The Multifaceted Functions of Neutrophils

TL;DR: Primordial neutrophil functions are discussed, and more recent evidence that interactions between neutrophils and adaptive immune cells establish a feed-forward mechanism that amplifies pathologic inflammation is presented.

Abstract: Neutrophils and neutrophil-like cells are the major pathogen-fighting immune cells in organisms ranging from slime molds to mammals. Central to their function is their ability to be recruited to sites of infection, to recognize and phagocytose microbes, and then to kill pathogens through a combination of cytotoxic mechanisms. These include the production of reactive oxygen species, the release of antimicrobial peptides, and the recently discovered expulsion of their nuclear contents to form neutrophil extracellular traps. Here we discuss these primordial neutrophil functions, which also play key roles in tissue injury, by providing details of neutrophil cytotoxic functions and congenital disorders of neutrophils. In addition, we present more recent evidence that interactions between neutrophils and adaptive immune cells establish a feed-forward mechanism that amplifies pathologic inflammation. These newly appreciated contributions of neutrophils are described in the setting of several inflammatory and autoimmune diseases.

Summary

INTRODUCTION

• Multicellular organisms face a constant challenge of surviving in an environment containing unicellular pathogens.
• Indeed, the survival of primitive organisms—for example, insects, which lack adaptive immune cells such as lymphocytes—relies on the function of their innate immune phagocytes (1).
• Thus, not surprisingly, 55% to 60% of the bone marrow is dedicated to their production (2).
• Traditional estimates based on ex vivo survival in culture or on half-life after adoptive transfer suggested that these cells survive for only 8–12 h in the circulation and up to 1–2 days in tissues, with their turnover delayed or accelerated during the inflammatory response (3–5).
• 8), these types of more updated immunological investigations are changing their overall perception of neutrophil function in immunity.

NEUTROPHIL HOMEOSTASIS

• Neutrophils are formed within the bone marrow during hematopoiesis in response to several cytokines, principally granulocyte colony–stimulating factor (G-CSF) (12).
• The IL-23–IL-17–G-CSF feedback loop is clearly not the only mechanism controlling neutrophil production, given that mice lacking T lymphocytes and NK cells (i.e., the major sources of IL-17) have normal neutrophil numbers (20, 21).
• That hematopoietic progenitors can ramp up production of granulocytes in response to inflammatory or pathogen challenge, even in the absence of canonical granulopoiesis-stimulating cytokines, indicates that other signaling pathways must exist to regulate granulopoiesis.
• Finally, the observation that mice lacking commensal organisms—that is, germ-free animals—have dramatic neutropenia, with neutrophil levels even lower than those in combined G-CSF and GM-CSF knockout mice, also suggests www.annualreviews.org Multifaceted Functions of Neutrophils 183 A nn u.
• Under any given condition, one pathway may dominate over the others, but a clear understanding of regulation of neutrophil numbers (especially during disease states) will require further research.

NEUTROPHIL RECRUITMENT

• After their birth in the bone marrow, mature neutrophils reach sites of tissue inflammation or infection via the vasculature.
• The neutrophil recruitment cascade is mediated by the sequential interaction of receptors present on neutrophils with ligands induced on the surface of the activated (i.e., inflamed) endothelium.
• IVM of murine neutrophils expressing human FcγRIIA and FcγRIIIB shows that these receptors trigger both slow rolling and adhesion in the presence of deposited immune complexes (33).
• IgG immune complexes can also trigger complement activation, leading to production of complement component 5a (C5a), a potent neutrophil chemoattractant.
• Neutrophils are unique among leukocytes in their ability to roll along vascular endothelium at significantly high shear stress (i.e., in larger vessels with higher blood pressure).

Rolling

• Selectin GPCR Integrin Arrest Chemokines Integrin ICAM-1 Crawling Integrin Transmigration JAMs PECAM CD99 Phagocytosis Reactive oxygen species Degranulation Opsonin: Immunoglobulin G Complement Basement membrane Interstitial migration Chemokines Slow rolling Selectin Integrin Selectin Figure 1 Multistep adhesion cascade of neutrophil recruitment.
• Neutrophils are guided into tissue by local gradients of chemoattractants (53, 256) in a process that requires them to switch from sensing chemokines on the apical endothelial surface to sensing those emanating from the tissue source of inflammation.
• Secondary granules also contain matrix metalloproteinases (e.g., MMP8, also known as collagenase).
• BPI, bactericidal/ permeability-increasing protein; CR, complement receptor; FcγR, Fcγ receptor; iNOS, inducible nitric oxide synthase; Mac-1, macrophage-1 antigen; NET, neutrophil extracellular trap; TLR, Toll-like receptor, also known as Abbreviations.
• Most transmigration occurs via the paracellular route, although the transcellular route is favored when endothelial expression of intracellular adhesion molecule (ICAM)-1 is high (38).

NEUTROPHIL ACTIVATION

• Circulating neutrophils are quiescent—their activation is a defining step in the inflammatory response.
• Neutrophil activation is usually a multistep process.
• It begins with the partial activation of cells as they transit through the vascular endothelium during the recruitment process.
• This effect, referred to as neutrophil priming, allows rapid and maximum neutrophil activation, including enhanced phagocytosis and radical oxygen generation (67–69).
• The activation mechanisms that are beneficial for pathogen killing can also be detrimental in the context of sterile injury such as autoimmune and other chronic inflammatory diseases.

Neutrophil Activation by Pathogen Molecules or Cell Damage–Associated Proteins

• Neutrophils recognize pathogens via classes of cell surface and intracellular receptors that bind to microbe-specific molecules.
• Neutrophils also use numerous receptors that recognize host proteins (such as IgG and complement) opsonizing the microbe.
• These findings predict that, in contrast to other immune cells, neutrophils regulate the intracellular signaling pathways of activating FcγRs via inhibitory receptors other than FcγRIIB.
• Signaling through GPCRs can also prime neutrophil activation in response to other activating agents or, at a high enough concentration, lead to full cellular activation.
• Some of these GPCRs, such as the formyl peptide receptors, also recognize host cellular products released during cell injury or death (93).

Disorders of neutrophil activation

• NETs provide antimicrobial function both by localizing and trapping pathogens within a sticky meshwork of chromatin and by exposing pathogens to highly concentrated antimicrobial peptides and enzymes trapped within the chromatin.
• What stimuli drive NETosis as opposed to apoptosis is not particularly clear, as ROS can play a major role in both processes.
• NETs are a rich source of pro-inflammatory molecules (such as chromatin–LL-37 complexes) and autoantigens (133).

DISORDERS OF NEUTROPHILS

• Given that immunological processes in mice and humans differ (136), and given the caveats with knockout approaches (e.g., compensation), it is important to validate in humans some of the concepts developed in murine systems.
• Congenital abnormalities in human patients involving leukocyte recruitment have helped define the molecular underpinnings of neutrophil recruitment and activation.
• Genetic deletions in components of the NADPH oxidase and MPO pathways have enriched their understanding of neutrophil cytotoxic functions, while deletions in PRRs such as Dectin-1 and downstream adapter molecules of TLRs have aided in their understanding of the relative contributions of these PRRs in neutrophil function and host defense.
• Table 1 summarizes some of these congenital abnormalities along with associated laboratory findings and clinical manifestations (137).
• One would predict from mouse studies that some of these mutations might protect patients from development of certain types of sterile inflammatory diseases, although proof for this concept is not forthcoming because of the rarity of these disorders.

Recruitment

• Inherited defects in neutrophil recruitment in patients with leukocyte adhesion deficiency (LAD) highlight the importance of selectins ; β2 integrins (LADI); and integrin activation, specifically Kindlin-3 (I) in getting cells to the site of inflammation.
• Patients with LADI, LADII, or LADIII present with infections without pus formation, a reflection of poor neutrophil accumulation.
• Importantly, mice with deficiencies in the β2 integrins, selectins, or integrin activation exhibit profound defects in neutrophil recruitment (28).
• Ironically, the initial confusion concerning the underlying gene mutations that cause LADIII (which was originally thought to be caused by mutations in a gene next to Kindlin-3) also taught us an important lesson about genotype-phenotype correlation (143).
• Finally, though extremely rare, patients have been found with mutations in the Rac2 GTPase, which lead to impairment in chemokine signaling and actin remodeling that result in recruitment defects (144).

Activation

• Numerous disorders linked to alterations either in pathogen sensing or in the molecules involved in intracellular signaling downstream of pathogen-sensing receptors have now been defined in patients.
• Patients with these defects manifest pyogenic infections.
• By contrast, patients with inborn deficiencies in the receptors or signaling molecules involved in fungal sensing [Dectin-1 and CARD9 (caspase recruitment domain 9)] present almost exclusively with fungal (often Aspergillus) infections.
• Mice deficient in these molecules have defects in neutrophil cytotoxic responses (70, 145).
• Indeed, increased susceptibility to herpesvirus encephalitis has been mapped to mutations in TLR3, the primary virus-sensing TLR, though these mutations affect antigenpresenting cells more than neutrophils (146).

IMMUNOMODULATORY FUNCTION OF NEUTROPHILS

• There is emerging evidence from a number of groups indicating that neutrophils not only are involved in the killing of extracellular pathogens but also contribute to the immune response to intracellular pathogens and viruses through complex cross talk with other immune cells, such as DCs, lymphocytes, and NK cells.
• Much of this cross talk is mediated by the ability of neutrophils to secrete a host of cytokines or express a large number of cell surface molecules that directly interact with other immune cells (9).
• These findings are changing their traditional view of neutrophils from terminally differentiated effectors to transcriptionally and functionally active partners in the entire immune response.

Regulation of Dendritic Cells

• Many neutrophil products, including lactoferrin, α-defensins, and chemokines (such as CCL3), are chemotactic for DCs and are required for rapid DC recruitment to sites of infection (150, 151).
• Direct binding of neutrophils to DCs promotes maturation of DCs into more effective antigen-presenting cells and provides DCs access to neutrophilcaptured pathogen products (152, 153).
• Neutrophil-DC interactions such as these have been defined in the colonic mucosa of Crohn’s disease patients (154).
• NETs, containing chromatin complexed with LL-37 peptides, induce IFN-α production from plasmacytoid DCs, which in turn drives formation of self-reactive lymphocytes that recognize chromatin–LL-37 complexes, leading to production of autoantibodies.
• Type I interferons, such IFN-α, also stimulate neutrophils, further promoting NET formation and thereby establishing a self-amplifying loop of inflammation between neutrophils and DCs.

Regulation of T and B Cell Function

• Neutrophils and T cells modulate each other at several levels.
• Interestingly, neutrophils can also carry antigens to lymph nodes by migrating through lymphatics, where they either directly present the antigen to T cells or deliver it to DCs (161, 162).
• They can inhibit the proliferation of IFN-γ-producing T cells through an NO-dependent mechanism.
• More recently, a subset of neutrophils was shown to inhibit T cell proliferation by releasing ROS in the immunological synapse (165).
• Activation of splenic neutrophils by microbial PAMPs leads to significant production of BAFF, APRIL, and IL-21, which in turn directly activate splenic marginal zone B cells and facilitate production of antibodies to T cell–independent antigens 198 Mayadas · Cullere · Lowell A nn u. R ev .

Regulation of Natural Killer Cells

• Numerous interactions between neutrophils and NK cells have recently been defined.
• Neutrophils regulate terminal NK cell maturation under steady-state conditions both in patients and in a mouse model of neutropenia (168).
• How this cross talk is mediated in the steady state remains to be determined.
• During infectious disease, release of cytokines from neutrophils directly activates NK cell function— for example, neutrophil-derived IL-18 is required for IFN-γ production by NK cells during Legionella infection in mice (169).
• IL12 production by DCs is also required for full activation, suggesting a three-way interaction between these cells.

Regulation of Macrophages

• Neutrophil-macrophage interactions are important in both the initiation and resolution phases of the inflammatory response.
• Neutrophil primary granule proteins also enhance the antimicrobial activity of macrophages by increasing their ability to phagocytose and elaborate cytokines (10, 171).
• A prime example is the demonstration that the murine cathelicidin peptide CRAMP (cathelicidin-related antimicrobial peptide; homologous to human LL-37) stimulates monocyte and macrophage recruitment into inflammatory atherosclerotic lesions by activation of formyl peptide receptors (172).
• During the resolution phase of inflammation, uptake of apoptotic neutrophils by macrophages leads to a decrease in IL-23 production by the macrophages, which diminishes IL-17 secretion by T cells and hence reduces G-CSF and neutrophil production (see Neutrophil Homeostasis, above).
• Phagocytosis of apoptotic neutrophils by macrophages also stimulates the macrophages to produce IL-10 and downmodulate IL-12, thus assuming an M2-like phenotype, to promote tissue repair during resolution of inflammation (173).

Effects on Endothelial and Epithelial Cells

• Interactions between neutrophils and endothelial or epithelial cells during inflammatory responses can have significant effects on the inflammatory and barrier functions of the latter cell types (174).
• The effects on epithelial barrier integrity during neutrophil transepithelial migration are particularly adverse and contribute to fluid accumulation on the luminal side of epithelial cells, such as in the lung (causing pulmonary edema) or the gut (exacerbating inflammatory diarrhea).
• Inactivation of HBP prevents neutrophils from inducing endothelial cell hyperpermeability both in cultured cells and in vivo (176).
• Indeed, the serum level of HBP has been used as a biomarker to predict clinical outcome in patients with pulmonary edema and severe sepsis or shock (177).

Neutrophil-Derived Microparticles as Modulators of Inflammation

• Recent studies have highlighted the potential immunomodulatory function of membrane microparticles derived from activated neutrophils in a variety of disease settings (180, 181).
• Microparticles are membrane fragments released from a variety of cell types by blebbing (similar to apoptotic blebs) during cellular activation.
• It is now clear that they contain functional ligands and receptors that can confer modulatory effects on other cells.
• Neutrophil-derived microparticles have both immune-activating functions (on platelets and endothelial cells) and immunesuppressive functions (on macrophages) (182, 183).
• The role of neutrophils in acute inflammation leading to tissue injury is well established.

Atherosclerosis

• Atherosclerosis is a chronic disease that results from the deposition of pro-inflammatory lipids in the vasculature, which leads to poorly controlled blood vessel inflammation (188).
• Until recently, the potential contribution of neutrophils was largely neglected in this disease because they were not detected in atherosclerotic lesions.
• MPO-generated ROS that promote endothelial cell apoptosis, tissue factor expression, low-density lipoprotein nitration, and lipid peroxidation may advance lesion development (193).
• Deposition of activated platelets in the vessel wall in turn attracts more neutrophils through the display of platelet adhesion molecules such as P-selectin or the production of platelet-derived chemokines such as CCL5.
• A group of antibodies against intracellular antigens in neutrophils and monocytes that present on the surface of activated cells, also known as Antineutrophil cytoplasmic antibodies.

Autoimmune Diseases

• Neutrophils have been implicated in the pathogenesis of numerous autoimmune diseases, both as effector cells that mediate tissue injury and, more recently, as immune-modulating cells that affect the function of other cells.
• The autoantigen MPO in the tissue-deposited NETs may instigate further immune-complex deposition and thus more inflammatory cell stimulation.
• Neutrophil activation in these diseases has only 202 Mayadas · Cullere · Lowell A nn u. R ev .
• For decades, rheumatoid factor (an IgG that recognizes self-IgM antibodies) was considered the main pathogenic autoantibody in RA.
• Several studies demonstrate that neutrophils from SLE patients readily undergo NETosis in response to numerous stimuli and have an enhanced ability to strongly activate type I interferon production by plasmacytoid DCs (155, 216).

Allergic Diseases

• Recent studies highlight a role for neutrophils in these diseases as well.
• These roles were recently demonstrated in a mouse model of anaphylaxis, both through passive administration of IgG and through administration of antigen to sensitized mice (222).
• In both models, depletion of neutrophils or neutrophil FcγRs protects mice from anaphylaxis.
• By contrast, neutrophil-derived histamine is the major contributor to pulmonary allergic inflammation in chronic mycoplasma infection (223).
• Neutrophils may also contribute to the sensitization phase of allergic skin diseases.

Inflammatory Bowel Disease

• Neutrophils play a clear role in the pathophysiology of inflammatory bowel disease (IBD), both Crohn’s disease and ulcerative colitis.
• Neutrophils make up a significant proportion of the inflammatory infiltrate in the intestinal walls of patients with IBD; the degree of neutrophil infiltration correlates with the clinical severity of the disease (225).
• Besides causing direct cellular damage, these proteases can degrade collagen to produce chemotactic peptides that further drive neutrophil recruitment to the bowel.
• Mice lacking the gp91phox subunit of NADPH oxidase are protected from experimental colitis (although curiously, gp47phox mutant animals respond normally) (230).
• Indeed, these antibodies, along with neutrophil secretion 204 Mayadas · Cullere · Lowell A nn u. R ev .

Cancer

• The relationship between neutrophils and cancer pathogenesis is a burgeoning area of research (233–235).
• Most clinical observations suggest that the presence of abundant neutrophils within a tumor bed is associated with increased tumor growth and hence a poor prognosis (236).
• Consistent with this effect, depletion of neutrophils in tumor-bearing mice can result in activation of CD8+ effector T cells to promote antitumor immunity (246).
• The presence of tumor-associated myeloid cells with immune-suppressor function has been recognized in mouse models for many years and was recently defined in human tumor isolates (234).
• Because many of these functions are associated with the pro-inflammatory, host-defense function of neutrophils, some investigators refer to these types of TANs as N1 neutrophils (246).

CONCLUSIONS AND PERSPECTIVES

• There has been little progress in specifically targeting these processes for therapeutic purposes.
• Advances in this area will require better tools.
• Neutrophil-specific targeting of signaling molecules downstream of activating receptors or approaches to changing the balance of activating versus suppressive function of these cells are interesting ideas.
• Killing of pathogens is achieved through the production of toxic metabolites and the release of nuclear contents.
• Neutrophils play a central role in coordinating the response of other immune effector cells.

Figures

Figure 3 Balancing apoptosis versus NETosis. In response to numerous inflammatory stimuli, including combinations of inflammatory cytokines, pathogens, immune complexes, and extracellular membrane (ECM) components, neutrophils can be activated to undergo either NETosis or apoptosis. We propose that the balance between these two outcomes is a function of the strength or presentation of the stimulus—neutrophils that are highly primed by cytokines and subsequently exposed to opsonized microbes may undergo apoptosis, whereas weaker stimuli may lead to NETosis. Whether neutrophils undergo NETosis or apoptosis dictates the subsequent immune responses. NETosis tends to be pro-inflammatory and prothrombotic and to lead to the release of novel self-antigens, such as deiminated proteins, which stimulate autoimmunity. By contrast, apoptosis is anti-inflammatory, in that it leads to the promotion of M2 macrophage development associated with wound healing and tissue repair processes.

Full text

UCSF
UC San Francisco Previously Published Works
Title
The multifaceted functions of neutrophils.
Permalink
https://escholarship.org/uc/item/9p7275kj
Journal
Annual review of pathology, 9(1)
ISSN
1553-4006
Authors
Mayadas, Tanya N
Cullere, Xavier
Lowell, Clifford A
Publication Date
2014
DOI
10.1146/annurev-pathol-020712-164023
Peer reviewed
eScholarship.org Powered by the California Digital Library
University of California

PM09CH09-Mayadas ARI 13 December 2013 14:45
The Multifaceted Functions
of Neutrophils
Tanya N. Mayadas,
1
Xavier Cullere,
1
and Clifford A. Lowell
2
1
Center for Excellence in Vascular Biology, Department of Pathology,
Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 20115;
email: tmayadas@rics.bwh.harvard.edu
2
Department of Laboratory Medicine, University of California, San Francisco,
California 94143; email: lowellc@labmed2.ucsf.edu
Annu. Rev. Pathol. Mech. Dis. 2014. 9:181–218
First published online as a Review in Advance on
September 16, 2013
The Annual Review of Pathology: Mechanisms of
Disease is online at pathol.annualreviews.org
This article’s doi:
10.1146/annurev-pathol-020712-164023
Copyright
c
 2014 by Annual Reviews.
All rights reserved
Keywords
recruitment, receptors, cytotoxic functions, adaptive immunity,
disorders, chronic diseases
Abstract
Neutrophils and neutrophil-like cells are the major pathogen-fighting
immune cells in organisms ranging from slime molds to mammals. Cen-
tral to their function is their ability to be recruited to sites of infection,
to recognize and phagocytose microbes, and then to kill pathogens
through a combination of cytotoxic mechanisms. These include the
production of reactive oxygen species, the release of antimicrobial pep-
tides, and the recently discovered expulsion of their nuclear contents
to form neutrophil extracellular traps. Here we discuss these primor-
dial neutrophil functions, which also play key roles in tissue injury, by
providing details of neutrophil cytotoxic functions and congenital dis-
orders of neutrophils. In addition, we present more recent evidence
that interactions between neutrophils and adaptive immune cells estab-
lish a feed-forward mechanism that amplifies pathologic inflammation.
These newly appreciated contributions of neutrophils are described in
the setting of several inflammatory and autoimmune diseases.
181
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PM09CH09-Mayadas ARI 13 December 2013 14:45
INTRODUCTION
Multicellular organisms face a constant chal-
lenge of surviving in an environment containing
unicellular pathogens. Phagocytes have evolved
as specialized cells that engulf and kill invad-
ing pathogens to protect the host against mi-
croorganisms. They are the major cellular arm
of the innate immune system, which is common
to species throughout the evolutionary tree. In-
deed, the survival of primitive organisms—for
example, insects, which lack adaptive immune
cells such as lymphocytes—relies on the func-
tion of their innate immune phagocytes (1). In
humans, neutrophils account for 50% to 70%
of all circulating leukocytes, and they are the
first line of host defense against a wide range of
infectious pathogens including bacteria, fungi,
and protozoa. Neutrophils are generated at a
rate of 10
11
per day, which can increase to 10
12
per day during bacterial infection. Thus, not
surprisingly, 55% to 60% of the bone mar-
row is dedicated to their production (2). Neu-
trophils are terminally differentiated and rela-
tively short lived. Traditional estimates based
on ex vivo survival in culture or on half-life af-
ter adoptive transfer suggested that these cells
survive for only 8–12 h in the circulation and up
to 1–2 days in tissues, with their turnover de-
layed or accelerated during the inflammatory
response (3–5). More modern approaches us-
ing deuterium labeling methods in vivo sug-
gest that under homeostatic conditions, human
neutrophils may have a circulatory life span up
to 5 days (6). Although this dramatically dif-
ferent view of neutrophil half-life is somewhat
controversial (7, 8), these types of more up-
dated immunological investigations are chang-
ing our overall perception of neutrophil func-
tion in immunity. Whereas researchers once
believed that neutrophils were present only
during the acute phase of the inflammatory re-
sponse, functioning only as pathogen killers,
we now appreciate that neutrophils can shape
the immune landscape by communicating with
macrophages, dendritic cells (DCs), and cells of
the adaptive immune response through direct
cell-cell contact or soluble mediators (9–11).
In this article, we aim both to revisit
well-established principles of neutrophil func-
tion during inflammation and host defense—
including their production, recruitment, and
killing capacities—and to illuminate many of
the newer findings on the contribution of these
cells to other aspects of immunity. The purpose
of this exercise is to renew and stimulate dis-
cussion on how processes fundamental to neu-
trophil function may uniquely influence disease
progression. We focus on those studies that re-
veal the growing appreciation of the complex-
ity of neutrophil function both in normal im-
mune responses and during immune-mediated
disease states.
NEUTROPHIL HOMEOSTASIS
Neutrophils are formed within the bone
marrow during hematopoiesis in response
to several cytokines, principally granulocyte
colony–stimulating factor (G-CSF) (12). The
major determinants of the total number of neu-
trophils in the body are their rate of production,
their storage in and egress from the bone mar-
row, and their survival in and clearance from the
blood. Entry into the tissues during inflamma-
tory responses can also affect overall neutrophil
numbers. The ability of an organism to main-
tain a balance between neutrophil production
and turnover, while adapting to environmental
challenge, implies that there must be a molec-
ular process for measuring neutrophil numbers
at any given time. The existence of some form
of a “neutrostat” that measures neutrophil
numbers and adjusts them accordingly remains
very controversial (13). However, recently
described feedback loops certainly contribute
to neutrophil homeostasis under resting and
inflammatory disease conditions.
One such feedback loop was discovered in
studies of adhesion molecule [CD18, E/P/L-
selectin, or CD11a (LFA-1)]–deficient mice.
In these animals, neutrophil egress from the
peripheral blood is reduced; hence, the animals
display significant neutrophilia. Under steady-
state conditions, senescent neutrophils are
182 Mayadas
·
Cullere
·
Lowell
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Granulocyte
macrophage
colony–stimulating
factor (GM-CSF):
a classical cytokine
that promotes
granulopoiesis and
primes neutrophils for
enhanced cytotoxic
responses
Toll-like receptors
(TLRs): a family of
intracellular and
extracellular PRRs that
engage the
intracellular
transcriptional
machinery to promote
synthesis and secretion
of pro-inflammatory
cytokines
engulfed by tissue macrophages [primarily in
the liver, spleen and bone marrow (14)], which
then initiate anti-inflammatory signals via ex-
pression of PPARγ (peroxisome proliferator–
activated receptor γ) and LXR (liver X recep-
tor) (15). These anti-inflammatory signals, in
turn, lower the steady-state production of inter-
leukin (IL)-23 by macrophages. IL-23 is a well-
established inflammatory cytokine that induces
IL-17 production by T lymphocytes, natural
killer (NK) cells, and natural killer T (NKT)
cells, which, in turn, induces production of
G-CSF and granulocyte macrophage colony–
stimulating factor (GM-CSF) by stromal cells,
driving granulopoiesis and inflammation (16).
Adhesion molecule–deficient mice have high
steady-state levels of IL-23 and IL-17, due
to reduced egress of neutrophils out of the
blood and hence reduced neutrophil uptake by
macrophages, leading to elevated G-CSF (17).
Genetic depletion of IL-23 in CD18 knockout
mice reverses their neutrophilia, supporting the
model that the rate of phagocytosis of apoptotic
neutrophils regulates their production via an
IL-23–IL-17 axis (18). Similarly, mice lacking
the LXRα and LXRβ receptors also display
significant neutrophilia associated with high
levels of IL-23- and IL-17-producing T cells
(19).
The IL-23–IL-17–G-CSF feedback loop
is clearly not the only mechanism controlling
neutrophil production, given that mice lacking
T lymphocytes and NK cells (i.e., the major
sources of IL-17) have normal neutrophil num-
bers (20, 21). Moreover, antibody-mediated
depletion of neutrophils in mice, through
use of the Gr-1 monoclonal antibody, leads
to a significant increase in serum GM-CSF
and G-CSF, which, in the absence of other
inflammatory cytokines, triggers neutrophil
progenitor proliferation (21). This observation
suggests that neutrophil numbers are regu-
lated, at least in part, simply according to the
available space within the bone marrow, in a
fashion referred to as density-dependent or
neutrophil-mass sensing. A molecular under-
standing of how bone marrow stroma, which
is the major site of G-CSF production, can
sense low neutrophil numbers remains unclear.
However, very recent results suggest that this
sensing pathway depends on the innate immune
receptor Toll-like receptor (TLR) 4 and its
signaling adapter Toll/interleukin-1 receptor
(TIR) domain–containing adapter-inducing
interferon-β (TRIF), because antibody-
mediated depletion of neutrophils fails to cause
G-CSF elevation or progenitor proliferation
in mice lacking TLR4 or TRIF (20).
Most studies have focused on G-CSF and
GM-CSF as the ultimate cytokines that reg-
ulate neutrophil production, but this is clearly
too simple a view, given that mice lacking one or
both of these cytokines still have approximately
20% of the normal level of mature neutrophils
in their blood (22). Indeed, these cytokine-
deficient animals can also mount increased
neutrophil production in response to inflam-
matory stimuli, a process termed emergency
granulopoiesis (23). That hematopoietic pro-
genitors can ramp up production of granulo-
cytes in response to inflammatory or pathogen
challenge, even in the absence of canonical
granulopoiesis-stimulating cytokines, indicates
that other signaling pathways must exist to
regulate granulopoiesis. New studies clearly
demonstrate that hematopoietic progeni-
tor cells proliferate upon sensing pathogen
molecules through a host of innate immune
receptors such as the TLRs and Nod-like re-
ceptors (24). The combination of consumption
of neutrophils in the periphery—in the process
of fighting pathogens—and exposure of pro-
genitor cells to pathogen molecules could lead
to a synergistic stimulation of granulopoiesis
during these emergency situations (25). The
observation that steady-state granulopoiesis
depends completely on the transcription
factor C/EBP (CCAAT/enhancer-binding
protein) α, whereas emergency granulopoiesis
requires C/EBPβ, also suggests that these
are two separate pathways (26, 27). Finally,
the observation that mice lacking commensal
organisms—that is, germ-free animals—have
dramatic neutropenia, with neutrophil levels
even lower than those in combined G-CSF
and GM-CSF knockout mice, also suggests
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Multifaceted Functions of Neutrophils 183
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PM09CH09-Mayadas ARI 13 December 2013 14:45
ROS: reactive oxygen
species
Neutrophil
extracellular traps
(NETs): formed by
the release from
neutrophils of
decondensed
chromatin that is
covered with
antimicrobial
components and
potential self-antigens
that hematopoietic progenitor cells are tuned
to respond directly to environmental cues
(20).
In the end, neutrophil homeostasis is likely
influenced by all these processes—phagocytic
uptake in the periphery, cell mass in the bone
marrow, and presence of pathogen (or com-
mensal) stimuli—in a complex concert. Under
any given condition, one pathway may domi-
nate over the others, but a clear understand-
ing of regulation of neutrophil numbers (espe-
cially during disease states) will require further
research.
NEUTROPHIL RECRUITMENT
After their birth in the bone marrow, mature
neutrophils reach sites of tissue inflammation
or infection via the vasculature. The exit of
neutrophils from the blood, primarily via post-
capillary venules, follows an ordered process
referred to as neutrophil recruitment (28–31).
The neutrophil recruitment cascade is medi-
ated by the sequential interaction of receptors
present on neutrophils with ligands induced
on the surface of the activated (i.e., inflamed)
endothelium. The classical multistep adhesion
cascade consists of the following steps: (a) initial
attachment of the neutrophil to the endothe-
lium (capture), (b) rolling of the neutrophil
along the endothelium, (c) firm arrest of the
neutrophil with accompanying cell spreading,
(d ) crawling of the neutrophil along the en-
dothelium, and (e) transmigration of the neu-
trophil into the tissue (Figure 1), where full
neutrophil activation leads to phagocytosis and
killing of pathogens through the production of
reactive oxygen species (ROS), degranulation
(Figure 2), and generation of neutrophil extra-
cellular traps (NETs) (Figure 3).
The molecular requirements for the mul-
tistep paradigm of neutrophil recruitment,
depicted in Figure 1, have primarily been
derived from the real-time analysis of postcap-
illary venules in the transparent cremaster mus-
cle or mesentery using intravital microscopy
(IVM) (32). However, in autoimmune dis-
eases, the contribution of humoral immunity
components, including immune complexes
and complement activation products, needs to
be taken into account. Fcγ receptors (FcγRs),
which are receptors for immunoglobulin (Ig) G
immune complexes, are a case in point. In mod-
els of antibody-mediated glomerulonephritis
and arthritis, mice that lack activating FcγRs
exhibit no neutrophil accumulation. However,
tissue recruitment is restored by the selec-
tive expression of human FcγRIIA and/or
FcγRIIIB on neutrophils (33, 34). IVM of
murine neutrophils expressing human FcγRIIA
and FcγRIIIB shows that these receptors trig-
ger both slow rolling and adhesion in the
presence of deposited immune complexes (33).
IgG immune complexes can also trigger com-
plement activation, leading to production of
complement component 5a (C5a), a potent
neutrophil chemoattractant. C5a lowers the
threshold of FcγR-mediated neutrophil ac-
tivation (34, 35) and increases macrophage-1
antigen (Mac-1) [complement receptor (CR) 3]
activity (36), which may potentially influence
neutrophil accumulation. Thus, in the presence
of immune complexes, the principles of neu-
trophil recruitment (i.e., slow rolling, adhesion,
and chemokine-directed migration) may not
fundamentally diverge from those that follow
simple cytokine stimulation but involve addi-
tional neutrophil receptors that link the inflam-
matory insult, in this case immune complexes
and complement, to neutrophil accumulation.
Neutrophils are unique among leukocytes in
their ability to roll along vascular endothelium
at significantly high shear stress (i.e., in larger
vessels with higher blood pressure). This ability
likely evolved to allow neutrophil recruitment
to occur in a broader range of tissue areas. Most
leukocytes roll along postcapillary venules at
shear stresses of 0–3 dyn/cm
2
, whereas neu-
trophils can roll on vessels at shear stresses of
6–10 dyn/cm
2
(31). Neutrophils accomplish
this resistance to fluid shear force by four
mechanisms: (a) flattening out over the en-
dothelium to engage more adhesive molecules,
(b) increased use of selectin family molecules
and their receptors to form catch bonds that
tend to become stronger with increasing force,
184 Mayadas
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Cullere
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Lowell
Annu. Rev. Pathol. Mech. Dis. 2014.9:181-218. Downloaded from www.annualreviews.org
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