Normal Structure, Function, and Histology of Lymph Nodes

Toxicologic Pathology, 34:409–424, 2006 C by the Society of Toxicologic Pathology Copyright  ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1080/01...
Author: Phyllis Cross
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Toxicologic Pathology, 34:409–424, 2006 C by the Society of Toxicologic Pathology Copyright  ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1080/01926230600867727

Normal Structure, Function, and Histology of Lymph Nodes CYNTHIA L. WILLARD-MACK Huntingdon Life Sciences, East Millstone, NJ, USA, 08875-2360 ABSTRACT Lymph nodes are traditionally regarded as having three compartments, the cortex, paracortex and medulla. B and T cells home to separate areas within these compartments, interact with antigen presenting cells, and undergo clonal expansion. This paper provides structural and functional details about how the lymph node brings lymphocytes and antigen presenting cells together. The concept of the lymphoid lobule as the basic functional and anatomic unit of the lymph node is developed and utilized to provide a framework for understanding lymph node pathobiology. Understanding the histomorphologic features of the lymphoid lobule and the role of the reticular meshwork scaffolding of the lymph node and how these related to the cortex, paracortex and medulla provides a unique approach to understanding lymph node structure and function. Keywords. Lymphoid lobule; reticular meshwork; deep cortical unit; paracortical sinus; sinuses; vasculature.

INTRODUCTION Lymph is derived from interstitial fluid and originates in the interstitial spaces of most of the body’s tissues. A vast system of converging lymphatic vessels funnels lymph to the thorax where it is returned to the circulation via the thoracic duct. When foreign antigens invade the body, antigenic material, antigen presenting cells known as dendritic cells (DCs) and inflammatory mediators generated by local immunological activity at the site of infection are all picked up by the lymphatic vessels and swept along in the flow of lymph. The system of lymphatic vessels has been called an “information superhighway” because lymph contains a wealth of information about local inflammatory conditions in upstream drainage fields (von Andrian and Mempel, 2003). At many sites along the lymphatic highways where lymphatic vessels converge, lymph flows through soft, pale tan, rather lumpy looking lymph nodes that contain large numbers of lymphocytes, macrophages and antigen presenting cells (APCs) (Tilney, 1971). Mice have 22 identifiable lymph nodes (Van den Broeck et al., 2006) while humans have about 450 (1989). Inside the lymph nodes, APCs and na¨ıve lymphocytes are brought together to initiate primary immune responses (Kaldjian et al., 2001); APCs display antigens to lymphocytes, reactive lymphocytes undergo clonal expansion to produce new lymphocytes and plasma cells, and the resulting plasma cells secrete antibodies into the lymph. These immunological processes take place in a specialized stromal structure called the reticular meshwork that supports, guides and organizes interactions between lymphocytes and APCs (Gretz et al., 1996, 1997). Particulate antigens are also filtered out of the lymph and destroyed by macrophages. Lymph nodes consist of multiple lymphoid lobules surrounded by lymph-filled sinuses and enclosed by a capsule. The complex three dimensional lobules and their surrounding sinuses present a variety of appearances in tissue sections depending on the plane of section (Sainte-Marie et al., 1990).

A detailed understanding of lobular architecture is helpful for recognizing the range of variation in normal lymph nodes due to anatomical location, age, diet and antigen exposure, compensating for the inevitable odd plane of section and accurately interpreting lymph node changes. The anatomy of the lymphoid lobule and the role of the reticular meshwork are emphasized in this paper, drawing on classical older works, particularly those of Saint-Marie, Belisle and Peng, on recent studies using modern molecular and imaging techniques and on personal observations made in a contract research laboratory setting. Detailed illustrations of an idealized lymphoid lobule (Figure 1) and lymph node (Figure 2) are provided to illustrate anatomical features discussed in the text.

LYMPH NODE DEVELOPMENT Webster’s New International Dictionary defines a node as “a body part resembling a knot; especially a discrete mass of one kind of tissue enclosed in tissue of a different kind.” Excluding its lymphocytes, which are transient residents, a lymph node is essentially a discrete mass of fibrovascular tissue enclosed within a dilated lymphatic vessel. This intimate juxtapositioning of the vascular and lymphatic systems begins during development when a mesenchymal bud invaginates into a lymphatic sac and compresses the sac lumen against the opposite wall (Bailey and Weiss, 1975; Eikelenboom et al., 1978; Mebius, 2003). The mesenchymal bud does not penetrate the lymphatic endothelium that lines the sac (and later the sinuses that arise from it) and instead becomes covered by the endothelial cells as it expands. The sac wall becomes the capsule and the point of mesenchymal invagination becomes the hilus. The mesenchymal tissue differentiates into lobules and the sac lumen develops into a system of sinuses that surround the lobules. Lymph flows through the sinuses and around the lobules. Lymphoid lobules are thus positioned right in the middle of the information superhighway where they can sample inflammatory mediators and collect DCs carried in the lymph stream and bring them into direct contact with lymphocytes imported from the circulation.

Address correspondence to: Cynthia L. Willard-Mack, Huntingdon Life Sciences, P.O. Box 2360 Mettlers Road, East Millstone, New Jersey, USA, 08875-2360; e-mail: [email protected]

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FIGURE 1.—Lymphoid lobule: The simplest possible lymph node containing a single lymphoid lobule is depicted in this illustration. Lymphocytes and other migratory cells have been omitted so that the underlying fibrovascular framework can be appreciated. The lobule has a bulbous apex and a base of slender medullary cords. It projects into and fills the lumen of a dilated lymphatic sac and is anchored by its blood vessels in the hilus. The lumen of the encapsulated lymphatic sac is divided into a system of sinuses that surround the lobule. Lymph from the afferent lymphatic vessel spreads over the lobule’s apical surface in the subcapsular sinus, moves down its sides through lateral transverse sinuses (see Figure 2) and then flows through medullary sinuses surrounding the medullary cords and exits via the efferent lymphatic vessel in the hilus. The sinuses are spanned by a delicate reticular meshwork, indicated here by a lacy background texturing. The lobule contains a denser reticular meshwork, shown here by a darker, more condensed background texturing. The reticular meshwork provides a three dimensional scaffold with spaces for lymphocytes, antigen presenting cells and macrophages to interact. B lymphocytes home to follicles in the superficial cortex where they interact with follicular dendritic cells. Three follicles are depicted by small spheres. Follicles are surrounded and separated by interfollicular cortex. In the deep cortex (paracortex), T lymphocytes home to the deep cortical unit (DCU) where they interact with dendritic cells. The DCU has a center and a periphery. The peripheral DCU and the interfollicular cortex are transit corridors that convey arterioles, high endothelial venules and paracortical sinuses. These structures are suspended in the reticular meshwork and can be seen more clearly in Figure 2 where the meshwork has been omitted. The follicles and central DCU, depicted here as a large sphere, do not contain these structures and their capillary beds (purple) and reticular meshwork are less dense than in the transit corridors.

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FIGURE 2.—Lymph node: An idealized midsagittal section of a small lymph node contains three lymphoid lobules. Each lobule is centered under its own afferent lymphatic vessel. Lymph node compartments: Taken together, the follicles and interfollicular cortex of these lobules constitute the superficial cortex of the lymph node, their deep cortical units constitute the paracortex (or deep cortex) and their medullary cords and medullary sinuses constitute the medulla. Left lobule: Arterioles (red) and venules (blue) are conveyed in the medullary cords. Arterioles arborize in the paracortical cords of the peripheral deep cortical unit (DCU) and interfollicular cortex and give rise to capillary beds (purple). Capillaries are present in the follicles and central DCU but are less dense than in the other areas. They are omitted from the medullary cords for clarity. Capillaries empty into high endothelial venules which condense repeatedly in the interfollicular cortex and peripheral DCU and then transition to medullary venules at the corticomedullary junction (see Figures 10–12). Center lobule: This lobule, with the reticular meshwork superimposed on the vasculature, is shown in Figure 1. Note the paracortical sinuses. The center lobule is separated from the left lobule by a transverse sinus. Right lobule: A micrograph from a rat mesenteric lymph node shows a lobule as it appears in histological section. Densely packed basophilic lymphocytes fill the lobular reticular meshwork. Five cortical follicles give the superficial cortex a lumpy appearance. Small empty paracortical sinuses are easily visible in the peripheral DCU. The medullary sinuses contain macrophages, lymphocytes and erythrocytes.

THE LYMPHOID LOBULE The lymphoid lobule is the basic anatomical and functional unit of the lymph node. The smallest lymph nodes may contain only a few lobules or even just one, while large lymph nodes may contain a great many. Lobules were described in lymph nodes as early as 1975 (Kelly, 1975) although some authors have described them as physiological compartments (Belisle and Sainte-Marie, 1990). Lobules have been mentioned in more recent work (Gretz et al., 1997), but they have not been described in detail and their relevance to understanding lymph node function and pathology is not widely recognized. Current best practice guidelines for the examination of a lymph node call for a detailed examination of the cortex, paracortex and medulla (Haley et al., 2005). These compartments contain specific lobular structures so changes in these compartments reflect alterations in the lobules. The lymphoid lobule is as potentially useful and necessary in understanding

lymph node function and pathology as the hepatic lobule is to understanding liver function and pathology. Lymphoid lobules are arranged side-by-side and radiate capsad from the hilus (Figure 2). Each lobule has a bulbous apex and a base of slender cords that gives it a medusoid appearance (Figure 1). Lobules are anchored in the hilus by their vascular roots but they are otherwise separated from the capsule by the subcapsular sinus. The apex forms part of the nodal cortex and the base forms part of the nodal medulla. The nodal cortex is bilayered and consists of a superficial cortex and a deep cortex. By common convention, pathologists usually apply the term cortex to the superficial cortex and refer to the deep cortex as the paracortex. The (superficial) cortex contains spherical follicles that are surrounded and separated by interfollicular (or diffuse) cortex. The paracortex consists of deep cortical units (DCUs). Each lobule has a single DCU that can be anatomically and functionally divided into a central

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DCU and a surrounding peripheral DCU (Sainte-Marie et al., 1982, 1990). DCUs of adjacent lobules often fuse into large multiunit complexes (Belisle and Sainte-Marie, 1981a). Subcompartmentalization of the lobule creates separate areas for T and B cells to interact with their APCs and to undergo clonal expansion. B lymphocytes home to primary follicles to survey follicular dendritic cells (FDCs). Stimulated B cells proliferate within the follicles forming distinctive germinal centers and the follicles are then referred to as secondary follicles. T lymphocytes home to the paracortex and interfollicular cortex to survey DCs. Stimulated T lymphocytes proliferate in the paracortex and enlarge it but do not produce structures analogous to germinal centers. The peripheral DCU and the interfollicular cortex also serve as transit corridors for lymphocytes migrating to and from the B and T cell areas. Plasma cell precursors produced by B cell proliferation migrate to the medullary cords where they mature and secrete antibodies that are released into the lymph. Each lobule is surrounded by a complex system of lymphatic sinuses that are divided into subcapsular, transverse and medullary sinuses. In large animals, lymph nodes with trabeculae also have trabecular sinuses. A single afferent lymphatic vessel delivers a constant stream of lymph to the subcapsular sinus over each lobule. Lymph spreads through the subcapsular sinus over the lobule’s apex, flows down the sides of the lobule through transverse sinuses and then flows into the medullary sinuses. Lymph from all the lobules drains into a single efferent lymphatic vessel that exits the node at the hilus (Sainte-Marie et al., 1982). Because each afferent lymphatic collects lymph from a different drainage field, each lobule is potentially exposed to a different set of antigens, APCs and inflammatory mediators (Sainte-Marie et al., 1982). As a result of varying immunological stimulation, lobules within the same lymph node may have different levels of immunological activity and the cortical, paracortical and medullary compartments composed of these lobules will not necessarily have a uniform appearance (Sainte-Marie et al., 1982). In particular, the size of the DCUs can vary widely so that the paracortex may have a very uneven appearance. RETICULAR MESHWORK The reticular meshwork is a delicate, porous, sponge-like tissue composed of stellate, spindle shaped or elongated fibroblastic reticular cells (FRCs) and their reticular fibers (Clark, 1962). The entire lymph node is filled with reticular meshwork. It forms the framework of the lobules and it criss crosses the lumens of the sinuses (Moe, 1963) (Figures 1, 3, and 4). The lobular reticular meshwork is composed of stellate FRCs whose processes subdivide the lobule into innumerable narrow channels and interstices that are occupied by lymphocytes, macrophages and APCs. FRCs have large, irregularly oval nuclei and pale cytoplasm and their cell bodies and larger processes can be seen in between and amongst the more basophilic lymphocytes that fill the channels created by their processes. The interstices are 10 to 20 microns wide, wide enough to allow lymphocytes to move through them freely but narrow enough that all the lymphocytes can remain in contact with the reticular cells (Kelly 1975; Gretz et al., 1996, 1997; Kaldjian et al., 2001). The surfaces of FRCs are coated with migration ligands, such as fibronectin, that facilitate lymphocyte adhesion and ameboid migration

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(Ruco et al., 1992; Gretz et al., 1996; Kaldjian et al., 2001). Lymphocytes move through the interstices by adhering to and crawling along the huge surface area of FRCs which serve as highways for the migrating cells (Gretz et al., 1996; Kaldjian et al., 2001). At the periphery of a lobule, lobular FRCs flatten out to form a layer that encloses and defines the lobule and separates it from the surrounding sinuses (Figures 3, 4, and 5). The lobule has been described as a labyrinthine space or chamber because of the complexity of the channels formed by the reticular meshwork (Clark, 1962; Kaldjian et al., 2001). The reticular meshwork that spans the sinuses has thinner, more delicate branches and correspondingly larger interstices than the lobular reticular meshwork (Luk et al., 1973) (Figures 3 and 4). Macrophages known as sinus histiocytes cling to the reticular meshwork like spiders on a web and snare bacteria, cell debris, red blood cells, carbon and other particulates suspended in the lymph as it flows through the meshes of this biological filter (Figure 6). They tend to occur in clusters, especially in transverse sinuses near the capsule, and are relatively infrequent in the subcapsular sinus (Sainte-Marie et al., 1982) (Figure 7). Sinus histiocytes increase in response to the need for particle clearance and may completely fill the sinuses (sinus histiocytosis) (Figure 8). Some sinus histiocytes originate in the tissues and migrate to the sinuses after antigenic stimulation (Grande et al., 1990). Mast cells in the peripheral tissues can also migrate to the sinuses in response to certain hypersensitivity conditions and express chemokines that regulate T cell recruitment (Tedla et al., 1998) (Figure 9). The sinuses are lined by a layer of flattened FRCs. The interface between a lobule and a sinus is a trilaminar membrane formed by the layer of flattened sinusal FRCs, the layer of flattened lobular FRCs and a layer of basment membrane or basal lamina sandwiched between them (Figures 3, 4, and 5) (Moe 1963; Farr et al., 1980; Kaldjian et al., 2001). This thin membrane can be difficult to appreciate by light microscopy, but it prevents lymph, cells and particulates from passively entering the lobules (Anderson and Anderson, 1975; SainteMarie et al., 1982; Gretz et al., 1996). Dendritic cells (DCs) do actively penetrate this barrier, however (see later). FRCs have some characteristics of epithelial cells. They can form flattened sheets that are morphologically indistinguishable from lymphatic endothelium (Ushiki et al., 1995). They express cytokeratins 8 and 18 (Franke and Moll, 1987) and form tight junctions with each other. FRCs are also have characteristics of fibroblasts. They secrete slender strands of extracellular matrix known as reticular fibers. Reticular fibers are composed of a core of collagen fibrils enveloped in a layer of basement membrane (Forkert et al., 1977). Components that have been identified in reticular fibers include collagens type I, III and IV, elastin, entactin, fibronectin, laminin-1, tenascin, vitronectin, and heparan sulfate (Sainte-Marie and Peng, 1986; Gretz et al., 1996; Kaldjian et al., 2001). FRC processes wrap around reticular fibers and enclose them in cytoplasmic tubes that cover 90% of the reticular fiber surface area, thus shielding lymphocytes from coming in direct contact with extracellular matrix components (Moe 1963; Hayakawa et al., 1988). The tubular cytoplasmic processes and reticular fibers within them form a system of miniature conduits or pipes that convey inflammatory mediators and soluble antigens from the sinuses into the interior

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FIGURE 3.—A cross-section of a small medullary cord in this rat mesenteric lymph node is centered around a small blood vessel. The medullary cord is surrounded by a medullary sinus. Branched sinusal fibroblastic reticular cells (FRCs) (arrows) create a loose reticular meshwork in the sinus. Lymph carrying densely basophilic lymphocytes flows through the interstices of the meshwork while sinus histiocytes (H) cling to its branches. Note the flattened nuclei of sinusal and lobular FRCs forming the sinus-lobule border (arrowheads). It is difficult to determine by light microscopy which flattened FRCs are associated with the lobular meshwork and which belong to the sinusal meshwork. 4.—A longitudinal section of a small medullary cord is surrounded by a medullary sinus. The medullary cord contains lymphocytes, plasma cells, macrophages and sections of two capillaries (C). Branched fibroblastic reticular cells (FRCs) (arrows) with long delicate processes create a loose reticular meshwork in the sinus lumen and flattened FRCs (sinus lining cells) line the sinus cavity and simultaneously cover the medullary cord (arrowheads). Some FRCs contribute processes to both the lining layer and the meshwork. Rat mesenteric lymph node.

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FIGURE 5.—At the corticomedullary junction of a rat mesenteric lymph node two paracortical sinuses (PS) filled with densely packed lymphocytes interdigitate with three medullary cords (MC). The sinus-lobule interface (arrow heads) can be seen as a thin eosinophilic line outlining the medullary cords. The paracortical sinuses are discharging their lymphocytes into a medullary sinus at the right side of the field. (HEV = High Endothelial Venule). 6.—Sinus histiocytes remove cells, cell debris and particulate antigens from the lymph as it flows through the sinus system. This filtration capacity is vividly demonstrated in this medullary sinus by sinus histiocytes that have rosetted and phagocytized large numbers of erythrocytes. Phagocytosis of erythrocytes, as in this case, may occur during euthanasia. Rat mesenteric lymph node.

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FIGURE 7.—Sinus histiocytes tend to cluster in transverse sinuses near the capsule. The eosinophilic areas in the transverse sinuses on either side of the lobule near the capsule (arrows) contain increased numbers of sinus histiocytes compared with the more sparsely populated medullary sinuses below the lobule. Rat mesenteric lymph node. 8.—In contrast to Figure 7, both transverse and medullary sinuses surrounding this lobule are filled with histiocytes.

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of the lobule (Sainte-Marie and Peng, 1986; Gretz et al., 1996, 2000; Sixt et al., 2005). A small percentage of the reticular fiber surface area is not covered by reticular cells and is instead in direct contact with APCs and macrophages (Hayakawa et al., 1988) that may allow APCs within the lobule to sample soluble antigens from conduit system (Sixt et al., 2005). Reticular fibers are dense in the medullary cords, peripheral DCUs and interfollicular cortex but are scarce in the follicles and central DCUs (Belisle and Sainte-Marie, 1981a; Sainte-Marie et al., 1982). CORDS The relationship between the reticular meshwork, the lobular blood vessels and the sinuses is fundamentally important to lymph node function and is most easily appreciated in the medulla. Branching medullary arterioles arise from the hilar artery and radiate centrifugally, and condensing medullary venules return centripetally to the hilar vein. A pair of arterioles and venules may run along the central axis of a cylindrical sheath of reticular meshwork surrounded by a dense network of capillaries (Okada et al., 2002), but vessels may also be ensheathed individually (see Fig 3). All the vessels are suspended in the meshwork by pericytic FRCs (Anderson et al., 1976; Gretz et al., 1996, 1997; Crivellato and Mallardi 1997; Okada et al., 2002). The interstices of the meshwork are filled with recirculating lymphocytes. These perivascular lymphocyte sheaths or cords are the basic repeating unit of the lobule (Gretz et al., 1997; Kelly, 1975). In the medulla they are called medullary cords. In the peripheral DCU they have been termed paracortical cords (Kelly, 1975) and they extend into the interfollicular cortex. Medullary cords are usually clearly delineated because their dense cellularity contrasts sharply against the sparse cellularity of the surrounding medullary sinuses. This contrast may be lessened if the sinuses contain large numbers of histiocytes and may be further obscured if the sinuses contain large numbers of lymphocytes (Figure 5). The peripheral DCU and interfollicular cortex are composed of closely packed paracortical cords that are difficult to distinguish individually. Paracortical cords are more numerous than medullary cords because they multiply in tandem with blood vessels which arborize extensively in the paracortex. They are reported to be 100 microns wide and 800–1500 microns long. They surround the central DCU and follicles like staves of a barrel. They become distended and elongated during immune responses and they decrease in diameter if lymphocytes are depleted (Gretz et al., 1997). Following a strong antigenic stimulus, the paracortex can undergo a 3- to 5-fold enlargement in 6 to 24 hours as a result of increased lymphocyte traffic into the lobules and decreased lymphocyte traffic out of the lobules http://www.geocities.com/artnscience/art-notes.html. THE PARENCHYMA (LYMPHOCYTES) Lymphocytes are the parenchymal cells of the lobules. They dominate the histological appearance of lymph nodes and obscure the reticular meshwork. The static appearance of lymphocytes in tissue sections belies their nomadic existence, however. Lymphocytes recirculate continually, entering lymphoid lobules via specialized blood vessels called high endothelial venules and exiting via specialized sinuses called paracortical sinuses. They leave the lymph node in the

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efferent lymph and are returned to the circulation via the thoracic duct to start the cycle again (Gowans and Knight, 1964; Rouse et al., 1984). Thus, quite unlike parenchymal cells in other organs, the lymphoid parenchymal cells move around inside the lymph nodes, relocate from one node to another and even move from one organ to another. The body’s pool of lymphocytes turns over 10 to 48 times every 24 hours http://www.geocities.com/artnscience/index.html. Lymph nodes are essentially information marketplaces where antigen presenting cells, the body’s scouts, come to display information they have gathered about antigens they have encountered in the field and patrolling lymphocytes come to look for evidence that their specific antigen has entered the body. The lobule provides the infrastructure for the marketplace and its activities. The reticular meshwork and its channels and interstices provide spaces for APCs and lymphocytes to meet and mingle and a three dimensional scaffold for them to move around on. T and B cells and their respective APCs meet in separate areas. The vascular and lymphatic systems, the body’s transportation systems, are intimately integrated into the lobule and provide portals for lymphocytes to enter and exit the reticular meshwork. When a lymphocyte encounters its designated antigen, clonal expansion occurs in specific areas of the lobule. This unique arrangement allows a relatively small population of lymphocytes to efficiently and effectively monitor antigens throughout the entire body. HIGH ENDOTHELIAL VENULES High endothelial venules (HEVs) are the gateways intravascular lymphocytes use to immigrate into the reticular meshwork from the closed circulation (De Bruyn and Cho, 1990). HEVs are so named because of their characteristic “high” cuboidal endothelial cells (Figure 10). These specialized endothelial cells bear receptors that bind intravascular lymphocytes as they pass by and facilitate their transmigration into the reticular meshwork (Sasaki et al., 1996). The cascade of adhesion events involved in transmigration is outside the scope of this paper and has been thoroughly characterized and described elsewhere (Anderson et al., 1976; Shimizu et al., 1992; Anderson and Shaw, 1993; Springer, 1995; von Andrian and Mempel, 2003). HEVs are located only in the interfollicular cortex and peripheral DCU (Figures 11 and 12) although HEVs that become trapped between two DCUs when lobules fuse can appear to be within a central DCU (Belisle and Sainte-Marie, 1981a). HEVs condense into progressively larger segments as they descend through the peripheral DCU. Lymphocytes exit an HEV along its entire length but transmigration is heaviest along the largest HEV segments (Okada et al., 2002) deep in the paracortex (Gretz et al., 1997). HEVs lose their high endothelium and revert to regular medullary venules lined by squamous endothelium at the corticomedullary junction (Figure 10). Lymphocyte recruitment in the HEVs can be modulated by remote inflammatory activity. Inflammatory mediators such as MCP-1 and IL-8 are carried to the local lymph node in the lymph. FRCs take up inflammatory mediators and soluble antigen from lymph in the subcapsular sinus and transfer them into the conduit system by transcytosis (Anderson and Shaw, 1993). Cells lining the sinuses have a great abundance of vesicles which are presumably involved in the transport process (Farr et al., 1980; Compton and Raviola, 1985).

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FIGURE 9.—Darkly basophilic mast cells are scattered throughout the subcapsular (SS) and medullary sinuses (MS) in this rat mesenteric lymph node. Mast cells can migrate from the drainage field into the lymph node sinuses via the lymph.

The conduit system conveys them directly to the perivenular spaces around HEVs where the inflammatory mediators can influence both the HEVs and the transmigrating lymphocytes (Gretz et al., 1996, 1997, 2000). These small molecules stimulate upregulation of adhesion molecules and can increase lymphocyte recruitment within minutes (Larsen et al., 1989; Palframan et al., 2001). These chemical messengers also maintain the high cuboidal endothelium phenotype. The plump cuboidal endothelial cells will revert to the typical flattened squamous endothelial cells following ligation of the afferent lymphatic vessels (Hendriks et al., 1987). Conversely, antigenic stimulation increases the height of the endothelial cells and the number of HEVs (Dabak and Ozturk, 2003). PARACORTICAL SINUSES Lymphocytes emigrate out of the lobule and into the sinus system by entering narrow sinuses less than 100 microns wide that finger in between the paracortical cords (Belisle and Sainte-Marie, 1981b; Ohtani et al., 2003). These sinuses originate as blind end cul de sacs in the interfollicular cortex, run through the peripheral DCU and discharge into the medullary sinuses (Figures 5 and 16). They have been termed unit sinuses (Sainte-Marie et al., 1981, 1990), cortical sinuses (Gretz et al., 1997), paracortical sinuses (Kelly, 1975), peripheral sinuses (Okada et al., 2002) and lymphatic labyrinths (He, 1985; Ohtani et al., 2003). They will be referred to as paracortical sinuses in this paper since this term underscores their association with the paracortical cords. When paracortical sinuses are filled with lymphocytes they are seen histologically as irregular, densely basophilic islands

and ribbons of closely packed lymphocytes enclosed by the thin pale line of the sinus-lobule membrane (see Figures 13– 16). At the corticomedullary junction the paracortical sinuses discharge their lymphocytes into the medullary sinuses (Belisle and Sainte-Marie, 1981b) (see Figures 5, 14, and 16). The molecular events controlling lymphocyte egress through the paracortical sinuses are not as well characterized as those controlling lymphocyte entry through the HEVs. Recently, however, sphingosine 1-phosphate (S1P) receptors located on the endothelial cells lining the sinuses have been shown to control the passage of lymphocytes through pores in the sinus wall. Activation of the receptors closes the endothelial gates so that lymphocytes are retained in the cords and the sinuses appear empty. Deactivation of the receptors opens the gates allowing lymphocytes to leave the cords and fill the sinuses (Wei et al., 2005). DENDRITIC CELLS Dendritic cells (DCs) are potent APCs that collect and process antigens from tissues, carry them to lymph nodes and present them to T-cells to initiate primary immune responses (Romani et al., 2001). DCs originate in the bone marrow and then spread to all the tissues of the body except the brain, eyes and testes (Hart, 1997). There are several different populations of DCs (Henri et al., 2001; Yrlid and Macpherson, 2003; Randolph et al., 2005; Villadangos and Heath, 2005), including the Langerhans cells of the dermis and a recently described population in the intestine that endocytizes apoptotic cells (Huang et al., 2000). In the tissues, immature DCs sample the local antigens and then migrate to lymphatic vessels

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FIGURE 10.—The high cuboidal endothelium lining a high endothelial venule in the upper center transitions to flattened endothelium as the vessel leaves the paracortex in the center of the field and enters a medullary cord in the lower right. 11.—A blood-filled high endothelial venule (HEV) descends through the peripheral deep cortical unit (pDCU) and transitions into a medullary venule. It is joined by a smaller HEV (arrows) running in the pDCU under the central deep cortical unit (cDCU).

to be transported in the lymph to the draining lymph node (Randolph et al., 2005). During their journey in the lymph, they lose their ability to collect antigen and gain the ability to present antigen to T cells. These maturing DCs have extensive cytoplasmic processes and are known as veil cells (Romani et al., 2001; Wacker et al., 1990). When they enter the subcapsular sinus, veil cells settle onto the sinus floor, actively migrate through the sinus-lobule membrane and home to the paracortex. Histologically, mature DCs in the paracortex have a broad rim of clear cytoplasm, a centrally located nucleus and abundant cytoplasmic processes that interdigitate with one another. These extensive processes provide large surface areas for contact with lymphocytes (Crivellato et al., 1993). The influx of DCs increases during antigenic stimulation. FOLLICULAR DENDRITIC CELLS The APC that present antigen to B cells are called follicular dendritic cells (FDCs) and they are distinctly different from the DCs that present antigen to T cells. The origin of FDCs has not been definitively established. They may develop in situ from preexisting reticular cells, or they may have a hematopoietic origin (Liu et al., 1996; Cyster et al., 2000). They trap antigen-antibody complexes through their complement and Fc receptors and retain these immune com-

plexes on their cell membranes for more than a year. They may collect antigen:antibody complexes from lymph that is carried into the follicle in the FRC conduit system. FDCs are found only in primary and secondary follicles. They have large irregularly shaped euchromatic nuclei and numerous fine cytoplasmic processes, and they form cellular networks in the center of the follicles (MacLennan, 1994). Their extensive cytoplasmic processes enfold B cells. LYMPHOCYTE INTERACTIONS WITH ANTIGEN PRESENTING CELLS When lymphocytes squeeze between endothelial cells and cross the HEV wall, they enter a narrow perivenular space between the endothelial basement membrane and the surrounding pericytic FRCs called the perivenular channel (Gretz et al., 1997). The perivenular channel receives inflammatory mediators from the conduit system. Lymphocytes spend 10 to 100 minutes in this space and their subsequent behaviour may be modified by exposure to inflammatory mediators during this period. Lymphocytes then move out into the corridors of the paracortical cords by crawling along FRCs (Gretz et al., 1996, 1997). T lymphocytes interact with dendritic cells positioned within the cords while B cells move through the cords to the follicles where they interact with FDCs (De Bruyn and Cho, 1990; Gretz et al., 1996,

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FIGURE 12.—Two high endothelial venules (arrows) are located in the interfollicular cortex immediately below a germinal center (GC) in this rat mediastinal lymph node. 13.—Two paracortical sinuses (PS) filled with densely packed lymphocytes lie on either side of a paracortical cord (all cut in cross section). Lymphocytes enter the paracortical cord by transmigrating across the wall of the high endothelial venule (arrowheads). After interacting with antigen presenting cells, lymphocytes enter the paracortical sinuses through ill-defined apertures and are discharged into the medullary sinuses.

1997). They make meandering random walks through the reticular meshwork at speeds of greater than 25 microns/ minute (Miller et al., 2003). Lymphocytes may spend a few hours to days searching for antigen (Gowans and Knight, 1964) and make contact with hundreds of APCs during their stay in the lobular reticular meshwork. In vivo videomicroscopy and confocal microscopy are providing rich details about the interactions between lymphocytes and APCs and footage of these encounters can be viewed on the web http://www.cbr.med.harvard.edu/labs/vonandrian/. Most lymphocytes do not find their specific antigen displayed on an APC and will eventually leave the lobule via the paracortical sinuses and recirculate to another lymph node to continue their search for antigen. LYMPHOCYTE PROLIFERATION B lymphocytes home to primary follicles where they interact with FDCs for about 24 hours (MacLennan, 1994). When a B lymphocyte encounters its antigen displayed on a FDC in a primary follicle, it is stimulated to undergo clonal expansion. The proliferating cells create a germinal center surrounded by a darker mantle or corona of displaced resting B cells that together are considered a secondary follicle (Figure 18). The germinal center reaction represents a complex interaction of the processes of activation, proliferation, differentiation and death of B lymphocytes (Hollowood and Goodlad, 1998). These processes have been extensively studied and reviewed (Kroese et al., 1990; MacLennan, 1994;

Kelsoe, 1995; Hollowood and Goodlad, 1998). Only the morphological appearance of germinal centers will be discussed here. The germinal center reaction is first visible 3–4 days after antigen exposure when a small group of dividing dark centroblasts appear in the center of primary follicles. On day 5 mitotic figures are present and tingible body macrophages appear giving the germinal center a starry sky appearance (Kroese et al., 1990). A very large number of B cells undergo apoptosis during the proliferation and differentiation processes which are eliminated by tingle body macrophages (Hollowood and Goodlad, 1998; Nakamura et al., 1996) Tingible bodies are phagolysosomes containing apoptotic cell debris. By day 7, lightly staining medium sized centrocytes, the progeny of the mitotic activity, start to accumulate on the capsad (apical) aspect of the germinal center with the darker centroblasts oriented hilad (basal). These two cell populations may be distinctly different or may be indistinguishable from each other depending on the species and the lymph node. The centrocytes are associated with a dense network of FDCs that makes them appear less densely packed than the centroblasts (MacLennan 1994). The peak of the immunological response occurs 7 to 10 days after antigenic stimulation when the germinal center has a basal proliferating dark zone of centroblasts and an apical nonproliferating light zone of centrocytes (Kroese et al., 1990) (Figure 17). If there is no new or ongoing stimulus, the germinal center will remains

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FIGURE 14.—Numerous paracortical sinuses filled with lymphocytes (arrows) are present in the peripheral deep cortical units (DCUs) beneath a multiunit complex of fused DCUs. Tingible body macrophages give the central DCUs a “starry sky” appearance. Rat mesenteric lymph node. 15.—A large number of paracortical sinuses (arrows) are visible in this tangential section through a peripheral deep cortical unit perpendicular to the area indicated by arrows in Figure 14. The sinuses are round to irregularly oval and are filled with densely packed lymphocytes. Note several cross sections of high endothelial venules with empty lumens (arrowheads). Rat mesenteric lymph node.

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FIGURE 16.—Two paracortical sinuses (PS) extend along a paracortical cord (PC) containing a high endothelial venule (arrowhead) and discharge their lymphocytes into a medullary sinus (MS) in the middle of the field. The closely packed lymphocytes highlight sinus histiocytes (H). Lymphocytes spread apart as they work their way through a medullary sinus towards the lower left (arrows). The medullary cords contain large numbers of plasma cells. 17.—A mature germinal center has a basal population of large, closely packed centroblasts (CB) and an apical population of smaller, less closely packed centrocytes (CC). The centrocytes are separated by, and enfolded in, eosinophilic cytoplasmic branches of the follicular dendritic cell network (arrows). Tingible body macrophages (arrowheads) phagocytize apoptotic lymphocytes. The mantle surrounding the germinal center is difficult to discern in this follicle. Rat lymph node.

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FIGURE 18.—Secondary follicle: Proliferating B-cell precursors in a germinal center displace mature B-cells to the periphery where they form a darkly basophilic mantle or corona. Dog mediastinal lymph node. 19.—Medullary cords are distended with plasma cells (arrows), a condition referred to as plamacytosis. Plasma cell precursors produced in secondary follicles migrate to the medullary cords and mature into plasma cells. Antibodies secreted by the plasma cells are picked up in the lymph flowing through the surrounding medullary sinuses and are conveyed to the circulation. Rat mesenteric lymph node.

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like this for 2–3 weeks after antigenic stimulation. Eventually the mitotic activity will subside leaving a small dark zone with little mitotic activity at the bottom and a larger, less densely populated light zone on the top. The centrocytes differentiate into memory B cells and plasma cell precursors (Arpin et al., 1995). Many of the plasma cell precursors migrate to the medullary cords and mature into plasma cells that secrete antibodies into the lymph (Figure 19). The germinal center reaction eventually disappears if there is no new antigenic stimulus. T cells are stimulated to proliferate 1.5 to 2 days after they encounter their antigens displayed on dendritic cells (Stoll et al., 2002). Unlike B cell proliferation, there is little information available in the literature about the histological appearance of T cell proliferation. The appearance of tingible body macrophages and a starry sky appearance in the paracortex are indicative of increased apoptosis and are suggestive of T cell production but may also occur with lymphocytolysis. Increased paracortical size occurs when lymphocyte ingress increases and egress decreases but presumably increased production could also contribute to an increase in the size of this compartment. CONCLUSION Lymph nodes are vital immunological organs. They are easily overlooked and rather featureless structures at the gross level but they are very complex structures at the microscopic level. The familiar cortex, paracortex and medulla are each composed of specific areas of the lymph node’s lobules and sinuses. The lobules lie together within the sinus system like islands in the middle of a stream. The body’s large archipelago of lymphoid islands is united by its nomadic lymphocytes which move back and forth between the lobules in an endless quest for antigens. This unique arrangement creates a very effective and efficient venue for antigen surveillance, lymphocyte production, antibody secretion and lymph filtration. This review paper was oriented toward helping the bench pathologist develop an appreciation of normal lymph node anatomy and function. A wealth of information is available regarding the mechanisms underlying lymphocyte recirculation, antigenic stimulation and proliferation and awaits the reader interested in learning more about the molecular control of lymphocyte activities in lymph nodes. ACKNOWLEDGEMENTS The author would like to thank Robert Maronpot for his invaluable support during manuscript preparation, David Sabio for his outstanding artistic contributions and Diane Creasy for her helpful suggestions about the manuscript. REFERENCES Anderson, A. O., and Anderson, N. D. (1975). Studies on the structure and permeability of the microvasculature in normal rat lymph nodes. Am J Pathol 80, 387–418. Anderson, A. O., and Shaw, S. (1993). T cell adhesion to endothelium: the FRC conduit system and other anatomic and molecular features which facilitate the adhesion cascade in lymph node. Semin Immunol 5, 271–82. Anderson, N. D., Anderson, A. O., and Wyllie, R. G. (1976). Specialized structure and metabolic activities of high endothelial venules in rat lymphatic tissues. Immunology 31, 455–73.

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