Leukocyte Development, Kinetics, and Functions PDF

9 Leukocyte Development, Kinetics, and Functions...

9 Leukocyte Development, Kinetics, and Functions Reeba A. Omman, Ameet R. Kini* OBJECTIVES After completion of this chapter, the reader will be able to: 1. Describe the pathways and progenitor cells involved in the 4. Describe the morphology of promonocytes, monocytes, derivation of leukocytes from the hematopoietic stem cell macrophages, T and B lymphocytes, and immature B cells to mature forms. (hematogones). 2. Name the different stages of neutrophil, eosinophil, and 5. Discuss the functions of monocytes, macrophages, T cells, basophil development and describe the morphology of B cells, and natural killer cells in the immune response. each stage. 6. Compare the kinetics of neutrophils and monocytes. 3. Discuss the important functions of neutrophils, eosinophils, 7. Discuss in general terms how the various types of lympho- and basophils. cytes are produced. OUTLINE Granulocytes Mast Cells Neutrophils Mononuclear Cells Eosinophils Monocytes Basophils Lymphocytes CASE STUDY After studying the material in this chapter, the reader should be able to respond 1. Which leukocytes are important in mediating the clinical symptoms in this to the following case study: patient? A 5-year-old girl presents with shortness of breath and wheezing. The patient 2. A complete blood count with differential was performed on this patient. What gives a history of similar symptoms in the last 6 months. After the patient was are the typical findings in such patients? given albuterol to control her acute symptoms, long-term control of her disease 3. How did monoclonal antibodies to IL-5 help in controlling her disease? was achieved through the use of corticosteroids, along with monoclonal antibod- ies to interleukin-5 (IL-5). Leukocytes (also known as white blood cells, or WBCs) are so whose nuclei are segmented or lobulated. Individually they named because they are relatively colorless compared to red include eosinophils, with granules containing basic proteins blood cells. The number of different types of leukocytes varies that stain with acid stains such as eosin; basophils, with gran- depending on whether they are being viewed with a light mi- ules that are acidic and stain with basic stains such as methy- croscope after staining with a Romanowsky stain (5 or 6 types) lene blue; and neutrophils, with granules that react with both or are identified according to their surface antigens using flow acid and basic stains, which gives them a pink to lavender cytometry (at least 10 different types). For the purposes of this color. Because nuclear segmentation is quite prominent in chapter, the classic, light microscope classification of leukocytes mature neutrophils, they have also been called polymorphonu- will be used. clear cells, or PMNs. Granulocytes are a group of leukocytes whose cytoplasm is Mononuclear cells are categorized into monocytes and lym- filled with granules with differing staining characteristics and phocytes. These cells have nuclei that are not segmented but *The authors extend appreciation to Woodlyne Roquiz, Sameer Al Daffalha, and Anne Stiene-Martin, whose work in prior editions provided the foundation for this chapter. 117 118 PART 2 Blood Cell Production, Structure, and Function Long-term self- Short-term self- renewing stem cell renewing stem cell Pluripotent hematopoietic stem cell Common Common myeloid progenitor lymphoid progenitor Granulocyte-monocyte Eosinophil-basophil Megakaryocyte-erythrocyte Dendritic Pre-B Pre-T Natural progenitor progenitor progenitor cell killer cell Myeloblast Monoblast Myeloblast Myeloblast Pronormoblast Megakaryoblast B lymphoblast T lymphoblast Neutrophil Monocyte Eosinophil Basophil Erythrocyte Megakaryocyte B cell T cell Macrophage Mast cell Platelets Plasma cell Figure 9.1 Diagram of Hematopoiesis Showing the Derivation Pathways of Each Type of Blood Cell from a Hematopoietic Stem Cell. are round, oval, indented, or folded. Leukocytes develop from but a typical reference interval is 4.5 ! 109/L to 11.5 ! 109/L hematopoietic stem cells (HSCs) in the bone marrow, where for adults. most undergo differentiation and maturation (Figure 9.1), and The overall function of leukocytes is in mediating immunity, then are released into the circulation. The number of circulat- either innate (nonspecific), as in phagocytosis by neutrophils, ing leukocytes varies with sex, age, activity, time of day, or specific (adaptive), as in the production of antibodies by and ethnicity; it also differs according to whether or not the lymphocytes and plasma cells. The term kinetics refers to the leukocytes are reacting to stress, being consumed, or being movement of cells through developmental stages, into the cir- destroyed, and whether or not they are being produced by the culation, and from the circulation to the tissues and includes bone marrow in sufficient numbers.1 Reference intervals for the time spent in each phase of the cell’s life. As each cell type is total leukocyte counts vary among laboratories, depending on discussed in this chapter, developmental stages, kinetics, and the patient population and the type of instrumentation used, specific functions will be addressed.  CHAPTER 9 Leukocyte Development, Kinetics, and Functions 119 GRANULOCYTES shows the presence of dispersed primary (azurophilic) gran- ules in the cytoplasm; the number of granules does not ex- Neutrophils ceed 20 per cell (Figure 9.3). Type III myeloblasts have Neutrophils are present in the peripheral blood in two forms a darker chromatin and a more purple cytoplasm, and according to whether the nucleus is segmented or still in a band they contain more than 20 granules that do not obscure the shape. Segmented neutrophils make up the vast majority of nucleus. Type III myeloblasts are rare in normal bone mar- circulating leukocytes. rows, but they can be seen in certain types of acute myeloid leukemias. Mufti and colleagues10 proposed combining type Neutrophil Development II and type III blasts into a single category of “granular blasts” Neutrophil development occurs in the bone marrow. Neutro- because of the difficulty in distinguishing type II blasts from phils share a common progenitor with monocytes and distinct type III blasts. from eosinophils and basophils, known as the granulocyte- Promyelocytes comprise 1% to 5% of the nucleated cells in monocyte progenitor (GMP).2 The major cytokine responsible the bone marrow. They are relatively larger than the myelo- for the stimulation of neutrophil production is granulocyte blast cells and measure 16 to 25 "m in diameter. The nucleus colony-stimulating factor, or G-CSF.3,4 is round to oval and is often eccentric. A paranuclear halo or There are three pools of developing neutrophils in the bone “hof ” is usually seen in normal promyelocytes but not in the marrow (Figure 9.2): the stem cell pool, the proliferation pool, malignant promyelocytes of acute promyelocytic leukemia and the maturation pool.5-8 The stem cell pool consists of HSCs (described in Chapter 31). The cytoplasm is evenly baso- that are capable of self-renewal and differentiation.9 The prolif- philic and full of primary (azurophilic) granules. These eration (mitotic) pool consists of cells that are dividing and granules are the first in a series of granules to be produced includes (listed in the order of maturation) common myeloid during neutrophil maturation (Box 9.1).11 The nucleus progenitors (CMPs), also known as colony-forming units– is similar to that described earlier for myeloblasts except granulocyte, erythrocyte, monocyte, and megakaryocyte (CFU- that chromatin clumping (heterochromatin) may be visible, GEMMs); granulocyte-monocyte progenitors (GMPs); myelo- especially around the edges of the nucleus. One to three blasts; promyelocytes; and myelocytes. The third marrow pool nucleoli can be seen but may be obscured by the granules is the maturation (storage) pool consisting of cells undergoing (Figure 9.4). nuclear maturation that form the marrow reserve and are Myelocytes make up 6% to 17% of the nucleated cells in the available for release: metamyelocytes, band neutrophils, and bone marrow and are the final stage in which cell division segmented neutrophils. (mitosis) occurs. During this stage, the production of primary HSCs, CMPs, and GMPs are not distinguishable with granules ceases and the cell begins to manufacture secondary the light microscope and Romanowsky staining and may (specific) neutrophil granules. This stage of neutrophil devel- resemble early type I myeloblasts or lymphoid cells. They can, opment is sometimes divided into early and late myelocytes. however, be identified through surface antigen detection by Early myelocytes may look very similar to the promyelocytes flow cytometry. (described earlier) in size and nuclear characteristics except Myeloblasts make up 0% to 3% of the nucleated cells in the that patches of grainy pale pink cytoplasm representing bone marrow and measure 14 to 20 "m in diameter. They are secondary granules begin to be evident in the area of the often subdivided into type I, type II, and type III myeloblasts. Golgi apparatus. This has been referred to as the dawn of The type I myeloblast has a high nucleus-to-cytoplasm (N:C) neutrophilia. Secondary neutrophilic granules slowly spread ratio of 8:1 to 4:1 (the nucleus occupies most of the cell, with through the cell until its cytoplasm is more lavender-pink very little cytoplasm), slightly basophilic cytoplasm, fine than blue. As the cell divides, the number of primary granules nuclear chromatin, and two to four visible nucleoli. Type I per cell is decreased and their membrane chemistry changes so blasts have no visible granules when observed under light that they are much less visible. Late myelocytes are somewhat microscopy with Romanowsky stains. The type II myeloblast smaller than promyelocytes (15 to 18 "m), and the nucleus Stem cell pool Proliferation (mitotic) pool Maturation (storage) pool GM-CSF IL-3 Hematopoietic GM-CSF stem cell IL-3 G-CSF Myelocyte Band CMP GMP Myeloblast Promyelocyte Metamyelocyte Neutrophil Figure 9.2 Neutrophil Development Showing Stimulating Cytokines and the Three Bone Marrow Pools. 120 PART 2 Blood Cell Production, Structure, and Function BOX 9.1 Neutrophil Granules Primary (Azurophilic) Granules Formed during the promyelocyte stage Last to be released (exocytosis) Contain: Myeloperoxidase Acid #-glycerophosphatase Cathepsins Defensins Elastase Proteinase-3 Others A Secondary (Specific) Granules Formed during myelocyte and metamyelocyte stages Third to be released Contain: #2-Microglobulin Collagenase Gelatinase Lactoferrin Neutrophil gelatinase-associated lipocalin Transcobalamin I Others Tertiary Granules Formed during metamyelocyte and band stages Second to be released Contain: Gelatinase B Collagenase Lysozyme Acetyltransferase #2-Microglobulin Secretory Granules (Secretory Vesicles) Formed during band and segmented neutrophil stages First to be released (fuse to plasma membrane) Contain (attached to membrane): CD11b/CD18 Alkaline phosphatase Vesicle-associated membrane-2 CD10, CD13, CD14, CD16 Cytochrome b558 Complement 1q receptor Complement receptor-1 C Figure 9.3 Myeloblasts. (A), Type I myeloblast (arrow). Note that no peanut shaped), and the chromatin is increasingly clumped. granules are visible in the cytoplasm. (B), Type II myeloblast (arrow) Nucleoli are absent. Synthesis of tertiary granules (also known with a few azure granules in the cytoplasm. (A, B, Bone marrow, as gelatinase granules) may begin during this stage. The size Wright-Giemsa stain, !1000.) (C), Electron micrograph of a myeloblast of the metamyelocyte is slightly smaller than that of the myelo- (!16,500). (C from Rodak, B. F., & Carr, J. H.. Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.) cyte (14 to 16 "m). The cytoplasm contains very little residual ribonucleic acid (RNA) and therefore little or no basophilia has considerably more heterochromatin. Nucleoli are difficult (Figure 9.6). to see by light microscopy (Figure 9.5). Bands make up 9% to 32% of nucleated marrow cells and Metamyelocytes constitute 3% to 20% of nucleated marrow 0% to 5% of the nucleated peripheral blood cells. All evidence cells. From this stage forward, the cells are no longer capable of RNA (cytoplasmic basophilia) is absent, and tertiary of division and the major morphologic change is in the shape granules continue to be formed during this stage. Secretory of the nucleus. The nucleus is indented (kidney bean shaped or granules (also known as secretory vesicles) may begin to be  CHAPTER 9 Leukocyte Development, Kinetics, and Functions 121 A A B B Figure 9.4 Promyelocytes. (A), Promyelocyte (arrow) with nucleoli and a large number of azure granules. (Bone marrow, Wright-Giemsa stain, !1000.) (B), Electron micrograph of a promyelocyte (!13,000). (B from Rodak, B. F., & Carr, J. H.. Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.) formed during this stage. The nucleus is highly clumped, and the nuclear indentation that began in the metamyelocyte stage now exceeds one half the diameter of the nucleus, but actual segmentation has not yet occurred (Figure 9.7). Over the past 70 years, there has been considerable controversy over the defi- nition of a band and the differentiation between bands and C segmented forms. There have been three schools of thought con- Figure 9.5 Myelocytes. (A), Two early neutrophil myelocytes. Note cerning identification of bands, from the most conservative— that they are very similar to the promyelocyte except for several light holding that the nucleus in a band must have the same diameter areas in their cytoplasm where specific granules are beginning to appear. (B), Arrows are pointing to three late myelocytes in the field. throughout its length—to the most liberal—requiring that a fila- Their cytoplasm has few if any primary granules, and the lavender sec- ment between segments be visible before a band becomes a ondary granules are easily seen. (A, B, Bone marrow, Wright-Giemsa segmented neutrophil. The middle ground states that when stain, !1000.) (C), Electron micrograph of a late neutrophil myelocyte doubt exists, the cell should be called a segmented neutrophil. (!16,500). (C from Rodak, B. F., & Carr, J. H.. Clinical Hematology An elevated band count was thought to be useful in the Atlas. [5th ed.]. St. Louis: Elsevier.) diagnosis of patients with infection. However, the clinical util- ity of band counts has been called into question,12 and most laboratories no longer perform routine band counts. The Segmented neutrophils make up 7% to 30% of nucleated cells Clinical and Laboratory Standards Institute (CLSI) recom- in the bone marrow. Secretory granules continue to be formed mends that bands should be included within the neutrophil during this stage. The only morphologic difference between count and not reported as a separate category because of the segmented neutrophils and bands is the presence of between difficulty in reliably distinguishing bands from segmented two and five nuclear lobes connected by thread-like filaments neutrophils.13 (Figure 9.8). Segmented neutrophils are present in the highest 122 PART 2 Blood Cell Production, Structure, and Function A A B B Figure 9.7 Neutrophil Bands. (A), Neutrophil band; note the nucleus Figure 9.6 Metamyelocytes. (A), Two neutrophil metamyelocytes is indented more than 50% of the width of the nucleus. (Peripheral (arrows). Note that there is no remaining basophilia in the cytoplasm, blood, Wright-Giemsa stain, !1000.) (B), Electron micrograph of a and the nucleus is indented. (Bone marrow, Wright-Giemsa stain, band neutrophil (!22,250). (B from Rodak, B. F., & Carr, J. H.. !1000.) (B), Electron micrograph of a neutrophil metamyelocyte Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.) (!22,250). (B from Rodak, B. F., & Carr, J. H.. Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.) Once in the peripheral blood, neutrophils are divided randomly into a circulating neutrophil pool (CNP) and a mar- numbers in the peripheral blood of adults (50% to 70% of ginated neutrophil pool (MNP). The neutrophils in the MNP leukocytes in relative numbers and 2.3 to 8.1 ! 109/L in abso- are loosely localized to the walls of capillaries in tissues such as lute terms). As can be seen from the table on the inside front the liver, spleen, and lung. There does not appear to be any cover, pediatric values are quite different; relative percentages functional differences between neutrophils of either the CNP or can be as low as 18% of leukocytes in the first few months of life the MNP, and cells move freely between the two peripheral and do not begin to climb to adult values until after 4 to 7 years pools.16 The ratio of these two pools is roughly equal overall7,17; of age. however, marginated neutrophils in the capillaries of the lungs make up a considerably larger portion of peripheral neutro- Neutrophil Kinetics phils.18 The half-life of neutrophils in the blood is relatively Neutrophil kinetics involves the movement of neutrophils and short at approximately 7 hours.7,19 neutrophil precursors between the different pools in the bone Integrins and selectins are of significant importance in al- marrow, the peripheral blood, and tissues. Neutrophil produc- lowing neutrophils to marginate as well as exit the blood and tion has been calculated to be on the order of between 0.9 and enter the tissues by a process known as diapedesis.20,21 Those 1.0 ! 109 cells/kg per day.14 neutrophils that do not migrate into the tissues eventually The proliferative pool contains approximately 2.1 ! 109 undergo programmed cell death or apoptosis and are removed cells/kg, whereas the maturation pool contains roughly 5.6 ! by macrophages in the spleen, bone marrow, and liver.22 109 cells/kg, or a 5-day supply.14 The transit time from the HSC Once neutrophils are in the tissues, their life span is variable, to the myeloblast has not been measured. The transit time from depending on whether or not they are responding to infectious myeloblast through myelocyte has been estimated to be roughly or inflammatory agents. In the absence of infectious or inflam- 6 days, and the transit time through the maturation pool is ap- matory agents, the neutrophil’s life span is measured in hours. proximately 4 to 6 days.7,14,15 Granulocyte release from the bone Spontaneous neutrophil apoptosis is regulated by pro- and marrow is stimulated by G-CSF.3,4 antiapoptotic members of the Bcl-2 family. Some products of  CHAPTER 9 Leukocyte Development, Kinetics, and Functions 123 nonstimulated marginated neutrophils. Rolling consists of transient adhesive contacts between neutrophil selectins and adhesive molecules on the surface of endothelial cells (P selec- tins and E selectins). Activation is facilitated by the rolling of neutrophils on endothelium surfaces by chemokines. When integrins bind to ligands on the neutrophil surface, an out- side-in signaling activates signaling pathways, stabilizes adhe- sion, and initiates cell motility.20,25 Once adhesion occurs, the neutrophil scans the region while tightly attached and does not always transmigrate at the location of adhesion.26 Active crawling depends on signaling between intercellular adhesion molecule-1 (ICAM-1) (expressed by endothelial cells) and A macrophage-1 antigen (MAC-1, expressed by neutrophils).27 Neutrophils then transmigrate in a directional manner either between endothelial cells (paracellular) or through endothelial cells (transcellular) toward the area of greatest concentration of chemotactic agents.24 Once at the site of infection or inflammation, neutrophils begin the process of phagocytosis (Box 9.2). They use their enormous inventory of surface receptors either to directly recognize the pathogen, apoptotic cell, or particle or to recog- nize opsonic molecules attached to the foreign particle such as antibodies or complement components. Surface cell receptors are primed when neutrophils are exposed to lipopolysaccha- ride, tumor necrosis factor-$ or granulocyte-macrophage col- B ony-stimulating factor (GM-CSF), which are recognized by Figure 9.8 Neutrophils. (A), Segmented neutrophils, also known as a Toll-like receptors.28 With recognition comes attachment and polymorphonuclear cell or PMN. (Peripheral blood, Wright-Giemsa stain, engulfment, in which cytoplasmic pseudopodia surround the !1000.) (B), Electron micrograph of a segmented neutrophil (!22,250). (B from Rodak, B. F., & Carr, J. H.. Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.) BOX 9.2 Phagocytosis Recognition and Attachment inflammation and infection such as Mcl-1 and myeloperoxidase Phagocyte receptors recognize and bind to certain foreign molecular patterns (MPO) tend to prolong the neutrophil’s life span through anti- and opsonins such as antibodies and complement components. apoptotic signals, whereas others such as MAC-1 trigger the death and phagocytosis of neutrophils.23 Ingestion Pseudopodia are extended around the foreign particle and enclose it within Neutrophil Functions a “phagosome” (engulfment). Neutrophils are part of the innate immune system. Character- The phagosome is pulled toward the center of the cell by polymerization istics of innate immunity include destruction of foreign organ- of actin and myosin and by microtubules. isms that is not antigen specific; no protection against reexpo- Killing and Digestion sure to the same pathogen; reliance on the barriers provided by Oxygen Dependent skin and mucous membranes, as well as phagocytes such as Respiratory burst through the activation of NADPH oxidase. H2O2 and neutrophils and monocytes; and inclusion of a humoral com- hypochlorite are produced. ponent known as the complement system. The major function of neutrophils is phagocytosis and Oxygen Independent destruction of foreign material and microorganisms. The pro- The pH within the phagosome becomes alkaline and then neutral, the pH at which digestive enzymes work. cess involves seeking (chemotaxis, motility, and diapedesis) and Primary and secondary lysosomes (granules) fuse to the phagosome destruction (phagocytosis and digestion). and empty hydrolytic enzymes and other bactericidal molecules into the Neutrophil extravasation involves rolling, adhesion, crawl- phagosome. ing, and finally transmigration.24 Neutrophil recruitment to an inflammatory site begins when chemotactic agents bind to Formation of Neutrophil Extracellular Traps neutrophil receptors. Chemotactic agents may be produced by Nuclear and organelle membranes dissolve, and activated cytoplasmic microorganisms, by damaged cells, or by other leukocytes enzymes attach to DNA. such as lymphocytes or other phagocytes. The first neutrophil The cytoplasmic membrane ruptures, and DNA with attached enzymes is response is to roll along endothelial cells of the blood expelled so that the bacteria are digested in the external environment. vessels using stronger adhesive molecules than those used by NADPH, Nicotinamide adenine dinucleotide phosphate (reduced form). 124 PART 2 Blood Cell Production, Structure, and Function particle, forming a phagosome within the neutrophil cyto- Eosinophil myelocytes are characterized by the presence of plasm.23 Formation of the phagosome allows the reduced nico- large (resolvable at the light microscope level), pale, reddish- tinamide adenine dinucleotide (NADH) oxidase complex, orange secondary granules, along with azure granules in blue NOX2, within the phagosome membrane to assemble; this cytoplasm. The nucleus is similar to that described for neutro- leads to the generation of reactive oxygen species such as hydro- phil myelocytes. Transmission electron micrographs of eosino- gen peroxide, which is converted to hypochlorite by MPO.29 phils reveal that many secondary eosinophil granules contain Likewise, a series of metabolic changes culminate in the fusion an electron-dense crystalline core (Figure 9.9).37 of primary (e.g., MPO, defensins) and/or secondary (e.g., Eosinophil metamyelocytes and bands resemble their neu- phagocytic receptors, nicotinamide adenine dinucleotide trophil counterparts with respect to their nuclear shape. Sec- phosphate [reduced form, NADPH] oxidase) granules to the ondary granules increase in number, and a third type of phagosome and the release of numerous bactericidal molecules granule is generated called the secretory granule or secretory into the phagosome.30 This combination of reactive oxygen vesicle. The secondary granules become more distinct and re- species and nonoxygen-dependent mechanisms is generally fractory. Electron microscopy indicates the presence of two able to destroy most pathogens. other organelles: lipid bodies and small granules (Box 9.3).38 In addition to emptying their contents into phagosomes, Mature eosinophils usually display a bilobed nucleus. Their tertiary granules degrade the extracellular matrix and act cytoplasm contains characteristic refractile, orange-red second- as chemotactic agents for extravasation and migration of ary granules (Figure 9.10). Electron microscopy of mature eo- additional neutrophils to the site of inflammation.11,24 sinophils reveals extensive secretory vesicles, and their number A second function of neutrophils is the generation of neu- increases considerably when the eosinophil is stimulated or trophil extracellular traps, or NETs.31,32 NETs are extracellular activated.37 thread-like structures believed to represent chains of nucleo- somes from unfolded nuclear chromatin material (DNA). Eosinophil Kinetics These structures have enzymes from neutrophil granules at- The time from the last myelocyte mitotic division to the emer- tached to them and have been shown to be able to trap and kill gence of mature eosinophils from the marrow is about 3.5 days. gram-positive and gram-negative bacteria as well as fungi. The mean turnover of eosinophils is approximately 2.2 ! 108 NETs are generated at the time that neutrophils die as a result cells/kg per day. There is a large storage pool of eosinophils in of antibacterial activity. The term NETosis has been used to the marrow consisting of between 9 and 14 ! 108 cells/kg.38 describe this unique form of neutrophil cell death that results in the release of NETs. A third and final function of neutrophils is their secretory function. Neutrophils are a source of transcobalamin I or R binder protein, which is necessary for the proper absorption of vitamin B12. In addition, they are a source of a variety of cytokines. Eosinophils Eosinophils make up 1% to 3% of nucleated cells in the bone marrow. Of these, slightly more than a third are mature, a quar- ter are eosinophilic metamyelocytes, and the remainder are eosinophilic promyelocytes or eosinophilic myelocytes. Eosino- phils account for 1% to 3% of peripheral blood leukocytes, with A an absolute number of up to 0.4 ! 109/L in the peripheral blood. Eosinophil Development Eosinophil development is similar to that described earlier for neutrophils, and evidence indicates that eosinophils arise from the CMP.33,34 Eosinophil lineage is established through the interaction between the cytokines interleukin-3 (IL-3), IL-5 (induced by IL-33), and GM-CSF and three transcription factors (GATA-1 (hematopoietic transcription factor), PU.1, B and c/EBP). IL-5 and IL-33 are critical for eosinophil growth and survival.35,36 Eosinophilic promyelocytes can be identified Figure 9.9 Immature Eosinophils. (A), Eosinophil myelocyte. Note the rounded nucleus and the cytoplasm in which there are numerous large, cytochemically because of the presence of Charcot-Leyden pale eosinophil granules. (Bone marrow, Wright-Giemsa stain, !1000.) crystal protein in their primary granules. The first maturation (B), Electron micrograph of eosinophil granules showing the central phase that can be identified as eosinophilic using light micros- crystalline core in some of the granules. (From Rodak, B. F., & Carr, J. H. copy and Romanowsky staining is the early myelocyte.. Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier).  CHAPTER 9 Leukocyte Development, Kinetics, and Functions 125 BOX 9.3 Eosinophil Granules Once in the circulation, eosinophils have a circulating half- life of roughly 18 hours39; however, the half-life of eosinophils Primary Granules is prolonged when eosinophilia occurs. The tissue destinations Formed during promyelocyte stage of eosinophils under normal circumstances appear to be under- Contain: Charcot-Leyden crystal protein lying columnar epithelial surfaces in the respiratory, gastroin- testinal, and genitourinary tracts. Survival time of eosinophils Secondary Granules in human tissues ranges from 2 to 5 days.40 Formed throughout remaining maturation Contain: Eosinophil Functions Major basic protein (core) Eosinophils have multiple functions. Eosinophil granules are Eosinophil cationic protein (matrix) full of a large number of previously synthesized proteins, in- Eosinophil-derived neurotoxin (matrix) cluding cytokines, chemokines, growth factors, and cationic Eosinophil peroxidase (matrix) proteins. There is more than one way for eosinophils to de- Lysozyme (matrix) Catalase (core and matrix) granulate in an inflammatory process. By classical exocytosis, #-Glucuronidase (core and matrix) granules move to the plasma membrane, fuse with the plasma Cathepsin D (core and matrix) membrane, and empty their contents into the extracellular Interleukin-2, -4, and -5 (core) space. Compound exocytosis is a second mechanism in which Interleukin-6 (matrix) granules fuse together within the eosinophil before fusing with Granulocyte-macrophage colony-stimulating factor (core) the plasma membrane. A third method is known as piecemeal Others degranulation, in which secretory vesicles remove specific pro- teins from the secondary granules. These vesicles then migrate Small Lysosomal Granules to the plasma membrane and fuse to empty the specific proteins Acid phosphatase Arylsulfatase B into the extracellular space.37 A fourth method of degranula- Catalase tion is cytolysis that occurs when extracellular intact granules Cytochrome b558 are deposited during cell lysis.41 Elastase Eosinophils play important roles in immune regulation. Eosinophil cationic protein They transmigrate into the thymus of the newborn and are believed to be involved in the deletion of double-positive Lipid Bodies thymocytes.42 Eosinophils are capable of acting as antigen- Cyclooxygenase presenting cells and promoting the proliferation of effector 5-Lipoxygenase T cells.43 They are also implicated in the initiation of either 15-Lipoxygenase Leukotriene C4 synthase type 1 or type 2 immune responses because of their ability Eosinophil peroxidase to rapidly secrete preformed cytokines in a stimulus-specific Esterase manner. They are also important factors in acute and chronic allograft rejection.44 Eosinophils regulate mast cell function Storage Vesicles through the release of major basic protein (MBP), which causes Carry proteins from secondary granules to be released into the extracellular mast cell degranulation as well as cytokine production, and medium they also produce nerve growth factor that promotes mast cell survival and activation. Eosinophil production is increased in infection by parasitic helminths, and in vitro studies have found that the eosinophil is capable of destroying tissue-invading helminths through the secretion of MBP and eosinophil cationic protein as well as the production of reactive oxygen species.43 There is also a sugges- tion that eosinophils play a role in preventing reinfection.45 Finally, eosinophilia is a hallmark of allergic disorders, of which asthma has been the best studied. The number of eosino- phils in blood and sputum correlates with disease severity. This has led to the suggestion that the eosinophil is one of the causes of airway inflammation and mucosal cell damage through se- cretion or production of a combination of basic proteins, lipid mediators, reactive oxygen species, and cytokines such as IL-5.43 Eosinophils have also been implicated in airway remodeling Figure 9.10 Mature Eosinophil. Note that the nucleus has only two (increase in thickness of the airway wall) through eosinophil- segments, which is usual for these cells. The background cytoplasm is colorless and filled with eosinophil secondary granules. (Peripheral derived fibrogenic growth factors, especially in steroid-resistant blood, Wright-Giemsa stain, !1000.) (From Rodak, B. F., & Carr, J. H. asthma.46-48 Treatment with an anti–IL-5 monoclonal antibody. Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.) has been found to reduce exacerbations in certain asthmatic 126 PART 2 Blood Cell Production, Structure, and Function patients.49 Eosinophil accumulation in the gastrointestinal tract clumped. Actual nuclear segmentation with visible filaments occurs in allergic disorders such as food allergy, allergic colitis, occurs rarely. The cytoplasm is colorless and contains large and inflammatory bowel disease such as Crohn’s disease and numbers of the characteristic large blue-black granules. If any ulcerative colitis.50,51 granules have been dissolved during the staining process, they often leave a reddish-purple rim surrounding what appears to Basophils be a vacuole (Figure 9.12). Basophils and mast cells are two cells with morphologic and functional similarities; however, basophils are true leukocytes Basophil Kinetics because they mature in the bone marrow and circulate in the Basophil kinetics is poorly understood because of their very blood as mature cells with granules, whereas mast cell precur- small numbers.56 This life span of basophils is relatively longer sors leave the bone marrow and use the blood as a transit sys- than that of the other granulocytes, 60 hours.56 This has been tem to gain access to the tissues where they mature. Basophils attributed to the fact that when they are activated by IL-3 and are discussed first. Basophils are the least numerous of the IL-25, antiapoptotic pathways are initiated that prolong the WBCs, making up between 0% and 2% of circulating leuko- basophil life span.57 cytes and less than 1% of nucleated cells in the bone marrow. Basophil Functions Basophil Development Basophil functions are also poorly understood because of the Basophils are derived from progenitors in the bone marrow and small numbers of these cells and the lack of animal models such spleen, where they differentiate under the influence of a num- as basophil-deficient animals. However, the recent development ber of cytokines, including IL-3 and TSLP (thymic stromal of a conditional basophil-deficient mouse model promises to lymphopoietin).52 Two basophil populations are identified: enhance the understanding of basophil function.58 Previously, IL-3 elicited basophils that are immunoglobulin E (IgE) depen- basophils were regarded as “poor relatives” of mast cells and dent and non-IgE dependent TSLP elicited basophils.53-55 The minor players in allergic inflammation because, like mast cells, type of mediator response is determined by the balance be- they have IgE receptors on their surface membranes that, when tween these two populations.54 Because of their very small cross-linked by antigen, result in granule release.59 Basophils numbers, the stages of basophil maturation are very difficult to functions in both innate and adaptive immunity. Basophils are observe and have not been well characterized. Basophils will capable of releasing large quantities of subtype 2 helper T cell therefore be described simply as immature basophils and ma- ture basophils. Immature basophils have round to somewhat lobulated nu- clei with only slightly condensed chromatin. Nucleoli may or may not be apparent. The cytoplasm is blue and contains large blue-black secondary granules (Figure 9.11). Primary azure granules may or may not be seen. Basophil granules are water soluble and therefore may be dissolved if the blood film is washed too much during the staining process. Mature basophils contain a lobulated nucleus that is often obscured by its granules. The chromatin pattern, if visible, is A B Figure 9.12 Basophils. (A), Mature basophil. Note that granules tend to obscure the nucleus and the background cytoplasm is only slightly Figure 9.11 Immature Basophil (arrow). Note that the background cyto- basophilic. (Peripheral blood, Wright-Giemsa stain, !1000.) (B), Electron plasm is deeply basophilic with few large basophilic granules and there micrograph of a basophil (!28,750). (B from Rodak, B. F., & Carr, J. H. appears to be a nucleolus. (Bone marrow, Wright-Giemsa stain, !1000.). Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.)  CHAPTER 9 Leukocyte Development, Kinetics, and Functions 127 (TH2) cytokines such as IL-4 and IL-13 that regulate the TH2 destination, complete maturation into mature mast cells occurs immune response.60,61 Basophils also induce B cells to synthe- under the control of the local microenvironment (Figure 9.13).72 size IgE.62 Whereas mast cells are the effectors of IgE-mediated Mast cells function as effector cells in allergic reactions chronic allergic inflammation, basophils function as initiators of through the release of a wide variety of lipid mediators, prote- the allergic inflammation through the release of preformed ases, proteoglycans, and cytokines as a result of cross-linking of cytokines.59 Basophil activation is not restricted to antigen- IgE on the mast cell surface by specific allergens. Mast cells can specific IgE cross-linking, but it can be triggered in nonsensitized also be activated independently of IgE, which leads to inflam- individuals by a growing list of parasitic antigens, lectins, viral matory reactions. They can function as antigen-presenting cells superantigens, cytokines, chemokines growth factors, proteases, to induce the differentiation of TH2 cells73; therefore mast cells and components of the complement system.62 act as mediators in both innate and adaptive immunity.71 Mast The contents of basophil granules are not well known. cells can have antiinflammatory and immunosuppressive func- Box 9.4 provides a short list of some of the substances released tions, and thus they can both enhance and suppress features of by activated basophils. Moreover, mature basophils are the immune response.74 Finally they may act as immunologic evidently capable of synthesizing granule proteins based on “gatekeepers” because of their location in mucosal surfaces and activation signals. For example, basophils can be induced to their role in barrier function.71 produce a mediator of allergic inflammation known as granzyme B.63 Mast cells can induce basophils to produce and MONONUCLEAR CELLS release retinoic acid, a regulator of immune and resident cells in allergic diseases.64 Basophils also play a role in angiogenesis Monocytes through the expression of vascular endothelial growth factor Monocytes make up between 2% and 11% of circulating leuko- (VEGF) and its receptors.65 cytes, with an absolute number of up to 1.3 ! 109/L. Along with eosinophils, basophils are involved in the control of helminth infections by enclosing toxic egg products with Monocyte Development granulomas and preventing tissue damage.66 They promote Monocyte development is similar to neutrophil development eosinophilia, are associated with hindering migration of larvae because both cell types are derived from the GMP (Figure. 9.1). to other organs, and contribute to efficient worm expulsion.67,68 Macrophage colony-stimulating factor (M-CSF) is the major Finally, data from the basophil-deficient mouse model indicate cytokine responsible for the growth and differentiation of that basophils play a nonredundant role in mediating acquired monocytes. The morphologic stages of monocyte development immunity against ticks.58 are monoblasts, promonocytes, and monocytes. Monoblasts in normal bone marrow are very rare and are difficult to distin- Mast Cells guish from myeloblasts based on morphology. Malignant Mast cells are not considered to be leukocytes. They are tissue monoblasts in acute monoblastic leukemia are described in effector cells of allergic responses and inflammatory reactions. A brief description of their development and function is included here because (1) their precursors circulate in the peripheral blood for a brief period on their way to their tissue destinations,69 and (2) mast cells have several phenotypic and functional similarities with both basophils and eosinophils.70 Mast cell progenitors (MCPs) originate from the bone marrow and spleen.69 The progenitors are then released to the blood before finally reaching tissues such as the intestine and lung, where they mediate their actions.69 The major cytokine responsible for mast cell maturation and differentiation is KIT ligand (stem cell factor).71 Once the MCP reaches its tissue BOX 9.4 Basophil Granules Secondary Granules Histamine Platelet-activating factor Leukotriene C4 Interleukin-4 Interleukin-13 Figure 9.13 Tissue Mast Cell in Bone Marrow. Note that the nucleus Vascular endothelial growth factor A is rounded and the cell is packed with large basophilic granules. Mast Vascular endothelial growth factor B cells tend to be a little larger than basophils (12 to 25 "m). (Wright- Heparan sulfate Giemsa stain, !1000.) (From Rodak, B. F., & Carr, J. H.. Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.) 128 PART 2 Blood Cell Production, Structure, and Function A Figure 9.14 Promonocyte (arrow). Note that the nucleus is deeply indented and should not be confused with a neutrophil band form (compare the chromatin patterns of the two). The cytoplasm is baso- philic with azure granules that are much smaller than those seen in promyelocytes. The azure granules in this cell are difficult to see and give the cytoplasm a slightly grainy appearance. (Bone marrow, Wright- Giemsa stain, !1000.) Chapter 31. Therefore only promonocytes and monocytes are described here. Promonocytes are 12 to 18 "m in diameter, and their nucleus is slightly indented or folded. The chromatin pattern is delicate, B and at least one nucleolus is apparent. The cytoplasm is blue Figure 9.15 Monocytes. (A), Typical monocyte. Note the vacuolated and contains scattered azure granules that are fewer and smaller cytoplasm, a contorted nucleus that folds on itself, the loose or lace-like than those seen in promyelocytes (Figure 9.14). Electron micro- chromatin pattern, and very fine azure granules. (Peripheral blood, scopic and cytochemical studies have found that monocyte Wright-Giemsa stain, !1000.) (B), Electron micrograph of a monocyte. Note that the villi on the surface are much greater in number than is azure granules are heterogeneous with regard to their content seen on neutrophils (!16,500). (B from Rodak, B. F., & Carr, J. H.. of lysosomal enzymes, peroxidase, nonspecific esterases, and Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.) lysozyme.75 Monocytes appear to be larger than neutrophils (diameter of intermediate monocyte cells reduce inflammation to support 15 to 20 "m) because they tend to stick to and spread out on healing.78 The exact role of monocytes in health and disease glass or plastic. Monocytes are slightly immature cells whose needs further exploration. ultimate goal is to enter the tissues and mature into macro- phages, osteoclasts, or dendritic cells. Monocyte/Macrophage Kinetics The nucleus may be round, oval, or kidney shaped but more The promonocyte pool consists of approximately 6 ! 108 cells/kg, often is deeply indented (horseshoe shaped) or folded on itself. and they produce 7 ! 106 monocytes/kg per hour. Under normal The chromatin pattern is looser than in the other leukocytes circumstances, promonocytes undergo two mitotic divisions in and has sometimes been described as lace-like or stringy. Nu- 60 hours to produce a total of four monocytes. Under conditions cleoli are generally not seen with the light microscope; however, of increased demand for monocytes, promonocytes undergo electron microscopy reveals nucleoli in roughly half of circulat- four divisions to yield a total of 16 monocytes in 60 hours. There ing monocytes. Their cytoplasm is blue-gray, with fine azure is no storage pool of mature monocytes in the bone marrow,79 granules often referred to as azure dust or a ground-glass and unlike neutrophils, monocytes are released immediately appearance. Small cytoplasmic pseudopods or blebs may be into the circulation upon maturation. Therefore, when the bone seen. Cytoplasmic and nuclear vacuoles may also be present marrow recovers from marrow failure, monocytes are seen in (Figure 9.15). Based on flow cytometry immunophenotyping, the peripheral blood before neutrophils and a relative monocy- three subsets of human monocytes have been described: the tosis may occur. There is recent evidence, however, that a rela- classical, intermediate, and nonclassical monocytes.76 Studies tively large reservoir of immature monocytes resides in the have found that certain subsets of monocytes expand with in- subcapsular red pulp of the spleen. Monocytes in this splenic fections and inflammatory and autoimmune conditions.77 It is reservoir appear to respond to tissue injury such as myocardial not clear whether the expansion is a cause or consequence of infarction by migrating to the site of tissue injury to participate these conditions. For example, in atherosclerosis it has been in wound healing.80 found that classical monocytes advance inflammation and Like neutrophils, monocytes in the peripheral blood can sift through necrotic debris, whereas nonclassical and possibly be found in a marginal pool and a circulating pool. Unlike  CHAPTER 9 Leukocyte Development, Kinetics, and Functions 129 BOX 9.5 Monocyte Destinations Differentiation into Macrophages In areas of inflammation or infection (inflammatory macrophages) As “resident” macrophages in: Liver (Kupffer cells) Lungs (alveolar macrophages) Brain (microglia) Skin (Langerhans cells) Spleen (splenic macrophages) Intestines (intestinal macrophages) Peritoneum (peritoneal macrophages) Bone (osteoclasts) Synovial macrophages (type A cell) Kidneys (renal macrophages) A Reproductive organ macrophages Lymph nodes (dendritic cells) with neutrophils, the marginal pool of monocytes is 3.5 times the circulating pool.81 Monocytes remain in the circulation approximately 3 days.82 Monocytes with different patterns of chemokine receptors have different target tissues and differ- ent functions. Box 9.5 contains a list of the various tissue destinations of monocytes.83 Once in the tissues, monocytes differentiate into macrophages, osteoclasts (Figure 9.16), or dendritic cells, depending on the microenvironment of the local tissues. Macrophages can be as large as 40 to 50 "m in diameter. They usually have an oval nucleus with a net-like (reticulated) chromatin pattern. Their cytoplasm is pale, of- B ten vacuolated, and often filled with debris of phagocytized Figure 9.16 Bone Marrow Cells. (A), Active marrow macrophage cells or organisms. (arrow). (B), Osteoclast with six nuclei. Both these cells are derived The life span of macrophages in the tissues depends on from monocytes. (A, B, Wright-Giemsa stain, !1000.) whether they are responding to inflammation or infection, or they are “resident” macrophages such as Kupffer cells or alveo- lar macrophages. Resident macrophages survive far longer than initiate the adaptive immune response. Dendritic cells are tissue neutrophils. For example, Kupffer cells have a life span of the most efficient and potent of the antigen-presenting cells. approximately 21 days.84 Inflammatory macrophages, on the Housekeeping functions: These include removal of debris and other hand, have a life span measured in hours. dead cells at sites of infection or tissue damage, destruction of senescent red blood cells and maintenance of a storage Monocyte/Macrophage Functions pool of iron for erythropoiesis, and synthesis of a wide vari- Functions of monocytes/macrophages are numerous and var- ety of proteins, including coagulation factors, complement ied. They can be subdivided into innate immunity, adaptive components, interleukins, growth factors, and enzymes.85 immunity, and housekeeping functions. Innate immunity: Monocytes/macrophages recognize a wide Lymphocytes range of bacterial pathogens by means of pattern recogni- Lymphocytes are divided into three major groups: T cells, tion receptors (Toll-like receptors) that stimulate inflamma- B cells, and natural killer (NK) cells. T and B cells are major play- tory cytokine production and phagocytosis. Macrophages ers in adaptive immunity. NK cells make up a small percentage of can synthesize nitric oxide, which is cytotoxic against vi- lymphocytes and are part of innate immunity. Adaptive immu- ruses, bacteria, fungi, protozoa, helminths, and tumor cells.30 nity has three characteristics: It relies on an enormous number of Monocytes and macrophages also have Fc receptors and distinct lymphocytes, each having surface receptors for a differ- complement receptors. Hence, they can phagocytize foreign ent specific molecular structure on a foreign antigen; after an organisms or materials that have been coated with antibod- encounter with a particular antigen, memory cells are produced ies or complement components. that will react faster and more vigorously to that same antigen Adaptive immunity: Both macrophages and dendritic cells on reexposure; and self-antigens are “ignored” under normal degrade antigen and present antigen fragments on their sur- circumstances (referred to as tolerance). faces (antigen-presenting cells). Because of this, they interact Lymphocytes can be subdivided into two major categories: with and activate both T lymphocytes and B lymphocytes to Those that participate in humoral immunity by producing 130 PART 2 Blood Cell Production, Structure, and Function antibodies and those that participate in cellular immunity by at- tacking foreign organisms or cells directly. Antibody-producing lymphocytes are called B lymphocytes or simply B cells because they develop in the bone marrow. Cellular immunity is accom- plished by two types of lymphocytes: T cells, so named because they develop in the thymus, and NK cells, which develop in both the bone marrow and the thymus.86-88 Lymphocytes are different from the other leukocytes in several ways, including the following: 1. Lymphocytes are not end cells. They are resting cells, and when stimulated, they undergo mitosis to produce both memory and effector cells. 2. Unlike other leukocytes, lymphocytes recirculate from the blood to the tissues and back to the blood. 3. B and T lymphocytes are capable of rearranging antigen Figure 9.17 Immature B Lymphocyte or Hematogone (arrow). Note the extremely scanty cytoplasm. This was taken from the bone marrow receptor gene segments to produce a wide variety of antibod- of a newborn infant. (Wright-Giemsa stain, !1000.) ies and surface receptors. 4. Although early lymphocyte progenitors such as the com- mon lymphoid progenitor originate in the bone marrow, cells. Effector B cells are antibody-producing cells known as T and NK lymphocytes develop and mature outside the plasma cells and plasmacytoid lymphocytes (Figure 9.18). bone marrow. Approximately 3% to 21% of circulating lymphocytes For these reasons, lymphocyte kinetics is extremely compli- are B cells. Resting B lymphocytes cannot be distinguished cated, not well understood, and beyond the scope of this morphologically from resting T lymphocytes. Resting lym- chapter. phocytes are small (around 9 "m in diameter), and the N:C Lymphocytes make up between 18% and 42% of circulating ratio ranges from 5:1 to 2:1. The chromatin is arranged in leukocytes with an absolute number of 0.8 to 4.8 ! 109/L. Lymphocyte Development For both B and T cells, development can be subdivided into antigen-independent and antigen-dependent phases. Antigen- independent lymphocyte development occurs in the bone mar- row and thymus (sometimes referred to as central or primary lymphatic organs), whereas antigen-dependent lymphocyte development occurs in the spleen, lymph nodes, tonsils, and mucosa-associated lymphoid tissue such as the Peyer’s patches in the intestinal wall (sometimes referred to as peripheral or secondary lymphatic organs). B lymphocytes develop initially in the bone marrow and go through three stages known as pro-B, pre-B, and immature A B cells. It is during these stages that immunoglobulin gene rearrangement occurs so that each B cell produces a unique immunoglobulin antigen receptor. The immature B cells, which have not yet been exposed to antigen (antigen-naive B cells), leave the bone marrow to migrate to secondary lym- phatic organs, where they take up residence in specific zones such as lymph node follicles. These immature B cells, also known as hematogones,89 have a homogeneous nuclear chro- matin pattern and extremely scanty cytoplasm (Figure 9.17). These cells are normally found in newborn peripheral blood and bone marrow and in regenerative bone marrows. Leuke- mic cells from patients with acute lymphoblastic leukemia (ALL) can sometimes resemble hematogones, but the leukemic cells can be distinguished from hematogones by flow cytome- B try immunophenotyping.90 Figure 9.18 Plasma Cell vs Plasmacytoid Lymphocyte. (A), Plasma It is in the secondary lymphatic organs or in the blood where cell. (B), Plasmacytoid lymphocyte. These are effector cells of the B cells may come in contact with antigen, which results in cell B lymphocyte lineage. (A, B, Peripheral blood, Wright-Giemsa stain, division and the production of memory cells as well as effector !1000.)  CHAPTER 9 Leukocyte Development, Kinetics, and Functions 131 C B A A Figure 9.20 Three Cells Representing Lymphocyte Activation. A small resting lymphocyte (A) is stimulated by antigen and begins to enlarge to form a medium to large lymphocyte (B). The nucleus reverts from a clumped to a delicate chromatin pattern with nucleoli (C). The cell is capable of dividing to form effector cells or memory cells. (A-C, Peripheral blood, Wright-Giemsa stain, !1000.) B Figure 9.19 Lymphocytes. (A), Small resting lymphocyte. (Peripheral blood, Wright-Giemsa stain, !1000.) (B), Electron micrograph of a small lymphocyte (!30,000). B from Rodak, B. F., & Carr, J. H.. Clinical Hematology Atlas. [5th ed.]. St. Louis: Elsevier.) blocks, and the nucleolus is rarely seen, although it is present (Figure 9.19). T lymphocytes develop initially in the thymus—a lymphoepi- thelial organ located in the upper mediastinum.91 Lymphoid progenitor cells migrate from the bone marrow to the thymic Figure 9.21 A Large Granular Lymphocyte That Could Be Either cortex, where, under the regulation of cytokines produced by a Cytotoxic T Lymphocyte or a Natural Killer Lymphocyte. (Periph- eral blood, Wright-Giemsa stain, !1000.) thymic epithelial cells, they progress through stages known as pro-T, pre-T, and immature T cells. During these phases they undergo antigen receptor gene rearrangement to produce T cell NK cells are a heterogeneous group of cells with respect to receptors that are unique to each T cell. T cells whose receptors their surface antigens. The majority are CD56%CD16%CD3& react with self-antigens are allowed to undergo apoptosis.92 In CD7% large granular lymphocytes (Figure 9.21).93 The mature addition, T cells are subdivided into two major categories, de- NK cell is relatively large compared with other resting lympho- pending on whether or not they have CD4 or CD8 antigen on cytes because of an increased amount of cytoplasm. Its cytoplasm their surfaces. Immature T cells then proceed to the thymic me- contains azurophilic granules that are peroxidase negative. Ap- dulla, where further apoptosis of self-reactive T cells occurs. The proximately 4% to 29% of circulating lymphocytes are NK cells. remaining immature T cells (or antigen-naive T cells) then leave the thymus and migrate to secondary lymphatic organs, where Lymphocyte Functions they take up residence in specific zones such as the paracortical Functions can be addressed according to the type of lympho- areas. T cells comprise 51% to 88% of circulating lymphocytes. cyte. B lymphocytes are essential for antibody production. In T cells in secondary lymphatic organs or in the circulating addition, they have a role in antigen presentation to T cells and blood eventually come in contact with antigens. This results in may be necessary for optimal CD4 activation. B cells also pro- cell activation and the production of either memory cells or duce cytokines that regulate a variety of T cell and antigen- effector T cells, or both (Figure 9.20). The transformation of presenting cell functions.94 resting lymphocytes into activated forms is the source of so- T lymphocytes can be divided into CD4% T cells and CD8% called medium and large lymphocytes that have increased T cells. CD4% effector lymphocytes are further subdivided into amounts of cytoplasm and usually make up only about 10% of TH1, TH2, TH17, and Treg (CD4%CD25% regulatory T) cells. TH1 circulating lymphocytes. The morphology of effector T cells cells mediate immune responses against intracellular pathogens. varies with the subtype of T cell involved, and they are often TH2 cells mediate host defense against extracellular parasites, referred to as reactive lymphocytes. including helminths. They are also important in the induction 132 PART 2 Blood Cell Production, Structure, and Function of asthma and other allergic diseases. TH17 cells are involved in activating apoptotic pathways in the target cell.97 These cells are the immune responses against extracellular bacteria and fungi. sometimes referred to as cytotoxic T lymphocytes. Treg cells play a role in maintaining self-tolerance by regulating NK lymphocytes function as part of innate immunity and are immune responses.95,96 capable of killing certain tumor cells and virus-infected cells CD8% effector lymphocytes are capable of killing target cells without prior sensitization. In addition, NK cells modulate the by secreting granules containing granzyme and perforin or by functions of other cells, including macrophages and T cells.98 SUMMARY Granulocytes are classified according to their staining char- Monocyte development can be subdivided into the pro- acteristics and the shape of their nuclei. Neutrophils are a monocyte, monocyte, and macrophage stages, each with major component of innate immunity as phagocytes; eo- specific morphologic characteristics. sinophils are involved in allergic reactions and helminth The majority of lymphocytes are involved in adaptive destruction; and basophils function as initiators of allergic immunity. B lymphocytes and plasma cells produce anti- reactions, helminth destruction, and immunity against ticks. bodies against foreign organisms or cells, and T lymphocytes Neutrophil development can be subdivided into specific mediate the immune response against intracellular and stages, with cells at each stage having specific morphologic extracellular invaders. Both B and T lymphocytes produce characteristics (myeloblast, promyelocyte, myelocyte, memory cells for specific antigens so that the immune metamyelocyte, band, and segmented neutrophil). Various response is faster if the same antigen is encountered again. granule types are produced during neutrophil development, Lymphocyte development is complex, and morphologic each with specific contents. divisions are not practical because a large number of lym- Eosinophil development can also be subdivided into specific phocytes develop in the thymus. Benign B-lymphocyte stages, although eosinophilic myeloblasts are not recogniz- precursors (hematogones) as well as B-lymphocyte effector able and eosinophil promyelocytes are rare. cells (plasma cells and plasmacytoid lymphocytes) have Basophil development is difficult to describe, and basophils been described. Natural killer (NK) lymphocytes and cyto- have been divided simply into immature and mature basophils. toxic T cells also have a distinct and similar morphology. Mononuclear cells consist of monocytes and lymphocytes. Monocytes are precursors to tissue cells such as osteoclasts, Now that you have completed this chapter, go back and read again macrophages, and dendritic cells. As a group, they perform the case study at the beginning and respond to the questions several functions as phagocytes. presented. REVIEW QUESTIONS Answers can be found in the Appendix. 5. Which of the following cells are important in immune regu- 1. Neutrophils and monocytes are direct descendants of a com- lation, allergic inflammation, and destruction of tissue in- mon progenitor known as: vading helminths? a. CLP a. Neutrophils and monocytes b. GMP b. Eosinophils and basophils c. MEP c. T and B lymphocytes d. HSC d. Macrophages and dendritic cells 2. The stage in neutrophilic development in which the nucleus 6. Basophils and mast cells have high-affinity surface receptors is indented in a kidney bean shape and the cytoplasm has for which immunoglobulin? secondary granules that are lavender in color is the: a. A a. Band b. D b. Myelocyte c. E c. Promyelocyte d. G d. Metamyelocyte 7. Which of the following cell types is capable of differentiating 3. Type II myeloblasts are characterized by: into osteoclasts, macrophages, or dendritic cells? a. The presence of fewer than 20 primary granules per cell a. Neutrophils b. Basophilic cytoplasm with many secondary granules b. Lymphocytes c. The absence of granules c. Monocytes d. The presence of a folded nucleus d. Eosinophils 4. Which one of the following is a function of neutrophils? 8. Macrophages aid in adaptive immunity by: a. Presentation of antigen to T and B lymphocytes a. Degrading antigen and presenting it to lymphocytes b. Protection against reexposure by same antigen b. Ingesting and digesting organisms that neutrophils cannot c. Nonspecific destruction of foreign organisms c. Synthesizing complement components d. Initiation of delayed hypersensitivity response d. Storing iron from senescent red cells  CHAPTER 9 Leukocyte Development, Kinetics, and Functions 133 9. Which of the following is the final stage of B cell maturation 10. The following is unique to both B and T lymphocytes and after activation by antigen? occurs during their early development: a. Large, granular lymphocyte a. Expression of surface antigens CD4 and CD8 b. Plasma cell b. Maturation in the thymus c. Reactive lymphocyte c. Synthesis of immunoglobulins d. Immunoblast d. 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