IMMUNOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ANTI-RNA POLYMERASE-I AUTOANTIBODIES PRODUCED BY TIGHT-SKIN MOUSE

Autoantibodies against nuclear proteins like RNA polymerase I (RNA pol I) are produced in a number of rheumatic autoimmune diseases. Product ion of antibodies specific for the 190-kD subunit of RNA pol I appears to be characteristic in the patients with systemic sclerosis. Previou s investigations have shown that the tight skin (TSK) mouse is an expe rimental model for systemic sclerosis. In the present study we show th at the TSK mice produce high titers of anti-RNA pol I antibodies, both of IgM and IgG classes. To characterize the immunochemical properties of these antibodies we obtained a large panel of hybridomas from thes e mice. Analysis of these hybridomas revealed that clonal frequency of autoreactive B cells specific for RNA pol I are higher in the TSK mic e than in the controls. mAbs obtained from the TSK mice were specific for the 190-kD subunit and cross-reacted with Esherichia coli and phag e T7 RNA polymerases (155-,150-, and 107-kD polypeptides). We have als o demonstrated that these antibodies bind better to the phosphorylated enzymes. The anti-RNA pol I mAbs were divided into three groups in te rms of their functional property. The first group of antibodies increa sed the catalytic activity of the enzyme whereas the antibodies of the second group inhibited the enzymatic activity. Competitive inhibition RIAs showed that these two groups of antibodies bound to distinct epi topes. The third group of antibodies was neutral and had no activity o n the enzyme function. These results suggest that TSK mouse anti-RNA p ol I antibodies recognize three or more conserved epitopes. To underst and the molecular basis of the generation of such autoreactive antibod ies we analyzed their V gene repertoire. Northern analysis of RNAs of 14 TSK hybridomas showed that the V(H) genes encoding these antibodies were mainly from V(H) J558 family. It is possible that these genes we re derived from a single germline gene or from a set of related genes of a single subgroup.