Access to the supplemental resources for this session is password-protected and restricted to University of Michigan students. If you are a University of Michigan student enrolled in a histology course at the University of Michigan, please click on the following link and use your Kerberos-password for access to download lecture handouts and the other resources.
|Atlas||Wheater's, pgs 215-233, Immune system|
Wheater's, pgs 107-214, Immune system (Optional)
|Text||Ross and Pawlina, Chapter 14 Lymphatic System|
There is a continuous production and recirculation of lymphocytes in the body. Many of the lymphocytes you see in blood vessels, lymphatic vessels or in tissue have been exposed to antigen and are thus poised to respond to specific antigenic stimuli. The lymph nodes and the spleen facilitate immunological surveillance of the host. They are the sites of 1) antigen trapping, 2) homing of cells of the immune system that have been exposed to their triggering antigens, and 3) subsequent expansion and release of lymphocyte clones capable of further response to specific antigens. The exposure of lymphocytes to antigens occurs in solitary lymph nodes (follicles), in aggregates of lymph nodes present beneath mucous membranes, and in the spleen. The palatine tonsils and the Peyer’s patches in the ileum (not seen in this laboratory) consist of multiple aggregates of lymph nodes. T lymphocytes, which participate in cell-mediated immunity or modulate the immune responses of other cells, differentiate in the thymus during childhood. B lymphocytes, which have the capacity to become plasma cells and secrete immunoglobulins, differentiate in the bone marrow throughout life.
Slide 027 Lymph node H&E Webscope Imagescope
Slide 026 Lymph nodes H&E Webscope Imagescope
Slide 026 Lymph node H&E Webscope Imagescope
Slide 142 Lymph node H&E Webscope Imagescope
Slide 030 Mesentery H&E Webscope Imagescope
Slide 028 Lymph node silver Webscope Imagescope
Use slide 27 to study the overall organization of a lymph node and identify the cortex, medulla and hilus (Slide 027 Webscope Imagescope). Some slides do not show a medulla or hilus, depending on the plane of section, especially, the hilus. The hilar region is rather small and will not be present in all slides. Using slide 27 and other slides, locate the capsule, lymphatic follicles or nodules (B cell-rich), the diffuse or deep cortical zone (T cell-rich), in addition to the medulla and the hilus. Recall that the deep cortex is the zone of antigen presentation, while T cell help occurs in the follicles. What is the distribution of B cells and T cells in the lymphatic tissue? answer
The thymus is entirely composed of T cells, epithelial reticular cells and some macrophages. Occasionally another type of cell will get into the thymus, but it does not belong there. The thymus consists of a cortex and medulla with no germinal centers. Immature T cells are found at the edges of the cortex. As they mature, they migrate deeper into the cortex and eventually leave as mature naÃ¯ve T cells through the venules of the medulla. In the lymph node, the cortex is made of primary follicles of resting B cells and secondary follicles with germinal centers. The germinal centers are where B cells are undergoing clonal expansion, after binding antigen and receiving T cell help. Most T cells are located deeper in the cortex. Antigen-stimulated helper T cells are formed when antigen is presented to them by dendritic cells (and possibly macrophages), deep in the cortex. B cells also bind their antigen in this region. B cells entering the lymph node travel through the cortex to a follicle. If they have bound antigen in the cortex, they attract antigen-stimulated T helper cells to the follicle, where T cell help occurs. T cell help in the spleen occurs in the PALS. Close to, if not touching, the PALS are follicles of resting B cells, a clone of which may give rise to a germinal center upon proper stimulation.
Generally the sinuses show up best beneath the capsule (subcapsular sinus) in either slide 26 #026 Webscope ImageScope orientation Image or #142 Webscope ImageScope Image or in the medulla (medullary sinuses, see below). Make sure you have found a real sinus and not just a separation artifact. You should be able to see: 1) reticular cells spanning the sinus, 2) some endothelial cell nuclei defining the inner wall of the sinus (adjacent to the lymphatic follicles), and 3) circulating lymphocytes and macrophages in the lumen. What is a fiber associated reticular cell? answerThese are the stellate shaped fibroblasts found in the lymph node that are involved in making reticular (type III collagen) fibers. They are NOT the same as the epithelial reticular cells found in the thymus: epithelial reticular cells s are true epithelial cells derived from the endodermal lining of the third pharyngeal pouch in the embryo (the third pouch is what gives rise to the thymus) whereas reticular cells, like most connective tissue fibroblasts, are derived primarily from mesoderm. Afferent lymphatic vessels that penetrate the capsule are difficult to identify, but can be observed in #030 Webscope ImageScope . If you see venule-sized vessels adjacent to or within the capsule you can make the provisional identification of an afferent lymphatic vessel, especially if these vessels contain lymphocytes and show valves.
With the low power objective find primary and secondary follicles (follicles with germinal centers, best seen in #27 Webscope ImageScope) . It is not always possible to distinguish primary and secondary follicles because a glancing section through a secondary follicle may miss the germinal center and thus mimic a primary follicle, and that sometimes the boundary between the follicles and the diffuse lymphatic cortex is indistinct. The germinal centers #27 Webscope ImageScope Image in these particular specimens are at an end stage; that is, blastogenesis has already occurred and most of the cells present in the center are simply reticular cells and a few macrophages. Recall that the germinal centers are the sites of T cell help, B lymphocyte proliferation and antigen-dependent differentiation of B cells into lymphoblasts. The lymphoblasts continue to differentiate into plasma cells and memory B cells. Where do the plasma cells in the lymph node go? answerNowhere. Once a plasma cell differentiates in the lymph node, it generally migrates to a medullary cord in the medulla of the lymph node and begins secreting antibodies that travel throughout the body (first through efferent lymphatics that then drain into venous blood). Once in the medullary cord, the plasma cell generally stays puts. It is worth noting that plasma cells are rarely observed in peripheral blood. The numerous plasma cells found in various tissues in the body traveled in the circulation as naive B lymphocytes until they were stimulated to enter the peripheral tissue and differentiate into a plasma cell locally. Make sure that you find the high endothelial venules (specialized cortical veins lined by high endothelium) especially visible in either slide 27 #27 Webscope ImageScope or #142 Webscope ImageScope. How do cells travel from the blood to the lymph node? answerThere are many adhesion molecules and receptors expressed both by the high endothelial post capillary venules of the lymph node and on the B and T lymphocytes that allow them to enter the lymph node. This process is called "lymphocyte homing". When appropriate signals are received, cells begin to cross the endothelial cells of the venules. Finally they reach the deep cortex where they can receive T cell help and continue on to the follicles.
With a low power objective locate the medullary cords and sinuses in either slide 26 #026 Webscope ImageScope or slide 142 #142 Webscope ImageScope. Find endothelial lining cells, reticular cells, circulating lymphocytes and macrophages (that may appear foamy after having engulfed some RBCs). Review the blood supply to the node. In slide 27, a number of efferent lymphatic vessels #27 Webscope ImageScope with valves can be found in the hilar connective tissue. Now look at slide 28 to review the role of reticular fibers in supporting the structure of the lymph node.
Slide 161 gastroduodenal junction H&E Webscope Imagescope
Slide 157b fundic stomach H&E Webscope Imagescope
Slide 175 Appendix H&E Webscope Imagescope
Slide UCSF 261 appendix H&E Webscope Imagescope (virtual slide courtesy of the University of California, San Francisco)
Slide 168 Small intestine, jejunum H&E Webscope Imagescope
Solitary lymphatic nodules are present throughout the GI tract, respiratory tract and, to a lesser extent, the urinary tract. Look for solitary nodules in slide 161 (the gastro-duodenal junction). At low power, localize the duodenum by identifying mucous glands (Brunner’s glands) in the submucosa and, then, look for discrete basophilic nodules (follicles) #161 Webscope Imagescope in the lamina propria or in the submucosa in the midst of mucous glands, some of which will have germinal centers. Solitary lymphatic nodules can be also seen in our slides of the stomach (#157b fundic stomach H&E Webscope Imagescope) and the appendix (#175 Webscope ImageScope and #175 Webscope ImageScope ). Many such nodules combined give rise to named structures, such as tonsils or "Peyer’s patches." The term, Peyer’s patches, specifically refers to aggregates of lymphatic nodules in the ileum, but often the term is used mistakenly to describe any aggregates of lymphatic nodules in other parts of the GI tract.
In addition to lymphoid aggregates and nodules present in the lamina propria and submucosa are intraepithelial lymphocytes which are T-cells that develop in the thymus and migrate into various epithelial tissues in the skin, GI, respiratory, and genitourinary tracts where they serve an important function as a first line of defense against pathogens. You can find intraepithelial lymphocytes in just about any of the slides that we've used to study these organ systems so far, but they are probably easiest to see in slide 168. To find them, look for small, dark, round nuclei #168 Webscope ImageScope situated between the epithelial cells.
Using slide 138L, observe the numerous aggregates of lymphatic follicles with germinal centers and the stratified squamous epithelium that covers the oral surface of these aggregates. The epithelium also lines crypts that dip into underlying lymphatic nodules. The epithelium lining some crypts is almost obliterated by infiltrating lymphocytes. Some crypts will probably be cut in cross section so continuity with the oral cavity will not be apparent. At 10X, locate a follicle with a germinal center. Note small, lightly stained areas (they almost look like small holes) distributed throughout the center. If you can’t find them look at another germinal center. These lightly stained areas are macrophages #138 Webscope ImageScope in the germinal center. Look at them with high power. You should see a large cell with a large, vesicular nucleus that contains at least one conspicuous nucleolus. You will have to work to see them, but once you’ve seen one you will be able to recognize them in most lymphatic tissue (e.g. #175 Webscope ImageScope and #175 Webscope ImageScope ). The cytoplasm of these macrophages will have a variable appearance but will usually include two or three hyperchromatic spheres that are ingested B lymphocytes that failed to differentiate properly and underwent apoptosis.
In slide 138_20x, locate the dense, irregular connective tissue capsule #138 Webscope ImageScope that separates the tonsil from the rest of the pharynx. In this section it does not completely enclose the tonsil. There are a few mucous glands and muscle fibers included in this section.
Of the two human slides, #147 is better in that it is more compact and the content of erythrocytes is reduced. With low power try to distinguish between the red and white pulp. Think about why these terms are employed. While the white pulp has an obvious role in the immune response, don’t forget the role of the red pulp in potential antigen trapping by macrophages. Find the capsule #147B Webscope ImageScope and trabeculae #147B Webscope ImageScope (containing trabecular arteries and veins—you may find just one or the other). Much of the white pulp you will see in your section of slide 147 is composed of B-cell dependent follicles, showing germinal centers. Slide 148 contains little easily recognizable white pulp (somewhat abnormal) but is helpful in seeing the periarteriolar lymphatic sheath (PALS) #148 Webscope ImageScope that surround central arteries (which range in size from small arteries to arterioles). Recall that the PALS contains mostly T lymphocytes and is homologous with the deep cortical zone of a lymph node.
Going back to slide 147, you'll see many instances where a PALS is expanded eccentrically to include a lymphatic follicle #147B Webscope ImageScope, usually with a germinal center; recall that these follicles are B-cell rich. With this expansion, the "central" artery is pushed off to one side and therefore not very "central." Many texts and atlases usually show this classic view of a the central artery and its PALS in cross section. However, you should realize that the PALS is a sheath that runs along the length of each central artery, and you can see this particularly well in #301 Webscope ImageScope. The marginal zone #148 Webscope ImageScope Image is a region surrounding the white pulp that is difficult to recognize unless the spleen is very well preserved and sectioned, or, as in slide 148, the red pulp is particularly obvious (here the red pulp is packed with erythrocytes). This region contains mostly reticular cells and antigen-presenting cells, as well as some lymphocytes that may play a role in antigen trapping or processing. Don't get too hung up on trying to definitively demarcate the marginal zone in these sections, but you should have a general idea of this transition zone between white pulp and red pulp and understand its function as a primary site of antigen presentation in the spleen.
In the red pulp, identify the splenic cords (tissue between the sinuses) and venous sinuses that are reasonably well preserved beneath the capsule in #147B Webscope ImageScope Image. The cords and sinuses show up particularly well in #148 Webscope ImageScope Image. The endothelial cell nuclei of the sinuses are characteristically plump and closely packed together. Note that the endothelial nuclei bulge toward the lumen of the sinuses --this may help you discern the cords from the sinuses.
With low power, look at slide 140 and distinguish between the cortex and the medulla Image. Why is the thymic medulla lighter than the cortex? answerWith their dark staining nuclei and little cytoplasm, the abundant T lymphocytes in the cortex give the thymus a dark color. The thymic medulla has far fewer lymphocytes in it and so stains much lighter. Locate Hassall’s corpuscles #140 Webscope ImageScope (which are aggregates of epithelial reticular cells) in the medulla. Try to find other epithelial reticular cells #140 Webscope ImageScope Image that are scattered throughout the cortex and medulla the key to identifying them is their large, pale nuclei. Of course, most of the cells with small round nuclei packed into the cortex and medulla are T lymphocytes (when immature, they are called “thymocytes”). Also present are numerous macrophages #140 Webscope ImageScope Image which will appear eosinophilic with a foamy cytoplasm or they may even contain engulfed lymphocytes. What are the differences among the various reticular cells and fibers of the lymphoid organs? answerLymph nodes are filled with reticular cells and fibers. The fibers, made by reticular cells (which are related to fibroblasts) and composed of type III collagen, form a supportive meshwork for lymphocytes and other cells in the cortex and medulla. Reticular cells in the lymph node wrap around the reticular fibers (they are extracellular fibers). This same reticular fiber support is found in the spleen and tonsil as well as the bone marrow. The thymus has epithelial reticular cells. Like reticular cells elsewhere, thymic epithelial reticular cells provide support in the cortex and medulla. Unlike reticular cells, epithelial reticular cells have keratin filaments and are joined by desmosomes. Epithelial reticular cells (which, unlike reticular cells, are true epithelial cells) help make the blood-thymus barrier in the thymic cortex and provide a microenvironment in the thymus for T cells to develop. They also play a crucial role in positive and negative selection of T-cells. How do lymphocytes leave the thymus? answerTo leave the thymus, lymphocytes must survive negative selection to enter the thymic medulla. From the medulla, they intravasate by crossing the walls of venules located at the cortico-medullary junction. Alternatively, they may leave the thymus via efferent lymphatic vessels. There are generally no afferent lymphatic vessels in the thymic cortex because this might allow free antigens to enter the cortex thereby impinging on the positive selection process.
Look at slide 141 and observe the histology of thymic involution, which is basically a replacement of lymphatic tissue by fat. The Hassal's corpuscles #141 Webscope ImageScope here are also quite large and a bit bizarre looking. The production of T cells by the thymus in the human adult is generally greatly reduced, but it can continue to produce T-cells even into adulthood.