The lymphatic system, which is composed of four components—lymphatic vessels, lymph fluid, lymph nodes or lymphoid aggregates, and lymphocytes or immunocytes—is a distinctive vasculature different from and yet similar to the blood vasculature. It is an integral component of the plasma–tissue fluid–lymph circulation (the “blood–lymph loop”), the key lipid “absorbent,” and the center of the immunoregulatory network.² Lymphology—or fashioned after current omes and omics terminology, “lymphatomics”—is the study of lymphatic system biology in health and disease.

Although there were scattered observations on lymphatic vessels and lymph dating back to the ancient Greeks, Gaspar Aselli of Padua is credited with the discovery of the system of chyliferous vessels of the mesentery, glistening after a fat meal in a living dog. His posthumous publication appeared in 1627, the same year that Harvey described the blood circulation.^3,4 Because of the difficulties in accessing and visualizing the lymphatic vessels during life, their collapse after death, and the meager physiologic understanding of the circulation of lymph, it was only during the past century, especially the past half-century, that the International Society of Lymphology founded lymphology as a distinct discipline in 1966. It was then that the integrated function of the four components of the lymphatic system—no longer viewed as “lymph nodes held together by strings”—was fully recognized. The peripheral and central lymphatic channels and nodes could be visualized in the living human by oily contrast lymphography, and the lymphatic system’s characteristic constellation of disorders of swelling, scarring, malnutrition, immunodysregulation, and disturbed angiogenesis gained attention.^2,5,6 Yet for centuries, clinicians had already viewed the lymphatic system as the stage on which key events in development, progression, and containment of cancer and of infections such as tuberculosis take place.^7,8 Recent advances in molecular lymphology (i.e., the discovery of lymphatic growth factors, endothelial receptors, transcription factors, genes, and highly specific immunohistochemical markers) and in clinical lymphology (refined tools for noninvasive dynamic lymphatic and soft tissue imaging, advanced microsurgical techniques, and putative therapeutic agents for lymphangio- and hemangiomodulation) are providing new opportunities for translational lymphology (i.e., taking these advances from “bench to bedside”).^9,10,11,12

Macroscopic Anatomy

Lymphatic vessels generally accompany venous trunks throughout the body except in the central nervous system, hepatic sinusoid, and cortical bony skeleton, where peri(blood)vascular spaces serve the function of prelymphatic vessels ^5,13,14,15 (Figure 3-1). Lymph from the lower torso, including the legs, pelvis, and viscera, is carried by the thoracic duct to the left subclavian–jugular venous junction (see Figure 3-1). Lymph from the head and neck and arms enters the central veins independently or by a common supraclavicular system. Numerous interconnections exist as well as significant topographical variants. The bulk of cardiac and pulmonary lymph along with transdiaphragmatic intraperitoneal fluid drains into substernal mediastinal collecting channels, which join to form the right lymphatic duct and empty independently into the central veins in the right neck.^5,9 Intestinal lymph, transporting cholesterol, long-chain triglycerides as chylomicra, and fat-soluble vitamins, flows retroperitoneally to the aortic hiatus to form the multichannel cisterna chyli.¹⁶ There, the bulk of hepatic lymph including contributions from the perisinusoidal space of Disse flows countercurrent to portal venous blood to join lymph flowing from the lower half of the body to form the thoracic duct, which courses through the posterior mediastinum crossing from the right to the left side of the chest to enter the systemic veins in the left neck. In addition, an extensive system of superficial lymphatic channels extends over the surface of the entire body, draining into communicating watersheds, regional lymph nodes, and ultimately the deep lymphatic system.

Microscopic Anatomy

The lymphatic vasculature is composed of a hierarchal network of initial and collecting lymphatic vessels that exhibit molecular, cellular, and functional differences.⁷ Initial lymphatic vessels consist of a single layer of oak leaf–shaped endothelial cells with end-to-end or overlapping junctions. They exhibit a discontinuous distribution of tight junction proteins at cell-to-cell interfaces and open gaps between endothelial cells in certain tissues.^16,17,18 Although a basement membrane is scant or lacking all together around initial lymphatic vessels, they are connected to the extracellular matrix by fibrillin containing anchoring filaments.^15,19,20 It is widely accepted, but not clearly proven, that anchoring filaments pull apart overlying lymphatic endothelial cells in response to increased interstitial fluid pressure to facilitate the uptake of fluid through gaps created between the cells.^19,20,21 Recently, a mechano- transduction mechanism has been proposed whereby anchoring filaments, attached to integrins on the abluminal surface of initial lymphatic vessels, activate intracellular signaling cascades in response to increased interstitial fluid.²² Therefore, anchoring filaments may modulate the uptake of interstitial fluid through molecular signaling in addition to pulling apart endothelial cells.²² Elastin fibers in the interstitium may also act as “guide wires” for the movement of fluid, macromolecules, and cells in the interstitium.²³

In contrast to initial lymphatic vessels, collecting lymphatic vessels are composed of elongated endothelial cells, exhibit a continuous distribution of tight junction proteins at cell–cell interfaces, and are surrounded by a well-defined basement membrane.^16,17,18 Furthermore, intraluminal bicuspid valves are present consisting of connective tissue between two layers of endothelial cells. Valves partition collecting lymphatic vessels into discrete contractile segments, termed lymphangions, which are surrounded by smooth muscle and contract to actively transport lymph through regional lymph nodes to the central lymphatic–venous junctions.¹⁵

Lymphatic Markers

Until recently, lymphatic vessels were distinguished from blood vessels largely by basic morphologic and ultrastructural characteristics. However, the discovery of distinctive lymphatic markers has facilitated the precise identification of lymphatic vessels in tissue sections and whole-mount preparations from normal as well as pathologic specimens (reviewed by Van der Auwera and coworkers²⁴; Figure 3-2). One of the most commonly used markers is LYVE-1, a receptor for the glycosaminoglycan hyaluronan. LYVE-1 is expressed by a subpopulation of macrophages and initial lymphatic vessels in normal as well as pathologic tissues.²⁵ However, LYVE-1 expression markedly decreases as initial lymphatic vessels transition to collecting lymphatic vessels.^26,27 Therefore, alternative markers are required to detect collecting lymphatic vessels. Initial and collecting lymphatic vessels express the transcription factor Prox1; however, its nuclear localization does not outline vessel structures.²⁴ It has been suggested by members of the First International Consensus on the Methodology of Lymphangiogenesis Quantification in Solid Human Tumors that markers such as podoplanin be included as a co-stain with Prox-1.²⁴ Podoplanin is a transmembrane glycoprotein expressed on the surface of lymphatic vessels and is recognized by the human antibody D2-40.²⁴ Additional lymphatic markers include the receptor tyrosine kinase vascular endothelial growth factor 3 (VEGFR3) and nucleotide salvage pathway enzyme 5′ nucleotidase.²⁸ Although these markers can readily distinguish lymphatic vessels from other cell types, they can be expressed by blood vessels in certain tissues (i.e., LYVE-1 by high endothelial venules and hepatic sinusoidal endothelium) or in pathologic settings (i.e., VEGFR3 by blood vessels in tumors).

Lymphatic System Development

Although much is known about the anatomy and physiology of the lymphatic system, its precise embryonic origin has been a subject of controversy for more than 100 years.^2,29 Much of this controversy has centered on the competing centrifugal and centripetal theories of lymphatic development. The centrifugal theory proposes that lymph sacs are the earliest anlage of the lymphatic system, arise from central embryonic veins, and give rise to the entire lymphatic vasculature of the body by a process of centrifugal sprouting.^30,31,32 The alternative centripetal theory proposes that numerous isolated lymphatic anlagen arise in the mesenchyme, fuse, and elongate to form lymphatic vessels.³³

Currently, the most accepted model of lymphatic development conforms to the centrifugal theory and is separated into the processes of lymphvasculogenesis, lymphangiogenesis, and remodeling (see sections below). Lymphvasculogenesis, the de novo formation of lymphatic structures by precursor cells from veins and mesenchyme, contributes to the formation of lymph sacs and vessels.³⁴ Lymph-angiogenesis, the sprouting of lymphatic endothelial cells from preexisting lymphatic vessels, is thought to give rise to a primitive network of lymphatic vessels that matures during the remodeling phase of lymphatic development.²⁷ (Figures 3-3 and 3-4).

Although recent molecular findings have bolstered the centrifugal theory, a centripetal component has been demonstrated to serve a role in the development of the lymphatic vasculature in chicken embryos and tadpoles. However, whether lymphangioblasts contribute to the development of the mammalian lymphatic system remains controversial.^34,35,36,37 Nevertheless, lymphangioblasts have been reported to contribute to pathologic lymphangiogenesis in mammals.³⁸

Despite an incomplete understanding of the embryonic development of the lymphatic vasculature, numerous genes and some chromosomal abnormalities have been identified that serve crucial functions in the sequential steps of lymphatic development (Tables 3-1 and 3-2).

Lymphvasculogenesis

Lymphvasculogenesis contributes to the development of lymphatic vessels and is the driving force behind the development of lymph sacs, the first obvious lymphatic structures in embryos. There are six lymph sacs in human embryos, two paired (jugular and posterior), and two unpaired (retroperitoneal and cisterna chyli).³² Of these, the jugular lymph sacs have been the most extensively characterized.

Jugular lymph sacs develop partly by the budding of lymphatic endothelial precursor cells from jugular veins. Observation on the development of the lymphatic system in pig embryos led Sabin to propose that the jugular lymph sacs arise from veins.³⁰ More recently, the expression pattern of the transcription factor Prox-1 (master regulator of lymphatic endothelial cell differentiation) during murine embryonic development has supported the venous origin of the jugular lymph sacs.^39,40 The transcription factor Sox18 drives Prox-1 expression by a subset of venous endothelial cells that bud from the cardinal vein in response to VEGF-C.^39,40,41,42 These budding cells form the jugular lymph sacs.^39,40,42 It has also been reported that cells expressing leukocyte and lymph-endothelial cell markers from the mesenchyme fuse with jugular lymph sacs in mouse embryos, suggesting a dual origin of these early lymphatic structures.⁴³ Molecular characterization of lymphatic development in chicks has also supported both a venous and mesenchymal origin of the jugular lymph sacs.³⁴

Although the development of the jugular lymph sacs has been extensively re-analyzed using molecular markers of the lymphatic system, the formation of the posterior and retroperitoneal lymph sacs as well as the cisterna chyli has not. Current understanding of the development of these structures is based on results obtained by basic histological techniques. The paired posterior lymph sacs develop near the sciatic veins whereas the retroperitoneal (mesenteric) lymph sac develops near the renal vein by the coalescence of small veins, which subsequently separate from the blood vasculature.^30,31,32,44 The retroperitoneal sac eventually connects to the cisterna chyli, which arises adjacent to inferior vena cava.³²

Lymphangiogenesis

Observations on living frog larva and transparent chambers in the rabbit ear revealed that lymphatic vessel networks expand by sprouting from preexisting lymphatic vessels, a process termed lymphangiogenesis.^45,46 Although lymphangiogenesis has been studied for more than a century, there has been a recent explosion of interest in this phenomenon because of the discovery of growth factors that stimulate this process. VEGF-C, a ligand for VEGFR-2 and -3, was the first lymphatic growth factor discovered.⁴⁷ The robust lymphangiogenic effect of VEGF-C was demonstrated by avian chorioallantoic membrane assays and transgenic overexpression in mice.^48,49 Indeed, Vegfc+/-, Chy-3 (Vegfc hemizygotes), and Chy (Vegfr3) mutant mice all exhibit lymphangiogenesis defects.^42,50,51.

Although VEGFC is an essential lymphatic growth factor, it does not act alone. The growth factors VEGF-D, insulin-like growth factor 1 (IGF-1), IGF-2, platelet-derived growth factor BB (PDGF-BB), HGF, angiopoietin-1, angiopoietin-2, and fibroblast growth factor 2(FGF-2) have all been demonstrated to stimulate lymphangiogenesis; however, the function of all these genes serve during the development of the lymphatic system has not been delineated.^{52,53,54,55,56,57,58} Furthermore, natural antagonists of lymphangiogenesis have been identified, suggesting that the balance between pro- and antilymphangiogenic factors could serve an important function during development and in pathologic settings (Table 3-3).

For the blood and lymphatic vascular systems to function as dual systems, it is critical for the two to be separated except at a few specific anatomical locations. Syk and the adapter molecule SLP-76 are proposed to inhibit the formation of aberrant connections between blood and lymphatic vessels during embryonic development.⁵⁹ Furthermore, fasting-induced adipose factor (fiaf) is required to maintain separation of the blood and lymphatic vasculatures during postnatal development.⁶⁰ Despite these observations, the repulsive mechanisms preventing the intermingling of blood and lymphatic vessels remain unclear.

Remodeling of the Lymphatic Vasculature

The processes of lymphvasculogenesis and lymphangiogenesis produce an immature network of lymphatic vessels that subsequently remodels into a hierarchal pattern of initial and collecting vessels (see Figures 3-3 and 3-4). Remodeling involves lymphangiogenesis, lymphatic vessel pruning, and the acquisition of a collecting vessel phenotype (development of valves and recruitment of smooth muscle cells) by specific lymphatic vessels. Sabin first described remodeling of the lymphatic vasculature more than 100 years ago in the skin of pig embryos.³¹ More recently, lymphatic remodeling was shown to occur postnatally in the skin of mice and to depend on the transmembrane ligand ephrinB2.²⁶ Mice expressing a mutant form of ephrinB2 display chylothorax, valveless collecting lymphatic vessels, and remodeling defects of the dermal lymphatic vasculature.²⁶ Interestingly, angiopoietin-2 (Ang2)–deficient mice exhibit similar defects of the lymphatic vasculature.^27,61

Ang2 is a ligand for the receptor tyrosine kinase Tie2 and is required for proper development of the blood vasculature in mice.⁶¹ Ang2-/- mice also display chylous ascites, peripheral lymphedema, and hypoplasia of the lymphatic vasculature.⁶¹ Furthermore, lymphatic vessels in Ang2-/- mice fail to mature resulting in a severe deficiency of collecting lymphatic vessels.^27,47 There is also a dramatic reduction in the number of lymphatic valves in the skin of Ang2-/- mice.²⁷

Foxc2 is a member of the forkhead family of transcription factors and was originally demonstrated to be involved in the development of the lymphatic system when mutations in FOXC2 were identified in patients with the disorder lymphedema–distichiasis.⁶² Lymphedema–distichiasis (LD) is characterized by pubertal onset of lymphedema, hyperplastic lymphatic vessels, and an extra row of eyelashes (distichiasis).⁶³ Imitating the human condition, Foxc2+/- mice have an extra row of eyelashes and hyperplastic lymphatic vessels.⁶⁴ Functional studies using Foxc2+/- mice suggested that collecting lymphatic vessels lacked valves or that valves were incompetent.⁶⁴ Analysis of Foxc2-/- mice revealed that Foxc2 is required for the development of lymphatic valves and to prevent the recruitment of smooth muscle cells to lymphatic vessels.⁶⁵

Despite the identification of mutant mice that lack lymphatic valves, mechanisms of valve development have not been fully elucidated. Ranvier (1865) proposed that valves arise when lymphatic sprouts fuse with recipient lymphatic vessels. Later, Kampmeier (1928) identified a similar mechanism and described it, as well as two other forms of lymphatic vessel valvulogenesis.⁶⁶ The first form of valvulogenesis proceeds by the growth of an endothelial extension along a recipient vessel (Figure 3-5). The extension acquires a lumen and invades the recipient vessel to form the valve. The second form of valvulogenesis takes place at the junction of two lymphatic vessels subsequent to the union of the two vessels (see Figure 3-5). These valves begin as thickened shelves of endothelium that elongate in the direction of lymph flow. After extensive proliferation, the endothelial cells form the mature valve. The third form of valvulogenesis proceeds by the formation of an endothelial girdle, which transforms into a valve (see Figure 3-5).

Although Kampmeier was a proponent of the centripetal theory, he noted that valve development proceeds in an anterior to posterior direction and in a centrifugal manner. Additionally, he noted variability in the number and position of lymphatic valves.⁶⁶ Despite the importance valves serve in the functioning of lymphatic system, these models of valvulogenesis have almost been entirely ignored or forgotten. Reevaluation of these models using immunohistochemical techniques is necessary to confirm these models of valve development.