peeking into the secret life of neutrophils

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

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

Singapore Immunology Network: SIgN (2012) 53:168181 169

123

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

170 Singapore Immunology Network: SIgN (2012) 53:168181

123

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


123

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


123

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


123

[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


123

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


123

[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.

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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

peeking into the secret life of neutrophils

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