Efficacy of B cell depletion therapy for murine joint arthritis flare is associated with increased lymphatic flow

Rheumatoid arthritis (RA) is a prevalent debilitating inflammatory joint disease characterized by exacerbations and remissions, whose etiology remains poorly understood (1). Reports that the inflammatory-erosive arthritis phenotype of tumor necrosis factor-transgenic (TNF-Tg) mice closely mimics human RA (2), and the remarkable clinical success of anti-TNF therapy for this disease (3-6), underscore a critical role for this pleiotropic cytokine in disease pathogenesis. More recently, the success of anti-CD20 (rituximab) B cell depletion therapy (BCDT) in RA suggests that B cells can also modulate synovial inflammation and matrix destruction (7-11). Interestingly, although a major rationale for BCDT is elimination of autoantibodies that are diagnostic for RA progression (i.e. rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA)) (12, 13), the efficacy of anti-CD20 in patients is not always associated with significant changes in autoantibody or total immunoglobulin levels (8, 14), and seronegative patients are responsive to anti-CD20 treatment (11, 15). Collectively, these findings support a non-antibody-mediated contribution of B cells to RA pathogenesis. However, the mechanism by which B cells trigger arthritic “flare,” as defined by the sudden exacerbation of synovitis and focal erosion in select joints during chronic-systemic inflammatory disease; and how BCDT ameliorates this condition, primarily by the indirect removal of macrophages from the inflamed synovium (16), remain enigmatic. Our approach to understand arthritic flare in TNF-Tg mice (3647 line in C57BL/6 background) has focused on longitudinal imaging studies using contrast enhanced (CE) MRI, near-infrared indocyanine green (NIR-ICG)-imaging and in vivo micro-CT, in order to visualize and quantify changes in soft tissues, lymphatic flow, and bone during inflammatory-erosive arthritis (17-23). These studies revealed that development of ankle and knee arthritis in TNF-Tg mice proceeds by two distinct mechanisms. Ankle arthritis begins as a symmetric tenosynovitis in the hindpaws characterized by macrophage and granulocyte infiltration of the tendon sheaths (24). This tenosynovitis rapidly progresses to an erosive pannus-like tissue that invades the mineralized cartilage and bone of the adjacent small bones in the foot. Interestingly, the pannus tissue is primarily comprised of macrophages and granulocytes (>90%), and very few lymphocytes (<1%) (24, 25). Conversely, the hematopoietic rich marrow that develops subchondral to the erosive pannus tissue contains large numbers of B cells (24, 25). This TNF-induced mechanism of arthritis in ankle joints is largely B cell-independent, as it has been shown that lymphocyte deficient TNF-Tg mice (TNFδARE × RAG1−/−) displayed full clinical and histopathological signs of chronic destructive inflammatory arthritis in their ankle joints (26). We have confirmed this finding with TNF-Tg × μMT−/− and TNF-Tg × RAG1−/− mice, and have found that anti-CD20 therapy does not ameliorate inflammatory-erosive arthritis in the ankles of TNF-Tg mice (Figure 1J, Supplemental Figure 1). Figure 1 LNCE is enhanced during effective anti-CD20 therapy for flaring knee synovitis. Untreated TNF-Tg mice with expanding (Exp) and collapsed (Col) (n=12 knees in each group) were identified by longitudinal CE-MRI (36). A separate Col group was treated with ... In contrast to arthritic progression in the hindpaws, arthritic flare in the knee of TNF-Tg mice is often asymmetric, and presents 2 to 4 months after the development of frank ankle arthritis without any detectable changes in immune activation (21, 23). Moreover, longitudinal imaging studies demonstrate a predictable temporal behavior in the draining lymph nodes (LN), which precede the onset of knee flare. Prior to detectable synvovial hyperplasia, the volume of popliteal LN (PLN) expands due to increased lymphangiogenesis, lymphatic fluid accumulation, CD11b+ macrophage infiltration, and the expansion of a unique subset of CD23+/CD21hi B cells in inflamed nodes (B-in) that was defined by a detailed phenotypic analysis (17-22). This expansion is followed by a sudden collapse with B-in translocation from the follicles into the LYVE-1+ lymphatic vessels of the paracortical sinusoids, and a significant decrease in lymphatic drainage as quantified by lymph node capacity (LNcap) and ICG clearance from footpad to draining LNs (17, 21, 23). Significantly, LN collapse occurs in series along the ipsilateral axis, such that the PLN lymphatics are also a biomarker of drainage from the knee to the iliac lymph node (ILN) (23). Taken together with the finding that anti-CD20 treatment of TNF-Tg mice with expanding PLN significantly protects against knee flare (21), we hypothesize that one mechanism of action of BCDT in this setting is B-in removal and “unclogging” of the lymphatic vessels within the LNs to restore inflammatory cell eggress from arthritic joints.