Chapter 60: Structure and Composition of Neutrophils, Eosinophils, and Basophils

In the normal adult human, the life of granulocytes is spent in three environments: marrow, blood, and tissues. Marrow is the site of differentiation of hematopoietic stem cells into granulocyte progenitors and of proliferation and terminal maturation (Fig. 60–1). Precursor cell proliferation, which consists of approximately five divisions, occurs only during the first three stages of maturation (blast, promyelocyte, and myelocyte). After the myelocyte stage, the cells are no longer capable of mitosis and enter a large marrow storage pool from which they are released into the blood where they circulate for a few hours before entering tissues.

NEUTROPHILS

LIGHT MICROSCOPY AND ELECTRON MICROSCOPY

The Myeloblast

The myeloblast is an immature cell with a large, oval nucleus, sizable nucleoli, and few or no granules. As the earliest precursor in the evolution of the neutrophil from the colony forming unit, it is an immature cell with a large nucleus and multiple nucleoli (Fig. 60–2). The nucleolus is the site of assembly of ribosomal proteins and ribosomal RNA, and is a prominent feature of early maturing cells. The scant cytoplasm contains reaction product for peroxidase within the rough-surfaced endoplasmic reticulum and Golgi cisternae and, sometimes, in early developing azurophilic granules. The dense product of the peroxidase reaction serves as a marker of azurophilic granules in human marrow and blood cells for electron and for light microscopy.^1,2,3,4

The Promyelocyte

In the promyelocyte stage, the azurophilic or primary granules, large peroxidase-positive granules that stain metachromatically (reddish-purple) with a polychromatic stain such as Wright stain, are formed. Figure 60–3 shows that the promyelocyte produces and accumulates a large population of peroxidase-positive granules. Most of the granules are spherical and have a diameter of 500 nm, but ellipsoid, crystalline forms and small granules connected by filaments also are present.⁵ As with other secretory cells, peroxidase is present throughout the secretory apparatus of the promyelocyte, including cisternae of the rough endoplasmic reticulum, all Golgi cisternae, some vesicles, and all developing granules.²

The Neutrophilic Myelocyte

During the myelocyte stage of maturation, the specific or secondary granules, which are peroxidase negative, are formed (see Fig. 60–2). At the end of the promyelocyte stage, peroxidase abruptly disappears from rough endoplasmic reticulum and Golgi cisternae, and the production of azurophilic granules ceases. The myelocyte stage begins with production of peroxidase-negative specific granules.²

The only peroxidase-positive elements at this stage are the azurophilic granules. The specific granules are formed by the Golgi complex. The granules vary in size and shape but typically are spherical (approximately 200 nm) or rod shaped (130 × 1000 nm). Figure 60–4 shows the cell also labeled with immunogold particles to illustrate the presence of lactoferrin, a specific granule marker. Approximately three cell divisions occur at this stage of maturation. Mitoses can be observed (Fig. 60–5), and the two types of granules appear to be distributed to the daughter cells in fairly equal numbers.

Metamyelocyte, Band, and Mature Neutrophil

The metamyelocyte and band neutrophils are nonproliferating cells that precede the development of the mature neutrophil (see Fig. 60–2). The mature, segmented neutrophilic cells contain primary, peroxidase-positive granules and specific peroxidase-negative granules in a 1:2 ratio. The nucleus of the circulating neutrophil is segmented, usually into two to four interconnected lobes. The late stages of maturation consist of nondividing cells that can be distinguished by their nuclear morphology, mixed granule populations, small Golgi regions, and accumulations of glycogen particles. On average, an electron micrograph of a neutrophil displays 200 to 300 granules, and approximately one-third are peroxidase-positive (Fig. 60–6).

The violet-colored granules seen with light microscopy in mature neutrophils on Wright-stained blood films are azurophilic granules whose staining characteristics altered during maturation (Fig. 60–7). Therefore, with light microscopy, the most reliable method for identifying azurophilic granules on blood films is staining the cells for peroxidase. The size of most of the peroxidase-negative granules (approximately 200 nm) is at the limit of resolution of the light microscope. The granules cannot be distinguished individually but are responsible for the pink background color of neutrophil cytoplasm during and after the myelocyte stage.

Peroxidase-negative granules are more numerous than peroxidase-positive granules during the myelocyte stage because peroxidase granule formation ceases after the promyelocyte stage, the number of oxidase-positive granules per cell is reduced by mitoses, and peroxidase-negative granules continue to be produced by each myelocyte generation.¹

The purpose of nuclear segmentation is not known. Fluorescence in situ hybridization with chromosome-specific probes has shown that chromosomes are randomly distributed among the nuclear lobes.⁶ Some mature neutrophils in women have drumstick- or club-shaped nuclear appendages. These appendages contain the inactivated X chromosome. An X-chromosome–specific nucleic acid probe has confirmed the position of the X chromosomes in the drumstick structure of leukocyte nuclei by in situ hybridization.⁷

NEUTROPHIL GRANULES

The diversity of neutrophil granules appears to be linked to the timing of biosynthesis during myelopoiesis. The hypothesis is that the different subsets of granules are the result of differences in the biosynthetic windows of the various granule proteins during maturation⁷ and not the result of specific sorting between individual granule subsets (Chap. 66). The control of biosynthesis is exerted by transcription factors that control the expression of the genes for the various granule proteins. Several transcription factors identified as important in the timing of granule protein synthesis, including the lineage-specific transcription factor GATA-1, the lineage-specific transcription factor PU.1, transcription factor for various hematologic lineages, AML1 (also known as runt-related transcription factor 1 [RUNX1] or core-binding factor subunit alpha-2 [CBFA2]), AML2 (also known as RUNX3), and AML3 (also known as RUNX2 or CBFA1), and regulating factor of gene expression C/EBPε.^7,8,9 The importance of C/EBPε has been emphasized by the recognition of mutations in this protein in patients with the rare syndrome called “specific granule deficiency,”^10,11,12 a condition that leads to increased susceptibility to bacterial infections. In neutrophils from these patients, total cellular content and release of the secondary and tertiary granule markers (e.g., lactoferrin, B₁₂ binding protein, and lysozyme) are diminished, although levels of primary granule constituents (e.g., myeloperoxidase, β-glucuronidase) generally are normal.

The granular constituents are released from the membrane-enclosed granules into phagosomes or transported to the cell surface by a process of exocytosis following stimulation of the neutrophil.¹³ The signal cascade following stimulation of specific receptors on the cytoplasmic membrane results in elevated intracellular Ca²⁺, lipid remodeling, and protein kinase activation, which culminate in fusion of granules with phagosomes or the cell surface membrane. The process is rapid, highly efficient, and involves families of docking proteins related to those found in neurons (e.g., vesicle-associated membrane protein [VAMP]-2, syntaxin-4, soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein [SNAP]-23).¹⁴

The granule subsets appear to have a significant differential sensitivity to undergo exocytosis, ranging from secretory vesicles to tertiary, secondary, and primary granules, with primary granules being most resistant. The significance of this differential release is incompletely understood, but some aspects are apparent in the functions of the constituents within the granules and granular membranes. For example, secretory vesicles and tertiary granules contain receptors, such as CD11b/CD18 (adhesion molecule, Mac-1), formyl peptide receptor (chemotactic receptor), FcγRIIIB (Fc receptor), and gelatinase (metalloproteinase [MMP]-9), which potentially enhance extracellular interactions of the neutrophil. Primary granules contain microbicidal proteins and acid hydrolases, and the acidic environment of the phagolysosome creates an optimal pH for these enzymes.

BIOACTIVE FACTORS IN GRANULES

Neutrophil granules are particularly rich in factors with antimicrobial activity. Some (e.g., myeloperoxidase) function in conjunction with the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, whereas others (e.g., defensins) exhibit activity independent of the oxidative burst. Table 60–1 lists the principal contents of the four granule types in neutrophils: primary (azurophilic), secondary (specific), tertiary, and secretory vesicles.^15–56

EOSINOPHILS

LIGHT MICROSCOPY OF EOSINOPHILS IN MARROW AND BLOOD FILMS

The earliest morphologically identifiable form of an eosinophilic leukocyte is as a late myeloblast or early promyelocyte (see Fig. 60–1). This cell is approximately 15 µm in diameter and has a large nucleus with nucleoli and a few blue or azurophilic granules in intensely basophilic cytoplasm. The later eosinophilic promyelocyte and myelocyte contain mostly acidophilic granules. A lineage-committed eosinophil progenitor has recently been identified that expresses high levels of interleukin (IL)-5 receptor α and is negative for myeloperoxidase.⁵⁷ The fully mature eosinophilic leukocyte has a bilobed nucleus (see Fig. 60–7), and its cytoplasm is filled with large eosinophilic granules whose rims stain for peroxidase and Sudan black. Multilobed nuclei, comparable to those of neutrophils, are rare. Eosinophils are susceptible to mechanical damage during preparation of blood films.

ELECTRON MICROSCOPY AND CYTOCHEMISTRY

Eosinophils of the promyelocyte and myelocyte stages stain positively for peroxidase in all cisternae of the rough-surfaced endoplasmic reticulum, including transitional elements and the perinuclear cisterna; clusters of smooth vesicles at the periphery of the Golgi complex; all cisternae of the Golgi complex; and all immature- and mature-specific granules.^4,58 Mature granules are completely filled with peroxidase except in areas occupied by centrally located crystals.

In the later stages of development, after granule formation has ceased, the eosinophils contain few of the organelles associated with the synthesis and packaging of secretory proteins. The endoplasmic reticulum is sparse or virtually nonexistent. The Golgi complex is small and inconspicuous. The cytoplasm of the mature eosinophil (Fig. 60–8) primarily contains granules and glycogen. Most of the granules are specific granules with crystals, which usually are centrally located. After the myelocyte stage, peroxidase can no longer be detected in the endoplasmic reticulum or Golgi elements of the eosinophil by any of the enzyme procedures; however, peroxidase can be found in the matrix of granules.^1,58

GRANULES

Contents

As with neutrophils, eosinophils contain distinct granular organelles: primary granules, crystalloid granules, small granules and secretory vesicles.⁵⁹ The crystalloid granules (see Fig. 60–8) are the largest, 0.5 to 0.8 µm in diameter, and contain much of the granular protein. The proteins packaged in these granules are highly basic proteins, with the crystalline core being mostly major basic protein (MBP).^60,61 The granule matrix contains eosinophil peroxidase, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). The primary granules contain Charcot-Leyden crystals. Charcot-Leyden crystals are bipyramidal crystals observed in fluids in association with eosinophilic inflammatory reactions. They possess lysophospholipase activity and compose 7 to 10 percent of total eosinophil protein.^62,63 The ultrastructural localization of this protein is in a large, crystal-free granule and supports the presence of a distinct primary granule population in mature eosinophils.^4,63,64 MBP consists of two homologues and is an abundant granular protein, 5 to 10 pg per cell. Mature eosinophils can no longer express this protein so all MBP is stored during development.⁶⁵ Eosinophil peroxidase is an abundant heme-containing protein (approximately 15 pg per cell) that catalyzes the peroxidation of halides together with hydrogen peroxide forming bactericidal hypohalous acids.^66,67 ECP is a bactericidal protein exists in two isoforms (ECP-1 and ECP-2) with activity toward helminthic parasites. EDN shares high sequence homology with ECP and is abundant, approximately 10 pg per cell. Other granule-stored proteins include several enzymes of potential importance in inflammation, including acid phosphatase, collagenase (MMP-8), matrix metalloproteases, histaminase, catalase, and phospholipase D.^68,69,70 Chapter 62 discusses the functional aspects of these granular proteins. In addition, mature eosinophils retain the ability to synthesize a diverse array of proteins including cytokines and chemokines,^71,72 adhesion molecules,^73,74,75,76 receptors for cytokines, complement components, lipid mediators, and immunoglobulins.^{77,78,79,80,81,82}

BASOPHILS AND MAST CELLS

Basophils (see Fig. 60–7) and mast cells were considered to be derived from distinct lineages, but recent data indicates a common basophil-mast cell progenitor exists from which mast cells exit the marrow as immature precursors and terminally differentiate in tissues; basophils mature in the marrow before entering the circulation.^83,84,85,86 This common progenitor is derived from the granulocyte–monocyte progenitor. The granules of the two cell types stain metachromatically but are distinct when examined by electron microscopy (Figs. 60–9 and 60–10). Identification of basophils in tissue at the light microscopic level is difficult without the use of cell specific antibodies.⁸⁷ Basophils and mast cells express the FcεR1 receptor. The cells can phagocytose sensitized red cells but are less active phagocytes than the other granulocytes. They lack significant amounts of antibacterial or lysosomal enzymes. Basophils are found in small numbers in blood (0.5 percent) and can be seen in tissues in which inflammation resulting from hypersensitivity to proteins, contact allergy, or skin allograft rejection is present. They have been shown to be rich sources of IL-4 and IL-13.^83,84,88

Mast cells are normal residents of connective tissue throughout the body. Mast cell granules contain various substances, including several preformed biologically active substances such as histamine, which causes increased vascular permeability; eosinophil chemotactic factor of anaphylaxis; and heparin, which has antithrombin activity.^89,90,91,92 This accounts for the metachromatic staining quality of the granules. The generation of anaphylatoxin (C3a, C5a) or the interaction of allergen with immunoglobulin (Ig) E receptors of plasma membrane can stimulate extracellular release of these granule contents and of several newly formed substances, such as slow-reacting substance of anaphylaxis, a leukotriene that causes contraction of human bronchioles and increased vascular permeability, and platelet-activating factor (PAF), which causes platelet aggregation and the subsequent release of serotonin. This phenomenon is called IgE-mediated mast cell degranulation.⁹⁰ Mast cells also have been implicated in various diseases that are accompanied by neovascularization (Chap. 63).

METABOLISM OF NEUTROPHILS

CARBOHYDRATE METABOLISM

Glycolysis

Glycolytic (Embden-Meyerhof) Pathway The main energy-producing pathway in the neutrophil is glycolysis, resulting in the conversion of glucose to lactate.^93,94,95 When intact or homogenized leukocytes are incubated with glucose uniformly labeled with ¹⁴C, approximately 80 percent of the radioactivity is recovered in lactic acid. Glycolysis is inhibited by cortisol.^96,97 In some cases, the conditions under which the neutrophils are disrupted have a significant effect on the activities measured.⁹⁸ Hexokinase is the rate-limiting enzyme of glycolysis in normal neutrophils.⁹⁴ The rate of glycolysis is not altered during phagocytosis,⁹⁵ but ATP levels, normally 1.9 nmol/10⁶ cells, fall to 0.8 nmol/10⁶ cells. Both the glycogen stores of neutrophils and the glucose of the plasma can serve as the source of glucose. Galactose, mannose, and fructose can also be metabolized by leukocytes.⁹⁹ Table 60–2 shows the glycolytic and other principal enzymes of the neutrophil.

Hexose Monophosphate Shunt Pathway Neutrophils also metabolize glucose by way of the hexose monophosphate shunt,^100,101,102 thus accounting for some of the oxygen consumption of the cells. In resting cells, the amount of glucose metabolized via this route amounts to only 2 to 3 percent of the total glucose consumed by the cell.^101,102,103 The operation of the hexose monophosphate shunt, however, is of special importance to the neutrophil, because this pathway provides the NADPH needed for generation of microbicidal oxidants.

Glycogen Metabolism Neutrophils contain a large quantity of glycogen arising mostly from glucose. Little net synthesis from substrates occurs at the triose phosphate level. Glycogen turnover increases when these cells are deprived of glucose, especially if they are engaged in phagocytosis, but resynthesis occurs when adequate glucose is added.^95,104,105 During phagocytosis by glucose-starved cells, glycogen phosphorylase activity rises, but phosphorylase kinase and glycogen synthase levels remain unchanged.¹⁰⁵ Glycogen first appears in myelocytes and increases with cell maturation.¹⁰⁶

PROTEIN SYNTHESIS BY MATURE NEUTROPHILS

Mature neutrophils have been classically viewed as terminally differentiated cells without the ability to synthesize proteins. This view has changed as a result of numerous investigations in vitro and in vivo showing that neutrophils can synthesize numerous proteins (e.g., cytokines, chemokines, growth factors, interferons) potentially important to the inflammatory process and the regulation of immune reactions. Table 60–3 lists some of the proteins expressed by mature neutrophils. The database for these observations has been extensively reviewed,^107,108 and some potentially important concepts are discussed here. As is evident from this list, the diversity is impressive, but the extent of production of each protein by individual neutrophils is limited when compared to mononuclear cells. However, because neutrophils make up the majority of infiltrating cells early in an acute inflammatory process, often emigrating in massive numbers, their aggregate synthetic ability may be significant to the course of the inflammatory or healing response. In vitro, an array of stimuli have been used to induce protein expression, including lipopolysaccharide (LPS), cytokines, chemotactic factors, adhesive ligands, opsonized particles, and modulatory cytokines such as IL-10 and IL-4.

The signaling pathways leading to new protein synthesis are subjects of extensive studies and are briefly described here. Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-10 have the ability to activate signal transducer and activator of transcription (STAT) proteins in neutrophils. Both STAT-1 and STAT-3 and the upstream kinase Janus-associated kinase 2 (JAK2) are rapidly tyrosine phosphorylated.^109,110 Neutrophils express nuclear factor κB-1 (NFκB1)/p50, p65/RelA, and c-Rel. Tumor necrosis factor-α (TNF-α), IL-1β, and IL-15 lead to the rapid loss of IκBα and the concomitant nuclear accumulation of NFκB/Rel proteins. This pathway is not activated by G-CSF, GM-CSF, IL-8, or IL-10. PU.1 is expressed in mature neutrophils and constitutively binds DNA, and the AP-1 transcription factor is evident. Several of the inflammatory mediators produced by mature neutrophils are AP-1 driven (e.g., TNF, IL-1, IL-8, intercellular adhesion molecule [ICAM]).⁶⁸ Production of the CXC chemokine IL-8 by neutrophils has been extensively studied, and a wide range of stimuli can induce its expression.¹¹¹ Cytokines such as TNF-α, IL-15, IL-1β, and GM-CSF; chemotactic factors such as C5a, PAF, and leukotriene B₄ (LTB₄); particles such as monosodium urate crystals; microbial products such as LPS and zymosan; interaction with antibody and complement opsonized bacteria and yeasts; and interactions with extracellular matrix molecules such as laminin and fibronectin, all have been shown to induce synthesis of IL-8 by neutrophils. In most studies, release of significant amounts of protein from the neutrophils and synthesis of mRNA have been demonstrated. Immunocytochemistry and in situ hybridization studies have provided evidence of IL-8 production in neutrophils infiltrating inflammatory sites.

Some of the stimuli that induce expression of IL-8 also stimulate production by mature neutrophils of other proinflammatory agents, such as growth-regulated protein (GRO)-α, TNF-α, IL-1β, oncostatin M, and C-C chemokines. In addition, neutrophils may produce antiinflammatory agents such as IL-1RA and transforming growth factor (TGF)-β, and of interest is the observation that cytokines such as IL-10 may have some selectivity with regard to induction of antiinflammatory factors in neutrophils. Thus, considerable evidence exists for protein synthesis capability in neutrophils, but because this field of study is relatively new, much work remains to define the importance of the various proteins to inflammation, immune reactions and healing, the selectivity of the conditions, and disease states linked to the synthetic activities of neutrophils. In keeping with its critical role in the inflammatory process, the neutrophil’s movement from blood to tissues requires surface adhesion molecules (Table 60–4), chemotactic receptors (Table 60–5), and the requirement to phagocytize microorganisms through opsonin receptors (Table 60–6).

PHENOTYPIC CHANGES

Phenotypic changes occur in neutrophils under specific conditions.^112,113 Degranulation results in marked changes in surface expression of an array of proteins arriving at the surface from the storage pools of granules (e.g., CD11b/CD18, CD66, some β₁-integrins). These phenomena can be seen in degrees in circulating neutrophils. Exposure of neutrophils to activating factors results in surface and functional changes as a result of new synthesis (e.g., Fc region of IgG [FcγR]I following elevations in interferon [IFN]-γ) or shedding (e.g., loss of L-selectin), also seen in circulating neutrophils. Cytokines (e.g., IL-15, IL-1, TNF) induce de novo synthesis of proteins (as noted in Table 60–1) to various degrees in blood neutrophils. Substantial changes occur once the neutrophil leaves the vasculature, increasing its expression of β₁-integrins, C-C chemokine receptors (CCRs), and protein synthesis.

Evidence indicates that in response to specific combinations of cytokines (e.g., GM-CSF, TNF-α, IFN-γ), neutrophils can acquire phenotypic and functional characteristics of immature dendritic antigen-presenting cells.¹¹² Thus, any consideration of the “composition” of neutrophils requires a detailed understanding of the stage of development and the environment to which the neutrophil is exposed in vivo. The neutrophil is a remarkably versatile cell.

Gene expression profiling has provided rich insights into the capacity of the mature neutrophil to change in response to environmental stimuli. Following exposure to 10 ng/mL Escherichia coli LPS, 307 genes are activated or repressed.¹¹⁴ These changes include transcription factors, cytokines, chemokines, interleukins, surface antigens, toll-like receptors, and members of immune mediator gene families. Major changes in gene expression occur following LPS,¹¹⁵ migration in wounds,¹¹⁶ activation by phagocytosis,¹¹⁷ or during the processes of apoptosis.¹¹⁸ These findings indicate that the neutrophil is a transcriptionally active cell responsive to environmental stimuli and capable of a complex series of both early and late changes in gene expression.

The neutrophil is particularly rich in glycogen. The concentration of this complex polysaccharide has been reported to average 7.36 mg/10⁹ cells.^119,120,121 The rate of glucose metabolism by neutrophils is affected by insulin in diabetics but not in normal subjects.^122,123 Inflammatory activation of normal neutrophils stimulates glucose uptake. The plasma membrane and the membranes of the intracellular organelles are rich in lipids. Five percent of the wet weight of neutrophils is lipid, which is distributed among various classes, as shown in Table 60–7.^124,125 The rare polyphosphoinositides are of special interest as sources of inositol 1,4,5-trisphosphate (a calcium-releasing mediator) and diacylglycerol (which activates protein kinase C).^126,127 The main glycolipid of neutrophils is lactosylceramide.¹²⁸

The reduced glutathione content of neutrophils is 9.8 nmol/10⁷ cells.¹²⁹ The protein content of the neutrophil is 74.2 ± 3.1 (mean ± 1 SE [standard error]) mg/10⁹ cells. These proteins include those of the structural matrix of the neutrophil; proteins required for its locomotion, chemotactic properties, and adhesiveness; and the many granule proteins with bactericidal, hydrolytic, and inflammatory functions. Table 60–8 summarizes the unbound amino acid concentration in neutrophils.

Table 60–9 summarizes the levels of nucleotides in the neutrophils.^130,131 Neutrophils contain all the forms of RNA needed for protein synthesis.^132,133 The DNA content of neutrophils is identical to that of all other haploid cells, at 0.7 pg DNA phosphorus per cell.¹³⁴

The average folic acid content of the leukocytes of normal subjects is 0.1 mcg/mL of packed leukocytes. Approximately 20 percent of the folic acid is free and the remainder conjugated. The cocarboxylate content of neutrophils is 340 mcg/10¹¹ cells,¹³⁵ pyridoxal phosphate 0.24 to 0.38 ng/10⁶ cells,¹³⁶ thiamine 67.5 ± 4.1 mcg/100 mL,¹³⁷ ascorbic acid 16.5 ± 5.1 mg/100 mL,¹³⁷ and folate 92 ng/mL.¹³⁸