IgVH genes from different anatomical regions, with different histopathological patterns, of a rheumatoid arthritis patient suggest cyclic re-entry of mature synovial B-cells in the hypermutation process

Abstract

In the present study 55 IgVH genes amplified from three different anatomical regions of a rheumatoid arthritis (RA) patient were analyzed, adding further information on synovial B-cell maturation and recirculation in RA. This analysis demonstrated somatically mutated IgVh genes in all regions studied, with amino acid deletions and mixed IgVh molecules, suggesting the existence of a novel pathway to generate (auto) antibody specificities. Comparison of amino acid sequences of amplified genes that belong to the VH1 family (with predominantly the same germline counterpart) exhibited strong homology, indicating an apparently conserved mutational pattern. This suggests that the number of antigens that activate B cells in different locations is restricted. The most striking result was the finding of clonally related sequences in different anatomical regions, indicating a recirculation of activated B cells between the different affected joints.

Figure 1

Comparison of the translated amino acid sequences of 19 VH1 segments with their closest germline counterparts. (b) Comparison of the translated amino acid sequences of 19 VH1 segments with germline gene IGHV1-18^*01. All sequences are deposited in Genbank database with the following accession numbers: AF209875-209902; AF211924-211932; AF241154-241156 and AF241186-241199.

Figure 2

Analyses of the VH4 family amplificates. (a) Comparison of the five nucleotide VH4 sequences with their germline counterpart IgHV4-30-1/4-31^*02. (b) Clonal relation between sequences k194/100 and k194/101; replacement (r) and silent (s) mutations on the VH segment when compared with the germline. (c) Clonal relation between sequence k194/23 amplified from the left peroneal tendon (lpt) and sequences k194/102 and k194/103 amplified from the cubita. Dashed arrows indicate the possibility of cyclic re-entry of the mature B cells into the hypermutation process.

Figure 3

Histopathology (haematoxylin and eosin) and immunohistochemistry (double staining: indirect immunoperoxidase and alcaline phosphatase in insert of parts a and b) of rheumatoid synovial tissue from three different anatomical locations of the RA patient. These are as follows: (a) right peroneal tendon sheath; (b) left peroneal tendon sheath with inserted figure showing Ki-M4 positive FDC network (brown) surrounded by CD20⁺ B lymphocytes (blue), representing a germinal center; and (c) synovial membrane from the right cubita. Arrows indicate enlarged synovial intima (original magnification 350×).

Figure 4

Total R/S ratios in the FR 1+2+3 and CDR 1+2 of B-cell clones from each different anatomical region of the RA patient: (a) right peroneal tendon sheath; (b) left peroneal tendon sheath; (c) cubita.

Figure 5

Amino acid replacement plot, calculated across translated amino acid sequences shown in Figure 3b. (a) Conservative replacement (Y D, E;↔ N D, E; L↔ V, A, F, I; S↔ N, T, Q; D↔ E; K↔ R); (b) indeterminant replacement (H↔ all amino acids; M↔ all amino acids; G↔ A, V, L, I, F, P; W↔ R, K, S, T, N, Q, D, E; P↔ A, V, I, L,F; K, R↔ N, Q, S, T, Y; C↔ D, E, K, N, Q, R, S, T, Y; Y↔ K, R, N, Q, S, T); and (c) nonconservative replacement (R, K↔ E, D; D, Y, E↔ A, I,L, P, F; S, T, Q, N↔ A, I, L, V, P, F; R, K↔ A, I, L, P,V, F; C↔ A, I, P, V, L, F; W↔ A, I, P, F, V, L; G↔ C, D, E, R, K, N, Q, S, T, Y). The type of replacement was defined on the basis of [45,46,47,48].

Figure 6

Generation of mixed molecules according to Maizels [37]. In RA synovial tissue mixed molecules could be the result of a templated or an untemplated hypermutation DNA repair process.