FormalPara Summary of Basic Concepts

The specialized structure of the lymphatic vasculature enables it to propel lymph through the body and back into blood circulation. The process starts with the extravasation of fluid from the blood capillaries. This excess fluid is taken up by the initial lymphatics and then propelled by the collective action of the initial and collecting lymphatic vessels back into the blood circulation at the subclavian veins.

  • The interstitium is composed of structural proteins and glycosaminoglycans that control its properties.

  • Extravasation of fluid into the interstitium occurs from the blood capillaries and is governed by Starling’s law.

  • The initial lymphatic vessels found throughout interstitial tissue are blind-ended structures composed only of endothelial cells and no basement membrane.

  • Fluid entering the lymphatic system is termed «lymph» and is prevented from escaping into the interstitium by the unique morphology of the initial lymphatics which form «primary lymphatic valves.»

  • The initial lymphatics play an important role in the uptake of lipid from the intestines and transport of macrophages to the lymph nodes.

  • Initial lymphatic function gets disrupted during chronic inflammation and lymphedema.

  • Collecting lymphatic vessels are formed of contractile units called lymphangions that actively pump lymph through the lymphatic network.

  • Lymphatic valves are present between the lymphangions that prevent the backflow of lymph.

  • Production of vasoactive substances, such as neurogenic and hormonal stimuli, allows the body to manipulate lymph flow rates by impacting lymphangion function and structure.

  • Mechanical factors, such as intramural pressure, stretch, and shear stress, can modulate and coordinate the function of the lymphatic muscle cells.

  • The presence of pacemaking cells has been suggested in collecting lymphatic vessels that might dictate the spontaneous contractility of the lymphangions.

  • Extrinsic factors like skeletal muscular contraction, arterial pulsations, and breathing can also contribute to the lymph flow.

  • Collecting lymphatic vessels might be compromised during lymphedema due to remodeling of the vessel wall and chronic pathological stimulation.

  • The lymph flows into lymph nodes through afferent collecting lymphatic vessels and exits through efferent collecting lymphatic vessels.

  • The lymph not only flows through the lymph node, but some of it is reabsorbed into the blood circulation at the lymph nodes.

1 Interstitium

1.1 Composition of the Interstitium and Maintenance of Interstitial Volume

The source of all lymphatic flow is in the interstitium, where the fluid and soluble interstitial proteins that are not absorbed by the venous circulation are returned to blood circulation by the lymphatic vasculature. The interstitial space is primarily composed of elastic fibers, collagens, and glycosaminoglycans (GAG) that combine to form larger protein complexes called proteoglycans. Proteoglycans are large negatively charged molecules that are essential for the structural properties of the interstitium. Their large negative charge makes them essential for maintaining interstitial volume by providing the interstitium with the capacity to resist compressive forces. Furthermore, the negative charge attracts other diffusible species which helps to maintain osmotic pressure and thus the fluid volume within the tissue. The osmotic pressure of the interstitium plays a significant role in determining the hydration of the tissue and effective porosity of the matrix and hence transport processes like convection, diffusion, etc. The porosity of the matrix and the extent that this porosity can be dynamically altered with mechanical loading depend also on the composition of the interstitium. This in turn dictates the hydraulic conductivity under various physiologic states. Interstitial properties are also highly variable between different parts of the body [6, 7]. Hence, the rates of interstitial fluid formation and drainage, and therefore the local demand on the lymphatic vessels, vary throughout the body.

1.2 Physics of Interstitial Fluid Formation

The circulatory system and the lymphatic system form a loop, regulating the amount of interstitial fluid by a continuous process of extravasation and reabsorption [8,9,10]. This process starts in the blood capillaries. Blood capillaries are composed of a single layer of endothelium with an almost nonexistent basement membrane, which makes it conducive to the exchange of fluid across its walls. The exact mechanism by which fluid is transferred across the blood capillary endothelium was first explained by physiologist Ernest Starling in what has come to be known as «Starling’s law».

Starling’s law, seen below, is an equation which states that the net rate of fluid transfer across a membrane is the weighted sum of the hydrostatic pressure difference across the membrane and the osmotic pressure of the solutes.

$$ {J}_{\mathrm{v}}={L}_{\mathrm{p}}\frac{S}{V}\left(\varDelta P+\sigma \varDelta \pi \right) $$

where J v is the fluid flux across the vessel wall (in or out), L p is the permeability, S is the surface area, V is the volume, ΔP is the hydrostatic pressure difference, Δπ is the osmotic pressure difference, and 𝜎 is the capillary osmotic reflection coefficient. In the context of fluid transport across blood capillaries, the blood capillary wall acts as a porous membrane, with blood on one side and interstitial fluid on the other side. There are two opposing forces acting on the blood capillary walls. Hydrostatic pressure drives fluid out of capillaries, while osmotic pressure drives fluid back in.

The rate of uptake of solutes by the blood capillaries is size and charge dependent, and it is found that small molecules and crystalloids are preferentially taken up, while transport of larger proteins is not permitted [11]. This is reflected as an osmotic reflection coefficient that is less than 1 in Starling’s law. This value means that there is a higher concentration of proteins within the blood capillaries than the interstitium. As the osmotic pressure is created primarily by the proteins, it is referred to as the oncotic pressure gradient. Fluid moves in the opposite direction of the oncotic pressure gradient, and therefore, the net effect is the movement of fluid from the interstitium into the blood capillaries. The mean capillary hydrostatic pressure is much greater than the mean interstitial hydrostatic pressure, which has the net effect of pushing fluid out of the capillaries [6, 9, 12].

The following example will help to better visualize this continuous exchange process. Let us start with an equilibrium where the contribution due to the hydrostatic pressure difference balances the contribution due to the oncotic pressure difference. Now consider an increase in the hydrostatic pressure in the capillary. This shift will cause movement of fluid out of the capillary. Under physiological conditions, the greater hydrostatic pressure of the capillary would lead to excess fluid flow into the interstitium. Fortunately, any reduction in capillary fluid increases the concentration of protein within the capillary, therefore increasing capillary oncotic pressure. This increase in oncotic pressure will cause movement of fluid into the capillary until an equilibrium is reached again.

In general, the rate at which fluid is squeezed out is slightly greater than the rate at which fluid is pushed back in. Thus, there is a net buildup of fluid in the interstitial space that needs to be drained in order to maintain fluid homeostasis in the interstitium. Additionally, it is essential that the lymphatics drain large proteins from the interstitial space to keep the protein concentration low and thus maintain an effective oncotic pressure gradient that favors fluid retention within the microcirculation. That is where the lymphatic system comes in, acting as a drainage system for the excess fluid or solutes while also serving many other important functions in the body like transportation of lipids from the intestine and the trafficking of immune cells to the lymph nodes [6, 7, 10, 13].

2 Initial Lymphatics

2.1 Structure

The initial lymphatics, also referred to as lymphatic capillaries, are blind-ended vessels characterized by endothelial cells with an attenuated cytoplasm, discontinuities in the basement lamina, and specialized junctions. The initial lymphatic endothelium is characterized by cells that are apposed to each other, forming valve-like structures that are commonly referred to as «primary lymphatic valves» since functionally they have been observed to allow fluid and large proteins to enter easily when the pressure gradient is favorable for lymphatic uptake and yet provide a large resistance to fluid and molecules leaving the capillary when lymphatic capillary pressure exceeds interstitial fluid pressure [1, 14,15,16,17,18]. This unique functional capacity of the initial lymphatics is thought to occur due to the specialized junctions formed by a unique configuration of VE-cadherin and CD31 commonly referred to as «buttons». The lymphatic endothelial cells form oak leaf-shaped structures that are characterized by discontinuous button-like junctions that form flap-like structures. These are characteristics of mature initial lymphatics, since immature initial lymphatics such as those observed during lymphangiogenesis induced by acute inflammation have zipper-like junctions [1]. The button-like junctions provide enough structural integrity to the initial lymphatics by being anchored at the side of the flaps while also forming points of entry at the tip of the flaps. Thus, the integrity of the initial lymphatic endothelium is maintained while also forming the valves that are necessary for entry of fluid into the lymphatics. While the size of many macromolecules and cells would restrict them from typically entering capillaries, the unique and ‘large’ entry junctions of the initial lymphatics enable passage into the lymphatics. The primary lymphatic valves are hence involved during the filling up of the lymphatic vessels with interstitial fluid and also prevent backflow into the interstitium. The initial lymphatic endothelial cells are connected to the surrounding connective tissues with anchoring filaments that play an important role in keeping the initial lymphatics from collapsing. Additionally, these filaments help in opening the primary lymphatic valves, leading to the formation of «lymph» [9, 14]. The structure of the initial lymphatic network and anatomy of the initial lymphatic vessels and valves are portrayed in ◘ Fig. 8.1.

Fig. 8.1
figure 1

a Structure of the initial lymphatic network and how it connects to the collecting lymphatic network is shown. The blowouts show the detailed structure of the initial lymphatic vessels, with the button-like junctions that form the flaps that act like valves. b The initial lymphatic vessels exhibit a discontinuous basement membrane and loose endothelial cell junctions, in contrast to blood capillaries that have a continuous basement membrane. This specialized structure of the initial lymphatics allows the paracellular transport of cells and proteins into the initial lymphatic lumen, but transport also happens through transcellular pathways. Anchoring filaments, which are responsible for pulling the initial lymphatic vessel open during the filling up stage, are also shown [19]

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2.2 Functional Role of Initial Lymphatics

2.2.1 Role in Lymph Formation and Transport

The initial lymphatics are the first stage of the lymphatic network that takes up the excess interstitial fluid. The average pressures in the interstitium and the initial lymphatics are similar. In fact, the average pressure in the interstitial space is usually slightly negative, thus being subatmospheric, while the average pressure in the initial lymphatic lumen is slightly positive. These measurements might indicate that the prevailing hydrodynamic forces are not conducive for the flow of interstitial fluid into the initial lymphatics. However, these are average pressures and do not account for the transient fluctuations in the interstitial and initial lymphatic pressures because of extrinsic and intrinsic factors. Extrinsic factors include skeletal muscle contractions, heart contractions, gastrointestinal muscle contractions, respiration, and arterial pulsations; intrinsic factors refer to the spontaneous contractions of the lymphatic network which will be described in greater detail later [13, 20].

During these cyclical pressure fluctuations, when the initial lymphatic pressure is lower than the interstitial pressure, the fluid flows into the initial lymphatics, thereby being referred to as «lymph.» The intrinsic properties of the initial lymphatics, such as anchoring filaments and «primary valves,» play a vital role here. An accumulation of fluid in the interstitium leads to swelling, which causes a tension in the anchoring filaments. This tension forces the primary lymphatic valves to open, allowing inflow. Once pressure inside the initial lymphatics is greater than the interstitial pressure, the primary valves in the initial lymphatics close, preventing the backflow of lymph into the interstitium [6, 20,21,22,23]. Recent research also suggests that apart from the aforementioned mechanism, transport of solutes can also occur through transcellular pathways which might be important in lymphatic solute transport [24]. Thus, the unique structure of the initial lymphatics coupled with the extrinsic and intrinsic factors leads to the formation of lymph.

2.2.2 Role in Lipid Transport

A special kind of initial lymphatic vessel is the lacteal. Lacteals are the initial lymphatic vessels found in the small intestine at the center of each villus. The main function of the lacteals, apart from the removal of water absorbed across the mucosal membrane [25], is the uptake of absorbed lipids in the form of high-density lipoproteins and chylomicrons from the small intestine. They are also shown to allow the migration of immune cells from the interstitium across the endothelium [26]. The lacteals then act as a conduit toward the mesenteric collecting lymphatic vessels, which then transports this unique, lipid-rich lymph to the systemic circulation [27]. For this reason, the lacteals also serve as an important channel for the uptake of lipophilic drugs that are orally ingested [28]. This uptake of lipoproteins and cells can happen paracellularly across the lymphatic endothelial cell junctions and also through transcellular pathways [29,30,31,32,33].

The lacteals are unique, because they show spontaneous contractility in conjunction with adjacent smooth muscles. This was first discovered by Howard Florey in 1927 [34, 35]. This unique physiology makes them exhibit properties of both the initial lymphatics in that they possess «button-like» junctions and also of the collecting lymphatics in that they possess spontaneous contractility that is regulated by the autonomic nervous system [36]. The function of lacteals, and hence the uptake of chylomicrons, is highly dependent on the prevailing forces in the interstitium, as lacteals have been observed as undergoing morphological changes in response to fasting and feeding and they also show sensitivity to lymph flow [37]. Experiments with mice having abnormal lymphatic lineage commitment due to a heterozygous mutations in the Prox-1 gene develop obesity due to compromised leaky lymphatic vessels [38]. VEGF-C, an important factor in lymphatic endothelial cell growth and proliferation, has been found to be extremely important in the maintenance of the function of the lacteals, as knockout of VEGF-C leads to atrophied intestinal lymphatics and impaired lipid transport [39].

2.2.3 Role in Transport of Immune Cells

The lymphatic vessels play a very important role in immune cell trafficking. However, the transmigration of leukocytes across the initial lymphatic endothelium is a matter of ongoing investigation. In the presence of pro-inflammatory cytokines, cultured lymphatic endothelial cells have been shown to upregulate the expression of leukocyte adhesion molecules [40]. Carbohydrate-binding proteins like galectin have also been implicated in this transmigration. The binding of the leukocytes to the adhesion molecules on the lymphatic endothelial cells causes them to crawl toward the cell junctions, where they interact with other adhesion molecules to promote entry into the initial lymphatic lumen. For example, gradients of the chemokine CCL21 have been implicated in the crawling of dendritic cells (DCs) across the initial lymphatic junctions [41, 42]. The transmigration can occur via both the button-like junctions and through the endothelial cells, although the exact point of entry is unresolved [1]. The amount of lymph drainage through the lymphatic network has also been implicated as a factor in regulating the transmigration of dendritic cells in the initial lymphatics [43]. There is also evidence for bidirectional transmigration of the leukocytes, which might be an important factor in effective immune surveillance [44].

2.3 Initial Lymphatic Disruption in Inflammatory Diseases and Lymphedema

Inflammation involves a series of responses initiated by the body in response to harmful stimuli like pathogens or external irritants causing damage to cells. Inflammation has several effects on the circulation, including increased permeability of the blood vasculature, leading to extravasation of fluid and tissue swelling. This increased permeability also allows leukocytes to enter the interstitium by extravasation. In the case of initial lymphatics, the effect of inflammation is similar, with an increase in permeability of the vessels. One probable cause for this increase in permeability might be the opening of the primary lymphatic valves due to the swelling of the tissue, which pulls on the anchoring filaments and holds the valves open. Other reasons proposed include the formation of pores in the lymphatic endothelium and rearrangement and loosening of the tight cell-cell junctions that are occasionally present in the initial lymphatic endothelium [1, 40]. It is hypothesized that there is a reduction in the effective functioning of the lymphatics during chronic inflammation because of compromised initial lymphatic junctions.

Inflammation and lymphedema influence each other in a complex manner, and this can be traced to altered function of the intial lymphatic system. Lymphedema induced in rodent models by artificial lymph stasis has been shown to induce inflammation and fibrosis [45, 46]. There is an overabundance of pro-inflammatory cells like macrophages, dendritic cells, and lymphocytes in the interstitium, leading to chronic inflammation and fibrosis [40, 44, 47]. Fibrosis caused by chronic inflammation can also worsen lymphedemic conditions [48, 49]. Lymphangiogenesis (the formation of new lymphatic vessels from pre-existing ones) induced by inflammation is thought to be involved in lymphedema, although whether the effect is benign or pathological is still a matter of contention [50, 51]. In the context of primary lymphedema, mutations in VEGFR-3 that lead to impaired lymphangiogenesis have been shown to induce lymphedema [52,53,54]. Hence, the use of lymphangiogenesis as a mean of therapeutics during lymphedema has also been suggested [55, 56]. However, lymphangiogenesis might not always have a benign effect on lymphedema. It has been shown that CD4+ T cells can interact with macrophages to produce VEGF-C which is essential for lymphangiogenesis. Inhibiting the interaction can lead to reduced lymphangiogenesis as well as reduced chances of later onset of secondary lymphedema [51]. The upregulation of VEGF-C during inflammation may also lead to loss of collecting lymphatic pumping function, although there are contradictory reports on the same [57, 58].

3 Collecting Lymphatic Vessels

3.1 Structure

As initial lymphatics connect downstream, they transition into a region of the lymphatic network known as the pre-collecting lymphatic vessels. Pre-collecting vessels are distinguished from the initial lymphatics by the presence of a basement membrane surrounding the lymphatic endothelial cells and the discontinuous arrangement of lymphatic smooth muscle cells [59, 60]. Pre-collectors have the continued presence of anchoring filaments, presumably to assist in lymph absorption and transport [59].

Pre-collecting lymphatic vessels transition into mature collecting lymphatic vessels. These collecting vessels are characterized by fully developed basement membranes, full coverage by lymphatic smooth muscle cells, and the presence of a second type of lymphatic valve. The basement membrane between the LEC monolayer and the muscle is primarily composed of collagen and elastin fibers which contribute significantly to the mechanical properties of the vessel. These two extracellular protein fibers also provide structural support to the bileaflet, unidirectional valves. The valves separate the collecting vessel into discrete segments known as lymphangions as seen in ◘ Fig. 8.2. These valves physically prevent backflow between lymphangions and allow for the directional flow of lymph toward the distal lymph nodes [15, 61]. The smooth muscle cells surrounding the collecting lymphatic vessels are highly specialized and are commonly referred to as lymphatic muscle cells (LMCs). Lymphatic muscle cells are capable of rapid, phasic contractions that are well coordinated with neighboring cells [2].

Fig. 8.2
figure 2

The mature lymphatic collecting vessel. The lymphatic endothelial cells (LECs) are supported by a layer of basement membrane composed primarily of extracellular fibrils of collagen and elastin (seen in orange and blue). These fibers also provide structural support to the bileaflet valves. The entire collecting vessel is surrounded by a layer of lymphatic muscle cells which contract to propel lymph flow downstream

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Unlike blood vasculature, there is no central pump to drive the flow of lymph through the lymphatic system. Lymph transport through the collecting vessels is therefore entirely dependent on the intrinsic contractility of the lymphatic muscle and extrinsic factors such as skeletal muscular contraction, arterial pulsation, passive movement, and respiration.

3.1.1 Lymphatic Valves

Functioning lymphatic valves within the collecting vessels are critical for functional lymph propulsion by either intrinsic or extrinsic factors. In humans, these valves are typically spaced every 1–3 mm [62]. To properly function, these valves must open and close under flow rates and pressure that are relatively low compared to pressure in the blood vasculature. Work with isolated vessels, where the inlet and outlet pressures can be manipulated independently, demonstrated that the pressure gradient across the valve needed for the valve to open is fairly consistent, but closing pressure gradient is dependent on transmural pressure of the adjacent lymphangion. In addition, there appears to be a slight bias in the valve to remain in the open position [3]. This likely explains why a small amount of retrograde flow is typically observed in the contraction cycle before valve closure [4].

While largely efficient, some regurgitation of lymph into an upstream lymphangion may occur before valve fully closes, specifically given their open bias [63]. Eliminating backflow is particularly critical at lymphovenous valves; unique valves present at the intersection of major collecting vessels, known as ducts, and the left or right subclavian vein where lymph is returned to the blood vasculature. At these locations, adverse pressure gradients can be significant, and several studies in mice demonstrate that pathologies of the lymphovenous valves lead to blood flowing into the lymphatic system [64]. Activation of platelets by specific receptors on the surface of lymphatic endothelial cells seems to play a critical role in preventing blood flow into the lymphatics particularly at these locations [65].

3.2 Intrinsic Contractility and Active Lymph Propagation

As previously stated, the lymphatic collecting vessel is composed of a chain of lymphangions, separated by unidirectional valves. These lymphangions are considered the contractile units of the vessel, working as a series of pumps to propel lymph. The rapid, phasic nature of this contraction is unique to LMCs and made possible by their unique molecular composition. While the LMCs are nonstriated muscle cells, they share contractile proteins that are otherwise limited to cardiomyocytes, which in part account for their unique pumping phenotype [2, 66]. This pumping mechanism in lymphatics is described using language consistent with the heart. For example, as a heart undergoes systole and diastole, so does the lymphangion. Systolic diameter of a lymphangions describes the minimum diameter in the lymphangion at the peak of a contraction, while the diastolic diameter is the maximal diameter between contractions. Ejection fraction refers to the fraction of end-diastolic volume ejected during lymphatic contraction.

The contractile frequency of a lymphangion is usually on the order of five to ten contractions per minute with amplitudes of up to 50% of the resting diameter for a major collecting vessel, but there is a significant amount of regional variability due to mechanical loading on the vessel [4, 67,68,69]. These relatively strong, regularly occurring contractions are critical for the collecting vessel to pump fluid against an adverse pressure gradient. In large animal models, experimental work has shown that short segments of isolated lymphatic vessels can overcome adverse pressure gradients of 10–30 cmH2O [70]. To overcome greater pressure gradients, it is important to remember that collecting lymphatic vessels work not as individual units, but as chains of pumps arranged in series. The extent that the coordination of the pumping activity of the chains regulates lymph flow is not well established, but computational modeling suggests that coordination could improve pumping [69], and it has also been observed experimentally [71]. Chains of multiple lymphangions are better suited to overcome larger adverse pressure gradients and appear to coordinate contractility to reduce pressure downstream before additional fluid arrives from upstream segments [72]. A chain of lymphatic vessels is able to overcome a rather large pressure gradient, with measurement in vivo reporting pumping pressures in excess of 30 mmHg (~40 cmH2O) in human legs [73], 40 mm Hg (~54 cmH2O) in human arms [74], and 35 mmHg (~47.5 cmH2O) in rats [75].

In addition to executing active phasic contractions, LMCs must modulate the vessel diameter in order to adjust the resistance to lymph flow through the lymphatics. Similar to the blood vasculature, regional changes in the lymphatic vessels’ diastolic diameter can locally regulate the quantity and velocity of transported lymph. This is often at odds with the vessels’ role as a pump, as relaxing the LMCs often impairs active contractility. Furthermore, active pumping by LMCs may increase the resistance to passive flow by transiently constricting the diameter of the vessel. Therefore, it is important that LMCs react to a variety of environmental cues that regulate their role as either an active pump or a passive conduit to lymph flow [76]. Factors that impact the coordination of these roles are outlined below.

3.2.1 Innervation and Sensitivity to Vasoactive Substances

Collecting lymphatic vessels have long been shown to be responsive to a variety of vasoactive peptides [77]. Noradrenergic, purinergic, cholinergic, and peptidergic neurons can be found throughout collecting vessel walls and manipulate various aspects of contractility [78]. Innervation of the lymphatics leads to altered function in response to a number of neurogenic and humoral stimuli that have been extensively researched [77,78,79,80,81,82,83]. Lymphatic regulation through by these vasoactive substances have been shown to influence resistance to flow, ejection rate, rate of formation of lymph, and lymphatic vessel permeability.

Recent work has explored the role of various vasoactive substances on the permeability of collecting vessels. Atrial and brain natriuretic peptides, hormones that are released to reduce stress on the heart, both reduce collecting vessel permeability. This may have the effect of temporarily reducing the reabsorption of lymph into blood circulation [84]. Similarly, immune cells that interact closely with or within the walls of the collecting lymphatic vessel play a significant role in modulating collecting vessel permeability and function [85]. Within adipose deposits, modulation of collecting vessel permeability has been shown to regulate local inflammation [86]. As a whole, the aforementioned studies in combination with others have demonstrated that excessive collecting vessel permeability impairs the ability of lymph, rich in antigens and dendritic cells, to reach downstream lymph nodes, significantly weakening the adaptive immune response [87].

Vasoactive substances released during inflammation appear to play competing roles in regulating lymph flow. Vasoactive substances released by myeloid cells during inflammation, such as nitric oxide and histamine, seem to both dilate collecting vessel walls and reduce frequency of spontaneous contractions. Furthermore, these substances seem to assist in DC trafficking to lymph nodes [88,89,90]. Other inflammatory cytokines, such as substance P, actually increase contractile frequency when incubated with isolated vessels [79]. How immune cells coordinate the release of these potentially competing vasoactive substances throughout the progression of inflammation is still poorly characterized.

3.2.2 Mechano-Regulation

As with the blood vasculature, shear stress elevated above a certain threshold due to fluid flow along the lymphatic lumen induces vasodilation of the collecting lymphatic vessels, reducing or fully inhibiting contractility [91]. The mechanism for this response is believed to be largely based on nitric oxide generation by LECs [92, 93], but recent research has implicated other vasodilators, such as histamine [94]. The shear stress magnitude within the lymphatic vasculature is much lower than that seen in blood vasculature and highly oscillatory [4]. A recent study demonstrated that the magnitude of shear needed to inhibit spontaneous contractions is much lower than that seen in the blood vasculature, depends on distention due to transmural pressure [95], and involves Ca2+ release by LEC [96, 97]. It is not clear what the molecular mechanisms are that allow lymphatic endothelial cells to respond to lower shear stresses than blood endothelial cells; however, recent work has suggested that this enhanced sensitivity involves VEGFR3, possibly through a mechanosensory complex formed at the cell junction [98]. Furthermore, shear stress along the wall appears to contribute to the coordination of contractility. Simply put, as lymph ejected from an upstream lymphangion moves into a downstream lymphangion, the increased shear stress along the wall of the downstream lymphangion dilates the diameter, reducing the resistance to the incoming flux of lymph [95, 99].

Contractility of the collecting vessel demonstrates a sensitivity to wall distension in accordance to the Frank-Starling’s law [63]. The length-tension relationship of lymphatic muscle cells determines the tension within the LMC at which force generation is maximal. It was determined in human thoracic ducts that average peak active wall tension was 6.24 ± 0.75 N/m at a corresponding transmural pressure of 47.3 ± 4.7 mmHg [100]. Increased transmural pressure increases wall tension, causing greater force generation by the LMCs, leading to an increase in pumping efficiency (e.g., the ejection fraction increases) up until a critical pressure is reached. Beyond this point, pumping efficiency diminishes as the LMCs, presumably, cannot generate more force [101, 102]. An early theory for contraction coordination along the collecting vessel was that the volume increase after a lymphangion takes on a bolus of lymph was the sole trigger for the contraction of locally distended muscle cells. This theory has been shown to be an oversimplification, as lymphangions can contract in the absence of lymph formation [103] and in the absence of apparent intraluminal distending force [104].

3.2.3 Electrophysiology and Pacemaking

Contractility of the collecting lymphatics in the absence of vessel distention indicates that contractile coordination depends, in part, on electrical mechanisms. Multiple ionic channels have been well characterized in LMCs [105,106,107]. Outward currents include calcium-dependent potassium channels and delayed rectifier potassium channels. Inward currents include fast sodium channels [108] and both L- and T-type calcium channels. The exact expression of these channels on LMCs seems to be heterogeneous, and it is hypothesized that different channels are responsible for specialized aspects of contractility. For example, work by Roizes and von der Weid demonstrated that the inhibition of L-type calcium channels primarily reduced the force generation during a contraction, while T-type calcium channel inhibition caused irregular contractile frequency [109]. Furthermore, a relationship between the resting membrane potential of LMCs and isometric tension has been established in animal models, further demonstrating the interplay between these regulatory factors [110].

The pacemaking behavior of lymphatic vessels was well demonstrated for decades, but the mechanisms by which this is possible were difficult to elucidate [111]. Are all LMCs capable of pacemaker-like spontaneous depolarization, or are there specialized pacemaker cells embedded within the vessel? More recently, it has been shown that a small population, approximately 5%, of LMCs isolated from sheep lymphatic collecting vessel walls may produce an inward current after hyperpolarization, similar to pacemaker cardiomyocytes. This inward current is similar to the «funny» pacemaker current seen in cardiac pacemaker cells [112]. Interstitial cells of Cajal are a cell type that is known to be responsible for pacemaking in the smooth muscle cells of the gut. Work by Boedtkjer has confirmed the presence of interstitial Cajal-like cells in human thoracic ducts through a variety of imaging modalities, but their exact functional roles need to be further investigated [113]. Earlier work seems to indicate that these pacemaking cells can coordinate over distances of at least 80 mm [114]. The mechanism behind this coordination and the differentiation of these specialized pacemakers within a developing collecting vessel are areas of active research.

3.3 Extrinsic Factors that Contribute to Lymph Flow

Under healthy conditions, external factors seem to improve lymph flow and interstitial fluid clearance by the lymphatics. Notably, exercise and manual massaging improve the rate of clearance of tracers injected within the interstitium via the lymphatics and the quantity of lymph flowing through the collecting vessels [115,116,117]. The exact means by which the extrinsic factors improve these metrics are contested. Amplitudes of pressure changes within subcutaneous lower leg lymphatic collecting vessels (3.2–4.7 mmHg) were unchanged by exercise [118]. In addition, one study demonstrated that muscular contractions did not independently generate flow, but did increase lymph flow during intrinsically generated contractions but muscular contractions did not independently generate flow [119].

Rates of lymph formation and reliance on extrinsic factors vary for collecting vessels found in different regions of the skin, subcutaneous tissue, fascia, or muscle. Lymphatics within highly mobile tissue such as skeletal muscle, lungs, and the heart have undergone special adaptations to adapt to their local environments. A review article by Negrini and Mariondo nicely summarizes some of these adaptations [23]. For instance, their research demonstrates how both the organization and properties of diaphragmatic lymphatics take advantage of the stresses exerted by the muscle fibers. Lymphatic orientation, both perpendicular and parallel to skeletal muscle fibers, allowed lymphatic vessels to exploit the full contractile cycle of lungs for lymph generation and propulsion. In addition, variations in the stiffness of diaphragmatic lymphatic walls allow compliant regions to act as a reservoir for lymph, while stiffer walled regions better allow local tissue displacement, leading to lymph propulsion [120]. The extent to which lymphatic orientation assists lymph propulsion in other tissue beds (e.g., cardiac tissue) is less known.

3.4 Collecting Lymphatics in Lymphedema

During lymphedema, lymph flow through the collectors appears to become increasingly dependent on extrinsic factors. Several studies demonstrate that intraluminal lymphatic pressure increases during lymphedema [121] and becomes more sensitive to contractions of surrounding skeletal muscle. The elevated sensitivity of lymphedemic limbs to manual massaging further highlights the reliance on extrinsic factors for lymph propulsion after the onset of lymphedema. Interstitial tissue pressures can increase to over 100 mmHg after application of manual massaging, creating a favorable pressure gradient for fluid to flow into the lymphatic system [122].

Structurally, it has been shown that the wall thickness of the lymphatic collecting vessel is strongly correlated with lymphedema disease severity. Rapid expansion of smooth muscle actin-positive cells and increased deposition of extracellular matrix elements such as collagen are hallmarks of vessels isolated from lymphedemic regions [123, 124]. While modeling indicates that these changes in vessel mechanics and dimensions would impact contractility [125], there is little work looking at functional pumping metrics of these remodeled vessels in vivo or in vitro. Many animal studies demonstrate that healthy collecting vessels exposed to edematous conditions, such as elevated pressures, have improved pumping metrics and lymph flow [70, 126, 127]. The extent to which these results translate to remodeled collecting vessels is unclear. In addition, it is difficult to determine if the increased lymph flow rates during edematous conditions are due to improved pumping of the collecting vessel or simply elevated rates of lymph formation.

Recent work by Mortimer et al. has shown that women with elevated lymphatic pumping are at a significantly higher risk of developing lymphedema later [74, 128]. They demonstrated that lymphatic pumping pressures before breast cancer surgery were 1.7-fold greater in patients who later developed lymphedema than those who did not. Clearance rates of tracers by the lymphatic system were consistently shown to be elevated by about 2.2-fold in patients who later developed lymphedema. The exact cause of this phenomena is still under investigation.

4 Lymph Nodes

4.1 Structure

Lymph moving through collecting vessels passes through at least one but often a series of lymph nodes. The human body contains hundreds of lymph nodes, which vary in size from 1 mm to 10 mm. Lymph nodes are vascularized for the exchange of nutrients and immune cells [5, 6]. Lymph, arriving from multiple afferent collecting vessels, contains a variety of antigen-presenting cells, cytokines, antigens, and exosomes from upstream tissue beds, which play a critical role in the priming and activation of the immune system. Lymph nodes are comprised of multiple «compartments» which house various lymphocytes, and the route and quantity of lymph that arrives in each compartment may potentially impact immune response. Lymph first arrives into the subcapsular sinus and can then be directed through either a «central» or «peripheral» path to the medulla sinus [129]. The path by which solutes travel within the lymph node is dictated in part by hydraulic conductivity, with different conduits within the node having variable densities. Therefore, size alone of arriving solutes can dictate their route through the lymph node [130]. While the peripheral path takes lymph directly to the medulla sinus, the central path has lymph travelling through B cell follicles and the T cell cortex. Within the T cell cortex are high endothelial venules (HEVs) which allow circulating T cells to enter the lymph node.

4.2 Lymph Flow and Fluid Exchange

Recent models estimate that about 90% of the fluid that is carried from the afferent collecting vessel into the lymph node flows through paths along the periphery of the lymph node [53, 131, 133]. The remaining fluid that travels along the central path, deeper into the lymph node, is reabsorbed into blood circulation by parenchymal HEVs. While the amount of fluid reabsorbed into circulation at an individual node is relatively small, given the hundreds of lymph nodes within the body, it is estimated that an additional 4 L of fluid is returned to circulation at lymph nodes in basal conditions. This doubles the amount of fluid the lymphatics is responsible for clearing from the interstitial tissue space from previous estimates, which were solely based on the flow rates out of the major lymphatic ducts at lymphovenous junctions. This fluid exchange has been shown to be dictated by Starling’s forces and is sensitive to changes in oncotic pressure of lymph or blood pressure at the HEVs [132]. Importantly, this fluid exchange within the node can alter the flow rate of lymph through the node as well as the composition of the efferent lymph. This alteration of lymph composition and flow implicates that the fluid exchange at the node not only regulates fluid recirculation but also activation of adaptive immunity.