Similar to other components of the hematopoietic system, the neutrophil is ultimately derived from a pluripotent hematopoietic stem cell. The development of the myeloid stem cell is largely determined by ambient cytokines and reflected in its surface markers, morphology, and functional characteristics. The myeloblast is fully committed to the neutrophil lineage and is the first morphologically distinct cell in neutrophil development. Subsequent stages of neutrophil development occur under the influence of granulocyte colony-stimulating factor (G-CSF) and granulocyte–macrophage colony-stimulating factor (GM-CSF). Four to six days are required for maturation through the mitotic phase to the myelocyte, and 5–7 days more for the myelocyte to develop into a mature neutrophil, including the metamyelocyte and band stages, before emerging as a fully developed neutrophil. Development of neutrophils through the myelocyte stage normally occurs exclusively in the bone marrow, which is composed of approximately 60% developing neutrophils. The mature neutrophil measures 10–12 μm and has a highly condensed, segmented, multilobulated nucleus, usually with three to five lobes. Although 1011 neutrophils are generated daily, this number can rise tenfold in the setting of infection. The calculated circulating granulocyte pool is 0.3 × 109 cells/kg blood and the marginated pool is 0.4 × 109 cells/kg blood, comprising only 3% and 4% of the total granulocyte pool, respectively. The bone marrow releases 1.5 × 109 cells/kg blood/day to this pool but keeps 8.8 × 109 cells/kg blood in the marrow in reserve. An additional reserve of immature and less competent neutrophils, 2.8 × 109 cells/kg blood, is also available.
G-CSF is critically important for neutrophil production.1 Mice deficient in G-CSF show reduced neutrophil numbers and cannot upregulate neutrophil numbers in response to infection. Interestingly, G-CSF production is under the influence of IL-17, a cytokine of importance in regulation of epithelial defenses.
Granule Content and Function
(Table 30-1.) Neutrophils are characterized by cytoplasmic granules and partially condensed nuclei. Granules are first found at the promyelocyte stage.2 Primary (azurophilic) granules are the first to arise, measure approximately 0.8 μm in diameter, and contain numerous antimicrobial products including lysozyme, myeloperoxidase, and defensins.3 Primary granules are only synthesized at the promyelocyte stage. The promyelocyte gives rise to the myelocyte, the last cell of the neutrophil lineage with proliferative potential. Therefore, cytokines or agents that increase total neutrophil production must act at or before the myelocyte stage. The smaller eosinophilic secondary (specific) granules appear during the myelocyte stage. These granules measure about 0.5 μm in diameter and contain lactoferrin, collagenase, gelatinase, vitamin B12-binding protein, and complement receptor 3 (CR3; CD11b/CD18). gp91phox and p22phox comprise the specific granule component cytochrome b558, defects in which cause chronic granulomatous disease (CGD), characterized by infections with particular catalase-producing bacteria. Gelatinase also cleaves and potentiates the activity of the chemokine interleukin-8 (IL-8). Because primary granules are synthesized early and distributed to daughter cells during division, they are eventually outnumbered by about 3:1 by the specific granules, which are produced throughout the myelocyte stage.
Table 30-1 Human Neutrophil Components ||Download (.pdf)
Table 30-1 Human Neutrophil Components
Other Cytoplasmic Organelles
|• Bactericidal/permeability-increasing protein|
|• Defensins||• p15s|
|• Lysozyme||• Lysozyme|
|• Cathepsin G|
|• Proteinase 3||• Proteinase 3|
|• Phospholipase A2|
|• Cathepsin B||• Cathepsin B||Cathepsin B|
|• Cathepsin D||• Cathepsin D||Cathepsin D|
Granules fuse in a sequential fashion with incoming phagocytic vacuoles, such as those containing ingested bacteria. Secondary granules fuse to the phagosome within the first 30 seconds after ingestion and release their enzymes, many of which function best at neutral or alkaline pH. By 3 minutes after ingestion, the primary granules have fused to the phagolysosome leading to rapid lowering of the intravacuolar pH. For objects too large to be ingested, or certain stimuli, degranulation to the cell surface occurs with release of granule contents into the surrounding environment. This can be inferred by detection of lactoferrin levels in blood.
An example of disordered granule biogenesis is Chédiak–Higashi syndrome (CHS), a rare autosomal recessive disorder with abnormal pigmentation due to a generalized abnormality of primary granule and lysosome formation (see Chapter 143).
Neutrophil-Specific Granule Deficiency
Neutrophil-specific granule deficiency is a rare, autosomal recessive condition clinically characterized by a profound susceptibility to bacterial infections. There is a paucity or absence of neutrophil-specific granules, specific granule proteins (e.g., lactoferrin) and their respective messenger RNAs, and very low levels of the primary granule products defensins and their messenger RNAs. Specific granule deficiency is due to loss of the transcriptional factor CCAAT/enhancer binding protein ε (CEBPε), which is essential in normal myeloid development. Acquired abnormalities of neutrophil granules are seen in some myeloid leukemias, in which primary granule contents may be aberrantly accumulated (e.g., Auer rods in acute myelogenous leukemia).
Chemoattractants and Chemotaxis
Metchnikoff discovered over a century ago that neutrophils move toward very slight gradients of chemical signals, now termed chemoattraction. The “classic” chemoattractants are N-formylmethionyl-leucyl-phenylalanine (fMLF), complement factor 5a (C5a), leukotriene B4, and platelet-activating factor (PAF). More recently, chemokines (chemoattractant cytokines), a class of small (<10 kDa) extremely active chemoattractant proteins have been identified (see Chapter 12).
IL-8 is a potent chemoattractant and neutrophil activator (see Chapter 12). Its blockade by neutralizing antibody in rabbit models of pulmonary inflammation prevented neutrophil accumulation and reperfusion injury, confirming its central role in neutrophil recruitment to sites of inflammation. Cellular sources for IL-8 include neutrophils, monocytes, T cells, B cells, natural killer cells, basophils, eosinophils, fibroblasts, endothelial cells, keratinocytes, and smooth muscle. Exudative neutrophils are particularly effective at synthesizing IL-8, probably through a calcium-regulated mechanism.
Exuberant neutrophil function is implicated in Sweet syndrome (acute febrile neutrophilic dermatosis) as shown by the frequent association of the syndrome with hematologic malignancies, dysmyelopoiesis, and sometimes with the use of G-CSF (see Chapter 32).
The classic chemoattractants and chemokines use similar receptors that have in common seven transmembrane regions, an extracellular amino terminal domain, and transduce their signals through pertussis toxin-sensitive heterotrimeric G proteins. These are the same type of receptors through which light, neuropeptides, and neurotransmitters signal. Chemoattractant receptor signaling entails exchange of bound guanine diphosphate for guanine triphosphate by the α subunit of the heterotrimeric G protein, which in turn leads to the dissociation of the β–γ subunit of the complex, stimulation of phospholipase C, and generation of inositol triphosphate and diacylglycerol from phosphatidylinositol bisphosphate. Inositol triphosphate stimulates the release of calcium from intracellular “calciosomes,” whereas diacylglycerol activates protein kinase C. Extracellular calcium also enters the cell, preparing it for subsequent movement, generation of oxidants, and secretion of vesicles.
Ras-related guanosine triphosphatases of the ρ superfamily are also involved in actin cytoskeletal regulation and adhesion. Phosphatidylinositol 3-kinase is activated by rho and ras and is necessary for the neutrophilic respiratory burst, adhesion, endothelial transmigration, and chemotaxis. Mitogen-activated protein (MAP) kinases are serine/threonine kinases that include p38, Erk1, Erk2, and Jnk and are involved in neutrophil signaling and adhesion. Inhibition of p38 impairs chemotaxis and tumor necrosis factor (TNF)-α-mediated superoxide production, adhesion, and release of secondary granules. Erk activation is required for neutrophil homotypic aggregation. Salicylates inhibit neutrophil adhesion through Erk inhibition.
Several chemokine receptors also function as requisite human immunodeficiency virus (HIV) coreceptors. Mutations in the chemokine receptor CCR5 that are protective against HIV infection led to development of chemokine receptor blockers for treatment of HIV infection (maraviroc and vicriviroc).
Neutrophils exist as free-flowing (those which are sampled on blood drawing) and marginated cells (those which are attached to the endothelium or are traversing the lung, skin, or other tissues). Neutrophils rolling along the endothelium recognize sites of activation (e.g., chemokine expression), adhere to those sites, and traverse the endothelium to enter the tissue and fight infection. Leukocyte physical interaction with endothelium and other leukocytes is mediated by integrins, selectins, and intercellular adhesion molecules (ICAMs; Fig. 30-1).
Tissue trafficking of neutrophils. The interaction of selectins and integrins on the leukocyte surface with their endothelial addressins is depicted (neutrophils on the top and eosinophils on the bottom). The leukocytes are in a laminar flow pattern. Following tissue signals that activate endothelial selectins and glycoproteins, a subset of leukocytes begin to roll along the vascular wall. For the neutrophil, this typically involves the interaction of CD15s with CD62P (P-selectin) and CD62E (e-selectin) on the endothelial cell or CD62L (L-selectin) with CD34 or glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) on the endothelial cell. Abnormalities in fucosylation of CD15s cause leukocyte adhesion deficiency 2 (LAD2). Activation of leukocytes by chemoattractants leads to the expression of integrins with higher avidity conformations. In the case of the neutrophil, the relevant integrins are the β2 family members [lymphocyte function-associated antigen 1 (LFA-1), Mac-1, and CR4]; whereas eosinophils express a more limited number of β2 family members (LFA-1 and Mac-1), but also express a β1 integrin VLA-4 and β7 integrin (α4β7). The β2 integrins bind to ICAM-1 and ICAM-2, whereas the β1 and β7 integrins bind VCAM-1 and mucosal addressin cell adhesion molecule-1 (MadCAM-1), respectively. Defects in CD18, the β chain of the β2 integrins, lead to the inability of neutrophils to exit the circulation at sites of infection and is called leukocyte adhesion deficiency 1. Interestingly, eosinophils, monocytes, and lymphocytes are observed at sites of infection in these patients, as these cells can use β1 integrins to mediate the firm adhesion step required for leukocyte transendothelial migration. See figure in Chapter 31 for a similar analysis of eosinophil function.
Elaboration of chemoattractants or display of activation markers on endothelium triggers leukocyte high affinity binding by β2 integrins, heterodimeric surface molecules largely stored in the secondary granules of neutrophils that are displayed on the cell surface upon leukocyte activation. There are three β2 integrin heterodimers comprised of different α chains, CD11a, -b, and -c, and a common β chain, CD18. Each CD11/CD18 complex has separate and overlapping activities. CD11a/CD18 [leukocyte function-associated molecule 1 (LFA-1)] binds to other leukocytes and mediates tight adhesion to the endothelium through ICAM-1 and ICAM-2. CD11b/CD18 (Mac-1, Mo-1, or CR3) binds to the inactivated form of the third component of complement (C3bi) and thereby facilitates complement-mediated phagocytosis. CD11b/CD18 also binds to bacteria directly, to fibrinogen, and to endothelium through ICAM-1. The divalent cations Ca2+ and Mg2+/Mn2+ mediate adhesion through β2 integrin “A” domains containing a metal ion-dependent adhesion site. CD11b/CD18 may also induce the expression of the β1 integrin very late antigen 6 [VLA-6 (CD49f/CD29)], derived from neutrophilic granules, to aid in tissue infiltration. The integrin-associated protein (CD47), expressed on neutrophils and endothelial and epithelial cells, is also involved in the transendothelial and transepithelial migration of neutrophils.4 Metalloproteinases may be involved in cleavage of L-selectin, allowing neutrophil migration through the basement membrane.
Absence of CD18 causes lack of CD18/CD11 heterodimers and is called leukocyte adhesion deficiency type 1 (LAD1). Neutrophils lacking CD18 roll normally along the endothelium but are unable to stick to the vessel wall or exit the circulation after chemotactic stimulation. Absence of LFA-1 (CD11a/CD18) makes neutrophils unable to bind tightly to and traverse activated endothelium to infected areas. Therefore, LAD1 patients have chronic neutrophil leukocytosis, partly from inability of neutrophils to bind tightly to endothelium and exit the circulation, thus leading to a reduction in the marginated pool and an increase in the circulating pool of neutrophils. Poor neutrophil penetration to sites of bacterial invasion leads to necrotic ulcers that lack neutrophils on biopsy. Absence of Mac-1 (CD18/CD11b or CR3) leads to inability to perform complement-mediated phagocytosis, although antibody-mediated phagocytosis remains intact.
LAD1 is diagnosed by fluorescent-activated cell sorting (FACS), which shows levels of CD18 and its coexpressed molecules CD11a, CD11b, and CD11c. Severe LAD1 (<0.5% of normal protein expression) is a disorder manifested by delayed umbilical stump separation, umbilical stump infection, persistent leukocytosis in the absence of active infection (>15,000/μL), and severe, destructive periodontitis with the loss of teeth and alveolar bone. Recurrent infections of the skin, upper and lower airway, bowel, perirectal area, and septicemia are common and usually due to Staphylococcus aureus or Gram-negative rods. Severe LAD1 patients should receive bone marrow transplantation in early childhood. Moderate LAD1 (2.5%–10.0% of normal protein expression) patients tend to be diagnosed later in life and have fewer life-threatening infections. Leukocytosis, delayed wound healing, and periodontal disease are still common.
Neutrophil “rolling” along the endothelium is mediated through selectins, surface glycoproteins on the endothelium, and sialyl-Lewis X (CD15s), a surface glycoprotein on neutrophils (see Fig. 30-1). Endothelial cells express e-selectin (CD62E) and P-selectin (CD62P), whereas leukocytes express L-selectin (CD62L). Although endothelial e-selectin and P-selectin bind to the sialyl-Lewis X (CD15s) antigen on neutrophils, the neutrophil molecule L-selectin probably binds to distinct antigens on endothelium, including CD34, and is highly sensitive to glycosylation. L-selectin is shed by neutrophils on activation, thereby allowing neutrophil migration into sites of inflammation. Cross-linking of L-selectin on neutrophils results in superoxide generation and may result in upregulation of TNF-α, IL-8, and tyrosine phosphorylation and activation of MAP kinase. LAD2 is the disease that occurs when CD15s is improperly fucosylated. These patients have neutrophilia, recurrentpulmonary, periodontal, and cutaneous infections, and abnormal chemotaxis. The defect is in the gene SLC35C1, a GDP-fucose transporter. Because the disease affects sugar transport it is now classified as a congenital disorder of glycosylation (CDG), and known as CDG IIc. Interestingly, although infections are common early in life, LAD2 (CDG IIc) patients appear to improve with age. Administration of oral fucose to some patients with LAD2 has been inconsistently therapeutic. Most patients also have mental retardation, short stature, distinctive facies, and the Bombay (hh) blood phenotype, indicating the multiple systems in which fucosylation is critical. LAD3 (previously known as LAD1 variant) is associated with a syndrome like Glanzmann's thrombasthenia, and is due to mutations in KINDLIN3 (FERMT3), a molecule responsible for β1, β2, and β3 integrin activation in leukocytes and platelets leading to recurrent infections and bleeding.
The CD18 integrin pathway is critical for inflammation in the skin, but not necessary for accumulation of neutrophils in the lung or peritoneum. Therefore, although CD18-dependent pathways are critical for cutaneous inflammation, CD18-independent pathways exist for pulmonary and peritoneal inflammation. Further, in the setting of congenital absence of CD18, compensatory pathways exist in the mouse to respond to peritoneal inflammation.
Phagocytosis is the culmination of object recognition, binding, signaling, adherence, cytoskeletal remodeling, engulfment, and membrane fusion. Two mechanisms are well characterized, one mediated by immunoglobulin and the other mediated by complement.
Receptors for the Fc portions of immunoglobulin G [IgG (FcγR)] are present on many components of the cellular immune response, including neutrophils, monocytes, macrophages, eosinophils, and basophils. FcγRI (CD64) is a receptor for IgG1 and IgG3 on monocytes, macrophages, and eosinophils and is upregulated on neutrophils after interferon-γ (IFN-γ) stimulation. FcγRII (CD32) binds IgG with rather low affinity, and prefers IgG1 and IgG3. FcγRIII (CD16) binds IgG1 and IgG3 with intermediate affinity. Neutrophil FcγRIIIB is bound to the membrane through a glycan phosphatidyl inositol linkage, which is largely cleavable by phosphatidyl inositol-specific phospholipase C. As FcγRIIIB has no cytoplasmic domain, its role in signal transduction may be through association with FcγRII. Cross-linking of these receptors by antibody leads to rapid engulfment of targets with the release of granule contents and oxygen metabolites into the phagolysosome. If the target is too large, degranulation occurs against the antibody-coated surface.
The complement receptors CR1 and CR3 are expressed on the surfaces of neutrophils, eosinophils, and basophils. Complement receptor 1 is designated CD35 and is found on many cell types. It binds C3b and enhances its degradation to C3dg by factor I, thereby removing that molecule from further activation of the alternative pathway. CR3, CD11b/CD18, binds iC3b and fibrinogen as well as certain bacteria, parasites, and fungi. CR1 and CR3 are not per se able to stimulate phagocytosis, but in the presence of a second signal, such as one given through FcγR or by cytokines, phagocytosis proceeds.
Neutrophil granules contain enzymes and proteins for killing ingested bacteria and fungi. Some of these bactericidal mechanisms are dependent on the generation of oxygen metabolites for microbicidal activity, but others are not. In addition to mobilizing their own resources, neutrophils produce a multitude of cytokines that stimulate and attract other phagocytes as well as lymphocytes.
Bactericidal/permeability-increasing protein (BPI) is a highly potent antibacterial granule protein synthesized and stored in the primary granules. It is a highly basic (isoelectric point >9.6) protein of 452 amino acids and approximately 58 kDa. Sequence homology to lipopolysaccharide (LPS)-binding protein, a critical endotoxin binding acute-phase reactant, suggests that it acts by directly binding to LPS. BPI is cytotoxic to Gram-negative bacteria at concentrations as low as 10−9 M, but much less effective against Gram-positive organisms. Binding to LPS leads to insertion of BPI into the outer membrane of the organism and eventual insertion into the inner membrane. Arrest of bacterial growth is solely dependent on the N-terminal half of the molecule. The C-terminal fragment serves as an anchor to the membrane. BPI appears to act inside the phagolysosome. Not all Gram-negative rods are sensitive to BPI, especially Burkholderia (Pseudomonas) cepacia and Serratia marcescens, pathogens in patients who lack oxidative killing.
Defensins are small (<4 kDa) cationic proteins in the primary granules of neutrophils involved in killing ingested Gram-positive and Gram-negative bacteria. Defensins are synthesized during the promyelocyte/myelocyte stage as prepropeptides, stored predominantly in a dense subset of the primary granules as propeptides, and released during neutrophil degranulation. Defensins prefer an actively metabolizing target and can kill transformed mammalian cells as well as prokaryotes and yeasts. Defensins and BPI act synergistically against Gram-negative bacteria. Defensins are reduced in CHS but absent in specific granule deficiency.
Proteinase 3 is the antigen against which the antineutrophil cytoplasmic antibody is directed in Wegener granulomatosis (see Chapter 164). It is found predominantly in primary granules but is also in secondary and secretory granules. Synthesis and surface display are upregulated by cytokines such as TNF-α. Elastase is a myeloid serine protease in primary granules that has remarkable roles in host defense. Mice with elastase defects have decreased resistance to Gram-negative bacteria. However, human neutrophil elastase deficiency causes the syndromes of severe congenital neutropenia and cyclic neutropenia. Phospholipase A2 is also found in neutrophil granules, where its various isoforms act both directly and synergistically with BPI to kill intracellular bacteria.
Lactoferrin is an iron-binding protein present in specific granules and in mucosal secretions. When released into the phagolysosome, lactoferrin binds iron and inhibits the growth of phagocytosed bacteria and some fungi. The mechanisms through which lactoferrin exerts its antimicrobial action probably include the depletion of iron from an organism's environment, but iron binding-independent antimicrobial activities and direct immunomodulatory effects are also reported. Lactoferrin is readily released into the circulation after burns and experimental endotoxemia, presumably from neutrophil degranulation.
Neutrophils can produce toxic oxygen metabolites through the reduced nicotinamide dehydrogenase phosphate (NADPH) oxidase complex, which typically is assembled in the wall of the phagolysosome. The NADPH oxidase catalyses the addition of an electron to molecular oxygen, leading to the formation of superoxide anion. Superoxide in turn is converted to hydrogen peroxide by superoxide dismutase. Inside the phagolysosome, the primary granule component myeloperoxidase converts hydrogen peroxide to hypohalous acid by the addition of a halogen (chloride in neutrophils forms bleach, bromide in eosinophils). The NADPH oxidase is a multiprotein complex, which is maintained in separate membrane-bound and cytosolic compartments. On cell activation, the cytosolic components translocate to the phagolysosome membrane, resulting in an active NADPH oxidase, which can produce the respiratory burst.
The components of the NADPH oxidase are named phox (phagocyte oxidase) proteins. p22phox and gp91phox are the α and β chains, respectively, of the cytochrome b558 complex, which resides in the wall of the secondary granule. The cytosolic compartment contains three factors, p47phox, p67phox, p40phox and the small guanine nucleotide (guanine triphosphate) binding protein rac. Assembly of the NADPH complex is caused by diverse stimuli. p47phox and p67phox contain src-homology type 3 domains (SH3 boxes), which bind to proline-rich targets in themselves and in other members of the NADPH complex. Stimulation leads to structural changes in p47phox that promote its interaction with p22phox through p47phox SH3 domains and p22phox C-terminal proline-rich sequences.
Pathologic mutations in the five required components impair the generation of phagocyte superoxide and cause CGD, a disease characterized by recurrent life-threatening infections with bacteria and fungi and exuberant granuloma formation (see Chapter 143).5
When neutrophils are overrecruited into neutrophilic dermatoses, they can produce reactive oxygen intermediates, activate proteinases, and release chemotactic cytokines, which can contribute to tissue injury and inflammation. Therefore, it makes sense to aim treatment to suppress the generation of reactive oxygen intermediates, to inhibit neutrophil adhesion and chemotaxis, and to suppress the release of lysosomal enzymes and chemotactic factors.
Dapsone is often used for chronic neutrophilic dermatoses such as dermatitis herpetiformis, subcorneal pustular dermatosis, or erythema elevatum diutinum (see Chapter 225). Dapsone suppresses neutrophil adherence and subsequent migration, is a successful scavenger of reactive oxygen intermediates, and interferes with the myeloperoxidase-halide system and leukocyte-mediated cytotoxicity. Thalidomide reduces inflammation through inhibition of TNF-α and subsequent neutrophil–endothelial adhesion and reactive oxidant generation (see Chapter 235). Dapsone and thalidomide both inhibit the proinflammatory cytokine TNF-α, which activates neutrophils through upregulation of complement receptors (CR3, CR4) and endothelial adhesion molecules, ICAM-1 and e-selectin. Colchicine inhibits neutrophil chemotaxis, release of lysosomal enzymes, and production of reactive oxidants and is therefore considered for Sweet syndrome and neutrophilic bullous dermatoses. Antibiotics such as tetracyclines, macrolides, and metronidazole also have antioxidant properties and interfere with neutrophil chemotaxis. In addition to its antioxidant activity, sulfasalazine induces neutrophil apoptosis and enhances adenosine release at sites of inflammation, making it especially useful in pyoderma gangrenosum.