Possible Implication of Intermolecular Epitope Spreading in the Production of Anti-Glomerular Basement Membrane Antibody in Anti-Neutrophil Cytoplasmic Antibody-associated Vasculitis (1)

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic small vessel vasculitis accompanied by the presence of ANCA in the serum. This disease entity includes microscopic polyangiitis (MPA), granulomatosis with polyangiitis, and eosinophilic granulomatosis with polyangiitis. The major antigens of ANCA are myeloperoxidase (MPO) and proteinase 3. Anti-glomerular basement membrane (GBM) disease is an organ-specic autoimmune disorder characterized by diffuse necrotizing crescentic glomerulonephritis and pulmonary hemorrhage with the production of anti-GBM antibody. The major epitopes recognized by anti-GBM antibodies are present in the α3-subunit NC1 domain. α3(IV)NC1 [α3-subunit NC1 domain of Col (IV)], which covers these epitopes, is structurally cryptic, owing to the hexamer formation-sulphilimine linkage of 3-4-5 α-subunit trimers—of the NC1 domain, and is revealed by cleaving the sulphilimine linkage. Approximately 30% of patients with anti-GBM disease possess ANCA in the serum and 5% of AAV patients are positive for circulating anti-GBM antibodies.

The retrieval of α3(IV)NC1 from FFPE normal kidney sections in IHC was examined using anti- α3(IV)NC1 mAb which recognizes a cryptic IPSTVKA amino acid sequence in the α3(IV)NC1 region. Although the anti-α3(IV)NC1 mAb did not bind to the epitope without antigen retrieval, a clear reaction with GBM and the basement membrane of Bowman’s capsule that connects to the glomerulus was observed when the sections were heated under acidic conditions. In contrast, no reaction appeared when the sections were heated under neutral conditions or alkaline conditions. Interestingly, the anti-α3(IV)NC1 mAb bound to GBM in FFPE normal kidney sections when the sections were exposed to proteases derived from S. griseus after heating under neutral conditions. α3(IV)NC1 was detected when Col (IV) was digested by an appropriate concentration (125 μg/ml) of the proteases derived from S. griseus. The reveal of α3(IV)NC1 was also observed when Col (IV) was digested by NE in a dose-dependent manner (at a concentration of 3500 μU/ml).

Any specific binding of the mAb to unaffected glomeruli did not appear when the sections were heated under alkaline conditions and not treated by proteases. In contrast, weak binding of the mAb to GBM was observed in crescentic glomeruli of MPA without any particular antigen retrieval. Weak binding of the mAb to GBM was also observed in crescentic glomeruli of IgA nephritis. Moreover, an obvious binding of the anti-α3(IV)NC1 mAb was observed in the sclerotic glomeruli of MPA, IgAN, and hypertensive arteriosclerosis.

WKY rats (n=23) were immunized with human MPO according to the original protocol and then divided into two groups, namely, Group 1 without PMA administration (original protocol, n=11) and Group 2 with PMA administration (modified protocol, n=12). Group 2 rats were given an intraperitoneal injection of PMA (1 μg) on days 7, 14, 21, 28, and 35. All rats in Groups 1 and 2 produced ANCA, and the titer was comparable between the two. Severe hematuria was detected in both groups of rats, whereas CRP levels were significantly higher in Group 2 than in Group 1. Correspondingly, the degree of renal tissue destruction represented by the number of erythrocyte casts in the kidney sections tended to be larger in Group 2 compared to Group 1. Infiltration of neutrophils into the upstream renal tubules of the erythrocyte casts was observed.

Anti-GBM antibody was not detected in all day 0 sera (n=23). Whereas neither rats in Group 1 nor rats given intraperitoneal PMA injection with mock-immunization produced anti-GBM antibody, two rats in Group 2 (#6 and #9) produced anti-GBM antibodies. Interestingly, chronological analysis of autoantibody production revealed that anti-GBM antibody production followed ANCA production in these rats. Also, the subclass of anti-GBM antibody produced in Group 2 AAV rats was determined as IgG2a, one of the pathogenic subclasses. Contrary to humans, Col (IV), including α3(IV)NC1, was distributed in the basement membrane of tubules as well as in glomeruli in rats. The reveal of α3(IV)NC1 in the affected renal interstitial tissues of Group 2 rats with antiGBM antibody production and infiltration of CD11c+ macrophages around the sites but not in Groups 1 and 2 rats without anti-GBM antibody production.

When the recombinant human MPO light chain, instead of native MPO, was used as an immunogen, ANCA was not detected by FCM, and renal tissue destruction was minimal regardless of PMA administration (unpublished results). Serum CRP levels were not elevated significantly in the rats immunized with the non-pathogenic recombinant human MPO light chain followed by PMA administration. In AAV, proteases released from neutrophils activated by ANCA digest Col (IV) and result in the reveal of α3(IV)NC1, AAV-specic CD11c+ macrophages catch and present the epitopes in the region, and then the host’s immune system produces anti-GBM antibodies.

All AAV patients do not produce an anti-GBM antibody regardless of the reveal of α3(IV)NC1 with a close association with CD11c+ macrophages in the kidneys. Other factors, e.g., genetic background such as MHC haplotypes, might be involved in the mechanism of anti-GBM antibody production in AAV patients. The subclass of anti-GBM antibody in AAV rats was examined by ELISA, and it was determined as IgG2a, one of the pathogenic subclasses. Also, one of the two AAV rats with anti-GBM antibody production developed more severe pulmonary hemorrhage than other rats.

1. Y. Nishibata, M. Nonokawa, Y. Tamura, R. Higashi, K. Suzuki, H. Hayashi, S. Masuda, D. Nakazawa, S. Tanaka, U. Tomaru, Others, Possible Implication of Intermolecular Epitope Spreading in the Production of Anti-Glomerular Basement Membrane Antibody in Anti-Neutrophil Cytoplasmic Antibody-associated Vasculitis (2021) (available at https://www.researchsquare.com/article/rs-322082/latest.pdf).