Monoclonal anti-IgG autoantibodies derived from lipopolysaccharide-activated spleen cells of 129/Sv mice

Abstract

In some colonies, 129/Sv mice produce, upon aging, a rheumatoid factor (RF) that is specific for mouse IgG2a but fails to react with IgG2a of the b allotype. It is not known whether this narrow specificity is due to the absence of other RF specificities in the repertoire of these mice or to the selective activation of the production of anti-IgG2a autoantibodies by a specific stimulus. To analyze the RF repertoire of 129/Sv mice, we have derived hybridomas from their spleen cells 3 d after an intraperitoneal injection of lipopolysaccharide. We have obtained 68 hybridomas secreting a monoclonal IgM with RF activity. This represents approximately 3 percent of the total number of hybridomas generated in four hybridizations. In addition, one monoclonal IgA RF was derived from unstimulated 129/Sv spleen cells. The specificities of these monoclonal RF were examined by testing their ability to bind to a panel of homologous and heterologous IgG preparations. The majority of the IgM RF reacted exclusively with a single mouse IgG subclass: 58 with IgG1, and 1 with IgG2a. Eight bound preferentially to IgG1 but cross-reacted to some extent with IgG2a and one was specific for a determinant shared by IgG1, IgG2a, and IgG3. The IgA RF derived from unstimulated spleen cells was primarily directed against IgG2a but cross- reacted somewhat with IgG2b. Identical results were obtained with two different monoclonal IgG1 and IgG2a proteins of the a allotype. No allotypic specificity was found for the anti-IgG1 RF, which all reacted well with IgG1 of the b allotype. In contrast, the IgM anti-IgG2a antibody exhibited such allotypic specificity because it failed to react with IgG2a of the b allotype. When tested on heterologous IgG preparations, all anti-IgG1 RF reacted better with rat IgG1, rat IgG2c, bovine IgG2, goat IgG2, and rabbit IgG than with mouse IgG1, demonstrating a particular homology between these Ig. On the basis of additional cross-reactions with other IgG, including rat IgG2a, rat IgG2b, bovine IgG1, goat IgG1, human IgG, and chicken IgG, seven different anti-IgG1 clonotypes could be identified. However, despite their heterogeneity, nearly all antigenic determinants recognized by anti-IgG 1 RF appeared to be located in the hinge region of the molecule. Total lack of binding to IgG1 Fab fragments was indeed observed, and only one antibody reacted with IgG1 Fc fragments. Unlike the anti-IgG1 RF, the IgM and the IgA anti-IgG2a antibodies did not cross-react with any heterologous IgG of the same panel. Altogether, t 1 different RF clonotypes could be distinguished on the basis of their fine specificity. The anti-IgG2a specificity of the RF spontaneously produced by 129/ Sv mice is thus not due to the absence of other RF specificities in the repertoire of these mice.