Increased lymphatic vessel formation has been reported in several animal models of inflammation (34
). However, it remains unclear if increased lymphangiogenesis only reflects the consequences of inflammation or if it plays an active role in the development and progression of inflammation. In the current study, we used TNF-Tg mice as a model of chronic inflammatory arthritis and demonstrated that blockade of VEGF-C/VEGFR-3 signaling by VEGFR-3 neutralizing antibody reduces lymphangiogenesis and lymphatic drainage and increases the severity of joint synovitis. In contrast, VEGFR-2 blockade reduces both lymphangiogenesis and synovitis. These findings indicate that lymphangiogenesis and lymphatic drainage actively contribute to the progression of inflammatory reactions in chronic inflammation. Improvement and maintenance of sufficient lymphatic drainage may represent a new therapeutic strategy for chronic inflammatory disorders.
Clinical studies in patients with RA have demonstrated high levels of pro-inflammatory cytokines in lymph draining arthritic joints and the enlargement of local draining lymph nodes in the ipsilateral limb (36
), indicating that local lymphatic circulation from inflamed joints to draining lymph nodes may affect the progression of inflammatory processes. It is predictable that lymph from arthritic joints and surrounding tissues carries large amounts of cytokines and immune cells. When this inflammatory lymph reaches lymph nodes, it could stimulate lymphangiogenesis. Thus inhibition of inflammation could reduce lymphangiogenesis. However, it is not clear whether or not lymphangiogenesis affects the natural progression of inflammation. This is an important point, because accumulated evidence from animal models (4
) and clinical studies (8
) has demonstrated increased lymphangiogenesis in inflammatory tissues.
Both VEGF-A and VEGF-C signaling pathways have been implicated in inflammatory lymphangiogenesis (38
). VEGFR-2 transduces signals in blood and lymphatic endothelial cells, while VEGFR-3 mediates signaling only in lymphatic endothelial cells. Thus, VEGFR-3 blockade affects lymphatics specifically (25
). We found that VEGFR-3 neutralizing antibody treatment of TNF-Tg mice reduces the number and area of joint and PLN lymphatic vessels, but it significantly increases the severity of joint inflammation. Decreased lymphangiogenesis is accompanied by reduced or slower transport of the lymphatic tracer, ICG, from paws to PLNs and reduced clearance of ICG from paws and PLNs. These findings strongly indicate that adequate lymph drainage from inflamed paws to local draining lymph nodes plays a beneficial role in the inflammatory process. Our findings are consistent with a recent report in which the same VEGFR-3 neutralizing antibody reduced the size of draining lymph nodes and increased lung weight, an outcome measure for lung inflammation, in a bacterial-induced airway inflammation model (17
). Thus sufficient lymphatic trafficking between the primary inflammation sites and local draining lymph nodes may play an important role in limiting the progression of inflammation.
We found that in contrast to VEGFR-3 blockage, VEGFR-2 neutralizing antibody significantly reduces both joint inflammation and lymphangiogenesis, indicating that VEGFR-2 and VEGFR-3 signal pathways have different mechanisms for reducing inflammatory lymphangiogenesis. Our explanation is that VEGFR-2 blockade-induced reduction in lymphatics is likely indirect through its inhibitory effect on angiogenesis. VEGF-A or VEGFR-1 inhibition reduces joint damage in various models of arthritis (39
). However, a previous study reported no effects of VEGFR-2 neutralization on arthritis in K/BxN arthritic mice model (39
). The discrepancy between our results and the previous report may reflect differences in the animal models used. K/BxN mice develop severe and aggressive joint lesions around 4 weeks of age (41
) and the disease is triggered following recognition of a NOD-derived MHC class II molecule by the transgenic TCR (43
). The Tg3647 line used in our study develop arthritis at a much slower pace (22
), and TNF is the pathogenic mediator. Thus, VEGFR-2 inhibition may be more effective in chronic inflammatory arthritis.
In this study, we started antibody treatment in 2.5-month-old TNF-Tg mice because our previous studies demonstrated that knee arthritis starts around this time and becomes more severe thereafter (22
), and we wanted to examine the effect of lymphatic blockade on the progression of joint lesions. Hayer et al have extensively characterized the initiation of ankle arthritis in this model, as well as its cellular make up (44
). However, the timing of the peak synovitis is unknown. Using a newly developed murine dual RF receiver coil that allows us to image knee and ankle joints in a single MRI scan, our preliminary results indicate that ankle synovitis starts at 1.5 months of age and peaks at 2.5 months of age (synovial volume mm3
: 12.5+3 in 1.5-month, 21+4.4 in 2.5 month and 21+3.4 in 4.5 month). In contrast, the synovial volume increases about 35% in the knee joints between 2.5 and 4.5 month of age (). These data suggest that the antibody treatment in this study likely started at the peak of the ankle synovitis and at the progression phase of the knee synovitis. Thus sufficient lymphatic function may not only slow the disease progression but also treat the disease. The effect of increased lymphangiogenesis on the joint lesions is currently under investigation”.
CD11b+ myeloid cells are the major source of VEGF-C in primary inflammatory sites and are responsible for promoting new lymphatic vessel formation. We found that in PLNs of TNF-Tg mice the majority of CD11b+ cells express VEGF-C, suggesting that VEGF-C in the lymph nodes is also produced by CD11b+ myeloid cells. Currently, the original of these CD11b+ cells is not clear. They may migrate from the blood circulation or through afferent lymphatics from joints or both. VEGFR3 antibody-treated mice have decreased CD11b+ cells in the PLNs. We believe that reduced CD11b+ cells in these mice is due to reduced CD11b+ cell drainage from the ankle and knee joints to the lymph nodes through lymphatic vessels because the antibody does not directly target these cells.
The mechanisms that regulate the recruitment and retention of CD11b+ cells in the PLNs are not known. The CCL19-CCL21/CCR7 axis is the major chemokine system expressing in the inflammatory lymphoid tissues and lymph nodes (45
). CCL19/CCL21 is produced by non-hematopoietic stromal and fibroblast-like cells and attracts CCR7 expressing dendritic cells to the lymphoid tissues and lymph nodes (46
). CD11b+ cells are composed of dendritic cells and their precursors, thus whether or not the CCL19-CCL21/CCR7 axis is responsible for increased CD11b+ cells in the TNF-Tg PLNs needs to be studied in future. Furthermore, the chemokine, CXCR12, maintains the CD11b+ hematopoietic cells in peripheral organs after they have been recruited from bone marrow by VEGF-A (48
) and bone marrow derived CD11b+ cells migrate to the CXCL12 gradients (49
). In our preliminary study, we found that CXCR12 mRNA levels are increased in TNF-Tg mouse PLNs compared to WT PLNS (data not shown), suggesting that the CXCL12/CXCR4 axis may also be involved in retaining CD11b+ cells in TNF-Tg PLNs.
Based on our findings, we have proposed a model to explain the importance of the lymphatic system in the development and progression of inflammatory arthritis (supplemental figure 4
). Joint inflammation recruits circulating CD11b+ myeloid cells from circulation. These cells produce lymphatic growth factors, such as VEGF-C, to stimulate lymphatic vessel formation. The functional lymphatic vessels transport inflammatory lymph carrying inflammatory cells, catabolic factors and cytokines to the draining lymph nodes and promote lymphangiogenesis, leading to an expansion of the lymph nodes and dilation of lymphatic sinuses containing inflammatory cells. Thus, sufficient lymphatic drainage could limit the degree of joint inflammation and the manipulation of the lymphatic system may represent a novel therapy for inflammatory disorders.