Lymphatic Vessel Network Structure and Physiology

Abstract

The lymphatic system is comprised of a network of vessels interrelated with lymphoid tissue, which has the holistic function to maintain the local physiologic environment for every cell in all tissues of the body. The lymphatic system maintains extracellular fluid homeostasis favorable for optimal tissue function, removing substances that arise due to metabolism or cell death, and optimizing immunity against bacteria, viruses, parasites, and other antigens. This article provides a comprehensive review of important findings over the past century along with recent advances in the understanding of the anatomy and physiology of lymphatic vessels, including tissue/organ specificity, development, mechanisms of lymph formation and transport, lymphangiogenesis, and the roles of lymphatics in disease. © 2019 American Physiological Society. Compr Physiol 9:207-299, 2019.

Fig. 1.

Diagram representing the blood and lymphatic circulation in mammals. Filtered plasma forms interstitial fluid that enters the initial lymphatics to become lymph. In the intestine, a significant amount of dietary lipids accompany the absorbed fluid, producing a milky lymph, or chyle. Lymph is transported through afferent collecting lymphatics to lymph nodes where for immune surveillance. Efferent collecting lymphatics then transport the postnodal lymph to larger trunks, which return it to the great veins. Arrows indicate the direction of transport.

Fig. 2.

Initial lymphatics (lymphatic capillaries) as the site of lymph formation. A. The intravital microscopic image shows the rat mesenteric microcirculation and lymphatics labeled with topically applied FITC-BSI-Lectin, which allows for easy visualization of vascular structures. The arrows show blind ends of initial lymphatic vessels. B. A cartoon of the image in panel A shows color labeling of the arterioles (red), capillaries (red/blue), and venules (blue), and the initial lymphatics (yellow) for easier view. C. This cartoon depicts the entry of interstitial fluid (blue arrows) into initial lymphatics, forming lymph that is then transported toward a precollector. The precollector is separated from the initial lymphatic network by a one-way intraluminal valve.

Fig. 3.

The oak leaf shape of endothelial cells of initial lymphatics. A. Silver nitrate labeling of the junctions between endothelial cells of initial lymphatics in the inner layer of the tunica vascularis from rat uterus. OJ = open junction formation. Scale bar = 45 μm. From reference (1220) with permission. B. Distribution of VE-cadherin and PECAM-1 at the junctions between initial lymphatic endothelial cells in the mouse trachea, identified by immunofluorescence microscopy. Scale bar = 5 μm. From reference (736) with permission.

Fig. 4.

Confocal microscopy image demonstrating the endothelial, smooth muscle, and adventitial layers of a rat mesenteric lymphatic vessel. The top panel shows labeling of the glycocalyx (red) with BSI-Lectin-TRITC. The middle panel shows smooth muscle actin (blue). The bottom panel shows an overlay, plus the nuclei labeled in white. Note the longitudinal orientation of the endothelial nuclei, versus the circular smooth muscle cell nuclei. Additional cells, nerve fibers, and vasa vasorum are common in the adventitia. Images from reference (568) with permission.

Fig. 5.

Secondary valve as seen in an isolated mouse mesenteric collecting lymphatic vessel. The vessel shown was cannulated on both ends and bathed in a Ca²⁺-free solution so that it was completely relaxed. A. When the fluid pressures are the same in both pipettes, the valve is in an open position. B. When the fluid pressure in the outflow pipette (right side) is raised higher than that of the inflow pipette (left side), the valve closes. The images were obtained in Dr. Joshua Scallan’s laboratory.

Fig. 6.

Antigen markers of the lymphatic endothelium have different labeling patterns in initial lymphatics, precollectors, collecting lymphatics, and intraluminal valves. In initial lymphatic networks (lymphatic capillaries), relatively high levels of Prox1, podoplanin, and Lyve1 are detected, plus these endothelial cells are also positive for Vegfr3. In Precollectors, the endothelial cells are positive for Prox1, Lyve1, and have relatively low levels of detectable podoplanin. Collecting lymphatic endothelium is positive for Prox1 and podoplanin, with extracellular reelin located between the endothelium and smooth muscle layer. The endothelium of intraluminal lymphatic valves (secondary valves) has high levels of Prox1 and FoxC2, and is also labels positively for podoplanin, Lyve1, and Vegfr3.

Fig. 7.

Lymphatic networks in the rat tongue. A. An image of a Mercox® corrosion cast of the rat tongue showing blind-ended initial lymphatics and impression patterns of “button” junctions and endothelial nuclei. B. The fine detail of oak leaf shaped endothelial cells, with (C) overlapping “button” junctions can also be observed in these images. Reproduced rom reference (165) with permission.

Fig. 8.

Lymphatic networks of the human tonsil. A. Lymphatic corrosion cast of human palatine tonsil viewed by scanning electron microscopy. Tubular lymphatic networks in the parafollicular area (P) connect to lymphatic sinuses (s) that surround the lower part of the follicle (110× magnification). B. Lymphatics (L) in the human palatine tonsil capsular region, with notches (arrowheads) showing locations of secondary valves (75× magnification). C. Schematic diagram of the organization of lymphatic networks in the human palatine tonsil, showing the epithelium(E) and epithelium infiltrated with lymphocytes (LS), mantle zone (M), germinal center (GC), and septum (S). The images are from reference (356), with permission.

Fig. 9.

Lymph lacteals of the rat small intestine. The image is of a corrosion cast of the rat upper small intestine viewed by scanning electron microscopy. Blind-ended lymph lacteals coalesce at the bottom, and this sinus (s) then connects to the submucosal lymphatic plexus (sl). Scale bar= 200 μm. The image is from reference (795) with permission.

Fig. 10.

Scanning electron micrograph of a lymphatic corrosion cast of rat cecum. The mucosal lymphatic (ml) capillaries form a network with many blind-ended vessels. These networks then drain in to thicker submucosal lymphatics (sl). The arrowhead indicates a constriction point indicative of a secondary valve. Scale bar = 500 μm. The image is from reference (795) with permission.

Fig. 11.

Collecting lymphatics of the rat mesentery contract intrinsically. Panels A and B show a typical mesenteric collecting lymphatic vessel of an anesthetized rat, observed by intravital microscopy during its phases of diastole (A) and systole (B). The thick white arrows denote the vessel walls, with the lumen in between. The black arrow indicates the site of a secondary valve separating two lymphangions, which prevents backflow of lymph. Panel C shows a trace of diameter versus time acquired from a mesenteric lymphatic vessel using intravital microscopic video recording. The trace shows cyclic changes in diameter. The points at which the end diastolic diameter (EDD) and end systolic diameter (ESD) for a single contraction cycle are shown. These images and data are from reference (128) with permission.

Fig. 12.

Lymphatic networks surrounding portal tracts in the rabbit liver. Corrosion casts were prepared after injection of resin into the bile ducts, which then leaked out and entered the lymphatic networks. Scanning electron microscopy was used to view the corrosion casts. The large panel (1a) shows a low power (30×) image of the rich lymphatic networks around the bile duct (B). The high power image (80×) in panel 1b shows where resin leaked from the bile duct (B) into the initial lymphatics (L). These images are from reference (1164) and reproduced with permission.

Fig. 13.

Diagram of fluid flow and cell migration pathways from the liver sinusoids to the portal lymphatics. Fluid draining from the liver sinusoids (S), indicated by the arrows, presumably passes through the space of Disse, channels of the limiting space, and through the portal tract interstitial space to reach the portal lymphatic vessels (L). Other cells and structures shown include dendritic cells (Dc), collagen fibers (C), interlobular artery (iA), interlobular vein (iV), interlobular bile duct (iB), fibroblasts (F), Ito (stellate) cell (I), Kuppfer cell (K), nerve (N), peribillary capillary plexus (PbP), afferent vessel of PbP (a), and efferent vessel of PbP (e). Reproduced from reference (794) with permission.

Fig. 14.

Lymphatic networks of the mouse ovary visualized in Prox1-EGFP reporter mice. A. Lymphatic networks (green) arise from the rete ovarii (RO), indicated by the arrow, and extend into the ovarian medulla (Om) and ovarian cortex (Oc). B. Blood vessels were labeled with an anti-endoglin (ENG) antibody immunofluorescence labeling (red). The arrow indicates the follicle(F). C. Lyve1 was also labeled (blue) and was localized mainly to lymphatics at the ovarian rete and extraovarian rete. Panel D shows a 3-dimensional representations of Prox1-EGFP-positive lymphatic vessels, while in panel E a similar 3-dimensional model shows Lyve1-positive lymphatics (blue) overlaid with endoglin-positive vessels. Panel F shows a composite image with Prox1-EGFP (green), endoglin (red), and Lyve1 (blue) labels, showing some overlap and also distinct patterns of the blood and lymphatic vessel networks. Scale bar = 1 mm. The images are reproduced from reference (1031) with permission.

Fig. 15.

Lymphatics in the testes of Prox1-EGFP reporter mice. A. During late gestation (E17.5), EGFP-positive lymphatics sprout from the spermatic cord across the surface of the testis (T). Lymphatics are also found on the head (E1) and tail (E2) of the epididymis. The scale bar = 500 μm and applies to panels A-C. B. Blood vessels were also visualized in the same specimen using an anti-endoglin antibody (ENG). C. The Prox1-EGFP-positive lymphatics (green) and ENG-labeled blood vessels (red) did not occupy the same space. Some yellow areas show overlap of the two fluorescence signals from different planes. D. A three-dimensional representation of the Prox1-EGFP-positive lymphatic network from panel A. Panel E shows a magnified region from panel C showing the lymphatic vessels (green) running parallel to the coelomic vessel (CV). Panel F shows a magnified region of the rete testes. The scale bar for panels E and F = 250 μm. G. Brightfield whole mount view of the adult mouse testis surface, showing blood vessels (BV) and the spermatic cord (asterisk). H. EGFP signal can be observed from the same surface view, however there is much background due to Prox1-EGFP expression within spermatids located in the testis. I. A confocal image better shows the Prox1-EGFP-positive lymphatic network located within the tunica albuginea of the adult testis. Panel I scale bar = 600 μm. The images are reproduced from reference (1031) with permission.

Fig. 16.

Endometrial lymphatics and blood vessels in women dilate in response to progestin treatment. Hysterectomy samples were obtained from women who either received no treatment, or intrauterine progestin therapy for heavy menstrual bleeding. Panels A and B are endometrial sections from controls, and C and D are from those treated with LNG-IUS. Sections were immunolabeled to identify either CD31 (A and C) or D2–40 (podoplanin, B and D). Reproduced from reference (277) with permission.

Fig. 17.

Lymphatic vessel networks of the skin. A. This image shows a translucent preparation of dorsal skin from the human foot in which the initial lymphatic network was labeled with India ink absorbed into the network after a subcutaneous injection. B. A view of the mouse tail with a fluorescent microscope, after intradermal injection at the distal tail with FITC-dextran-2000kDa reveals the polygonal lymphatic capillary network. C. A block diagram of the cutaneous lymphatic and blood vessel networks in human skin. D-J. Confocal microscopic images of human skin. D. Only PECAM-1-positive capillary networks are visible 0–25 μm below the dermoepipermal junction. E. At 25–50 μm both capillary networks and initial lymphatics identified by LYVE1 labeling are visible. F. Three-dimensional reconstruction of these networks. G. Decrease in LYVE1-labeling in a lymphatic network at the interface where collecting lymphatics appear in the skin. H and I. Podoplanin-positive endothelial cells within lymphatics remain, despite the decrease in LYVE1 labeling. J. The appearance of smooth muscle actin-positive smooth muscle cells at the collecting lymphatic interface. The images are from references (562, 597, 1122) and reproduced here with permission.

Fig. 18.

Lymphatic networks of the diaphragm. A. Subpleural lymphatics in the diaphragm from a 23-week old rat, stained with 5’Nase and viewed by light microscopy. Scale bar = 200 μm. L= lymphatic capillaries; C = collecting lymphatic vessel. The arrows indicate circular smooth muscle on the collecting lymphatic. B. Lyve1 immunolabeling of lymphatic lacunae, a lattice-like network featuring irregular and wide shapes, of the peritoneal side of the rat diaphragm. Scale bar = 50 μm. The images are reproduced from references (795, 973) with permission.

Fig. 19.

Mouse tracheal microvascular and lymphatic vessel networks, and changes in response to M. pulmonis infection. A. Blood vessel (green) and lymphatic (red) networks in a flat whole mount of the trachea from a pathogen-free C57BL/6 mouse. Capillaries (arrows) cross the cartilage, but lymphatics do not cross. B. After 7 days of M. pulmonis infection, capillaries (arrows) crossing the cartilage are widened. C. After 14 days of M. pulmonis infection, the blood vessels appear larger, and abundant lymphatic sprouts (arrows) are apparent. Panels D-F show enlargements of the boxed regions in panels A-C. In panel D, Lyve1-positive lymphatic sprouts are absent, but there is Lyve1 expression on some leukocytes (arrows). E. An influx of leukocytes, many labeling for PECAM-1 (short arrows) accompanies the vessel changes. The large arrows indicate lymphatic sprouts. F. Enlarged blood vessels and abundant lymphatic sprouts (arrows) are present. The scale bar for the upper panels = 200 μm, and for the lower panels = 50 μm. Reproduced from reference (64) with permission.

Fig. 20.

Scanning electron micrographs of corrosion casts of lymphatics in the rat lung visceral pleura. A. In the first image (320× magnification) he initial lymphatics are flat and ribbon-like (termed prelymphatics in the original paper; PL). These connect to conduit lymphatics (CL, denoted by arrowheads). B. In the second image (640 × magnification), the arrows denote the connections between the flat, ribbon-like lymphatics and conduit lymphatic vessel. Reproduced from reference (17) with permission.

Fig. 21.

A cross-sectional view of a secondary valve in a pulmonary lymphatic vessel. The pair of leaflets originates along the circumference of the inner lymphatic wall, projecting to the lumen of the vessel. Reproduced from reference (583) with permission.

Fig. 22.

A schematic diagram of lymphatics in the kidney, in relation to the local microvasculature and nephrons. Intralobular lymphatic capillaries (IaL) originate near renal tubules (T), renal corpuscles (RC), or afferent arterioles (AA). These lymphatics feed into interlobular lymphatics (IeL). The capsular lymphatics (CL) receive lymph from the perforating lymphatics (PL) in the superficial cortex, and from communicating lymphatics (CmL) that follow arteries (A) or veins (V) that occasionally pierce the renal capsule. The interlobular and communicating lymphatics both drain into arcuate lymphatics (AL) with valves. The arcuate lymphatics coalesce into interlobar lymphatics (IL), which drain into the hilar lymphatics, which are contractile collecting lymphatics. Reproduced from reference (777) with permission.

Fig. 23.

Schematic diagrams of early development of the mammalian lymphatic vasculature and different origins of the organ-specific lymphatic vessels. A. Sagittal view of the key steps during the formation of the first lymphatic structure-lymph sac along the cardinal vein (CV) from E9.0 to E11.5 in the mouse embryo. Around E9.5, the transcription factors CoupTFII and Sox18 induce Prox1 expression in a subpopulation of venous ECs in the CV and intersomitic vessels (ISV). The appearance of the Prox1-expressing LEC progenitors indicates that LEC specification has commenced. Most of those progenitors start to leave the CV and ISV and migrate in the surrounding mesenchyme in response to VEGFC gradient. The differentiating LECs maintain the expression of Vegfr3 mediated by Prox1. As LEC migrate and proliferate in an interconnected manner, they assemble together to form lymph sacs around E11.5. B. Both the venous derived LEC (green) and non-venous derived LEC (light green) contribute to the formation of the lymphatic vasculature in the dermis, the heart, and the mesentery while the lymph sacs are formed by only the venous derived LEC. C. As LEC proliferate and sprout out of the lymph sacs, they start to remodel into the collecting lymphatic vessels and the lymphatic capillaries around E14.5. In the course of the maturation of the lymphatic vessel network, the collecting lymphatic vessels form intraluminal valves to prevent lymph backflow and recruit smooth muscle cells to the outside of the vessels to facilitate pumping during lymph transport. The lymphatic capillaries develop button-like junctions to serve as the primary valves for fluid and cell entry into the lymphatic vessels.

Fig. 24.

Representative images of initial lymphatic network patterns in adult rat mesenteric tissues. PECAM labeling identifies both lymphatic and blood endothelial cells across the hierarchy of intact networks. Lymphatic vessels are distinguishable based on a lighter labeling intensity and increased diameter. A) Image of a microvascular network containing both lymphatic and blood vessels. B) An image of a microvascular network region containing only lymphatic vessels. Note even in the lymphatic only region, disconnected endothelial segments characteristic of blood capillary sprouts can be observed. C) Example of apparent blood and lymphatic capillary patterning coordination. D) Example of an apparent blood-to-lymphatic capillary connection. Scale bars: A, B = 500 μm, C = 100 μm, D = 200 μm.

Fig. 25.

Representative image of simultaneous lymphatic and blood microvascular network growth in the same adult rat mesenteric tissue. The angiogenic blood microvascular network (top) and the lymphangiogenic lymphatic network (bottom) appear to be growing toward the same avascular interstitial space. This example motivates the emerging area of lymphatic biology research focused on the common cellular and molecular dynamics involved in the coordination of growth between the two systems. Scale bar = 500 μm.

Fig. 26.

The Starling Forces and microvascular leakage. The Starling forces include the hydrostatic and osmotic pressures that drive fluid flow across the microvascular wall. A. The net hydrostatic pressure gradient, determined as the capillary hydrostatic pressure (P_c) minus the surrounding tissue’s interstitial hydrostatic pressure (P_i) favors fluid flux out of the capillary. Note that P_c, which is determined by the upstream arterial and downstream venous hydrostatic pressures, decreases along the length of the capillary when moving away from the arterial side (left) and getting closer to the venous side (right), while p_i. is equivalent along the entire length of the capillary. B. The capillary osmotic pressure (∏_c) is determined primarily by plasma proteins, while the interstitial osmotic pressure (∏_i) is determined by the protein content in the interstitial space. The osmotic (or oncotic) pressure gradient, determined by the difference between ∏_c and ∏_i, favors fluid entry into the capillary, and is generally constant along the length of the capillary. C. The resulting net fluid flux when considering both the hydrostatic and osmotic pressure gradients favors extravasation, although the flux decreases along the length of capillary. D. The current equation utilized to describe these forces also includes additional factors, including the hydraulic conductivity of water (L_p), the surface area for exchange (S), and the reflection coefficient for plasma proteins (σ). These factors take into account changes that may occur due to increased blood flow and capillary recruitment (which affects S), and changes in microvascular wall integrity (which affects L_p and σ).

Fig. 27.

CCL21 depositions located in specific regions of initial lymphatic endothelial cells. A. Confocal image rendering of triple labeling for Lyve1, CCL-21, and VE-cadherin of a mouse ear initial lymphatic show the distinct sites of CCL21 deposition. Panel B shows a closer view. Reproduced from reference (1050) with permission.

Fig. 28.

The pie chart shows the protein composition of rat mesenteric lymph described in the proteomic study by Mittal et al (705). The percentages are based on the number of identified, non-redundant proteins, classified into their functional groups. Reproduced from reference (705) with permission.

Fig. 29.

Action potentials measured from human mesenteric lymphatic vessels. A. The frequency distribution shows measurements of resting potential from 18 lymphatic smooth muscle impalements from 10 different vessels. B. The lower trace shows changes in membrane potential over time, including action potentials. The upper trace shows contractions of the lymphatic vessel over the same time frame. These occur immediately after the start of each action potential. C. The two traces show greater detail of the changes in membrane potential and force. Note the transient hyperpolarizations that occur just prior to the upstroke of the action potential. This figure is reproduced from reference (1062) with permission.

Fig. 30.

Impact of increasing preload at constant afterload on contractions of isolated rat mesenteric lymphatic vessels. A. Time course of lymphatic pressure (P_L, black trace) and diameter when the inflow pressure (P_in, blue trace) is raised to various levels while the outflow pressure (P_out, red trace) is held constant. After each step increase in Pin, the frequency of the phasic contractions initially increases and gradually becomes slower. The amplitude of the phasic contractions initially decreases but then becomes larger (arrow). In panel B, pressure-volume (P-V) loops were plotted from the same data. The blue trace represents three consecutive contraction cycles prior to the upward steps in P_in, at the time point indicated by the blue dot in panel A. The black traces show single contraction cycles at the times indicated by the black dots shown in panel A, which correspond to the time points immediately after the upward steps in P_in. The gold traces show single contraction cycles corresponding to the times indicated by the gold dots in panel A, each about 1 min after the upward pressure step. Evaluation of the end-systolic P-V relationship (ESPVR) between the black and gold traces shows a leftward shift, while the end-systolic P-V relationship (EDPVR) was unchanged, indicating an increase in lymphatic contractility. This figure is modified from reference (961) with permission.

Fig. 31.

Impact of increasing afterload at constant preload on contractions of isolated rat mesenteric lymphatic vessels. A. Time course of lymphatic pressure (P_L, black trace) and diameter when the outflow pressure (P_out, red trace) is raised to various levels while the inflow pressure (P_in, blue trace) is held constant. The open circles denote spike artifacts in the P_L recording due to table vibration of the tip touching the vessel wall. The dotted horizontal line indicates the level of the ESD for the initial phasic contraction after the step increase in P_out. The solid horizontal line indicates the ESD for the 8^th - 12^th phasic contractions.. B. P-V plots were drawn for the each of the pressure steps in panel A. The P-V loops corresponding to each pressure step are plotted. The color-coding corresponds to the two phasic contractions prior to the upward pressure (dark brown) proceeding to the last phasic contraction prior to the downward pressure step (yellow). Linear fits to the ESPVR are shown for the first P-V loop after the pressure step (ESV early) and the last P-V loop (ESV late). The shift in ESPVR indicates an increase in lymphatic contractility. This figure is modified from reference (249) with permission.

Fig. 32.

Signaling Cascades leading to contraction in lymphatics. Several ion channels affect membrane potential and allow for transient increases in intracellular free Ca²⁺ ([Ca²⁺]_i), which subsequently can activate signaling pathways that cause phasic and tonic contractions. Plasma membrane channels that contribute to oscillations in membrane potential (“Oscillators”) include T-type Ca²⁺ channels (Cav3.2), HCN channels, and CaCl channels. Voltage-gated channels that allow for rapid influx of Na⁺ and Ca²⁺ to generate action potentials include Nav1.3 and Cav1.2. Hyperpolarizing channels include K_ATP and BK channels. Release of Ca²⁺ from the sarcoplasmic reticulum involves IP₃-receptor channels and possibly ryanodine-receptor channels. Elevations in [Ca²⁺]_i allow for activation of troponins and binding of Ca²⁺ to calmodulin. Troponins facilitate brief actin-myosin interactions that cause phasic contractions. Ca²⁺-calmodulin activates myosin light chain kinase (MLCK), causing specific phosphorylation of myosin light chains (MLC) allowing for sustained interaction of myosin with actin and generation of tone. This pathway may be modulated by Ca²⁺-independent signaling by CPI-17 or ROCK, which inhibit the MLC phosphatase, which in turns allows for sustained phosphorylation of MLC.

Fig. 33.

Changes in Ca²⁺-dependent fluorescence tension of an isolated rat thoracic duct in response to stretch. The arrows depict the time points at which the vessel was stretched. Reproduced from reference (981) with permission.

Fig. 34.

Determination of solute permeability of isolated, cannulated collecting lymphatic vessels. A. Mouse mesenteric lymphatic vessels were isolated and cannulated onto glass micropipettes. The inflow pipette (left) is a “theta” pipette with a septum that allows for perfusion with solutions from two different reservoirs, indicated by the blue and green colors (i). Flow is controlled by raising inflow pressure in either the blue (no tracer) or green (fluorescent tracer) reservoirs. When tracer is infused, it initially is only observed in the lumen of the vessel (ii), but over a short amount of time some of the tracer leaks across the vessel wall into the surrounding bath (iii). Panel B shows a brightfield image of a cannulated lymphatic (top), a fluorescent image during perfusion with physiological salt solution with no tracer (middle), and during perfusion with solution containing fluorescent albumin (bottom). The red background in the fluorescent images is due to an infrared filter that allows for measurement of diameter throughout recording. C. A trace of the fluorescence intensity of the vessel and surrounding area is recorded with a photometer. Initially, baseline fluorescence intensity is recorded, corresponding to image (i) in panel A. When the fluorescent albumin is infused, corresponding to image (ii) in panel A, there is a step increase in the intensity, followed by a gradual increase (iii) due to the flux of the tracer across the vessel wall. After washout, the fluorescence intensity returns to baseline. The initial rise (ii in Panel C) and the slope (iii in panel C) represent the initial concentration difference across the vessel wall and the solute flux of the tracer, which along with the area of the vessel wall can be utilized to solve for the solute permeability coefficient using Fick’s First Law of Diffusion. Reproduced from reference (958) with permission.