Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

Abstract

Anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.

Figure 1. Study of autoantibody epitope specificity within an MPO-ANCA–positive cohort.

(A) Heat map demonstrating high-level positive epitope binding (bright red), weakly positive epitope binding (low titer) detected only with very sensitive H₂¹⁸O labeling (dark red), and negative detection (black). The y axis includes samples from active disease patients (n = 52), patients in remission (n = 35), and healthy subjects (HS) (n = 10). The x axis includes 25 epitopes classified as exclusive to active disease, persistent during remission if detected in active disease, in remission, and rarely, at low level, in healthy subjects. Epitopes were designated asymptomatic or natural if present in healthy subjects (required H₂¹⁸O labeling for detection). (B) Analysis of epitope specificity by MS was performed on Ig from a UNC cohort (n = 97) and an NL cohort (n = 20). (B–D) Distribution of autoantibody epitopes identified. UNC–active disease (n = 52), NL–active disease (n = 20), UNC–clinical remission (n = 35), UNC–healthy subjects (n = 10). Unique epitopes strictly associated with ANCA disease were identified in all 72 active disease samples (B and C). Extremely low-level anti-MPO autoantibodies from healthy subjects were negative for reactivity with disease-specific epitopes (D).

Figure 2. MPO-ANCA reactive with epitope aa–447-459 are exclusively associated with active disease.

(A and B) ELISA results testing for reactivity against 2 linear epitopes identified by MS. Anti-MPO^447–459 autoantibodies correlated with disease activity in both the UNC and NL cohorts (A). Anti-MPO^516–524 autoantibodies were present in active disease and remission but were absent in healthy subjects in both cohorts (B). (Note: ELISAs of NL cohorts were conducted at the UMCG using their specific protocol and reagents, except for synthetic peptides provided by UNC). (C) Location of epitopes on the MPO molecule. Disease-associated epitopes (blue) (including aa 447–459) exist in tandem with epitopes recognized by natural autoantibodies (green). Amino acids predicted to be required for autoantibody binding are highlighted in red.

Figure 3. Epitope excision/MS detects autoantibodies in patients with an ANCA-negative serology.

Ig from seronegative UNC patients (n = 10) and NL patients (n = 12) was incubated with leukocyte protein lysates as depicted (A). Reactive antigens were captured by autoantibodies. Sites of contact between the autoantigen and the Ig (epitope) were protected from digestion. Peptides remaining bound to Ig after digestion were eluted and analyzed by MALDI-TOF/TOF MS/MS. A search for autoantigens recognized by Ig purified from patients with pauci-immune vasculitis and ANCA-negative serology revealed a single MS peak determined to be an MPO epitope aa 447–459 (B). MS results were validated by ELISA indicating Ig reactivity against native MPO and MPO peptide aa 447–459 (C and D). Analysis of longitudinal samples (UNC cohort, n = 4) (NL cohort, n = 5) indicated a correlation between active disease and the presence of Ig reactive with native MPO and MPO peptide aa 447–459 (C and D).

Figure 4. MPO epitope aa 447–459 is masked by a proteolytic fragment of a common serum protein.

Sera from ANCA-negative vasculitis patients (n = 8) was negative for reactivity against native MPO, while Ig was positive by direct ELISA (A). (B) Similar results when testing for reactivity against MPO peptide aa 447–459 by direct ELISA. (C) Inhibitory effects of serum spiked into purified Ig. (D–F) Data from protein studies to identify the masking factor in serum. Affinity purification of serum proteins that complex with peptide aa 447–459 identified an approximately 50-kDa protein by SDS-PAGE, Coomassie-stained gel (D). MS analysis identified the protein as CP. Identity was confirmed by Western blot (E) probed with an anti-CP antibody. Purified CP was purchased and digested with plasmin in vitro to produce a 50-kDa fragment SDS-PAGE, Coomassie-stained gel (samples were run on the same gel but were not contiguous) (F). ELISA results (G) indicated that full-length CP (151 kDa) did not mask the epitope, while CP cleaved by plasmin was effective in blocking reactivity by 30%–50%. Reactivity appears unaffected by addition of undigested CP to MPO-ANCA IgG (polyclonal) from 4 patients (H), indicating that specificity of the CP fragment effect on aa 447–459. Error bars represent the mean ± SEM.

Figure 5. In vitro and in vivo pathogenic potential of anti-MPO^447–459.

Affinity-purified anti-MPO^447–459 autoantibodies were capable of activating neutrophils, as measured by their ability to induce release of reactive oxygen species, while nonpathogenic (MPO^516–524) and natural (MPO^579–590, MPO^237–248, and MPO^530–536) anti-MPO autoantibodies were not. Neutrophils isolated from healthy subjects (n = 4) were exposed to purified autoantibodies from unique individuals (n = 4). Results represent the mean ± SEM. No further statistics were done due to the limited sample size.

Figure 6. Passive transfer of IgG from MPO^442–460–immunized mice is nephritogenic.

Independently, a T cell MPO epitope had been identified, and DR2 transgenic mice were injected with the overlapping MPO peptide aa 442–460 (LYQEARKIVGAMVQIITYR) that includes the human MPO epitope aa 447–459 (RKIVGAMVQIITY). Albuminuria and hematuria (A and B) were measured on days 1 and 6 and BUN on day 6 (C). Abnormal glomeruli (day 6, D, E, G, and H) were assessed based on capillary wall thickening and mesangial hypercellularity on formalin-fixed PAS–stained kidney sections. Scale bar: 45 μm). Neutrophil recruitment (F) was assessed based on immunohistochemistry by anti–Gr-1 antibodies on PLP-fixed frozen kidneys.