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Histology_Ross. Thermogenic activity of brown adipose tissue is facilitated by UCP-1 that is found at the inner mitochondrial membrane. The mitochondria in eukaryotic cells produce and store energy as an electrochemical proton gradient across the inner mitochondrial membrane. As described earlier (see page 55), this energy is used to synthesize ATP when the protons return to the mitochondrial matrix through the ATP synthase enzyme located at the inner mitochondrial membrane.


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Histology_Ross. The mitochondria in the cytoplasm of brown adipose tissue cells contain uncoupling protein (UCP-1), which uncouples the oxidation of fatty acids from the production of ATP. Therefore, protons are allowed to travel from the inter-membrane space back to the mitochondrial matrix along the gradient without passing through ATP synthase and thus without producing ATP. This can occur since an alternative pathway for the protons return is available through an UCP-1 that facilitates proton transport across the inner mitochondrial membrane. The movement of protons from the inner mitochondrial compartment dissipates the mitochondrial proton gradient, thus uncoupling respiration from ATP synthesis. The energy produced by the mitochondria is then dissipated as heat in process known as thermogenesis. The metabolic activity of brown adipose tissue is regulated by the sympathetic nerve system and is related to ambient outdoor temperature.


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Histology_Ross. The metabolic activity of brown adipose tissue is largely regulated by norepinephrine released from the sympathetic nerve terminals, which stimulates lipolysis and hydrolysis of triglycerydes as well as increases mitochondrial expression and activity of UCP-1 molecules. In experimental animals, chapter 9 Adipose Tissue B ROWN AD I POSE TISS U E TABLE Summary of Adipose Tissue Features 9.2 Features White Adipose Tissue Brown Adipose Tissue Location Subcutaneous layer, mammary gland, Large amounts in newborn greater omentum, mesenteries, Remnants in adults at the retroperitoneal retroperitoneal space, visceral space, deep cervical and supraclavicular pericardium, orbits (eye sockets), regions of the neck, interscapular, bone marrow cavity paravertebral regions of the back, mediastinum Function Metabolic energy storage, insulation, Heat production (thermogenesis) cushioning, hormone production, source of metabolic water Adipocyte morphology Unilocular, spherical, flatten nucleus,


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Histology_Ross. Function Metabolic energy storage, insulation, Heat production (thermogenesis) cushioning, hormone production, source of metabolic water Adipocyte morphology Unilocular, spherical, flatten nucleus, Multilocular, spherical, round rim of cytoplasm eccentric nucleus Large diameter (15–150 m) Smaller diameter (10–25 m) Transcription factors “master PPAR-/RXR PRDM16/PGC-1 switch” in differentiation UCP-1 genes expression No Yes (unique to brown fat) Mitochondria Few, poorly developed Many, well developed Innervation Few sympathetic nerve fibers High density of sympathetic nerve fibers Vascularization Few blood vessels Highly vascularized tissue Response to environmental Decreased lipogenesis Increased lipogenesis stress (cold exposure) Increased lipoprotein lipase activity Decreased lipoprotein lipase activity Growth and differentiation Throughout entire life from Only during fetal period stromal-vascular cells Decreases in adult life (exception: individuals with pheochromocytoma and


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Histology_Ross. lipase activity Growth and differentiation Throughout entire life from Only during fetal period stromal-vascular cells Decreases in adult life (exception: individuals with pheochromocytoma and hibernoma)  FOLDER 9.3 Clinical Correlation: PET Scanning and Brown Adipose Tissue Interference


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Histology_Ross. Positron emission tomography, also called a “PET scan,” is a diagnostic tool that can locate malignant cells in the body. PET is based on the detection of high-energy gamma rays created when positrons (subatomic particles of antimatter), produced during decay of radioactive mate-rials, are encountered by electrons. The procedure re-quires the injection of a radioactive tracer, most commonly 18-fuorine-2-fuoro-2-deoxy-D-glucose (18F-FDG). This radioactive glucose isotope is used in PET imaging because malignant cells metabolize glucose at a greater rate than normal cells. After injection of the isotope, a de-tector scans the entire body and records radiation emitted by the 18F-FDG tracer as it becomes incorporated within the body’s cells. A computer reassembles the signals into images, which are, in effect, biological maps of 18F-FDG distribution in the body. Recently, due to greater diagnostic accuracy and improved biopsy methods, combined positron emission tomography and computed


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Histology_Ross. which are, in effect, biological maps of 18F-FDG distribution in the body. Recently, due to greater diagnostic accuracy and improved biopsy methods, combined positron emission tomography and computed tomography (PET/CT) scanners are utilized more frequently. One drawback to PET imaging is that many normal tissues and benign lesions also show increased glucose metabolism and can thus be misinterpreted as malignant. For example, brown adipose tissue, with its increased glucose uptake me-diated by increased activity of glucose transporters, can be a potential source of false-positive interpretation of PET scans. Since brown adipose tissue is present in the neck, supraclavicular regions, and mediastinum (see page 260) it is commonly observed on PET scans, especially in underweight patients and during winter months, when brown adipose tis-sue is more predominant. This 18F-FDG uptake most likely represents activated brown adipose tissue during increased sympathetic nerve activity related to


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Histology_Ross. during winter months, when brown adipose tis-sue is more predominant. This 18F-FDG uptake most likely represents activated brown adipose tissue during increased sympathetic nerve activity related to cold stress. A typical PET image of brown fat is usually bilateral and symmetric; however, in the mediastinum, the image may be asymmetric or focal and can mimic malignancy. False-positive results from brown fat 18F-FDG uptake in these areas have been reported in young women undergoing scans for diagnosis and staging of breast cancer. Therefore, under-standing that brown fat can show that increased radioac-tive tracer uptake is crucial for establishing an accurate diagnosis and avoiding false-positive results (Fig. F9.3.1). FIGURE F9.3.1 • Coronal positron emission tomography/ computed tomography (PET/CT) image of a healthy young woman. This upper part of the coronal section of this whole-body PET/CT scan shows extensive bilateral increased 18F-FDG uptake (red color) in the neck,


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Histology_Ross. tomography (PET/CT) image of a healthy young woman. This upper part of the coronal section of this whole-body PET/CT scan shows extensive bilateral increased 18F-FDG uptake (red color) in the neck, supraclavicular, and upper axillary regions. Note that moderate increased radioactive tracer uptake is also detectable in the myocardium (yellow color). Regions of extensive metabolic activity correlate with the distribution pattern of low-density brown adipose tissue. PET/CT imaging allows for precise localization of increased 18F-FDG uptake areas and differentiation between brown adipose tissue tracer uptake and malignant tumor findings. (Courtesy of Dr. Jolanta Durski.)


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Histology_Ross. UCP-1 activity has been shown to increase during cold stress. In addition, cold stimulates glucose utilization in brown adipocytes by overexpression of glucose transporters (Glut-4). Recent clinical studies using PET scans in adults have shown a direct relationship between outdoor temperature and the amount of brown fat accumulated in the body. An increase in the amount of brown adipose tissue has been reported on the neck and supraclavicular regions during the winter months especially in lean individuals. This is supported by autopsy findings of larger amounts of brown fat in outdoor workers exposed to cold. Modern molecular imaging techniques now allow clinicians to precisely locate where brown fat is distributed in the body, which is essential for proper diagnosis of cancerous lesions (see Folder 9.3). This page intentionally left blank.


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Histology_Ross. ADI POS E TI SS U E 266 Adipose tissue is widely distributed throughout the body and in varying amounts in different individuals. It is a specialized connective tissue consisting of fat-storing cells, adipocytes, and has a rich blood supply. Two types of adipose tissue are recognized. The more common and abundant is referred to as white adipose tissue. Its adipocytes are very large cells whose cytoplasm contains a single large vacuole in which the fat is stored in the form of triglycerides. When observed in a typical H&E section, white adi-pose tissue appears as a mesh-like structure (see orientation micrograph). The second type is brown adipose tissue. It consists of smaller cells. Their cytoplasm is characterized by numerous vesicles that occupy much of the cells’ volume. It also is very richly vascu-larized. Brown adipose tissue is found in human newborns where it assists in maintaining body temperature. ORIENTATION MICROGRAPH: Shown here is white adipose tissue from the


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Histology_Ross. is very richly vascu-larized. Brown adipose tissue is found in human newborns where it assists in maintaining body temperature. ORIENTATION MICROGRAPH: Shown here is white adipose tissue from the hypo-dermis of skin. It consists of numerous adipocytes closely packed in lobules. Dense con-nective tissue (DICT) surrounds the adipose tissue. The loss of the fat within the cell gives the adipose tissue a mesh-like appearance. Note the small blood vessels (BV) ob-served at the periphery of the tissue. They provide a rich capillary network within the adi-pose tissue. Several sweat gland ducts (SGD) are also present in the dense connective tissue. DICTBVSGDDICT BV SGD


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Histology_Ross. White adipose tissue, human, H&E, ×363; inset ×700.


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Histology_Ross. This is a higher magnification micrograph of white adipose tissue from the specimen shown in the orientation micro-graph. It reveals portions of several lobules of adipose cells. Dense connective tissue (DICT) separates the lobules from surrounding structures. In well preserved specimens, the adipocytes (A) have a spherical profile in which they exhibit a very thin rim of cytoplasm surrounding a single, large fat-containing vacuole. Because the fat is lost during tissue preparation, one only sees the rim of cytoplasm and an almost clear space. Between the cells, there is an extremely thin, delicate connective tissue stroma holding the adipocytes together and within this stroma are small blood vessels (BV), mostly capillaries and venules. The majority of nuclei that are observed within the adipose tissue belong to fibroblasts, adipocytes, or cells of small blood vessels. However, distinguishing between fibroblast nuclei and adipocyte nuclei is often difficult. The inset shows an


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Histology_Ross. the adipose tissue belong to fibroblasts, adipocytes, or cells of small blood vessels. However, distinguishing between fibroblast nuclei and adipocyte nuclei is often difficult. The inset shows an adipocyte whose nucleus (N) is relatively easy to identify. It appears to reside within the rim of cytoplasm (Cy), giving the adipocyte the classic “signet ring” appearance. A second nucleus (N’), partially out of the plane of section, appears to reside between the cytoplasmic rim of two adjacent cells. This is probably the nucleus of a fibroblast. Because of the relatively large size of the adipocyte, it is very infrequent that the nucleus of the cell is included in the plane of section of a given cell. Other cells that may be seen within the delicate connective tissue stroma are mast cells (MC).


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Histology_Ross. Brown adipose tissue, human, H&E, ×450; inset ×1100.


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Histology_Ross. The brown adipose tissue shown here consists of small fat cells that are very closely packed with minimal intercellular space. Because of this arrangement, it is hard to define individual cells at this magnification. At higher magnification (not shown), it is possible to identify some individual cells. One cell, whose boundaries could be identified at higher magnification is circumscribed by a dotted line. Each cell contains many small, fat-containing vacuoles surrounded by cytoplasm. Included in this cell is its nucleus (N). As noted, brown adipose tissue is highly vascularized, and in this specimen, one can see numerous blood vessels (BV) as evidenced by the red blood cells that they contain. It is even more difficult to distinguish fibroblasts within the lobule from nuclei of the fat cells. Even at higher magnification (inset), it is difficult to determine which nuclei belong to which cells. A capillary (C) can be identified in the inset. Again, it is recognized by the presence of


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Histology_Ross. Even at higher magnification (inset), it is difficult to determine which nuclei belong to which cells. A capillary (C) can be identified in the inset. Again, it is recognized by the presence of red blood cells. Where the lobules are slightly separated from one another (arrows), small elongate nuclei can be recognized. These belong to fibroblasts in the connective tissue forming the septa.


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Histology_Ross. KEY DICT, dense irregular connective tissue A, adipocytes BV, blood vessels N, nucleus Cy, cytoplasm MC, mast cells C, capillary SGD, sweat gland ducts


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Histology_Ross. OVERVIEW OF BLOOD / 268 PLASMA / 269 ERYTHROCYTES / 270 LEUKOCYTES / 275 Neutrophils / 275 Eosinophils / 280 Basophils / 282 Lymphocytes / 283 Monocytes / 286 THROMBOCYTES / 286 FORMATION OF BLOOD CELLS (HEMOPOIESIS) / 289 Monophyletic Theory of Hemopoiesis / 289 Development of Erythrocytes (Erythropoiesis) / 293 Kinetics of Erythropoiesis / 295 Development of Thrombocytes (Thrombopoiesis) / 295 Development of Granulocytes (Granulopoiesis) / 295 Kinetics of Granulopoiesis / 296 Development of Monocytes / 298 Development of Lymphocytes (Lymphopoiesis) / 298 BONE MARROW / 298 Folder 10.1 Clinical Correlation: ABO and Rh Blood Group Systems / 273 Folder 10.2 Clinical Correlation: Hemoglobin in Patients with Diabetes / 274 Folder 10.3 Clinical Correlation: Hemoglobin Disorders / 276 Folder 10.4 Clinical Correlation: Inherited Disorders of Neutrophils; Chronic Granulomatous Disease (CGD) / 281 Folder 10.5 Clinical Correlation: Hemoglobin Breakdown and Jaundice / 281 Folder 10.6 Clinical


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Histology_Ross. 10.4 Clinical Correlation: Inherited Disorders of Neutrophils; Chronic Granulomatous Disease (CGD) / 281 Folder 10.5 Clinical Correlation: Hemoglobin Breakdown and Jaundice / 281 Folder 10.6 Clinical Correlation: Cellularity of the Bone Marrow / 300


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Histology_Ross. Blood is a fluid connective tissue that circulates through the cardiovascular system. Like the other connective tissues, blood consists of cells and an extracellular component. Total blood volume in the average adult is about 6 L or 7% to 8% of total body weight. The heart’s pumping action propels blood through the cardiovascular system to the body tissues. Blood’s many functions include:  delivery of nutrients and oxygen directly or indirectly to cells, transport of wastes and carbon dioxide away from cells, delivery of hormones and other regulatory substances to and from cells and tissues,  maintenance of homeostasis by acting as a buffer and participating in coagulation and thermoregulation, and  transport of humoral agents and cells of the immune system that protect the body from pathogenic agents, foreign proteins, and transformed cells (i.e., cancer cells). Blood consists of cells and their derivatives and a protein-rich fluid called plasma.


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Histology_Ross. Blood consists of cells and their derivatives and a protein-rich fluid called plasma. Blood cells and their derivatives include:  erythrocytes, also called red blood cells (RBCs), leukocytes, also known as white blood cells (WBCs), and thrombocytes, also termed platelets. Plasma is the liquid extracellular material that imparts fluid properties to blood. The relative volume of cells and plasma in whole blood is approximately 45% and 55%, respectively. The TABLE Formed Elements of the Blood 10.1 Cells/L Formed Elements Male Female % Erythrocytes 4.3– 5.7 1012 3.9–5.0 1012 Leukocytes 3.5–10.5 109 3.5–10.5 109 100 Agranulocytes Lymphocytes 0.9–2.9 109 0.9–2.9 109 25.7–27.6a Monocytes 0.3–0.9 109 0.3–0.9 109 8.6a Granulocytes Neutrophils 1.7–7.0 109 1.7–7.0 109 48.6–66.7a Eosinophils 0.05–0.5 109 0.05–0.5 109 1.4–4.8a Basophils 0–0.03 109 0–0.03 109 0–0.3a Thrombocytes (platelets) 150–450 109 150–450 109 aPercentage of leukocytes.


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Histology_Ross. volume of packed erythrocytes in a sample of blood is called the hematocrit. The hematocrit is measured by centrifuging a blood sample to which anticoagulants have been added, and then calculating the percentage of the centrifuge tube volume occupied by the erythrocytes compared with that of the whole blood. A normal hematocrit reading is about 39% to 50% in men and 35% to 45% in women; thus, 39% to 50% and 35% to 45% of the blood volume for men and women, respectively, consists of erythrocytes. Low hematocrit values often reﬂect reduced numbers of circulating erythrocytes (a condition called anemia) and may indicate significant blood loss caused by internal or external bleeding.


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Histology_Ross. Leukocytes and platelets constitute only 1% of the blood volume. In a blood sample that has been centrifuged, the cell fraction (the part of the sample that contains the cells) consists mainly of packed erythrocytes (99%). The leukocytes and platelets are contained in a narrow layer at the upper part of the cell fraction called the buffy coat. As Table 10.1 indicates, there are nearly 1,000 times more erythrocytes (5 1012 cells/L of blood) than leukocytes (7 109/L of blood). Although the blood cells are the major objects of interest in histology, a brief examination of plasma is also useful. The composition of plasma is summarized in Table 10.2. More than 90% of plasma by weight is water, which serves as the solvent for a variety of solutes, including proteins, dissolved gases, electrolytes, nutrients, regulatory substances, and waste materials. The solutes in the plasma help maintain homeostasis, a steady state that provides optimal pH and osmolarity for cellular metabolism.


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Histology_Ross. Plasma proteins consist primarily of albumin, globulins, and fibrinogen. Albumin is the main protein constituent of the plasma, accounting for approximately half of the total plasma proteins. It is the smallest plasma protein (about 70 kilodaltons) and is made in the liver. Albumin is responsible for exerting the concentration gradient between blood and extracellular tissue fluid. This major osmotic pressure on the blood vessel wall, called the colloid osmotic pressure, maintains the correct proportion of blood to tissue fluid volume. If a significant amount of albumin leaks out of the blood vessels into the loose connective tissue or is lost from the blood to urine in the kidneys, then the colloid osmotic pressure of the blood decreases, and ﬂuid accumulates in the tissues. (This increase in tissue ﬂuid is most


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Histology_Ross. TABLE Composition of Blood Plasma10.2 Component % Water 91–92 Protein (albumin, globulins, fibrinogen) 7–8 Other solutes: 1–2 • Electrolytes (Na, K, Ca2, Mg2, Cl, HCO3 , PO4 3, SO4 2) • Nonprotein nitrogen substances (urea, uric acid, creatine, creatinine, ammonium salts) • Nutrients (glucose, lipids, amino acids) • Blood gases (oxygen, carbon dioxide, nitrogen) • Regulatory substances (hormones, enzymes) readily noted by swelling of the ankles at the end of a day.) Albumin also acts as a carrier protein; it binds and transports hormones (thyroxine), metabolites (bilirubin), and drugs (barbiturates). Globulins include the immunoglobulins (-globulins), the largest component of the globulin fraction, and nonimmune globulins (-globulin and -globulin). The immunoglobulins are antibodies, a class of functional immune-system molecules secreted by plasma cells. (Antibodies are discussed in Chapter 14, Lymphatic System.)


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Histology_Ross. Nonimmune globulins are secreted by the liver. They help maintain the osmotic pressure within the vascular system and also serve as carrier proteins for various substances such as copper (by ceruloplasmin), iron (by transferrin), and the protein hemoglobin (by haptoglobin). Nonimmune globulins also include fibronectin, lipoproteins, coagulation factors, and other molecules that may exchange between the blood and the extravascular connective tissue. Fibrinogen, the largest plasma protein (340 kilodaltons), is made in the liver. In a series of cascade reactions with other coagulation factors, soluble fibrinogen is transformed into the insoluble protein fibrin (323 kilodaltons). During conversion of fibrinogen to fibrin, fibrinogen chains are broken to produce fibrin monomers that rapidly polymerize to form long fibers. These fibers become cross-linked to form an impermeable net at the site of damaged blood vessels, thereby preventing further blood loss.


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Histology_Ross. With the exception of these large plasma proteins and regulatory substances, which are small proteins or polypeptides, most plasma constituents are small enough to pass through the blood vessel wall into the extracellular spaces of the adjacent connective tissue. In general, plasma proteins react with common fixatives; they are often retained within the blood vessels in tissue sections. Plasma proteins do not possess a structural form above the molecular level; thus, when they are retained in blood vessels in the tissue block, they appear as a homogeneous substance that stains evenly with eosin in hematoxylin and eosin (H&E)–stained sections. Serum is the same as blood plasma except that clotting factors have been removed.


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Histology_Ross. For laboratory purposes, samples of blood are often drawn from a vein (the procedure called venipuncture). When blood is removed from the circulation, it immediately clots. A blood clot consists mostly of erythrocytes entangled in a network of fine fibers composed of fibrin. To prevent clotting of a blood sample, an anticoagulant such as citrate or heparin is added to the blood specimen as it is obtained. Citrate binds calcium ions, which are essential for triggering the cascade of coagulation reactions; heparin deactivates the clotting factors in the plasma. Plasma that lacks coagulation factors is called serum. For many biochemical laboratory tests, plasma and blood serum can be used interchangeably. Serum is preferred for several specific tests because the anticoagulants in plasma can interfere with the results. However, tests of clotting ability require that all coagulation factors be preserved; therefore, serum is inappropriate for these tests.


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Histology_Ross. The interstitial fluid of connective tissues is derived from blood plasma. The fluid that surrounds tissue cells, called interstitial ﬂuid, not surprisingly, has an electrolyte composition that reflects that of blood plasma, from which it is derived. The composition of interstitial fluid in nonconnective tissues, however, is subject to considerable modification by the absorptive and secretory activities of epithelia. Epithelia may create special microenvironments conducive to their function. For example, a blood–brain barrier exists between the blood and nerve tissue. Barriers also exist between the blood and the parenchymal tissue in the testis, thymus gland, eye, and other epithelial compartments. Fluids, barriers, and their functions are discussed in subsequent chapters that describe these particular organs. Examination of blood cells requires special preparation and staining.


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Histology_Ross. Examination of blood cells requires special preparation and staining. The preparation method that best displays the cell types of peripheral blood is the blood smear. This method differs from the usual preparation seen in the histology laboratory in that the specimen is not embedded in paraffin and sectioned. Rather, a drop of blood is placed directly on a slide and spread thinly over the surface of the slide (i.e., “pulled” with the edge of another slide) to produce a monolayer of cells (Fig. 10.1a). The preparation is then air dried and stained. Another difference in the preparation of a blood smear is that instead of H&E, special mixtures of dyes are used to stain the blood cells. The resulting preparation may then be examined with a high-power oil-immersion lens, with or without a coverslip (Fig. 10.1b and Plate 17, page 302).


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Histology_Ross. The modified Romanovsky-type stain commonly used for blood smears consists of a mixture of methylene blue (a basic dye), related azures (also basic dyes), and eosin (an acidic dye). On the basis of their appearance after staining, leukocytes are traditionally divided into granulocytes (neutrophils, eosinophils, and basophils) and agranulocytes (lymphocytes and monocytes). Although both cell types may contain granules, the granulocytes possess obvious, specifically stained granules in their cytoplasm. In general, the basic dyes stain nuclei, granules of basophils, and the RNA of the cytoplasm, whereas the acidic dye stains the erythrocytes and the granules of eosinophils. Scientists originally thought that the fine neutrophil granules were stained by a “neutral dye” that formed when methylene blue and its related azures were combined with eosin. The mechanism by which the specific neutrophil granules are stained is still not clearly understood. Some of the basic dyes (the azures) are


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Histology_Ross. blue and its related azures were combined with eosin. The mechanism by which the specific neutrophil granules are stained is still not clearly understood. Some of the basic dyes (the azures) are metachromatic and may impart a violet to red color to the material they stain.


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Histology_Ross. Erythrocytes are anucleate, biconcave discs. Erythrocytes or red blood cells (RBCs), are anucleate cells devoid of typical organelles. They function only within the bloodstream to bind oxygen for delivery to the FIGURE 10.1  Blood smear: Preparation technique and overview photomicrograph. a. Photograph showing the method of producing a blood smear. A drop of blood is placed directly on a glass slide and spread over its surface with the edge of another slide. b. Photomicrograph of smear from peripheral blood stained with Wright’s stain, showing the cells evenly distributed. The cells are mainly erythrocytes. Three leukocytes are present. Platelets are indicated by arrows. 350. tissues and, in exchange, bind carbon dioxide for removal from the tissues. Their shape is that of a biconcave discs with a diameter of 7.8 m, an edge thickness of 2.6 m, and a central thickness of 0.8 m. This shape maximizes the cell’s surface area (140 m2), an important attribute in gas exchange.


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Histology_Ross. 7.8 m 0.8 m 2.6 m The life span of erythrocytes is approximately 120 days, after which most (90%) of them are phagocytosed by macrophages in the spleen, bone marrow, and liver. The remaining aged erythrocytes (10%) break down intravascularly, releasing insignificant amounts of hemoglobin into the blood. In H&E–stained sections, erythrocytes are usually 7 to 8 m in diameter. Because their size is relatively consistent in fixed tissue, they can be used to estimate the size of other cells and structures in tissue sections; in this role, erythrocytes are appropriately referred to as the “histologic ruler.”


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Histology_Ross. Because both living and preserved erythrocytes usually appear as biconcave discs, they can give the impression that their form is rigid and inelastic (Fig. 10.2). They are, in fact, extremely deformable. They pass easily through the narrowest capillaries by folding over on themselves. They stain uniformly with eosin. In thin sections viewed with the transmission electron microscope (TEM), the contents of an erythrocyte appear as a dense, finely granular material. The shape of the erythrocyte is maintained by membrane proteins. The cell membrane of an erythrocyte is composed of a typical lipid bilayer and contains two functionally significant groups of proteins.


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Histology_Ross. The cell membrane of an erythrocyte is composed of a typical lipid bilayer and contains two functionally significant groups of proteins.  Integral membrane proteins represent most of the proteins in the lipid bilayer. They consist of two major families: glycophorins and band 3 proteins. The extracellular domains of these integral membrane proteins are glycosylated and express specific blood group antigens. Glycophorin C, a member of the glycophorin family of transmembrane proteins, plays an important role in attaching the underlying cytoskeletal protein network to the cell membrane. Band 3 protein binds hemoglobin and acts as an additional anchoring site for the cytoskeletal proteins (Fig. 10.3).


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Histology_Ross.  Peripheral membrane proteins reside on the inner surface of the cell membrane. They are organized into a two-dimensional hexagonal lattice network that laminates the inner layer of the membrane. The lattice itself, which is positioned parallel to the membrane, is composed mainly of cytoskeletal proteins including spectrin tetramers, actin, band 4.1 protein, adducin, band 4.9 protein, and tropomyosin (see Fig. 10.3) that form a network or mesh. The lattice is anchored to the lipid bilayer by the globular protein ankyrin, which interacts with band 4.2 protein as well as with band 3 integral membrane protein. glycophorin C adductin spectrin ankyrin actin tropomyosin band 3 protein 4.2


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Histology_Ross. glycophorin C adductin spectrin ankyrin actin tropomyosin band 3 protein 4.2 FIGURE 10.2  Erythrocyte morphology. a. Photomicrograph of three capillaries (Cap) joining to form a venule (V ), as observed in adipose tissue within a full-thickness mesentery spread. The erythrocytes appear in single file in one of the capillaries (the other two are empty). The light center area of some of the erythrocytes results from their biconcave shape. Erythrocytes are highly plastic and can fold on themselves when passing through very narrow capillaries. The large round structures are adipose cells (A). 470. b. Scanning electron micrograph of erythrocytes collected in a blood tube. Note the concave shape of the cells. The stacks of erythrocytes in these preparations are not unusual and are referred to as rouleau. Such formations in vivo indicate an increased level of plasma immunoglobulin. 2,800.


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Histology_Ross. This unique cytoskeletal arrangement contributes to the shape of the erythrocyte and imparts elastic properties and stability to the membrane. The cytoskeleton is not static; it undergoes continuous rearrangement in response to various protein 4.9 protein 4.1 FIGURE 10.3  Erythrocyte membrane organization. The rectangle in the sectioned erythrocyte (upper left) represents the area of the membrane in the larger diagram. The large diagram shows the arrangement of the peripheral and integral membrane proteins. The peripheral proteins form a cytoskeletal lattice on the interior surface of the plasma membrane; the predominant protein is spectrin. The lattice is anchored to the plasma membrane by a number of protein complexes.


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Histology_Ross. physical factors and chemical stimuli as the cell moves through the vascular network. Any defect in the expression of genes that encode these cytoskeleton proteins can result in abnormally shaped and fragile erythrocytes. For instance, hereditary spherocytosis is caused by a primary defect in spectrin gene expression that results in spherical erythrocytes. Hereditary elliptocytosis is caused by a deficiency in band 4.1 proteins that results in elliptical erythrocytes. In both conditions, erythrocytes are unable to adapt to changes in their environment (e.g., osmotic pressure and mechanical deformations), which results in premature destruction of the cells, or hemolysis. Erythrocytes contain hemoglobin, a protein specialized for the transport of oxygen and carbon dioxide.


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Histology_Ross. Erythrocytes contain hemoglobin, a protein specialized for the transport of oxygen and carbon dioxide. Erythrocytes transport oxygen and carbon dioxide bound to the protein hemoglobin (68 kilodaltons). A monomer of hemoglobin is similar in composition and structure to myoglobin, the oxygen-binding protein found in striated muscle. A high concentration of hemoglobin is present within erythrocytes and is responsible for their uniform staining with eosin and the cytoplasmic granularity seen with the TEM. The disc shape of the erythrocyte facilitates gas exchange because more hemoglobin molecules are closer to the plasma membrane than they would be in a spherical cell. Thus, gases have less distance to diffuse within the cell to reach a binding site on the hemoglobin.  FOLDER 10.1 Clinical Correlation: ABO and Rh Blood a life-threatening complication, blood for transfusion must


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Histology_Ross. An important factor in blood transfusion is the ABO blood group system, which essentially involves three antigens called A, B, and O (Table F10.1.1). These antigens are glycoproteins and glycolipids and differ only slightly in their composition. They are present on the surface of erythrocytes and are attached to the extracellular domains of integral membrane proteins called glycophorins. The presence of A, B, or O antigens determines the four primary blood groups: A, B, AB, and O. All humans have enzymes that catalyze the synthesis of the O antigen. Individuals with A blood group have an additional enzyme (N-acetylgalactosamine transferase or Aglycosyltransferase) that adds N-acetylgalactosamine to the O antigen. Individuals with B blood group have an enzyme (galactose transferase or B-glycosyltransferase) that adds galactose to the O antigen (Fig. F10.1.1). Individuals with the AB blood group express both enzymes, whereas individuals with type O blood group lack both enzymes. In


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Histology_Ross. B-glycosyltransferase) that adds galactose to the O antigen (Fig. F10.1.1). Individuals with the AB blood group express both enzymes, whereas individuals with type O blood group lack both enzymes. In humans, ABO genes consist of at least seven exons, and they are located on chromosome 9. The O allele is recessive, whereas A and B alleles are codominant.


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Histology_Ross. The differences in the carbohydrate molecules of these antigens are detected by specific antibodies against either A or B antigens. Individuals with A antigens possess serum anti-B antibodies that are directed against the B antigen. Individuals with B antigens possess serum anti-A antibodies that are directed against A antigen. Individuals with blood group AB do not have antibodies directed against A or B antigens. Thus, they are universal acceptors of any blood type. Group O individuals have both anti-A and anti-B antibodies in their serum and neither A nor B antigens on their erythrocytes. Thus, these individuals are universal blood donors.


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Histology_Ross. If an individual is transfused with blood of an incompatible type, the recipient’s antibodies will attack the donor erythrocytes, causing a hemolytic transfusion reaction, or destruction of the transfused erythrocytes. To prevent such be always cross-matched to the blood of a recipient. In this procedure, serum from the recipient is tested against the donor’s erythrocytes. If there is no reaction to this cross-match test, then the donors’ blood can be used for the transfusion.


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Histology_Ross. Rh blood group system The other important blood group system, the Rh system, is based on the Rhesus (Rh) antigen. In humans, this system is represented by a 40-kilodalton transmembrane nonglycosylated Rh30 polypeptide that shares antigenic sites with rhesus monkey erythrocytes. Rh30 polypeptide is a component of a larger (90-kilodalton) erythrocyte integral membrane protein complex that includes Rh50 glycoprotein. Although the Rh30 polypeptide expresses many antigen sites on its extracellular domain, only three of them—D, C, and E antigens—have clinical significance. Interactions between Rh30 and Rh50 molecules are essential for the expression of D, C, and E antigens. An individual who possesses only one of these three antigens is referred to as Rh positive (Rh). All three antigens stimulate production of anti-Rh antibodies in individuals without the same antigens.


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Histology_Ross. Rh incompatibility may induce a hemolytic transfusion reaction and in newborns causes the hemolytic disease erythroblastosis fetalis. Erythroblastosis fetalis occurs in Rh(D) newborns delivered by Rh(D ) mothers and results from an immune reaction of anti-D immunoglobulins passed across the placenta from the mother. The anti-D antibodies are produced by the mother in response to the D antigen expressed on the fetal erythrocytes that leak into her circulation during pregnancy. Administration of anti-D antibodies (RhoGAM) to the mother during pregnancy and after parturition destroys any circulating Rh(D) fetal erythrocytes that persist in the mother’s blood, thus preventing Rh-incompatibility reactions in future pregnancies.


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Histology_Ross. Blood Type Erythrocyte Surface Antigen Serum Antibody Can Give Blood to Can Receive Blood From A A antigen Anti-B A and AB A and O B B antigen Anti-A B and AB B and O AB A and B antigens No antibodies Only AB A, B, AB, and O (universal blood recipient) O O antigen (no A or B antigens) Anti-A and anti-B A, B, AB, and O (universal blood donor) Only O TABLE ABO Blood Group System F10.1.1 Hemoglobin consists of four polypeptide chains of globin , , ,and , each complexed to an iron-containing heme group (Fig. 10.4). The structure of the polypeptide chains varies. Depending on the particular polypeptides present, the following types of hemoglobin can be distinguished:  Hemoglobin HbA is most prevalent in adults, accounting for about 96% of total hemoglobin. It is a tetramer with two and two chains (2 2).  Hemoglobin HbA2 accounts for 1.5% to 3% of total hemoglobin in adults. It consists of two and two chains (22).


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Histology_Ross.  Hemoglobin HbA2 accounts for 1.5% to 3% of total hemoglobin in adults. It consists of two and two chains (22).  Hemoglobin HbF accounts for less than 1% of total hemoglobin in adults. It contains two and two chains (22) and is the principal form of hemoglobin in the fetus. HbF production falls dramatically after birth; however, in some individuals HbF is produced throughout their entire lives. Although HbF persists in slightly higher percentages than normal in those with sickle cell disease and thalassemia, it does not appear to have a pathologic role. Mutations in the genes encoding the globin chains can cause disorders in hemoglobin production. An example of a mutation in the gene encoding hemoglobin is discussed in the Folder 10.3. Interestingly, more than 550 types of abnormal hemoglobin molecules have been identified, but the majority of them have no clinical significance.  FOLDER 10.2 Clinical Correlation: Hemoglobin in Patients with Diabetes


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Histology_Ross. As mentioned in the text, about 96% of total hemoglobin in adults is represented by hemoglobin type HbA. Approx-imately 8% of HbA consists of several subtypes that dis-play slight chemical differences. These subtypes are hemoglobins HbA1a1, HbA1a2, HbA1b, and HbA1c. Of these subtypes, hemoglobin type A1c is of clinical sig-nificance because it binds irreversibly to glucose. It is re-ferred to as glycated or glycosylated hemoglobin. Levels of this hemoglobin subtype are used to monitor an individual’s blood glucose levels over the previous 2 to 3 months (clinically referred to as an A1c test). Individuals with diabetes have increased levels of glycated hemoglobin HbA1c in the blood because of their elevated blood glu-cose. Since the normal life span of erythrocytes is about 120 days (see page 295), glycated hemoglobin can only be eliminated when the RBCs containing it are de-stroyed. Thus, HbA1c values are directly proportional to the concentration of glucose in the blood over the


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Histology_Ross. page 295), glycated hemoglobin can only be eliminated when the RBCs containing it are de-stroyed. Thus, HbA1c values are directly proportional to the concentration of glucose in the blood over the entire life span of the erythrocyte. In healthy individuals and in those with diabetes that is being effectively controlled, HbA1c levels should not be higher than 7% of the total hemoglobin. Since HbA1c values are not subject to the short-term fluctuations in blood glucose levels that are seen, for example, after meals or during fasting, blood for HbA1c testing can be obtained without regard to when food is consumed.


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Histology_Ross. FIGURE 10.4  Structural diagram of the hemoglobin molecule. Each hemoglobin molecule is composed of four subunits. Each subunit contains a heme, the iron-containing portion of hemoglobin, embedded in a hydrophobic cleft of a globin chain. The folding of the globin chain places the heme near the surface of the molecule, where it is readily accessible to oxygen. There are four different types of globin chains: , , , and occurring in pairs. The types of globin chains present in the molecules determine the type of hemoglobin. The figure illustrates hemoglobin A (HbA), which is composed of two and two chains.


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Histology_Ross. Leukocytes are subclassified into two general groups. The basis for this division is the presence or absence of prominent specific granules in the cytoplasm. As previously noted, cells containing specific granules are classified as granulocytes (neutrophils, eosinophils, and basophils) (Plate 17, page 302), and cells that lack specific granules are classified as agranulocytes (lymphocytes and monocytes) (Plate 18, page 304). However, both agranulocytes and granulocytes possess small number of nonspecific azurophilic granules, which are lysosomes. The relative number of the various leukocytes is given in Table 10.1. Neutrophils are the most numerous WBCs as well as the most common granulocytes.


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Histology_Ross. Neutrophils are the most numerous WBCs as well as the most common granulocytes. Neutrophils measure 10 to 12 m in diameter in blood smears and are obviously larger than erythrocytes. Although named for their lack of characteristic cytoplasmic staining, they are also readily identified by their multilobal nucleus; thus, they are also called polymorphonuclear neutrophils or polymorphs. Mature neutrophils possess two to four lobes of nuclear material joined by thinner nuclear strands (Plate 17, page 302). The arrangement is not static; rather, in living neutrophils the lobes and connecting strands change their shape, position, and even number.


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Histology_Ross. The chromatin of the neutrophil has a characteristic arrangement. Wide regions of heterochromatin are located chiefly at the periphery of the nucleus in contact with the nuclear envelope. Regions of euchromatin are located primarily at the center of the nucleus with relatively smaller regions contacting the nuclear envelope (Fig. 10.5). In women, the Barr body (the condensed, single, inactive X chromosome) forms a drumstick-shaped appendage on one of the nuclear lobes. Neutrophils contain three types of granules. The cytoplasm of a neutrophil contains three kinds of granules. The different types of granules reflect the various phagocytotic functions of the cell.


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Histology_Ross. Neutrophils contain three types of granules. The cytoplasm of a neutrophil contains three kinds of granules. The different types of granules reflect the various phagocytotic functions of the cell.  Specific granules (secondary granules) are the smallest granules and are at least twice as numerous as azurophilic granules. They are barely visible in the light microscope; in electron micrographs, they are ellipsoidal (see Fig. 10.5). Specific granules contain various enzymes (i.e., type IV collagenase, phospholipase) as well as complement activators and other antimicrobial peptides (i.e., lysozymes, lactoferrins).


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Histology_Ross.  Azurophilic granules (primary granules) are larger and less numerous than specific granules. They arise early in granulopoiesis and occur in all granulocytes, as well as in monocytes and lymphocytes. The azurophilic granules are the lysosomes of the neutrophil and contain myeloperoxidase (MPO) (a peroxidase enzyme), which appears as a finely stippled material with the TEM. Myeloperoxidase helps to generate highly reactive bactericidal hypochlorite and chloramines. In addition to containing a variety of the typical acid hydrolases, azurophilic granules also contain cationic proteins called defensins, which function analogously to antibodies and the antimicrobial peptide cathelicidin to kill pathogens.


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Histology_Ross.  Tertiary granules in neutrophils are of two types. One type contains phosphatases (enzymes that remove a phosphate group from a substrate) and is sometimes called a phosphasome. The other type contains metalloproteinases, such as gelatinases and collagenases, which are thought to facilitate the migration of the neutrophil through the connective tissue. Aside from these granules, membrane-bounded organelles are sparse. A small Golgi apparatus is evident in the center of the cell, and mitochondria are relatively few in number (see Fig. 10.5). Neutrophils are motile cells; they leave the circulation and migrate to their site of action in the connective tissue.


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Histology_Ross. Neutrophils are motile cells; they leave the circulation and migrate to their site of action in the connective tissue. An important property of neutrophils and other leukocytes is their motility. Neutrophils are the most numerous of the first wave of cells to enter an area of tissue damage. Their migration is controlled by the expression of adhesion molecules on the neutrophil surface that interact with corresponding ligands on endothelial cells (Fig. 10.6) and are often involved in cell binding. The initial phase of neutrophil migration occurs in the post-capillary venules and is regulated by a mechanism involving neutrophil–endothelial cell recognition. Selectins (a type of cell adhesion molecule) on the surface of the circulating neutrophil (CD62L) interact with receptors (GlyCAM-1) on the surface of the endothelial cells. The neutrophil becomes partially tethered to the endothelial cell as a result of this  FOLDER 10.3 Clinical Correlation: Hemoglobin Disorders


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Histology_Ross. Anemia Anemia is defined clinically as a decrease in the concentration of hemoglobin in the blood for the age and sex of an individual. Although in certain anemias this decreased concentration of hemoglobin is caused by a decrease in the amount of hemoglobin in each cell, most anemias are caused by a reduction in the number of erythrocytes. Causes of anemia include loss of blood (hemorrhage), insufficient production of erythrocytes, or accelerated destruction of erythrocytes in the circulation. Insufficient dietary iron or deficiencies of vitamins such as vitamin B12 or folic acid can lead to decreased production of erythrocytes. Gastric atrophy, as a result of autoimmune disease, with concomitant destruction of the parietal cells that secrete intrinsic factor, a molecule essential for absorption of vitamin B12 by cells in the ileum, is the cause of a form of anemia called pernicious anemia. The clinical symptoms of anemia vary, depending on the type of anemia, the underlying cause,


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Histology_Ross. of vitamin B12 by cells in the ileum, is the cause of a form of anemia called pernicious anemia. The clinical symptoms of anemia vary, depending on the type of anemia, the underlying cause, and other associated medical conditions. The common symptoms of even mild anemia include weakness, fatigue, and loss of energy. The other symptoms associated with anemia are shortness of breath, frequent headaches, difficulty concentrating, mental confusion, loss of sexual drive, dizziness, leg cramps, insomnia, and pale skin.


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Histology_Ross. interaction, which slows the neutrophil and causes it to roll on the surface of the endothelium. In the second phase, another group of adhesion molecules on the neutrophil surface, called integrins (VLA-5), are activated by chemokine signals from the endothelial cells. In the third phase, integrins and other adhesion molecules from the immunoglobulin superfamily (e.g., intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) expressed on the neutrophil surface engage with their specific receptors on the endothelial cells,


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Histology_Ross. Sickle Cell Disease Sickle cell disease is caused by a single-point mutation in the gene that encodes the -globin chain of hemoglobin A. The result of this mutation is an abnormal -globin chain in which the amino acid valine is substituted for glutamic acid in position 6. Hemoglobin containing this abnormal -globin chain is designated sickle hemoglobin (HbS). The substitution of the hydrophobic valine for the hydrophilic glutamic acid causes HbS to aggregate under conditions of reduced oxygen tension. Instead of the normal biconcave disc shape, many of the erythrocytes become sickle-shaped at low oxygen tension, hence the name of this disease (Fig. F10.3.1). Sickled erythrocytes are more rigid than normal cells and adhere more readily to the endothelial surface. Thus, sickled erythrocytes may pile up in the smallest capillaries, depriving portions of tissues and organs of oxygen and nutrients. Large-vessel obstruction may also occur, which in children frequently leads to stroke.


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Histology_Ross. may pile up in the smallest capillaries, depriving portions of tissues and organs of oxygen and nutrients. Large-vessel obstruction may also occur, which in children frequently leads to stroke. Sickled erythrocytes are also more fragile and break down or are destroyed more quickly (after 20 days) than normal erythrocytes.


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Histology_Ross. Sickle cell disease is a homozygous recessive genetic disorder. However, heterozygous individuals with sickled cell trait may occasionally have clinical consequences at high altitude or when under extreme physical stress. FIGURE F10.3.1 • Photomicrograph of a sickle cell anemia blood smear. Blood smear stained with Wright’s stain shows abnormal “boat”and “sickle”-shaped cells from an individual with sickle cell anemia. 400. attaching the neutrophil to the endothelial cell. The neutrophil then extends a pseudopod to an intercellular junction. Histamine and heparin released at the injury site by perivascular mast cells open the intercellular junction, allowing the neutrophil to migrate into the connective tissue. With the TEM, the cytoplasmic contents of a neutrophil pseudopod appear as an expanse of finely granular cytoplasmic matrix with no membranous organelles (see Fig. 10.5). The finely granular appearance is attributable to the presence of actin filaments, some


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Histology_Ross. FIGURE 10.5 • Electron micrograph of a human mature neutrophil. The nucleus shows the typical multilobed configuration with the heterochromatin at the periphery and the euchromatin more centrally located. A small Golgi apparatus (G) is present; other organelles are sparse. The punctate appearance of the cytoplasm adjacent to the convex aspect of the nuclear profile is caused by glycogen particles. Adjacent to the concave aspect of the nuclear profile are numerous granules. Specific granules appear less dense and more rounded than azurophilic granules. The latter are fewer in number and are extremely electron dense. 22,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) For comparison, the inset shows a neutrophil from a blood smear observed in the light microscope. 1,800. FIGURE 10.6 • Diagram of events in the migration of a neutrophil from a postcapillary venule into the connective tissue. a.


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Histology_Ross. Circulating neutrophils are slowed down by the interaction of their surface adhesion molecules, selectins (CD62L), with the endothelium of the venule (b). c. As the result of this interaction, the cell rolls on the surface of the endothelium. The neutrophil then adheres to the endothelium and responds to chemokines secreted by the endothelial cells. d. Their secretion induces the expression of other adhesion molecules on the surface of the neutrophil such as integrins (VLA-5) and the immunoglobulin superfamily of adhesion molecules (e.g., intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]). e. These adhesion molecules allow the neutrophil to bind to adhesion molecule receptors on the endothelial cells. f. The neutrophil then extends a pseudopod to an intercellular junction previously opened by histamine and heparin released from the mast cells in the connective tissue, allowing the neutrophil to migrate through the vessel wall (g).


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Histology_Ross. microtubules, and glycogen, which are involved in the extension of the cytoplasm to form the pseudopod and the subsequent contraction that pulls the cell forward. Once the neutrophil enters the connective tissue, further migration to the injury site is directed by a process known as chemotaxis, the binding of chemoattractant molecules and extracellular matrix proteins to specific receptors on the surface of the neutrophil. Neutrophils are active phagocytes that utilize a variety of surface receptors to recognize bacteria and other infectious agents at the site of inflammation.


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Histology_Ross. Once at the site of tissue injury, the neutrophil must first recognize any foreign substances before phagocytosis can occur. Like most phagocytic cells, neutrophils have a variety of receptors on their cell membrane that can recognize and bind to bacteria, foreign organisms, and other infectious agents (Fig 10.7). Some of these organisms and agents bind directly to neutrophils (no modifications of their surfaces are required), whereas others must be opsonized (coated with antibodies or complement) to make them more attractive to the neutrophil. The most common receptors used by neutrophils during phagocytosis include the following.  Fc receptors on the neutrophil surface bind to the ex posed Fc region of IgG antibodies that coat bacterial surfaces (see Fig 10.7). Binding of IgG-coated bacteria activates the neutropil’s phagocitic activity and causes a rapid surge in intracellular metabolism.


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Histology_Ross.  Complement receptors (CRs) facilitate binding and uptake of immune complexes that are opsonized by active C3 complement protein, namely, C3b. Binding of bacteria or other C3b-coated antigens to CRs triggers phagocytosis, resulting in activation of a neutrophil’s lytic pathways and respiratory burst reactions.  Scavenger receptors (SRs) are a structurally diverse group of transmembrane glycoproteins that bind to modified (acetylated or oxidized) forms of low-density lipoproteins (LDLs), polyanionic molecules that are often on the surface of both Gram-positive and Gram-negative bacteria and apoptotic bodies. Binding of these receptors increases the phagocitic activity of neutrophils.


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Histology_Ross.  Toll-like receptors, also known as pattern recognition receptors (PRRs), are neutrophil receptors that recognize pathogenic molecules such as endotoxins, lipopolysaccharides, peptidoglycanes, and lipoteichoic acids that are arranged in predictable pathogen-associated molecular patterns (PAMPs) and are commonly expressed on bacterial surfaces and other infectious agents. Like other phagocytic cells, neutrophils possess a variety of toll-like receptors that recognize PAMPs. Binding of bacterial


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Histology_Ross. FIGURE 10.7 • Neutrophil phagocytosis. a. Phagocytosis begins with recognition and attachment of foreign material (antigen), mainly by Fc receptors that interact with the Fc region of antibodies bound to the antigen. b. The antigen is then engulfed by pseudopods of the neutrophil. c. As the pseudopods come together and fuse, the antigen is internalized. d. Once the phagosome is formed, digestion is initiated by activation of membrane-bounded oxidases of the phagosome. e. Next, both specific and azurophilic granules fuse with the phagosome and release their contents, forming a phagolysosome. This fusion and release of granules is called degranulation. f. The enzymatic contents of the granules are responsible for killing and digesting the microorganism. The entire digestive process occurs within the phagolysosome, which protects the cell from self-injury. g. The digested material is either exocytosed into the extracellular space or stored as residual bodies within the neutrophil.


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Histology_Ross. antigens to these receptor causes phagocytosis and the release of cytokines such as interleukin-1 (IL-1), interleukin-3 (IL-3), and tumor necrosis factor (TNF-) from the neutrophil. IL-1, historically known as a pyrogen (fever-causing agent), induces synthesis of prostaglandins, which in turn act on the thermoregulatory center of the hypothalamus to produce fever. Therefore, fever is a consequence of acute reaction to invading pathogens that cause a massive neutrophilic response. Phagocytosed bacteria are killed within phagolysosomes by the toxic reactive oxygen intermediates produced during respiratory burst.


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Histology_Ross. Phagocytosis begins when the neutrophil recognizes and attaches to the antigen. Extended pseudopods of the neutrophil engulf the antigen and internalize it to form a phagosome (see Fig. 10.7). Specific and azurophilic granules fuse with the phagosome membrane, and the lysosomal hydrolases of the azurophilic granules digest the foreign material. During phagocytosis, the neutrophil’s glucose and oxygen utilization increases noticeably and is referred to as the respiratory burst. It results in synthesis of several oxygen-containing compounds called reactive oxygen intermediates (ROIs). They include free radicals such as oxygen and hydroxyl radicals that are used in immobilizing and killing live bacteria within the phagolysosomes. By definition, free radicals possess an unpaired electron within their chemical structure, which makes them highly reactive and therefore capable of damaging intracellular molecules, including lipids, proteins, and nucleic acids. The process by which


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Histology_Ross. within their chemical structure, which makes them highly reactive and therefore capable of damaging intracellular molecules, including lipids, proteins, and nucleic acids. The process by which microorganisms are killed within neutrophils is termed oxygen-dependent intracellular killing. In general, two biochemical pathways are involved in this process: the first is the phagocyte oxidase (phox) system that utilizes the phagocyte’s nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in the phagolysosome membrane; the second is associated with the lysosomal enzyme myeloperoxidase (MPO) found in the azurophilic granules of neutrophils (Fig 10.8).


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Histology_Ross. Within the phagocyte oxidase pathway, or phox system, phagocytosis proceeds by signaling the cell to produce sufficient amounts of NADPH needed to generate superoxide anions. Increased glucose uptake and shunting of NADPH metabolism is achieved via the pentose phosphate pathway (also known as pentose shunt). The cytosolic NADPH becomes an electron donor: The NADPH oxidase enzyme complex transports electrons across the membrane to molecular O2 inside the phagolysosome to generate the free radical superoxide anions (O–2). These superoxide anions are converted into ROIs. The superoxide dismutase converts superoxide anions to singlet oxygen (1O2) and hydrogen peroxide (H2O2), which further reacts with superoxide anions to produce bactericidal hydroxyl radicals (OH–) (the neutral form of the hydroxyl ion) and more singlet oxygen molecules (see Fig 10.8).


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Histology_Ross. Oxygen-dependent killing with MPO involvement occurs when azurophilic granules containing MPO fuse with phagosomes containing phagocytosed bacteria. During the neutrophil’s respiratory burst, MPO, using heme as a cofactor, catalyzes a reaction that produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and a chloride anion (Cl–). Hypochlorous acid, which is about 1,000 times more effective in bacterial killing than hydrogen peroxide, is further metabolized to a highly toxic hypochlorite OCl– (bleach) and chlorine (Cl2). Some of the hypochloride may spontaneously break down to yield toxic singlet oxygen (1O2) and chloride ions (Cl–) (see Fig. 10.8).


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Histology_Ross. In addition, nitric oxide (NO) and other reactive nitrogen intermediates (RNIs) have also been implicated in the intracellular microbial killing mechanisms. NO has been found in neutrophils; however, it is believed that RNI-mediated killing mechanisms do not appear to have a critical role in humans. The main role of neutrophil-derived NO is to induce vasodilatation, which in turn facilitates the migration of neutrophils from blood vessels to surrounding connective tissue. Phagocytosed bacteria can also be killed by a diverse arsenal of oxygen-independent killing mechanisms utilizing bacteriolytic enzymes and antimicrobial peptides.


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Histology_Ross. In addition to the oxygen-dependent respiratory burst reactions, microorganisms can be killed by bacteriolytic enzymes and cationic antimicrobial peptides that are stored within the granules of the neutrophil’s cytoplasm. These oxygen-independent killing mechanisms are directed toward the bacterial cell membrane, causing its breakdown and leakage. Neutrophils contain particularly large amounts of cationic antimicrobial proteins such as defensins and antimicrobial peptides called cathelicidins. Similar to lysozymes and cathepsins stored in the specific granules, these cationic antimicrobial proteins break down the bacterial wall. In addition, lysosomal hydrolytic enzymes that digest bacterial proteins and lactoferrins that chelate iron from nutritional bacterial pathways contribute to the destruction of the invading bacteria. These mechanisms are not as efficient as oxygendependent killing pathways. Neutrophils from patients with defects in oxygen-dependent pathways, such as those with


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Histology_Ross. of the invading bacteria. These mechanisms are not as efficient as oxygendependent killing pathways. Neutrophils from patients with defects in oxygen-dependent pathways, such as those with chronic granulomatous disease (see Folder 10.4), are still able to destroy phagocytosed bacteria to some degree. However, because of the low efficiency of these processes, individuals with these defects are more susceptible to serious infections.


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Histology_Ross. After intracellular digestion by the neutrophil, the remnants of degraded material are stored in residual bodies or exocytosed. Most neutrophils die in this process; the accumulation of dead bacteria and dead neutrophils constitutes the thick exudate called pus. The yellow–green color of the pus and of mucus secretions (e.g., from infected lungs) comes from the heme pigment of MPO enzyme in azurophilic granules of neutrophils. Inflammation and wound healing also involve monocytes, lymphocytes, eosinophils, basophils, and fibroblasts. Monocytes also enter the connective tissue as a secondary response to tissue injury. At the site of tissue injury, they transform into macrophages that phagocytose cell and tissue debris, fibrin, remaining bacteria, and dead neutrophils. Normal wound healing depends on the participation of FIGURE 10.8 • Pathways leading to synthesis of reactive oxygen intermediates during neutrophil’s respiratory burst reactions.


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Histology_Ross. This schematic diagram shows a phagolysosome that contains already pagocytosed bacterium. Two oxygen-dependent killing mechanisms are depicted in this drawing. The first mechanism depends on a phagocyte oxidase (phox) system that utilizes the NADPH oxidase complex (contains five subunits). This complex transports excess electrons across the membrane of the phagolysosome, where they interact with molecular oxygen to generate free superoxide anions. These anions are converted into reactive oxygen intermediates. Another enzyme superoxide dismutase converts superoxide anions to singlet oxygen and hydrogen peroxide, which further reacts with superoxide anions to produce bactericidal hydroxyl radicals and more singlet oxygen molecules. The second mechanism involves lysosomal enzyme myeloperoxidase (MPO) found in the azurophilic granules of neutrophils. MPO catalizes the production of hypochlorous acids from hydrogen peroxide and chloride anions. Hypochlorous acid is further metabolized to a


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Histology_Ross. (MPO) found in the azurophilic granules of neutrophils. MPO catalizes the production of hypochlorous acids from hydrogen peroxide and chloride anions. Hypochlorous acid is further metabolized to a highly toxic hypochlorite (bleach) and chlorine. Some of the hypochloride may spontaneously break down to yield toxic singlet oxygen and chloride ions. All molecules produced during oxygen bursts in neutrophils (associated with red arrows) are highly effective in killing ingested bacteria.


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Histology_Ross. macrophages in the inflammatory response; they become the major cell type in the inflammatory site after the neutrophils are spent. At the same time that the macrophages become active at the site of inflammation, fibroblasts near the site and undifferentiated mesenchymal cells in the adventitia of small vessels at the site begin to divide and differentiate into fibroblasts and myofibroblasts that then secrete the fibers and ground substance of the healing wound. Like neutrophils, monocytes are attracted to the inflammatory site by chemotaxis. Lymphocytes, eosinophils, and basophils also play a role in inﬂammation, but they are more involved in the immunologic aspects of the process (see Chapter 14, Lymphatic System). Eosinophils and lymphocytes are more commonly found at sites of chronic inﬂammation.


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Histology_Ross. Eosinophils are about the same size as neutrophils, and their nuclei are typically bilobed (Fig. 10.9; Plate 17, page 302). As in neutrophils, the compact heterochromatin of eosinophils is chiefly adjacent to the nuclear envelope, whereas the euchromatin is located in the center of the nucleus. Eosinophils are named for the large, eosinophilic, refractile granules in their cytoplasm. The cytoplasm of eosinophils contains two types of granules: numerous, large, elongated specific granules and azurophilic granules (otherwise, the eosinophil contains only a sparse representation of membranous organelles).  FOLDER 10.4 Clinical Correlation: Inherited Disorders of Neutrophils; Chronic Granulomatous Disease (CGD)


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Histology_Ross. A primary example of genetic immunodeficiency that af-fects oxygen-dependent killing mechanisms is chronic granulomatous disease (CGD). In this inherited disor-der of neutrophils and other phagocytic cells, one of the components of the NADPH oxidase complex (phox system) has mutated or is absent. As a result, neutrophils cannot produce reactive oxygen intermediates (ROIs). The NADPH oxidase complex consists of five molecules. Two of them, glycoprotein 91 (gp91) and protein 22 (p22), are part of a membrane-bound cytochrome called cy-tochrom B558 (see Fig. 10.8). Three other cytosolic com-ponents—protein 47 (p47), protein 67 (p67), and protein 40 (p40)—are components of Rac-2 GTPase, which is required for oxidase activity. Neutrophil activation and stimulation from phagocytosis cause cytosolic pro-teins to translocate to the plasma membrane of the phagolysosome to assemble the active NADPH oxidase complex. The assembled enzyme transports electrons from cytosolic NADPH across the


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Histology_Ross. cytosolic pro-teins to translocate to the plasma membrane of the phagolysosome to assemble the active NADPH oxidase complex. The assembled enzyme transports electrons from cytosolic NADPH across the membrane to molecular O2 residing inside the phagolysosome, generating bactericidal superoxide anions (O2 –) and other ROIs. Approximately 50% to 70% of all CGD cases are caused by a mutation in the CYBB (cytochrome B, b subunit) gene located on the X chromosome. This gene encodes glycoprotein 91 (gp91), which is necessary for proper function of the NADPH oxidase complex. Since gp91 deficiency is an X-linked disease, CGD caused by this mutation is often re-ferred as X91 disease. Another 20% to 40% of those with CGD have mutations in the NCF1 gene on chromo-some 7 that codes for protein 47. Mutations in gene NCF2 (which encodes protein 67) and CYBA (which encodes protein 22) are rare, accounting for fewer than 10% of all cases of CGD. Mutations in the NCF1, NCF2, and CYBA genes produce


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Histology_Ross. Mutations in gene NCF2 (which encodes protein 67) and CYBA (which encodes protein 22) are rare, accounting for fewer than 10% of all cases of CGD. Mutations in the NCF1, NCF2, and CYBA genes produce autosomal recessive forms of CGD. CGD decreases the ability of neutrophils to kill certain types of bacteria and fungi. Individuals with this disease are frequently affected by recurrent life-threatening bacte-rial and fungal infections and chronic inflammatory condi-tions. The most common pathologic changes occur in tissues and organs that form barriers against the entry of microorganisms from the external environment. They in-clude skin (skin infections), gingiva (swollen inflamed gums), lungs (pneumonia), lymph nodes (lymphoadenitis), gastrointestinal tract (enteritis, diarrhea), liver, and spleen. Another characteristic feature of CGD is the development of enlarged, tumorlike masses called granulomas. The presence of granulomas may cause serious problems in the gastrointestinal tract


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Histology_Ross. spleen. Another characteristic feature of CGD is the development of enlarged, tumorlike masses called granulomas. The presence of granulomas may cause serious problems in the gastrointestinal tract by obstructing the passage of food and in the genitourinary tract by blocking the flow of urine from the kidneys and bladder.


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Histology_Ross.  Specific granules of eosinophils contain a crystalloid body that is readily seen with the TEM, surrounded by a less electron-dense matrix. These crystalloid bodies are responsible for the refractivity of the granules in the light microscope. They contain four major proteins: an arginine-rich protein called major basic protein (MBP), which accounts for the intense acidophilia of the granule; eosinophil cationic protein (ECP); eosinophil peroxidase (EPO); and eosinophil-derived neurotoxin (EDN). MBP is localized in the crystalloid body; the other three proteins are found in the granule matrix. MBP, ECP, and EPO have a strong cytotoxic effect on protozoans and helminthic parasites; EDN causes nervous system dysfunction in parasitic organisms; histaminase neutralizes the activity of histamine; and arylsulfatase neutralizes leukotrienes secreted by basophils and mast cells (see Chapter 6, Connective Tissue,  FOLDER 10.5 Clinical Correlation: Hemoglobin Breakdown and Jaundice


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Histology_Ross. If the conjugation of bilirubin or its excretion into bile by the liver cells is inhibited, or if blockage of the bile duct system occurs, bilirubin may reenter the blood, causing a yellow appearance of the sclera of the eye and the skin. This con-dition is called jaundice. Jaundice can be caused by the destruction of circulating erythrocytes. An example of such a condition is a hemolytic transfusion reaction when ABO-incompatible blood is administered to a patient, usu-ally because of a clerical error. Massive hemolysis of the transfused erythrocytes may be associated with severe systemic complications such as hypotension (decreased blood pressure), renal failure, and even death. Jaundice is also characteristic in a variety of hemolytic anemias that result from either inherited defects in the erythrocyte (e.g., hereditary spherocytosis) or external fac-tors such as pathogenic microorganisms, animal venoms, chemicals, and drugs. Some jaundice is common in new-born infants (physiologic


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Histology_Ross. the erythrocyte (e.g., hereditary spherocytosis) or external fac-tors such as pathogenic microorganisms, animal venoms, chemicals, and drugs. Some jaundice is common in new-born infants (physiologic jaundice) because of ineffi-ciency of the bilirubin-conjugating system in the newborn liver.


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Histology_Ross. page 186). Specific granules also contain histaminase, arylsulfatase, collagenase, and cathepsins.  Azurophilic granules are lysosomes. They contain a variety of the usual lysosomal acid hydrolases and other hydrolytic enzymes that function in destruction of parasites and hydrolysis of antigen–antibody complexes internalized by the eosinophil. Eosinophils are associated with allergic reactions, parasitic infections, and chronic inflammation. Eosinophils develop and mature in the bone marrow. Once released from the bone marrow, they circulate in peripheral blood and then migrate to the connective tissue. Eosinophils are activated by interactions with IgG, IgA, or secretory IgA antibodies.


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Histology_Ross. The release of arylsulfatase and histaminase by eosinophils at sites of allergic reaction moderates the potentially deleterious effects of inﬂammatory vasoactive mediators. The eosinophil also participates in other immunologic responses and phagocytoses antigen–antibody complexes. Thus, the count of eosinophils in blood samples of individuals with allergies and parasitic infections is usually high. Eosinophils play a major role in host defense against helminthic parasites. They are also found in large numbers in the lamina propria of the intestinal


Histology_Ross

Histology_Ross. FIGURE 10.9 • Electron micrograph of a human eosinophil. The nucleus is bilobed, but the connecting segment is not within the plane of section. The granules are of moderate size, compared with those of the basophil, and show a crystalline body (Cr) within the less electron-dense matrix of the granule. M, mitochondria. 26,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) Inset. Light microscopic image of an eosinophil from a blood smear. 1,800. tract and at other sites of potential chronic inﬂammation (i.e., lung tissues in patients with asthma). Basophils are about the same size as neutrophils and are so named because the numerous large granules in their cytoplasm stain with basic dyes (Plate 17, page 302). Basophils are the least numerous of the WBCs, accounting for less than 0.5% of total leukocytes.


Histology_Ross

Histology_Ross. Basophils are the least numerous of the WBCs, accounting for less than 0.5% of total leukocytes. Often, several hundred WBCs must be examined in a blood smear before one basophil is found. The lobed basophil nucleus is usually obscured by the granules in stained blood smears, but its characteristics are evident in electron micro-graphs (Fig. 10.10). Heterochromatin is chiefly in a peripheral location, and euchromatin is chiefly centrally located; typical cytoplasmic organelles are sparse. The basophil plasma membrane possesses numerous high-affinity Fc receptors for IgE antibodies. In addition, a specific 39-kilodalton protein called CD40L is expressed on the basophil’s surface. CD40L interacts with a complementary receptor (CD40) on B lymphocytes, which results in increased synthesis of IgE.


Histology_Ross

Histology_Ross. FIGURE 10.10 • Electron micrograph of a human basophil. The nucleus appears as three separate bodies; the connecting strands are not in the plane of section. The basophil granules (B) are very large and irregularly shaped. Some granules reveal myelin figures (MF ). M, mitochondria. 26,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) Inset. Light microscopic appearance of a basophil from a blood smear. 1,800. The basophil cytoplasm contains two types of granules: specific granules, which are larger than the specific granules of the neutrophil, and nonspecific azurophilic granules.


Histology_Ross

Histology_Ross. The basophil cytoplasm contains two types of granules: specific granules, which are larger than the specific granules of the neutrophil, and nonspecific azurophilic granules.  Specific granules exhibit a grainy texture and myelin figures when viewed with the TEM. These granules contain a variety of substances, namely, heparin, histamine, heparan sulfate, leukotrienes, IL-4, and IL-13. Heparin, a sulfated glycosaminoglycan, is an anticoagulant. Histamine and heparan sulfate are vasoactive agents that among other actions cause dilation of small blood vessels. Leukotrienes are modified lipids that trigger prolonged constriction of smooth muscles in the pulmonary airways (see page 186). Interleukin-4 (IL-4) and interleukin-13 (IL-13) promote synthesis of IgE antibodies. The intense basophilia of these specific granules correlates with the high concentration of sulfates within the glycosaminoglycan molecules of heparin and heparan sulfate.


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Histology_Ross.  Azurophilic granules are the lysosomes of basophils and contain a variety of the usual lysosomal acid hydrolases that are similar to those in other leukocytes. The function of basophils is closely related to that of mast cells.


Histology_Ross

Histology_Ross. Basophils are functionally related to, but not identical with, mast cells of the connective tissue (see Table 6.6, page 187). Both mast cells and basophils bind an antibody secreted by plasma cells, IgE, through high-affinity Fc receptors expressed on their cell surface. The subsequent exposure to, and reaction with, the antigen (allergen) specific for IgE triggers the activation of basophils and mast cells and the release of vasoactive agents from cell granules. These substances are responsible for the severe vascular disturbances associated with hypersensitivity reactions and anaphylaxis. Furthermore, both basophils and mast cells are derived from the same basophil–mast cell progenitor (BMCP) cell. If a specific BMCP expresses the granulocyte-related transcription factor CCAAT/enhancer-binding protein (C/EBP), the cell becomes committed to differentiate into a basophil progenitor (BaP) cell. Basophils develop and differentiate in the bone marrow and are released to the peripheral


Histology_Ross

Histology_Ross. protein (C/EBP), the cell becomes committed to differentiate into a basophil progenitor (BaP) cell. Basophils develop and differentiate in the bone marrow and are released to the peripheral blood as mature cells. In the absence of C/EBP transcription factor, a BMCP cell migrates to the spleen and after further differentiation travels as a mast cell precursor (MPC) to the intestine, where it becomes a mature mast cell.


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Histology_Ross. Lymphocytes are the main functional cells of the lymphatic or immune system.


Histology_Ross

Histology_Ross. Lymphocytes are the most common agranulocytes and account for about 30% of the total blood leukocytes. In understanding the function of the lymphocytes, one must realize that most lymphocytes found in blood or lymph represent recirculating immunocompetent cells (i.e., cells that have developed the capacity to recognize and respond to antigens and are in transit from one lymphatic tissue to another). In the tissues associated with the immune system (see Chapter 14, Lymphatic System), three groups of lymphocytes can be identified according to size: small, medium, and large lymphocytes, ranging in diameter from 6 to 30 m. The large lymphocytes are either activated lymphocytes, which possess surface receptors that interact with a specific antigen, or natural killer (NK) lymphocytes. In the bloodstream, most lymphocytes are small or medium sized, 6 to 15 m in diameter. The majority—more than 90%—are small lymphocytes.


Histology_Ross

Histology_Ross. In blood smears, the mature lymphocyte approximates the size of an erythrocyte. When observed in the light microscope in a blood smear, small lymphocytes have an intensely staining, slightly indented, spherical nucleus (Plate 17, page 302). The cytoplasm appears as a very thin, pale blue rim surrounding the nucleus. In general, there are no recognizable cytoplasmic organelles other than an occasional fine azurophilic granule. The TEM reveals that the cytoplasm primarily contains free ribosomes and a few mitochondria. Other organelles are so sparse that they are usually not seen in a thin section. Small, dense lysosomes that correspond to the azurophilic granules seen in the light microscope are occasionally observed; a pair of centrioles and a small Golgi apparatus are located in the cell center, the area of the indentation of the nucleus.


Histology_Ross

Histology_Ross. In the medium lymphocyte, the cytoplasm is more abundant, the nucleus is larger and less heterochromatic, and the Golgi apparatus is somewhat more developed (Fig. 10.11). Greater numbers of mitochondria and polysomes and small profiles of rough endoplasmic reticulum are also seen in these medium-sized cells. The ribosomes are the basis for the slight basophilia displayed by lymphocytes in stained blood smears. Three functionally distinct types of lymphocytes are present in the body: T lymphocytes, B lymphocytes, and NK cells. The characterization of lymphocyte types is based on their function, not on their size or morphology. T lymphocytes (T cells) are so named because they undergo differentiation in the thymus. B lymphocytes (B cells) are so named because they were first recognized as a separate population in the bursa of Fabricius in birds or bursa-equivalent organs (e.g., bone marrow) in mammals. Natural killer (NK) cells


Histology_Ross

Histology_Ross. FIGURE 10.11 • Electron micrograph of a medium-sized lymphocyte. The punctate appearance of the cytoplasm is caused by the presence of numerous free ribosomes. Several mitochondria (M) are evident. The cell center or centrosphere region of the cell (the area of the nuclear indentation) also shows a small Golgi apparatus (G) and a centriole (C). 26,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) Inset. Light microscopic appearance of a medium-sized lymphocyte from a blood smear. 1,800. develop from the same precursor cell as B and T cells and are so named because they are programmed to kill certain types of transformed cells.


Histology_Ross

Histology_Ross. develop from the same precursor cell as B and T cells and are so named because they are programmed to kill certain types of transformed cells.  T cells have a long life span and are involved in cell-mediated immunity. T cells are characterized by the presence of cell-surface recognition proteins called T-cell receptors (TCRs), which in a majority of T cells comprise two glycoprotein chains called and -TCR chains. They express CD2, CD3, CD5, and CD7 marker proteins on their surface; however, they are subclassified on the basis of the presence or absence of CD4 and CD8 proteins. CD4 T lymphocytes possess the CD4 marker and recognize antigens bound to major histocompatability complex II (MHC II) molecules. CD8 T lymphocytes possess the CD8 marker and recognize antigen bound to MHC I molecules.


Histology_Ross

Histology_Ross.  B cells have variable life spans and are involved in the production of circulating antibodies. Mature B cells in blood express IgM and IgD and MHC II molecules on their surface. Their specific markers are CD9, CD19, CD20, and CD24.  NK cells are programmed during their development to kill certain virus-infected cells and some types of tumor cells. They also secrete an antiviral agent, interferon (IFN-). NK cells are larger than B and T cells (15 m in diameter) and have a kidney-shaped nucleus. Because NK cells have several large cytoplasmic granules easily seen by light microscopy, they are also called large granu lar lymphocytes (LGLs). Their specific markers include CD16, CD56, and CD94.


Histology_Ross

Histology_Ross. T and B cells are indistinguishable in blood smears and tissue sections; immunocytochemical staining for different types of markers and receptors on their cell surface must be used to identify them. NK lymphocytes can be identified in the light microscope by size, nuclear shape, and presence of cytoplasmic granules; however, immunocytochemical staining for their specific markers is used to confirm microscopic identification. T and B lymphocytes express different surface molecules.


Histology_Ross

Histology_Ross. T and B lymphocytes express different surface molecules. Although the T and B cells cannot be distinguished on the basis of their morphology, their distinctive surface proteins (CD proteins) can be used to identify the cells with immunolabeling techniques. In addition, immunoglobulins are expressed on the surface of B cells that function as antigen receptors. In contrast, T cells do not have antibodies but express TCRs. These recognition proteins appear during discrete stages in the maturation of the cells within the thymus. In general, the surface molecules mediate or augment specific T-cell functions and are required for the recognition or binding of T cells to antigens displayed on the surface of target cells.


Histology_Ross

Histology_Ross. In human blood, 60% to 80% of lymphocytes are mature T cells, and 20% to 30% are mature B cells. Approximately 5% to 10% of the cells do not demonstrate the surface markers associated with either T or B cells. These are NK cells and the rare circulating hemopoietic stem cells (see below). The size differences described above may have functional significance; some of the large lymphocytes may be cells that have been stimulated to divide, whereas others may be plasma cell precursors that are undergoing differentiation in response to the presence of antigen. Several different types of T lymphocytes have been identified: cytotoxic, helper, suppressor, and gamma/delta (). The activities of cytotoxic, helper, suppressor, and gamma/delta T lymphocytes are mediated by molecules located on their surface. Immunolabeling techniques have made it possible to identify specific types of T cells and study their function.


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Histology_Ross.  Cytotoxic CD8 T cells serve as the primary effector cells in cell-mediated immunity. CD8 cells are specifically sensitized T lymphocytes that recognize antigens through the TCRs on viral or neoplastic host cells. Cytotoxic CD8 T lymphocytes only recognize antigens bound to MHC I molecules. After the TCR binds the antigen–MHC I complex, the cytotoxic CD8 T cells secrete lymphokines and perforins that produce ion channels in the membrane of the infected or neoplastic cell, leading to its lysis (see Chapter 14, Lymphatic System). Cytotoxic CD8 T lymphocytes play a significant role in rejection of allografts and in tumor immunology.


Histology_Ross

Histology_Ross.  Helper CD4 T cells are critical for induction of an immune response to a foreign antigen. Antigen bound to MHC II molecules is presented by antigen-presenting cells such as macrophages to a helper CD4 T lymphocyte. Binding of the TCR to the antigen–MHC II complex activates the helper CD4 T cells. The activated helper CD4 T lymphocytes then produce interleukins (mainly IL-2), which act in an autocrine mode to simulate the proliferation and differentiation of more helper CD4 T lymphocytes. Newly differentiated cells synthesize and secrete lymphokines that affect function as well as differentiation of B cells, T cells, and NK cells. B cells differentiate into plasma cells and synthesize antibody.


Histology_Ross

Histology_Ross.  Regulatory (suppressor) T-cells represent a phenotypically diverse population of T lymphocytes that can functionally suppress an immune response to foreign and self-antigen by influencing the activity of other cells in the immune system. The CD4CD25FOXP3 regulator T cells represent a classical example of cells that can down-regulate the ability of T lymphocytes to initiate immune responses. The FOXP3 marker indicates an expression of forkhead family transcription factors that are characteristic of many T cells. Furthermore, tumor-associated CD8CD45RO T suppressor cells secrete IL-10 and also suppress T-cell activation. The suppressor T cells may also function in suppressing B-cell differentiation and in regulating erythroid cell maturation in the bone marrow.


Histology_Ross

Histology_Ross.  Gamma/delta () T cells represent a small population of T cells that possess a distinct TCR on their surface. As discussed previously, most T cells have a TCR receptor composed of two glycoprotein chains called and TCR chains. In contrast, T cells possess TCR receptors made up of one -chain and one -chain. These cells develop in the thymus and migrate into various epithelial tissues (e.g., skin, oral mucosa, intestine, and vagina). Once they colonize an epithelial tissue, they do not recirculate between blood and lymphatic organs. They are also known as intraepithelial lymphocytes. Their location within the skin and mucosa of internal organs allows them to function in the first line of defense against invading organisms. Monocytes are the precursors of the cells of the mononuclear phagocytotic system.


Histology_Ross

Histology_Ross. Monocytes are the precursors of the cells of the mononuclear phagocytotic system. Monocytes are the largest of the WBCs in a blood smear (average diameter, 18 m). They travel from the bone marrow to the body tissues, where they differentiate into the various phagocytes of the mononuclear phagocytotic system— that is, connective tissue macrophages, osteoclasts, alveolar macrophages, perisinusoidal macrophages in the liver (Kupffer cells), and macrophages of lymph nodes, spleen, and bone marrow among others (see Chapter 6, Connective Tissue). Monocytes remain in the blood for only about 3 days.


Histology_Ross

Histology_Ross. The nucleus of the monocyte is typically more indented than that of the lymphocyte (Fig. 10.12 and Plate 18, page 304). The indentation is the site of the cell center where the well-developed Golgi apparatus and centrioles are located. Monocytes also contain smooth endoplasmic reticulum, rough endoplasmic reticulum, and small mitochondria. Although these cells are classified as agranular, they contain small, dense, azurophilic granules. These granules contain typical lysosomal enzymes similar to those found in the azurophilic granules of neutrophils. Monocytes transform into macrophages, which function as antigen-presenting cells in the immune system.


Histology_Ross

Histology_Ross. Monocytes transform into macrophages, which function as antigen-presenting cells in the immune system. During inﬂammation, the monocyte leaves the blood vessel at the site of inﬂammation, transforms into a tissue macrophage, and phagocytoses bacteria, other cells, and tissue debris. The monocyte–macrophage is an antigen-presenting cell and plays an important role in immune responses by partially degrading antigens and presenting their fragments on the MHC II molecules located on the macrophage surface of helper CD4 T lymphocytes for recognition. Thrombocytes are small, membrane-bounded, anucleate cytoplasmic fragments derived from megakaryocytes.


Histology_Ross

Histology_Ross. Thrombocytes are small, membrane-bounded, anucleate cytoplasmic fragments derived from megakaryocytes. Thrombocytes (platelets) are derived from large polyploid cells (cells whose nuclei contain multiple sets of chromosomes) in the bone marrow called megakaryocytes (Fig. 10.13). In platelet formation, small bits of cytoplasm are separated from the peripheral regions of the megakaryocyte by extensive platelet demarcation channels. The membrane that lines these channels arises by invagination of the plasma membrane; therefore, the channels are in continuity with the extracellular space. The continued development and fusion of the platelet demarcation membranes result in the complete partitioning of cytoplasmic fragments to form individual platelets. After entry into the vascular system from the bone marrow, the platelets circulate as discoid structures about 2 to 3 m in diameter. Their life span is about 10 days.


Histology_Ross

Histology_Ross. Structurally, platelets may be divided into four zones based on organization and function. The TEM reveals a structural organization of the thrombocyte cytoplasm that can be categorized into the following four zones (Fig. 10.14).  The peripheral zone consists of the cell membrane covered by a thick surface coat of glycocalyx. The glyco calyx consists of glycoproteins, glycosaminoglycans, and FIGURE 10.12 • Electron micrograph of a human mature monocyte. The nucleus is markedly indented, and adjacent to this site, a centriole (C) and several Golgi profiles (G) are evident. The small dark granules are azurophilic granules, the lysosomes (L) of the cell. The slightly larger and less dense profiles are mitochondria (M). 22,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) Inset. Light microscopic appearance of a monocyte from a blood smear. 1,800.


Histology_Ross

Histology_Ross. FIGURE 10.13 • Electron and light micrographs of a megakaryocyte. This electron micrograph shows a portion of a megakaryocyte from a bone marrow section. Two lobes of the nucleus and the surrounding cytoplasm are visible. The cell border is indicated by the dotted line (upper right). The cytoplasm reveals evidence of platelet formation as indicated by the extensive platelet demarcation channels. 13,000. Left inset. Light micrograph showing an entire megakaryocyte from a marrow smear. Its nucleus is multilobed and folded on itself, giving an irregular outline. The “foamy” peripheral cytoplasm of the megakaryocyte represents areas in which segmentation to form platelets is occurring. The smaller surrounding cells are developing blood cells. 1,000. Right inset. Higher-power electron micrograph showing a section of cytoplasm that is almost fully partitioned by platelet demarcation channels (arrows). It also shows mitochondria (M), a very dense granule, and glycogen particles. For


Histology_Ross

Histology_Ross. showing a section of cytoplasm that is almost fully partitioned by platelet demarcation channels (arrows). It also shows mitochondria (M), a very dense granule, and glycogen particles. For comparison, Figure 10.14a shows a mature circulating platelet. 30,000.


Histology_Ross

Histology_Ross. several coagulation factors adsorbed from the plasma. The integral membrane glycoproteins function as receptors in platelet function.  The structural zone comprises microtubules, actin filaments, myosin, and actin-binding proteins that form a network supporting the plasma membrane. From 8 to 24 microtubules reside as a bundle immediately below the actin filament network. They are circumferentially arranged and are responsible for maintaining the platelet’s disc shape.


Histology_Ross

Histology_Ross.  The organelle zone occupies the center of the platelet. It consists of mitochondria, peroxisomes, glycogen particles, and at least three types of granules dispersed within the cytoplasm. The most numerous granules are granules (300 to 500 nm in diameter) that contain mainly fibrinogen, coagulation factors, plasminogen, plasminogen activator inhibitor, and platelet-derived growth factor. The contents of these granules play an important role in the initial phase of vessel repair, blood coagulation, and platelet aggregation. The smaller, denser, and less numerous granules mainly contain adenosine diphosphate (ADP), adenosine triphosphate (ATP), serotonin, and histamine, which facilitate platelet adhesion and vasoconstriction in the area of the injured vessel. The granules are similar to lysosomes found in other cells and contain several hydrolytic enzymes. The contents of granules function in clot resorption during the later stages of vessel repair.


Histology_Ross

Histology_Ross.  The membrane zone consists of two types of membrane channels. The open canalicular system (OCS) is the first type of membrane channel. The OCS is a developmental remnant of the platelet demarcation channels and is simply a membrane that did not participate in subdividing the megakaryocyte cytoplasm. In effect, open canaliculi are invaginations into the cytoplasm from the plasma membrane. The dense tubular system (DTS) is the


Histology_Ross

Histology_Ross. FIGURE 10.14 • Platelet electron micrograph and diagram. a. High-magnification electron micrograph of a platelet situated between an erythrocyte on the left and an endothelial cell on the right. Visible organelles include a mitochondrion, microtubules, a single profile of the surface-connected open canalicular system, profiles of the dense tubular system, the moderately dense granules, a single very dense granule, and glycogen particles. The microfilaments are not evident against the background matrix of the platelet. b. Diagram of a platelet showing the components of the four structural zones.


Histology_Ross

Histology_Ross. second type of membrane channel. The DTS contains an electron-dense material originating from the rough endoplasmic reticulum of the megakaryocyte, which serves as a storage site for calcium ions. DTS channels do not connect with the surface of the platelet; however, both the OCS and DTS fuse in various areas of the platelet to form membrane complexes that are important in regulation of the intraplatelet calcium concentration. Platelets function in continuous surveillance of blood vessels, blood clot formation, and repair of injured tissue. Platelets are involved in several aspects of hemostasis (control of bleeding). They continuously survey the endothelial lining of blood vessels for gaps and breaks. When a blood vessel wall is injured or broken, the exposed connective tissue at the damaged site promotes platelet adhesion. Adhesion of the platelets at the damaged site triggers their degranulation and release of serotonin, ADP, and thromboxane A2.