Polymorphism in the immunoglobulin VH gene V1-69 affects susceptibility to rheumatoid arthritis in subjects lacking the HLA-DRB1 shared epitope.

OBJECTIVE: To investigate the contribution of polymorphism in the immunoglobulin heavy chain variable region V1-69 gene set to genetic susceptibility to rheumatoid arthritis (RA) in Czech and British patients. METHODS: We used V1-69 gene sequence-specific polymerase chain reaction (PCR) and restriction enzyme digestion to study polymorphism in the V1-69 gene set in germline DNA of 109 Czech and 159 British RA patients and 164 ethnically matched controls. Polymorphism was further studied by nucleotide sequencing of the V1-69 gene locus in germline DNA. RESULTS: We found that all patients and controls had at least one V1-69 gene copy. In the Czech RA cohort, the dimorphic nucleotide in codon 73 of V1-69 (GAA or AAA) was present in the homozygous form 73(A/A) in 31 of 109 (28.4%) RA patients vs 12 of 79 (15.2%) controls [odds ratio (OR)=2.22, P<0.001]. When the RA patients and controls were classified according to HLA shared epitope (SE) status, 73(A/A) was found in 18 of 76 (23.7%) SE(+) patients compared with 13 of 38 (34.2%) SE(-) patients, four of 12 (18.2) SE(+) controls and eight of 57 (14%) SE(-) controls. This suggests that homozygosity for the dimorphic sequence 73(A) contributed to susceptibility to RA in SE(-) Czech individuals (OR=3.2, P<0.001). The most striking observation was that none of the 38 SE(-) Czech patients, compared with 11 of 76 (14.5%) SE(+) RA patients, three of 22 (13.6%) SE(+) and 11 of 57 (19.3%) SE(-) ethnically matched controls, were homozygous for the alternative dimorphic sequence 73(G/G) (OR=9.1, P<0.05). These data, however, were not replicated in a Caucasoid British RA population. CONCLUSION: The dimorphic sequence at codon 73 (73(A/A)) of the V1-69 gene contributes to genetic susceptibility in SE(-) Czech RA patients.

Rearrangement of the human heavy chain variable region gene V3-23 in transgenic mice generates antibodies reactive with a range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain variable region.

To formulate a 'logic' for how a single immunoglobulin variable region gene generates antibodies with different antigen specificity and polyreactivity, we analysed chimeric antibodies produced in transgenic mice carrying the germ-line human V3-23 gene, multiple diversity (D) and joining (J) gene segments. Hybridomas producing antibodies encoded by the V3-23 gene in combination with different mouse Vkappa genes were obtained by fusion of splenocytes from transgenic mice. All antibodies had human mu-chains and mouse light chains, were multimeric in structure and expressed the human V3-23 gene. Nucleotide sequence analyses of genes encoding the heavy and light chains of 12 antibodies in relation to antigen specificity highlighted the importance of heavy chain variable region CDR3 in determining reactivity with different antigens. However, the results also suggest that non-CDR3 sequences intrinsic to the V3-23 gene itself may be involved in, or determine, the binding of the chimeric antibodies to some of the antigens tested in the current study.

Rheumatoid factor isotype switch and somatic mutation variants within rheumatoid arthritis synovium.

The presence of clonally-related B-lymphocyte aggregates within synovial lining tisue of rheumatoid arthritis (RA) patients suggests a germinal centre-like reaction, which may hold implications for disease pathogenesis and the causes of chronic inflammation. We studied 250 rheumatoid factor (RF) heavy-chain sequences cloned from the synovium of three patients with RA, to determine whether they undergo both somatic mutation and isotype switching consistent with this hypothesis. Size analysis of immunoglobulin heavy-chain cDNAs from synovial RF+ B cells revealed oligoclonal RF+ populations and identically-sized VH-D-JH transcripts of different immunoglobulin isotypes. Sequencing of individual inserts selected from cloned immunoglobulin heavy-chain cDNAs demonstrated a clonal relationship between immunoglobulin M (IgM) RF and IgA RF, suggesting that this isotype switch occurred in synovium. Furthermore, most somatic mutations were found to have occurred after this isotype switch. This finding suggests that the RA synovial microenvironment sustains somatic mutation and isotype switching in RF-specific B lymphocytes akin to secondary lymphoid organs.

Loss of original antigenic specificity in T cell hybridomas transduced with a chimeric receptor containing single-chain Fv of an anti-collagen antibody and Fc epsilonRI-signaling gamma subunit.

T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen type II, when engrafted with a chimeric cell surface receptor, scC2Fv/gamma, which includes the single-chain Fv domain (scFv) of the anti-collagen type II mAb C2 and the signaling gamma subunit of the Fc epsilonRI. When transduced into MD.45 cells, scC2Fv/gamma or its mutated form lacking immunoreceptor tyrosine-based activation motif (ITAM), scC2Fv/gammaIC-, formed mainly homodimers. A small proportion of these molecules formed heterodimers with endogenous CD3zeta in these hybridoma cells. By contrast, in HCQ6 cells, the majority of scC2Fv/gamma and scC2Fv/gammaIC- molecules formed heterodimers with CD3zeta, and only a small proportion of them was expressed as homodimers. Stimulation with plastic-immobilized collagen induced IL-2 production in scC2Fv/gamma-transduced MD.45 cells, but not in MD.45 cells transduced with the ITAM-less chimera scC2Fv/gammaIC-. HCQ6 cells transduced with scC2Fv/gamma responded to plastic-bound collagen. Due to the high content of CD3zeta-associated chimeras, HCQ6 cells transduced with the ITAM-less scC2Fv/gammaIC- chimera were also responsive to plastic-bound collagen. When cells were stimulated with collagen in solution, MD.45 cells transduced with scC2Fv/gamma produced IL-2, whereas transduced HCQ6 cells were unresponsive, hence suggesting that the ability of cells transduced with scC2Fv chimeras to respond to soluble collagen correlated with predominant expression of divalent scC2Fv/gamma homodimers, but not monovalent scC2Fv/gamma-CD3zeta or scC2Fv/gammaIC(-)-CD3zeta heterodimers. Of interest, expression of CD3 subunits in hybridomas transduced with scC2Fv chimeras was reduced, resulting in decreased response to cognate Ags.

Differential use of immunoglobulin light chain genes and B lymphocyte expansion at sites of disease in rheumatoid arthritis (RA) compared with circulating B lymphocytes.

The presence of germinal centre-like structures and clonotypic expansion of lymphocytes in RA synovia may indicate a site-specific immune response to local antigens, rather than passively entrapped immune cells, that sustains synovial inflammation. In this study we compare the nature of immunoglobulin light chain variable region gene use in the synovium of RA patients with peripheral B cells to determine the nature of the synovial immune response. Using Vlambda and Vkappa gene fingerprinting, which relies on differences in CDR3 length, we demonstrate differences in the pattern of Vlambda and Vkappa use and clonotypic expansion of B cells between the synovium and peripheral blood of RA patients. Further, we show that some synovial rearrangements with long CDR3 are selectively expanded. These longer than usual CDR3 were generated by a number of mechanisms including N-additions. However, the observed differences were not uniform in different patients. These observations suggest that local synovial antigens drive significant numbers of T and B lymphocytes selected from an existing repertoire shaped by genetic and environmental factors. Further, the data argue against passive retention of most B cells in the synovium of RA patients.

Gene therapy for rheumatoid arthritis. Theoretical considerations.

Current understanding of the pathogenesis of rheumatoid arthritis has provided evidence that therapeutic benefit can be achieved by using antagonists targeted to the inflammatory cytokines involved, mainly tumour necrosis factor-alpha and interleukin-1. Gene delivery of antagonists, which can inhibit the production or action of these cytokines and other mediators, has been achieved in experimental animal models. This new method of delivery can produce therapeutic effects at lower concentrations and in a local environment, overcoming the adverse effects that often accompany protein therapy. However, several technological and biological restraints preclude the immediate adaptation of this method to human treatment. Based on the experimental evidence, possible target therapeutic genes, cell types and vector systems that could be used are discussed in this article.

Rheumatoid factor autoantibodies in health and disease.

Recent advances in molecular biological and human cell hybridization technology have significantly advanced the knowledge of mechanisms that underlie human rheumatoid factor (RF) production. These advances have provided insight into the etiopathogenesis of synovial inflammation and lymphocyte recruitment in rheumatoid arthritis (RA) joints. We have examined the mechanisms that lead to RF production in RA patients and those that regulate RF production in normals. The studies revealed structural features that distinguish RF produced in normals from those produced in RA synovial tissue. There are significant differences in the use of VL and VH genes between the two RF populations. Furthermore, IgV genes encoding synovial RF in RA have extensive evidence for nucleotide changes, leading to amino acid replacement in the complementarity determining regions (CDRs). In addition, RF produced in RA synovia show evidence for affinity maturation, isotype switch to IgG RF, and repertoire shift indicative of a continued recruitment of B cells. Together with computer modeling and crystallographic studies, our data suggest that the mechanisms that operate on RF selection in RA synovia are similar to immune responses to exogenous antigens. In contrast, RF established from human immunized donors (HID) are characterized by a very low ratio of replacement to silent (R:S) nucleotide changes in the CDR1+2. In addition, there is little increase in affinity with increasing numbers of mutations. There is thus evidence for regulatory mechanisms that limit affinity maturation of RF in normals.

Analysis of V kappa genes in rheumatoid arthritis (RA) synovial B lymphocytes provides evidence for both polyclonal activation and antigen-driven selection.

To define mechanisms of sustained activation of synovial B lymphocytes in RA, we studied hybridomas established from the local synovial B cell repertoire of two RA patients for V kappa gene expression and for antigen-binding specificity. The analyses revealed that members of the main V kappa families (I, II and III) were utilized at frequencies consistent with random V kappa gene family use. Furthermore, although the hybridomas expressed genes frequently seen in response to other self- and exogenous antigens, only one V kappa I- and two of three V kappa III-expressing hybridomas exhibited reactivity with self-antigens. Nucleotide sequence analysis revealed that all hybridomas, with the exception of rheumatoid factor (RF)-producing hybridomas, expressed V kappa genes highly related to known germ-line genes (99.3-100% homology) and that diversity was generated by deletions and random nucleotide insertions at the V kappa-J kappa junction. Examination of the few nucleotide changes seen with the V kappa genes revealed a predominance of silent to replacement changes. Moreover, most of these changes can be attributable either to allotypic variations or to limited random nucleotide replacements independent of antigen selection. In contrast, one IgG-RF (B4D8) exhibited predominantly replacement nucleotide changes in the complementarity-determining regions, suggestive of antigen-driven selection. The random expression of immunoglobulin variable region genes with no, or little, evidence of mutation in the synovial B lymphocyte repertoire, including natural polyreactive antibodies, alongside mutated IgG-RF, suggest that both polyclonal activation and antigen-driven responses occur in RA synovia.

Sequence analysis of immunoglobulin heavy-chain variable region genes from the synovium of a rheumatoid arthritis patient shows little evidence of mutation but diverse CDR3.

To gain insight into the nature of B-lymphocyte responses in the synovium of rheumatoid arthritis (RA) patients, we amplified and sequenced immunoglobulin heavy-chain variable region genes expressed in seven IgM and three IgG-secreting synovial-derived hybridomas established from one patient. Each hybridoma V-region was encoded by unique VH-D-JH combination demonstrating that none of these hybridomas derived from clonally related B-lymphocytes in vivo. The expressed VH genes closely resembled (95.6%-100% homology) known germline VH genes in most hybridomas, including VH genes frequently used to encode autoantibodies. The antibodies produced by these hybridomas, with the exception of one IgM rheumatoid factor, did not bind to any of a large panel of autoantigens in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluoresence, suggesting that frequent expression of 'autoantibody-associated' VH genes does not correlate with detectable autoreactivity in this patient. Hybridoma CDR3 DNA was diverse in length and gene composition. Conserved heavy-chain cross-reactive idiotypes were expressed on 4/7 IgM- and 2/3 IgG-secreting hybridomas. The close similarity of expressed VH genes to germline counterparts of these hybridomas suggests that polyclonal activation is a prominent mechanism in B-lymphocyte activation in the synovium of this rheumatoid arthritis patient.