Does lymphatic growth rely on immune cell function ?

Abstract Introduction Lymphatic vessels found in most of the tissues in the body form a network that is tremendously important in macromolecular and cellular transport from peripheral tissues. In the past decade, a massive number of studies have focused on the role of the lymphatic system in immune cell trafficking during both physiological and pathological conditions. Concomitantly, immune cells, such as dendritic cells, monocytes, and T and B lymphocytes, have been described to be the major source of lymphangiogenic factors. Put together, it has been hypothesised that immune cells not only travel through lymphatic vessels from point A to point B but also regulate lymphatic vessel expansion and function per se. Several findings suggest a convincing give-and-take interaction between the circulating immune cells and the lymphatic vessels. In this review, we have reported the newly described impacts of immune cells on lymphatic growth and function in developmental and pathological contexts related to inflammatory events. Conclusion We believe that mutual interactions between the lymphatic vessels and immune cells is critical, and that understanding the specific role of different populations of immune cells during lymphangiogenesis may provide a useful approach for modulating the lymphatic network in inflammatory diseases.


Introduction
The lymphatic network is part of a system that extends its vessels in nearly every tissue of the body.The main functions of this tightly regulated system are to allow fluid haemostasis, and permit immune cell trafficking and lipid transport.In addition, the physiological relevance of the lymphatic circulation in removing cholesterol from peripheral tissues has been recently described 1,2 .Whether lymphatic network expansion is a friend or foe in the onset and/or progression of numerous diseases still requires attention.In the past decade, a growing number of studies have been exploring the 'regulation of lymphangiogenesis' not only during developmental, but also inflammatory settings, together with the pleiotropic activities of several classes of immune cells on the regulation of lymphangiogenesis during pathological and non-pathological contexts.
In this review, we focus on the roles of immune cells, such as macrophages, B and T lymphocytes, and dendritic cells (DCs), played in lymphangiogenesis and lymphatic vessels remodelling, during developmental and pathological contexts related to inflammatory events.

Discussion
The authors have referenced some of their own studies in this review.The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.Animal care was in accordance with the institution guidelines.

Lymphangiogenic factors
Lymphangiogenesis during development and inflammation is a tightly regulated process depending on several specific transcription factors, including Prox1,Sox18 and COUP-TFII(1-3) vascular endothelial growth factors (VEGF).VEGF-A, C and D play a critical role in the development of the lymphatic system through the binding and activation of VEGFR-2 and 3 signalling.VEGF-A exercises its biological function through binding and signalling via VEGFR-2, whereas VEGF-C and D act via VEGFR-3.As immune cells are the main source of lymphangiogenic factors, it has been predicted that modulating these cells function could have a strong impact on lymphangiogenesis and results in a defective lymphatic network (Figure 1).

T lymphocytes
Although other immune cell types are known to positively correlate with lymphangiogenesis, recent reports demonstrate that T cells are negatively regulating the development of lymphatic vessels.In athymic nude mice, in which functional T cells are absent, a high density of Lyve1 + lymphatic vessels can be observed in the lymph nodes (LNs) 6 .In this model, Kataru et al. 6 show that adoptive transfer of naive CD4 + or CD8 + T cells abrogates the LN lymphatic vessel density, a consequence that was even more prominent when activated T cells were injected.But how can T cells, in both inflammatory and steady state, mediate this negative regulation?Because adoptively transferred interferon-γ (IFNγ)-deficient T cells failed to negatively regulate lymphatic vessel formation, Kataru et al. 6 state that IFNγ is responsible for the crucial role of T

Dendritic cells
Lymphatic entry of DCs has been studied extensively for more than a decade [10][11][12][13][14] .As DCs have a crucial role in the initiation of immune responses within the draining LNs, the afferent lymphatic vessels serve as active conduits by which DC migration from peripheral tissues is tightly regulated 15 .More recently, Johnson et al. 16,17 have shown evidence that numerous chemokines and adhesion molecules are involved in a multistep process regulating the entry of DCs to activated lymphatic vessels.
Since the DCs must cross the endothelial barrier to enter the lymphatics, interactions occur between DCs and the lymphatic endothelium.Thus, in addition to the role of the lymphatic vessels in the transport of those key players in immunity, is it possible that there could be 'giveand-take' interactions between DCs and the lymphatic vessels?In a recent review, Russo et al. 18 describe the current state of knowledge about the cellular interactions that occur between DCs and lymphatic endothelial cells (LECs).They summarise our current knowledge on the impact of DCs/LECs interactions on the process of DC migration through the lymphatics and also, of great interest, on lymphatic vessels growth.

B lymphocytes
Is there a role for B cells in lymphangiogenesis occurring during inflammation?In 2006, Angeli et al. 19 have unravelled the interrelationship between B cells, lymphatic vessels and migratory DCs, demonstrating that DC migration from the periphery is amplified by B cell-dependent signals.In a mouse model of immunisation with keyhole limpet haemocyanin/CFA, they have shown that B cells are key actors in LN swelling.While assessing the consequences of a lack of B cells on the regulation of factors known to promote lymphangiogenesis, Angeli et al. 19 described that the production of Lymphangiogenic factors are produced at steady state and during inflammation mostly by immune cells that are using lymphatic vessels as conduits.Question marks represent the possible, but still unknown, interaction between lymphatic endothelial cells and understudied immune cell types in lymphatic growth.cells in maintaining the homeostatic balance of lymphatic vessels, at least in the LNs.
To better characterise the inflammatory response occurring in secondary lymphoedema, Zampell et al. 7,8 used a model of mouse-tail lymphoedema and antibody depletion techniques.They assessed a diminished propensity to develop tail oedema and lymphatic stasis in absence of CD4+ T cells, but not CD8+ T cells.In CD4-depleted mice, the lymphatic function was improved, while production of IFNγ TGF-β1 was decreased, thereby increasing inflammatory lymphangiogenesis.Those mice failed to develop lymphoedema and maintained normal lymphatic function.In a subsequent publication, Avraham et al. 9 demonstrate that progression of lymphoedema is associated with high production of Th2-related cytokines (IL-4 and IL-13).According to their findings, blocking those cytokine functions by using neutralising antibodies would improve lymphatic function and inhibit the consequent inflammation and fibrosis.Furthermore, when immunising the mice by complete Freund's adjuvant (CFA)/ ovalbumin intradermal injection, IL-4 neutralisation significantly increased lymphatic vessel density in the corresponding draining LN.All together, these data demonstrate that both CD4 + and CD8 + T cells negatively regulate lymphatic vessel growth via production of soluble factors, such as IFNγ and IL-4.
point out the effect of NK and NK T cells on lymphatic growth.Mouse models and depleting antibodies are currently available to specifically address the role of these cells during lymphangiogenesis.Other cell types are also understudied in the process: the role of Th17 cytokines or γδ T cells, major cell population involved in the production of IL-17 and IL-22, and on LEC is also unknown.Considering the role of eosinophils, a population of cells found in blood and tissues in both human and mouse, in modulating macrophage functions 26 we hypothesise that this cell type could also modulate the production of pro-lymphangiogenic signals.

Conclusion
We believe that mutual interactions between the lymphatic vessels and immune cells is critical, and that understanding the specific role of different populations of immune cells during lymphangiogenesis may provide a useful approach for modulating the lymphatic network in inflammatory diseases.
strongly inhibits the formation of new lymphatic vessels 23 .

Neutrophils
The role of neutrophils during lymphangiogenesis was not addressed until very recently.A recent report elegantly demonstrated the role of these cells during inflammatory lymphangiogenesis 24 .In their recent manuscript, Tan et al. 24 reveal that, in B cells-deficient animals, neutrophils take over the role of B cells and orchestrate the development of the lymphatic network.They further emphasise the function for neutrophils as organisers of lymphangiogenesis during inflammation by depleting neutrophils in wild-type mice developing skin inflammation in response to immunisation or contact hypersensitisation.In this model, they show that lymphangiogenesis is decreased and local inflammation is increased.According to their findings, neutrophil-derived matrix met-alloproteinase9 and heparanase are the main factors involved in this phenomenon, by controlling VEGF-A bioavailability in the inflamed tissues.Moreover, as neutrophils can secrete lymphangiogenic factors, notably VEGF-D, they consequently directly regulate lymphangiogenesis 24,25 .

Perspectives
Despite the tremendous recent progress that we described herein, the role of immune cells during lymphangiogenesis remains poorly understood: very little is still known on the direct and indirect impact of immune cells on the development of the lymphatic network.For instance, we reported studies showing that soluble factors such as IFNγ and IL-4 play a critical role during both developmental and inflammatory lymphangiogenesis.As IFNγ can not only be produced from conventional T cells, as described, but also by innate lymphocyte populations such as natural killer (NK) cells and NK T cells, it would be interesting to VEGF-A was dramatically decreased in both immunised mice and µMT mice, lacking B cells.In agreement with this early report, VEGF-A tg mice, overexpressing VEGF-A have increased number of LEC in LNs 20 and specific overexpression of VEGF-A by B cells (CD19Cre/hVEGF-Afl mice) induced increased lymphangiogenesis and angiogenesis in LNs 21 .Despite these interesting findings on the connection between B cells and VEGF-A in lymphangiogenesis during immunisation, the role of B cells and B cell-derived factors on the development of lymphatic vessels in steady state is still unclear: no direct characterisation of the lymphangiogenic pattern has been assessed in mice lacking B cells compared with wild-type mice.Comparing the regulation of lymphatic growth in wild-type mice and in mice lacking B cells (µMT or injected with a neutralising antibody directed against CD20, for instance) would provide further insights on role of B cells in lymphangiogenesis.

Macrophages
Macrophages constitute an important cell population typically involved in the host defence against pathogens and wound repair.As postnatal lymphangiogenesis is primarily associated with chronic inflammation, a process that is predominantly regulated, at the cellular level, by macrophages, it is not surprising that a growing body of evidence is revealing new functions of macrophages in lymphangiogenesis.Kubota et al. 22 described a reduction of the lymphatic network in trachea and ears in osteopetrotic mice bearing a mutation in csf1 gene and therefore a decreased abundance of tissue resident macrophages 22 .The recruitment of macrophages (VEGF-A dependent) to the site of injury, and their production of VEGF-C and VEGF-D, plays a critical step in lymphangiogenesis during inflammation.Depletion of macrophages with clodronate liposomes

Figure 1 :
Figure 1: Interactions between immune cells lymphatic endothelial cells.Lymphangiogenic factors are produced at steady state and during inflammation mostly by immune cells that are using lymphatic vessels as conduits.Question marks represent the possible, but still unknown, interaction between lymphatic endothelial cells and understudied immune cell types in lymphatic growth.