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ASH 50th anniversary review
The phagocytes: neutrophils and monocytes
David C. Dale,1 Laurence Boxer,2 and W. Conrad Liles3
1Department
of Medicine, University of Washington School of Medicine, Seattle; 2Women’s Hospital, University of Michigan, Ann Arbor; and 3University of
Toronto, Toronto General Hospital, ON
The production and deployment of phagocytes are central functions of the hematopoietic system. In the 1950s, radioisotopic studies demonstrated the high
prodution rate and short lifespan of neutrophils and allowed researchers to follow the monocytes as they moved from
the marrow through the blood to become
tissue macrophages, histiocytes, and dendritic cells. Subsequently, the discovery
of the colony-stimulating factors greatly
improved understanding the regulation
of phagocyte production. The discovery
of the microbicidal myeloperoxidaseH2O2-halide system and the importance
of NADPH oxidase to the generation of
H2O2 also stimulated intense interest in
phagocyte disorders. More recent research has focused on membrane receptors and the dynamics of the responses
of phagocytes to external factors including immunoglobulins, complement proteins, cytokines, chemokines, integrins,
and selectins. Phagocytes express tolllike receptors that aid in the clearance of
a wide range of microbial pathogens
and their products. Phagocytes are also
important sources of pro- and antiinflammatory cytokines, thus participating in host defenses through a variety of
mechanisms. Over the last 50 years, many
genetic and molecular disorders of phagocytes have been identified, leading to
improved diagnosis and treatment of conditions which predispose patients to the
risk of recurrent fevers and infectious
diseases. (Blood. 2008;112:935-945)
Introduction
The importance of “laudable pus” in the response to injury and
infection was recognized in ancient times. In the book The Healing
Hand: Man and Wound in the Ancient World, Majno wrote:
Pus is therefore a noble substance: it is made of brave cells that
never sneak back into the blood vessels to escape; they all die in the
line of duty. Note also the double meaning of suppuration: it
indicates that there is an infection, but also that the body is fighting
it well. The outcome of the battle can be predicted, to some extent,
from the aspect of the pus, as was observed even in ancient times.
The whitish, creamy kind (and therefore rich in polys) is “preferable,” because it indicates that an infection is being fought
effectively. Hence its ancient Latin name of pus bonum et laudable,
“good and laudable pus.” Thin or malodorous pus suggests a poor
defense of especially vicious bacteria.1p4
Much of what we know about the cellular components of the
inflammatory response was gradually discovered in the 19th and
early 20th centuries.2 Landmark reports include the following:
(1) In 1841, William Addison compared colorless corpuscles in
the blood with those of inflamed tissues and proposed that
leukocytes get to the tissues by diapedesis.3 (2) In 1873,
Cohnheim reported the margination of leukocytes along vessel
walls and leukocyte protrusion and extravasation through the
vessel wall (ie, transmigration) to reach the extravascular
tissues.4 (3) In 1880, Ehrlich developed the staining techniques
that facilitated identification of developing phagocytes in the
bone marrow, blood, and tissues.5 Using Erhlich’s stains,
leukocyte counting and microscopic observations of blood cells
became common, leading to clear definitions of normal counts,
leukemia, leukopenia, neutropenia, and agranulocytosis.6 (4) In
1884, Metchnikov microscopically observed the phagocytic
process, first in ameboid cells of the marine sponge and later in
higher species. He is credited with the origin of the terms
phagocyte and phagocytosis.7 (5) In 1904, Arneth introduced the
counting of the lobes of neutrophils as an index for the maturity
of neutrophils and coined the term shift to the left to describe an
abnormal number of immature neutrophils on a blood smear.8
(6) In 1911, Schilling developed the current format of the
leukocyte differential count. He coined the term, regenerative
shift, to designate the outpouring of neutrophils in response to
infection. The term, degenerative shift, was used to describe a
failure of this response.9 (7) In 1942, the first edition of
Wintrobe’s text, Clinical Hematology, was published and contained clear descriptions and excellent illustrations of normal
and abnormal leukocytes. The text described neutrophil chemotaxis, phagocytosis, the proteolytic enzymes of the neutrophil
granules, and “toxic granules.” Wintrobe also described physiological leukocytosis and the changes in leukocyte, neutrophil,
and monocyte counts with age. He gave excellent descriptions
of leukemia, agranulocytosis, infectious mononucleosis, and
leukopenia with splenomegaly.10 (8) In 1948, Beeson described
the release of “endogenous pyrogen” from leukocytes exposed
in vitro to particles. This pioneering study was instrumental in
our understanding the association of fever, leukocytosis, and
neutrophilia. Furthermore, findings from his studies provided
the fundamental basis for future studies on the physiological
effects of endogenous cytokines.11
The fifth edition of Wintrobe’s Clinical Hematology, published
soon after the beginning of Blood, had extensive chapters on
phagocytes. The text described their abundant glycogen in the
cytoplasm of neutrophils and aerobic glycolysis with oxygen
consumption, glucose utilization, and lactic acid. It also described
leukocyte alkaline phosphatase and its variation in chronic myeloproliferative diseases.
Submitted December 13, 2007; accepted February 19, 2008; DOI 10.1182/blood2007-12-077917.
© 2008 by The American Society of Hematology
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
935
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936
DALE et al
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
Figure 1. Model for the total blood granulocyte pool (TBCG) in normal subjects.
CGP indicates circulating granulocyte pool; MGP, marginal granulocyte pool; and
BM, bone marrow. Illustration by Kenneth Probst. This research was originally
published in Blood. Cartwright GE, Athens JW, Wintrobe MM. The kinetics of
granulopoiesis in normal man. Blood. 1964;24:780-803. ©American Society of
Hematology.16
By 1960, hematology sections in general medical texts, such as
Harrison’s Principles of Internal Medicine, contained specific
sections on leukocyte disorders, particularly leukemia, agranulocytosis, and the diseases causing pancytopenia.
This brief review outlines some of the pivotal discoveries in
phagocyte biology during the past 50 years. These discoveries have
significantly increased our understanding of host defense and
immunity, leading to important advances in the practice of medicine.
Neutrophils (polymorphonuclear leukocytes)
Production and kinetics
Radioisotopic tracers were first introduced to study the origin and
fate of hematopoietic cells in the early 1950s.12 Results from this
line of investigation and the growing understanding of hematopoietic stem cells by Osgood, Lajtha, Fliedner, McCullough, and
others provided important foundations for many major advances in
hematology and oncology in the 1960s and the years that followed.
At the beginning of this era, studies of bone marrow and blood
counts after radiation-induced injury or administration of early
chemotherapeutic agents, such as nitrogen mustard, suggested that
neutrophil precursors have a very high proliferative rate and that
mature neutrophils have a short lifespan. Craddock et al used 32P to
investigate replicating marrow cells in dogs and defined the mature
nondividing compartment of neutrophils in the normal marrow, as
well as the effects of leukopheresis, endotoxin, and inflammation
on developing and mature neutrophils in the marrow.13 Cronkite
and Fliedner quantified the high proliferative rate of neutrophil
precursors using tritiated thymidine.14 In this same time period,
Athens et al used 32P-diisopropylfluorophosphate (32P DFP), which
binds irreversibly to the serine proteases of the neutrophil granules,
to investigate and quantify the production and fate of blood
neutrophils in dogs and in humans.15 These studies confirmed early
reports that neutrophils have a short half-life and high turnover rate
in peripheral blood and that the circulating and marginal pools of
blood neutrophils are in a dynamic equilibrium16 (Figure 1).
Quantitation of marrow production of neutrophils and monocytes took another step forward when Finch et al, using radioactive
iron to determine the erythropoietic mass,17 and his trainees,
Dancey, Dubelbeiss, and coworkers quantified the absolute and
relative size and turnover of myeloid cell compartments in the
marrow and described the “effectiveness” of production based on
the ratio of marrow production versus peripheral cell turnover
(Dancey and Deubelbeiss18 and Deubelbeiss et al19). Price et al then
Figure 2. Colony assay led to the identification of the colony-stimulating
factors beginning in the late 1960s. “Feeder” layers, containing various types of
white blood cells in a semisolid medium, were placed in a small laboratory dish (1).
Bone marrow cells (including stem cells) were added to form a second layer (2).
When the dish was incubated, colonies of white blood cells formed in the second
layer (3). The colonies were counted and the cells identified (4). When the contents of
the first layer were varied, different types of colonies formed, implying the existence of
a range of colony-stimulating factors. Illustration by Kenneth Probst. Adapted with
permission from original artist Patricia J. Wynne. Golde DW, Gasson JC. Hormones
that stimulate the growth of blood cells. Sci Am. 1998;259:67.25
extended these studies and showed that patients with chronic
neutropenia have “ineffective production” with cell loss along the
developmental pathway.20 This work presaged the concept of
accelerated apoptosis as a common mechanism for chronic neutropenia and myelodysplasia. Although kinetics and turnover of
radiolabeled blood cells are now rarely done, they were instructive
in the 1990s in the development of clinical applications and
understanding of the effects of the hematopoietic growth factors,
granulocyte colony-stimulating factor (G-CSF), and granulocytemacrophage colony-stimulating factor (GM-CSF).21,22 Nuclear
medicine studies with labeled cells are also now frequently used to
detect occult infections and abscesses.
Hematopoietic growth factors and the regulation of neutrophil
production
Prior to the 1960s, there were many efforts to define leukopoietins,
the myeloid equivalent of erythropoietin, but essentially all of these
efforts were unsuccessful. In the mid 1960s, Bradley and Metcalf23
in Australia and Pluzink and Sachs24 in Israel independently
developed in vitro culture techniques for hematopoietic cells and
discovered the colony-stimulating factors (Figure 2).25 It was soon
learned that these factors were endogenously produced and released during neutropenia and infection and after endotoxin
administration. Subsequently, rapid advances in molecular biology
during the late 1970s allowed cloning of the genes for these growth
factors and their receptors. These studies led to fundamental
insights into the mechanisms responsible for physiological regulation of neutrophil production. Targeted genetic disruption (“knockout”) experiments in mice clearly showed that G-CSF and its
receptor are essential for the maintenance of normal levels of blood
neutrophils,26,27 similar to findings in dogs made neutropenic by
immunologic neutralization of their endogenous G-CSF.28 Moreover, G-CSF was demonstrated to play a critical role in the
leukocytosis caused by bacterial infection.29
Therapeutically, the development of the colony-stimulating
factors as therapeutic agents has had a major impact on the practice
of hematology and oncology. Both G-CSF and GM-CSF have a
multitude of pharmacological effects, including increasing the
proliferative activity of progenitor cells, shortening the time for
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BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
THE PHAGOCYTES: NEUTROPHILS AND MONOCYTES
937
neutrophil production and maturation in the marrow, accelerating
the release of maturing cells from the marrow to the blood,
augmenting the production of neutrophil granule proteins, and
stimulating the release of proteases and perhaps other constituents
from the cells to their surroundings. Somewhat serendipitously, it
was learned that these factors, as part of their effect to expand the
hematopoietic tissue mass and the production and deployment of
phagocytes, stimulate the release of progenitor cells from the
marrow to the blood. The harvest of these cells (ie, CD34⫹
hematopoietic stem cells) by leukapheresis has dramatically changed
the practice of hematopoietic stem cell transplantation.
Neutrophil granules and granule-associated proteins
The presence of granules in neutrophils, monocytes, and eosinophils was recognized by Metchnikov and Ehrlich,6,7 and the
proteins associated with neutrophils were first defined through
biochemical and histochemical studies beginning early in the 20th
century. In 1952, Chediak, a Cuban physician, described patients
with an autosomal recessive disease with several distinctive
characteristics, including abnormal leukocyte granules.30 This
disease subsequently became known as the Chediak-HigashiSteinbrinck syndrome. In pioneering work first reported in the early
1960s, Cohn and Hirsch, building on earlier studies of de Duve et
al,31 isolated and characterized granules from rabbit neutrophils
and described the fusion of the neutrophil granule with ingested
particles to form a “digestive vacuole” or “phagosome.”32 Hirschhorn and Weissmann then extended these studies to human
neutrophils,33 thus providing a dynamic description of the process
of phagocytosis described earlier by Metchnikov.7
Advances in electron microscopy in this same era allowed
dissection of the phagocytic process, and phase contrast microscopy allowed visualization of the killing of microbes.34,35 Extension of this work has defined organisms that are relatively readily
killed by neutrophils and monocytes, such as streptococci and
yeast, and microorganisms that are relatively protected and survive
in phagocytes, such as Mycobacterium and Salmonella.
Purification, quantification, and understanding of the role of
each of the phagocyte granule proteins have proved a complex task.
From the work of Bainton et al, it was learned that granule proteins
are produced in sequence, with the earliest proteins produced in
myeloid progenitors and packaged in primary granules.34 Subsequently, phagocytes produce proteins that are packaged in secondary and tertiary granules, under control of genes that in turn are
regulated by distinct transcription factors.36
Neutrophil granules serve as reservoirs for digestive and
hydrolytic enzymes prior to delivery into the phagosome. Pioneering studies by Spitznagel,37 Elsbach and Weiss,38 and Ganz et al39
indicated that azurophilic granule contents possess microbicidal
activity and may play an important role in the tissue destruction
observed during inflammatory reactions.
Probably because of their overlapping functions, natural deficiencies or “knockouts” of the granule proteins are not necessarily
associated with enhanced susceptibility to infection. The condition
termed specific granule deficiency, a disorder in which all the
secondary granules of the neutrophil are absent secondary to the
functional loss of the transcription factor C/EBP ⑀40 or Gfi-I,41 is
associated with recurrent bacterial infections caused by Staphylococcus aureus and Pseudomonas spp. In contrast, congenital
deficiency of myeloperoxidase from the primary granules of
neutrophils is generally not associated with an increased risk of
serious infections.42 More recently, researchers found that mutations in the gene for another primary granule protein, neutrophil
Figure 3. The MPO-H2O2-chloride antimicrobial system (taken from Goebel and
Dinauer50). NADPH indicates reduced nicotinamide adenine dinucleotide phosphate; O2, superoxide anion; and HOCI, hypochlorous acid. Illustration by Kenneth
Probst. Adapted with permission from Journal of Leukocyte Biology. Klebanoff SJ.
Myeloperoxidase: a friend and foe. J Leukoc Biol. 2005;77:598-625.46
elastase or ELA-2, are the cause for most cases of cyclic and severe
congenital neutropenia.43,44
Microbicidal mechanisms of neutrophils
The killing of microbes is a critical physiological function of
phagocytes. How this occurs was perhaps the most interesting
and important observation related to these cells of the last half
century. Before the 1950s, the general aspects of the process of
phagocytosis—from the rich glycogen supply of the neutrophil
cytoplasm to the enzymatic contents of the neutrophil granules—
were already recognized. In the early 1950s, Valentine and Beck
described glycolysis by leukocytes45 and Sbarra and Karnovsky
described the burst of glycolysis that occurs associated with
phagocytosis.46 Klebanoff then discovered that myeloperoxidase, known from work in the 1920s to be released from
granules during phagocytosis, was involved in generating
hydrogen peroxide and proposed that it is potentially the critical
antimicrobial substance generated within neutrophils.47 Further
experimentation clarified that another oxidase, NADPH oxidase,
also played an important role in the generation of hydrogen
peroxide and other reactive oxygen species.48 This observation
led Klebanoff to pursue further experiments demonstrating that
hydrogen peroxide interacted with myeloperoxidase and a
halide, predominantly chloride, to generate the potent antibacterial substance, hypochlorous acid, within the phagosome49
(Figure 3). Although phagocytes have other microbicidal mechanisms, including antimicrobial peptides (eg, defensins) and
broadly acting proteases, phagocytosis with generation of
reactive oxygen species and hypochlorous acid is still regarded
as the critical killing mechanism for most invading pathogens.51,52
It had been known since 1932 that a marked increase in
neutrophil oxygen consumption, termed the respiratory burst,
occurred during phagocytosis.53 Stimulated neutrophils oxidize
NADPH through a reaction yielding hydrogen peroxide.54 The
clinical significance of these findings was recognized by Baehner
and Nathan,55 Holmes et al,56 and Quie et al57 who observed that
neutrophils from chronic granulomatous disease (CGD) patients
failed to generate the products of the respiratory burst.
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DALE et al
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
Figure 4. Model of NADPH oxidase assembly.
Activation/phosphorylation (P)-induced conformational changes in p47phox release autoinhibitory interactions to unmask essential binding domains and
exposure of PX domains that facilitate membrane
targeting and binding of SH3-mediated and non–SH3mediated binding events. Final interaction of the
p67phox and Rac with flavocytochrome b induces
conformational change, resulting in electron flow.
Illustration by Kenneth Probst. Adapted with permission from Journal of Leukocyte Biology. Quinn MT,
Gauss KA. Structure and regulation of the neutrophil
respiratory burst oxidase: comparison with nonphagocyte oxidases. J Leukoc Biol. 2004;76:760-781.71
The neutrophil metabolic burst and chronic granulomatous
disease
Following the discovery of the importance of NADPH oxidase,
mentioned above, investigators elucidated its components and
the effects of mutations in the NADPH complex over a period of
several decades.58 Segal et al59 and Segal and Jones60 identified a
cytochrome within phagocytic vacuoles that was missing or
functioned abnormally in cell homogenetics from CGD patients.
It was referred to as cytochrome b558 and was later found to be
composed of a heavy and light chain.59 The gene for X-linked
CGD at p2.1 was identified by the first use of positional cloning
by the Orkin laboratory (Royer-Pokora et al61), and in complementary studies Parkos et al62 and Segal63 purified the peptide
constituents of cytochrome b, which led to the identification of
the gene product as the heavy chain of cytochrome b558 that was
lacking in X-linked CGD. Further studies by Roos documented a
number of different mutations responsible for X-linked CGD.64
The disease caused by mutations in NADPH oxidase, CGD, was
first described in 1957 by 2 groups, Berendes et al65 and Landing
and Shirkey.66 In 1967, Quie et al reported that a microbicidal
defect in the neutrophils of affected children caused recurrent
bacterial and fungal infections, associated with early mortality.67 It is now recognized that CGD can be caused by genetic
alterations in one of the components of NADPH oxidase,
including gp91phox, p47phox, p67phox, and p22phox. Nunoi et al68
and Volpp et al69 identified 2 forms of autosomal CGD in which
either a 47-kDa or a 67-kDa protein was genetically altered.
Subsequently, defects in the light chain of cytochrome b558
known as p22phox were found to account for some CGD cases.59
The lack of function of any of the 4 components of the NADPH
oxidase leads to a failure to generate hydrogen peroxide in
response to bacterial or fungal infection with catalase-positive
organisms.70,71 Mouse knockout models by Goebel and Dinauer50 and Jackson et al72 of CGD have provided further insight
on pathogen virulence factors (Figure 4). Unraveling the details
of the molecular biology, biochemistry, and potential therapies
for this disorder have been a major focus of phagocyte research
for more than 40 years.59 Such studies have shed light on the role
of prophylactic therapies with gamma-interferon and itraconazole as well as the use of curative therapies with stem cell
transplantation and gene therapy.59,73-75
The neutrophil surface and its receptors
The surface of the neutrophil is complex, with myriads of folds,
crevices, and sites for interaction of the neutrophil with its
surroundings.76 Receptors for interaction with opsonins (eg, receptors Fc R-I, -II, and -III, and fragments of the third component of
complement) are expressed on both neutrophils and monocytes that
are critical surface receptors for facilitating phagocytic movement
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BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
and ingestion through pathways affecting cytoskeletal reorganization.77,78 The complement-mediated reactions that generate chemotactic factors in plasma also produce by-products that coat microbes and opsonize them such as C3b as shown by Alper and
Rosen79 and Muller-Eberhard80 to be critical in preventing infection in patients with C3 deficiency. Antibody participation in
opsonization including IgM and in some circumstances IgG was
elegantly demonstrated by Brown et al to activate complement
components, which resulted in the deposition of C3 on the surface
microbes to initiate complement-dependent opsonization of encapsulated virulent pathogens.81
The birth of understanding of immunodeficiency came with the
report of the first case of X-linked agammaglobulinemia in 1952 by
Bruton et al.82 The primary abnormality in this disorder is the
B-cell failure to produce gamma-globulins, but the functional
abnormality rests with the failure of the phagocytic process to halt
bacterial invasion of tissues and to clear bacterial pathogens from
the blood.83 Abnormalities in the complement system (ie, deficiency in components C3 and C5) that were discovered later led to
defective opsonization and chemotaxis of neutrophils, respectively,
because of deficiencies of these proteins.84
Neutrophils and monocytes also express cell-adhesion molecules, such as selectins and integrins, which, if mutated as seen
in leukocyte adhesion deficiency 2 and leukocyte adhesion
deficiency 1, respectively, affect the trafficking of neutrophils
by impeding their rolling and subsequent adhesion to the
capillary vascular wall in the process of diapedesis and eventual
migration into tissue.85-87 As first described by Hayward et al88
and Crowley et al,89 patients with mutations impairing the
expression of CD18, which affects the function of the leukocyte
integrin CD11/CD18, are usually recognized by having neutrophilia with the inability to produce “laudable pus.”90 It is now
well recognized that there are several variants of leukocyte
adhesion deficiency, each illustrating the critical importance of
neutrophil adherence for normal host defenses.91
Neutrophils bear a family of receptors that facilitate the
migration of phagocytes after they leave the vascular compartment. This critical response can be triggered in a multiplicity of
ways, and the development of the Boyden chamber was
strategically important for dissecting the specific roles of
individual chemotactic factors, as demonstrated by Ward and
Becker.92 In addition to the complement receptors (ie, receptors
for C5a and C3b) and C3bi, neutrophils have several other
chemotactic receptors. These include receptors for bacterially
derived or synthesized N-formyl peptides, platelet activating
factor (PAF), leukotriene B-4 (LTB-4), and a variety of other
chemokines and ligands for Toll-like receptors.93-95
The importance of the chemokine receptors is illustrated by
findings in patients with the myelokathexis syndrome, also referred
to as WHIM (warts, hypogammaglobulinemia, infections, and
myleokathexis) syndrome, first described in the 1960s by Zuelzer.96
Patients who have this disorder have severe leukopenia and
neutropenia, with accumulation of neutrophils in the bone marrow,
and often have many surface warts. This syndrome is now
attributable to a defect in the chemokine receptor CXCR-4.97
Observations in these patients have suggested an important role for
the ligand, stromal-derived factor 1 (SDF-1), in regulating neutrophil migration as well as the trafficking of lymphocytes and
hematopoietic progenitor cells.98
Neutrophils also bear surface receptors for the colonystimulating factors granulocyte colony-stimulating factor (G-CSF)
and granulocyte-macrophage colony-stimulating factor (GM-CSF)
THE PHAGOCYTES: NEUTROPHILS AND MONOCYTES
939
from early in development to the mature circulating neutrophil.99
This was a surprising finding, in light of the fact that the CSFs were
discovered as growth factors and that receptors for the parallel
growth factor, erythropoietin, are not expressed on mature erythrocytes. Experimentally, G-CSF and GM-CSF enhance the responsiveness of mature neutrophils to other stimuli, priming them for an
enhanced metabolic burst.100 In addition, these growth factors have
an antiapoptotic effect on mature neutrophils, prolonging their
survival.101 Thus at a tissue site of an infection, where cytokines
and growth factors are generated, the inflammatory milieu promotes the survival and function of arriving phagocytes and
promotes further recruitment of phagocytes from the bone marrow
and blood.
Phagocyte membrane dynamics and stimulus-response
coupling
The neutrophil is responsive to chemotactic factors and ingested
particles, and undergoes metabolic and morphologic changes.
Ligand binding to neutrophil surface induces hyperpolarization and calcium fluxes increase, and cyclic AMP rises
transiently.102-104 Zigmond observed chemotactic factors polarize and orient attached neutrophils for locomotion.105 Hirsch
found that neutrophils in response to a chemotactic source
acquire a characteristic asymmetric shape.106 In the front of the
cell is a pseudopodium that advances before the body of the cell
containing the nucleus and the cytoplasmic granules. In the
posterior of the moving cell is a knoblike tail. The formation of
the pseudopodium is essential for neutrophil locomotion. Contributions of Stossel in describing cytoskeletal chemistry provided
insight into the basis of neutrophil locomotion, particle ingestion, and digestion.77 The hyaline pseudopodia of the neutrophil
contains filament networks composed of actin filaments and
other regulatory proteins. The actin in the pseudopodia exists as
a gel and is concentrated at the cell periphery along with
myosin, which upon being engaged permits phagocytosis to
occur. A rise in calcium concentration dissolves the actin gel by
activating the protein gelsolin, which shortens actin filaments
and allows for sol formation permitting neutrophil movement
and directionality.
When a neutrophil comes in contact with a particle, the
pseudopodium flows round the particle. Its extensions fuse and
thereby it compasses the particle within the phagosome. Silverstein observed the phagocyte membrane adheres firmly to
particles it ingests and surrounds the particle in a zipperlike
fashion.107
Leukocyte chemoattractants induce a series of metabolic changes
including activation of trimeric G-proteins followed by enhancing
intracellular calcium levels, lipid remodeling, and protein kinase
activations. These events culminate in fusion of granule membranes with phagosomes or with the plasma membrane.108 Leukocyte chemoattractants stimulate signaling pathways that evolve are
Rho GTPases, including Rac-dependent NADPH oxidase activation, Rac- and Rho-dependent phospholipase D activity, and Racand CDC-42–regulated p21-activated protein kinases.109 The studies by Bokoch109 have illuminated the role of the GTPases
especially the necessary requirement for Rac-dependent and
NADPH oxidase activation. The importance of Rac-2 function in
human neutrophil chemotaxis and NADPH oxidase activation has
been highlighted by the discovery of a toddler with a naturally
occurring dominant-negative Rac-2 mutation. This individual
suffered from severe, recurrent infection, a markedly reduced
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DALE et al
neutrophil migration, and NADPH oxidase–dependent
activation.110,111
Studies by Greenberg and Grinstein in macrophages have
contributed to the understanding of the mechanism of phagocytosis
initiated by Fc receptor engagement.112 This includes cytoskeletal
alterations and membrane trafficking. The Fc receptors require
phosphorylation of the receptors themselves or associated immunoreceptor tyrosine-based activating motif (ITAM)–containing subunits, by members of the Src family. Phosphorylated receptor/
subunit ITAMs serve as docking sites for SyK processes in
phagocytosis mediated by the engagement of the Fc receptor.
Pseudopodia extension in turn requires a wave of lipid remodeling
in which phosphatidylinositol 3 kinase (PI3K) is generated at the
phagosomal cup. Cessation of PI3 kinase is abrupt and in part is
due to the recruitment of lipid phosphotase (SHIP) to the phagocytic cup.113,114 The synthesis of phosphatidyl 1-4.5, bisphosphate
is also accelerated during phagocytosis. This lipid serves as a
substrate of PI3K. It is also the target of phospholipase C, which
generates diacylglycerol during phagocytosis. The latter mediator
can activate protein kinase C, which can be recruited to the
phagosome and participate in particle uptake. Other kinases
implicated in phagocytosis include MEK-1, which may be selectively involved in Fc ␥R-mediated phagocytosis in neutrophils but
not in macrophages.115 Both phospholipase A2 and phospholipase
D are also activated and are thought to participate in the phagocytic
process. The former product may participate in degranulation
during phagocytosis as well as amplify the production of leukotrienes that amplify the phagocytic signal.
Co-opting phagocytic machinery by invasive pathogens
The initial response to most bacterial and fungal pathogens is
phagocytosis through distinct receptors. Listeria is able to co-opt a
receptor kinase to invade the host cells.116 The Listeria infection
can be fatal in immunocompromised patients, neonates, and
pregnant women. Southwick and Purich observed that Listeria can
co-opt the receptor tyrosine kinase to invade host cells. In turn,
Listeria can usurp the cytoskeletal system of the host cell and
survive and thrive within the host.117 Actin assembly is essential for
the cell-to-cell spread of Listeria, which allows it to move through
the cytoplasm of host cells and to be transferred from one host cell
to another, thereby invading the host immune system.
Other pathogens synthesize antiphagocytic factors. For an
example, Yersinia secretes YopH, a tyrosine phosphotase that
dephosphorylates a focal adhesion protein Cas.118 Other pathogens
such as Microbacterium tuberculosis can suppress calcium signaling thereby inhibiting phagolysosome fusion and allowing for
survival in the phagosome.119
Monocytes
The production and life cycle for mononuclear phagocytes is more
complex than for neutrophils. Neutrophils follow a relatively
simple pathway from the marrow to the blood and the tissues.
Although they share some similar physiological capacities as
neutrophils, marrow and blood monocytes retain a proliferative
capacity and can differentiate into resident phagocytic cells,
broadly termed macrophages and histiocytes in the spleen, liver,
and lungs and other tissues. During chronic inflammatory conditions including sarcoidosis and tuberculosis, these cells can fuse to
form giant cells. Historically, this system of tissue-based mononuclear phagocytes was called the reticuloendothelial system.120
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
Life cycle and progeny
The complex life cycle of monocytes led Virchow and other
prominent 19th century pathologists to believe that macrophages
were derived from mesenchymal tissue, rather than blood cells.
Only in the modern era, using radioisotopic labeling of blood and
marrow cells, has it been possible to establish that circulating
monocytes are the precursors for macrophages in all tissues. The
studies of Lewis and Lewis,121 Cohn and Benson,122 van Furth and
Cohn,123 and Nichols et al124 were landmark papers regarding
monocyte development and differentiation. Later work revealed
that monocytes are not homogeneous but actually represent at least
2 distinct subsets of mononuclear phagocytes.125-127 Recently, it has
been conclusively demonstrated that monocytes also serve as the
precursors of dendritic cells, which play an important role in host
defense as potent antigen-presenting cells during T-lymphocyte
activation.128-131
Mononuclear phagocytes of the blood and tissues survive far
longer than neutrophils. This feature of phagocytes is clinically
very important. It protects patients from an overwhelming risk of
fatal infections when neutrophil production is transiently interrupted, as occurs with cancer chemotherapy, idiosyncratic reactions
to many drugs, and hematopoietic stem cell transplantation.
More comparisons of monocytes and neutrophils
Over the past 50 years, researchers have continuously focused on
defining the similarities and differences between neutrophils and
monocytes. Mononuclear phagocytes share many properties with
neutrophils, but they also have distinctive morphologic and functional properties, depending upon their state of differentiation. For
example, the granule-associated proteins are similar to those of
neutrophils, but there are some distinctive differences.132 Monocytes have a preserved capacity to augment production of granule
proteins through new protein synthesis, a feature that is lost in
mature neutrophils. There are also significant differences in their
chemotactic responses and metabolic burst activity during phagocytosis.132 At a site of acute inflammation, monocytes accumulate
more slowly, but persist longer. Their metabolic burst is less
extreme, but their capacity to kill many microbes is more diverse
compared with that of neutrophils. Monocytes have Fc receptors
and express the IgG receptor Fc␥RI (CD64) constitutively in
contrast to neutrophils, which express this receptor only in
response to inflammatory stimuli.132
An important difference between neutrophils and monocytes is
also in their capacity to produce new proteins, including a variety
of cytokines associated with enhancement of the inflammatory
response. Of historical interest in this regard, the “endogenous
pyrogen,” discovered by Beeson and thought for many years to be
primarily a product of neutrophils, is now known to be predominantly produced by mononuclear phagocytes, because of their far
greater capacity for cytokine production.133,134
Physiological functions
The human immune system has been divided traditionally into
innate immunity and acquired (adaptive) immunity. Monocytes and
their differentiated progeny play important regulatory and effector
roles in both arms of the human immune system.135-137 Mononuclear phagocytes have at least 3 major functions: presentation of
antigens as discussed above, phagocytosis, and immunomodulation. Mononuclear phagocytes ingest material for 2 purposes: to
eliminate waste and debris and to kill invading pathogens. Mononuclear phagocytes dispose of effete and aged red cells and remove
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BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
THE PHAGOCYTES: NEUTROPHILS AND MONOCYTES
941
Figure 5. Overview of the pattern-recognition receptor system of phagocytes, including the TLR family.
Illustration by Kenneth Probst. Adapted with permission
from: Trinchieri G, Sher A. Cooperation of Toll-like receptor signals in innate immune defense. Nat Rev Immunol.
2007;7:179-190.147
red cell inclusions in the spleen. They also clean up debris at sites
of infection or tissue damage.138,139
Activated monocytes and macrophages also release IL-1, IL-6,
TNF, and INF-␣/␤—cytokines that are involved in the regulation
of hematopoiesis.140 Monocytes are also subject to immune modulation through the role of chemokines. The chemokines MIP-1␣,
MIP-1␤, and RANTES produced by CD8⫹ T cells inhibit human
immunodeficiency virus infection by monocyte trophic-1 strains.141
Macrophages as shown by Nathan and colleagues can activate
nitric oxide synthase, which leads to the synthesis of nitric oxide
(MacMicking et al142). In turn, sustained production of nitric oxide
endows macrophages with cytostatic or cytotoxic activity against
viruses, bacteria, fungi, protozoa, helminthes, and tumor cells.
Recent studies by Tall and colleagues suggest that the macrophage
contributes to the pathogenesis of both atherosclerosis and insulin
resistance (Liang et al143). Insulin-resistant macrophages in the
arterial wall undergo increased apoptosis, which may lead to larger
lipid-rich cores, increased inflammation, and more plaque formation.
Monocytes, macrophages, and dendritic cells express a large
number of cell surface proteins that play crucial functional roles
in phagocyte biology. Microbial pattern-recognition receptors
are an essential component of innate immunity, in which they
recognize and detect pathogen-associated molecular patterns,
resulting in activation of monocytes, macrophages, and dendritic cells (and neutrophils) as part of the host response to
eradicate invading pathogens.
An important class of pattern-recognition receptors is the
recently described mammalian Toll-like receptor (TLR) family,
which recognizes a wide range of microbial pathogens and
pathogen-related products (Figure 5).144-147 TLRs are expressed to a
far higher degree by monocytes than neutrophils. Upon binding of
specific ligands, TLRs signal via a pathway involving the adaptor
protein MyD88, or via a MyD88-independent pathway involving
TRIF, to activate NF-␬B and stimulate proinflammatory cytokine
production from monocytes and macrophages.148,149 Other cellbased receptors may cooperate with specific TLRs to enhance
pathogen recognition. For example, CD14 binds LPS and interacts
with TLR4 to facilitate recognition and enhance eradication of
Gram-negative bacilli from the circulation and tissue sites.150
Congenital defects in monocyte-macrophage function
Recent progress in the elucidation of the genetic and molecular
basis of congenital immunodeficiency disorders has defined critical
factors regulating monocyte/macrophage-mediated innate immune
responses. Functional mutations in the interleukin-12 (IL-12)/
interleukin-23/interferon-␥ (IFN␥) axis are associated with nontuberculous mycobacterial infections due to the inability of monocytes and macrophages to kill these organisms of relatively low
pathogenicity following phagocytosis.151-153 X-linked susceptibility to mycobacterial disease has recently been attributed to
functional mutations in NEMO, the ␥-subunit of I␬B (inhibitor of
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942
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
DALE et al
NF-␬B) kinase (IKK) complex, that regulates the activation of the
NF-␬B signaling pathway.154 Deficiency of IL-1 receptor–
associated kinase 4 (IRAK4), a component of the TLR/IL-1–
mediated MyD88-dependent signaling pathway described above,
has been shown to predispose affected individuals to invasive
pneumococcal disease.155,156
Finally, several groups have recently announced the discovery
of the mutations that appear to be responsible for the pathogenesis
of hyper-IgE syndrome (formerly Job syndrome)—a severe multisystemic disease associated with recurrent and severe staphylococcal infections of the lungs and skin.157,158 These findings highlight
the fundamental and essential role of the cytokine-induced JAKSTAT (Janus kinase–signal transducer and activator of transcription) pathway in phagocyte-mediated host defense against microbial pathogens.
Final comments
This brief historical review only touches on the many significant
events in advancing our understanding of phagocytes over the past
50 years. Only a few of the thousands of landmark papers are
mentioned, and the authors regret having to omit numerous other
worthy and important citations. Nevertheless, on this 50th anniversary of Blood, it is valuable to reflect on the major progress that has
been made in understanding phagocyte biology. These advances
provide an excellent foundation for further research to find
improved therapeutic strategies and options for our patients.
Authorship
Contribution: D.C.D. is the primary author, and was assisted in
writing and editing by W.C.L. and L.B.
Conflict-of-interest disclosure: D.C.D. has served as consultant
and speaker for and has received research funds from Amgen and
Anormed (now Genzyme). He has also served during the past
2 years as a consultant for Cellerant, Xenon, Maxygen, ScheringPlough, and Merck. L.B. has ownership of Amgen stock. W.C.L.
declares no competing financial interests.
Correspondence: David C. Dale, Department of Medicine,
University of Washington, Box 356422, 1959 NE Pacific St, Seattle
WA 98195; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
THE PHAGOCYTES: NEUTROPHILS AND MONOCYTES
My interest in hematology began in the early 1960s when Drs David Nathan and Frank Gardner
were inspiring second-year teachers in the hematology course at the Harvard Medical School. I
then had Drs William Castle and Jim Jandl as advisors and teachers in a medicine clerkship at the
Boston City Hospital, and they stimulated my interest in hematology and research. I began studying host defense mechanisms, neutrophils, and the problem of neutropenia under the mentorship
of Dr Sheldon Wolff in the Laboratory of Investigation of the NIAID at the NIH in the late 1960s.
Shelly, as we affectionately called him, got me interested in cyclic neutropenia in humans and
gray collie dogs, the study of the colony-stimulating factors, the formation and fate of neutrophils,
and the search for treatments to improve the well-being of patients with neutropenia. My enjoyment of the study of phagocytes has come through caring for patients who are susceptible to infections and through friendships with fellow researchers in this interesting field. My coauthors, Drs Laurence Boxer and Conrad Liles, are special friends with whom I have enjoyed working for many years.
David C. Dale
Beginning with my tour of duty in the United States Army I became interested in the pathophysiology of neutropenic disorders. When I entered my fellowship in the laboratory of Tom Stossel at the
Children’s Hospital Medical Center in Boston, I was afforded the opportunity to build a better
mousetrap to identify antibodies directed against neutrophils. While a fellow, I also had the opportunity to unravel some disorders of phagocyte function, which became another passion of mine
throughout my career. The subsequent study of neutrophil abnormalities obtained from patients
with recurrent bacterial infections paved the way over the years to a better understanding of the
components of oxidative and nonoxidative microbial killing mediated by neutrophils. The discovery and eventual clinical application of granulocyte colony-stimulating factor for the treatment of
patients with severe chronic neutropenia have provided the opportunity to engage in translational
research and explore the mechanisms underlying the basis for the production abnormalities associated with severe chronic neutropenia disorders. All in all, it has been highly gratifying to engage in research that has improved the understanding of the disorders of the phagocyte and in
many instances improved the lives of many patients.
W. Laurence Boxer
My interest in phagocytes began during my fellowship in Infectious Diseases at the University of
Washington under the mentorship of Seymour Klebanoff, MD, PhD. His meticulous and insightful
studies of the mechanisms of killing of microbes by phagocytes were an inspiration to me and all
of his trainees. During my fellowship, I became interested in the role and regulation of apoptosis
and began to study the importance of Fas/Fas ligand system in innate immunity. Over the years,
my research interests have expanded to include broader studies of inflammation, sepsis, congenital immunodeficiency disorders, and immunomodulatory therapies. In 2006, I moved from the
University of Washington to become Director of the Division of Infectious Diseases and Vice Chair
of the Department of Medicine at the University of Toronto, where I also serve as the Canada Research Chair in Inflammation and Infectious Diseases and am a member of the McLaughlinRotman Center for Global Health.
W. Conrad Liles
945
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2008 112: 935-945
doi:10.1182/blood-2007-12-077917
The phagocytes: neutrophils and monocytes
David C. Dale, Laurence Boxer and W. Conrad Liles
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