A MURINE NEPHRITOGENIC MONOCLONAL-ANTIBODY BINDS TO BOTH SINGLE-STRANDED DEOXYRIBONUCLEIC-ACID AND GLOMERULUS

BACKGROUND: Autoantibodies such as anti-DNA and antimyeloperoxidase (M PO) antibodies have been shown to cause glomerulonephritis in experime ntal animal models. To analyze pathogenic autoantibodies, we developed hybridomas from spleen cells of nontreated FGS mice, in which focal s egmental glomerular sclerosis develops spontaneously. EXPERIMENTAL DES IGN: Reactivity and specificity of a monoclonal antibody (FG1H5) were examined using enzyme-linked immunosorbent assay and cryosections of m ouse organs as substrates. Immunoprecipitation was performed to analyz e reactive antigens. Hybridoma cells were injected ip into severe comb ined immunodeficiency (SCID) mice to examine their nephritogenicity in vivo. RESULTS: The binding of FG1H5 to single-stranded DNA (ssDNA) wa s inhibited by ssDNA and also MPG. The binding of FG1H5 to MPO was wea k, not inhibited by MPG, and markedly enhanced by the presence of ssDN A. This marked enhancement of the binding to MPO was abolished by DNas e I-treatment of the mixture of FG1H5 and ssDNA. When MPO was introduc ed into ssDNA-coated wells, the binding of FG1H5 to ssDNA was inhibite d. On the other hand, when ssDNA was introduced into MPO-coated wells, the binding of FG1H5 to MPO was markedly enhanced. Inhibition tests u sing double-stranded DNA revealed that FG1H5 is specific for ssDNA. Hi stologic examination of FG1H5-reactive antigen using SCID mouse kidney showed positive stainings in the nucleus and glomerulus (mainly the m esangium). These positive stainings were abolished after the incubatio n of FG1H5 with ssDNA. The DNase I treatment of kidney sections marked ly reduced the nuclear staining, but the staining of the glomerulus wa s preserved. Immunoprecipitation of a soluble fraction of SCID mouse k idney with FG1H5 revealed that FG1H5-reactive antigen in the glomerulu s is an approximately 28-kilodalton molecule. When FG1H5 hybridoma cel ls were injected ip into SCID mice, the mice showed glomerluonephritis with the increases in mesangial cells and matrix as well as immunoglo bulin M deposition mainly in the mesangium. CONCLUSIONS: Data demonstr ate that FG1H5 binds strongly and specifically to ssDNA (but weakly an d nonspecifically to MPO), and that ssDNA and MPO bind to each other. One monoclonal antibody reacts with both the nucleus and glomerulus (m ainly the mesangium), and glomerular staining is not caused by nonspec ific DNA binding. FG1H5, which binds to ssDNA, can induce glomerulonep hritis, probably because of a direct crossreactivity to glomerular com ponents.