 5, 2016 Published online ahead of print. Publication date available at www. cjasn.org. This article contains supplemental material online at http://cjasn. asnjournals.org/lookup/suppl/doi:10.2215/CJN.05270515/-/ DCSupplemental.