Toxicologic Pathology, 34:409424, 2006

Copyright

C by the Society of Toxicologic Pathology

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 A detailed understanding of lobular architecture is helpful

Lymph is derived from interstitial uid and originates in
the interstitial spaces of most of the bodys 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 mate-
rial, antigen presenting cells known as dendritic cells (DCs)
and inammatory mediators generated by local immunolog-
ical activity at the site of infection are all picked up by the
lymphatic vessels and swept along in the ow of lymph. The
system of lymphatic vessels has been called an informa-
tion superhighway because lymph contains a wealth of in-
formation about local inammatory conditions in upstream
drainage elds (von Andrian and Mempel, 2003).
At many sites along the lymphatic highways where lym-
phatic vessels converge, lymph ows through soft, pale tan,
rather lumpy looking lymph nodes that contain large num-
bers of lymphocytes, macrophages and antigen presenting
cells (APCs) (Tilney, 1971). Mice have 22 identiable lymph
nodes (Van den Broeck et al., 2006) while humans have about
450 (1989). Inside the lymph nodes, APCs and nave lym-
phocytes are brought together to initiate primary immune re-
sponses (Kaldjian et al., 2001); APCs display antigens to lym-
phocytes, reactive lymphocytes undergo clonal expansion to
produce new lymphocytes and plasma cells, and the result-
ing 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 ltered
out of the lymph and destroyed by macrophages.
Lymph nodes consist of multiple lymphoid lobules sur-
rounded by lymph-lled sinuses and enclosed by a capsule.
The complex three dimensional lobules and their surrounding
sinuses present a variety of appearances in tissue sections de-
pending on the plane of section (Sainte-Marie et al., 1990).
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: Willard-mackc@princeton.huntingdon.com
409
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 ac-
curately 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 labora-
tory 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
Websters New International Dictionary denes 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 brovascular
tissue enclosed within a dilated lymphatic vessel. This in-
timate juxtapositioning of the vascular and lymphatic sys-
tems 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 dif-
ferentiates into lobules and the sac lumen develops into a
system of sinuses that surround the lobules. Lymph ows
through the sinuses and around the lobules. Lymphoid lob-
ules are thus positioned right in the middle of the information
superhighway where they can sample inammatory media-
tors and collect DCs carried in the lymph stream and bring
them into direct contact with lymphocytes imported from the
circulation.

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410 WILLARD-MACK TOXICOLOGIC PATHOLOGY

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 brovascular framework can be appreciated. The lobule has a bulbous apex and a base of slender medullary
cords. It projects into and lls 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 lobules apical surface in the subcapsular
sinus, moves down its sides through lateral transverse sinuses (see Figure 2) and then ows 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 supercial 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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Vol. 34, No. 5, 2006 STUDY OF LYMPH NODES 411

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 supercial 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 1012). 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 ll the lobular reticular
meshwork. Five cortical follicles give the supercial 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 con-
tain 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 men-
tioned in more recent work (Gretz et al., 1997), but they have
not been described in detail and their relevance to understand-
ing lymph node function and pathology is not widely recog-
nized. 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 compart-
ments contain specic lobular structures so changes in these
compartments reect 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 ap-
pearance (Figure 1). Lobules are anchored in the hilus by their
vascular roots but they are otherwise separated from the cap-
sule 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 supercial cortex and a
deep cortex. By common convention, pathologists usually
apply the term cortex to the supercial cortex and refer to the
deep cortex as the paracortex. The (supercial) cortex con-
tains 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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412 WILLARD-MACK
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). Stimu-
lated B cells proliferate within the follicles forming distinc-
tive 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 lym-
phocytes 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 ma-
ture and secrete antibodies that are released into the lymph.
Each lobule is surrounded by a complex system of lym-
phatic sinuses that are divided into subcapsular, transverse
and medullary sinuses. In large animals, lymph nodes with
trabeculae also have trabecular sinuses. A single afferent lym-
phatic vessel delivers a constant stream of lymph to the sub-
capsular sinus over each lobule. Lymph spreads through the
subcapsular sinus over the lobules apex, ows down the sides
of the lobule through transverse sinuses and then ows 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 affer-
ent lymphatic collects lymph from a different drainage eld,
each lobule is potentially exposed to a different set of anti-
gens, APCs and inammatory mediators (Sainte-Marie et al.,
1982). As a result of varying immunological stimulation, lob-
ules 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 -
broblastic reticular cells (FRCs) and their reticular bers
(Clark, 1962). The entire lymph node is lled 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 in-
numerable narrow channels and interstices that are occupied
by lymphocytes, macrophages and APCs. FRCs have large,
irregularly oval nuclei and pale cytoplasm and their cell bod-
ies and larger processes can be seen in between and amongst
the more basophilic lymphocytes that ll the channels cre-
ated 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 bronectin,
that facilitate lymphocyte adhesion and ameboid migration
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TOXICOLOGIC PATHOLOGY
(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 atten
out to form a layer that encloses and denes 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 thin-
ner, more delicate branches and correspondingly larger inter-
stices 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 ows through the
meshes of this biological lter (Figure 6). They tend to oc-
cur in clusters, especially in transverse sinuses near the cap-
sule, and are relatively infrequent in the subcapsular sinus
(Sainte-Marie et al., 1982) (Figure 7). Sinus histiocytes in-
crease in response to the need for particle clearance and may
completely ll 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 attened FRCs. The in-
terface between a lobule and a sinus is a trilaminar membrane
formed by the layer of attened sinusal FRCs, the layer of
attened 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 difcult to appreciate by light microscopy,
but it prevents lymph, cells and particulates from passively
entering the lobules (Anderson and Anderson, 1975; Sainte-
Marie 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 attened sheets that are morphologically indistin-
guishable 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 broblasts. They secrete slender strands of
extracellular matrix known as reticular bers. Reticular bers
are composed of a core of collagen brils enveloped in a layer
of basement membrane (Forkert et al., 1977). Components
that have been identied in reticular bers include collagens
type I, III and IV, elastin, entactin, bronectin, 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 bers and enclose
them in cytoplasmic tubes that cover 90% of the reticular
ber surface area, thus shielding lymphocytes from com-
ing in direct contact with extracellular matrix components
(Moe 1963; Hayakawa et al., 1988). The tubular cytoplasmic
processes and reticular bers within them form a system of
miniature conduits or pipes that convey inammatory medi-
ators and soluble antigens from the sinuses into the interior
Vol. 34, No. 5, 2006 STUDY OF LYMPH NODES 413

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 broblastic reticular cells (FRCs) (arrows) create a loose reticular meshwork in the sinus. Lymph carrying densely basophilic
lymphocytes ows through the interstices of the meshwork while sinus histiocytes (H) cling to its branches. Note the attened nuclei of sinusal and lobular FRCs
forming the sinus-lobule border (arrowheads). It is difcult to determine by light microscopy which attened 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 broblastic reticular cells (FRCs) (arrows) with long delicate processes create
a loose reticular meshwork in the sinus lumen and attened 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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414 WILLARD-MACK TOXICOLOGIC PATHOLOGY

FIGURE 5.At the corticomedullary junction of a rat mesenteric lymph node two paracortical sinuses (PS) lled 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 eld. (HEV = High Endothelial Venule). 6.Sinus histiocytes remove cells,
cell debris and particulate antigens from the lymph as it ows through the sinus system. This ltration 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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Vol. 34, No. 5, 2006 STUDY OF LYMPH NODES 415

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 lled with histiocytes.

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416 WILLARD-MACK
of the lobule (Sainte-Marie and Peng, 1986; Gretz et al.,
1996, 2000; Sixt et al., 2005). A small percentage of the
reticular ber 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 bers 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 lob-
ular 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 arte-
rioles and venules may run along the central axis of a cylin-
drical 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 lled 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 com-
posed of closely packed paracortical cords that are difcult
to distinguish individually. Paracortical cords are more nu-
merous than medullary cords because they multiply in tan-
dem with blood vessels which arborize extensively in the
paracortex. They are reported to be 100 microns wide and
8001500 microns long. They surround the central DCU
and follicles like staves of a barrel. They become dis-
tended and elongated during immune responses and they
decrease in diameter if lymphocytes are depleted (Gretz
et al., 1997). Following a strong antigenic stimulus, the para-
cortex can undergo a 3- to 5-fold enlargement in 6 to 24
hours as a result of increased lymphocyte trafc into the
lobules and decreased lymphocyte trafc 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 ex-
istence, however. Lymphocytes recirculate continually, en-
tering 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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TOXICOLOGIC PATHOLOGY
efferent lymph and are returned to the circulation via the tho-
racic 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 bodys 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 bodys scouts, come to dis-
play information they have gathered about antigens they have
encountered in the eld and patrolling lymphocytes come
to look for evidence that their specic antigen has entered
the body. The lobule provides the infrastructure for the mar-
ketplace and its activities. The reticular meshwork and its
channels and interstices provide spaces for APCs and lym-
phocytes to meet and mingle and a three dimensional scaffold
for them to move around on. T and B cells and their respec-
tive APCs meet in separate areas. The vascular and lymphatic
systems, the bodys transportation systems, are intimately in-
tegrated 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
specic areas of the lobule. This unique arrangement allows a
relatively small population of lymphocytes to efciently and
effectively monitor antigens throughout the entire body.
HIGH ENDOTHELIAL VENULES
High endothelial venules (HEVs) are the gateways in-
travascular 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 special-
ized endothelial cells bear receptors that bind intravascular
lymphocytes as they pass by and facilitate their transmigra-
tion into the reticular meshwork (Sasaki et al., 1996). The cas-
cade of adhesion events involved in transmigration is outside
the scope of this paper and has been thoroughly character-
ized 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 pro-
gressively larger segments as they descend through the pe-
ripheral 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 inammatory activity. Inammatory mediators such
as MCP-1 and IL-8 are carried to the local lymph node in
the lymph. FRCs take up inammatory mediators and solu-
ble antigen from lymph in the subcapsular sinus and trans-
fer them into the conduit system by transcytosis (Anderson
and Shaw, 1993). Cells lining the sinuses have a great abun-
dance of vesicles which are presumably involved in the trans-
port process (Farr et al., 1980; Compton and Raviola, 1985).
Vol. 34, No. 5, 2006 STUDY OF LYMPH NODES
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 eld into the lymph node sinuses via the lymph.
The conduit system conveys them directly to the perivenu-
lar spaces around HEVs where the inammatory mediators
can inuence both the HEVs and the transmigrating lympho-
cytes (Gretz et al., 1996, 1997, 2000). These small molecules
stimulate upregulation of adhesion molecules and can in-
crease 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 at-
tened squamous endothelial cells following ligation of the af-
ferent 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 si-
nus system by entering narrow sinuses less than 100 microns
wide that nger 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 cor-
tex, 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 si-
nuses (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 lled with lymphocytes they
are seen histologically as irregular, densely basophilic islands
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417
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 si-
nuses 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 ll the
sinuses (Wei et al., 2005).
DENDRITIC CELLS
Dendritic cells (DCs) are potent APCs that collect and pro-
cess 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 popula-
tions of DCs (Henri et al., 2001; Yrlid and Macpherson, 2003;
Randolph et al., 2005; Villadangos and Heath, 2005), includ-
ing the Langerhans cells of the dermis and a recently de-
scribed population in the intestine that endocytizes apoptotic
cells (Huang et al., 2000). In the tissues, immature DCs sam-
ple the local antigens and then migrate to lymphatic vessels
418 WILLARD-MACK TOXICOLOGIC PATHOLOGY

FIGURE 10.The high cuboidal endothelium lining a high endothelial venule in the upper center transitions to attened endothelium as the vessel leaves the
paracortex in the center of the eld and enters a medullary cord in the lower right. 11.A blood-lled 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 abil-
ity 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 oor, ac-
tively migrate through the sinus-lobule membrane and home
to the paracortex. Histologically, mature DCs in the paracor-
tex have a broad rim of clear cytoplasm, a centrally located
nucleus and abundant cytoplasmic processes that interdig-
itate with one another. These extensive processes provide
large surface areas for contact with lymphocytes (Crivellato
et al., 1993). The inux of DCs increases during antigenic
stimulation.
FOLLICULAR DENDRITIC CELLS
The APC that present antigen to B cells are called follic-
ular dendritic cells (FDCs) and they are distinctly different
from the DCs that present antigen to T cells. The origin of
FDCs has not been denitively established. They may de-
velop 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
ne cytoplasmic processes, and they form cellular networks
in the center of the follicles (MacLennan, 1994). Their ex-
tensive 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 be-
tween the endothelial basement membrane and the surround-
ing pericytic FRCs called the perivenular channel (Gretz
et al., 1997). The perivenular channel receives inamma-
tory mediators from the conduit system. Lymphocytes spend
10 to 100 minutes in this space and their subsequent be-
haviour may be modied by exposure to inammatory medi-
ators 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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Vol. 34, No. 5, 2006 STUDY OF LYMPH NODES 419

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) lled 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-dened 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 nd their specic antigen displayed
on an APC and will eventually leave the lobule via the para-
cortical sinuses and recirculate to another lymph node to con-
tinue their search for antigen.
LYMPHOCYTE PROLIFERATION
B lymphocytes home to primary follicles where they in-
teract 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 rest-
ing B cells that together are considered a secondary folli-
cle (Figure 18). The germinal center reaction represents a
complex interaction of the processes of activation, prolifera-
tion, 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 mor-
phological appearance of germinal centers will be discussed
here.
The germinal center reaction is rst visible 34 days after
antigen exposure when a small group of dividing dark cen-
troblasts appear in the center of primary follicles. On day 5
mitotic gures 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 un-
dergo 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 centro-
cytes, 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 indistin-
guishable 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 immunolog-
ical 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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420 WILLARD-MACK TOXICOLOGIC PATHOLOGY

FIGURE 14.Numerous paracortical sinuses lled 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 lled with densely packed lymphocytes. Note several cross sections of high endothelial venules with empty lumens (arrowheads).
Rat mesenteric lymph node.

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Vol. 34, No. 5, 2006 STUDY OF LYMPH NODES 421

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 eld. 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 difcult to discern in this follicle. Rat lymph node.

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422 WILLARD-MACK TOXICOLOGIC PATHOLOGY

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 owing through the surrounding medullary sinuses and are conveyed to the circulation. Rat mesenteric lymph node.

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Vol. 34, No. 5, 2006 STUDY OF LYMPH NODES
like this for 23 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 differenti-
ate 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 infor-
mation available in the literature about the histological ap-
pearance of T cell proliferation. The appearance of tingible
body macrophages and a starry sky appearance in the paracor-
tex 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 pro-
duction 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 mi-
croscopic level. The familiar cortex, paracortex and medulla
are each composed of specic areas of the lymph nodes lob-
ules and sinuses. The lobules lie together within the sinus
system like islands in the middle of a stream. The bodys
large archipelago of lymphoid islands is united by its no-
madic lymphocytes which move back and forth between the
lobules in an endless quest for antigens. This unique ar-
rangement creates a very effective and efcient venue for
antigen surveillance, lymphocyte production, antibody se-
cretion and lymph ltration. This review paper was oriented
toward helping the bench pathologist develop an apprecia-
tion of normal lymph node anatomy and function. A wealth
of information is available regarding the mechanisms un-
derlying 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 in-
valuable support during manuscript preparation, David Sabio
for his outstanding artistic contributions and Diane Creasy for
her helpful suggestions about the manuscript.
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