We examined mature CD27+ peripheral blood B lymphocytes for the expression of RAG
genes on a single‐cell level. RAG
gene expression is detectable in mature B cells. About one third of 777 B lymphocytes examined from all populations together had detectable levels of any of the RAG
genes. Our results challenge recent views that RAG1
expression physiologically occurs only in a coordinate 1:1 relationship.28
This hypothesis, however, has been generated in the analysis of “primary” V(D)J recombination of early B cell progenitors in bone marrow. Recently, evidence is emerging that receptor revision in the periphery may be an important process that ensures tolerance.16,29
Our findings give new insights into divergent RAG1
expression, which are partly supported by findings that engagement of the BCR can have contrary effects in distinct developmental stages of B lymphocytes.30
We found a more than twofold and fourfold decrease in the frequency of RAG2‐expressing B cells isolated from patients with o‐JIA in CD27+ CD5+ and CD27+ CD5− populations when compared with healthy people. In contrast, RAG1 frequencies tended to be unchanged or slightly increased in those with o‐JIA. Thus, we hypothesised that RAG2 is differentially expressed in patients with o‐JIA when compared with healthy people. Finally, this resulted in a loss of coordinate RAG expression in CD5− but not in CD5+ B cells.
The argument that an increased bone marrow lymphopoiesis results in a major fraction of immature RAG
‐expressing B cells in the periphery during a systemic inflammatory process14
is not applicable to our results, as we studied CD27+ mature B cells. Arguably, the effects seen are caused by cell cycle dependence of RAG
expression, as a larger fraction of proliferating lymphocytes can be assumed in inflammation. Relative percentages of CD27+ among CD19+ cells were comparable in patients with o‐JIA and in controls in a current flow‐cytometric analysis, with a mean of about 16–18% (data not shown). No evidence in our analysis and in previous publications23
showed an increase in total B cells isolated from patients with o‐JIA. Thus, considerably differing absolute CD27 counts are unlikely. Although the RAG2 protein is down regulated at least 20‐fold before a cell enters the S phase to protect against unselective recombination under vulnerable conditions,31,32
these changes in the RAG protein level occur primarily at a post‐translational level, with RAG2 mRNA levels remaining nearly unaffected.31
So far, it cannot be excluded that drugs had an effect on the composition of lymphocyte subsets, as we have previously shown for cyclophosphamide in SLE in children.33
However, the completely missing coordinate RAG
expression in CD5− B cells isolated from patients with JIA, despite a broad spectrum in disease activity compared with a solid presence in two of three in controls, would argue for a disease‐related phenomenon.
Our results from human peripheral CD27+ B cells are concordant with recent reports on individual tonsil B cells from healthy children, in whom distinct developmental stages of germinal centre B cells were examined for RAG
gene expression, with a special focus on CD5 expression29
: on CD38+ IgD+ CD23+ B cells, induction of surface CD5 after stimulation with anti‐IgM and CD40L was described, followed by coordinate RAG mRNA expression. Interestingly, RAG2
transcripts appeared later than RAG1
transcripts, supporting the hypothesis of a differential regulation of these two genes, and of RAG2
as a limiting factor.29
In contrast, among early tonsil memory B cells (CD38− IgD−), nearly three times as many double‐RAG
‐positive cells were found in the CD5− subgroup as in the CD5+ population (16.6 v
6%). These findings are comparable to our results in peripheral CD19+ CD27+ B cells of controls: the relationship was similar, with 4.9% and 1.3% in CD5− and CD5+ B cells, respectively (p<0.05). Interestingly, the absolute frequencies of cells expressing RAG1
were lower in our study on peripheral B cells. Thus, a small subset of B cells expressing RAG1
seems to persist physiologically in the peripheral blood of children, as we have described previously in the case of adults.7
Reduced coordinate RAG
expression in CD27+ CD5− B cells isolated from patients with o‐JIA may be relevant in pathogenesis, possibly disabling these B cells to carry out sufficient receptor revision.
The regulation of RAG
genes is complex and tightly regulated, owing to the possible disastrous consequences of unselective DNA cleavage. It can happen at the level of transcription, post‐transcriptional mRNA and protein modification or DNA accessibility (reviewed by Schlissel34
). It has been difficult so far to assess factors influencing RAG
expression: it is known that BCR engagement itself can turn off the expression of V(D)J recombinase genes in mature B cells.35,36RAG
can be induced again in these cells via a combination of CD40L or lipopolysaccharide and IL4 (or IL7) signalling, imitating T cell help.9,37
In contrast, in immature B cells, the opposite reaction has been documented. RAG
genes have been induced after BCR
Among CD5+ B cells, low coordinate RAG transcripts were detected in patients and in controls. In the CD5− children with JIA, we found no coordinate RAG
expression at all. The CD5 molecule negatively regulates antigen receptor‐mediated growth signals by recruiting SH2‐domain‐containing protein tyrosine phosphatase‐1 into the BCR.39
Thus, CD5 action may have a stimulating role on the final coordinate RAG
expression in patients with JIA by inhibiting a BCR
‐mediated suppression of RAG
transcription in those with JIA. This may be due to a strong challenge of autoantigens via the BCR, which, on the other hand, suppresses RAG
transcription, especially in B cells lacking the CD5 molecule in patients with JIA, as shown in our analysis. Thus, we hypothesise that pathogenetic processes are imposed on mature B cells, which become engaged in autoimmunity.
In earlier studies, we have shown increased coordinate RAG
expression in IgD+ B cells from peripheral blood of patients with SLE,17,33
proposing a prolonged receptor editing in B cells from patients with SLE, which have prematurely left the bone marrow. As these cells represent an earlier stage in B cell development, comparability to the currently analysed post‐switch memory B cells in patients with JIA is limited.
Indeed, these findings may show two different mechanisms proposed for the role of secondary rearrangements in the pathogenesis of autoimmune diseases, both promoting the disease by an uncontrolled creation of autoreactive antibodies. In contrast, an impaired receptor revision may result in ineffective deletion of B cells that have acquired autoreactive receptors in patients with o‐JIA. However, the consequences of these immunoregulatory abnormalities on the pathogenesis of autoimmune diseases cannot be defined yet.
In conclusion, we found comparable low frequencies of RAG double‐positive CD27+ B cells in healthy children and CD5+ populations with JIA, whereas the CD5− populations showed a complete absence of RAG1‐positive and RAG2‐positive lymphocytes in peripheral memory B cells in children with o‐JIA. We obtained evidence for a lack of receptor revision in the periphery as a feature for CD27+ CD5− B cells in children with JIA, which may contribute to the autoimmune pathogenesis of the disease.