Experimental Anti-GBM Glomerulonephritis Induced in Rats by Immunization With Synthetic Peptides Based on Six Α Chains of Human Type Iv Collagen

Abstract

The anti‐glomerular basement membrane (GBM)‐nephritis‐inducing activity of six synthetic peptides having an amino acid sequence consisting of the six α chains of human type IV collagen was examined by injecting the peptides into rats. The peptides consisted of 27 amino acid residues from the non‐collagenous domain (NC1) of the α1 to α6 chains and were non‐consensus sequences sandwiched between two consensus sequences near the carboxyl terminus. Each peptide was coupled to keyhole limpet haemocyanin and injected with adjuvant into the footpads of 20 female WKY–Crj rats. The number of rats with proteinuria (over 10·0 mg of urinary protein/15 h) and haematuria was 2 with the α3 peptide, 8 with the α4 peptide, and 1 with the α5 peptide. Histological changes seen in the glomeruli were characteristic of those in anti‐GBM nephritis. Linear deposition of rat IgG along the GBM was observed in five rats injected with the α4 peptide. A nephritogenic monoclonal antibody against the α4 peptide was established using lymph node cells from a rat injected with the α4 peptide. The results indicate that α4(IV)NC1 is a potent nephritogenic antigen like α3(IV)NC1, which has already been recognized as a primary target antigen in Goodpasture's syndrome.

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

“…6D). This nding corresponded to the literature reporting that the α3subunit of Col (IV) is distributed in the basement membrane of renal tubules in rats [19]. IHC demonstrated the reveal of α3(IV)NC1 in the affected renal interstitial tissues of Group 2 rats with anti-GBM antibody production and in ltration of CD11c + macrophages around the sites but not in Groups 1 and 2 rats without anti-GBM antibody production (Fig.…”

High nephritogenicity of monoclonal antibodies belonging to IgG2a and IgG2b subclasses in rat anti-GBM nephritis

“…The antigen is derived from solubilized GBM [13], or a purified fraction containing a(IV)NC1 domains [14]. Synthetic a3(IV)NC1 or a4(IV)NC1 peptide sequences [15] or recombinant a3(IV)NC1 or a4(IV)NC1 proteins [16][17][18] have also be used. Studies on actively induced anti-GBM nephritis have revealed the target antigen of nephritis to be a3(IV)NC1 and a4(IV)NC1.…”

“…However, only three clones of nephritogenic monoclonal antibodies have been characterized [15,21], and this number is too small to know the nature and function of nephritogenic autoantibodies. In 1995 we reported a new monoclonal-antibody-producing method called the rat lymph node method [23].…”

Purification and Characterization of Human Nephritogenic Antigen That Induces Anti-GBM Nephritis in Rats

“…Since then, improved active type models induced in rats by a single injection of an emulsion of soluble nephritogenic antigen from GBM and adjuvant have become widely applied. [3][4][5][6][7][8] Passive types of experimental anti-GBM nephritis can also be induced by injections of homologous polyclonal 9 or monoclonal 10,11 antibodies to the GBM or type IV collagen.…”

“…31 In addition, injection of a synthetic peptide that contains part of the human 4(IV)NC1 sequence can induce experimental anti-GBM nephritis in rats. 11 We developed a novel method of generating rat monoclonal antibodies that uses the enlarged medial iliac lymph nodes (as a source of B cells) from rats injected with emulsified antigen into their hind footpads. 33 The frequency of positive hybridomas producing antibodies to the immunogen is about ten times higher than that obtained by the conventional method using mouse or rat splenic lymphocytes as a source of B cells.…”