IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 3. The Lymphoid System

SECONDARY LYMPHOID ORGANS AND STRUCTURES

From the Primary lymphoid Organs, the lymphocytes generated there migrate to secondary or peripheral lymphoid Tissues—densely structured, encapsulated organs (the Spleen and Lymph Nodes) and unencapsulated accumulations. Unencapsulated islands of lymphoid tissue, located mostly in mucous membranes, are referred to as mucosa-associated lymphoid tissue (MALT).

The Functions of mucosa-associated lymphoid tissue differ from those of other lymphoid organs in the Immune Response

The spleen responds to Blood-borne Antigens (surgical removal of this organ increases a patient's susceptibility to blood-borne pathogens). Lymph nodes protect the body against antigens penetrating through the Skin or mucous membranes and subsequently transported via Lymphatic vessels. The Immune Response to antigens entering through these pathways comprises the secretion of Antibodies into the bloodstream and local cellular reactions. In contrast, mucosa-associated lymphoid tissue specifically protects the mucous membranes. MALT is the site of priming—the initial contact between immune Cells and antigens originating from the epithelial surface. Lymphoid tissue is present in the mucosa of the gut, respiratory tract (particularly the Bronchi), and urogenital tract. The primary effector mechanism of the local immune response at the mucosal level is the secretion and transport of secretory IgA antibodies (sIgA) directly onto the epithelial surface. It is not surprising that the majority of the body's lymphoid tissue (>50%) resides in the mucous membranes and is particularly abundant in the intestine, given that external antigens predominantly enter through mucosal surfaces. For the same reason, IgA antibodies are present in the body in the highest relative Abundance compared to other antibody isotypes.

Encapsulated Secondary Lymphoid Organs

Spleen. The spleen is situated in the upper left quadrant of the Abdominal cavity, posterior to The Stomach and adjacent to the Diaphragm. In an adult, it measures approximately 13 x 8 cm and weighs 180–250 g. Externally, the spleen is enclosed by a Connective Tissue capsule composed of bundles of Collagen fibers that extend into the organ parenchyma, forming short partitions (trabeculae). Together with the reticular stroma, they provide a structural framework for the mass of diverse cells that populate the spleen (Fig. 3.5). The spleen comprises two MAIN TYPES OF tissue: red pulp and white pulp.

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Fig. 3.5. Histological section of the spleen showing the reticular stroma of the red pulp. Reticulin staining reveals annularly arranged reticular fibers (R) that support the endothelial Cells of the venous sinusoids. The walls of these vessels consist of a discontinuous basement membrane, with gaps between the endothelial cells that allow free plasma flow into the vessel lumen as well as the selective passage of cellular elements from the red pulp cords (RC), x 125.

White pulp. The white pulp consists of lymphoid tissue forming periarteriolar lymphoid sheaths (PALS) around the central arterioles (Fig. 3.6). The PALS contain T- and B-Cell zones: T-cells directly surround the central arteriole, whereas B-cells may form primary, unstimulated follicles (aggregates of naive lymphocytes that have never encountered an antigen) or secondary, stimulated follicles containing germinal centers with memory cells (Figs. 3.6 and 3.7). Germinal centers also harbor follicular dendritic cells and phagocytic macrophages. The marginal zone, located peripheral to the mantle, contains specialized macrophages and B-cell subpopulations that respond to type II T-independent antigens, such as Polysaccharides (see Chapter 11). Macrophages and follicular dendritic cells in the spleen present antigens to B-cells. Through capillary Branches of the central arteriole entering the marginal zone, B-cells and other lymphocytes can freely enter the PALS and leave the spleen. Specific lymphocyte subpopulations, notably maturing plasmablasts, can traverse the marginal zone into the red pulp via vascular bridges.

Fig. 3.6. Histological section of the spleen (white pulp lymphoid tissue). A secondary lymphoid follicle with a germinal center (GC) and mantle (M) is surrounded by the marginal zone (MZ) and red pulp (RP). An arteriole (A) runs close to the follicle, surrounded by the periarteriolar lymphoid sheath (PALS), which consists mainly of T cells. The marginal zone is present on only one side of the secondary follicle. (Micrograph kindly provided by Prof. I. MacLennan.)

Red pulp. This tissue is formed by venous sinusoids and cellular cords (reticulum), the spaces between which are filled with resident macrophages (Fig. 3.8), erythrocytes, platelets, granulocytes, lymphocytes, and numerous plasma cells. Notably, aside from its immunological functions, the spleen acts as a reservoir for platelets, erythrocytes, and granulocytes. It is also the site where senescent platelets and erythrocytes are destroyed. This process, termed hemocataberesis, takes place in the red pulp. Storage and hemocataberesis are facilitated by the specialized vascular architecture of the spleen (Fig. 3.7). The central arterioles, surrounded by PALS, terminate in capillaries that open freely into the red pulp cords. Consequently, circulating cells are trapped upon reaching the cords. Here, macrophages recognize and phagocytose aged platelets and erythrocytes. Uncaptured and undamaged Blood Cells return to the Circulation by squeezing through the discontinuous endothelial lining of the venous sinusoids (Fig. 3.8) via gaps between cells that freely permit plasma flow.

Fig. 3.7. The white pulp consists of periarteriolar lymphoid sheaths (PALS), many of which contain lymphoid follicles. It is surrounded by a marginal zone filled with numerous macrophages, antigen-presenting cells, slowly recirculating B lymphocytes, and natural killer cells. The red pulp contains venous channels (sinusoids) separated by splenic cords. Blood enters the splenic tissues via trabecular Arteries, which give rise to branching central arterioles. Some of these arterioles terminate in the white pulp, supplying the germinal centers and the marginal zone of the follicle, but the majority reach the marginal zone or adjacent areas. Certain arteriolar branches penetrate directly into the red pulp, ending within the splenic cords. From the venous sinusoids, blood collects into pulp Veins, then into trabecular veins, and ultimately into the splenic vein.

Fig. 3.8. Venous sinusoids and cords of the red pulp. Immunoperoxidase staining for cathepsin D demonstrates macrophages in the sinusoid walls (S) and splenic cords (SC). (Micrographs kindly provided by Dr. A. Stevens and Prof. J. Lowe.)

LYMPH NODES AND The Lymphatic system. Lymph nodes form part of the system that "traps" antigens from tissue fluid and lymph as it flows from the periphery to the Thoracic duct via major lymphatic collectors (Fig. 3.9). Lymph nodes are typically located at the branching points of lymphatic vessels. At strategic sites within the system—the cervical, axillary, and inguinal regions, the Mediastinum, and the abdominal cavity—they form clusters that collect lymph from corresponding superficial and deep body regions. Superficial lymph nodes, referred to as subcutaneous, protect the skin. Visceral (deep) lymph nodes provide defense for the mucous membranes of the respiratory, digestive, and urogenital tracts.

Fig. 3.9. Lymph nodes are located at the confluence of lymphatic vessels, forming a network that collects and filters interstitial tissue fluid. They are classified into superficial and visceral nodes, which drain deeply situated tissues and Internal Organs, respectively. Ultimately, the lymph reaches the thoracic duct, which empties into the left subclavian vein, thereby returning to the bloodstream.

Human lymph nodes are round or bean-shaped structures, 2–8 mm in diameter, featuring an indentation for the entry and exit of Blood Vessels known as the hilum. Lymph enters a node via several afferent Lymphatic vessels and exits through a single efferent lymphatic vessel at the hilum. Externally, the lymph node is covered by a collagen fiber capsule (Fig. 3.10). Radially arranged partitions, or trabeculae, together with reticular fiber cords, support the diverse cells filling the node. The lymph node is structurally divided into an outer B-cell cortex, a T-cell (paracortical) area, and a central medulla (Figs. 3.11 and 3.12). The medulla is composed of cellular cords containing T AND B lymphocytes, plasma cells, and macrophages.

Fig. 3.10. Histological section of a lymph node. The lymph node is enclosed in a connective tissue capsule and exhibits three main regions: the B-cell cortex (C), the T-cell paracortex (P), and the medulla (M), formed by cords of lymphoid tissue (a T- and B-cell region rich in plasma cells and macrophages). Hematoxylin and eosin (H&E) staining, x 10. After Zucker-Franklin D., Greaves V.F., Grossi C.E. et al. 1988. Atlas of Blood Cells: Function and Pathology. Vol. II. 2nd edn. Philadelphia: Lea and Febiger.

Fig. 3.11. Histological Structure of a lymph node. The cortical (C), paracortical (P), and medullary (M) regions are visible. The section is stained to reveal T-cell localization. They are most abundant in the paracortical region, with some present in the germinal center (GC) of the secondary lymphoid follicle, as well as in the cortex and medullary cords (MC). (Photo kindly provided by Dr. A. Stevens and Prof. J. Lowe.)

Fig. 3.12. Beneath the capsule formed by collagen fibers lies the subcapsular sinus, lined with endothelium and macrophages. Afferent lymphatic vessels deliver lymphocytes and antigens to it from the intercellular spaces of surrounding tissues and adjacent lymph nodes. The cortical region is populated mainly by B cells, which form primary and, more frequently, secondary follicles (i.e., those containing germinal centers). The paracortical region contains predominantly T cells. Each lymph node is supplied by its own artery and vein. Lymphocyte entry from the bloodstream occurs in the paracortical region via functionally specialized high endothelial venules (HEVs). The medullary region contains not only the majority of the lymph node's plasma cells, but also T and B cells forming cords of lymphoid tissue. Lymphocytes leave the lymph node via the efferent lymphatic vessel.

The paracortical region contains numerous interdigitating dendritic cells expressing high levels of class II MHC surface antigens. These cells gather here by migrating from the skin (Langerhans cells) or mucous membranes (dendritic cells), transporting processed antigens from the body's outer surfaces and mucosae into the lymph nodes (Fig. 3.13). The bulk of the lymph node's lymphoid tissue is concentrated in the cortical and paracortical regions. The medullary region is formed by cords that delineate the lymphatic (medullary) sinuses, which collect lymph into the marginal sinus and further into the efferent lymphatic vessel (Fig. 3.12). Along the sinuses, mostly in the medullary region, are debris-phagocytosing cells. As lymph flows through the lymph node from the afferent to the efferent lymphatic vessels, these phagocytic cells capture particulate antigens from it and transport them into the proper lymphoid tissue of the lymph node.

Fig. 3.13. Branched, interdigitating dendritic cells (IDCs) in the paracortical region of a lymph node in contact with each other and with T lymphocytes. (Photo kindly provided by Dr. A. Stevens and Prof. J. Lowe.)

The cortical region contains aggregates of B cells forming Primary and secondary follicles, whereas the paracortical region houses predominantly T cells. Consequently, following the injection of any T-dependent antigen into the skin or mucous membrane (see ch. II), active T-cell proliferation is observed in the paracortical region of the lymph node draining the injection site. Further evidence for this specific T-cell localization comes from patients with congenital thymic aplasia (DiGeorge syndrome), whose lymph node paracortical regions contain fewer cells than normal. Similar phenomena are observed in neonatally thymectomized or congenitally athymic (nude) mice and rats.

Germinal centers can be found in the secondary follicles of antigen-stimulated lymph nodes. They resemble the germinal centers in the B-cell regions of splenic PALS and LTs. The large and small cells of follicular germinal centers are termed centroblasts and centrocytes. The nuclei of proliferating B cells within germinal centers exhibit a characteristic cleaved shape, serving as a valuable diagnostic marker for certain malignant lymphoproliferative disorders, such as centroblastic-centrocytic lymphomas arising from these cells.

Germinal centers are surrounded by a lymphocyte mantle (Fig. 3.14). B cells in the mantle zone co-express surface IgM and IgD (Fig. 3.15). The mantle of most secondary follicles exhibits a thickening (corona) on the side facing the lymph node capsule (Fig. 3.15). In addition to B cells, secondary follicles contain follicular dendritic cells (FDCs) (Fig. 3.16), A number of macrophages (Fig. 3.17), and a small number of CD4⁺ T cells that interact with the dendritic cells of the germinal centers (see ch. 2). All these cells, together with specialized marginal sinus macrophages, presumably facilitate the B-cell immune response, particularly The Development of immunological B-cell memory—one of the primary functions of germinal centers (the cellular architecture of the germinal center is detailed in ch. 12).

Fig. 3.14. Structure of a secondary lymph node follicle. A large germinal center (GC) is surrounded by the mantle (M).

Fig. 3.15. Distribution of B cells in the cortical region of a lymph node. Immunohistochemical staining for surface IMMUNOGLOBULINS revealed B cells concentrated most heavily in the secondary follicle—the germinal center (GC) and mantle (M)—as well as between the follicle and the node capsule in the subcapsular zone (SC). Some B cells are present in the paracortical region (P), which consists primarily of T cells (see Fig. 3.11).

Fig. 3.16. Section of a secondary lymphoid follicle demonstrating a network of dendritic cells (stained using enzyme-conjugated Monoclonal Antibodies specific for FDCs).

Fig. 3.17. Germinal center macrophages. Enzyme-linked immunohistochemical staining specific for cathepsin D revealed several macrophages localized in the germinal center (GC) of the secondary follicle. When these cells phagocytose apoptotic B lymphocytes, they are referred to as tingible body macrophages (TBMs).

Mucosa-Associated Lymphoid System

Mucosa-Associated Lymphoid Tissue. Aggregates of unencapsulated lymphoid tissue can be found in the lamina propria and submucosa of the gastrointestinal, respiratory, and urogenital tracts (Fig. 3.1). Lymphoid cells form solitary or aggregated clusters with germinal centers (secondary follicles) within them. For instance, in human Tonsils, where lymphoid tissue is abundant, large secondary follicles are frequently present; they alternate with T-cell zones traversed by high endothelial venules. The tonsils—named palatine, pharyngeal (adenoids), and lingual according to their Location—form Waldeyer's ring (Fig. 3.1). A histological section of the lingual tonsil is shown in Fig. 3.18. Similar lymphoid formations are associated with the mucosa lining the bronchi and urogenital tract. The mucosal linings of the digestive, respiratory, and urogenital systems themselves contain dendritic cells essential for the uptake, Processing, and transport of antigens to regional lymph nodes. Aggregates of lymphoid tissue located in the lamina propria of the gastrointestinal tract often extend into the submucosa. They appear as solitary follicles (Fig. 3.19) or aggregated nodules, such as those in the cecal Appendix (Fig. 3.20). Peyer's patches are typically found in the lower ileum. The intestinal epithelium covering them (follicle-associated epithelium) is capable of transporting antigens and microbes into the lymphoid tissue. This specialized function is performed by unique epithelial cells interspersed among enterocytes, termed M cells because their luminal surface forms numerous microfolds (Fig. 3.21). In the basolateral region of M cells, there are deep Plasma Membrane invaginations—pockets that house B AND T lymphocytes, dendritic cells, and macrophages (Fig. 3.22). Antigens and microbes undergo transcytosis into these pockets and further into the organized subepithelial mucosal lymphoid tissue. M cells are found not only in the regions of Peyer's patches, but also in other mucosal lymphoid structures.

Fig. 3.18. STRUCTURE OF THE lingual tonsil. The lingual tonsil is located in the posterior third of the Tongue and consists of aggregates of lymphoid tissue (L) containing large secondary follicles associated with the mucosa, which forms deep cleft-like invaginations in this area (arrow). Mucus-secreting Salivary Glands (SG) are visible around the tonsil. All other tonsils share this same structure. (Photo kindly provided by Dr. A. Stevens and Prof. J. Lowe.)

Fig. 3.19. Solitary lymphoid follicle in the Large Intestine. This lymphoid tissue nodule is located in the mucosa and submucosa of the intestinal wall (arrow). (Photo kindly provided by Dr. A. Stevens and Prof. J. Lowe.)

Fig. 3.20. Lymphoid nodules in the human vermiform appendix. 1. Appendix of a 10-year-old child; large lymphoid nodules in the mucosa extending into the submucosa. 2. Appendix of a 36-year-old male; a marked reduction in lymphoid tissue is evident, with virtually complete disappearance of follicles—age-related lymphoid atrophy characteristic not only of the vermiform appendix. (Photo kindly provided by Dr. A. Stevens and Prof. J. Lowe.)

Fig. 3.21. Dome-shaped protrusion formed by the intestinal mucosa in a region devoid of villi. The superficial epithelium in this area, known as follicle-associated epithelium (FAE), contains M cells. Deep within the mucosa lies a cluster of secondary lymphoid follicles with prominent germinal centers. The surrounding Thymus-dependent interfollicular zones contain interdigitating cells and high endothelial venules. The dome area between the FAE and the follicles is populated predominantly by B cells, the majority of which are memory cells.

Fig. 3.22. The intracellular pocket houses lymphocytes and frequently macrophages (Mφ). Antigens endocytosed by the M cell gain access to the subepithelial lymphoid tissue via this pocket. (FAE - follicle-associated epithelium.)

In the local HUMORAL IMMUNE RESPONSE at the mucosal level, antibodies of the IgA isotype are predominantly produced. Secretory IgAs are antibodies capable of penetrating epithelial cell membranes to provide protection against pathogenic microbes (Fig. 3.23).

Fig. 3.23. IgA dimers (sIgA) secreted by plasma cells in the lamina propria of the intestinal mucosa bind to poly-Ig receptors on the basolateral surface of the epithelium. Subsequently, the sIgA-receptor complexes are delivered by endocytosis and transcellular transport in transport vesicles (to whose membrane they remain bound) to the luminal surface of the epithelium. Here, the transport vesicles fuse with The Plasma Membrane of the epithelial cells, releasing the IgA dimers with the attached secretory component (a receptor fragment) into the lumen, where the secretory component protects the IgA dimers from Cleavage by Proteolytic Enzymes.

Mucosal lymphocytes. In addition to the organized lymphoid tissue that forms the integrated mucosal-associated lymphoid tissue (MALT) system, numerous scattered lymphocytes and plasma cells are present in the mucosa of the stomach, intestines, upper and lower respiratory tracts, and several other organs. Lymphocytes are found in the connective tissue of the lamina propria and within the epithelial lining.

Among the lamina propria lymphocytes (LPLs), activated T cells predominate, although activated B cells and plasma cells are also present in significant numbers. These plasma cells secrete antibodies predominantly of the IgA isotype, which are transported across the epithelial cells and released into the organ lumen (Fig. 3.23).

Intraepithelial lymphocytes (IELs) consist mainly of T cells that differ phenotypically from LPLs (Fig. 3.24).

Fig. 3.24. Phenotypic markers of lamina propria lymphocytes of the intestinal mucosa (LPLs) are generally similar to those of peripheral blood lymphocytes. A significant proportion of intraepithelial lymphocytes (IELs) belong to γδ T cells, many of which express CD8, likely as an activation marker.

The majority of LPLs and IELs are memory cells bearing the CD45RO marker. They respond poorly to stimulation with anti-CD3 antibodies but are sensitive to other activation mechanisms, such as those mediated by CD2 or CD28.

Resting peripheral blood T cells do not express the HML-1 integrin α chain (CD103), but stimulation with phytohemagglutinin (PHA) induces its synthesis. Antibodies against this chain are mitogenic for the same cells and induce the expression of the low-affinity IL-2 receptor α chain (CD25). The HML-1 polypeptide is an α chain of the integrin family that, upon interaction with the β₇ chain, forms the αHML-1-β₇ heterodimer—an integrin synthesized by IELs and other activated lymphocytes.

Intraepithelial lymphocytes secrete a number of cytokines, including IFNγ and IL-5. One of the functions of IELs is presumably immune surveillance aimed at eliminating mutant and virus-infected cells.