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SINGAPORE IMMUNOLOGY NETWORK
Peeking into the secret life of neutrophils
Jackson LiangYao Li Lai Guan Ng
Published online: 11 March 2012
Springer Science+Business Media, LLC 2012
Abstract The migration of neutrophils between tissue compartments is an important aspect of innate immune surveil-
lance. This process is regulated by a cascade of cellular and molecular signals to avoid unnecessary crowding of neu-
trophils at the periphery, to allow rapid mobilization of neutrophils in response to inflammatory stimuli, and to return to a
state of homeostasis after the response. Intravital microscopy approaches have been fundamental in unraveling many
aspects of neutrophil behavior, providing important mechanistic information on the processes involved in basal and disease
states. Here, we provide a broad overview of the current state of research on neutrophil biology, describing the processes in
the typical life cycle of neutrophils, from their first appearance in the bone marrow until their eventual destruction. We will
focus on novel aspects of neutrophil behavior, which had previously been elusive until their recent elucidation by advanced
intravital microscopy techniques.
Keywords Neutrophil Intravital microscopy Bone marrow Blood Vessel Interstitium Effector Clearance Basal Inflammation
Introduction
Neutrophils represent a major subset of innate immune
cells and play a key role in the defense against invading
pathogens. It is well established that neutrophils are rapidly
recruited into sites of inflammation or infection, where they
can destroy bacterial and fungal pathogens by several
mechanisms including the release of enzymes and reactive
oxygen species, as well as through direct phagocytosis.
Proteolytic enzymes and oxidants released by neutrophils
can however also cause collateral damage to the sur-
rounding tissue. Due to the highly destructive nature of
these cells, neutrophil homeostasis is maintained through
tight regulation of neutrophil production in the bone
marrow (granulopoiesis), distribution between different
compartments, and migration through interstitial tissues.
One of the most fundamental characteristics of neutro-
phils is their ability to shuttle between different body
compartments in response to inflammatory stimuli. Under
homeostatic conditions, bone marrow serves as the major
reservoir for neutrophils, and only 12% of neutrophils are
present in the circulation in mice [1]. During acute
inflammatory responses, neutrophils can be rapidly
released from bone marrow into the bloodstream, resulting
in a dramatic increase in circulating neutrophil numbers.
To enter sites of inflammation in the tissue, neutrophils
need to move across the blood vessel walls. The cascade of
events leading to neutrophil extravasation has been exam-
ined extensively, and these studies have provided a wealth
of information about the mechanisms underlying the
recruitment of neutrophils to sites of inflammation. In
contrast, the subsequent cellular and molecular events
involved in neutrophil migration within interstitial tissue
are not well defined. This aspect of neutrophil mobilization
has been overlooked due, at least partly, to the lack of
J. L. Li L. G. Ng (&)Singapore Immunology Network (SIgN), Agency for Science,
Technology and Research (A*STAR), 8A Biomedical Grove,
#03 Immunos, Biopolis, Singapore 138648, Singapore
e-mail: [email protected]
Lai Guan Ng
123
Immunol Res (2012) 53:168181
DOI 10.1007/s12026-012-8292-8
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suitable technologies to address this type of questions in
the past. It is also possible that neutrophils were typically
considered to be too short-lived to play a significant role in
the immune response after entering interstitial tissues (few
hours) [2, 3]. However, a recent study, which used deute-
rium to label neutrophils in vivo, has shown that non-
activated neutrophils may have a longer lifespan than
previously thought ([half a day in mice, 5.4 days inhumans) [4]. In contrast to the general belief that neutro-
phils can only migrate into tissues in response to inflam-
matory stimuli, we recently showed that neutrophils are
present in the interstitial tissue of the skin even at resting
state [5]. This indicates that neutrophils may have an
important role in maintaining tissue homeostasis and
shaping immune responses. Thus, a better understanding of
neutrophil behavior and function in living tissues may
reveal new knowledge about neutrophil biology.
Historically, intravital microscopy (IVM) techniques
have been instrumental in unraveling the complex mech-
anisms of neutrophil behavior. The first description of the
leukocyte extravasation process was provided by an IVM
study performed more than 100 years ago [6]. The con-
tinued advancement in microscopy technology coupled
with the availability of a wide array of genetically modified
mice has helped to further define the molecular basis of
neutrophil migration. One microscopy approach that has
received a lot of attention in recent years is multiphoton
(MP) microscopy. This technique has overcome previous
limitations of conventional microscopy by allowing a
greater depth of penetration as a result of its localized
nonlinear laser signal generation. As such, it is now rou-
tinely used to perform dynamic, multidimensional imaging
to simultaneously track cell populations at the single-cell
level in living tissues or organs. For more detailed tech-
nical information about the general setup of an MP
microscope, please refer to these comprehensive review
articles [7, 8].
The availability of new technology will often open up
new avenues of research, and this is certainly true in the
case of MP microscopy. Immunologists have long adopted
IVM techniques to address biological questions relating to
leukocyte trafficking. However, IVM approaches using
linear-absorption microscopy techniques such as bright-
field, epifluorescence, and laser scanning confocal
microscopy are limited to the imaging of translucent tissues
or superficial regions of non-translucent organs due to the
lack of tissue penetration. Since the first application of MP
imaging for the study of immune cells in intact organs or
tissues in early 2000 [912], this technology has become an
increasingly important tool in the field of immunology.
Notably, adaptive immune cells are relatively better-char-
acterized by MP imaging studies than innate immune cells.
T cells arguably represent the most studied immune cells
by MP imaging, and important information has been
obtained about their behavior at resting stage and during
various pathological settings within their native microen-
vironment (e.g., in tumors and infections, see reviews
[1315]). In contrast, MP microscopy has only recently
been used to study neutrophil dynamics in vivo. Despite
this, these studies have already provided us with important
clues as to how neutrophil migration is regulated. In this
review, we will provide a broad description of neutrophil
behavior and their immune surveillance strategies, includ-
ing granulopoiesis, trafficking, adhesion, crawling, trans-
migration, swarming, effector functions, clearance, among
others. In particular, we will focus on discussing the results
obtained from IVM imaging of neutrophils (especially
those from MP microscopy) in various mouse models,
highlighting the new perspectives offered by these studies.
The behavior of neutrophils in different tissue
compartments
Bone marrow
Neutrophils make their first appearance in the hematopoi-
etic cords of the bone marrow [16]. A series of proliferating
myeloid precursors generate non-dividing neutrophilic
metamyelocytes, which then differentiate linearly into
mature segmented neutrophils [17, 18] (See Fig. 1). Pro-
duction and maturation of neutrophils appear to be con-
fined to the bone marrow, and this is in line with the role of
neutrophils as the major innate effector cells of the immune
systemunlike B or T lymphocytes, which require highly
specialized training in additional tissues for maturation.
Within the bone marrow, neutrophils are the most abundant
leukocytes producedapproximately 107 neutrophils are
generated each day in mice [19], and 1011 neutrophils per
day in humans [2]. A high constitutive neutrophil produc-
tion rate is important because an effective neutrophil
response, when initiated, typically requires the concerted
action of very large numbers of neutrophilsa high total
threshold number thus needs to be maintained even during
physiological basal conditions. Production of neutrophils
takes approximately 2.3 days to complete in mice, with at
least 16 h spent in the mitotic pool (promyelocytes and
myelocytes) [4], and thus, requirements for neutrophil
supply during an infectious episode are unlikely to be
achieved without a large reserve pool. In addition, neu-
trophils have a relatively short half-life compared to other
leukocytes, thus necessitating high production rates to
maintain a constant turnover [19]. The short-lived nature of
neutrophils might be an evolutionary defense against the
development of parasites (that might otherwise adapt to
colonizing neutrophils) [2022], but may simply reflect a
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more stringent functional requirement for neutrophil
maintenance [23] (e.g., since the unplanned necrosis of
aging neutrophils in the periphery would release noxious
mediators that are highly damaging to the surrounding
tissue).
The bone marrow acts as an important reservoir to hold
excess neutrophils, maintaining the high total threshold
number of neutrophils and preventing the unnecessary
crowding of neutrophils in the periphery. At the physio-
logical state in mice, it is estimated that more than 90% of
the total neutrophils are held within the bone marrow, with
only 12% present in the circulation [1, 24]. The majority
of mature neutrophils localize in the extravascular com-
partments between the sinusoids of the bone marrow. A
small proportion of neutrophils (30%) are motile and crawl
randomly within the space (mean migration veloc-
ity = 1.5 lm/min) [25]. An even smaller percentage ofneutrophils may spontaneously exit the bone marrow by
entering the surrounding sinusoids, accounting for the basal
blood neutrophil counts. However, upon acute tissue injury
or other forms of inflammation, endothelia or other
immune cells at the peripheral site such as macrophages
may release granulocyte colony-stimulating factor (G-
CSF), a glycoprotein growth factor that maintains granu-
locyte survival and increases granulocyte proliferation
(granulopoiesis) [16]. In addition to stimulating granulo-
poiesis, G-CSF also functions as a mobilizing agent that
triggers the release of bone marrow neutrophils into the
bloodstream [1]. Despite the well-established role of
G-CSF in neutrophil mobilization from the bone marrow,
the spatiotemporal regulation of this process remains
poorly defined. Recently, an MP-IVM study by Gunzers
laboratory [25] showed that a single dose of G-CSF
intraperitoneally injected in mice triggered neutrophil
mobilization by increasing both neutrophil motility and
directionality in the bone marrow. Mobilized neutrophils
were found to migrate toward the nearest sinusoids and exit
the bone marrow, leading to a dramatic increase in the
number of circulating neutrophils. Blood neutrophil counts
were found to peak 2 h after G-CSF injection, while the
increased neutrophil mean migration velocity in the bone
marrow persisted for up to 4 h post-injection, and the
increased proportion of motile neutrophils persisted for
greater than 16 h.
The mechanisms regulating the number of neutrophils
being released from the bone marrow has been proposed to
involve the chemokine receptors CXCR4 and CXCR2
(IL8-R beta) expressed on neutrophils, in which CXCR4 is
important for the retention of neutrophils in the bone
marrow, whereas CXCR2 is important for neutrophil
release from the bone marrow [26]. The major binding
partner for CXCR4 is CXCL12 (SDF-1) [27], which is
expressed by reticular cells [28] of the bone marrow. On
the other hand, the major binding partners for CXCR2 are
CXCL1 (KC) and CXCL2 (MIP-2a), which can be secretedby endothelial cells [29]. Neutrophil retention in the bone
marrow is believed to be dependent on CXCR4CXCL12
interactions between neutrophils and bone marrow reticular
cells. Consistent with this hypothesis, neutrophil numbers
are reduced in the bone marrow and elevated in the
peripheral blood of CXCR4-deficient mice [30], and anti-
CXCR4 blocking antibodies have been shown to mobilize
Fig. 1 A schematicrepresentation of neutrophil
development during
granulopoiesis. Top right Az-projection multiphoton imageof skull bone marrow in
Lysozyme-GFP C57BL/6
mouse injected with TRITC
dextran for blood vessel
labeling. Skull bone collagen
shows up as second harmonic
generation (SHG). Image
courtesy of Dr. Yilin Wang
(SIgN, Singapore). Left andbottom Development ofneutrophils in the bone marrow,
showing maturation stages
commonly described in
hematology. Relationships with
other hematopoietic cells are
also shown
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bone marrow neutrophils [31]. Previously, G-CSF was
proposed to mobilize neutrophils from the bone marrow by
activating proteases that cleave CXCL12 [32]. However, a
study using protease-deficient mice demonstrated that these
mice retain the ability to mobilize bone marrow neutrophils
[33], and another study showed the down-regulation of
both CXCR4 and CXCL12 by G-CSF [34], providing a
simpler explanation of the relationship between G-CSF and
CXCR4-mediated retention. On the other hand, CXCR2
activation by its ligands CXCL1 or CXCL2 is proposed to
be an important chemotactic signal for neutrophil emigra-
tion. G-CSF injections were unable to mobilize neutrophils
from the bone marrow of CXCR2-deficient mice, thus
demonstrating that G-CSF-induced mobilization is cen-
trally dependent on CXCR2 [25]. In agreement with this
observation, CXCR2 ligands are elevated in blood during
G-CSF-induced mobilization, which thus provides a simple
mechanistic explanation for neutrophil mobilization from
bone marrow into the sinusoids. During sepsis, however,
CXCL12 concentrations become elevated in blood [35],
providing a reverse gradient described earlier for CXCR4
CXCL12 binding retention, indicating that the actual
mechanisms of regulation may be more complex than
conceptualized here.
Perhaps of more novelty is the discovery that megakary-
ocytes, the precursors of platelets, are able to produce
CXCR2 ligands in response to G-CSF, mediated by throm-
bopoietin (TPO) [25]. Megakaryocytes, which derive from a
common myeloid progenitor, reside in the bone marrow and
are usually found lining the sinusoids. An MP imaging study
from von Andrians laboratory [36] has provided the first
dynamic view of megakaryocyte activities in vivo. The
authors showed that perisinusoidal megakaryocytes were
able to send out cytoplasmic projections through the vessel
walls that break off from their transendothelial stems to form
platelets. Although not conclusively proven, the fact that
portions of the megakaryocytes cross the vessel walls sug-
gests the enticing idea that these cells can release CXCL1
and/or CXCL2 directly into the bloodstream in very close
vicinity to bone marrow neutrophils and thus provide or
regulate directional cues for CXCR2-mediated mobilization
[25] (See Fig. 2). This might help to explain the mechanism
behind the seemingly stochastic neutrophil mobilization
during basal state, since systemic CXCR2 ligands generated
from a distant source would be expected to quickly dilute in a
homogeneous manner such that chemokine gradients would
be detected almost equally well by the majority of bone
marrow neutrophils.
Blood
Neutrophils patrol the body by circulating in the blood,
which allows them to respond quickly to signals of
pathogen entry or loss of tissue integrity. To detect
pathogens, neutrophils express innate pathogen recognition
receptors (PRRs), of which Toll-like receptors (TLRs)
feature prominently [37]. These receptors detect evolu-
tionarily conserved signature molecules of commonly
encountered pathogens, which are collectively known as
pathogen-associated molecular patterns (PAMPs). Neutro-
phils express many of the known TLRs [38] and, upon
activation, typically undergo pro-inflammatory changes,
including increased propensities for phagocytosis and
cytokine secretion [39]. For detecting tissue injury, analo-
gous damage-associated molecular patterns (DAMPs) [37]
are thought to mediate inflammatory responses of neutro-
phils. DAMPs are molecules associated with necrotizing
cells, and examples include ATP, uric acid, heat-shock
proteins, and mitochondrial DNA [40]. At basal level, there
are approximately 5001,000 neutrophils per microliter of
blood, although the actual numbers can vary among mouse
strains [41]. This basal level of neutrophils in the periphery
allows prompt responses since the earliest neutrophils can
arrive at the target site without the need to travel vast
distances from the bone marrow in order to establish the
inflammatory response. This also allows a graded response
to injury or pathogen, since minor insults need only trigger
a localized response that draws nearby circulating neutro-
phils, without unnecessarily mobilizing neutrophils from
the bone marrow. The surveillance program of neutrophils
takes advantage of the ubiquitous perfusion of blood
throughout the body, allowing neutrophils to extend their
reach of response into most tissues, although immune-
privileged sites such as the brain have other specialized cell
types (i.e., microglia and astrocytes) to carry out similar
Fig. 2 Relationship between CXCR2 and CXCR4 in neutrophilmobilization and retention in bone marrow. Neutrophils are retained
in the bone marrow by the interaction between CXCR4 and its only
known ligand CXCL12. During mobilization, GCSF reduces the
strength of this interaction by down-regulating CXCR2 and CXCL12
expression levels, while CXCR2 ligands in the blood, which may
include CXCL1 and CXCL2, provide the chemokine gradients for
neutrophil extravasation into sinusoids. Also shown is the hypothet-
ical contribution of CXCR2 ligands by megakaryocytes into the
bloodstream, as megakaryocytes have been shown to be able to
express them under GCSF stimulation via TPO
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duties [42]. In accordance with this theory, an MP-IVM
study in mice from Dustins laboratory showed that laser
injury to the brain parenchyma triggered the rapid exten-
sion of microglia dendrites and polarization of the astrocyte
cytoplasm toward the injury site, without causing any
neutrophil infiltration [43].
Peripheral neutrophils are also known to spend addi-
tional time transiting certain highly vascularized organs
such as the lungs and liver [44], although it is unclear
whether this reflects an increased need for the patrolling of
these regions or whether these organs represent secondary
storage depots for additional fine-tuning of blood neutro-
phil counts. In fact, large numbers of extravascular neu-
trophils can be observed in healthy lung tissue [45] despite
the fact that neutrophils are thought to rarely exit blood
vessels except during activation. Using a combination of
MP-IVM and functional approaches, we have recently
shown that even under homeostatic conditions, a very small
percentage of neutrophils can be found in the mouse skin
interstitium, as well as within lymph fluid (demonstrated in
sheep) [5]. This perhaps signifies that the neutrophil patrol
routes incorporate not only the blood circulation but
interstitial and lymphoid paths as well.
However, it is clear that neutrophils primarily employ
blood vessels of healthy tissue as highways to access various
locations of injury or pathogen detection in the interstitium. In
mice, blood is pumped at approximately 10 beats per second
[46] and can flow at approximately 1 mm/s in a mid-sized
venule [47]this velocity represents approximately a hun-
dred neutrophil lengths each second. While such high speeds
are useful for transporting neutrophils rapidly from site to site,
this compromises the ability of neutrophils to sense and
respond to subtle cues presented on the vascular walls. Thus,
upon activation, neutrophils must exit the central bloodstream
and roll along the vessel walls in the boundary layer (where
shear forces dictate lower fluid flow velocities) in order to
sample the more precise chemotactic signals. Under basal
conditions in the skin, blood vessels are shown to express
CD62E and CD62P (E- and P-selectins) [48], which allow
neutrophils to exhibit constitutive rolling. This may represent
a more nuanced patrolling strategy for skin, given the high
correlation between the loss of its integrity and subsequent
pathogen invasion, although it is unclear whether other sites at
high risk of pathogen influx also exhibit constitutive neutro-
phil rolling [49]. In a large venule (*30 lm diameter), con-stitutive rolling in the skin occurs at approximately 10 lm persecond, with approximately 50% of total leukocytes display-
ing such behavior [50].
Blood vessel walls
Neutrophils are able to respond to pathogens and tissue
injury through specialized receptors described earlier, but
as the majority of patrolling neutrophils are found flowing
in the blood, they are usually unable to directly make
contact with the pathogen or injured tissue. As such, they
often depend on other sentinel cells to provide the distress
signals for their recruitment. Many cell types can trigger
neutrophil recruitment directly or indirectly. In the skin,
these cells can be endothelial cells or other sentinel cells
such as dendritic cells [51, 52], mast cells [53], macro-
phages [54], basophils [55], and monocytes [56], with the
actual mechanisms used varying according to the context.
Endothelial cells of the blood vessel walls constitute a very
important sentinel cell type, but are generally overlooked
due to their non-leukocyte nature, and are often assumed to
be passive scaffolding for neutrophil adhesion. Impor-
tantly, however, they have been shown to express classical
molecules involved in pathogen detection (which are more
often associated with leukocytes), including CD14, TLR2,
TLR4, TLR9, MD2, and MyD88 [49], and thus provide a
large surface area for pathogen detection, which can lead to
neutrophil activation. Endothelial cells are also crucially
important for neutrophil function, since neutrophils require
their active cooperation for successful tethering, rolling,
adhesion, crawling, and transmigration in order to exit the
bloodstream and travel to the target site.
Since chemokine signals that enter the blood would be
quickly flushed away and potentially scavenged (e.g., by
DARCs on erythrocytes), it was somewhat of a mystery as
to how a neutrophil could recognize when and where to
exit the blood stream in order to migrate to the injury site.
A seminal study by Kubess laboratory [57] used a sterile
hepatic focal injury IVM model in which well-defined
thermal injuries could be generated and examined the
subsequent neutrophil recruitment processes from liver
sinusoids. They found that contrary to common perception,
ATP (in its role as a DAMP) did not function in vivo to
trigger direct neutrophil activation, and neither did it serve
as a chemoattractant. Instead, ATP triggered inflamma-
some-dependent cell signaling processes within the
microenvironment that culminated in the activation of
endothelial cells, which upregulated CD54 (ICAM-1) to
provide neutrophils with a surface conducive for activation
and recruitment. Modifications of glycocalyx thickness
might also be necessary to expose the adhesion molecules
on the endothelia cell surface [58]. In addition, they also
found that CXCL2 formed a gradient on the luminal side of
the blood vessel walls and was mainly responsible for
CXCR2-mediated neutrophil chemotaxis there. CXCL1
played a minor role in this process. The chemokine gra-
dient was made possible by the immobilization of che-
mokines on the vessel walls, presumably mediated by
endothelial surface heparan sulfate [59]. However, this
gradient started abruptly approximately 150 lm from theinjury site although proximal endothelial cells were still
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intact. Despite the lack of a CXCL2 gradient, neutrophils
were still able to travel into the center of the necrotic tissue.
This was attributed to a second gradient of formylated
peptides that overrode the CXCL2 signal [57]. Formylated
peptides are DAMPs of mitochondrial origin recognized by
formyl-peptide receptor 1 (FPR-1) [60]. Thus, the current
paradigm on neutrophil recruitment consists of a series of
sequential signals that act at different ranges, and these
signals may progressively override earlier signals [6164]
(e.g., G-CSF at ultra-long range, ATP at mid-range via
endothelial cells, CXCL2 at short range, and formylated
peptides at immediate range)neutrophils thus start off
heading in the general direction where they are needed, but
once they get closer, they make decisional switches and
respond to progressively more precise cues in order to
finally arrive at the intended site. Recently, hydrogen per-
oxide was shown to function as a tissue-scale chemoat-
tractant in the zebrafish for the recruitment of leukocytes to
injury [65] and thus might represent another layer of sig-
naling for neutrophils, but it remains to be seen whether
hydrogen peroxide also has a function in the mammalian
system.
As previously mentioned, in order to cross the blood
vessel walls from high initial flow velocity, activated
neutrophils have to slow down and come to a stop before
transmigration can take place. This process, commonly
termed the leukocyte adhesion cascade (since similar pro-
cesses also occur for other leukocytes), has been exten-
sively studied [66], but many of the details have only been
elucidated relatively recently, with the advent of IVM
technology. The initial step triggering the slowing down of
fast-flowing neutrophils is termed tethering or capture and
is largely mediated by selectins expressed on both the
neutrophils and the endothelial cells, which have corre-
sponding ligands on the opposing cell for attachment.
CD162 (PSGL-1) is an important selectin ligand expressed
on the neutrophil that interacts with CD62E and CD62P
(E- and P-selectin) expressed on the endothelial cells [49].
However, in the scenario that immune complexes form and
deposit on the endothelium, neutrophils are also able to
employ a selectin-independent tethering process through
the use of their constitutively expressed Fc-gamma recep-
tor IIIB in a low-affinity binding interaction with immune
complexes [67]. The tethering process is thus the initial
contact between the neutrophil and the vessel wall that
allows the neutrophil to slow down and marginalize to the
vessel wall where rolling can then take place. During
rolling, the decrease in velocities allows neutrophils to
come into stronger contact with the endothelial cells and
further decelerate. Neutrophils then activate integrins to
mediate strong binding interactions with the integrin
ligands presented on the endothelial cells for firm adhesion
and crawling.
For neutrophils, the most important integrins are CD11a/
CD18 (LFA-1) and CD11b/CD18 (Mac-1), and both inte-
grins bind CD54 (ICAM-1). Previously, CD11a and
CD11b, both of which partner CD18, were thought to
possess similar and overlapping roles in neutrophil adhe-
sion. An elegant study by Hentzen et al. [68] over a decade
ago using in vitro flow chamber assays and mathematical
modeling proposed that CD11a and CD11b function
sequentially for neutrophil initial capture and subsequent
adhesion, based on their capture efficiency and adhesion
stability kineticswhile CD11a was much more efficient
than CD11b in binding CD54, CD11b was able to maintain
that binding for a much longer period of time. It is
important to note that the calculations of capture efficiency
were based on end-point measurements determined by flow
cytometry, and the authors were thus unable to place the
relevance of their findings in the scheme of the exact
adhesion cascade. The roles of CD11a and CD11b in the
adhesion cascade were later validated by Phillipson et al.
[69] in IVM experiments on the cremaster muscle in vivo.
However, rather than playing sequential roles during the
initial tethering and adhesion process, CD11a and CD11b
were found to be crucial for the steps of firm adhesion and
subsequent intraluminal crawling, respectively. In their
study, blocking CD11a on neutrophils resulted in a pro-
nounced inability for these cells to adhere, but those few
that managed to adhere displayed normal crawling on the
vessel walls. In contrast, when CD11b was blocked, neu-
trophils could still adhere normally but were unable to
perform intraluminal crawling. Interestingly, in monocytes,
CD11a/CD18 is known to additionally bind CD102
(ICAM-2) and has been demonstrated to be important for
intraluminal crawling [70], but curiously, neutrophils uti-
lize only CD11b/CD18 for this purpose. Antibody block-
ade of CD54 in wild-type mice still allowed neutrophils to
adhere, but not crawl [69], and this suggests that CD11a/
CD18 may also utilize CD102 as a substitute for CD54
during firm adhesion. Other molecules may also be relevant
in certain contexts. For example, in an in vivo Escherichia
coli infection model, neutrophils in the liver exhibited
dependence on CD44 for adhesion instead [71].
Activated neutrophils almost always display intraluminal
crawling behavior after firm adhesion. One explanation
would be the need for the neutrophils to follow the chemo-
kine gradients in order to exit the blood vessels at the location
closest to the injury or pathogen site, and indeed, this was
observed in the hepatic focal injury model earlier described
[57]. However, in many other cases, most neutrophils
already land at locations very close to their eventual emi-
gration site and do not appear to follow intravascular che-
motactic gradients. Instead, this crawling behavior appears
to be necessary for a more mundane reasonthe sampling of
the blood vessel wall for an optimal endothelial cellcell
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junction for emigration. In the CD11b-deficient mice
described earlier, neutrophils were still able to transmigrate
after adhesion, but they did so with greatly reduced effi-
ciency [69], presumably since they were forced to exit in situ
at non-optimal sites without the ability to crawl. Indeed,
these neutrophils were later found to transmigrate predom-
inantly through transcellular [72] routes (directly through the
center of an endothelial cell) [73], which would be expected
to be less efficient than paracellular routes (via the junctions
at the borders of endothelial cells). In line with this thinking,
a recent dynamic imaging study showed that the main route
of neutrophil transmigration during inflammation was
paracellularonly approximately 10% of neutrophils
transmigrated transcellularly in their model [74]. Interest-
ingly, another study demonstrated that after firm adhesion,
most neutrophils start spontaneous crawling perpendicular to
the blood flow [75]. This perpendicular movement was
observed only under conditions of fluid flow and could be
reproduced in in vitro experiments without the need for
chemokines [75], suggesting that this behavior was me-
chanotactic in natureshear stress was necessary for the
neutrophil to orient itself with respect to blood flow in order
to crawl perpendicularly on the vessel wall. The perpendic-
ular movement allows neutrophils to quickly sample the
vessel wall for the longitudinal border junctions between two
endothelial cells, so that efficient paracellular transmigration
can occur in vivo. Perpendicular sampling is more efficient
because endothelial cells under shear flow conditions form
vessel walls in a brick-like manner, such that their width
(perpendicular to the vessel) is only one-fifth that of their
length (parallel to the vessel) [75]. Neutrophils are thus more
likely to arrive at a cellcell junction traveling perpendicu-
larly than traveling longitudinally along the length of an
endothelial cell. In fact, the authors also found that after
reaching their first cellcell junction through perpendicular
movement, most neutrophils switch crawling direction to
follow the junctions parallel to the vessel and were able to
travel against blood flow just as easily as traveling with blood
flow. In the same study, Vav-1 deficient neutrophils, which
display an inability to perform perpendicular crawling or
travel against blood flow, were also found to have impaired
transmigration efficiency, presumably due to their inability
to find optimal endothelial junctions for transmigration. The
fact that neutrophils continue to crawl longitudinally after
having already found an endothelial cellcell junction indi-
cates that certain portions along the endothelial borders must
provide even more optimal emigration. An early in vitro
study found that neutrophils preferentially transmigrate at
tricellular endothelial junctions and might thus represent the
optimal emigration sites [76].
Emigration, which involves the traversing of large cells
across an essentially gap-free wall, is a highly complex
process and involves several classes of molecules such as
CD31 (PECAM-1), CD99, ICAMs, cadherins, junctional
adhesion molecules, and integrins [77]. For example,
directionality of transmigration was recently found to be
dependent on JAM-C [74]. Despite this complexity, one
picture that emerges from studies on transmigration is that
the endothelial cells are not just passive gate-keepers, but
are actively involved in getting the neutrophil across [77].
As mentioned earlier, transmigration may occur through
paracellular or transcellular routes. In both routes, neutro-
phil adhesion induces the formation of endothelial adhesive
platforms (EAPs), which are pro-adhesive domains on the
endothelial surface [78]. Projections from the endothelial
cell membrane, termed docking structures, form and
facilitate subsequent transmigration [79]. During the
paracellular route, in addition to the forces generated by the
neutrophils when squeezing through the tight junctions,
endothelial cells rapidly alter their cell surface adhesion
molecule profiles to weaken endothelial cellcell attach-
ment in order to smooth the way for neutrophil passage and
will then rapidly re-establish these junctions once the
neutrophils have crossed [80]. This is necessary to maintain
the integrity of the endothelial barrier, but during severe
inflammatory responses, junctional disruptions may ensue
and result in increased vascular permeability, potentially
causing edema [77]. In the case of the transcellular route,
endothelial cells have to play an even more active role, as it
would be inconceivable for neutrophils to punch holes
through the endothelial cell centers without causing severe
losses in vascular integrity. Leukocytes are proposed to
form short actin-rich protrusions (termed podosomes) at
their contact regions with the endothelial cell, which results
in corresponding deformations in the endothelial cell
membrane [81]. This has been proposed to be a probing
mechanism for the leukocytes to identify suitable trans-
migration spots [82]. Next, endothelial cells form migra-
tory cups [83] that essentially allow neutrophils to sink
into the endothelial cell. This process is analogous to a
sequential phagocytosis and exocytosis process, because
endothelial dome structures [73] immediately close behind
the neutrophils after their initial entry into the endothelial
cell, preserving the integrity of the endothelial wall before
membrane closure after neutrophil exit (see Fig. 3).
Interstitium
Perhaps one of the biggest recent advances in the field of
neutrophil research is the ability to directly visualize neu-
trophil activities within the interstitium by MP-IVM. Using
this approach, several studies have revealed that a charac-
teristic behavior of neutrophils is their propensity to form
dynamic swarms. This behavior has been characterized in
pathogen infection models, including Toxoplasma gondii in
the lymph node [84] and Listeria monocytogenes in the lung
174 Singapore Immunology Network: SIgN (2012) 53:168181
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[45], as well as in sterile models, including laser injury model
in ear dermis [5] and ischemiareperfusion injury in lung
explants [45], indicating that swarming may be a general
neutrophil effector strategy. Typically, multiple neutrophils
will enter the interstitium in response to a specific stimulus
and swarm around the stimulus, for example parasite or
injury location. Of note, in the ischemic-reperfusion injury
model, tissue damage is expected to be uniform, yet
swarming behavior was still observed [45]. In the T. gondii
model, swarms may be either small and transient or large and
persistent [84]. Small swarms were observed to contain
approximately 150 neutrophils and last for 1040 min before
dissipating. This dissipation occurred as a result of the neu-
trophils leaving the swarm to join other swarm clusters. On
the other hand, when a swarm consisted of more than 300
neutrophils, they grew larger in size instead of dissipating
and remained stable for extended periods of time up to sev-
eral hours. This increase in size could be due to the continued
migration of neutrophils into the swarm cluster, or it could
also arise from their merging with smaller nearby swarms.
Large clusters may include up to 2,000 neutrophils. This
behavior suggests a mechanism whereby neutrophils secrete
their own chemotactic signals to induce swarming in a
positive feedback cycle, where a certain threshold concen-
tration exists that allows swarm numbers to be stably
maintained [84]. In the L. monocytogenes model, the authors
detected small, transient swarms that dissipated in
1020 min [45], but also observed the dynamic nature of the
swarms in which clusters grow and shrink.
In the sterile injury model of mouse ear skin, neutrophils
formed a single cluster at the localized laser injury site, and
the cluster remained stable for at least an hour [5]. In this
model, a three-phase cascade of cluster formation was
observed, which included scouting, amplification, and
stabilization phases. The accumulation of neutrophils at the
injury site was observed to be initiated by a few scouting
neutrophils. These scouting neutrophils appeared to move
randomly within the tissue at basal speeds, but upon their
arrival at the injury foci, additional waves of neutrophils
rapidly moved through the interstitium with markedly
increased velocity and directionality toward the injury foci.
The amplification of the neutrophil numbers persisted for
approximately 30 min, before numbers stabilized and
plateaued off. The scouting phase was found to be depen-
dent on Gai-mediated signaling, suggesting the involve-ment of chemokine receptors, whereas the amplification
phase depended on cADPR-mediated signaling, which is
known to be involved in the recognition of certain DAMPs,
including those mediated by formyl-peptide receptors [85,
86]. Similarly, in the T. gondii model, amplification phases
were described to occur after the arrival of pioneer neu-
trophils [84]. Before swarming occurred, some neutrophils
were observed to meander randomly across the cluster
focus, but after the arrival and arrest of pioneer neutrophils,
those earlier neutrophils were observed returning to con-
tribute to swarm formation. Swarming behavior may thus
operate similarly in the context of a pathogen invasion and
during sterile injury. A role for monocytes in initiating
swarming behavior was proposed in the ischemic-reperfu-
sion injury model [45], and thus neutrophil swarming
behavior may be initiated by various mechanisms and
potentially involve different signals.
At the injury foci
Upon reaching their target site, neutrophils can perform
several effector functions. Neutrophils are able to secrete the
contents of their preformed granules, which contain a
cocktail of antibacterial peptides and enzymes [87]. Granule
types include azurophilic (primary), specific (secondary),
and gelatinase (tertiary), in addition to vesicles [88]. Neu-
trophils can also generate and release toxic reactive oxygen
species (ROS) through NADPH oxidase in a respiratory
burst mechanism [89]. Additionally, neutrophils are profi-
cient phagocytes and are able to hunt down and engulf
opsonized bacteria and parasites, which include those
opsonized by either complement or immunoglobulins [49].
Engulfed contents are then subjected to enzymatic degra-
dation in the phagolysosomes, which contain additional
antibacterial peptides and toxic chemicals as a decontami-
nation measure. More recently, there has been growing
evidence for an additional method of pathogen defense
involving the release of neutrophil extracellular traps (NETs)
[90], which comprise of dense strands of DNA and proteins
released from within the cell body. The DNA found within
NETs is believed to be nuclear chromosomal DNA, but some
studies have shown mitochondrial DNA to be possible as
well [91]. NETs contain several highly positively charged
molecules such as histones and are able to trap negatively
charged bacteria that make contact. In addition to DNA,
other antibacterial peptides and proteases are extruded,
including elastase, cathepsins, and lactoferrins among many
others. Most of these studies about NETs relied on in vitro
Fig. 3 Schematic showing paracellular and transcellular routes oftransmigration by neutrophils across endothelial wall
Singapore Immunology Network: SIgN (2012) 53:168181 175
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approaches. To the best of our knowledge, a study from
Gunzers laboratory represents the first description of using
MP imaging to study the formation of NETs in mice in vivo.
Using the lung slice approach, they were able to image NETs
and neutrophil behavior in Aspergillus fumigatus-infected
lungs [92]. The process of forming NETs is not an uncon-
trolled cell burst and appears to follow a well-defined pro-
gram. More than one program exists, depending on the
context of activation, and may include steps such as the
systematic breakdown of nuclear envelope, modification of
the DNA for increased toxicity, and packaging of DNA into
vesicles for release [61]. Conceivably, NETs function most
effectively within the blood, since shear forces would flush
circulating bacteria directly into the mesh of DNA for trap-
ping and killing, whereas the other three defense mecha-
nisms mentioned earlier are unlikely to be of any use in this
setting. Indeed, TLR4-activated platelets trigger the forma-
tion of NETs by neutrophils [93]since platelets require a
much higher threshold of TLR4 signaling than neutrophils to
become activated, their activation signifies that a severe
infection has already occurred within the blood compartment
[61]. Thus, neutrophils possess a large variety of weapons in
their arsenal and can employ very different tactics in
response to a pathogen invasion.
In addition, neutrophils are also able to secrete a variety of
cytokines and thus participate in shaping the immune land-
scape. For example, during T. gondii infection, neutrophils
engulf the parasite but are unable to perform killing, thereby
acting as potential hosts for facilitating pathogen spread.
Interestingly, however, the depletion of neutrophils resulted
in a worse prognosis [94], and the protective effect of neu-
trophils was attributed to their shaping of the subsequent
adaptive immune response via cytokine production [95].
Other major functions that neutrophils can perform include
tissue remodeling [84], as well as antigen presentation to
specialized cells of the adaptive immune system [96].
On the other hand, the exact roles of neutrophils at sites
of sterile injury are not well understood. The recruitment of
neutrophils to sterile injury sites may be an evolutionary
response to the strong association between pathogen
invasion and tissue injury. In fact, in addition to triggering
similar inflammatory responses, many DAMPs can be
recognized by the same receptors as PAMPs [40]. Thus,
neutrophil recruitment during sterile conditions may pri-
marily represent a precautionary measure against patho-
gens. There could be advantages to the pre-emptive
recruitment of neutrophils prior to pathogen entry, such as
the initiation of speedier and more robust responses upon
pathogen arrival, which would reduce the likelihood of
successful countermeasures by the pathogen. The presence
of injury may also signify invisible pathogens that
neutrophils are unable to detect, which may include hidden
intracellular viruses. Consequently, a pro-inflammatory
microenvironment ensues around the injury site, estab-
lished largely by TNFa and IL-1 [40]. The exact contri-bution made by neutrophils under this condition is
uncertain. Neutrophils are thought to release granule con-
tents (which include proteolytic enzymes such as elastase,
gelatinase B, and collagenase [87]) and phagocytose cel-
lular debris for tissue remodeling, but the released granule
contents also possess some pathogen extermination ability.
Neutrophil depletion has been associated with the accel-
erated closure of wounds in the epidermis [97], and this
phenomenon can be attributed to the ability of neutrophils
to induce keratinocyte differentiation, which slows down
keratinocyte hyperproliferation [98]. The increased rates of
keratinocyte differentiation possibly protect against tumor
formation, while the delayed wound-healing process may
be necessary to buy time for the adaptive immune system
to establish an effective response during events for which
neutrophils have no satisfactory responses against, such as
viral infections [98]. This delay may also be necessary for
the neutrophils to buffer enough time for their own clear-
ance of the infection, since wound closure might be fol-
lowed by a period of hypoxia where neutrophils may not
act effectively (e.g., in producing ROS) [98]. Curiously
though, neutrophil depletion had no apparent effect on the
rate of wound healing in the dermis [97, 99].
Neutrophil clearance
Many of the effector functions of neutrophils result in
damage to themselvessecreted granules and ROS are
toxic to the neutrophils, phagocytic neutrophils become
unable to continue the process above a certain threshold
[100], and the release of NETs entails the loss of tran-
scriptional capability necessary for continued cell survival.
Consequently, in most cases, activated neutrophils have
short life spans and will undergo apoptosis and clearance
by tissue macrophages. This uptake of apoptotic neutro-
phils by macrophages instructs macrophages to undergo a
gradual switch from inflammatory to anti-inflammatory
cytokine production and may thus promote the resolution
of inflammation [101].
As for the non-activated neutrophils that fulfill their
designated patrolling lifetimes, although the majority will
retire in the liver (29%) and spleen (31%), many will return
to the bone marrow (32%) for clearance [16, 23]. The
clearance of these neutrophils is mediated by specialized
macrophages within the various organs [102]. Homing of
neutrophils to the liver occurs independently of Gai-med-iated signaling. In contrast, for the spleen, half of the
homing was attributed to a Gai-dependent pathway [16],and thus at least two distinct pathways regulate neutrophil
clearance, one of which might be chemokine dependent
176 Singapore Immunology Network: SIgN (2012) 53:168181
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[102]. On the other hand, neutrophil homing to the bone
marrow appears almost fully dependent on CXCR4
CXCL12 interaction. Cell surface expression of CXCR4 on
neutrophils gradually increases as they age [26, 103] and
enables neutrophils that are nearing the end of their
patrolling lifetimes to return to the bone marrow via
CXCL12 binding. These neutrophils can then transmigrate
out of the sinusoids back into the extravascular cavities of
the bone marrow. However, this sinusoid-to-bone-marrow
transmigration is CD18 independent [104], which is in
contrast to transendothelial migration in the periphery. The
adhesion molecules responsible for sinusoid-to-bone-mar-
row transmigration are as yet unknown, but CD49d
(VLA-4 subunit a) has been implicated in the bone-mar-row-to-sinusoid transmigration [105]. Upon entry back into
the bone marrow, neutrophils initiate apoptosis and depend
on macrophages for their removal. At this stage, the jour-
ney of a neutrophil would be concluded, but even then, a
final role awaits themapoptotic neutrophils stimulate the
bone marrow macrophages that engulf them to produce
G-CSF [44], which in turn induces the production and
survival of successive neutrophil populations. Hence,
neutrophil production rates are pegged to neutrophil utili-
zation rates, providing a mechanism for the bone marrow
to regulate total neutrophil numbers. Therefore, an intricate
feedback system exists, at least partly mediated by the
neutrophils themselves, to maintain the integrity of the
immune surveillance program, ensuring their continued
vigilance against pathogen attack.
Conclusion
A fundamental characteristic of the immune system lies in
the highly dynamic nature of its cellular components. This
is perhaps best represented by the in vivo behavior of
neutrophils, which have the ability to rapidly shuttle
between different body compartments to carry out their
effector functions in a timely manner. The studies dis-
cussed in this review have employed different IVM
approaches to study neutrophil behavior in various tissues,
uncovering some of the previously unknown or underap-
preciated aspects of neutrophil functions. Current and
emerging evidence implies strongly that many of the
complexities of neutrophil behavior can only be revealed
when these processes are studied in real-time in vivo and in
stimulus- and tissue-specific manners. We thus have reason
to believe that IVM, especially that of MP-IVM, will
continue to play a vital role in future investigations of the
cellular functions of neutrophils in vivo.
Acknowledgments We would like to thank Dr. Jo Keeble for hercritical comments on the manuscript and proofreading.
References
1. Semerad CL, Liu F, Gregory AD, Stumpf K, Link DC. G-CSF is
an essential regulator of neutrophil trafficking from the bone
marrow to the blood. Immunity. 2002;17(4):41323. Epub
2002/10/22. PubMed PMID: 12387736.
2. Dancey JT, Deubelbeiss KA, Harker LA, Finch CA. Neutrophil
kinetics in man. J Clin Invest. 1976;58(3):70515. Epub
1976/09/01. doi:10.1172/JCI108517. PubMed PMID: 956397;
PubMed Central PMCID: PMC333229.
3. Babior BM, Golde DW. Production, distribution, and fate of
neutrophils. In: Beutler E, Coller BS, Lichtman MA, Kipps TJ,
Seligsohn U, editors. Williams Hematology. New York, USA:
McGraw-Hill; 2001. pp. 7539.
4. Pillay J, den Braber I, Vrisekoop N, Kwast LM, de Boer RJ,
Borghans JA, et al. In vivo labeling with 2H2O reveals a human
neutrophil lifespan of 5.4 days. Blood. 2010;116(4):6257.
Epub 2010/04/23. doi:10.1182/blood-2010-01-259028. PubMed
PMID: 20410504.
5. Ng LG, Qin JS, Roediger B, Wang Y, Jain R, Cavanagh LL,
et al. Visualizing the neutrophil response to sterile tissue injury
in mouse dermis reveals a three-phase cascade of events.
J Invest Dermatol. 2011;131(10):205868. Epub 2011/06/24.
doi:10.1038/jid.2011.179. PubMed PMID: 21697893.
6. Waller A. Microscopic examination of some principal tissues of
the animal frame as observed in the tongue of the living frog,
toad, etc. Philos Mag. 1846;29:27187.
7. Germain RN, Miller MJ, Dustin ML, Nussenzweig MC.
Dynamic imaging of the immune system: progress, pitfalls and
promise. Nat Rev Immunol. 2006;6(7):497507. Epub 2006/06/
27. doi:10.1038/nri1884. PubMed PMID: 16799470.
8. Cahalan MD, Parker I, Wei SH, Miller MJ. Two-photon tissue
imaging: seeing the immune system in a fresh light. Nat Rev
Immunol. 2002;2(11):87280. Epub 2002/11/05. doi:10.1038/
nri935. PubMed PMID: 12415310; PubMed Central PMCID:
PMC2749751.
9. Miller MJ, Wei SH, Parker I, Cahalan MD. Two-photon imaging
of lymphocyte motility and antigen response in intact lymph
node. Science. 2002;296(5574):186973. Epub 2002/05/23.
doi:10.1126/science.1070051. PubMed PMID: 12016203.
10. Mempel TR, Henrickson SE, Von Andrian UH. T-cell priming
by dendritic cells in lymph nodes occurs in three distinct phases.
Nature. 2004;427(6970):1549. Epub 2004/01/09. doi:10.1038/
nature02238. PubMed PMID: 14712275.
11. Bousso P, Robey E. Dynamics of CD8?T cell priming by
dendritic cells in intact lymph nodes. Nat Immunol. 2003;
4(6):57985. Epub 2003/05/06. doi:10.1038/ni928. PubMed
PMID: 12730692.
12. Bousso P, Bhakta NR, Lewis RS, Robey E. Dynamics of thy-
mocyte-stromal cell interactions visualized by two-photon
microscopy. Science. 2002;296(5574):187680. Epub 2002/06/
08. doi:10.1126/science.1070945. PubMed PMID: 12052962.
13. Cahalan MD, Parker I. Choreography of cell motility and
interaction dynamics imaged by two-photon microscopy in
lymphoid organs. Annu Rev Immunol. 2008;26:585-626. Epub
2008/01/05. doi:10.1146/annurev.immunol.24.021605.090620.
PubMed PMID: 18173372; PubMed Central PMCID: PMC27
32400.
14. Ng LG, Mrass P, Kinjyo I, Reiner SL, Weninger W. Two-photon
imaging of effector T-cell behavior: lessons from a tumor
model. Immunol Rev. 2008;221:14762. Epub 2008/02/16.
doi:10.1111/j.1600-065X.2008.00596.x. PubMed PMID: 18275
480.
15. Mueller SN, Hickman HD. In vivo imaging of the T cell
response to infection. Curr Opin Immunol. 2010;22(3):2938.
Singapore Immunology Network: SIgN (2012) 53:168181 177
123
-
Epub 2010/01/19. doi:10.1016/j.coi.2009.12.009. PubMed PMID:
20080040.
16. Furze RC, Rankin SM. The role of the bone marrow in neu-
trophil clearance under homeostatic conditions in the mouse.
Faseb J. 2008;22(9):31119. Epub 2008/05/30. doi:10.1096/
fj.08-109876. PubMed PMID: 18509199; PubMed Central
PMCID: PMC2593561.
17. Dale DC, Liles WC. Neutrophils and monocytes: Normal
physiology and disorders of neutrophil and monocyte produc-
tion. In: Handin RI, Lux SE, Stossel TP, editors. Blood:
principles and practice of hematology. Philadelphia, USA:
Lippincott Williams & Wilkins; 2003.
18. Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for
stem cell biology. Cell. 2008;132(4):63144. Epub 2008/02/26.
doi:10.1016/j.cell.2008.01.025. PubMed PMID: 18295580; Pub-
Med Central PMCID: PMC2628169.
19. Boxio R, Bossenmeyer-Pourie C, Steinckwich N, Dournon C,
Nusse O. Mouse bone marrow contains large numbers of func-
tionally competent neutrophils. J Leukoc Biol. 2004;75(4):
60411. Epub 2003/12/25. doi:10.1189/jlb.0703340. PubMed
PMID: 14694182.
20. Ashtekar AR, Saha B. Polys plea: membership to the club of
APCs. Trends Immunol. 2003;24(9):48590. doi:10.1016/s1471-
4906(03)00235-7.
21. Sabroe I, Prince LR, Jones EC, Horsburgh MJ, Foster SJ, Vogel
SN, et al. Selective roles for Toll-like receptor (TLR)2 and
TLR4 in the regulation of neutrophil activation and life span.
J Immunol. 2003;170(10):526875. Epub 2003/05/08. PubMed
PMID: 12734376.
22. John B, Hunter CA. Immunology. Neutrophil soldiers or Trojan
Horses? Science. 2008;321(5891):9178. Epub 2008/08/16.
doi:10.1126/science.1162914. PubMed PMID: 18703727.
23. Suratt BT, Young SK, Lieber J, Nick JA, Henson PM, Worthen
GS. Neutrophil maturation and activation determine anatomic
site of clearance from circulation. Am J Physiol Lung Cell Mol
Physiol. 2001;281(4):L91321. Epub 2001/09/15. PubMed
PMID: 11557595.
24. Athens JW, Haab OP, Raab SO, Mauer AM, Ashenbrucker H,
Cartwright GE, et al. Leukokinetic studies. IV. The total blood,
circulating and marginal granulocyte pools and the granulocyte
turnover rate in normal subjects. J Clin Invest. 1961;40:98995.
Epub 1961/06/01. doi:10.1172/JCI104338. PubMed PMID:
13684958; PubMed Central PMCID: PMC290816.
25. Kohler A, De Filippo K, Hasenberg M, van den Brandt C, Nye
E, Hosking MP, et al. G-CSF-mediated thrombopoietin release
triggers neutrophil motility and mobilization from bone marrow
via induction of Cxcr2 ligands. Blood. 2011;117(16):434957.
Epub 2011/01/13. doi:10.1182/blood-2010-09-308387. PubMed
PMID: 21224471; PubMed Central PMCID: PMC3087483.
26. Martin C, Burdon PC, Bridger G, Gutierrez-Ramos JC, Williams
TJ, Rankin SM. Chemokines acting via CXCR2 and CXCR4
control the release of neutrophils from the bone marrow and
their return following senescence. Immunity. 2003;19(4):58393.
Epub 2003/10/18. PubMed PMID: 14563322.
27. Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, So-
droski J, et al. The lymphocyte chemoattractant SDF-1 is a
ligand for LESTR/fusin and blocks HIV-1 entry. Nature.
1996;382(6594):82933. Epub 1996/08/29. doi:10.1038/
382829a0. PubMed PMID: 8752280.
28. Sugiyama T, Kohara H, Noda M, Nagasawa T. Maintenance of
the hematopoietic stem cell pool by CXCL12-CXCR4 chemo-
kine signaling in bone marrow stromal cell niches. Immunity.
2006;25(6):97788. Epub 2006/12/19. doi:10.1016/j.immuni.
2006.10.016. PubMed PMID: 17174120.
29. Bozic CR, Gerard NP, von Uexkull-Guldenband C, Kolakowski
LF, Jr., Conklyn MJ, Breslow R, et al. The murine interleukin 8
type B receptor homologue and its ligands. Expression and
biological characterization. J Biol Chem. 1994;269(47):
293558. Epub 1994/11/25. PubMed PMID: 7961909.
30. Eash KJ, Means JM, White DW, Link DC. CXCR4 is a key
regulator of neutrophil release from the bone marrow under
basal and stress granulopoiesis conditions. Blood. 2009;
113(19):47119. Epub 2009/03/07. doi:10.1182/blood-2008-09-
177287. PubMed PMID: 19264920; PubMed Central PMCID:
PMC2680371.
31. Suratt BT, Petty JM, Young SK, Malcolm KC, Lieber JG, Nick
JA, et al. Role of the CXCR4/SDF-1 chemokine axis in circu-
lating neutrophil homeostasis. Blood. 2004;104(2):56571.
Epub 2004/04/01. doi:10.1182/blood-2003-10-3638. PubMed
PMID: 15054039.
32. Delgado MB, Clark-Lewis I, Loetscher P, Langen H, Thelen M,
Baggiolini M, et al. Rapid inactivation of stromal cell-derived
factor-1 by cathepsin G associated with lymphocytes. Eur J
Immunol. 2001;31(3):699707. Epub 2001/03/10. doi:10.1002/
1521-4141(200103)31:33.0.CO;2-6. PubMed PMID: 11241273.
33. Levesque JP, Liu F, Simmons PJ, Betsuyaku T, Senior RM,
Pham C, et al. Characterization of hematopoietic progenitor
mobilization in protease-deficient mice. Blood. 2004;104(1):
6572. Epub 2004/03/11. doi:10.1182/blood-2003-05-1589.
PubMed PMID: 15010367.
34. Kim HK, De La Luz Sierra M, Williams CK, Gulino AV, Tosato G.
G-CSF down-regulation of CXCR4 expression identified as a
mechanism for mobilization of myeloid cells. Blood. 2006;
108(3):812-20. Epub 2006/03/16. doi:10.1182/blood-2005-10-
4162. PubMed PMID: 16537807; PubMed Central PMCID:
PMC1895847.
35. Delano MJ, Kelly-Scumpia KM, Thayer TC, Winfield RD,
Scumpia PO, Cuenca AG, et al. Neutrophil mobilization from the
bone marrow during polymicrobial sepsis is dependent on CXCL12
signaling. J Immunol. 2011;187(2):9118. Epub 2011/06/22.
doi:10.4049/jimmunol.1100588. PubMed PMID: 21690321.
36. Junt T, Schulze H, Chen Z, Massberg S, Goerge T, Krueger A,
et al. Dynamic visualization of thrombopoiesis within bone
marrow. Science. 2007;317(5845):176770. Epub 2007/09/22.
doi:10.1126/science.1146304. PubMed PMID: 17885137.
37. Seong SY, Matzinger P. Hydrophobicity: an ancient damage-
associated molecular pattern that initiates innate immune
responses. Nat Rev Immunol. 2004;4(6):46978. Epub 2004/06/
03. doi:10.1038/nri1372. PubMed PMID: 15173835.
38. Hayashi F, Means TK, Luster AD. Toll-like receptors stimulate
human neutrophil function. Blood. 2003;102(7):26609. Epub
2003/06/28. doi:10.1182/blood-2003-04-1078. PubMed PMID:
12829592.
39. Parker LC, Whyte MK, Dower SK, Sabroe I. The expression and
roles of Toll-like receptors in the biology of the human neu-
trophil. J Leukoc Biol. 2005;77(6):88692. Epub 2005/02/25.
doi:10.1189/jlb.1104636. PubMed PMID: 15728244.
40. Chen GY, Nunez G. Sterile inflammation: sensing and reacting
to damage. Nat Rev Immunol. 2010;10(12):82637. Epub
2010/11/23. doi:10.1038/nri2873. PubMed PMID: 21088683;
PubMed Central PMCID: PMC3114424.
41. von Vietinghoff S, Ley K. Homeostatic regulation of blood neu-
trophil counts. J Immunol. 2008;181(8):51838. Epub 2008/10/
04. PubMed PMID: 18832668; PubMed Central PMCID:
PMC2745132.
42. Pachter JS, de Vries HE, Fabry Z. The blood-brain barrier and
its role in immune privilege in the central nervous system.
J Neuropathol Exp Neurol. 2003;62(6):593604. Epub 2003/07/
02. PubMed PMID: 12834104.
43. Kim JV, Kang SS, Dustin ML, McGavern DB. Myelomonocytic
cell recruitment causes fatal CNS vascular injury during acute
178 Singapore Immunology Network: SIgN (2012) 53:168181
123
-
viral meningitis. Nature. 2009;457(7226):1915. Epub 2008/11/
18. doi:10.1038/nature07591. PubMed PMID: 19011611; Pub-
Med Central PMCID: PMC2702264.
44. Summers C, Rankin SM, Condliffe AM, Singh N, Peters AM,
Chilvers ER. Neutrophil kinetics in health and disease. Trends
Immunol. 2010;31(8):31824. Epub 2010/07/14. doi:10.1016/
j.it.2010.05.006. PubMed PMID: 20620114; PubMed Central
PMCID: PMC2930213.
45. Kreisel D, Nava RG, Li W, Zinselmeyer BH, Wang B, Lai J,
et al. In vivo two-photon imaging reveals monocyte-dependent
neutrophil extravasation during pulmonary inflammation. Proc
Natl Acad Sci USA. 2010;107(42):180738. Epub 2010/10/07.
doi:10.1073/pnas.1008737107. PubMed PMID: 20923880;
PubMed Central PMCID: PMC2964224.
46. Mitchell GF, Jeron A, Koren G. Measurement of heart rate and
Q-T interval in the conscious mouse. Am J Physiol. 1998;274
(3 Pt 2):H74751. Epub 1998/04/08. PubMed PMID: 9530184.
47. Kamoun WS, Chae SS, Lacorre DA, Tyrrell JA, Mitre M,
Gillissen MA, et al. Simultaneous measurement of RBC
velocity, flux, hematocrit and shear rate in vascular networks.
Nat Methods. 2010;7(8):65560. Epub 2010/06/29. doi:10.1038/
nmeth.1475. PubMed PMID: 20581828; PubMed Central
PMCID: PMC2921873.
48. Mazo IB, Gutierrez-Ramos JC, Frenette PS, Hynes RO, Wagner
DD, von Andrian UH. Hematopoietic progenitor cell rolling in
bone marrow microvessels: parallel contributions by endothelial
selectins and vascular cell adhesion molecule 1. J Exp Med.
1998;188(3):46574. Epub 1998/08/04. PubMed PMID:
9687524; PubMed Central PMCID: PMC2212463.
49. Hickey MJ, Kubes P. Intravascular immunity: the host-pathogen
encounter in blood vessels. Nat Rev Immunol. 2009;9(5):364
75. Epub 2009/04/25. doi:10.1038/nri2532. PubMed PMID: 193
90567.
50. Weninger W, Ulfman LH, Cheng G, Souchkova N, Quacken-
bush EJ, Lowe JB, et al. Specialized contributions by alpha(1,3)-
fucosyltransferase-IV and FucT-VII during leukocyte rolling in
dermal microvessels. Immunity. 2000;12(6):66576. Epub
2000/07/14. PubMed PMID: 10894166.
51. Bohannon J, Cui W, Sherwood E, Toliver-Kinsky T. Dendritic
cell modification of neutrophil responses to infection after burn
injury. J Immunol. 2010;185(5):284753. Epub 2010/08/04.
doi:10.4049/jimmunol.0903619. PubMed PMID: 20679533;
PubMed Central PMCID: PMC3100157.
52. Ng LG, Hsu A, Mandell MA, Roediger B, Hoeller C, Mrass P,
et al. Migratory dermal dendritic cells act as rapid sensors of
protozoan parasites. PLoS Pathog. 2008;4(11):e1000222. Epub
2008/12/02. doi:10.1371/journal.ppat.1000222. PubMed PMID:
19043558; PubMed Central PMCID: PMC2583051.
53. Malaviya R, Ikeda T, Ross E, Abraham SN. Mast cell modulation
of neutrophil influx and bacterial clearance at sites of infection
through TNF-alpha. Nature. 1996;381(6577):7780. Epub
1996/05/02. doi:10.1038/381077a0. PubMed PMID: 8609993.
54. Beck-Schimmer B, Schwendener R, Pasch T, Reyes L, Booy C,
Schimmer RC. Alveolar macrophages regulate neutrophil
recruitment in endotoxin-induced lung injury. Respir Res.
2005;6:61. Epub 2005/06/24. doi:10.1186/1465-9921-6-61.
PubMed PMID: 15972102; PubMed Central PMCID: PMC118
8075.
55. Mukai K, Matsuoka K, Taya C, Suzuki H, Yokozeki H,
Nishioka K, et al. Basophils play a critical role in the devel-
opment of IgE-mediated chronic allergic inflammation inde-
pendently of T cells and mast cells. Immunity. 2005;23(2):
191202. Epub 2005/08/23. doi:10.1016/j.immuni.2005.06.011.
PubMed PMID: 16111637.
56. Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, Kayal S,
et al. Monitoring of blood vessels and tissues by a population of
monocytes with patrolling behavior. Science. 2007;317(5838):
66670. Epub 2007/08/04. doi:10.1126/science.1142883. Pub-
Med PMID: 17673663.
57. McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I,
Waterhouse CC, et al. Intravascular danger signals guide neu-
trophils to sites of sterile inflammation. Science. 2010;330
(6002):3626. Epub 2010/10/16. doi:10.1126/science.1195491.
PubMed PMID: 20947763.
58. Chappell D, Westphal M, Jacob M. The impact of the glycocalyx on
microcirculatory oxygen distribution in critical illness. Curr Opin
Anaesthesiol. 2009;22(2):15562. Epub 2009/03/25. doi:10.1097/
ACO.0b013e328328d1b6. PubMed PMID: 19307890.
59. Massena S, Christoffersson G, Hjertstrom E, Zcharia E,
Vlodavsky I, Ausmees N, et al. A chemotactic gradient seques-
tered on endothelial heparan sulfate induces directional intra-
luminal crawling of neutrophils. Blood. 2010;116(11):192431.
Epub 2010/06/10. doi:10.1182/blood-2010-01-266072. PubMed
PMID: 20530797; PubMed Central PMCID: PMC3173988.
60. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, et al.
Circulating mitochondrial DAMPs cause inflammatory respon-
ses to injury. Nature. 2010;464(7285):1047. Epub 2010/03/06.
doi:10.1038/nature08780. PubMed PMID: 20203610; PubMed
Central PMCID: PMC2843437.
61. Phillipson M, Kubes P. The neutrophil in vascular inflammation.
Nat Med. 2011;17(11):138190. Epub 2011/11/09. doi 10.1038/
nm.2514. PubMed PMID: 22064428.
62. Heit B, Robbins SM, Downey CM, Guan Z, Colarusso P, Miller
BJ, et al. PTEN functions to prioritize chemotactic cues and
prevent distraction in migrating neutrophils. Nat Immunol.
2008;9(7):74352. Epub 2008/06/10. doi:10.1038/ni.1623. Pub-
Med PMID: 18536720.
63. Foxman EF, Campbell JJ, Butcher EC. Multistep navigation and
the combinatorial control of leukocyte chemotaxis. J Cell Biol.
1997;139(5):134960. Epub 1998/01/07. PubMed PMID: 938
2879; PubMed Central PMCID: PMC2140208.
64. Sogawa Y, Ohyama T, Maeda H, Hirahara K. Inhibition of
neutrophil migration in mice by mouse formyl peptide receptors
1 and 2 dual agonist: indication of cross-desensitization in vivo.
Immunology. 2011;132(3):44150. Epub 2010/11/03. doi:10.
1111/j.1365-2567.2010.03367.x. PubMed PMID: 21039475;
PubMed Central PMCID: PMC3044910.
65. Niethammer P, Grabher C, Look AT, Mitchison TJ. A tissue-
scale gradient of hydrogen peroxide mediates rapid wound
detection in zebrafish. Nature. 2009;459(7249):9969. Epub
2009/06/06. doi:10.1038/nature08119. PubMed PMID: 19494
811; PubMed Central PMCID: PMC2803098.
66. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the
site of inflammation: the leukocyte adhesion cascade updated.
Nat Rev Immunol. 2007;7(9):67889. Epub 2007/08/25.
doi:10.1038/nri2156. PubMed PMID: 17717539.
67. Coxon A, Cullere X, Knight S, Sethi S, Wakelin MW, Stavrakis G,
et al. Fc gamma RIII mediates neutrophil recruitment to immune
complexes. a mechanism for neutrophil accumulation in immune-
mediated inflammation. Immunity. 2001;14(6):693704. Epub
2001/06/23. PubMed PMID: 11420040.
68. Hentzen ER, Neelamegham S, Kansas GS, Benanti JA, McIntire
LV, Smith CW, et al. Sequential binding of CD11a/CD18 and
CD11b/CD18 defines neutrophil capture and stable adhesion to
intercellular adhesion molecule-1. Blood. 2000;95(3):91120.
Epub 2000/01/29. PubMed PMID: 10648403.
69. Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne CM,
Kubes P. Intraluminal crawling of neutrophils to emigration
sites: a molecularly distinct process from adhesion in the
recruitment cascade. J Exp Med. 2006;203(12):256975. Epub
2006/11/23. doi:10.1084/jem.20060925. PubMed PMID:
17116736; PubMed Central PMCID: PMC2118150.
Singapore Immunology Network: SIgN (2012) 53:168181 179
123
-
70. Schenkel AR, Mamdouh Z, Muller WA. Locomotion of
monocytes on endothelium is a critical step during extravasa-
tion. Nat Immunol. 2004;5(4):393400. Epub 2004/03/17.
doi:10.1038/ni1051. PubMed PMID: 15021878.
71. Menezes GB, Lee WY, Zhou H, Waterhouse CC, Cara DC,
Kubes P. Selective down-regulation of neutrophil Mac-1 in
endotoxemic hepatic microcirculation via IL-10. J Immunol.
2009;183(11):755768. Epub 2009/11/18. doi:10.4049/jimmu
nol.0901786. PubMed PMID: 19917697.
72. Feng D, Nagy JA, Pyne K, Dvorak HF, Dvorak AM. Neutrophils
emigrate from venules by a transendothelial cell pathway in
response to FMLP. J Exp Med. 1998;187(6):90315. Epub
1998/04/04. PubMed PMID: 9500793; PubMed Central PMCID:
PMC2212194.
73. Phillipson M, Kaur J, Colarusso P, Ballantyne CM, Kubes P.
Endothelial domes encapsulate adherent neutrophils and mini-
mize increases in vascular permeability in paracellular and
transcellular emigration. PLoS One. 2008;3(2):e1649. Epub
2008/02/26. doi:10.1371/journal.pone.0001649. PubMed PMID:
18297135; PubMed Central PMCID: PMC2250804.
74. Woodfin A, Voisin MB, Beyrau M, Colom B, Caille D, Diapouli
FM, et al. The junctional adhesion molecule JAM-C regulates
polarized transendothelial migration of neutrophils in vivo. Nat
Immunol. 2011;12(8):7619. Epub 2011/06/28. doi:10.1038/
ni.2062. PubMed PMID: 21706006; PubMed Central PMCID:
PMC3145149.
75. Phillipson M, Heit B, Parsons SA, Petri B, Mullaly SC,
Colarusso P, et al. Vav1 is essential for mechanotactic crawling
and migration of neutrophils out of the inflamed micro-
vasculature. J Immunol. 2009;182(11):68708. Epub 2009/05/
21. doi:10.4049/jimmunol.0803414. PubMed PMID: 19454683.
76. Burns AR, Walker DC, Brown ES, Thurmon LT, Bowden RA,
Keese CR, et al. Neutrophil transendothelial migration is inde-
pendent of tight junctions and occurs preferentially at tricellular
corners. J Immunol. 1997;159(6):2893903. Epub 1997/09/23.
PubMed PMID: 9300713.
77. Schmidt EP, Lee WL, Zemans RL, Yamashita C, Downey GP.
On, around, and through: neutrophil-endothelial interactions in
innate immunity. Physiology (Bethesda). 2011;26(5):33447.
Epub 2011/10/21. doi:10.1152/physiol.00011.2011. PubMed
PMID: 22013192.
78. Barreiro O, Zamai M, Yanez-Mo M, Tejera E, Lopez-Romero P,
Monk PN, et al. Endothelial adhesion receptors are recruited to
adherent leukocytes by inclusion in preformed tetraspanin
nanoplatforms. J Cell Biol. 2008;183(3):52742. Epub 2008/10/
29. doi:10.1083/jcb.200805076. PubMed PMID: 18955551;
PubMed Central PMCID: PMC2575792.
79. Barreiro O, Yanez-Mo M, Serrador JM, Montoya MC, Vicente-
Manzanares M, Tejedor R, et al. Dynamic interaction of
VCAM-1 and ICAM-1 with moesin and ezrin in a novel endo-
thelial docking structure for adherent leukocytes. J Cell Biol.
2002;157(7):123345. Epub 2002/06/26. doi:10.1083/jcb.20011
2126. PubMed PMID: 12082081; PubMed Central PMCID:
PMC2173557.
80. Muller WA. Leukocyte-endothelial-cell interactions in leuko-
cyte transmigration and the inflammatory response. Trends
Immunol. 2003;24(6):32734. Epub 2003/06/18. PubMed
PMID: 12810109.
81. Carman CV, Sage PT, Sciuto TE, de la Fuente MA, Geha RS,
Ochs HD, et al. Transcellular diapedesis is initiated by invasive
podosomes. Immunity. 2007;26(6):78497. Epub 2007/06/16.
doi:10.1016/j.immuni.2007.04.015. PubMed PMID: 17570692;
PubMed Central PMCID: PMC2094044.
82. Carman CV. Mechanisms for transcellular diapedesis: probing
and pathfinding by invadosome-like protrusions. J Cell Sci.
2009;122(Pt 17):302535. Epub 2009/08/21. doi:10.1242/
jcs.047522. PubMed PMID: 19692589.
83. Carman CV, Springer TA. A transmigratory cup in leukocyte
diapedesis both through individual vascular endothelial cells and
between them. J Cell Biol. 2004;167(2):37788. Epub 2004/10/
27. doi:10.1083/jcb.200404129. PubMed PMID: 15504916;
PubMed Central PMCID: PMC2172560.
84. Chtanova T, Schaeffer M, Han SJ, van Dooren GG, Nollmann
M, Herzmark P, et al. Dynamics of neutrophil migration in
lymph nodes during infection. Immunity. 2008;29(3):48796.
Epub 2008/08/23. doi:10.1016/j.immuni.2008.07.012. PubMed
PMID: 18718768; PubMed Central PMCID: PMC2569002.
85. Partida-Sanchez S, Cockayne DA, Monard S, Jacobson EL,
Oppenheimer N, Garvy B, et al. Cyclic ADP-ribose production
by CD38 regulates intracellular calcium release, extracellular
calcium influx and chemotaxis in neutrophils and is required for
bacterial clearance in vivo. Nat Med. 2001;7(11):120916. Epub
2001/11/02. doi:10.1038/nm1101-1209. PubMed PMID: 116
89885.
86. Partida-Sanchez S, Iribarren P, Moreno-Garcia ME, Gao JL,
Murphy PM, Oppenheimer N, et al. Chemotaxis and calcium
responses of phagocytes to formyl peptide receptor ligands is
differentially regulated by cyclic ADP ribose. J Immunol.
2004;172(3):1896906. Epub 2004/01/22. PubMed PMID:
14734775.
87. Borregaard N, Cowland JB. Granules of the human neutrophilic
polymorphonuclear leukocyte. Blood. 1997;89(10):350321.
Epub 1997/05/15. PubMed PMID: 9160655.
88. Lacy P. Mechanisms of degranulation in neutrophils. Allergy
Asthma Clin Immunol. 2006;2(3):98108. Epub 2006/09/15.
doi:10.1186/1710-1492-2-3-98. PubMed PMID: 20525154;
PubMed Central PMCID: PMC2876182.
89. de Oliveira-Junior EB, Bustamante J, Newburger PE, Condino-
Neto A. The human NADPH oxidase: primary and secondary
defects impairing the respiratory burst function and the micro-
bicidal ability of phagocytes. Scand J Immunol. 2011;73(5):
4207. Epub 2011/01/06. doi:10.1111/j.1365-3083.2010.025
01.x. PubMed PMID: 21204900.
90. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann
Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria.
Science. 2004;303(5663):15325. Epub 2004/03/06. doi:10.
1126/science.1092385. PubMed PMID: 15001782.
91. Yousefi S, Mihalache C, Kozlowski E, Schmid I, Simon HU.
Viable neutrophils release mitochondrial DNA to form neutro-
phil extracellular traps. Cell Death Differ. 2009;16(11):
143844. Epub 2009/07/18. doi:10.1038/cdd.2009.96. PubMed
PMID: 19609275.
92. Bruns S, Kniemeyer O, Hasenberg M, Aimanianda V, Nietzsche
S, Thywissen A, et al. Production of extracellular traps against
Aspergillus fumigatus in vitro and in infected lung tissue is
dependent on invading neutrophils and influenced by
hydrophobin RodA. PLoS Pathog. 2010;6(4):e1000873. Epub
2010/05/06. doi:10.1371/journal.ppat.1000873. PubMed PMID:
20442864; PubMed Central PMCID: PMC2861696.
93. Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, Kelly
MM, et al. Platelet TLR4 activates neutrophil extracellular traps
to ensnare bacteria in septic blood. Nat Med. 2007;13(4):4639.
Epub 2007/03/27. doi:10.1038/nm1565. PubMed PMID: 173
84648.
94. Bliss SK, Gavrilescu LC, Alcaraz A, Denkers EY. Neutrophil
depletion during Toxoplasma gondii infection leads to impaired
immunity and lethal systemic pathology. Infect Immun. 2001;
69(8):4898905. Epub 2001/07/12. doi:10.1128/IAI.69.8.4898-
4905.2001. PubMed PMID: 11447166; PubMed Central PMCID:
PMC98580.
180 Singapore Immunology Network: SIgN (2012) 53:168181
123
-
95. Denkers EY, Butcher BA, Del Rio L, Bennouna S. Neutrophils,
dendritic cells and Toxoplasma. Int J Parasitol. 2004;34(3):
41121. Epub 2004/03/09. doi:10.1016/j.ijpara.2003.11.001. Pub-
Med PMID: 15003500.
96. Shen L, Rock KL. Priming of T cells by exogenous antigen
cross-presented on MHC class I molecules. Curr Opin Immunol.
2006;18(1):8591. Epub 2005/12/06. doi:10.1016/j.coi.2005.11.
003. PubMed PMID: 16326087.
97. Dovi JV, He LK, DiPietro LA. Accelerated wound closure in
neutrophil-depleted mice. J Leukoc Biol. 2003;73(4):44855.
Epub 2003/03/28. PubMed PMID: 12660219.
98. Dovi JV, Szpaderska AM, DiPietro LA. Neutrophil function in
the healing wound: adding insult to injury? Thrombosis and
Haemostasis. 2004. doi:10.1160/th03-11-0720.
99. Simpson DM, Ross R. The neutrophilic leukocyte in wound
repair a study with antineutrophil serum. J Clin Invest.
1972;51(8):200923. Epub 1972/08/01. doi:10.1172/JCI107007.
PubMed PMID: 5054460; PubMed Central PMCID: PMC292
357.
100. Simon SI, Schmid-Schonbein GW. Biophysical aspects of
microsphere engulfment by human neutrophils. Biophys J. 1988;
53(2):16373. Epub 1988/02/01. doi:10.1016/S0006-3495(88)
83078-9. PubMed PMID: 3345329; PubMed Central PMCID:
PMC1330137.
101. Fadok VA, McDonald PP, Bratton DL, Henson PM. Regulation
of macrophage cytokine production by phagocytosis of apop-
totic and post-apoptotic cells. Biochem Soc Trans. 1998;
26(4):6536. Epub 1999/02/27. PubMed PMID: 10047800.
102. Rankin SM. The bone marrow: a site of neutrophil clearance.
J Leukoc Biol. 2010;88(2):24151. Epub 2010/05/21. doi:10.
1189/jlb.0210112. PubMed PMID: 20483920.
103. Nagase H, Miyamasu M, Yamaguchi M, Imanishi M, Tsuno
NH, Matsushima K, et al. Cytokine-mediated regulation of
CXCR4 expression in human neutrophils. J Leukoc Biol. 2002;
71(4):7117. Epub 2002/04/03. PubMed PMID: 11927659.
104. Stark MA, Huo Y, Burcin TL, Morris MA, Olson TS, Ley K.
Phagocytosis of apoptotic neutrophils regulates granulopoiesis
via IL-23 and IL-17. Immunity. 2005;22(3):28594. Epub
2005/03/23. doi:10.1016/j.immuni.2005.01.011. PubMed PMID:
15780986.
105. Burdon PC, Martin C, Rankin SM. The CXC chemokine MIP-2
stimulates neutrophil mobilization from the rat bone marrow in a
CD49d-dependent manner. Blood. 2005;105(6):25438. Epub
2004/11/16. doi:10.1182/blood-2004-08-3193. PubMed PMID:
15542579.
Singapore Immunology Network: SIgN (2012) 53:168181 181
123
Peeking into the secret life of neutrophilsPeeking into the secret life of neutrophilsIntroductionThe behavior of neutrophils in different tissue compartmentsBone marrowBloodBlood vessel wallsInterstitiumAt the injury foci
Neutrophil clearanceConclusionAcknowledgmentsReferences