Abstract

In order to search for further evidence for a pathogenetic role of recirculating, antigen-driven B cell clones in rheumatoid arthritis (RA) rearranged VH genes were analysed for clonal relationship and somatic mutations from synovial tissue and peripheral blood of a patient with RA undergoing synovectomy of several finger joints. DNA was prepared from the synovial tissue of two finger joints and blood. PCR for the different VH families was performed with one specific oligonucleotide for each VH family and a mixture of JH-specific oligonucleotides. The PCR products were separated on a high resolution acrylamide gel differentiating one base pair difference of length. Transfer of the products onto a nylon membrane and hybridization with an oligonucleotide specific for the FR3 region revealed a polyclonal representation of rearranged VH1, VH3, VH4 and VH5 genes. The VH6 family, which is encoded by a single germline gene, was represented by few distinct bands, with some bands of identical height for both joints and blood. DNA from these bands of interest was eluted, reamplified by PCR, cloned and sequenced. Sequence analysis of 27 independent bacterial colonies allowed distribution of the different VH genes to seven B cell clones (A-G). Members of clone A were found in both joints and blood, clones B and C in one joint and blood, clone D in both joints, and clones E, F and G only in one joint. The VH regions were somatically mutated with characteristic patterns for the different clones. In conclusion, our findings confirm the systemic character of RA, because they show that not only expansion and affinity maturation of B cells occur in synovial membranes but antigen-specific B cells recirculate between different joints and blood.