Patients with RA often accompany with enlarged draining lymph nodes [42
] and increased lymph flow rates [43
] in affected limbs, which are correlated with inflamed synovial volume. However, a cellular and molecular mechanism to explain these changes has yet to be postulated. In the present study, we demonstrated that CD11b+
OCPs from joints of TNF-Tg mice produce high levels of the lymphatic growth factor, VEGF-C, and that joints from mice in two models of RA have increased numbers of lymphatic vessels and enlargement of draining popliteal lymph nodes. Thus, lymphangiogenesis is also significantly increased in joints of mice with inflammatory arthritis. To date, most studies of inflammation-induced lymphangiogenesis have been performed in animal models in which the examined tissues have included cornea, lung, and skin [15
], because frozen sections from these tissues can be prepared readily for immunostaining to identify lymphatic vessels and because dye injection can be used to examine the lymphatic drainage. However, it is difficult to apply these methods to joints. Here, we used a combination of microarrays, FACS, immunohistochemistry, and a novel in vivo
contrast-enhanced-MRI technique to demonstrate that the lymphatic vasculature in inflamed joints and draining lymph nodes are significantly increased in mice with TNF and SIA. Our findings are consistent with those in clinical studies demonstrating increased VEGF-C expression in RA joints [45
] and increased size of lymph nodes [43
]. Furthermore, based on increased volume of collected lymph [42
] and our demonstration of increased VEGF-C production by joint OCPs, we propose that the development of large lymphatic vessels in the pannus results from proliferation of lymphatic endothelial cells and their distention by increased amounts of lymph.
While the origin of lymphatic endothelia cells remains an area of active research, several studies on inflamed corneas, skin, and lung have reported the presence of CD11b+
myeloid cells expressing VEGF-C in these tissues [15
]. These studies speculated that inflammatory cytokines stimulate VEGF-C production by these CD11b+
cells based on previous work in human lung fibroblasts and human umbilical vein endothelial cells [18
]. Here, we demonstrated that TNF and IL-1 upregulate VEGF-C expression in CD11b+
OCPs. To validate our microarray findings and provide insight into the mechanism of TNF-induced VEGF-C expression in OCPs, we provide preliminary evidence indicating that this response is mediated by NF-κB-dependent transcription. Since other signal transduction pathways could also be involved and TNF could be acting indirectly through prostaglandins [48
], which also mediate VEGF-C
transcription in cancer cells [49
], future studies are needed to elucidate the mechanisms of inflammation-induced VEGF-C expression in OCPs.
Considering the cellular heterogeneity of joint pannus, it is important to determine the primary source of VEGF-C in arthritic joints. While our studies focused on OCPs, others have shown that TNF and/or IL-1 stimulates VEGF-C expression by human lung fibroblasts, blood vascular endothelial cells, and synovial cells [19
]. We found that TNF also stimulates VEGF-C expression in NIH3T3 and C2C12 fibroblast cell lines (data not shown), suggesting that fibroblast-like cells in synovium could perhaps be another source of VEGF-C in arthritic pannus. However, these results were obtained from in vitro
treatment of cells with cytokines and may not precisely reflect the in vivo
situation, particularly in the joint local microenvironment. Our immunocytochemistry studies using cells freshly isolated from joints demonstrated that most of the VEGF-C-expressing cells are CD11b+
(Figure ). One potential concern with this approach is that primary joint cells are composed of a mixture of cell types. To address this limitation, we cultured these cells with M-CSF and used only adherent cells for further study. Under these culture conditions, more than 90% of adherent cells are CD11b+
OCPs (Q. Zhang, Y. Lu, S. Proulx, R. Guo, Z. Yao, E.M. Schwarz, B.F. Boyce, L. Xing, unpublished data). We found that these M-CSF-dependent joint cells express much higher levels of VEGF-C than cells treated in vitro
(20- to 30-fold increase in joint cells in Figure versus 3- to 5-fold increase in TNF-treated cells in Figure ). Thus, although other cell types may also be VEGF-C-producing cells, CD11b+
OCPs likely are one of the major sources of VEGF-C in joint pannus.
Our findings demonstrated that increased lymphangiogenesis is associated with the progression of joint inflammation, which occurs not only in dysregulated TNF-induced arthritis (Figure ) but also in mice with SIA (Figure ). A recent study reported that, in joint sections of collagen-induced arthritis, the number of LYVE-1+
lymphatic vessels is increased [50
]. Thus, elevated lymphangiogenesis likely is a common feature of inflammatory arthritis. Inflammation-induced lymphangiogenesis in joints appears to be a slow process, taking 2 to 3 weeks (Figure ). Our explanation is that OCPs or other VEGF-C-producing cells may need to migrate to the inflamed joints first and then respond to elevated cytokine levels to produce VEGF-C, which then stimulates formation of lymphatic endothelial cells.
Interestingly, increased lymphatic vasculature persists even after serum-induced inflammation has resolved (Figure ). This is consistent with our observation of no change in lymphatic vessel area or number in TNF-Tg mice with a significant reduction in their joint inflammation after anti-TNF therapy treatment (see Additional File 1
). Persistent lymphangiogenesis was also reported in a mouse model of chronic respiratory tract infection in which inflammation has been cured by antibiotics [17
]. Currently, there is no explanation why these lymphatic vessels do not disappear along with the reduction in inflammation. One speculation is that lymphatic enlargement makes affected tissues more susceptible to later inflammation by facilitating the accumulation of immune cells at the site of injury or infection [51
]. However, it is also possible that an increased lymphatic network will prime tissues to respond to acute inflammation.
A central question that arises from our study and other recent studies regarding inflammation-induced lymphangiogenesis is whether lymphangiogenesis in arthritis is beneficial or harmful to affected joints. Early clinical studies proposed that inflammation-driven lymphangiogenesis induces the expansion of the lymphatic network in an exacerbated manner such that the lymphatic vessels may be dysfunctional, as reported in psoriasis and Crohn disease [52
]. Recently, VEGF-C or VEGFR-3 antagonists have been used to directly stimulate or block VEGF-C/VEGFR-3-mediated lymphangiogenesis in several models of inflammation. In general, the effects of manipulating lymphangiogenesis in inflammatory conditions are not clear and appear to be largely dependent on the type of model used. For example, in the corneal transplantation model, lymphangiogenesis and angiogenesis are increased by grafting, and blockade of either form of new vessel formation has a similar beneficial effect and prevents graft rejection [55
]. In contrast, UVB-induced skin inflammation is exacerbated by VEGF-C antagonism [56
], suggesting that stimulation of the lymphatic system might reduce disease severity. Thus, the functional importance of lymphangiogenesis in the pathogenesis of RA remains to be determined.