Although the identification of autoantibodies in the serum of RA patients dates back to the 1950s, the role that these autoantibodies and B cells may play in the disease's pathogenetic processes are still poorly understood. One of the main reasons for this uncertainty is the underlying heterogeneity of the human patient population, which provides a strong rationale for the use of genetically and etiologically homogeneous mouse models of disease to tease out possible contributing factors. Among the many available arthritis models, the hTNF-tg strain stands out for sharing several key characteristics with human RA, including the spontaneous, progressive nature of the disease, and the well-recognized key pathogenetic role of TNFα. Although experiments with the TNF-overexpressing TNF ΔARE strain in a RAG-deficient background indicated that B and T lymphocytes are not required for arthritis onset (19
), the experiments detailed above highlight several key features that accompany arthritis progression in hTNF-tg mice which implicate B cells in at least some aspects of pathogenesis.
First, we show that onset of arthritic disease is paralleled by a dramatic increase in the B cell component of the draining lymph nodes, which involves most markedly a population with a unique CD23+, CD21-high, IgM-high, IgD+, CD1d+ phenotype. These B cells are preferentially restricted to LNs draining arthritic tissues, suggesting that their accumulation is dependent on signals coming from the affected joints. However, the expansion of the same population in K/BxN mice clearly indicates that this Bin population is not a unique feature of the TNF-transgenic microenvironment. The rapid and significant expansion of B cells, and particularly of the Bin population, in the early stages of disease in hTNF-tg mice does not appear to be dependent on one or a few autoantigens. Whether Bin cell expansion is equally polyclonal in K/BxN mice, in which a large proportion of lymph node B cells are known to be expressing anti-gpi antibodies (33
), remains to be determined. Our clonality analysis cannot rule out the possibility that hTNF-tg PLN B cells are more broadly autoreactive (beyond the limit of detection of oligoclonal expansion by spectratyping), or that clonal populations may be selected as disease progresses, but the lack of expression of activation and plasma cell markers on these cells implies that, regardless of their antigen specificity, they are not directly involved in a conventional immune responses within the node. Thus, it seems more likely that Bin cells arise as a polyclonal, possibly antigen-independent population that is associated with arthritis, regardless of the primary etiology and nature of autoantigen, and may exert additional roles in the context of disease progression.
Interestingly, B cells with a range of phenotypes that resemble marginal zone precursors, are CD1d+, and in some cases CD21-high have been defined as a regulatory, anti-inflammatory subset in a number of murine autoimmune conditions, including arthritis models (34
, reviewed in 38
). The common feature to these B-regulatory (B-reg) subsets is their ability to produce IL10, but according to our preliminary observations, hTNFtg CD23+ CD21-high B cells do not seem to be capable to prominently express this cytokine, or pro-inflammatory cytokines such as TNFα (human or mouse) and IFNγ (Supplemental Fig.3
). Thus, while it is tempting to speculate that Bin cells may be a regulatory subset specifically recruited/differentiated at sites of ongoing inflammation, further analysis will be required to directly address this possibility. Interestingly, it was recently reported that T-reg cells are inherently unstable and can transition to a pathogenetic state and accumulate at inflammation sites (40
), highlighting the fluid nature of regulatory populations and their potential to contribute to pathogenesis. If Bin cells do play a regulatory role in pathogenesis, however, a central function on the progression of inflammatory processes at the lymph node level seems more likely than local effects at the inflamed sites, because minimal if any lymphoid infiltrates are known to be present in the arthritic joints of these mice (20
The second key observation we have made here is that a close correlation exists between the exacerbation of knee disease in the hTNF-tg strain, and significant changes in the structure of the ipsilateral PLN, with a marked reduction in the node capacitance and a massive migration of B cells into the expanded lymphatic spaces in the node (“collapse” phase). In support of this correlation we have observed hTNF-tg knees of 1-year-old mice with expanding PLN >20mm3
(10x WT) that never developed inflammatory-erosive arthritis. However, this correlation is not absolute, as we have also observed some hTNF-tg knees (~20%) with expanding PLN and inflammatory-erosive arthritis. Thus, PLN collapse appears to be a prominent, but not necessary component for the initiation of inflammatory-erosive arthritis of the knee in mice. To better understand lymphatics in this model, we have reported that arthritis in mice is accompanied by an increase in lymphoangiogenesis and that lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis (43
). These results are consistent with expansion of the sinusoids in the draining lymph nodes and the higher PLNcap that we have shown by CE-MRI and histology, which is associated with the earlier stages of disease (23
, and ). Thus, we hypothesize that the reduction in LNcap and an open sinusoidal spaces caused by B cell migration would correlate with a reduction in the lymphatic flow capacity of the draining lymph nodes, with resulting reduction in the clearance of inflammatory cells and factors from the drained sites. In the case of knee arthritis in the hTNFtg mice, this would result in the “flare” in synovitis and bone erosion that is observed in the PLN collapse phase. Because human RA is well known to alternate between moderate stages of inflammation and acute flares, the origin of which is yet unexplained, this may also represent an intriguing candidate for a more general mechanism of disease behavior.
Two critical questions with regard to the collapse process are the nature of the signals that induce migration of B cells from the follicular sites to the sinusoidal spaces, and whether B cell migration is causal to the collapse, or simply associated with it. Based on immunohistological analysis, the migrating cells are preferentially of an IgM-high phenotype, suggesting they may be the same CD23+, CD21-high cells that are observed accumulating during the expansion stage. However, only adoptive transfer experiments of purified, identifiable cells of the various subsets can answer the question of direct lineage relationship. Regardless, the distinct phenotype of the migrating population renders it amenable to specific functional studies aimed at identifying the potential chemotactic signals responsible for their unusual localization.
Finally, we have shown that BCDT is effective in ameliorating disease in hTNFtg mice. This is a startling observation, because of the commonly accepted paradigm that arthritis in TNF-overexpressing mouse models does not require adaptive immunity. Several possibilities can reconcile these findings. First, it is likely that the levels of TNF overexpression in the Tg3647 strain used here are lower than those in the TNF ΔARE mice used by Kontoyannis and co-workers, making disease in Tg3647 mice more dependent on additional mechanisms. Certainly, TNF ΔARE mice display a far more aggressive disease phenotype than Tg3647 mice, and die by 3 months of age (18
). An additional and more interesting possibility is that we are looking at two different stages of disease, with potentially different proximal causal mechanisms. Both Tg3647 and TNF ΔARE first develop arthritis in the ankle, where disease commences with infiltration of the tendon sheaths by granulocytes and macrophages, and the formation of osteoclasts next to the inflamed tendon sheaths (20
). Then, the tenosynovitis rapidly progresses into pannus-like tissue largely void of lymphocytes, with osteoclasts mediating focal erosions. While this earlier stage is dominated by innate immunity components, we would like to suggest here that there exists a second stage, associated with knee “flare” and PLN collapse, which is B cell-dependent. If this is the case, the variability in clinical effectiveness of BCDT in RA patients may be in part due to the type/stage of disease primarily responsible for that patient's symptoms. Future pre-clinical and clinical studies prospectively designed to assess the cause-effect relationship of BCDT on lymphatic flow are warranted to test this hypothesis.