Immunostimulatory sequence up-modulate the interaction between immunoglobulins and plasmid DNA at acidic pH

To identify specific receptors for immunostimulatory sequence (ISS), a cDNA library of human bone marrow was screened by one-hybrid system using ISS as bait. Among four duel-positive clones isolated so far, two encode light chain of immunoglobulin (Ig) homologous to anti-DNA and anti-HBsAg (HBsAb), respectively, suggesting a direct interaction between ISS and Igs. ELISA revealed that HBsAb, as well as Igs in normal mouse serum and a monoclonal anti-v5 antibody, bound to plasmid DNA in a pH-dependent way; more importantly, the plasmids inserted with more copies of ISS shown higher reactivity with these Igs than their blank vectors. Electrophoretic mobility shift assay (EMSA) showed HBsAb bound to oligonucleotides (ODN) also in a pH-dependent and ISS-specific way. Furthermore, ISS-containing ODN could enhance the binding of Igs to plasmid DNA at acidic pH. These results support existence of direct ISS-Ig interaction and demonstrated that DNA-Ig interaction could be up-modulated by ISS. In consideration of the crucial role of membrane Ig (mIg) in B cell activation, these results suggest mIg may be a candidate ISS receptor in ISS-induced B cell activation.