Inhibition of lymphangiogenesis and lymphatic drainage via VEGFR-3 blockade increases the severity of inflammation in chronic inflammatory arthritis

doi:10.1002/art.24764

Arthritis Rheum. Author manuscript; available in PMC 2010 September 1.

Published in final edited form as:

Arthritis Rheum. 2009 September; 60(9): 2666–2676.

doi: 10.1002/art.24764

PMCID: PMC2810533

NIHMSID: NIHMS167129

Inhibition of lymphangiogenesis and lymphatic drainage via VEGFR-3 blockade increases the severity of inflammation in chronic inflammatory arthritis

Ruolin Guo, Ph.D.,¹^* Quan Zhou, M.D.,¹^* Steven T. Proulx, Ph.D.,² Ronald Wood, Ph.D.,³ Rui-Cheng Ji, Ph.D.,⁴ Christopher T. Ritchlin, M.D.,² Bronislaw Pytowski, Ph.D.,⁵ Zhenping Zhu, Ph.D., M.D.,⁵ Yong-Jun Wang, Ph.D., M.D,⁶ Edward M. Schwarz, Ph.D.,² and Lianping Xing, Ph.D.^1,^2,⁷

¹ Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA

² Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY 14642, USA

³ Department of Urology, University of Rochester Medical Center, Rochester, NY 14642, USA

⁴ Department of Anatomy, Biology and Medicine, Oita University Faculty of Medicine, Oita 879-5593, Japan

⁵ Imclone Systems, New York City, NY 10014, USA

⁶ Shanghai University of Chinese Medicine, Shanghai, China

⁷ Address correspondence to: Lianping Xing, Department of Pathology and Laboratory Medicine, 601 Elmwood Ave, Box 626, Rochester, NY 14642, Tel: 585-273-4090; Fax: 585-756-4468; Email: Lianping_xing/at/urmc.rochester.edu

^*These authors contribute equally to this work.

The publisher's final edited version of this article is available free at Arthritis Rheum

See other articles in PMC that cite the published article.

Abstract

Object

Investigation of the effect of lymphatic inhibition on joint and draining lymph node pathology during the course of arthritis progression in mice.

Method

TNF transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Mice received contrast enhanced MRI to obtain ankle and knee joint synovial volumes and draining popliteal lymph node (PLN) volumes before and 8 weeks after treatment with VEGFR-3 or VEGFR-2 neutralizing antibodies, or isotype IgG. The animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining PLNs prior to sacrifice. Lymphatic vessel formation and morphology of joints and PLNs were examined by histology, immunohistochemistry, and RT-PCR.

Results

Compared to IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of PLNs, the number of lymphatic vessels in joints and PLNs, the lymphatic drainage from paws to PLNs, and the number of VEGF-C expressing CD11b+ myeloid cells in PLNs. However, it increased the synovial volumes and inflammatory area in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation.

Conclusion

Lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation.

Keywords: Lymphatic drainage, lymphangiogenesis, inflammation, lymph nodes, in vivo imagining

Lymphatic vessels are present in almost all tissues of the body. They are composed of an extensive network of thin-walled capillaries that drain protein-rich lymph from extracellular spaces (1). Under normal conditions, the major functions of the lymphatic system include maintenance of tissue fluid homeostasis, absorption of fatty acids, and mediation of the afferent immune response (2, 3). Recent studies show increasing evidence that the lymphatic system also plays key roles in disease processes such as cancer metastasis, lymphedema, obesity, and inflammation (4, 5).

Lymphatic endothelial cell proliferation and lymphatic hyperplasia are reported in psoriatic skin lesions in humans and in chronic skin inflammation in mice (6). Kidney transplant rejection is frequently accompanied by enhanced lymphangiogenesis and production of lymphatic endothelial cell-derived chemokines in grafted tissues (7). Synovial specimens from patients with rheumatoid arthritis (RA) and osteoarthritis have an increased number of lymphatic vessels and increased expression of the lymphatic growth factor, vascular endothelial growth factor-C (VEGF-C) (8, 9). Furthermore, clinical reports have described larger lymph nodes (10) and increased lymphatic flow rates in lymphatic vessels draining arthritic joints in RA patients (11). Similarly, recent studies in animal models of RA demonstrated increased lymphatic vessel formation in inflamed joints and in draining popliteal lymph nodes (PLN) (12–14). These clinical and preclinical studies have demonstrated that inflammation stimulates proximal lymphangiogenesis in the joints and distal lymphangiogenesis in the draining lymph nodes. Thus, inflammation is the primary cause of lymphangiogenesis in arthritis. However, the effects of inflammation-induced lymphangiogenesis on joint inflammation in RA have yet to be determined.

VEGF-A- and VEGF-C-mediated signaling pathways are centrally involved in inflammatory lymphangiogenesis. VEGF-A signals through VEGF receptors (VEGFR)-1 and VEGFR-2. The effect of VEGF-A on lymphatics is mediated by VEGFR-2. Blockade of VEGF-A/VEGFR-2 interaction, using VEGFR-2 neutralizing antibody, reduces adjuvant-induced (15) and delayed-type hypersensitivity reaction-induced (16) lymphangiogenesis in lymph nodes. VEGF-C signals primarily through VEGFR-3 on lymphatic endothelial cells. VEGFR-3 blockade specifically inhibits the effects of VEGF-C, but not VEGF-A, on lymphatics. VEGFR-3 neutralizing antibody reduces bacterial infection associated-airway inflammation (17) and surgical blockade-induced lymphangiogenesis (18, 19). These studies have established a direct role for VEGF-C:VEGFR3 signaling in inflammation-induced lymphangiogenesis. However, most published studies have used acute inflammation models in which inflammation is triggered within hours to days. The effects of lymphangiogenesis on the natural course of chronic inflammation, such as that occurring in RA, have not been addressed. Specifically, it is not known how newly-generated lymphatic vessels affect drainage from inflamed joints, or if reduced lymphatics exacerbates inflammation. These questions are even more important in chronic inflammation where monocytes/macrophages are the major infiltrating cell type, given the fact that macrophages are the main source of VEGF-C in response to pro-inflammatory cytokines TNF and IL-1 (12, 20, 21)

In the current study, we used TNF transgenic (Tg) mice as a model of chronic inflammatory arthritis (22), and examined the effect of lymphatic inhibition by VEGFR-2 and VEGFR-3 neutralizing antibodies on lymphatic drainage and the severity of joint inflammation. Contrast enhancement (CE) MRI (13, 23) and indocyanine green-near infrared (ICG-NIR) in vivo imaging technologies were used to monitor the changes of synovial and PLN volumes during the 8-week treatment period. We found that VEGFR-3 neutralizing antibody significantly decreased joint and PLN lymphangiogenesis, lymphatic drainage from inflamed paws to PLNs, and the number of VEGF-C expressing CD11b+ myeloid cells in the PLNs. However, it significantly exacerbated joint inflammation. In contrast, VEGFR-2 neutralization inhibited both joint inflammation and lymphangiogenesis. These data indicate that inflammation-induced lymphangiogenesis is an important compensatory mechanism to limit joint inflammation during the course of chronic arthritis, and that improving lymphatic drainage represents a new potential therapeutic strategy for chronic inflammatory disorders.

MATERIAL AND METHODS

Animal experiments

TNF-Tg mice (Tg3647) were originally obtained from Dr. G. Kollias, and were bred with C57BL/6 mice for 8 generations. TNF-Tg mice (2.5-months-old) were treated with anti-mouse VEGFR-2 (DC101), anti-mouse VEGFR-3 (mF4-31C1) neutralizing antibody (ImClone, New York, NY) or rat IgG (Innovative, Southfield, MI) at a dose of 0.8 mg/mouse, twice a week, by intra-peritoneal injection, for 8 weeks. DC101 is a rat monoclonal antibody which specifically blocks the binding of VEGF-A to VEGFR-2 and inhibits tumor growth in mice through an anti-angiogenic mechanism (24). mF4-31C1 is a rat monoclonal antibody which specifically antagonizes the binding of VEGF-C to VEGFR-3 and potently blocks both VEGF-C-enhanced physiological and tumor-induced lymphangiogenesis in a murine tail skin lymphatic generation model (25). The rationale for choosing 2.5 month-old TNF-Tg mice for 8-week treatment is based on our previous experience using antibody therapy in this model. We have shown that 8-week anti-RANK or anti-TNF treatment of 2.5-month-old TNF-Tg mice significantly reduces joint lesions (22, 26).

FITC-dextran footpad injection and PLN confocal microscopy

TNF-Tg mice (4–6-months-old) and wild type (WT) littermates were injected with 10 μl (10 mg/ml) of FITC-dextran (molecular weight, 2,000Kd, Sigma) into the footpad intra-dermally. This size of dextran is too large to enter the blood stream and is routinely used in lymphatic studies (27, 28). The entire PLNs were scanned one hour later under a Confocal microscope. A total of 100 slices about 600~800 μm deep were taken for each node, and 4 nodes from TNF-Tg mice or WT littermates were examined.

CE-MRI

CE-MRI was performed before and after the antibody treatment as previously described (13, 23). Mice were positioned with a hind leg in a newly-developed custom-designed murine dual RF receiver coil (one coil enclosing the ankle joint and another for the knee joint and PLN). Mice were scanned in a Siemens 3 Tesla clinical magnet (Siemens AG, Munich, Germany) as we previously described (13, 23). Analysis was performed with Amira 3.1 (Mercury Computer Systems, Chelmsford, MA). An image registration and subtraction algorithm was used on the pre- and post-contrast images in Amira in order to generate an image of the voxels of contrast enhancement. From this image, a three-dimensional region of interest of muscle tissue was used as a measure of delivered contrast agent, and a threshold of enhancing synovial tissue was generated from this value. Lymph nodes were traced manually and thresholded to define the margin between the node and surrounding fat. Tissue volumes were reconstructed using a surface generation module in Amira.

ICG-NIR lymphatic imaging

To examine the status of lymphatic draining function in the legs of TNF-Tg mice, we established a protocol using ICG-NIR imaging technology according to published literature (29, 30) and our past experience with large animals and human subjects (detailed methods will be published separately). In brief, on the first day, ICG solution (1 μg in 10 μl) was injected into the footpads intra-dermally. Five minutes later, the dynamics of ICG fluorescence over the entire leg region, including PLNs and paws, was visualized under an infrared laser and recorded. The NIR imaging was repeated at 24 hours post ICG injection (suppl fig. 1). These images were read into the Image J software. Regions of interest (ROI) defining the PLNs and injection site were identified, yielding 4 outcome measures of lymphatic function: 1) T-initial (Ti), which is the time that it takes for the ICG to be detected in the draining PLN; 2) S-max, which is the maximum ICG signal intensity observed in the PLN during the first day imaging session; 3) T-max, which is the time it takes for a PLN to achieve maximal ICG signal intensity; and 4) % Clearance, which is an assessment of ICG wash out through the lymphatics and is quantified as the percent difference of ICG signal intensity between the two NIR scans from the ROI of the PLNs or footpad at 1) S-max (first day) and 2) 24 hours post ICG injection.

To observe the ICG distribution within the lymphatic network of PLNs, ICG was injected intra-dermally into footpads, and PLNs were harvested 1 hour later. Frozen sections (10 μm) were observed under a microscope with an infrared filter. After taking pictures to record the distribution of ICG fluorescence, the same section was stained with rabbit anti-Lymphatic Vessel Endothelial Receptor 1 (LYVE-1) antibody (Abcam Inc. Cambridge. MA) followed by Alexa Fluor 488 F(ab′)₂ fragment goat anti-rabbit IgG (H+L), as we described previously (12).

Immunostaining and histomorphometric analysis

PLN, ankle, and knee specimens were fixed in formalin and embedded in paraffin. For immuno-fluorescence staining and data analysis, sections were stained with a mixture of PE-anti-CD11b or PE-anti-CD11c and anti-VEGF-C antibody followed by Alexa Fluor 488 goat anti-rabbit IgG and by To-Pro-3 iodide, as we described previously (12). Three pictures were taken from each section (x40) from different fields. The analysis was performed by counting the To-Pro-3 iodide+ cells as the total cell number in each field, and the numbers of CD11b+ and VEGF-C+ cells in the same field. The data were presented as the % of CD11b+ or VEGF-C+ cells over the total cell number. For immunohistochemistry staining, adjacent serial sections were stained with anti-LYVE-1 or anti-CD31 antibodies. Lymphatic vessels were quantified by a point-counting method, as we described previously (31). For each mouse, 2 sections were cut at 250 μm apart and the area and size of LYVE-1+ lymphatic vessels or CD31+ blood vessels were measured within the synovial tissue and expressed per square millimeter of synovium, as we previously described (12). The % of inflammatory area was measured from H&E-stained joint sections. The data were presented as the mean from 3 levels of each ankle joint.

Quantitative real time RT-PCR

RNA was extracted using TRIzol Reagent and cDNA synthesis was performed using GeneAmp RNA PCR core kit. Quantitative PCR amplification was performed with gene-specific primers (suppl table) using an iCycler real-time PCR machine and iQ SYBR Green supermix. The relative standard curve method was used to calculate the amplification difference for each primer set, as we described previously (32). The quantity of the target gene mRNA was then obtained by division of each value by the actin value. Standards and samples were run in triplicate.

Statistics

Data are presented as means ± standard deviation, and all experiments were performed at least twice with similar results. Statistical analyses were performed with Stat view statistical software. Differences between two groups were compared using unpaired Student t-test and more than two groups were compared using one-way ANOVA, followed by a Bonferroni/Dunnet test. p values less than 0.05 were considered to be statistically significant.

RESULTS

Increased lymphatic drainage in draining lymph nodes of TNF-Tg mice

We reported previously that 4–6-month-old TNF-Tg mice have increased lymphangiogenesis in their PLNs, as evidenced by numerous dilated LYVE-1+ vessels (13). These large lymphatic vessels are associated with severe ankle arthritis and variable knee synovitis in all mice at this age. To determine if these dilated LYVE-1+ sinuses in the PLNs are functional and capable of draining afferent lymph, we intra-dermally injected FITC-dextran as a lymphatic tracer into the footpads of 4–6-month-old TNF-Tg mice and wild type (WT) littermate controls. The distribution of FITC fluorescence within the PLNs was observed by scanning confocal microscope. In WT mice, a thin layer of the FITC fluorescence was visualized primarily in marginal sinuses, which receives lymph from the afferent lymphatic vessels (Fig. 1a). The majority of the FITC fluorescence was concentrated at the medullary region, from which lymph exits through efferent lymphatic vessels (33). In contrast, the FITC signals were broadly distributed throughout the TNF-Tg PLNs (Fig. 1b). TNF-Tg PLNs were markedly expanded and displayed disorganized T and B cell zones compared to WT (Fig. 1c & d). The distribution pattern of actively draining lymph was corroborated by ICG-NIR imaging, as intra-dermal injection of ICG, another lymphatic tracer, into the footpad generated the same results as FITC-dextran (Fig. 1e & f). To confirm that the ICG was within lymphatic vessels we performed LYVE-1 immunostaining in adjacent sections (Fig. 1g & h), which demonstrated that ICG was contained within numerous dilated lymphatic sinuses. These findings indicate that chronic inflammation in the lower extremities of TNF-Tg mice leads to significantly increased lymphangiogenesis and functional dilation of lymphatic sinuses in the PLNs, which actively drain fluid from the foot.

Figure 1

Increased lymphangiogenesis and lymphatic flow in draining popliteal lymph nodes of TNF-Tg mice. Popliteal lymph nodes (PLNs) from 4–6 month-old TNF-Tg mice and WT littermates (N=4) were used. All photos were taken at x4. FITC-dextran was injected (more ...)

Blockade of VEGFR-3 signal pathway prevents increased lymph node enlargement and lymphangiogenesis but increases the severity of joint synovitis in TNF-Tg mice

Inflammation-induced lymphangiogenesis in various animal models has been demonstrated to be mediated by inflammatory cytokine induced VEGF-A and/or VEGF-C signaling pathways (15–17, 34). To investigate the involvement of these VEGF members in our model, we performed quantitative RT-PCR on mRNA isolated from WT and TNF-Tg PLNs and examined the expression levels of VEGF-A, B, C, and D mRNA (Fig. 2). Consistent with other models, we found that VEGF-A and VEGF-C levels were significantly increased in TNF-Tg PLNs compared to WT nodes, while VEGF-B and VEGF-D levels were unchanged.

Figure 2

Increased VEGF-A and VEGF-C expression in lymph nodes of TNF-Tg mice. Total RNA was extracted from pooled PLNs (n= 3–4) obtained from 4–6-month-old TNF-Tg mice or WT littermates, and the expression levels of VEGF family members were determined (more ...)

In order to further elucidate the roles of VEGF-A and VEGF-C signaling in TNF-induced inflammation and lymphangiogenesis during the development and progression of chronic arthritis in our model, we chose to perform VEGFR blockade experiments with neutralizing antibodies. Our hypothesis for these experiments was that blockade of VEGFR-2 would inhibit both inflammation and lymphangiogenesis by reducing angiogenesis as has been show for VEGF-A antagonism (15), while interruption of VEGF-C/VEGFR-3 signaling specifically blocks lymphangiogenesis, but not angiogenesis or inflammation (25). To test this hypothesis we treated 2.5 month-old TNF-Tg mice with anti-VEGFR-2, VEGFR-3 or irrelevant IgG control antibodies for 8 weeks, and assessed the treatment effects on PLN volume and lymphangiogenesis via CE-MRI obtained before and after therapy. Compared to the baseline values, the PLN volume in placebo-treated mice increased by more than 100%, while it was unchanged or slightly decreased in VEGFR-2 and VEGFR-3 antibody-treated mice (Fig. 3A & B). Accordingly, the PLNs in anti-VEGFR treated mice weighed significantly less than those of IgG-treated animals (Fig. 3C). LYVE-1 immunostaining and histomorphormetric analysis of the PLN sections revealed that VEGFR-2 and 3 antibodies significantly reduced the size, number, and area of LYVE-1+ lymphatic capillaries compared to placebo controls (Fig. 3D, suppl fig. 2).

Figure 3

VEGFR-2 and VEGFR-3 neutralizing antibodies prevent TNF-induced PLN enlargement and lymphatic vessel formation. TNF-Tg mice (2.5-month-old, N=5/group) received CE-MRI of ankle and knee joints to obtain baseline value of synovial and PLN volume. Animals (more ...)

Having demonstrated the effects of VEGFR blockade on lymphangiogenesis in PLNs, we next examined its effects on joint inflammation to establish the direct relationship between lymphatic vessel formation and synovitis. 3D analyses of the CE-MRI data revealed dramatic differences in changes in ankle synovitis among all three treatment groups (Fig. 4A, suppl fig. 3). The synovial volumes of the placebo group demonstrated an insignificant change, those of the anti-VEGFR-3 group were significantly increased, and those of the anti-VEGFR-2 group were significantly decreased. The mean percent changes also reflected this, with a 45% increase in the anti-VEGFR-3 group and a 35% decrease in the anti-VEGFR-2 group. Volumetric assessment of knee synovitis also demonstrated that anti-VEGFR-3 treatment exacerbated inflammatory arthritis (Fig. 4A). These findings were corroborated by histomorphometric assessment of the inflammatory tissue in the ankle joints of H&E stained histology sections, which also demonstrated a significant increase and decrease in ankle inflammation in the VEGFR-3 vs. VEGFR-2 groups, respectively (Fig. 4B & C).

Figure 4

Differential effects of VEGFR-2 and VEGFR-3 blockade on ankle synovial volume in TNF-Tg mice. (A) The ankle (upper panel) and knee (lower panel) synovial volumes of the mice described in Figure 3 before and after antibody treatment are shown as the mean (more ...)

As these results support our hypothesis regarding the differential effects of VEGFR-2 vs. VEGFR-3 blockade during chronic arthritis, we next investigated the changes in the vasculature within joints via immunohistochemistry. Consistent with our PLN data, both VEGFR-2 and VEGFR-3 antibody-treated mice had fewer LYVE-1+ lymphatic vessels compared to placebo treated controls (Fig. 4D). In contrast, only the anti-VEGFR-2 treatment reduced the number of CD31+ blood vessels (Fig. 4E), indicating that this treatment inhibits angiogenesis, an essential factor for inflammation. Collectively, our interpretation of these findings is that VEGFR-2-induced lymphangiogenesis is indirect through its stimulatory effects on angiogenesis and its recruitment of VEGF-C-producing inflammatory cells to the joint. This triggers VEGFR-3 signals to stimulate lymphangiogenesis in the synovium and draining lymph nodes.

Exacerbation of inflammatory arthritis by VEGFR-3 blockade is associated with decreased afferent lymph flow to draining nodes

In order to test the hypothesis that VEGFR-3 signals are responsible for increased lymphatic drainage of inflamed joints during arthritis progression we performed ICG-NIR lymphatic imaging on the legs of TNF-Tg mice treated with anti-VEGFR-3 or placebo. Figure 5 demonstrates the dramatic differences of local lymphatic flow between placebo and anti-VEGFR-3 treatment after 8-weeks of therapy. At one hour after ICG injection, placebo PLNs displayed a bright fluorescent signal at S-max (Fig. 5A-a) compared to anti-VEGFR-3 treated PLNs (Fig. 5A-c). This indicates that less ICG was traveling to the PLNS through lymphatic vessels as a result of VEGFR-3 blockade reduced lymphangiogenesis. At 24 hours after ICG injection, placebo PLNs (Fig. 5A-b) and paws (Fig. 5A-d) had very little ICG fluorescence, indicating more ICG had been cleared away from these sites through the lymphatics. In contrast, at the same time point, the fluorescent signal in anti-VEGFR-3 treated paws remained strong, an indicator of slower ICG clearance. Quantitative analysis of the ICG-NIR images confirmed the significant increases in T-initial and T-max, and significant decreases in S-max and ICG clearance in anti-VEGFR-3 vs. placebo treated mice (Fig. 5B). Thus, these results demonstrate for the first time that VEGFR-3 induced lymphangiogenesis protects joints from accelerated arthritis by increasing the afferent flow of lymph from the inflamed tissues.

Figure 5

VEGFR-3 blockade inhibits lymph flow from the foot to draining popliteal lymph nodes. TNF-Tg mice (2.5-month-old, N=4) were treated with VEGFR-3 neutralizing antibody or IgG as described in Figure 3, and received an ICG intra-dermal injection in their (more ...)

VEGFR-3 blockade decreases VEGF-C expressing CD11b+ cells in draining lymph nodes of TNF-Tg mice

Since it is known that CD11b+ macrophages and dendritic cells are the primary infiltrating cells that produce VEGF-C in inflammatory tissues (17, 35), we investigated this cell population in PLNs of TNF-Tg mice with inflammatory arthritis vs. WT controls using two color immunofluorescence microscopy. The results demonstrated that the number of CD11b+ and VEGF-C+ cells was significantly increased in TNF-Tg PLNs vs. WT, and many of these cells were CD11b/VEGF-C double positive (Fig. 6A&B). The expression of TNF and IL-1 mRNA was increased in TNF-Tg PLNs (Fig. 6C). Interestingly, this CD11b+ population was markedly reduced in PLNs of anti-VEGFR-3 treated TNF-Tg mice as evidenced by a significant decrease in immunoreactive cells and CD11b transcript levels (Fig. 6D). These data indicate that increased expression of inflammatory cytokines and the presence of myeloid cells in draining lymph nodes may contribute to lymph node lymphangiogenesis in TNF-Tg mice through a VEGF-C/VEGFR-3 mediated event.

Figure 6

VEGFR-3 blockade decreases VEGF-C-expressing CD11b+ cells in lymph nodes of TNF-Tg mice. (A) PLNs from TNF-Tg mice (4–6-month-old) and WT littermates were double immuno-stained with PE-anti-CD11b and FITC-anti-VEGF-C antibodies. Representative (more ...)

DISCUSSION

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, 37), 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, 12, 17) and clinical studies (8, 9, 14) 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, 40). 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, 42) 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, 23), 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 (Fig. 4). 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, 47). 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.

Supplementary Material

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Acknowledgments

The authors would like to thank Edmund Kwok and Zhigang You for developing the dual coil; and Ms. Yan Lu, Xiaoyun Zhang and Yanyun Li for technical assistance with the histology. This work was supported by research grants from the National Institutes of Health PHS awards (AR48697 and AR53586 to LX, DE17096 and AR54041 to ES, DK075036 to RW). Part of Dr. Zhou, Quan’s salary was supported by a grant from the National Science Fund for Distinguished Young Scholars of China (30625043 to YJW).

Footnotes

AUTHOR CONTRIBUTIONS

Dr. Lianping Xing has full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Drs. Guo, Zhou, Proulx, Wood, Schwarz, and Xing.

Acquisition of data. Drs. Guo, Zhou, Proulx, Wood, and Xing.

Analysis and interpretation of data. Drs. Guo, Zhou, Proulx, Wood, Ji, Ritchlin, Wang, Pytowski, Zhu, Schwarz, Xing.

Manuscript preparation. Drs. Guo, Zhou, Proulx, Wood, Ji, Ritchlin, Pytowski, Zhu, Wang, Schwarz, and Xing.

Statistical analysis. Drs. Guo, Zhou, and Proulx.

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