IMMUNE AND HEMOPOIETIC ORGANS

Immune and hemopoietic organs

1. Functions of the hemopoietic system system.

2. Distinguishing features of the lymphoid organs.

3. Difference between central and peripheral hemopoietic organs.

4. Description of the thymus in terms of its general functions, its location in the body and the type of reticular cells it contains and their embryonic germ layer of origin.

5. Comparison of the cortex and medulla of the thymus in terms of the packing density of lymphocytes and presence of Hassall’s corpuscles.

6. Location of the blood-thymus barrier, the layers through which a substance in the blood would have to pass to cross the blood-thymus barrier.

7. The probable function of the blood-thymus barrier.

8. Describtion of lymph nodes in terms of their general functions, their location in the body and their embryonic germ layer of origin.

9. Beginning with the afferent lymphatic vessels and ending with the efferent lymphatic vessels, trace the path of lymph through a lymph node. What percentage of the lymph actually penetrates the nodules?

10. Cells and structures commonly found in the lumens of a lymph node’s sinuses.

11. The type of cells of lymph node paracortical zone.

12. Main features in structure of cortex and medulla.

13. Basic function of each cell type: B-lymphocyte, T-lymphocyte, memory cell, plasma cell, follicular dendrites cell, macrophage, reticular cell.

14. Hemolymph nodes.

15. Classification of lymphoid tissues and organs.

16. Characteristic of the lymphoid system main cells.

17. Major components of the white pulp, the predominant type of lymphocytes which they contains.

18. Describtion of the sequence of events in the life cycle of B lymphocyte following its encounter with an appropriate antigen in the marginal zone of the white pulp.

19. Major components of red pulp.

20. Comparison of the white and red pulp of the spleen in term of the predominant cell type present.

21. Comparison of the red pulp sinusoids with common capillaries.

22. Peculiarities of the splenic circulation, open and closed theory.

23. Macrophagic system of haemopoietic organs.

 

In human body there are special structures, which prove the homeostasis of blood and immune system, they are the organs of hematopoiesis and immune protection. Such organs belong to this system: red bone marrow, thymus, spleen, lymph nodes, and lymph nodules (mucosa-associated lymphoid tissue of tonsil’s, intestine, respiratory system).

There are central and peripheral organs. Red bone marrow and thymus are the central ones. Erythrocytes, platelets, granulocytes and lymphocytes precursors take their origin in the red bone marrow. Thymus is the central lymphopoietic organ. It is necessary to underline that lymphocytes antigenindipendent prolipheration takes place in the central hematopoietic organs.

Spleen, lymph nodes, lymph and hemolymph nodules, tonsilles and Payer’s patches belong to the peripheral hemopoietic organs.

T- and B-lymphocytes from central organs pass to the spleen, lymph nodes and nodules and continue their further development into the effectors cells under the influence of antigens. Thus antigendependent proliferation of lymphocytes occurs in the peripheral hematopoietic organs, which lead to appearance of active special cells and memory cells. Besides the old and destroying cells are finishing their life span there.

All the hematopoietic organs are functioning simultaneously and prove the constant compounds of the blood and immune homeostasis. Coordination and regulation of their functions is performed by the humoral and nerve influences.

Despite on structural differences of hematopoietic organs, all of them have the similar morphofunctional signs: they consist of stroma and parenchyma. First of all reticular tissue (connective tissue with special properties) form their stroma and create the special microenvironment to the developing hemopoietic cells and lymphocytes. Reticular cells have long processes, which form the special meshwork together with reticular fibers. These cells produce hemopoietins – regulatory factors.

Capillaries of hemopoietic organs (sinusoids in red bone marrow and fenestrated in other organs) have the electoral permeability to the mature cells. Thanks to the numerous phagocyzing cells (macrophages of different types: free, dendritic, interdigital) and immune cells hematopoietic organs have protectory function, they can purify the blood or lymph from foreign particles, bacteria, abnormal and remnant of dead cells.

 

 

A reminder that bone marrow is one place where you find a stroma of reticular tissue. Here the tissue has been silvered so that you can see the network of fine reticular fibers that support all the blood forming cells. Large spaces represent fat cells.

Parenchyma of hemopoietic organs may have mainly lymphocytes or different cells – lymphoid or myeloid tissue adequately. Thus red bone marrow has myeloid parenchyma, the other organs – lymphoid one.

 

Bone marrow (medulla osseum)

 

Bone Marrow– is the central hematopoietic organ, which contain the population of self-supporting stem cells and produces both cells types –myeloid and lymphoid.

Bone marrow originates in embryo clavicle at the 2^nd month of embryogenesis, at the 3^rd month it appears in the lamellar bones – shoulder blade, pelvis, occipital bone, ribs and vertebras, at the beginning of 4^th month – in the tubular bones of extremities. Till the 11^th weeks it is osteoblastic bone marrow, which has osteogenic function. At this period bone marrow accumulates stem cells and stromal cells form the micromedium to hematopoietic stem cells differentiation. In 12-14-weeks embryo hematopoietic cells are developing around the blood vessels. Since that time red bone marrow become the universal hemopoietic organ. In 20-28-week fetus bone marrow is developing very actively, bone trabeculas are resorbed by osteoclasts, thus bone channel appear and red bone marrow is growing to the epiphysis. Till that time red bone marrow becomes the principal hematopoietic organ with mainly erythropoiesis.

 

 

Bone marrow seen with low power (to the left). This marrow cavity lies within a shaft of compact bone (middle, pink band). Attaching muscle is to the right.

 

In 36-week embryo adipose cells are found in diafisial medulla and hemopoietic islets are observed in epiphyses.  

In adult human being bone marrow is one of the large organs of the body and the main site of hematopoiesis. Under normal conditions, the production of blood cells by the marrow is perfectly adjusted to the organism’s functions. It can adjust rapidly to the body’s needs, increasing its activity several fold in a very short time if required. Bone marrow is found in the medullar canals of long bones and in the cavities of spongious bones. Two types have been described according to their appearance on gross examination: red, or hematogenous, whose color is due to the pres­ence of blood and blood-forming cells, and yellow bone marrow, whose color is due to the presence of a great number of adipose cells.

 

RED BONE MARROW (medulla osseum rubra)

 

Red bone marrow is the hemopoietic portion of the bone marrow. It is the semifluid organ of dark red colour. This allows preparing the red bone marrow smear for investigation, which may be stained likes a blood smear with Romanovsky-Himsa method.

 Red bone marrow is composed of a stroma (from Greek, bed), parenchyma of hematopoietic cords and sinusoidal capillaries. The stroma is a 3-dimensional meshwork of reticular cells with phagocytotic properties and a delicate web of reticular fibers containing hematopoietic cells and macrophages. Reticular cells have extensive branched cytoplasmic processes, which enclose well over 50 % of the outer surface area of the sinusoid wall. It is the reticular cells, which also ramify throughout the hemopoietic spaces forming a regular sponge-like matrix, a meshwork to support the hemopoietic cells, they also produce special growth factors, which influence on the hematopoiesis.

 

 

Bone marrow surrounding a pink, Y-shaped piece of spongy bone. Notice the small osteocytes scattered within the bony matrix. In the marrow there are clumps of small blood-forming cells scattered among the large round fat cells. (The lipid content of the fat cells has been dissolved out in fixation of the tissue). The elements formed most abundantly in marrow are r.b.c.’s, granular leukocytes, and platelets. Lymphocytes and monocytes are also formed here, but go elsewhere to proliferate. Another name for blood-forming tissue such as this is hemopoietic or myeloid tissue.

 

  The extracellular matrix of bone marrow in the hemopoietic compartment contains fibers of col­lagen types I and III, as well as fibronectin, laminin, and proteoglycans. Laminin, fibronectin, and another cell-binding substance, hemonectin, interact with cell receptors to bind cells to the matrix. The associated proteoglycans, chondroitin sulfate, hyaluronic acid and heparan sulfate, may also bind growth factors, which control hematopoiesis.

 

 

Sinusoid of bone marrow seen here in longitudinal section. There is a good nucleus of a lining endothelial cell near the lower center of the field. Junctions between lining cells are loose so that newly formed blood cells can enter the vessels.

 

The sinu­soids in red bone marrow are formed by a of endothelial cells with fenestrae and discontinuous basement membrane. Thanks to such structure endothelial cells directly contact with hemopoietic and stromal cells. Fenestrated thin regions of the endothelium are the sites for migration of mature cells from the stroma into the sinusoids. Endothelial cells are capable for contractile movement, which helps to blood cells exfusion from the red bone marrow parenchyma into the capillaries blood stream. After the cells passage fenestrae are closed. Endotheliocytes produce colony-stimulying factors and fibronectin, special protein antigen properties, which ensures cells adhesion one to each other and with substratum.

 

 

          Bone marrow showing the typical cellular masses of developing blood cells lying between the round, empty fat cells. There are two large megakaryocytes in the field, one just about in the center and the other to the extreme right. Notice orange-colored rbc’s in thin-walled sinusoids.

 

The sinusoidal capillaries are reinforced by an ex­ternal discontinuous layer of reticular cells and a loose net of reticular fibers. The release of mature bone cells from the marrow is controlled by releasing factors produced in response to the needs of the organism. Several substances with releasing activity have been described and include the C3 component of complement (a series of immunologically active blood proteins), hormones (glucocorticoids and androgens), and some bacterial toxins. The main functions of red bone marrow are the production of blood cells, destruction of red blood cells, and storage (in macrophages) of iron derived from the breakdown of hemoglobin.

Now some others cells are concerned to the hemopoietic cells microenvironment. They are: osteogenic cells, adipocytes, adventitial cells, endothelial cells and macrophages.

By accumulating lipid, the reticular support cells may transform into the adipocytes. Thus such cells are the constant compounds of the red bone marrow. They are oval-shaped and contain large vacuole of the lipid inclusions; their flattened nuclei are disposed peripherally under the cell membrane.

Group of osteogenic cells includes the stem cells of the supporting tissues like’s osteoblasts and their precursor cells. Osteogenic cells are disposed in the endosteum and may be found in the bone marrow spaces. These cells are capable to produce growth factors and to stimuli the blood stem cells to proliferation and differentiation in their site. Thus the most active hematopoietic processes take place near the endosteum, where the amount of stem cells are three times numerous then inside in the middle of bone marrow space.

Adventitial cells are the frequent cells of blood vessels wall, very often they cower the sinusoids from outside. They may contract under the influence of some hemopoietic factors (for example, erythropoietin). Thus they promote cells migration into the blood stream.

Inhomogeneous cells, which are a little bit differ in structure and functional possibilities, present macrophages in the red bone marrow but all of them are reach in lisosomes and phagosomes. Some of macrophages populations are secreting biologic active substances (erythropoietin, colony stimulying factor, interleukins, prostaglandins, interferon and others).  By means of their processes, macrophages can introduce into blood stream through the capillaries wall and extract ironcontaining substance transferrin from the blood, and transfer it to the maturing erythroid cells. This matter is utilized for the hemoglobin gemin construction.

Red bone marrow parenchyma consists of hematopoietic cells differons, which are organized into four principle groups or hematopoietic islets: erythropoietic, granulocytopoietic, agranulocytopoietic and poiesis of platelets.

Erythropoiesis is tightly connected with macrophages. Red cells are formed in small erythroblastic islet. Such islet consists of one or two specialized phagocyte surrounded by red progenitor cells, which arise from colony forming unite that touch in contact with bone marrow macrophage. The macrophages have long cytoplasmic processes and deep invaginations to accommodate the dividing erythroid cells. All these cells are combined with him by his receptors – syaloadhesins.

 

 

          The cells shown here are all stages in the development of erythrocytes. Generally in the red blood cell line: (1) the cells become progressively smaller, (2) the cytoplasm changes from blue to pink, and (3) the nucleus becomes smaller and more condensed and ultimately is lost altogether. Cells shown here include (in developmental order):

• Top cell – proerythroblast

• Lower row:  left = basophilic normoblast or erythroblast. It is still blue, but is smaller; the nucleus is more condensed

• middle = polychromatophilic normoblast or erythroblast. Cytoplasm is grayer or muddier; nucleus is even more condensed.

• right = orthochromatic (or eosinophilic) normoblast. Cytoplasm is pinker and cell is smaller; nucleus is pyknotic.

 

 

          Diagrammatic summary of the events that take place during maturation of red blood cells (erythropoiesis). Staining is with special blood stains (Giemsa, etc.). Primitive status is on the left; mature status is on the right.

• Top line: there’s a decrease in cell size (from left to right) and a decrease in basophilia (blueness) of cytoplasm. At the same time, hemoglobin increases, making the cytoplasm more and more acidophilic (pink). Basophilia is due to presence of abundant polyribosomes.

• Second line: there’s a decrease iuclear size and ultimately extrusion and loss of the nucleus.

• Third line: there’s increased condensation of nuclear chromatin and eventual pyknosis of the nucleus (very dark, compact, dying). Also the nucleoli, evident at first, are soon lost.

 

 

Reticulocytes with polyribosomal remnants (RNA) staining dark in their cytoplasm. They are slightly larger than the completely mature erythrocytes and are often found in the peripheral bloodstream at times when blood cells are being formed unusually rapidly (as during or after certain blood diseases). Remember not to confuse reticulocytes of the blood with reticular cells of connective tissue!

 

Macrophages “feed” erythroblasts with iron and some compounds for ferritin. They also remove the nuclei of erythroblasts, which are exfused during their maturation. Erythroid cells migrate outwards along the macrophages cytoplasmic processes. When mature, the red cells contact nearby sinusoidal endothelium, separate from islets and pass out to enter the circulation through the capillaries wall, which consists of fenestrated endothelial cells. There are a lot of fixed macrophages among the endothelial cells. Numerous phagocytes that are present in blood islets and capillaries wall allow controlling the maturation of erythrocytes and their passage into the blood stream.

 Granulopoiesis includes the formation of granulated cells. This takes place under the influence of cytokines. Granulocytopoietic cells are organized into islets, which lie in the periphery of the bone medullar cavity. Nonmature cells are surrounded with proteoglycans, which are necessary to their normal development.  Maturing granulocytes are stored in the red bone marrow; there are three times more granulocytes then erythrocytes here, and 20 times more granulocytes in the red bone marrow than in the peripheral blood.

 

 

          Maturational stages in development of granular leukocytes:

• Extreme left – myeloblast (the most primitive stage)

• Next – promyelocyte (these first two stages are undifferentiated precursors of all three granulocyte types)

• Next four: myelocyte, metamyelocyte, band cell, and mature cell

The top row represents the eosinophilic cell line, the middle row represents the neutrophilic line, and the bottom row represents the basophilic line. Note the decrease in cell size, the decrease in cytoplasmic basophilia (meaning decrease in polyribosomes), the increase in cytoplasmic granules (these first become specific and distinguishable as eosinophilic or basophilic at the myelocyte stage), and an increase in lobulation of the nucleus.

A neutrophilic series showing changes in cell size and nuclear shape:

•  early neutrophilic myelocyte (large cell, rounded nucleus)

• late neutrophilic myelocyte or early metamyelocyte (nucleus beginning to indent)

• neutrophilic metamyelocyte (indented nucleus)

• neutrophilic band cell (much thinner nucleus)

• segmented (mature) neutrophil

• polychromatophilic erythroblast (muddy colored cytoplasm and not very much of it)

 

 

There is a large “blast” cell in the upper left group and a large promyelocyte at upper center. The latter is recognizable by the non-specific azurophilic granules in its cytoplasm, foretelling that it is heading toward one of the granulocyte lines. A basophilic normoblast with blue cytoplasm is in lower center. To the right of it are two early orthochromatic normoblasts. At bottom center is a late polychromatophilic normoblast with muddy cytoplasm. A slightly younger polychromatophilic cell is in the extreme lower left corner, with a slightly larger and less condensed nucleus. A neutrophilic metamyelocyte with indented nucleus also lies near the lower edge of the field.

Megakaryoblasts and megakaryocytes huge multinuclear cells are disposed near the capillaries wall in such way that their pseudopodia introduce through the wall pores into the blood.  Platelets are formed by separation of cytoplasm portions (by demarcation membranes) directly into the blood stream.

 

 

Megakaryocyte as seen in an H & E stained section. Note its multilobed nucleus and its comparatively giant cell size. (Remember that the other giant cell of bone, the osteoclast, has multiple separate nuclei. The osteoclast lies next to bone, while the megakaryocyte lies out in the middle of the marrow.)

Myeloid tissue of red bone marrow includes small group of bone marrow lymphocytes and macrophages, which surround the blood capillary. Monocytes leave the marrow after formation with no marrow pool. They spend about 3 days in the blood before migration into the tissues in an apparently random fashion; they are then unable to re-enter the circulation.

 

The bone marrow is the site of formation of primitive lymphocyte precursors, which subsequently give rise to both T- and B-lymphocytes at different sites. B cells undergo initial maturation in bone marrow and move on to colonize peripheral lymphoid tissues. T cells migrate to the thymus gland where they undergo initial maturation before moving on to colonize peripheral lymphoid tissues. Lymphoid cells are capable of division in adult life, when expansion of selected clones is desirable to mount a specific immune response. Lymphoblasts are recognizable dividing lymphocytes, having large opeucleus, prominent nucleolus, and small amount of cytoplasm. This cell division takes place in the specialized peripheral lymphoid tissues.

YELLOW BONE MARROW (medulla osseum flava)

 

In newborns, all bone marrow is red and is therefore active in the production of blood cells. As the child grows, most of the bone marrow changes gradually into the yellow variety in the diaphysis of tubular bones. It has yellowish colour due to the presence of numerous adipocytes with pigment likes lipochromes.

As a rule iormal condition yellow bone marrow has no hematopoietic function but under certain conditions, such as severe bleeding or hypoxia, yellow bone marrow converts back into red bone marrow and myelopoietic islets appear from the bringing stem and hemistem cells.

There is no distinct difference between the yellow and red bone marrow. Some adipocytes may be observed in the red bone marrow constantly. Correlation between red and yellow bone marrow is changed in accordance with age, feed, nerve and endocrine regulation.

BONE MARROW BLOOD SUPPLYING

 

Red bone marrow possesses its blood supplying from the nutritive vessels, which introduce through the periosteum into the special opening in the compact bone. In the red bone marrow these arteries are branching into the ascending and descending branches from which arterioles are passing radially. They continue into narrow capillaries (2-4 мm), and then in the endosteum are transforming into the sinuses (10-14 мm in diameter). From these capillaries blood is collecting into the central venule. Hydrostatic pressure in the sinuses is higher because of smaller diameter of carrying out venule compare to arteriole size that’s why sinuses are constantly gaping and have chinks in the endothelial layer. Discontinuous layer of adventitial cells outside perform favourable conditions the red bone marrow cells migration into the blood. Less amount of the blood is passing from the periosteum into the osteons channels and then into endosteum and sinuses. Being in touch with bone tissue blood is enriched with mineral salts and hematopoiesis regulators.

Blood vessels comprise 50 % of bone marrow weight and 30 % of them – sinusoids. In the bone marrow of different bones arteries have a well-developed muscular tunica and adventitia, numerous venules with a thin wall, by the way, as a rule arteries and veins are passing separately. There are two types of capillaries in the bone marrow: the thin ones (6-20 мm) of somatic type and wide typical sinusoids 200-500 мm in diameter. The thin capillaries have nutritive function, while the larger ones are the place of erythrocytes maturation and place of mature blood cells passage into the blood stream. 

Innervation. Bone marrow is innervated from the nearest blood vessels plexuses, muscular nerves and special nerves to the bone marrow. Nerves enter the bone marrow with the blood vessels through the bones channels. Then they leave vessels and proceeded with independent branches into the medullar parenchyma of the spongy bone. They are ramifying into thin fibers, which touch to the walls of bone marrow vessels or lye free among the bone marrow cells.

Age changes. In the childhood red bone marrow fulfills epiphyses and diaphyses of tubular bones and in the lamellar bones. At about 12–18 years red bone marrow is transformed into yellow one in the diaphyses. In the old age bone marrow (both red and yellow) has a mucous constituency and is called gelatinous bone marrow. It is necessary to say, that this type of bone marrow may be found somewhere early (for example, in the developing bones of skull and face).

Regeneration. Red bone marrow has well prominent physiologic regeneration and good capability for reparative one. Stem cells in tight connection with reticular stroma are the source of hemopoietic cells formation. The speed of the red bone marrow regeneration much depends on the microenvironment and growth stimulating factors of hematopoiesis.

THYMUS (thymus)

 

In man the thymus or thymic gland is the central immune and lymphocytopoietic organ. It proves antigenindipendent differentiation of T-lymphocytes from their precursors. During this process, the immune system distinguishes self from foreign antigens and develops self-tolerance.

 It is an unpaired organ situated in the anterior mediastinum, in close connection with the pericardium and the great veins at the base of the heart. The thymus presents marked variations in its structure, depending on the age and on the condition of the organism as a whole. The organ is closed related lo lymphatic tissue; it produces lymphocytes and occasional lymphatic nodules develop within it. Other functions of this organ are the next: regulation of T-lymphocytes proliferation in the peripheral organs by means of thymosin production and their selection. Thymus also performs endocrine function, producing such hormones as: insulinlike factor, which decrease the sugar level in the blood, calcitoninlike factor, which decrease the calcium level and growth factor.

Histogenesis. The thymus is the first lymphoid organ to develop. In man the primordium of the thymus is an outgrowth of the third pharyngeal pouch on each side of the median line: the fourth pharyngeal pouch often gives rise to some thymic tissue. It has a cleft-like lumen and a wall of several layers of cylin­drical epithelium. The epithelial bud proliferates, the lumen disappears, and anatomizing strands extend into the mesenchyme. The future lobules arise at the ends of these branches.

At the 7^th week in an embryo of about 20 mm, the first small lymphocytes (thymocytes) appear. These cells develop from the inwandering of lymphoid cells. At the 8-11^th week of embryogenesis, mesenchyme with vessels introduces inside and divides thymus into lobules. At the 11-12^th week lymphocytes undergo differentiation and special receptors and antigens appear on the cells surface. Some of them are large, others are small, and there are numerous transitions between the two types. The number of lymphocytes increases greatly, partly from inwandering and in part through their own prolifera­tion. The small lymphocyte type gradually predomi­nates.

The epithelium is converted into a reticular cell mass whose meshes is occupied by the lympho­cytes and is penetrated here and there by blood vessels.

The definitive medulla arises late (at the 3^rd month) in the main stem and deeper portions of the lobules by hypertrophy of the epithelium, while most of the lymphocytes move from these areas or degenerate. Later, the Hassall bodies arise.

Newly formed T-lymphocytes then migrate to the lymph nodes and others peripheral lymphoid organs. During 3-5^th month stromal cells are developing and different types of T-lymphocytes appear. They are killers, suppressors and helpers, which are capable to lymphokines production. By the 6^th month thymus formation is ended and his epitheliocytes begin to secrete the hormones. During 15-17 days after the birth T-lymphocytes   migrate from thymus very actively and their activity increase too.

In relation to body weight, the thymus is largest during embryonic life and in child­hood up to the period of puberty. At birth the thymus weighs 12 to 15 gm. This increases to about 30 to 40 gm at puberty, after puberty the thymus begins to decrease in weight, so that at sixty years it weighs only 10 to 15 gm.

Structure. The thymus consists of two main lobes, one on each side of the median line, which is closely joined by connective tissue, but is not actually fused. Each of these lobes is divided into a number of macroscopic lobules varying from 0.5 to 2 mm in diame­ter. The lobules are separated from one another by the interlobular connective tissue and are divided into a darkly staining, peripheral cortical area and a lighter staining, inner medullary portion. With the study of serial sections one can trace a continuity of the medullary tissue from one lobule to another. That is, the medulla con­sists of a central stalk from which arise pro­jections of medullary tissue; these are almost completely surrounded by a zone of cortical tissue.

 

 

Panoramic view of the infant thymus, showing its lobulated structure. Within each lobule is a dark cortex and pale medulla. There are no round nodules or germinal centers in the cortex, just diffusely and densely packed lymphocytes. The thymus is seeded by lymphocytic stem cells very early in life and is particularly active in the production of lymphocytes in the young person. Those lymphocytes which resided in the thymic cortex early in life are forever after “thymus dependent”-cells , no matter where they reside in the body later on.

 

The difference between the cortex and medulla is the fact that the cortex consists mainly of densely parked small lymphocytes with their dark nuclei and, in between them, only a relatively few reticular cells, with pale-staining nuclei. In the medulla it is the reverse. As one proceeds from the cortex toward the medulla, the number of lympho­cytes drops rather abruptly, but there is no sharp line of demarcation between the two zones. The medulla is more vascular that the cortex.

 

 

Survey micrograph of the thymic cortex of the rat. 1= small lymphocytes; 2= medium-sized lymphocytes.

 

As parenchymatous organ the thymus consists of the stroma and parenchyma. The stromal epithelioreticular cells are elongated and have pale, round or oval nuclei. In most cases they contain predominantly euchromatin and one or two nucleoli. In the cortex it is difficult to follow the outlines of their cytoplasm, since the surrounding lymphocytes (thymocytes) are closely packed around them. However, in the medulla, where the lymphocytes are less numerous, it can be seen that the reticular cells form a network with its meshes filled with lymphocytes.

 

 

          Thymus cortical zone showing epithelial reticular cells with visible nucleoli (arrowheads) surrounded by dark-stained T lymphocytes undergoing differentiation. PT stain. Medium magnification.

 

In the embryo the epithelial nature of many of the reticular cells is quite obvious. However, as the organ becomes more and more heavily infiltrated with lym­phocytes, these epithelial cells become flat­tened, and it is difficult to distinguish them from the nuclei of connective tissue reticular cells. A peculiarity of the thymic cellular reticulum is the fact that in vitally stained animals these cells in the thymus do not take up any of the dyestuff, whereas in cer­tain diseases of malnutrition in infants they store large quantities of iron and fat, and in lipoid histiocytosis (Newmann-Pick dis­ease) they become swollen with lipid drop­lets. In experimental accidental involution the epithelial cells become loaded with dead lymphocytes.

The epithelial nature of the great mass of reticular cells becomes prominent when x-rays have destroyed the lymphocytes and the epithelium begins to develop. It becomes even more prominent in trans­plants and tissue cultures of the thymus. Certain tumors of clearly epi­thelial nature arise in this gland.

Different type of epithelioreticulocytes may be recognized due to their disposition, size, shape, hyaloplasm density, amount of organelles and inclusions. There are secretory cortical and medullary cells, nonsecretory (supporting) cells, and cells of Hassall’s body.

 Secretory cells contain vacuoles and secretory inclusions, which are composed of hormone-like factors: a-thymosin, thymulin, thymiopoietins. In subcapsular zone and outer cortex lymphocytes are rested in deep invaginations of epithelial cells. As a rule from 10 to 20 thymocytes may be disposed in such nurse-cell, thus cytoplasmic layer between them are very thin. Lymphocytes may “exfuse” and enter the invaginations and form the tight contacts with stromal cells.

The other types of supporting cells in the cortex are macrophages: typical free and their derivatives – dendrite cells.

The lymphocytes of the thymus are mor­phologically identical with the small lympho­cytes in the lymph node and other lymphatic tissues. Some of them are identical with medium-sized and large lymphocytes. Thymocytes arise from the mesenchyme in red bone marrow, and are lymphocytes, which have wandered into the epithelium. In addition, these small cells show the same susceptibility to x-ray injury as do ordinary lymphocytes. Both are cytolyzed by sera ob­tained by the injection of thymus cells into rats, and both show the same type of amoeboid motion and ability to transform into macrophages. Transplants of the thymus con­sist only of epithelium if lymphocytes are prevented by mechanical means from mi­grating into them. Small T-lymphocytes are disposed more peripherally, while lymphoblasts occupy the subcapsular zone of the cortex.

Clones of T cells are produced by cell division in the outer part of the thymic cortex and undergo maturation as they are pushed deep into the cortex toward the medulla.

In the medulla, the maturing T cells enter blood vessels and lymphatics to join the pool of circulating T cells. They subsequently populate peripheral lymphoid tissues, where they reach full immunologic maturity. The amount of the cells is much more less then in cortical zone of the lobule. Cell divisions are seen very rare (15 times less then in cortex).  Only a small minority of lymphocytes generated in the thymus are believed to reach maturity. These are clones of T cells with the ability to recognize foreign antigens.

The rest of the lymphocytes are thought to recognize self antigens and are eliminated: this results in immunologic self-tolerance.

Hassall’s bodies. The medulla con­tains the bodies of Hassall, which are charac­teristic of the thymus. They are rounded mild acidophilic structures, which vary from 30 to 100 microns in diameter. They are composed of concentrically arranged cells, many of which show evidences of degeneration and hyalinization. Reticular cells are con­nected at one or more places with the periphery of each Hassall body. The cells of the central part of Hassall body may de­generate completely, so that small cysts may develop in the center. In other cases calcium may be deposited in them.

 

The paler thymic medulla shows some bright pink Hassall’s corpuscles. They are diagnostic features for thymus.

 

Detail of Hassall’s corpuscle, with concentric layers of keratinizing epithelial cells. Although the significance of the corpuscles is uncertain, they are apparently formed from the epithelioid stromal cells.

 

Involution of the thymus

 

The thymus reaches its maximum weight at puberty after which it begins to involute—a process, which pro­ceeds gradually and continuously through­out life under normal conditions. It is declining so that in old age it may be so small as to be unrecognizable. This change in its structure is spoken of as age involution.

 

 

Panoramic view of adult thymus, largely replaced with adipose tissue. There are recognizable remnants of thymic lymphatic tissue, however, and Hassall’s corpuscles are still present in the medulla.

 

During the course of infectious and cachectic diseases the normal slow in­volution may be greatly accelerated. This is called accidental involution, and explains many of the contradictory reports on the size of the thymus, since the organ did not have a chance to regenerate in those persons who died as a result of severe infection.

The clear-cut separation of the thymus into cortex and medulla obtains normally in the later em­bryonic periods and in childhood. Normally, involution begins as a gradual thinning out of the lymphoid cells of the cortex; at about four years the epithelial reticular cells be­come coin pressed, and the area occupied by them is gradually replaced by adipose tis­sue (fatty infiltration), which is thought to arise in the inter-lobular connective tissue. The medulla be­gins to atrophy at puberty. Adipocytes increase iumber in the perivascular compartment. Initially this is most apparent in the septa, thus the cortex is involved first and then the process is extended into the medulla. This process continues throughout life. Lymphocytes depletion begins after one year of age and thereafter continues at constant rate independent of puberty. It results in progressive collapse of the spong-like epitheliocyte framework, which nevertheless remains intact, so that cords of epitheliocytes can be seen histologically even in the most atrophic thymic remnants. Such cells probably continue to secrete thymic hormones into old age.

Despite the progressive decrease in their number with involution, thymic lymphocytes continue to differentiate and proliferate, thus maintaining a supply of T cells throughout life.

 The last elements to be replaced are the Hassall bodies, but even in very old persons there are scattered Hassall bodies surrounded by a few reticular cells and lymphocytes. This process of nor­mal or age involution may be complicated by the rapid changes of “accidental involu­tion”. Histological picture of the thymus are similar to the above mentioned age involution but it is connected with rapid exfusion of T lymphocytes into the blood stream and their mass destruction inside in the thymus especially in the cortical zone. At the same time epitheliocyte are enlarged. Their cytoplasm contains numerous droplets, which are glycoproteid-positive. Sometimes these inclusions are organized into follicular structures between the cells.

The thymus is tightly interconnected with adrenal gland. The increase of the adrenal cortex hormones, especially glucocorticoids, may produce very active and well prominent accidental involution of the thymus.

But sometimes, age involution of the thymus does not occur. Such situation is known as “status thymico-lymphaticus”, it is the evidence of serious problem of adrenal cortex deficiency. In such men there is enlarged lymphoid tissue: enlarged lymph nodes and even in adult well prominent thymus but there are no enough hormones of adrenal glands. They are very sensitive to different influences from outside and require a special attention of medical stuff.

Vessels and nerves. The thymus has a reach vascular supply, which allows migration of lymphoid cells. The arteries supplying the thymus arise from the internal thoracic and the inferior thyroid arteries and are first distributed to the cortical tissue via the interlobular septa.

In the region of the corticomedullary junction the vessels give rise to small radially arranged arterioles and capillary loops to supply the cortex and medulla. The cortical capillaries have continuous endothelium, whereas those of the medulla and septa are of fenestrated type. Continuous basement membrane and a layer of epithelial cells, which border pericapillary space, surround cortical capillaries. In this space a lot of macrophages and some lymphocytes are observed. All this structures are named “the hematothymical barrier”. It includes the wall of hemocapillary, which consists of endothelial cells and continuous basement membrane, pericapillary space with lymphocytes, macrophages and intercellular substance and epithelioreticular cells with their basement membrane. This barrier has selective permeability as for the antigens.  It preserves the lymphocytes from antigens influence and thus promotes their antigen independent prolipheration. In the case of barrier been damaged isolated plasma cells, granular leucocytes and must cells are observed. Sometimes foci of extramedullary myelopoiesis appear in the cortex.

 At the corticomedullary junction, which is the site of lymphocyte migration into the thymus, postcapillary venules have a taller endothelium with features of high endothelial venules. Lymphocytes of this zone may migrate out of thymus and return back (recirculate).

Venous tributaries follow the course of the arterial vessels in the septa, some forming a plexus within the thymic capsule before draining via the thymic veins into the left brachiocephalic, internal thoracic and inferior thyroid veins.

Thus, the blood drainage from thymic lobules cortex and medulla is separating.

 The thymus receives no afferent lymphatics. But the medulla and corticomedullary area give rise to efferent lymphatics. There are superficial (capsular and subcapsular) and deep (parenchymatous) network of efferent capillaries. Parenchymatous network is especially well developed in the cortical layer, while in medulla they are found around the Hassall’s bodies. Lymph capillaries are gathered into the interlobular vessels, which follow the course of the arteries and veins and run mainly in the interlobular connective tissue and empty into the anterior mediastinal and tracheo-bronchial lymph nodes.

The thymus receives a few branches from the vagus and sympathetic nerves; these are probably mainly of vasomotor nature.

Thymus functions. As a central hematopoietic organ thymus is responsible for the antigenindependent proliferation of T lymphocytes, it also participates in the lymphocytes selection and regulates their proliferation and differentiation in the peripheral hematopoietic organs by means of hormone thymosin.

The change from a large organ during embryonic development, infancy, and child­hood, into a gradually disappearing organ with the development of sexual maturity, has led many authors to ascribe an endocrine function to this gland.

This function includes thymosin production and synthesis of some others biologically active factors as insulinlike factor, which decrease the sugar level in the blood, calcitoninlike factor, which decrease the calcium concentration in the blood and growth factor.

Of course, endocrine function of the thymus is tightly connected with endocrine system function. Purified adrenocorticotrophic hormone causes a striking reduction in weight and size of the thymus in male rats. Repeated in­jection of horse gonadotrophic hormone cause’s atrophy of the thymus, but this does not occur in castrated rats. On the contrary, castration causes hyperplasia of the involuted gland in the rat. Selye has found that fasting, toxins, and morphine cause a rapid atrophy of the thymus and enlargement of the adrenal cortex in rats, and that atrophy of the thymus does not take place in adrenalectomized rats; hypophysectomy hastens thymic atrophy. The claims that the injection of extracts of the¹ thymus into parent rats causes a pre­cocious growth of their progeny, which is cumulative in succeeding generations, and that thymectomy causes retardation in growth of the progeny, have not been sub­stantiated.

 

Peripheral hemopoietic system

LYMPH NODES

 

These are the smallest, but most numerous encapsulated lymphoid organs. Scattered in groups along lymphatic vessels, they act as inline filters of the lymph, removing antigens and cellular debris and adding immunoglobulins.

Lymph nodes are encapsulated round or kidney-shaped organs composed of lymphoid tissue. They are distributed throughout the body, always along the course of the lymphatic vessels, which transport lymph into the thoracic and the right lymphatic ducts. They are found in the axillas and in the groin, along the great vessels of the neck, and in large numbers, in the thorax, the abdomen, and especially in the mesentery.

Lymph node has both convex and concave surfaces. The parenchyma (lymphoid tissue) consists of a peripheral cortex, adjacent to the convex surface, and a central medulla lying near the depression (hilum) in the concave surface. The connective tissue capsule that covers the node’s surface gives off trabeculae that penetrate between the cortical nodules and subdivide the cortex. Blood vessels enter and leave through the hilum.

 

 

The darker cortex at the periphery of the node has nodules, some of which show pale, mitotically active germinal centers. In the middle of the lymph node is the medulla with its dark cords of dense lymphocyte population. The medullary cords are surrounded by paler lymph channels (the medullary sinuses) which have relatively fewer lymphocytes. Lymphatic afferent vessels enter the node at the periphery, through the capsule (blue c.t. here), pouring lymph into the sinus system of the node. The lymph “percolates” through the cortical and medullary sinuses and leaves via the efferent lymphatics near the hilus of the node. While looking at this picture, be reminded that the nodules of the outer cortex and the cords of the medulla are included among the “homing areas” for B-cells, plasma cells, and helper T-cells. The thymic dependent area, where the majority of T-cells are found, is the deeper part of the cortex, seen here as the dense, diffuse lymphoid area lying below the cortical nodules and above the medullary cords. It is within this inner cortex that the postcapillarv venules also lie; they have an unusually high endothelium, and it is through their walls that lymphocytes of the bloodstream enter the substance of the node.

 

 

Detail of outer surface of lymph node, with pale c.t. capsule on the outside, sending an extension (trabecula) down into the substance of the node as a frame-work. Pale sinus channels can be seen lying immediately beneath the capsule (subcapsular sinus) and surrounding the trabecula (cortical or trabecular sinuses). From these superficial sinuses, lymph then flows into the deep medullary sinuses (not pictured here). Stellate reticular cells can be seen spanning the sinuses, where lymphocytes are less dense. Reticular cells support the lymphocytes of the denser lymphoid tissue as well, but are harder to see there.

 

Cortex in active lymph nodes, the cortex is dark-staining because of the presence of tightly packed lymphocytes. These are suspended in a reticular connective tissue network and arranged as a layer of typical secondary lymphoid nodules (containing primarily В lymphocytes) with germinal centers. The cortex also contains reticular cells, antigen-presenting follicular dendritic ceils, a few plasma cells, and some helper T cells. Secondary lymphoid nodules are covered by an incomplete lining of reticuloendothelial cells. These cells combined structure of reticular cells with function of epithelial cells, because they cover sinuses of the lymph nodes. Bach germinal center contains В lymphoblasts and it is light, here is proliferation of lymphocytes. In dark peripheral portion of the germinal center there are a lot of small and middle-sized lymphocytes.

Medulla lighter staining than the cortex, the medulla is composed of cords of lymphoid tissue (medullary cords). The В lymphocytes are mainly small, less numerous than in the cortex, and concentrated in the cords. The cords are also rich in macrophages and contain many plasma cells that have migrated from the cortex. Medullary cords are covered by reticuloendothelial cells as secondary lymphoid nodules in the cortex.

 

 

Pale medullary sinuses surrounding darker medullary cords. You can see many stellate reticular cells which, with reticular fibers, make a meshwork through the sinuses. Lymph “percolates” through the meshes of the sinuses while debris of foreign matter in it is phagocytized. Lymph nodes thus act as filters for the connective tissue fluid compartment of the body.

 

Paracortical zone. This is the T-dependent region, lying between the cortical lymphoid nodules and the medulla. It is poorly defined morphologically but functionally distinct. It contains mainly T-lymphocytes suspended in a reticular connective tissue network. В lymphocytes, plasma cells, and antigen-presenting interdigitating cells may also be present. Antigen-presenting interdigitating cells connected together by their processes and produce substances which stimulate the proliferation of T lymphocytes.

This zone is also characterized by the presence of many high-endothelial postcapillary venules. T lymphocytes leave the blood to enter the paracortical zone by passing between the cuboidal endothelial cells of these vessels.

Lymphocytes found in the paracortical zone dissapear when an animal has its thymus removed, especially if this is dine at birth. This shows that they are thyraus-deperident, belonging to the population of T lymphocytes. They derive from precursor cells that inigrate from the bone marrow to the thymus, where they divide by mitosis. Г lymphocytes are most numerous in blood and lymph and are found also in the periarterial sheaths of the spleen and other lymphoid organs. They are responsible for cell-mediated immune responses such as delayed immune reactions and graft rejections. These cells colonize the thymus-dependent zones in the peripheral lymphoid organs with long-lived lymphocytes that recirculate and are capable of further replication under proper stimulation. The remaining lymphocytes, which constitute the greater mass of cells in the cortex, are the В cells, responsible for the synthesis and secretion of immunoglobulins (antibodies).

Lymphatic vessels associated with lymph nodes are of 2 types. Both contain valves to ensure a unidirectional flow of lymph through the node. Afferent lymphatic vessels deliver lymph by penetrating the capsule at several points on the node’s convex surface. Efferent lymphatic vessels carry filtered lymph away from the node, exiting through the hilum on the concave surface.

Sinuses. The lymphatic sinuses are the spaces incompletely lined by reticuloendothelial cells. The sinuses of the lymph nodes filter the lymph passing through them and direct its flow. Partly lined by reticular cells and many macrophages, they are not simply open spaces, but are traversed by a meshwork of reticular cells and fibers, macrophages, and follicular dendritic cells. The complex seiving action slows lymph flow to facilitate the removal of antigens. Lymph is delivered by the afferent vessels to the cuplike subcapsular sinus between the capsule and cortical secondary nodules. From here it passes directly into the peritrabecular sinuses on either side of the trabeculae. It then flows through the anastomotic network of medullary sinuses which are between medullary cords that converge on the efferent lymphatic vessels exiting through the hilum.

Filtration of lymph. Cellular debris and antigens carried by incoming lymph are removed by the macrophages and follicular dendritic cells of the sinuses (similar ceils are found in the cortical nodules and medullary cords). The lymphocytes carried by the lymph may flow through the nodes, contacting antigen-presenting cells and macrophages in the sinuses, they may instead leave the sinuses and enter the parenchyma. By the time the lymph reaches the efferent lymphatic vessels, more than 90% of the antigens and cellular debris have been removed. More than 99% of the lymph remains in the sinuses as it passes through the nodes.

Almost all of the lymph traverses the nodule by flowing through the sinuses. However, a small amount, less than 1 % of the volume, will penetrate the nodules. As the lymph filters through the nodules, the bulk of the antigen is processed by macrophages. Some antigen, however, is trapped on the surface of specialized reticular cells known as dendritic cells. This bound antigen is not phagocytosed but is exposed on the dendritic cell surface where it may be recognized and acted upon by immunologically competent lymphocytes. If а В cell recognized the antigen, under appropriate conditions (which may necessiate the involvement of T cells), the В lymphocytes may be activated. Activated В lymphocytes migrate to the germinal center and undergo a series of transitions and cell divisions that lead to the production of immature immunoblasts. These in turn divide and give rise to plasma cells and activated В lymphocytes. The plasma cells leave the germinal center and migrate into the medullary cords. Here, the cells actively synthesize specific antibodies and release them into the lymph flowing through the medullary sinuses. Activated В cells, which can secrete some antibody and also bind some to their surface, leave the nodule and flow with the lymph to reenter the circulatory system. If in its travel the В cell encounters more of the stimulating antigen, it may leave the blood, enter the connective tissue, and differentiate into an immotile, secretory plasma cell. As a consequense of infection and antigenic stimulation, affected lymph nodes exhibit swelling, reflecting the formation of multiple germinal centers and active cell proliferation. In resting nodes, plasma cells constitute 1 – 3 % of the cell population; however, their numbers are greatly increased and they partially account for the enlargement of stimulated 1 mph nodes.

 

SPLEEN

 

          The spleen is the largest accumulation of lymphatic tissue in the organism, and in humans it is the largest lymphatic organ in the circulatory system. It is a visceral organ lying between the gastric fundus and the diaphragm. The spleen is 12 cm long, 7 cm wide, and 4 cm thick and weighs 100-150 g in a healthy adult.

The largest lymphoid organ, spleen’s functions include lymphopoiesis, immunoglobulin production, and filtering the blood for cellular debris and antigens. Because it serves as the immunologic filter of the blood, its blood supply and circulation are especially important. The spleen is encapsulated in a dense irregular connective tissue capsule covered by mesothelium. Unlike other lymphoid organs, the spleen lacks a definitive cortex and medulla. The parenchyma (splenic pulp) lacks true lobules; however, the dense connective tissue capsule gives rise to trabeculae that divide the splenic pulp into incomplete compartments (white and red pulp).

The cornective tissue of the capsule and of the trabeculae contains some smooth muscle cells. In humans, these cells are not numerous. In certain other mammals (cat, dog, horse) they are quite abundant, and their contraction causes the expulsion of accumulated blood from the spleen, which has a spongy structure and serves to store blood cells.

Splenic pulp. This is composed of many erythrocytes, leukocytes and macrophages, as well as a variety of blood vessels, all suspended within a supporting meshwork of reticular cells and fibers, the main function of which is of support. Unstained slide of splenic pulp exhibit numerous whitish islands of lymphoid tissue (white pulp) embedded in a sea of dark red erythrocyte-rich tissue (red pulp).

White pulp (20 %) is composed of collections of lymphocytes similar to the primary and secondary nodules of lymph nodes. These nodules are surrounded by reticuloendothelial cells. These nodules are easily distinguished from other nodules encountered in the various lymphatic organs because of the presence of the central artery.

 

 

           Closer examination of white pulp with two eccentrically placed “central arterioles” (quite pink here). A germinal center has formed here, displacing the artery to the edge of the area. As always, the germinal center indicates active production of plasma cells from B-lymphocytes. Helper T-cells mingle with the plasma cells at the rim of the nodule. Meanwhile, the majority of splenic T-cells reside in the non-nodular portions of the periarterial sheath immediately surrounding the central arteriole. Macrophages tend to congregate in the marginal zone between white pulp and red pulp, and here they begin to process red blood cells for possible breakdown and phagocytosis.

 

Nodules of the spleen white pulp have four major components: 1 well-formed germinal centers; 2. periarterial lymphatic sheaths; 3. peripheral white pulp; 4. marginal zone.

Germinal centers have the same structure and functions as lymphatic nodules of the lymph nodes. They contain В lymphoblasts, dendritic and reticular cells. The appearence of germinal centers is the reaction for antigen stimulation.

The sleeves of lymphoid tissue immediately surrounding each central artery tire called periarterial lymphatic sheaths (PALS). These contain mainly T lymphocytes and constitute the thymus -dependent zones of the spleen. Surrounding each PALS or appended to one of its sides is the third component, the marginal zone.

Marginal zone Forming the border between white and red pulp, this zone consists of a moatlike arrangement of blood sinuses and loose lymphoid tissue containing few T and В lymphocytes, but with many macrophages, having branching processes and showing active pfagocytosis. The marginal zone retains great amounts of blood antigens and thus plays a major role in the imrnulogic activity of the spleen. Many of the pulp arterioles, derived from a central vein, extend out and away from the white pulp but then turn back and empty into the moatlike sinuses of the marginal zone that encircles the nodules. As a consequence of this drainage, which includes the added blood flow from vessles within the white pulp that also terminate in the marginal zone, this area plays a significant role in filtering the blood and launching an immune response. A large number of macrophages end reticuloendothelial cells serve to phagocytose and remove antigenic debris. Dendritic cells in the marginal zone trap and present antigens to immunologically competent cells. The marginal zone is an idea! site for this activity since not only the antigens are removed from the blood here but also the T and В lymphocytes. All these lymphocytes leave the systemic circulation to penetrate the white pulp, they pass by the dendritic cells, presenting exposed antigen. If the appropriate В cells. T ceils, and antigen are present, an immune response will be initiated. Activated В cells migrate to the center of the v/hite pulp nodule, the germinal center, and give rise to immunoblasts, plasma cells, and activated В cells. The latter 2 cells enter the red pulp, where the plasma cells remain in the cords and release antibody into the blood of the sinuses. Activated В cells leave the red pulp and return to the general circulation.

The lymphocytes of the periarterial lymphatic sheaths are thymus-depended, whereas the marginal zones and periphery of the nodules – the peripheral white pulp – are populated by В lymphocytes. Thus, the splenic white pulp has В and T lymphocytes segregated in 2 different sites.

The splenic reticulum (stroma). Reticular cells and reticular fibers similar to those in lymph nodes exist throughout much of the parenchyma of the spleen. These cells and fibers form a scaffolding that supports both red and white pulp of the spleen. Reticular cells and fibers around central arteries are arranged in concentric layers (as opposed to a sponge mesh) to support the PALS.

Red pulp (80 %) is composed of elongated structures, the splenic red pulp cords which lie between the splenic sinusoids. Red pulp cords (Billroth’s cords; are irregular sheets of reticular connective tissue, these cords branch and anastomose to surround the sinuses. They vary in thickness according to the distention of the adjacent sinusoids. In addition to reticular cells and fibers, the cores contain many cell types, including all the formed elements of blood (erythrocytes, platelets, and granulocytes), dendritic cells, macrophages, plasma cells, and lymphocytes.

 

 

Panoramic view of spleen, staining bright pink throughout the “red pulp” because of the presence of so many filled and distended blood sinusoids. Scattered at random throughout the red pulp are aggregates of lymphocytes (“white pulp”, also known as Malpighian bodies). Notice the pink structures within the white pulp. These are the central arterioles, and they are diagnostic features of the spleen. The arteriole characteristically has a very small lumen but a definite smooth muscle coat. Actually, white pulp is arranged in long “sleeves” of lymphatic tissue (PALS: periarterial lymphatic sheaths) following along the length of the arteries; here we are cutting the arteries and their sheaths in cross-section. Arteries leave the trabeculae quite early to travel through the pulp, while veins tend to remain within the trabeculae. The very large, mostly empty vessel seen here is a trabecular vein.

 

Splenic sinuses are peculiar vascular channels, which are lined by tapered cells elongated parallel to the long axis of the sinus. These cells contain many contractile microfilaments and lack attachment specializations between them. Fixed macrophages between them are known to be macrophages (waterside cells)

The splenic sinuses are surrounded by a fenestrated basement membrane and by adventitial reticular cells that comprise part of the splenic cords. Blood cells in the splenic cords pass through the wall of a splenic sinus between the lining cells, into the lumen of the splenic sinus, and then out of the spleen through the splenic veins.

Splenic sinusoids. These differ from common capillaries in 3 ways: 1. they have a dilated, large irregular lumen; 2. between their lining endothelial cells are spaces that facilitate exchange between the sinusoids and lying tissues; 3. the basement membrane-like material is not continuous but forms barrel hooplike rings around the endothelial walls.

The endothelial cells that line the splenic sinusoids are elongated, with their long axis parallel tj the sinusoids. These cells are enveloped in reticular fibers set mainly in a transverse direction, similar to the loops of a barrel. The transverse fibers and those oriented in various directions join to form a network enveloping the sinusiod cells. It was formerly thought that the endothelial cells of sinuses were phagocytic; however, it appears that the observed phagocytosis was due to processes of macrophages that has penetrated into the spaces between adjacent endothelial cells. The spaces between the cells of the splenic sinusoids can be 2- 3 μm in diameter or even larger, so that erythrocytes are able to pass easily from the lumen of the sinusoids to the red pulp cords.

The slitlike spaces between the endothelial cells permit extensive exchange of fluids, solutes, and flexible cells between the sinusoids and cords. Macrophages in the cords extend their processes through the slits and phagocytose material in the sinusoid lumen.

Splenic circulation

 

         Arterial supply. The spleen receives blood from the splenic artery (a branch of the celiac trunk of the aorta). Near the hilum, the splenic artery branches into a series of trabecular arteries. These enter the spleen through the trabeculae and branch to enter the parenchyma as the numerous central arteries around which the white pulp is organized. After passing through the white pulp, the arteries give rise to many penicilar arterioles, which in turn give rise to many capillaries and sheathed arterioles. Near their termination, the sheathed arterioles have localized wall thickenings that consist of macrophages. Many of the capillaries arising from the central artery loop back toward the white pulp to feed the marginal sinuses. Others, including those arising from the penicilar and sheathed arterioles, are. the sinusoids of the red pulp.

         Open and closed theories of splenic circulation. The way in which blood in the splenic capillaries reaches the sinusoid lumens is not clear. The closed theory of circulation holds that the capillary walls are continuous with the walls of the sinusoids and the capillaries empty directly into the sinusoid lumens. The open theory holds that the capillaries end abruptly in the red pulp cords and that blood reaches the sinusoid lumens by percolating through the cords and passing through openings in the sinusoid walls. For humans, current evidence favors the open theory.

         Venous drainage from the sinusoids, blood flows into red pulp veins that converge on the trabeculae and empty into trabecular veins; these are unusual in that walls lack a distinct tunica media. The trabecular veins exit the spleen through the hilum, emptying into the splenic vein, which joins the inferior mesenteric vein and empties into the hepatic portal vein just before it enters the liver.

 

Functions of the spleen

 

 1. Elimination of old and abnormal blood cells (e.x.) erythrocytes.

 2. Hemopoiesis (formation of lymphocytes in adult, all blood cells in embryo).

3. Immune defence of organism against foreign particles that enter the bloodstream.

 4. Storage of the blood.

5. Endocrine (splenin synthesis).

1. The spleen is a complex filter. In the cords and sinuses, aged and damaged erythrocytes are examined, removed from the circulation, and destroyed. Toward the end of their average 120-da> lifespan, erythrocytes become less flexible, they become less plastic due, in part, to age-dependent changes in the chemical composition of the plasma membrane, they can fragment before or during their passage through the spleen. Young, healthy cells can make their way through the phagocyte-laden cords into sinuses and out of the spleen. Aging cells are retarded in the cords and eventually are engulfed by macrophages and destroyed. Splenic macrophages and phagocytic reticular cells also clear particulate material from the blood. The engulfed erythrocytes are altered and digested by the lysosomes of the phagocytes. The hemoglobin they contain is broken down into several components, forming a pigment, bilirubin, which contains no iron, and a protein, ferritin, which does contain iron. The protein portion (globin) is hydrolysed to amino acids for reuse in protein synthesis. Iron released from the heme is transported, with transferrin, to the bone marrow for reuse in erythropoiesis. These compounds are then returned to the blood. Bilirubin is excepted by the hepatic cells together with the bile. Ferritin, which represents a form of mobile iron, is used by the erythrocytes of the bone marrow, which draw iron from it for the synthesis of new hemoglobin.

2.       The white pulp of the spleen produces lymphocytes that migrate to the red pulp and reach the lumens of sinusoids, where they are incorporated into the blood that is present; there. A constant flow of lymphocytes is observed from the splenic parenchyma to the bloodstream as well as in the opposite direction. Radioautography after intravenous injection of radioactive lymphocytes has demonstrated that many of these labeled lymphocytes appear in the white pulp
of the spleen. In the fetus, the spleen also produces granulocytes (neutrophils, basophils and eosiniphils) and erythrocytes, but this activity ceases at the end of the fetal phase. In certain pathologic conditions (eg, leukemia), the spleen may recommence the production of granulocytes and erythrocytes, thus undergoing a process known as myeloid metaplasia (pathologic transformation of one kind of cell into another). In some cases, the liver and lymph nodes resume similar functions. Since it contains both T and В lymphocytes and macrophages, the spleen is important in body defence. In the same way that lymph nodes “filter” the lymph, the spleen is considered as a “filter” for the blood.

4. Blood deposition in venous sinuses.

5. Endocrine function – splenin synthesis (antierithropoietin).

The T lymphocytes found in the periarterial sheats of the white pulp proliferate and enter the bloodstream. They participate in cell-mediated immune mechanisms.

Radioautographic studies with labeled antigens injected into the blood show that the antigens are preferentially retained by the surface of cells found in the nodules and in the marginal zone. These ceils have many long processes that branch profusely, thus increasing the cell surface. Because of their shape they are called dendritic cells.

Under the stimulus of antigens, splenic В lymphocytes proliferate and give rise to antibody-producing plasma cells.

Of all the macrophagic cells of the organism, those of the spleen are most active in the phagocytosis of living particles (bacteria and viruses) and inert particles that find their way into the bloodstream. After the injection of trypan blue, the macrophages of the spleen are among the first to accumulate this dye.

When there is an excess of lipids in the blood plasma (hyperlipidemia), the macrophages of the spleen accumulate considerable quantities of these substances. In diabetes, hyperlipidemia is frequent, and for this reason large macrophages, their cytoplasm containing numerous lipid droplets, are common in the spleens of diabetics.

4.  Owing to the spongy structure of the red pulp, the spleen stores blood, which can returned to the circulation to increase the volume of circulating blood. In animals with spleens composed of a capsule and trabeculae rich in smooth muscle, the organ is emptied by muscular contraction. Because the haman spleen is poor in smooth muscle fibers, the storage and expulsion of зіоос dependes on changes in the diameter of the blood vessels. It has been
demonstrated tha: in humans the blood storage capacity of the spleen is very small.

5.   Lymphopoiesis.   Both  В   and  T  lymphocytes  are   activated   in   by  the   spleen. Lymphocyte-antigen interactions are more intense in the white pulp, particularly near- the marginal zone, but they also occur in red pulp. T lymphocyte effector cells formed in the PALS migrate through the pulp cords to the sinusoids to enter the circulation. В  lymphocytes stimulated in the marginal zone move to germinal centers of the PWP, where they divide. Plasma cells generated in this way leave the white pulp. They migrate and remain in red pulp cords, producing immunoglobulins that percolate into the sinusoids and leave the spleen in the venous blood.

Splenectomy. Although it has important functions, the spleen can be removed without serious damage to the individual. Other organs with cells similar to those found in the spleen will compensate for its loss.

After spleenectomy, a temporary increase in the number of lymphocytes is observed in the blood. This is due to excessive compensatory lymphocyte production by other lymphatic organs (lymph nodes, isolated nodules). The number of platelets also increases

Spleenectomy is beneficial in diseases where there is a deficiency in bone marrow function. In these cases, spleenectomy is followed by bone marrow activation. This permits the conclusion that the spleen inhibits bone marrow function in such cases. This inhibiting effect has not been proved under normal conditions, but many investigators argue that the spleen has a regulating effect on bone marrow. This effect would be more pronounced in certain pathologic states.

 

LYMPHATIC NODULES

 

also called lymphatic follicles – can be found isolated in the loose connective tissue of several organs. These aggregations consist of lymphocytes that occur in small clusters. The classic example of these structures is Peyer’s patches that are found in the lamina propria of the small intestine (ileum). Nodule clusters also occur in the appendix, and there are scattered solitary nodules beneath the epithelium in the walls of the digestive, respiratory, urinary, and genital passageways. These occur especially at branchpoints and the sites at which one organ joins another (e.g., esophageal cardiac-stomach junction, recto-anal junction). While lacking the connective tissue capsule that surrounds lymphoid organs, nodules may be covered by a layer of flattened reticular cells.

 

 

 

This slide shows an isolated, single primary lymph nodule in the wall of the stomach stained dark purple because of the small lymphocyte accumulation. Such nodules are often found in the wall of tracts that exit to the outside and represent a first line of defense against foreign substances that might enter the body via these routes. Nodules like this in the GI tract are thought to be one source of B-type lymphocytes producing IgA antibodies.

 

Each lymphatic nodule is a round structure, in histologic sections they are strongly stained by hematoxylin as a consequence of the presence of a dense population of lymphocytes, which possess a basophilic nucleus with condensed chromatin and a narrow corona of basophilic cytoplasm. The interior of the nodule often shows a less densely stained region called the germinal center. This difference in staining of the central region of many nodules is due to the presence of activated lymphocytes (immunoblasts) that exhibit a large, pale-staining cytoplasm and large, active, euchromatic nucleus; for this reason, it contrasts with smaller lymphocytes, which have darker nuclei and predominate in the periphery of the nodule, which is not well defined.

The presence of the germinal center may appear and disappear in a nodule according to its functional state. The activity of the lymphatic nodules depends upon several factors including the effects of the bacterial flora. In animals kept in sterile conditions, nodules with germinal centers are rare. The opposite situation occurs in some infections, where the production of lymphocytes increases and germinal centers become frequent. In newborns as well as in animals grown in aseptic environments, lymphatic nodules are very rare., which indicates that their formation depends on antigenic stimuli. In localized inflammations, there is an increase in the number of limphatic nodules close to the inflamed site, and most nodules have a germinal center.

Lymphoid nodules are commonly divided into 2 classes:

Primary Nodules: Containing only small lymphocytes, primary lymphoid nodules are present prenatally and in the absence of antigens (eg, in animals housed in sterile surroundings). Primary nodules, which lack a germinal center, become secondary nodules on exposure to antigens.

Secondary Nodules: These nodules, which appear after birth, are primary nodules that have been activated by exposure to antigens; their size and number are proportional to the amount of antigenic stimulation. They occur in the same sites as the primary nodules. Structurally, the nodule is characterized by a narrow, dark-staining peripheral hale of small lymphocytes surrounding a larger, lighter-staining germinal center that contains mainly lymphoblasts. The dark periphery often shows a cap, a localized crescent-shaped thickening. The size of the germinal center gradually decreases when antigenic stimuli are removed, and the nodules eventually assume a form more like that of a primary nodule. Although thin sections through a cap or periphery of an active secondary nodule may resemble primary nodules, the presence of primary nodules is doubtful if nearby nodules contain germinal centers.

TONSILS

 

These incompletely encapsulated lymphoid aggregates contain many lymphoid nodules; they underlie the mucous membranes (epithelial lining) of the mouth and pharynx. Together with the diffuse subepithelial lymphoid tissue that connects them to form a ring, they guard the common entrance to the digestive and respiratory tracts. The 3 types of tonsils (named according to their locations) differ iumber, epithelial covering, presence (or absence) and number of epithelial invaginations or crypts, and presence (or absence) of a definitive partial capsule

Palatine tonsils. These are 2 dense lymphoid aggregates found in the lateral walls of the oral pharynx, below the back of the soft palate. Each tonsil is covered by nonkeratinized stratified squamous epithelium and partly penetrated by 10-20 crypts. The tonsils are separated from underlying tissues by a partial capsule of dense connective tissue that acts as a barrier against the spread of tonsillar infections.

 

 

 

Palatine tonsil made up basically of epithelial crypts extending into the connective tissue coats of the pharyngeal wall. You can see the luminal lining of stratified squamous epithelium to the right, continuing down into the crypts. This epithelium plus many lymphatic nodules is diagnostic for tonsil. The nodules pictured here show pale germinal centers. Tonsils are a first line of defense in the GI tract.

 

 

 

            H&E stain of tonsil with stratified squamous epithelium lining the lumen of crypts. Often such epithelium is all but hidden by lymphocytes wandering through it. Note lymphatic nodules with germinal centers, indicating active production of plasma cells from B-lymphocytes.

Pharyngeal tonsil. A single small lymphoid aggregation at the back of the nasopharynx above the soft palate, this is covered by ciliated pseudostratified columnar epithelium. Its surface may be pleated, but it has no crypts. A thin partial capsule separates it from underlying structures.

Lingual tonsils. The smallest and most numerous tonsils, these are located at the back of the tongue. Covered by partly keratinized stratified squamous epithelium, each tonsil has a single crypt but no definitive capsule.

 

 

 

 

 

 

 

 

Students’ Practical Activities

Students must know and illustrate such histological specimens:

Specimen 1. Bone marrow smear.

Romanowsky-Giemsa stain.

 

 

Intertrabecular spaces of all bones are filled with bone marrow containing the primitive stem cells from which all the cellular elements of blood are derived. In section, bone marrow is seen to consist of a mass of nucleated blood cell precursors pervaded by broad blood sinuses. This bone marrow smear illustrates several stages in erythropoiesis and granulopoiesis. Reticulocytes are usually slightly larger than the surrounding mature erythrocytes, and their cytoplasm contains granullar structures – remains of nuclei and organells. Erythrocytes are stained pink due to their high content of haemoglobin. The pale staining of the central region of the erythrocyte is a result of its unusual biconcave disc shape.

In this speciment you also see three phases of neutrophil granulocyte development. A neutrophil myelocyte is recognized by a large, eccentrically located nucleus, a prominent Golgi apparatus and cytoplasm containing many azurophilic (primary) granules. The next stage towards maturity, the metamyelocyte is a smaller cell characterized by indentation of the nucleus and loss of prominence of the azurophilic granules. The final stage before maturity, the stab cell, has a more highly segmented nucleus approaching that of the mature neutrophil.

Megakaryocytes – are the cells of bone marrow where they are responsible for production of platelets. Megakaryocytes are huge cells with a single, highly irregular polyploid nucleus. The extensive cytoplasm appears finely granular due to a profusion of organelles. Platelets are formed by budding from the megakaryocyte cytoplasm.

Illustrate and indicate: 1.Proerythroblast. 2.Polichromatic erythroblast. 3.Neutrophil metamyelocyte. 4.Megakaryocyte. 5.Sinusoidal capillaries: a)erythrocytes; b)leucocytes.

 

Specimen 2. Thymus.

Haematoxylin and Eosin.

 

 

            The thymus is a highly lobulated organ invested by a loose connective tissue capsule from which short septa containing blood vessels radiate into the substance of the organ. Thymic tissue is divided into two distinct zones, a dense outer cortex and an inner, pale-stained medulla.

The thymic cortex is predominantly populated by lymphocytes and, as seen in this speciment, those of the outer cortex are larger than those deeper in the cortex. The large lymphocytes of the outer cortex represent lymphoblasts which divide by mitosis to produce large numbers of smaller lymphocytes which are pushed into the deeper layers. The thymic cortex also contains numerous pale-stained, vacuolated macrophages responsible for engulfing dead lymphocytes but which also may be involved in ‘processing’ antigens before presentation to the lymphocytes. Note also a small capillary, lined by flattened endothelial cells, entering the cortex from the capsule. Around the capillary can be seen a distinct basement membrane constituting the blood-thymus barrier.

In the thymic medulla, cells of the epithelial framework can be more readily identified by their relatively large, pale-stained nuclei, eosoinophilic cytoplasm and prominent basement membranes. A feature of the thymic medulla are the concentrically lamellated eosinophilic structures known as Hassal’s corpuscles which first appear in fetal life and increase iumber throughout life. Initially, the corpuscles begin as a single medullary epithelial cell which enlarges and then degenerates to form a vacuolated eosinophilic mass. Further epithelial cells become similarly involved to form a lamellated hyaline mass surrounded by flattened degenerating epithelial cells as seen in this speciment. Nearby is a small mass of large atypical degenerate epithelial cells which may represent an early Hassal’s corpuscle.

 

 

Illustrate and indicate: I. Lobule of the gland: 1.Thymic cortex: a) T – lymphocytes. 2.Thymic medulla: a) epithelioreticulocytes; b) Haasal’s corpuscles. II. Connective tissue between lobules: 3.Blood vessels.

 

Specimen 3. Lymph node.

Haematoxylin and Eosin.

 

The lymph node is encapsulated by dense connective tissue from which trabeculae extend for variable distances into the substance of the node. The cortex consists of densely-packed lymphocytes and forms extensions called medullary cords which project into the medulla between the medullary sinuses.

The lymphocytes of the outer cortex are mainly arranged in spheroidal lymphoid follicles and these are the major sites in which lymphocytes localize and proliferate. Traditionally, lymphoid follicles have been classified as ‘primary follicles’ if a central pale area is absent and ‘secondary follicles’ if such an area is present. The pale central areas are termed germinal centers and the ‘primary’ follicles probably merely represent quiescent ‘secondary’ follicles. Cells of a germinal centre are a mixed population of lymphocytes and the plasma cell precursors, plasmablasts and proplasmacytes.

The deep cortical zone, or paracortex, consists mainly of T-lymphocytes which are never arranged as follicles. The medullary cords mainly contain lymphocytes and their derivatives.

Illustrate and indicate: 1. Dense connective capsule. 2. Trabeculae. 3. Cortex: a) lymphoid follicle. 4. Medulla: a) medullary cords 5. Subcapsular sinus. 6. Trabecular sinus. 7. Medullary sinuses. 8. Hilum of the node. 9. Reticular tissue.

 

Specimen 4. Spleen.

Heamatoxylin and Eosin.

 

          At low magnification spleen appears to consist of discrete white nodules, the  (white pulp), embedded in a red matrix called the red pulp. At high magnification the white pulp is seen to consist of lymphoid aggregations.

          Like lymph nodes, the spleen has a dense, fibro-elastic outer capsule which is thickened at the hilum and gives rise to supporting connective tissue trabeculae. In some mammals, the capsule and trabeculae contain smooth muscle which exerts a rhythmic pumping action, clearing the spleen of blood and allowing the spleen to act as a reservoir. In humans only a few smooth muscle cells persist.

          The capsule and the trabeculae provide a robust framework, which supports a fine reticulin meshwork ramifying throughout the organ in the red pulp. The reticular skeleton is almost absent in the centre of the white pulp but is well developed at the white pulp margins and around the central arteriole.

          The white pulp or periarteriolar lymphoid sheats contain populations of both T and B lymphocytes, the central region containing predominantly B lymphocytes, which may form germinal centers if a humoral response is stimulated in the spleen by blood-borne antigen. The outer marginal zone consists mainly of closely packed T lymphocytes.

The red pulp consists of irregular anastomosing plates, separated by broad interconnected venous sinuses.

 

Illustrate and indicate: 1. Spleen capsule. 2.Connective tissue trabeculae. 3.White pulp (lymphatic follicles): a) central artery. 4.Red pulp: a) reticular tissue; b) blood cells. 5. Venous sinuses.

 

References:

 

A-Basic:

1.     Practical classes materials:http://intranet.tdmu.edu.ua/data/kafedra/internal/histolog/classes_stud/English/medical/III%20term/15%20Immune%20and%20hemopoietic%20organs.htm

2.     Lecture presentations: http://intranet.tdmu.edu.ua/ukr/kafedra/index.php?kafid=hist&lengid=eng&fakultid=m&kurs=2&discid=Histology,%20cytology%20and%20embryology

3.     Stevens A. Human Histology / A. Stevens, J. Lowe. – [second edition]. –Mosby, 2000. – P. 117-136.

4.     Wheter’s Functional Histology: A Text and Colour Atlas / [Young B., Lowe J., Stevens A., Heath J.]. – Elsevier Limited, 2006. – P. 207-234.

5.     Singh I. Textbook of Human Histology with colour atlas / I. Singh. – [fourth edition]. – Jaypee Brothers Medical Publishers (P) LTD, 2002. – P. 178-192.

6.     Ross M. Histology : A Text and Atlas / M. Ross W.Pawlina. – [sixth edition]. – Lippincott Williams and Wilkins, 2011. – P. 440-488.

 

B – Additional:

1.     Eroschenko V.P. Atlas of Histology with functional correlations / Eroschenko V.P. [tenth edition]. – Lippincott Williams and Wilkins, 2008. – P. 191-211.

2.     Junqueira L. Basic Histology / L. Junqueira, J. Carneiro, R. Kelley. – [seventh edition]. – Norwalk, Connecticut : Appleton and Lange, 1992. – P.285-306.

3.     Charts: http://intranet.tdmu.edu.ua/index.php?dir_name=kafedra&file_name=tl_34.php#inf3

4.     Disk:  http://intranet.tdmu.edu.ua/data/teacher/video/hist/  

5.     Volkov K. S. Ultrastructure of cells and tissues – Ternopil : Ukrmedknyha, 1999. – P. 40-47.  http://intranet.tdmu.edu.ua/data/books/Volkov(atlas).pdf

6.     http://en.wikipedia.org/wiki/Immune

7.     http://en.wikipedia.org/wiki/Immune-system

8.     http://www.meddean.luc.edu/LUMEN/MedEd/Histo/frames/histo_frames.html

9.      http://www.udel.edu/biology/Wags/histopage/histopage.htm