Woodlyne Roquiz, Sameer Al Diffalha, Ameet R. Kini*
After completion of this chapter, the reader will be able to:
1. Describe the pathways and progenitor cells involved in the derivation of leukocytes from the hematopoietic stem cell to mature forms.
2. Name the different stages of neutrophil, eosinophil, and basophil development and describe the morphology of each stage.
3. Discuss the important functions of neutrophils, eosinophils, and basophils.
4. Describe the morphology of promonocytes, monocytes, macrophages, T and B lymphocytes, and immature B cells (hematogones).
5. Discuss the functions of monocytes, macrophages, T cells, B cells, and natural killer cells in the immune response.
6. Compare the kinetics of neutrophils and monocytes.
7. Discuss in general terms how the various types of lymphocytes are produced.
After studying the material in this chapter, the reader should be able to respond to the following case study:
A 5-year-old girl presents with shortness of breath and wheezing. The patient gives a history of similar symptoms in the last 6 months. After the patient was given albuterol to control her acute symptoms, long-term control of her disease was achieved through the use of corticosteroids, along with monoclonal antibodies to IL-5.
1. Which leukocytes are important in mediating the clinical symptoms in this patient?
2. A complete blood count with differential was performed on this patient. What are the typical findings in such patients?
3. How did monoclonal antibodies to IL-5 help in controlling her disease?
Leukocytes (also known as white blood cells, or WBCs) are so named because they are relatively colorless compared to red blood cells. The number of different types of leukocytes varies depending on whether they are being viewed with a light microscope after staining with a Romanowsky stain (5 or 6 types) or are identified according to their surface antigens using flow cytometry (at least 10 different types). For the purposes of this chapter, the classic, light microscope classification of leukocytes will be used.
Granulocytes are a group of leukocytes whose cytoplasm is filled with granules with differing staining characteristics and whose nuclei are segmented or lobulated. Individually, they include eosinophils, with granules containing basic proteins that stain with acid stains such as eosin; basophils, with granules that are acidic and stain with basic stains such as methylene blue; and neutrophils, with granules that react with both acid and basic stains, which gives them a pink to lavender color. Because nuclear segmentation is quite prominent in mature neutrophils, they have also been called polymorphonuclear cells, or PMNs.
Mononuclear cells are categorized into monocytes and lymphocytes. These cells have nuclei that are not segmented but are round, oval, indented, or folded. Leukocytes develop from hematopoietic stem cells (HSCs) in the bone marrow, where most undergo differentiation and maturation (Figure 12-1), and then are released into the circulation. The number of circulating leukocytes varies with sex, age, activity, time of day, and ethnicity; it also differs according to whether or not the leukocytes are reacting to stress, being consumed, or being destroyed, and whether or not they are being produced by the bone marrow in sufficient numbers.1 Reference intervals for total leukocyte counts vary among laboratories, depending on the patient population and the type of instrumentation being used, but a typical reference interval is 4.5 × 109/L to 11.5 × 109/L for adults.
FIGURE 12-1 Diagram of hematopoiesis showing the derivation pathways of each type of blood cell from a hematopoietic stem cell.
The overall function of leukocytes is in mediating immunity, either innate (nonspecific), as in phagocytosis by neutrophils, or specific (adaptive), as in the production of antibodies by lymphocytes and plasma cells. The term kinetics refers to the movement of cells through developmental stages, into the circulation, and from the circulation to the tissues and includes the time spent in each phase of the cell’s life. As each cell type is discussed in this chapter, developmental stages, kinetics, and specific functions will be addressed.
Neutrophils are present in the peripheral blood in two forms according to whether the nucleus is segmented or still in a band shape. Segmented neutrophils make up the vast majority of circulating leukocytes.
Neutrophil development occurs in the bone marrow. Neutrophils share a common progenitor with monocytes, known as the granulocyte-monocyte progenitor (GMP). The major cytokine responsible for the stimulation of neutrophil production is granulocyte colony-stimulating factor, or G-CSF.2, 3
There are three pools of developing neutrophils in the bone marrow (): the stem cell pool, the proliferation pool, and the maturation pool.Figure 12-24-7 The stem cell pool consists of hematopoietic stem cells (HSCs) that are capable of self-renewal and differentiation.8 The proliferation (mitotic) pool consists of cells that are dividing and includes (listed in the order of maturation) common myeloid progenitors (CMPs), also known as colony-forming units–granulocyte, erythrocyte, monocyte, and megakaryocyte (CFU-GEMMs); granulocyte-macrophage progenitors (GMPs); myeloblasts; promyelocytes; and myelocytes. The third marrow pool is the maturation (storage) pool consisting of cells undergoing nuclear maturation that form the marrow reserve and are available for release: metamyelocytes, band neutrophils, and segmented neutrophils.
FIGURE 12-2 Neutrophil development showing stimulating cytokines and the three bone marrow pools.
HSCs, CMPs, and GMPs are not distinguishable with the light microscope and Romanowsky staining and may resemble early type I myeloblasts or lymphoid cells. They can, however, be identified through surface antigen detection by flow cytometry.
Myeloblasts make up 0% to 3% of the nucleated cells in the bone marrow and measure 14 to 20 μm in diameter. They are frequently subdivided into type I, type II, and type III myeloblasts. The type I myeloblast has a high nucleus-to-cytoplasm (N:C) ratio of 8:1 to 4:1 (the nucleus occupies most of the cell, with very little cytoplasm), slightly basophilic cytoplasm, fine nuclear chromatin, and two to four visible nucleoli. Type 1 blasts have no visible granules when observed under light microscopy with Romanowsky stains. The type II myeloblast shows the presence of dispersed primary (azurophilic) granules in the cytoplasm; the number of granules does not exceed 20 per cell (Figure 12-3). Type III myeloblasts have a darker chromatin and a more purple cytoplasm, and they contain more than 20 granules that do not obscure the nucleus. Type III myeloblasts are rare in normal bone marrows, but they can be seen in certain types of acute myeloid leukemias. Recently, Mufti and colleagues9 proposed combining type II and type III blasts into a single category of “granular blasts” due to the difficulty in distinguishing type II blasts from type III blasts.
FIGURE 12-3 A, Type I myeloblast (arrow). Note that no granules are visible in the cytoplasm. B, Type II myeloblast (arrow) with a few azure granules in the cytoplasm. C, Electron micrograph of a myeloblast. Source: (C from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.)
Promyelocytes comprise 1% to 5% of the nucleated cells in the bone marrow. They are relatively larger than the myeloblast cells and measure 16 to 25 μm in diameter. The nucleus is round to oval and is often eccentric. A paranuclear halo or “hof” is usually seen in normal promyelocytes but not in the malignant promyelocytes of acute promyelocytic leukemia (described in Chapter 35). The cytoplasm is evenly basophilic and full of primary (azurophilic) granules. These granules are the first in a series of granules to be produced during neutrophil maturation (Box 12-1).10 The nucleus is similar to that described earlier for myeloblasts except that chromatin clumping (heterochromatin) may be visible, especially around the edges of the nucleus. One to three nucleoli can be seen but may be obscured by the granules (Figure 12-4).
FIGURE 12-4 A, Promyelocyte. Note the large number of azure granules and the presence of nucleoli. B, Electron micrograph of a promyelocyte. Source: (B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.)
Primary (azurophilic) granules
Formed during the promyelocyte stage
Last to be released (exocytosis)
• Acid β-glycerophosphatase
Secondary (specific) granules
Formed during myelocyte and metamyelocyte stages
Third to be released
• Neutrophil gelatinase-associated lipocalin
• Transcobalamin I
Formed during metamyelocyte and band stages
Second to be released
Secretory granules (secretory vesicles)
Formed during band and segmented neutrophil stages
First to be released (fuse to plasma membrane)
Contain (attached to membrane):
• Alkaline phosphatase
• Vesicle-associated membrane-2
• CD10, CD13, CD14, CD16
• Cytochrome b558
• Complement 1q receptor
• Complement receptor-1
Neutrophil myelocytes make up 6% to 17% of the nucleated cells in the bone marrow and are the final stage in which cell division (mitosis) occurs. During this stage, the production of primary granules ceases, and the cell begins to manufacture secondary (specific) neutrophil granules. This stage of neutrophil development is sometimes divided into early and late myelocytes. Early myelocytes may look very similar to the promyelocytes (described earlier) in size and nuclear characteristics except that patches of grainy pale pink cytoplasm representing secondary granules begin to be evident in the area of the Golgi apparatus. This has been referred to as the dawn of neutrophilia. Secondary neutrophilic granules slowly spread through the cell until its cytoplasm is more lavender-pink than blue. As the cell divides, the number of primary granules per cell is decreased, and their membrane chemistry changes so that they are much less visible. Late myelocytes are somewhat smaller than promyelocytes (15 to 18 μm), and the nucleus has considerably more heterochromatin. Nucleoli are difficult to see by light microscopy (Figure 12-5).
FIGURE 12-5 A, Two early neutrophil myelocytes. Note that they are very similar to the promyelocyte except for several light areas in their cytoplasm where specific granules are beginning to appear. B, Arrows are pointing to three late myelocytes in the field. Their cytoplasm has few if any primary granules, and the lavender secondary granules are easily seen. C, Electron micrograph of a late neutrophil myelocyte. Source: (C from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.)
Neutrophil metamyelocytes constitute 3% to 20% of nucleated marrow cells. From this stage forward, the cells are no longer capable of division, and the major morphologic change is in the shape of the nucleus. The nucleus is indented (kidney bean shaped or peanut shaped), and the chromatin is increasingly clumped. Nucleoli are absent. Synthesis of tertiary granules (also known as gelatinase granules) may begin during this stage. The size of the metamyelocyte is slightly smaller than that of the myelocyte (14 to 16 μm). The cytoplasm contains very little residual ribonucleic acid (RNA) and therefore little or no basophilia (Figure 12-6).
FIGURE 12-6 A, Two neutrophil metamyelocytes (arrows). Note that there is no remaining basophilia in the cytoplasm, and the nucleus is indented. B, Electron micrograph of a neutrophil metamyelocyte. Source: (B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.)
Neutrophil bands make up 9% to 32% of nucleated marrow cells and 0% to 5% of the nucleated peripheral blood cells. All evidence of RNA (cytoplasmic basophilia) is absent, and tertiary granules continue to be formed during this stage. Secretory granules (also known as secretory vesicles) may begin to be 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 12-7). Over the past 70 years, there has been considerable controversy over the definition of a band and the differentiation between bands and segmented forms. There have been three schools of thought concerning identification of bands, from the most conservative—holding that the nucleus in a band must have the same diameter throughout its length—to the most liberal—requiring that a filament between segments be visible before a band becomes a segmented neutrophil. The middle ground states that when doubt exists, the cell should be called a segmented neutrophil. An elevated band count was thought to be useful in the diagnosis of patients with infection. However, the clinical utility of band counts has been called into question,11 and most laboratories no longer perform routine band counts. The Clinical and Laboratory Standards Institute (CLSI) recommends that bands should be included within the neutrophil counts and not reported as a separate category, due to the difficulty in reliably distinguishing bands from segmented neutrophils.12
FIGURE 12-7 A, Neutrophil band; note the nucleus is indented more than 50% of the width of the nucleus. B, Electron micrograph of a band neutrophil. Source: (B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.)
Segmented neutrophils make up 7% to 30% of nucleated cells in the bone marrow. Secretory granules continue to be formed during this stage. The only morphologic difference between segmented neutrophils and bands is the presence of between two and five nuclear lobes connected by threadlike filaments (Figure 12-8). Segmented neutrophils are present in the highest numbers in the peripheral blood of adults (50% to 70% of leukocytes in relative numbers and 2.3 to 8.1 × 109/L in absolute terms). As can be seen from the table on the inside front cover, pediatric values are quite different; relative percentages can be as low as 18% of leukocytes in the first few months of life and do not begin to climb to adult values until after 4 to 7 years of age.
FIGURE 12-8 A, Segmented neutrophil (also known as a polymorphonuclear cell or PMN). B, Electron micrograph of a segmented neutrophil. Source: (B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.)
Neutrophil kinetics involves the movement of neutrophils and neutrophil precursors between the different pools in the bone marrow, the peripheral blood, and tissues. Neutrophil production has been calculated to be on the order of between 0.9 and 1.0 × 109 cells/kg per day.13
The proliferative pool contains approximately 2.1 × 109 cells/kg, whereas the maturation pool contains roughly 5.6 × 109 cells/kg or a 5-day supply.13 The transit time from the HSC to the myeloblast has not been measured. The transit time from myeloblast through myelocyte has been estimated to be roughly 6 days, and the transit time through the maturation pool is approximately 4 to 6 days.4, 13, 14 Granulocyte release from the bone marrow is stimulated by G-CSF.2, 3
Once in the peripheral blood, neutrophils are divided randomly into a circulating neutrophil pool (CNP) and a marginated neutrophil pool (MNP). The neutrophils in the MNP are loosely localized to the walls of capillaries in tissues such as the liver, spleen, and lung. There does not appear to be any functional differences between neutrophils of either the CNP or the MNP, and cells move freely between the two peripheral pools.15 The ratio of these two pools is roughly equal overall;4, 16 however, marginated neutrophils in the capillaries of the lungs make up a considerably larger portion of peripheral neutrophils.17 The half-life of neutrophils in the blood is relatively short at approximately 7 hours.4, 18
Integrins and selectins are of significant importance in allowing neutrophils to marginate as well as exit the blood and enter the tissues by a process known as diapedesis.19, 20 Those neutrophils that do not migrate into the tissues eventually undergo programmed cell death or apoptosis and are removed by macrophages in the spleen.21
Once neutrophils are in the tissues, their life span is variable, depending on whether or not they are responding to infectious or inflammatory agents. In the absence of infectious or inflammatory agents, the neutrophil’s life span is measured in hours. Some products of inflammation and infection tend to prolong the neutrophil’s life span through anti-apoptotic signals, whereas others such as MAC-1 trigger the death and phagocytosis of neutrophils.20
Neutrophils are part of the innate immune system. Characteristics of innate immunity include destruction of foreign organisms that is not antigen specific; no protection against reexposure to the same pathogen; reliance on the barriers provided by skin and mucous membranes, as well as phagocytes such as neutrophils and monocytes; and inclusion of a humoral component known as the complement system.
The major function of neutrophils is phagocytosis and destruction of foreign material and microorganisms. The process involves seeking (chemotaxis, motility, and diapedesis) and destruction (phagocytosis and digestion).
Neutrophil recruitment to an inflammatory site begins when chemotactic agents bind to neutrophil receptors. Chemotactic agents may be produced by microorganisms, by damaged cells, or by other leukocytes such as lymphocytes or other phagocytes. The first neutrophil response is to roll along endothelial cells of the blood vessels using stronger adhesive molecules than those used by nonstimulated marginated neutrophils. Rolling consists of transient adhesive contacts between neutrophil selectins and adhesive molecules on the surface of endothelial cells. At the same time, secretory granules containing additional adhesive molecules are fused to the neutrophil’s plasma membrane. β2 integrins such as CD11b/CD18 from secretory granules contribute to tight stationary binding between neutrophils and endothelial cells. This is followed by diapedesis or transmigration of neutrophils either between or through endothelial cells—a process that is also mediated by integrins and integrin-associated proteins. Tertiary granules containing gelatinase and collagenase are released by transmigrating neutrophils. Gelatinase degrades denatured collagen as well as types IV and V collagen and activates chemokines such as interleukin-8 (IL-8).22Neutrophils then migrate in a directional manner toward the area of greatest concentration of chemotactic agents.
Once at the site of infection or inflammation, neutrophils begin the process of phagocytosis (). They utilize their enormous inventory of surface receptors either to directly recognize the pathogen, apoptotic cell, or particle, or to recognize opsonic molecules attached to the foreign particle such as antibodies or complement components. With recognition comes attachment and engulfment, in which cytoplasmic pseudopodia surround the particle, forming a phagosome within the neutrophil cytoplasm.Box 12-223 Formation of the phagosome allows the reduced nicotinamide adenine dinucleotide (NADH) oxidase complex within the phagosome membrane to assemble; this leads to the generation of reactive oxygen species such as hydrogen peroxide, which is converted to hypochlorite by myeloperoxidase. Likewise, a series of metabolic changes culminate in the fusion of primary and/or secondary granules to the phagosome and the release of numerous bactericidal molecules into the phagosome.24 This combination of reactive oxygen species and non-oxygen-dependent mechanisms is generally able to destroy most pathogens.
Recognition and attachment
Phagocyte receptors recognize and bind to certain foreign molecular patterns and opsonins such as antibodies and complement components.
Pseudopodia are extended around the foreign particle and enclose it within a “phagosome” (engulfment).
The phagosome is pulled toward the center of the cell by polymerization of actin and myosin and by microtubules.
Killing and digestion
Respiratory burst through the activation of NADPH oxidase. H2O2 and hypochlorite are produced.
The pH within the phagosome becomes alkaline and then neutral, the pH at which digestive enzymes work.
Primary and secondary lysosomes (granules) fuse to the phagosome and empty hydrolytic enzymes and other bactericidal molecules into the phagosome.
Formation of neutrophil extracellular traps
Nuclear and organelle membranes dissolve, and activated cytoplasmic enzymes attach to DNA.
The cytoplasmic membrane ruptures, and DNA with attached enzymes is expelled so that the bacteria are digested in the external environment.
NADPH, Nicotinamide adenine dinucleotide phosphate (reduced form).
In addition to emptying their contents into phagosomes, secondary and primary granules may fuse to the plasma membrane, which results in release of their contents into the extracellular matrix. These molecules can then act as chemotactic agents for additional neutrophils and as stimulating agents for macrophages to phagocytize dead neutrophils, as well as inflammatory agents that may cause tissue damage.
A second function of neutrophils is the generation of neutrophil extracellular traps, or NETs.25, 26 NETs are extracellular threadlike structures believed to represent chains of nucleosomes from unfolded nuclear chromatin material (DNA). These structures have enzymes from neutrophil granules attached to them and have been shown to be able to trap and kill gram-positive and gram-negative bacteria as well as fungi.NETs are generated at the time that neutrophils die as a result of antibacterial activity. The term NETosis has been used to 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 make up 1% to 3% of nucleated cells in the bone marrow. Of these, slightly more than a third are mature, a quarter are eosinophilic metamyelocytes, and the remainder are eosinophilic promyelocytes or eosinophilic myelocytes. Eosinophils account for 1% to 3% of peripheral blood leukocytes, with an absolute number of up to 0.4 × 109/L in the peripheral blood.
Eosinophil development is similar to that described earlier for neutrophils, and evidence indicates that eosinophils arise from the common myeloid progenitor (CMP).27, 28 Eosinophil lineage is established through the interaction between the cytokines IL-3, IL-5, and GM-CSF and three transcription factors (GATA-1, PU.1, and c/EBP). IL-5 is critical for eosinophil growth and survival.29 Whether or not there exist myeloblasts that are committed to the eosinophil line has not been established. Eosinophilic promyelocytes can be identified cytochemically due to the presence of Charcot-Leyden crystal protein in their primary granules. The first maturation phase that can be identified as eosinophilic using light microscopy and Romanowsky staining is the early myelocyte.
Eosinophil myelocytes are characterized by the presence of large (resolvable at the light microscope level), pale, reddish-orange secondary granules, along with azure granules in blue cytoplasm. The nucleus is similar to that described for neutrophil myelocytes. Transmission electron micrographs of eosinophils reveal that many secondary eosinophil granules contain an electron-dense crystalline core (Figure 12-9).30
FIGURE 12-9 A, Eosinophil myelocyte. Note the rounded nucleus and the cytoplasm in which there are numerous large, pale eosinophil granules. B, Electron micrograph of eosinophil granules showing the central crystalline core in some of the granules. Source: (A, B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.).
Eosinophil metamyelocytes and bands resemble their neutrophil counterparts with respect to their nuclear shape. Secondary granules increase in number, and a third type of granule is generated called thesecretory granule or secretory vesicle. The secondary granules become more distinct and refractory. Electron microscopy indicates the presence of two other organelles: lipid bodies and small granules (Box 12-3).31
Formed during promyelocyte stage
• Charcot-Leyden crystal protein
Formed throughout remaining maturation
• Major basic protein (core)
• Eosinophil cationic protein (matrix)
• Eosinophil-derived neurotoxin (matrix)
• Eosinophil peroxidase (matrix)
• Lysozyme (matrix)
• Catalase (core and matrix)
• β-Glucuronidase (core and matrix)
• Cathepsin D (core and matrix)
• Interleukins 2, 4, and 5 (core)
• Interleukin-6 (matrix)
• Granulocyte-macrophage colony-stimulating factor (core)
Small lysosomal granules
Eosinophil cationic protein
Leukotriene C4 synthase
Carry proteins from secondary granules to be released into the extracellular medium
Mature eosinophils usually display a bilobed nucleus. Their cytoplasm contains characteristic refractile, orange-red secondary granules (Figure 12-10). Electron microscopy of mature eosinophils reveals extensive secretory vesicles, and their number increases considerably when the eosinophil is stimulated or activated.30
FIGURE 12-10 Mature eosinophil. Note that the nucleus has only two segments, which is usual for these cells. The background cytoplasm is colorless and filled with eosinophil secondary granules. Source: (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)
The time from the last myelocyte mitotic division to the emergence of mature eosinophils from the marrow is about 3.5 days. The mean turnover of eosinophils is approximately 2.2 × 108 cells/kg per day. There is a large storage pool of eosinophils in the marrow consisting of between 9 and 14 × 108 cells/kg.31
Once in the circulation, eosinophils have a circulating half-life of roughly 18 hours;32 however, the half-life of eosinophils is prolonged when eosinophilia occurs. The tissue destinations of eosinophils under normal circumstances appear to be underlying columnar epithelial surfaces in the respiratory, gastrointestinal, and genitourinary tracts. Survival time of eosinophils in human tissues ranges from 2 to 5 days.33
Eosinophils have multiple functions. Eosinophil granules are full of a large number of previously synthesized proteins, including cytokines, chemokines, growth factors, and cationic proteins. There is more than one way for eosinophils to degranulate. By classical exocytosis, granules move to the plasma membrane, fuse with the plasma membrane, and empty their contents into the extracellular space. Compound exocytosis is a second mechanism in which granules fuse together within the eosinophil prior to fusing with the plasma membrane. A third method is known as piecemeal degranulation, in which secretory vesicles remove specific proteins from the secondary granules. These vesicles then migrate to the plasma membrane and fuse to empty the specific proteins into the extracellular space.30
Eosinophils play important roles in immune regulation. They transmigrate into the thymus of the newborn and are believed to be involved in the deletion of double-positive thymocytes.34 Eosinophils are capable of acting as antigen-presenting cells and promoting the proliferation of effector T cells.35 They are also implicated in the initiation of either type 1 or type 2 immune responses due to their ability to rapidly secrete preformed cytokines in a stimulus-specific manner.36 Eosinophils regulate mast cell function through the release of major basic protein (MBP) that causes mast cell degranulation as well as cytokine production, and 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 shown that the eosinophil is capable of destroying tissue-invading helminths through the secretion of major basic protein and eosinophil cationic protein as well as the production of reactive oxygen species.35 There is also a suggestion that eosinophils play a role in preventing reinfection.37
Finally, eosinophilia is a hallmark of allergic disorders, of which asthma has been the best studied. The number of eosinophils 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 secretion or production of a combination of basic proteins, lipid mediators, reactive oxygen species, and cytokines such as IL-5.35 Eosinophils have also been implicated in airway remodeling (increase in thickness of the airway wall) through eosinophil-derived fibrogenic growth factors.38 Treatment with an anti-IL-5 monoclonal antibody has been shown to reduce exacerbations in certain asthmatic patients.39 Eosinophil accumulation in the gastrointestinal tract occurs in allergic disorders such as food allergy, allergic colitis, and inflammatory bowel disease such as Crohn’s disease and ulcerative colitis.40, 41
Basophils and mast cells are two cells with morphologic and functional similarities; however, basophils are true leukocytes because they mature in the bone marrow and circulate in the blood as mature cells with granules, whereas mast cell precursors leave the bone marrow and use the blood as a transit system to gain access to the tissues where they mature. Basophils are discussed first. Basophils are the least numerous of the WBCs, making up between 0% and 2% of circulating leukocytes and less than 1% of nucleated cells in the bone marrow.
Basophils are derived from progenitors in the bone marrow, where they differentiate under the influence of a number of cytokines, including IL-3.42, 43 Due to their very small numbers, the stages of basophil maturation are very difficult to observe and have not been well characterized. Basophils will therefore be described simply as immature basophils and mature basophils.
Immature basophils have round to somewhat lobulated nuclei with only slightly condensed chromatin. Nucleoli may or may not be apparent. The cytoplasm is blue and contains large blue-black secondary granules (Figure 12-11). 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.
FIGURE 12-11 Immature basophil (arrow). Note that the background cytoplasm is deeply basophilic with few large basophilic granules and there appears to be a nucleolus.
Mature basophils contain a lobulated nucleus that is often obscured by its granules. The chromatin pattern, if visible, is clumped. Actual nuclear segmentation with visible filaments occurs rarely. The cytoplasm is colorless and contains large numbers of the characteristic large blue-black granules. If any granules have been dissolved during the staining process, they often leave a reddish-purple rim surrounding what appears to be a vacuole (Figure 12-12).
FIGURE 12-12 A, Mature basophil. Note that granules tend to obscure the nucleus and the background cytoplasm is only slightly basophilic. B, Electron micrograph of a basophil. Source: (B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.)
Basophil kinetics is poorly understood because of their very small numbers. According to a recent study, the life span of a mature basophil is 60 hours.44 This life span of basophils is relatively longer than that of the other granulocytes. This has been attributed to the fact that when they are activated by the cytokine IL-3, anti-apoptotic pathways are initiated that cause the prolongation of the basophil life span.45
Basophil functions are also poorly understood because of the small numbers of these cells and the lack of animal models such as basophil-deficient animals. However, the recent development of a conditional basophil-deficient mouse model promises to enhance the understanding of basophil function.46 In the past, basophils have been regarded as the “poor relatives” of mast cells and minor players in allergic inflammation because, like mast cells, they have immunoglobulin E (IgE) receptors on their surface membranes that, when cross-linked by antigen, result in granule release.47 Today, something of a reawakening has occurred regarding basophils and their functions in both innate and adaptive immunity. Basophils are capable of releasing large quantities of subtype 2 helper T cell (TH2) cytokines such as IL-4 and IL-13 that regulate the TH2 immune response.48, 49 Basophils also induce B cells to synthesize IgE.50 Whereas mast cells are the effectors of IgE-mediated chronic allergic inflammation, basophils function as initiators of the allergic inflammation through the release of preformed cytokines.47 Basophil activation is not restricted to antigen-specific IgE cross-linking, but it can be triggered in nonsensitized individuals by a growing list of parasitic antigens, lectins, and viral superantigens binding to nonspecific IgE antibodies.51
The contents of basophil granules are not well known. provides a short list of some of the substances released by activated basophils. Moreover, mature basophils are evidently capable of synthesizing granule proteins based on activation signals. For example, basophils can be induced to produce a mediator of allergic inflammation known as Box 12-4 granzyme B.52 Mast cells can induce basophils to produce and release retinoic acid, a regulator of immune and resident cells in allergic diseases.53 Basophils also play a role in angiogenesis through the expression of vascular endothelial growth factor (VEGF) and its receptors.54
Vascular endothelial growth factor A
Vascular endothelial growth factor B
Chondroitin sulfates (e.g., heparan)
Along with eosinophils, basophils are involved in the control of helminth infections. They promote eosinophilia, are associated with the differentiation of alternatively activated macrophages in the lung, and contribute to efficient worm expulsion.44 Finally, data from the basophil-deficient mouse model indicate that basophils play a nonredundant role in mediating acquired immunity against ticks.46
Mast cells are not considered to be leukocytes. They are tissue 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,55 and (2) mast cells have several phenotypic and functional similarities with both basophils and eosinophils.56
Mast cell progenitors (MCPs) originate from the bone marrow and spleen.55 The progenitors are then released to the blood before finally reaching tissues such as the intestine and lung, where they mediate their actions.55 The major cytokine responsible for mast cell maturation and differentiation is KIT ligand (stem cell factor).57 Once the MCP reaches its tissue destination, complete maturation into mature mast cells occurs under the control of the local microenvironment (Figure 12-13).58
FIGURE 12-13 Tissue mast cell in bone marrow. Note that the nucleus is rounded and the cell is packed with large basophilic granules. Mast cells tend to be a little larger than basophils (12 to 25 μm). Source: (From Rodak BF, Carr JH: Clinical hematology atlas, ed 4, Philadelphia, 2013, Saunders, Elsevier.)
Mast cells function as effector cells in allergic reactions through the release of a wide variety of lipid mediators, proteases, proteoglycans, and cytokines as a result of cross-linking of IgE on the mast cell surface by specific allergens. Mast cells can also be activated independently of IgE, which leads to inflammatory reactions. Mast cells can function as antigen-presenting cells to induce the differentiation of TH2 cells;59therefore, mast cells act in both innate and adaptive immunity.60 In addition, mast cells can have anti-inflammatory and immunosuppressive functions, and thus they can both enhance and suppress features of the immune response.61
Monocytes make up between 2% and 11% of circulating leukocytes, with an absolute number of up to 1.3 × 109/L.
Monocyte development is similar to neutrophil development because both cell types are derived from the granulocyte-monocyte progenitor (GMP) (see Figure 12-1). Macrophage colony-stimulating factor (M-CSF) is the major cytokine responsible for the growth and differentiation of monocytes. The morphologic stages of monocyte development are monoblasts, promonocytes, and monocytes. Monoblasts in normal bone marrow are very rare and are difficult to distinguish from myeloblasts based on morphology. Malignant monoblasts in acute monoblastic leukemia are described in Chapter 35. Therefore, only promonocytes and monocytes are described here.
Promonocytes are 12 to 18 μm in diameter, and their nucleus is slightly indented or folded. The chromatin pattern is delicate, and at least one nucleolus is apparent. The cytoplasm is blue and contains scattered azure granules that are fewer and smaller than those seen in promyelocytes (Figure 12-14). Electron microscopic and cytochemical studies have shown that monocyte azure granules are heterogeneous with regard to their content of lysosomal enzymes, peroxidase, nonspecific esterases, and lysozyme.62
FIGURE 12-14 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 basophilic with azure granules that are much smaller than those seen in promyelocytes. The azure granules in this cell are hard to see and give the cytoplasm a slightly grainy appearance.
Monocytes appear to be larger than neutrophils (diameter of 15 to 20 μm) because they tend to stick to and spread out on glass or plastic. Monocytes are slightly immature cells whose ultimate goal is to enter the tissues and mature into macrophages, osteoclasts, or dendritic cells.
The nucleus may be round, oval, or kidney shaped, but more frequently is deeply indented (horseshoe shaped) or folded on itself. The chromatin pattern is looser than in the other leukocytes and has sometimes been described as lacelike or stringy. Nucleoli are generally not seen with the light microscope; however, electron microscopy reveals nucleoli in roughly half of circulating monocytes. Their cytoplasm is blue-gray, with fine azure granules often referred to as azure dust or a ground-glass appearance. Small cytoplasmic pseudopods or blebs may be seen. Cytoplasmic and nuclear vacuoles may also be present (Figure 12-15). Based on flow cytometry immunophenotyping, three subsets of human monocytes have been described: the classical, intermediate, and nonclassical monocytes.63 The roles of these monocyte subsets in health and disease are currently being characterized.
FIGURE 12-15 A, Typical monocyte. Note the vacuolated cytoplasm, a contorted nucleus that folds on itself, loose or lacelike chromatin pattern, and very fine azure granules. B, Electron micrograph of a monocyte. Note that the villi on the surface are much greater in number than is seen on neutrophils. Source: (B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.)
The promonocyte pool consists of approximately 6 × 108 cells/kg, and they produce 7 × 106 monocytes/kg per hour. Under normal circumstances, promonocytes undergo two mitotic divisions in 60 hours to produce a total of four monocytes. Under conditions of increased demand for monocytes, promonocytes undergo four divisions to yield a total of 16 monocytes in 60 hours. There is no storage pool of mature monocytes in the bone marrow,64 and unlike neutrophils, monocytes are released immediately into the circulation upon maturation. Therefore, when the bone marrow recovers from marrow failure, monocytes are seen in the peripheral blood before neutrophils and a relative monocytosis may occur. There is recent evidence, however, that a relatively large reservoir of immature monocytes resides in the subcapsular red pulp of the spleen. Monocytes in this splenic reservoir appear to respond to tissue injury such as myocardial infarction by migrating to the site of tissue injury to participate in wound healing.65
Like neutrophils, monocytes in the peripheral blood can be found in a marginal pool and a circulating pool. Unlike with neutrophils, the marginal pool of monocytes is 3.5 times the circulating pool.66Monocytes remain in the circulation approximately 3 days.67 Monocytes with different patterns of chemokine receptors have different target tissues and different functions. Box 12-5 contains a list of the various tissue destinations of monocytes.68 Once in the tissues, monocytes differentiate into macrophages, osteoclasts (Figure 12-16), or dendritic cells, depending on the microenvironment of the local tissues. Macrophages can be as large as 40 to 50 μm in diameter. They usually have an oval nucleus with a netlike (reticulated) chromatin pattern. Their cytoplasm is pale, frequently vacuolated, and often filled with debris of phagocytized cells or organisms.
FIGURE 12-16 A, Active marrow macrophage (arrow). B, Osteoclast with 6 nuclei. Both of these cells are derived from monocytes.
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)
• Reproductive organ macrophages
• Lymph nodes (dendritic cells)
The life span of macrophages in the tissues depends on whether they are responding to inflammation or infection, or are “resident” macrophages such as Kupffer cells or alveolar macrophages. Resident macrophages survive far longer than tissue neutrophils. For example, Kupffer cells have a life span of approximately 21 days.69 Inflammatory macrophages, on the other hand, have a life span measured in hours.
Functions of monocytes/macrophages are numerous and varied. They can be subdivided into innate immunity, adaptive immunity, and housekeeping functions.
• Innate immunity: Monocytes/macrophages recognize a wide range of bacterial pathogens by means of pattern recognition receptors (toll-like receptors) that stimulate inflammatory cytokine production and phagocytosis. Macrophages can synthesize nitric oxide, which is cytotoxic against viruses, bacteria, fungi, protozoa, helminths, and tumor cells.24 Monocytes and macrophages also have Fc receptors and complement receptors. Hence, they can phagocytize foreign organisms or materials that have been coated with antibodies or complement components.
• Adaptive immunity: Both macrophages and dendritic cells degrade antigen and present antigen fragments on their surfaces (antigen-presenting cells). Because of this, they interact with and activate both T lymphocytes and B lymphocytes to initiate the adaptive immune response. Dendritic cells are the most efficient and potent of the antigen-presenting cells.
• Housekeeping functions: These include removal of debris and dead cells at sites of infection or tissue damage, destruction of senescent red blood cells and maintenance of a storage pool of iron for erythropoiesis, and synthesis of a wide variety of proteins, including coagulation factors, complement components, interleukins, growth factors, and enzymes.70
Lymphocytes are divided into three major groups: T cells, B cells, and natural killer (NK) cells. T and B cells are major players in adaptive immunity. NK cells make up a small percentage of lymphocytes and are part of innate immunity. Adaptive immunity has three characteristics: it relies on an enormous number of distinct lymphocytes, each having surface receptors for a different specific molecular structure on a foreign antigen; after an encounter with a particular antigen, memory cells are produced that will react faster and more vigorously to that same antigen upon reexposure; and self-antigens are “ignored” under normal circumstances (referred to as tolerance).
Lymphocytes can be subdivided into two major categories: those that participate in humoral immunity by producing antibodies and those that participate in cellular immunity by attacking 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 accomplished 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.71-73
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 receptor gene segments to produce a wide variety of antibodies and surface receptors.
4. Although early lymphocyte progenitors such as the common lymphoid progenitor originate in the bone marrow, T and NK lymphocytes develop and mature outside of the bone marrow.
For these reasons, lymphocyte kinetics is extremely complicated, not well understood, and beyond the scope of this chapter.
Lymphocytes make up between 18% and 42% of circulating leukocytes with an absolute number of 0.8 to 4.8 × 109/L.
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 marrow 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 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 lymphatic organs, where they take up residence in specific zones such as lymph node follicles. These immature B cells, also known as hematogones,74 have a homogeneous nuclear chromatin pattern and extremely scanty cytoplasm (Figure 12-17). These cells are normally seen in newborn peripheral blood and bone marrow and in regenerative bone marrows. Leukemic cells from patients with acute lymphoblastic leukemia (ALL) can sometimes resemble hematogones, but the leukemic cells can be distinguished from hematogones by flow cytometry immunophenotyping.75
FIGURE 12-17 Immature B lymphocyte or hematogone (arrow). Note the extremely scanty cytoplasm. This was taken from the bone marrow of a newborn infant.
It is in the secondary lymphatic organs or in the blood where B cells may come in contact with antigen, which results in cell division and the production of memory cells as well as effector cells. Effector B cells are antibody-producing cells known as plasma cells and plasmacytoid lymphocytes (Figure 12-18).
FIGURE 12-18 A, Plasma cell. B, Plasmacytoid lymphocyte. These are effector cells of the B lymphocyte lineage.
Approximately 3% to 21% of circulating lymphocytes are B cells. Resting B lymphocytes cannot be distinguished morphologically from resting T lymphocytes. Resting lymphocytes are small (around 9 μm in diameter), and the N:C ratio ranges from 5:1 to 2:1. The chromatin is arranged in blocks, and the nucleolus is rarely seen, although it is present (). Figure 12-19
FIGURE 12-19 A, Small resting lymphocyte. B, Electron micrograph of a small lymphocyte. Source: (B from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders, Elsevier.)
T lymphocytes develop initially in the thymus—a lymphoepithelial organ located in the upper mediastinum.76 Lymphoid progenitor cells migrate from the bone marrow to the thymic cortex, where, under the regulation of cytokines produced by 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 receptors that are unique to each T cell. T cells whose receptors react with self-antigens are allowed to undergo apoptosis.77 In addition, T cells are subdivided into two major categories, depending on whether or not they have CD4 or CD8 antigen on their surfaces. Immature T cells then proceed to the thymic medulla, where further apoptosis of self-reactive T cells occurs. The remaining immature T cells (or antigen-naive T cells) then leave the thymus and migrate to secondary lymphatic organs, where they take up residence in specific zones such as the paracortical areas. T cells comprise 51% to 88% of circulating lymphocytes.
T cells in secondary lymphatic organs or in the circulating blood eventually come in contact with antigen. This results in cell activation and the production of either memory cells or effector T cells, or both (). The transformation of resting lymphocytes into activated forms is the source of so-called medium and large lymphocytes that have increased amounts of cytoplasm and usually make up only about 10% of circulating lymphocytes. The morphology of effector T cells varies with the subtype of T cell involved, and they are often referred to as Figure 12-20reactive or variant lymphocytes.
FIGURE 12-20 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.
NK cells are a heterogeneous group of cells with respect to their surface antigens. The majority are CD56+CD16+CD3− CD7+ large granular lymphocytes. (Figure 12-21).78 The mature NK cell is relatively large compared with other resting lymphocytes because of an increased amount of cytoplasm. Its cytoplasm contains azurophilic granules that are peroxidase negative. Approximately 4% to 29% of circulating lymphocytes are NK cells.
FIGURE 12-21 A large granular lymphocyte that could be either a cytotoxic T lymphocyte or a natural killer lymphocyte.
Functions can be addressed according to the type of lymphocyte. B lymphocytes are essential for antibody production. In addition, they have a role in antigen presentation to T cells and may be necessary for optimal CD4 activation. B cells also produce cytokines that regulate a variety of T cell and antigen-presenting cell functions.79
T lymphocytes can be divided into CD4+ T cells and CD8+ T cells. CD4+ effector lymphocytes are further subdivided into TH1, TH2, TH17, and Treg (CD4+CD25+ regulatory T) cells. TH1 cells mediate immune responses against intracellular pathogens. TH2 cells mediate host defense against extracellular parasites, including helminths. They are also important in the induction of asthma and other allergic diseases. TH17 cells are involved in the immune responses against extracellular bacteria and fungi. Treg cells play a role in maintaining self-tolerance by regulating immune responses.80, 81
CD8+ effector lymphocytes are capable of killing target cells by secreting granules containing granzyme and perforin or by activating apoptotic pathways in the target cell.82 These cells are sometimes referred to as cytotoxic T lymphocytes.
NK lymphocytes function as part of innate immunity and are capable of killing certain tumor cells and virus-infected cells without prior sensitization. In addition, NK cells modulate the functions of other cells, including macrophages and T cells.83
• Granulocytes are classified according to their staining characteristics and the shape of their nuclei. Neutrophils are a major component of innate immunity as phagocytes; eosinophils are involved in allergic reactions and helminth destruction; and basophils function as initiators of allergic reactions, helminth destruction, and immunity against ticks.
• Neutrophil development can be subdivided into specific stages, with cells at each stage having specific morphologic characteristics (myeloblast, promyelocyte, myelocyte, metamyelocyte, band, and segmented neutrophil). Various granule types are produced during neutrophil development, each with specific contents.
• Eosinophil development can also be subdivided into specific stages, although eosinophilic myeloblasts are not recognizable and eosinophil promyelocytes are rare.
• Basophil development is difficult to describe, and basophils have been divided simply into immature and mature basophils.
• Mononuclear cells consist of monocytes and lymphocytes. Monocytes are precursors to tissue cells such as osteoclasts, macrophages, and dendritic cells. As a group, they perform several functions as phagocytes.
• Monocyte development can be subdivided into the promonocyte, monocyte, and macrophage stages, each with specific morphologic characteristics.
• The majority of lymphocytes are involved in adaptive immunity. B lymphocytes and plasma cells produce antibodies against foreign organisms or cells, and T lymphocytes mediate the immune response against intracellular and extracellular invaders. Both B and T lymphocytes produce memory cells for specific antigens so that the immune response is faster if the same antigen is encountered again.
• Lymphocyte development is complex, and morphologic divisions are not practical because a large number of lymphocytes develop in the thymus. Benign B-lymphocyte precursors (hematogones) as well as B-lymphocyte effector cells (plasma cells and plasmacytoid lymphocytes) have been described. NK lymphocytes and cytotoxic T cells also have a distinct and similar morphology.
Answers can be found in the Appendix.
1. Neutrophils and monocytes are direct descendants of a common progenitor known as:
2. The stage in neutrophilic development in which the nucleus is indented in a kidney bean shape and the cytoplasm has secondary granules that are lavender in color is the:
3. Type II myeloblasts are characterized by:
a. Presence of fewer than 20 primary granules per cell
b. Basophilic cytoplasm with many secondary granules
c. Absence of granules
d. Presence of a folded nucleus
4. Which one of the following is a function of neutrophils?
a. Presentation of antigen to T and B lymphocytes
b. Protection against reexposure by same antigen
c. Nonspecific destruction of foreign organisms
d. Initiation of delayed hypersensitivity response
5. Which of the following cells are important in immune regulation, allergic inflammation, and destruction of tissue invading helminths?
a. Neutrophils and monocytes
b. Eosinophils and basophils
c. T and B lymphocytes
d. Macrophages and dendritic cells
6. Basophils and mast cells have high-affinity surface receptors for which immunoglobulin?
7. Which of the following cell types is capable of differentiating into osteoclasts, macrophages, or dendritic cells?
8. Macrophages aid in adaptive immunity by:
a. Degrading antigen and presenting it to lymphocytes
b. Ingesting and digesting organisms that neutrophils cannot
c. Synthesizing complement components
d. Storing iron from senescent red cells
9. Which of the following is the final stage of B cell maturation after activation by antigen?
a. Large, granular lymphocyte
b. Plasma cell
c. Reactive lymphocyte
10. The following is unique to both B and T lymphocytes and occurs during their early development:
a. Expression of surface antigens CD4 and CD8
b. Maturation in the thymus
c. Synthesis of immunoglobulins
d. Rearrangement of antigen receptor genes
1. von Vietinghoff S, Ley K. Homeostatic regulation of blood neutrophil counts. J Immunol; 2008; 181(8):5183-5188.
2. Price T.H, Chatta G.S, Dale D.C. Effect of recombinant granulocyte colony-stimulating factor on neutrophil kinetics in normal young and elderly humans. Blood; 1996; 88(1):335-340.
3. Chaiworapongsa T, Romero R, Berry S.M, et al. The role of granulocyte colony-stimulating factor in the neutrophilia observed in the fetal inflammatory response syndrome. J Perinat Med; 2011; 39(6):653-666.
4. Summers C, Rankin S.M, Condliffe A. M, et al. Neutrophil kinetics in health and disease. Trends Immunol; 2010; 31(8):318-324.
5. Iwasaki H, Akashi K. Hematopoietic developmental pathways on cellular basis. Oncogene; 2007; 26(47):6687-6696.
6. Terstappen L. W, Huang S, Safford M, et al. Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD341CD38– progenitor cells. Blood; 1991; 77(6):1218-1227.
7. Manz M. G, Miyamoto T, Akashi K, Weissman I. L. Prospective isolation of human clonogenic common myeloid progenitors. Proc Natl Acad Sci U S A; 2002; 99(18):11872-11877.
8. Adams G. B, Scadden D. T. The hematopoietic stem cell in its place. Nat Immunol; 2006; 7(4):333-337.
9. Mufti G. J, Bennett J. M, Goasguen J, et al. Diagnosis and classification of myelodysplastic syndrome International Working Group on Morphology of Myelodysplastic Syndrome (IWGM-MDS) consensus proposals for the definition and enumeration of myeloblasts and ring sideroblasts. Haematologica; 2008; 93(11):1712-1717.
10. Faurschou M, Borregaard N. Neutrophil granules and secretory vesicles in inflammation. Microbes Infect; 2003; 5(14):1317-1327.
11. Cornbleet P. J. Clinical utility of the band count. Clin Lab Med; 2002; 22(1):101-136.
12. Reference leukocyte (WBC) differential count (proportional) and evaluation of instrumental methods; approved standard-Second Edition. (H20-A2). : Vol 27(4): Clinical and Laboratory Standards Institute (CLSI) 2007.
13. Dancey J. T, Deubelbeiss K. A, Harker L. A, Finch C. A. Neutrophil kinetics in man. J Clin Invest; 1976; 58(3):705-715.
14. Athens J. W, Haab O. P, Raab S. O, et al. Leukokinetic studies. IV. The total blood, circulating and marginal granulocyte pools and the granulocyte turnover rate in normal subjects. J Clin Invest; 1961; 40:989-995.
15. Hetherington S. V, Quie P. G. Human polymorphonuclear leukocytes of the bone marrow, circulation, and marginated pool function and granule protein content. Am J Hematol; 1985; 20(3):235-246.
16. Cartwright G. E, Athens J. W, Wintrobe M. M. The kinetics of granulopoiesis in normal man. Blood; 1964; 24:780-803.
17. Cowburn A. S, Condliffe A. M, Farahi N, et al. Advances in neutrophil biology clinical implications. Chest; 2008; 134(3):606-612.
18. Saverymuttu S. H, Peters A. M, Keshavarzian A, et al. The kinetics of 111indium distribution following injection of 111indium labelled autologous granulocytes in man. Br J Haematol; 1985; 61(4):675-685.
19. Zarbock A, Ley K, McEver R. P, Hidalgo A. Leukocyte ligands for endothelial selectins specialized glycoconjugates that mediate rolling and signaling under flow. Blood; 2011; 118(26):6743-6751.
20. Ley K, Laudanna C, Cybulsky M. I, Nourshargh S. Getting to the site of inflammation the leukocyte adhesion cascade updated. Nat Rev Immunol; 2007; 7(9):678-689.
21. Mayadas T. N, Cullere X. Neutrophil beta2 integrins moderators of life or death decisions. Trends Immunol; 2005; 26(7):388-395.
22. Burg N. D, Pillinger M. H. The neutrophil function and regulation in innate and humoral immunity. Clin Immunol; 2001; 99(1):7-17.
23. Stuart L. M, Ezekowitz R. A. Phagocytosis elegant complexity. Immunity; 2005; 22(5):539-550.
24. Dale D. C, Boxer L, Liles W.C. The phagocytes: neutrophils and monocytes. Blood; 2008; 112(4):935-945.
25. Brinkmann V, Zychlinsky A. Beneficial suicide why neutrophils die to make NETs. Nat Rev Microbiol; 2007; 5(8):577-582.
26. Brinkmann V, Zychlinsky A. Neutrophil extracellular traps is immunity the second function of chromatin. J Cell Biol; 2012; 198(5):773-783.
27. Mori Y, Iwasaki H, Kohno K, et al. Identification of the human eosinophil lineage-committed progenitor revision of phenotypic definition of the human common myeloid progenitor. J Exp Med; 2009; 206(1):183-193.
28. Uhm T. G, Kim B. S, Chung I. Y. Eosinophil development, regulation of eosinophil-specific genes, and role of eosinophils in the pathogenesis of asthma. Allergy Asthma Immunol Res; 2012; 4(2):68-79.
29. Takatsu K, Nakajima H. IL-5 and eosinophilia. Curr Opin Immunol; 2008; 20(3):288-294.
30. Melo R. C, Spencer L. A, Perez S. A, et al. Vesicle-mediated secretion of human eosinophil granule-derived major basic protein. Lab Invest; 2009; 89(7):769-781.
31. Giembycz M. A, Lindsay M. A. Pharmacology of the eosinophil. Pharmacol Rev; 1999; 51(2):213-340.
32. Steinbach K. H, Schick P, Trepel F, et al. Estimation of kinetic parameters of neutrophilic, eosinophilic, and basophilic granulocytes in human blood. Blut; 1979; 39(1):27-38.
33. Park Y. M, Bochner B. S. Eosinophil survival and apoptosis in health and disease. Allergy Asthma Immunol Res; 2010; 2(2):87-101.
34. Throsby M, Herbelin A, Pleau J. M, Dardenne M. CD11c1 eosinophils in the murine thymus developmental regulation and recruitment upon MHC class I-restricted thymocyte deletion. J Immunol; 2000; 165(4):1965-1975.
35. Hogan S. P, Rosenberg H. F, Moqbel R, et al. Eosinophils biological properties and role in health and disease. Clin Exp Allergy; 2008; 38(5):709-750.
36. Spencer L. A, Szela C. T, Perez S. A, et al. Human eosinophils constitutively express multiple Th1, Th2, and immunoregulatory cytokines that are secreted rapidly and differentially. J Leukoc Biol; 2009; 85(1):117-123.
37. Hagan P, Wilkins H. A, Blumenthal U. J, Hayes R. J, Greenwood B. M. Eosinophilia and resistance to Schistosoma haematobium in man. Parasite Immunol; 1985; 7(6):625-632.
38. Phipps S, Benyahia F, Ou T. T, et al. Acute allergen-induced airway remodeling in atopic asthma. Am J Respir Cell Mol Biol; 2004; 31(6):626-632.
39. Robinson D. S. Mepolizumab for severe eosinophilic asthma. Expert Rev Respir Med; 2013; 7(1):13-17.
40. Walsh R. E, Gaginella T. S. The eosinophil in inflammatory bowel disease. Scand J Gastroenterol; 1991; 26(12):1217-1224.
41. Hogan S. P, Waddell A, Fulkerson P. C. Eosinophils in infection and intestinal immunity. Curr Opin Gastroenterol; 2013; 29(1):7-14.
42. Min B, Brown M. A, Legros G. Understanding the roles of basophils breaking dawn. Immunology; 2012; 135(3):192-197.
43. Ohmori K, Luo Y, Jia Y, et al. IL-3 induces basophil expansion in vivo by directing granulocyte-monocyte progenitors to differentiate into basophil lineage-restricted progenitors in the bone marrow and by increasing the number of basophil/mast cell progenitors in the spleen. J Immunol; 2009; 182(5):2835-2841.
44. Ohnmacht C, Voehringer D. Basophil effector function and homeostasis during helminth infection. Blood; 2009; 113(12):2816-2825.
45. Didichenko S. A, Spiegl N, Brunner T, Dahinden C. A. IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils. Blood; 2008; 112(10):3949-3958.
46. Wada T, Ishiwata K, Koseki H, et al. Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks. J Clin Invest; 2010; 120(8):2867-2875.
47. Obata K, Mukai K, Tsujimura Y, et al. Basophils are essential initiators of a novel type of chronic allergic inflammation. Blood; 2007; 110(3):913-920.
48. Sullivan B. M, Locksley R. M. Basophils a nonredundant contributor to host immunity. Immunity; 2009; 30(1):12-20.
49. Schroeder J. T, MacGlashan D. W, Jr. Lichtenstein L. M. Human basophils mediator release and cytokine production. Adv Immunol; 2001; 77:93-122.
50. Gauchat J. F, Henchoz S, Mazzei G, et al. Induction of human IgE synthesis in B cells by mast cells and basophils. Nature; 1993; 365(6444):340-343.
51. Falcone F. H, Zillikens D, Gibbs B. F. The 21st century renaissance of the basophil? Current insights into its role in allergic responses and innate immunity. Exp Dermatol; 2006; 15(11):855-864.
52. Tschopp C. M, Spiegl N, Didichenko S, et al. Granzyme B, a novel mediator of allergic inflammation its induction and release in blood basophils and human asthma. Blood; 2006; 108(7):2290-2299.
53. Spiegl N, Didichenko S, McCaffery P, et al. Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid. Blood; 2008; 112(9):3762-3771.
54. de Paulis A, Prevete N, Fiorentino I, et al. Expression and functions of the vascular endothelial growth factors and their receptors in human basophils. J Immunol; 2006; 177(10):7322-7331.
55. Hallgren J, Gurish M. F. Mast cell progenitor trafficking and maturation. Adv Exp Med Biol; 2011; 716:14-28.
56. Valent P. The phenotype of human eosinophils, basophils, and mast cells. J Allergy Clin Immunol; 1994; 94(6 Pt 2):1177-1183.
57. Valent P, Spanblochl E, Sperr W. R, et al. Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture. Blood; 1992; 80(9):2237-2245.
58. Kambe N, Hiramatsu H, Shimonaka M, et al. Development of both human connective tissue-type and mucosal-type mast cells in mice from hematopoietic stem cells with identical distribution pattern to human body. Blood; 2004; 103(3):860-867.
59. Nakano N, Nishiyama C, Yagita H, et al. Notch signaling confers antigen-presenting cell functions on mast cells. J Allergy Clin Immunol; 2009; 123(1):74-81 e71.
60. Heib V, Becker M, Taube C, Stassen M. Advances in the understanding of mast cell function. Br J Haematol; 2008; 142(5):683-694.
61. Galli S. J, Grimbaldeston M, Tsai M. Immunomodulatory mast cells negative, as well as positive, regulators of immunity. Nat Rev Immunol; 2008; 8(6):478-486.
62. Nichols B. A, Bainton D. F, Farquhar M. G. Differentiation of monocytes. Origin, nature, and fate of their azurophil granules. J Cell Biol; 1971; 50(2):498-515.
63. Ziegler-Heitbrock L, Ancuta P, Crowe S, et al. Nomenclature of monocytes and dendritic cells in blood. Blood; 2010; 116(16):e74-80.
64. Meuret G, Bammert J, Hoffmann G. Kinetics of human monocytopoiesis. Blood; 1974; 44(6):801-816.
65. Swirski F. K, Nahrendorf M, Etzrodt M, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science; 2009; 325(5940):612-616.
66. Meuret G, Batara E, Furste H. O. Monocytopoiesis in normal man pool size, proliferation activity and DNA synthesis time of promonocytes. Acta Haematol; 1975; 54(5):261-270.
67. Whitelaw D. M. Observations on human monocyte kinetics after pulse labeling. Cell Tissue Kinet; 1972; 5(4):311-317.
68. Kumar S, Jack R. Origin of monocytes and their differentiation to macrophages and dendritic cells. J Endotoxin Res; 2006; 12(5):278-284.
69. Crofton R. W, Diesselhoff-den Dulk M. M, van Furth R. The origin, kinetics, and characteristics of the Kupffer cells in the normal steady state. J Exp Med; 1978; 148(1):1-17.
70. Nathan C. F. Secretory products of macrophages. J Clin Invest; 1987; 79(2):319-326.
71. Lotzova E, Savary C. A, Champlin R. E. Genesis of human oncolytic natural killer cells from primitive CD341CD33– bone marrow progenitors. J Immunol; 1993; 150(12):5263-5269.
72. Res P, Martinez-Caceres E, Cristina Jaleco A, et al. CD341CD38dim cells in the human thymus can differentiate into T, natural killer, and dendritic cells but are distinct from pluripotent stem cells. Blood; 1996; 87(12):5196-5206.
73. Di Santo J. P, Vosshenrich C. A. Bone marrow versus thymic pathways of natural killer cell development. Immunol Rev; 2006; 214:35-46.
74. Sevilla D. W, Colovai A. I, Emmons F. N, et al. Hematogones a review and update. Leuk Lymphoma; 2010; 51(1):10-19.
75. McKenna R. W, Washington L. T, Aquino D. B, et al. Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4-color flow cytometry. Blood; 2001; 98(8):2498-2507.
76. Haynes B. F. The human thymic microenvironment. Adv Immunol; 1984; 36:87-142.
77. von Boehmer H, The H. S, Kisielow P. The thymus selects the useful, neglects the useless and destroys the harmful. Immunol Today; 1989; 10(2):57-61.
78. Grossi C. E, Cadoni A, Zicca A, et al. Large granular lymphocytes in human peripheral blood ultrastructural and cytochemical characterization of the granules. Blood; 1982; 59(2):277-283.
79. LeBien T. W, Tedder T. F. B lymphocytes how they develop and function. Blood; 2008; 112(5):1570-1580.
80. Zhu J, Paul W. E. CD4 T cells fates, functions, and faults. Blood; 2008; 112(5):1557-1569.
81. Vignali D. A, Collison L. W, Workman C. J. How regulatory T cells work. Nat Rev Immunol; 2008; 8(7):523-532.
82. Rufer N, Zippelius A, Batard P, et al. Ex vivo characterization of human CD81 T subsets with distinct replicative history and partial effector functions. Blood; 2003; 102(5):1779-1787.
83. Vivier E, Tomasello E, Baratin M, et al. Functions of natural killer cells. Nat Immunol; 2008; 9(5):503-510.
*The authors acknowledge the contributions of Anne Stiene-Martin, author of this chapter in the previous edition.