A murine nephritogenic monoclonal antibody binds to both single-stranded deoxyribonucleic acid and glomerulus.
BACKGROUND
Autoantibodies such as anti-DNA and antimyeloperoxidase (MPO) antibodies have been shown to cause glomerulonephritis in experimental animal models. To analyze pathogenic autoantibodies, we developed hybridomas from spleen cells of nontreated FGS mice, in which focal segmental glomerular sclerosis develops spontaneously.


METHODS
Reactivity and specificity of a monoclonal antibody (FG1H5) were examined using enzyme-linked immunosorbent assay and cryosections of mouse organs as substrates. Immunoprecipitation was performed to analyze reactive antigens. Hybridoma cells were injected ip into severe combined immunodeficiency (SCID) mice to examine their nephritogenicity in vivo.


RESULTS
The binding of FG1H5 to single-stranded DNA (ssDNA) was inhibited by ssDNA and also MPO. The binding of FG1H5 to MPO was weak, not inhibited by MPO, and markedly enhanced by the presence of ssDNA. This marked enhancement of the binding to MPO was abolished by DNase I-treatment of the mixture of FG1H5 and ssDNA. When MPO was introduced into ssDNA-coated wells, the binding of FG1H5 to ssDNA was inhibited. On the other hand, when ssDNA was introduced into MPO-coated wells, the binding of FG1H5 to MPO was markedly enhanced. Inhibition tests using double-stranded DNA revealed that FG1H5 is specific for ssDNA. Histologic examination of FG1H5-reactive antigen using SCID mouse kidney showed positive stainings in the nucleus and glomerulus (mainly the mesangium). These positive stainings were abolished after the incubation of FG1H5 with ssDNA. The DNase I treatment of kidney sections markedly reduced the nuclear staining, but the staining of the glomerulus was preserved. Immunoprecipitation of a soluble fraction of SCID mouse kidney with FG1H5 revealed that FG1H5-reactive antigen in the glomerulus is an approximately 28-kilodalton molecule. When FG1H5 hybridoma cells were injected ip into SCID mice, the mice showed glomerulonephritis with the increases in mesangial cells and matrix as well as immunoglobulin M deposition mainly in the mesangium.


CONCLUSIONS
Data demonstrate that FG1H5 binds strongly and specifically to ssDNA (but weakly and nonspecifically to MPO), and that ssDNA and MPO bind to each other. One monoclonal antibody reacts with both the nucleus and glomerulus (mainly the mesangium), and glomerular staining is not caused by nonspecific DNA binding. FG1H5, which binds to ssDNA, can induce glomerulonephritis, probably because of a direct crossreactivity to glomerular components.
First Department of Pathology, Kansai Medical University, Osaka, Japan.
N
N
Nagata
S
S
Miyashima
S
S
Taketani
J
J
Toki
N
N
Hosaka
T
T
Tomita
H
H
Fujishima
R
R
Tokunaga
S
S
Ikehara
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000737:chemistry; D001327:Autoimmune Diseases / Q000276:immunology*; D004277:DNA, Single-Stranded / Q000276:immunology*; D005921:Glomerulonephritis / Q000276:immunology*; D007678:Kidney Glomerulus / Q000276:immunology*; D051379:Mice; D008815:Mice, Inbred Strains; D016513:Mice, SCID; D008970:Molecular Weight; D009195:Peroxidase / Q000276:immunology
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D004277:DNA, Single-Stranded; D009195:Peroxidase