1 neutrófilos - primeira apresentação.pdf

IY30CH19-Zychlinsky ARI 27 December 2011 13:33

Review in Advance first posted online
V I E W
E on January 3, 2012. (Changes may
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still occur before final publication

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online and in print.)

C E
I N

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A
D V A

Neutrophil Function:
From Mechanisms to Disease
Borko Amulic, Christel Cazalet,
Garret L. Hayes, Kathleen D. Metzler,
and Arturo Zychlinsky∗
Annu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org
by Universidade Federal do Amazonas on 03/21/12. For personal use only.

Department of Cellular Microbiology, Max Planck Institute for Infection Biology,
Charit´ platz 1, 10117 Berlin, Germany; email: amulic@mpiib-berlin.mpg.de,
e
cazalet@mpiib-berlin.mpg.de, hayes@mpiib-berlin.mpg.de, metzler@mpiib-berlin.mpg.de,
zychlinsky@mpiib-berlin.mpg.de

Annu. Rev. Immunol. 2012. 30:459–89 Keywords
The Annual Review of Immunology is online at
immunol.annualreviews.org inflammation, antimicrobial, granule, phagocytosis, NET
This article’s doi: Abstract
10.1146/annurev-immunol-020711-074942
Neutrophils are the most abundant white blood cells in circulation,
Copyright c 2012 by Annual Reviews.
All rights reserved and patients with congenital neutrophil deficiencies suffer from severe
infections that are often fatal, underscoring the importance of these
0732-0582/12/0423-0459$20.00
cells in immune defense. In spite of neutrophils’ relevance in immunity,
∗
All authors contributed equally to the work and research on these cells has been hampered by their experimentally in-
are listed alphabetically.
tractable nature. Here, we present a survey of basic neutrophil biology,
with an emphasis on examples that highlight the function of neutrophils
not only as professional killers, but also as instructors of the immune
system in the context of infection and inflammatory disease. We focus
on emerging issues in the field of neutrophil biology, address questions
in this area that remain unanswered, and critically examine the experi-
mental basis for common assumptions found in neutrophil literature.

459

Changes may still occur before final publication online and in print


INTRODUCTION early and enthusiastic evolutionary biologist in-
terested in the phagocytic capacity of cells.
In the late nineteenth century, Paul Ehrlich,
Metchnikoff demonstrated that injury of
dissatisfied with what he considered an in-
starfish embryos resulted in recruitment of
excusable disinterest in the white blood cell,
phagocytic cells to the site of injury (3). He
began to utilize newly developed cell-staining
theorized (correctly) that these cells migrate to
techniques to examine subpopulations of leuko-
injured sites and participate in microbe diges-
cytes. His experimentation led to a new appreci-
tion. Remarkably, this prescient view of neu-
ation for the heterogeneity of white blood cells
trophil action still aptly summarizes, more than
and to the discovery of several novel leukocyte
a century later, the basic role of neutrophils
subpopulations. Ehrlich named one of these
in immunity. The uniquely lobulated nucleus
newly discovered cell types, characterized by a
of the neutrophil also inspired Metchnikoff to
“polymorphous nucleus” and a tendency to re-
rename these cells: He called them polymor-
tain neutral dyes, the “neutrophil” (1) (see also
phonuclear leukocytes (or PMNs), a title that
the sidebar, A Natural History of Neutrophils).

still enjoys frequent use and that is used inter-

The function of neutrophils was initially
changeably with neutrophil throughout this re-
shrouded in considerable mystery; their con-
view. Together with two other developmentally
spicuous presence during infections led several
related cell types, the eosinophils and basophils
researchers to arrive hastily at a rather ironic
(also discovered by Ehrlich), PMNs form the
conclusion: They surmised that neutrophils
granulocyte family of white blood cells, a fam-
promote infection, serving as cellular shuttles
ily whose hallmark is the presence of “granules,”
for bacteria (2). Their actual function, that of
unique storage structures important in antimi-
antimicrobial actors in the immune response,
crobial functions (see section on Granules and
was eventually demonstrated conclusively by a
Degranulation, below).
contemporary of Ehrlich, Elie Metchnikoff, an
Neutrophils were discovered at the dawn
of the immunological sciences; consequently,
elucidation of their role in the immune re-
sponse has been an ongoing process stretching
A NATURAL HISTORY OF NEUTROPHILS over more than a century. We now know that
they are key components of the innate immune
Phagocytes are ancient cells that evolved to allow multicellular response and vital in immune function; unfor-
organisms to thrive in the face of constant competition with mi- tunately, their importance has often been over-
crobes for resources. Metchnikoff ’s seminal theory of cellular shadowed by breakthroughs in the study of the
immunity was based on comparative embryology and observa- adaptive immune response (4). Admittedly, this
tions of phagocytes in various simple organisms, including the mi- situation is exacerbated by neutrophils’ notori-
croscopic crustacean Daphnia. Remarkably, even the slime mold ous experimental intractability: They exhibit a
Dictyostelium discoideum has phagocytic cells that protect it from short life span and are terminally differentiated,
infection (200). The short-lived neutrophil with a lobulated nu- preventing growth in tissue culture. The stan-
cleus and granule-packed cytoplasm is a more recent evolutionary dard tools of molecular biology, such as trans-
adaptation. In insects, phagocytes are long lived and have round fection and RNA interference, are of little use
nuclei. They do, however, produce hydrogen peroxide and carry when applied to these cells, and immortalized
distinct classes of granules (201). Bony fish and frogs have bona “neutrophil-like” cell lines rarely reflect the
fide neutrophils that are functionally similar to mammalian ones functional diversification of neutrophils. Fur-
(202, 203). In both zebrafish and rodents, neutrophils are less thermore, neutrophil-like cells studied in the
abundant than in humans, comprising only 15–20% of immune isolation of a culture dish most certainly do not
cells. In chimpanzees, neutrophils account for more than 50% of mimic the complex biological reality in tissues
the differential blood count (204). or circulation. Conclusions from in vitro stud-
ies should, therefore, be carefully interpreted.

460 Amulic et al.



Unfortunately, in vivo studies of neutrophil infection. Indeed, the number of neutrophils
function also raise concerns. Mouse neu- drastically increases during infection and some
trophils, the preferred model for in vivo diseases. Interestingly, neutrophils circulate
studies, differ in important aspects from their for only approximately 6–8 h and are among
human equivalents. This is perhaps best the shortest-lived cells in the human body.
exemplified by the differences in the respective Although the reason for this short life is unclear,
antimicrobial repertoires and the numbers of it may ensure neutrophil integrity; this hypoth-
PMNs in circulation (30% versus 70% in mice esis is bolstered by observations that apoptosis
and humans, respectively). prevents the release of noxious molecules.
Despite these difficulties, no picture of the Still, the question of why evolution opted for
immune response can be complete without eliminating neutrophils quickly as opposed
a comprehensive understanding of the neu- to reducing leakage of their dangerous cargo
trophil and its functions. The extensive nature remains an unanswered and intriguing mystery.
of neutrophil research, however, precludes a Mature neutrophils emerge from the bone

comprehensive review of the subject matter. marrow intent on pursuing one simple, yet

In this review, we intend to provide a survey essential, question: Has host integrity been
of basic neutrophil biology and function, while compromised by potentially harmful invaders?
emphasizing recent advances in neutrophil re- Should the answer prove to be “yes,” the
search and providing a critical assessment of neutrophil must swiftly enact a carefully
some current reports on PMN action. choreographed process to locate, attack, and
Our survey of the neutrophil begins in destroy the potential threat. At its disposal is
adult bone marrow where, under the in- an impressive arsenal of antimicrobial weapons
struction of growth factors and cytokines, that are deadly, indiscriminate, and brutish in
pluripotent hematopoietic cells differentiate their application. Although effective in their
into myeloblasts, a developmental cell type destructive capacity, these weapons can prove
committed to becoming granulocytes. As these to be just as dangerous to the host cells as to
precursor cells mature to neutrophils, they syn- their intended targets, the microbial invaders.
thesize proteins that are sorted into different Therefore, their deployment must be executed
granules (5). Traditionally, granules have been with exquisite precision and timing, at locations
subdivided into three different classes based where they are both contained and effective.
on their resident cargo molecules: azurophilic, How then does the neutrophil locate and
specific, and gelatinase granules. Although this identify infections? How does it transition
subdivision is practical, these designations are at the correct time and place from an in-
largely artificial. Granules are formed through a active cellular bystander to a fully activated
continuous process; vesicles bud from the Golgi microbial killing machine? This transition
apparatus and fuse, producing granular struc- process, during which the neutrophil inte-
tures. The content of these structures is dic- grates a complex barrage of environmental
tated by the transcriptional program active at cues and translates them into specific actions,
the time of their formation. As the maturing is known as neutrophil “activation.” As it
neutrophil sequentially alters its transcriptional pursues microbes, the neutrophil will enact an
profile, granule content changes, resulting in a impressive multitude of cellular mechanisms:
continuum of granule species with overlapping It will mobilize secretory vesicles and granules,
cargoes (6). identify chemotactic gradients and traverse
The release of neutrophils from the bone them through destruction and reorganization
marrow is tightly regulated in healthy in- of the actin skeleton, penetrate the endothelial
dividuals: Chemokines control the passage barrier and navigate a course through the
of PMNs into circulation and maintain a basement membrane, and begin transcription
pool of cells ready for release in case of of cytokines for recruitment of new immune

www.annualreviews.org • Neutrophil Functions 461



cells. Ultimately, upon arriving at the infection family kinases, Syk, phosphoinositide 3-kinase
site, it will seek the insulting pathogens and (PI3K), and p38 mitogen-activated protein
unleash its extensive arsenal of antimicrobial kinase (11–13). This cascade initiates a number
Selectins:
transmembrane weapons. The initiation of these processes oc- of changes in neutrophil biology and sets the
glycoproteins that curs in the bloodstream, where the neutrophil stage for integrin activation and firm adhesion.
mediate cell adhesion acts as a monitor for host distress, patrolling After selectin-mediated rolling, neutrophils
via binding to sugar vessels and vigilantly seeking out indications of enter a “firm adhesion” state mediated by the
moieties
an incipient inflammatory response. β2 integrin family of proteins (LFA-1 and
Integrins: Mac-1 proteins on the neutrophil); firm adhe-
transmembrane
sion is characterized by the arrest of neutrophil
receptors that mediate NEUTROPHIL ACTIVATION
attachment to the rolling in preparation for transendothelial
extracellular matrix, as At inflammatory sites, bacterial-derived and migration (13, 14). As the neutrophil rolls
well as direct cell-cell host-produced inflammatory signals are along the endothelium, interaction with
interaction and abundant; these compounds stimulate the selectins, chemoattractants, cytokines, and
signaling

endothelial cells near the inflammatory site. bacterial products results in activation and

Oxidative/respiratory These stimulants, such as the bacterial-derived clustering of the β2 integrins on the surface of
burst: a rapid increase
lipopolysaccharide (LPS) and fMLP, as well the neutrophil (15, 16). The β2 integrins then
in oxygen
consumption upon as the classical chemoattractants and cytokines engage their endothelial ligands, members of
neutrophil activation tumor necrosis factor (TNF)-α, interleukin the ICAM-1 immunoglobulin superfamily,
due to production of (IL)-1β, and IL-17, prompt endothelial cells to resulting in arrest of neutrophil rolling and
ROS by the NADPH produce adhesion molecules on their luminal firm adhesion. This integrin engagement, as
oxidase
side: the P-selectins, E-selectins, and several well as continuing input from inflammatory
members of the integrin superfamily, the chemoattractants and cytokines, prepares the
ICAMs (5). As neutrophils traverse the circu- neutrophil for its final chemotactic pursuit: The
latory system, they continuously and randomly cell spreads, producing a leading-edge lamel-
probe the vessel wall; the postcapillary venules, lipodium where chemokine and phagocytic
where flow dynamics and the constricted space receptors are concentrated, the cytoskeleton is
are particularly amenable to increased random rebuilt and targeted toward movement along
probing, are often the best-suited location chemotactic gradients, and initiation of the
for neutrophils to encounter the stimulated neutrophil oxidative burst begins (17, 18).
endothelial cells (7, 8). Now firmly adhered, the neutrophil must
On the surface of neutrophils, two constitu- negotiate a path through the endothelium into
tively expressed proteins are critical for recog- the underlying tissue. In a process dependent
nition of the endothelial inflammatory signals: on β2 integrins and ICAMs, neutrophils
the glycoprotein P-selectin glycoprotein crawl along the vessel wall until a preferred
ligand-1 (PSGL-1) and L-selectin (9, 10). Upon site of transmigration is reached (19–21).
random contact with the endothelium, these Upon arrival at an endothelial cell junction, a
molecules engage the P- and E-selectins of complex interaction between (a) the neutrophil
endothelial cells, resulting in selectin-mediated integrins and their endothelial partners and
tethering of neutrophils to the vessel wall. (b) neutrophil surface proteins and various
This is followed by a characteristic “rolling” of endothelial junction molecules results in trans-
neutrophils along the endothelium. It is here migration through the endothelial junction
that the complex activation cascade begins (13). Once through the endothelial lining,
and the neutrophil commitment to microbial the neutrophil must navigate the basement
killing commences. What changes occur in the membrane, a protein mesh consisting largely
neutrophil at this early time point? The engage- of laminins and collagen type IV. Speculation
ment of PSGL-1 and L-selectin on neutrophils abounds that granule proteases assist in this
activates a variety of kinases, including Src migration by digesting the protein mesh

462 Amulic et al.



subsequent to degranulation; however, conclu- point of high chemoattractant concentration,
sive experimental evidence for this is lacking. where no discernible gradient exists, the
Once the endothelial barrier has been neutrophil halts and begins the final release of
traversed, the neutrophil finds itself in a its antimicrobial arsenal; the neutrophil is now
much different inflammatory milieu: Here, the fully in an antimicrobial attack state.
environment is awash in a soup of chemoat- The complex signaling cascade leading to
tractants and inflammatory stimulants, both final neutrophil activation has several facets
host derived and of pathogenic origin. These worthy of note. The movement to ever-higher
compounds will now be the primary dictators concentrations of chemoattractant is key in
of neutrophil behavior and assume respon- this process, as individual chemoattractants
sibility for initiating the concluding steps of may have very different effects on neutrophil
neutrophil activation. In the interstitial space, physiology at different concentrations, a
the neutrophil follows chemotactic gradients phenomenon exemplified by one of the key
toward the invading microbes, pursuing host- neutrophil-recruiting chemokines and ac-

produced cytokines (e.g., IL-8) and, in parallel, tivators, IL-8. At low concentrations, IL-8

pathogen-derived chemoattractants (e.g., stimulates L-selectin shedding and increased
fMLP). During this process, these chemoat- expression of β2 integrins; slightly higher
tractants bind to their respective neutrophil concentrations result in initiation of the
receptors (often G protein–coupled receptors, oxidative burst. At the highest concentrations,
as is the case with the fMLP receptor FPR1 or IL-8 induces degranulation of neutrophils (27).
the chemokine receptors), which initiate a sig- In addition, many chemoattractant molecules
naling cascade dominated by the MAPK/ERK exert a “priming” effect. That is, alone they
pathway (22, 23). Downstream molecules stimulate the oxidative response only mildly,
prompt assembly of the oxidative burst ma- but they dramatically enhance the subsequent
chinery, a hallmark of neutrophil activation. response to other stimuli. A notable example of
Furthermore, the stimulation of FPR1 triggers this phenomenon is the strong priming effect
the release of ATP, whose autocrine action of LPS on the fMLP response (28). In this case,
through activation of purinergic receptors is exposure of the neutrophil to LPS induces
critical for the initiation of effective functional assembly of the NADPH oxidase machinery on
responses in neutrophils (24). Concomitantly, the membrane; fMLP stimulation then induces
a family of molecules, the pattern-recognition activation of this machinery (29). In contrast to
receptors, is activated through recognition of receptor priming, another critical feature of the
specific nonself patterns present on many mi- stimulation process is the desensitization to pre-
crobes (25). Perhaps the best-known example viously encountered ligands. Stimulation of the
of this family is the Toll-like receptors (TLRs); neutrophil by a chemoattractant often results
they are responsible for recognizing a number in endocytosis of the corresponding receptor,
of pathogen-derived compounds, collectively thus leading to a desensitization of the neu-
called pathogen-associated molecular patterns trophil to repeated stimulation with the same
(PAMPs), including LPS (TLR4), bacterial molecule (30, 31). The rich and varied input
lipopeptides (TLR2), flagellin (TLR5), and received by a neutrophil during this final leg of
DNA (TLR9). In neutrophils, all but one the activation process is complex, and the exact
of these receptors (TLR3) are constitutively effects of priming, desensitization, and signal-
expressed, and their stimulation contributes ing are incompletely understood. Regardless,
to further activation, e.g., induction of the the end result of this signaling cacophony is
oxidative burst (25, 26). As the neutrophil nears unambiguous: The neutrophil begins to imple-
its target, continued activation by chemoattrac- ment its regime of microbial killing, executing
tants further stimulates the oxidative response programs of phagocytosis, degranulation, and
and degranulation. Upon finally reaching a NETosis (i.e., the process of setting neutrophil




extracellular traps) (see the section on Neu- parsed by the complex neutrophilic signaling
trophils and the Elimination of Microbes, mechanisms, a process that gradually leads
below). to complete activation and culminates in the
The initiation of these microbicidal actions premiere killing functions of phagocytosis,
indicates the final stage of the neutrophil’s degranulation, and NETosis. It is, therefore,
journey through the activation process. How- more insightful to view neutrophil activation
ever, a prominent question remains largely as a continuum of processes, priming steps,
unanswered by the preceding exposition: What and signal cascades with varying effects and
exactly is meant by the (admittedly ambiguous) outcomes, all focused on the realization of
phrase “neutrophil activation”? A quick scan one goal: the transition of naive, circulating
of the literature presents the inexperienced neutrophils to their microbe-eliminating,
reader with a sometimes rather conflicting (and tissue-resident counterparts (Figure 1).
overwhelming) view of neutrophil activation.
In fact, one could be (erroneously) led to
NEUTROPHILS AND THE

believe that neutrophil activation refers only to

direct stimulation of the oxidative burst, as this
ELIMINATION OF MICROBES
has been the canonical in vitro activation assay The basic instruction set of the activated
for decades. This is, however, an oversimpli- neutrophil is both effective and ruthless in
fied view of a complex process. The myriad its simplicity: (1) kill microbes, (2) do no
interactions that occur during a neutrophil’s harm to the host, and (3) when in doubt, see
journey toward an inflammatory site must be rule 1. To fulfill this antimicrobial agenda,

a Capture b Rolling c Firm adhesion

Neutrophil

Integrin
P-selectin and ICAM
PSGL-1, E-selection
L-selectin

Phagocytosis
Endothelial cell Degranulation

Cytokine secretion
NETs

Figure 1
Neutrophil recruitment to sites of inflammation. The circulating neutrophil must recognize signs of
inflammation and migrate to areas where its antimicrobial arsenal is needed for the elimination of infection.
(a) Close to the inflammatory sites, stimulated endothelial cells expose a class of molecules, the selectins,
which serve to capture circulating neutrophils and tether them to the endothelium. (b) Selectin-mediated
rolling along chemoattractant gradients then ensues, followed by (c) integrin-mediated firm adhesion.
Subsequently, the neutrophil traverses through the endothelium and arrives at the site of inflammation.
Here, the neutrophil releases cytokines that recruit other immune cells, and it begins to implement its
antimicrobial agenda. Among the processes employed are engulfment of microbes via receptor-mediated
phagocytosis, release of granular antimicrobial molecules through degranulation, and formation of
neutrophil extracellular traps (NETs).

464 Amulic et al.



neutrophils possess an array of toxic weapons needs of neutrophils. Granules are, however,
that are carefully regulated through controlled far more than just latent repository organelles
mechanisms. These antimicrobial weapons for dangerous substances; they are active and in-
Inflammation:
vary considerably in their methods of action dispensable participants in almost all neutrophil recruitment and
and thus reflect the neutrophil’s attempt to activities during inflammation. activation of immune
exploit any and all weaknesses that microbes As mentioned above, there are three cells upon infection or
might present during the course of infection. fundamental types of granules in neutrophils injury; when
uncontrolled it leads to
An understanding of these weapons, their (Figure 2). Azurophilic granules (also known
tissue damage
action, and their method of release is critical as peroxidase-positive or primary granules) are
to understanding neutrophil function. the largest, measuring approximately 0.3 μM
in diameter, and are the first formed during
neutrophil maturation. They are named for
Granules and Degranulation their ability to take up the basic dye azure A and
The neutrophil must safely transport a plethora contain myeloperoxidase (MPO), an enzyme

of dangerous substances through the blood- critical in the oxidative burst (32, 33). Other

stream and then correctly deploy them at the cargo of this granule class include the defensins,
appropriate time. Therefore, it comes as no lysozyme, bactericidal/permeability-increasing
surprise that a specialty storage organelle has protein (BPI), and a number of serine proteases:
evolved in neutrophils: the granule. Expect- neutrophil elastase (NE), proteinase 3 (PR3),
edly, these structures are replete with specifi- and cathepsin G (CG) (34). As such, these
cally tuned mechanics that address the unique granules are brimming with antimicrobial

Primary Secondary Tertiary Secretory
Granule type (azurophilic) (specific) (gelatinase) vesicles

Stage of Myeloblast Promyelocyte Myelocyte Metamyelocyte Band cell
formation PMN

Degranulation
propensity

Characteristic Lysozyme Complement receptor 1
proteins
Myeloperoxidase Lactoferrin FcγRIII

Elastase Gelatinase

Defensin

Other Cathepsin G, PR3, Gp91phox/p22phox, Gp91phox/p22phox, Gp91phox/p22phox,
proteins BPI, azurocidin, CD11b, collagenase, CD11b, MMP25, CD11b, MMP25, C1q-R,
sialidase, hCAP18, NGAL, B12BP, arginase-1, FPR, alkaline
β-glucuronidase SLPI, haptoglobin, β2-microglobulin, phosphatase, CD10,
pentraxin 3, CRISP3 CD13, CD14,
oroscomucoid, plasma proteins
β2-microglobulin,
heparanase, CRISP3

Figure 2
Neutrophil granules. Neutrophil granules carry a rich variety of antimicrobials and signaling molecules. They are typically divided into
three types (primary or azurophilic, secondary or specific, and tertiary or gelatinase). Additionally, structures called secretory vesicles
are also considered to be a granule subset. Considerable overlap exists in the cargo of the different granules, and their contents seem
determined by the timepoint during hematopoiesis at which they are produced (5). Granules also differ in their ability to mobilize, with
secretory vesicles being the first to fuse with the plasma membrane and the azurophilic granules demonstrating the least degranulation
propensity.




compounds and function as a primary reposi- granule subset has been traditionally associated
tory for the molecular weaponry of neutrophils. with a particular stage of neutrophil activation.
The second class of granules, the specific (or After neutrophils contact the endothelium,
secondary) granules, are smaller (0.1 μM stimulation through selectins and chemoattrac-
diameter), do not contain MPO, and are char- tants induces mobilization of secretory vesi-
acterized by the presence of the glycoprotein cles, whose membranes are rich in key factors
lactoferrin. These granules are formed after necessary for continued activation of the neu-
azurophilic granules; they also contain a wide trophil, including, among others, the β2 inte-
range of antimicrobial compounds including grins, complement and fMLP receptors, as well
NGAL, hCAP-18, and lysozyme (33, 35). The as the FcγRIII receptor CD16 (5, 38, 39, 42).
third class, the gelatinase (tertiary) granules, are Fusion of the secretory vesicles with the plasma
also MPO-negative, are smaller than specific membrane exposes these components to the ex-
granules, and contain few antimicrobials, ternal environment. This results in the transi-
but they serve as a storage location for a tion to firm adhesion, mediated by β2 integrin

number of metalloproteases, such as gelatinase interaction with the endothelium. As they pro-

and leukolysin. These granules are also the ceed through the endothelium, neutrophils are
last population of granules formed during exposed to further activation signals that initiate
neutrophil maturation (5). Finally, a fourth set mobilization of gelatinase granules, thereby re-
of structures, the secretory vesicles, are also leasing metalloproteases. The activity of these
commonly considered part of the neutrophil proteases may help neutrophils traverse the
granule family. In contrast to the classical basement membrane, although this has not
granules, these do not bud from the Golgi, been conclusively demonstrated (43, 44).
but instead are formed through endocytosis At the inflammatory site, complete acti-
in the end stages of neutrophil maturation vation of the neutrophil ensues, prompting
(36). Consequently, their cargo consists pre- initiation of the oxidative burst and mobiliza-
dominantly of plasma-derived proteins such as tion of the azurophilic and specific granules.
albumin. The membrane of secretory vesicles These granules either fuse with the phagosome
serves as a reservoir for a number of important (see section on Phagocytosis, below), con-
membrane-bound molecules employed during tributing to the antimicrobial activities of this
neutrophil migration. compartment, or fuse with the plasma mem-
As a neutrophil proceeds through activation, brane, releasing their potent antimicrobials
granules are mobilized and fuse with either the into the tissue. The fusion of specific granules
plasma membrane or the phagosome, releasing with the plasma or phagosomal membrane is of
their contents into the respective environment. particular importance for the oxidative burst,
In both cases, the membrane of the granule as flavocytochrome b558, a component of the
becomes a permanent part of the target mem- NADPH oxidase machinery, resides in the
brane, thus altering its molecular composition specific granule membrane (45). This fusion
(6). The different classes of granules demon- permits assembly of the NADPH oxidase com-
strate varying propensities for mobilization in plex and allows reactive oxygen species (ROS)
response to inflammatory signals: Azurophilic production both inside the phagolysosome and
granules are the most difficult to mobilize, fol- outside of the cell. Degranulation of primary
lowed by specific granules, gelatinase granules, and secondary granules contributes to the
and finally, secretory vesicles (37–41). The creation of an antimicrobial milieu at the in-
underlying mechanisms for this differential flammatory site and produces an environment
mobilization are not entirely understood, al- inhospitable to invading pathogens.
though regulation of intracellular calcium levels The release of granular proteins during de-
appears to play a salient role (32, 39). Because granulation presents the astute observer with
of this varying mobilization propensity, each a tempting proposition: Could these granular

466 Amulic et al.



components also serve as signaling molecules whereas others may be redundant. One of the
for subsequent inflammatory cell recruitment? challenges in understanding the neutrophil’s
Recent studies have provided experimental evi- antimicrobial mechanisms is to study their
dence suggesting this does seem to be the case: function during concerted action and in con-
Granule proteins from neutrophils, including ditions that mimic an infection site. Therefore,
PR3 and azurocidin, can induce monocyte re- testing the relevance of antimicrobials in vivo
cruitment. Furthermore, neutrophil granule is essential. This is, however, particularly chal-
proteins may increase macrophage bacterial lenging; ablation of a single antimicrobial gene
clearance by enhancing phagocytosis (46). This may only subtly affect immune defense. In ad-
could be advantageous in situations in which the dition, much biochemical identification of neu-
extracellular concentration of released granule trophil antimicrobials has been performed in
proteins is insufficient to exert extensive micro- rabbits and humans, species with abundant neu-
bicidal effects. In such cases, the granule pro- trophils. Mice, which are genetically tractable,
teins would instead operate as signaling and re- have neutrophils that function differently from

cruitment factors (see section on Neutrophils those of other species. Indeed, as already men-

in Immune Cell Cross Talk, below). tioned, mice lack the genes for some antimicro-
By necessity, most data on neutrophil bials identified in humans. Interestingly, there
degranulation and its effects on neutrophil ac- are few clinically relevant innate immune de-
tivity have been acquired through biochemical ficiencies that directly link antimicrobial activ-
approaches performed exclusively in vitro. A ity with a particular mutation. Thus, with few
pertinent question therefore presents itself: Is exceptions, evidence for clinical or biological
this process truly relevant during the in vivo relevance of these molecules is still lacking.
inflammatory response? The data here are There are three main types of antimicro-
sparse, and understandably so: Historically, the bials: (a) cationic peptides and proteins that
possibilities for such an in vivo observation have bind to microbial membranes, (b) enzymes,
been restrained by technical limitations. Most and (c) proteins that deprive microorganisms
evidence for in vivo degranulation relies on of essential nutrients. Here we present an
observation of increased levels of extracellular overview of this rich field of investigation.
granular proteins at inflammatory sites. Even There are more than 800 antimicrobial
so, release of granular components could occur peptides described in nature, some of them
primarily through other means, most notably highly conserved throughout evolution (47).
through formation of neutrophil extracellular These peptides are often charged, a feature that
traps, cell damage, or cell lysis. With the probably promotes their initial interaction with
advent of intravital microscopy techniques, microbial surfaces. Under artificial conditions,
direct observation of the degranulation process many of these peptides disrupt the membrane
in vivo may soon be realized. integrity. Because in vitro tests are often exe-
cuted at high antimicrobial concentrations to
obtain maximal microbial killing in the shortest
Antimicrobial Proteins possible time, it is unclear whether this disrup-
Neutrophils produce a plethora of peptides and tion reflects their mechanism of action under
proteins that directly or indirectly kill microbes physiological conditions. Alternatively, some
(Table 1). Many of these antimicrobials were antimicrobials are thought to disrupt essential
identified through biochemical fractionation of microbial functions, such as DNA replication,
neutrophil extracts, and their in vitro activity transcription, or production of energy. Little
is easily demonstrated in optimized conditions; is known about antimicrobial concentrations
nonetheless, showing in vivo relevance is chal- achieved at inflammatory sites or in the phago-
lenging. The diversity of antimicrobials sug- some. This information, as well as information
gests that some of them evolved to act together, about the synergistic interactions of different




Table 1 Mechanism of action of neutrophil antimicrobial proteins
Antimicrobial peptide Antimicrobial mechanisma
Cationic antimicrobial peptides
α-defensins (HNP-1, HNP-2, Permeabilize membrane bilayers containing negatively charged
HNP-3, HNP-4) phospholipids
Inhibit DNA, RNA as well as protein biosynthesis
Inhibition of bacterial cell wall synthesis
LL-37 Transmembrane pore-forming
BPI Increase bacterial permeability and hydrolysis of bacterial
phospholipids by binding to LPS
Histones Unknown mechanism
Proteolytic enzymes
Lysozyme Degrades bacterial cell wall
Proteinase 3 (PR3) Mechanism independent of a proteolytic activity by binding to the

bacterial membrane
Neutrophil elastase (NE), Cleaves bacterial virulence factors and outer membrane
cathepsin G (CG) proteins
Mechanism independent of a proteolytic activity by binding to
the bacterial membrane
Azurocidin Mechanism independent of a proteolytic activity by binding to the
bacterial membrane
Metal chelator proteins
Lactoferrin Alters bacterial growth by binding to iron, an essential bacterial
nutrient
Binds to the lipid A part of LPS, causing a release of LPS from
the cell wall and an increase in membrane permeability
Calprotectin Alters bacterial growth by sequestering manganese and zinc

a
Only direct actions of neutrophil antimicrobial proteins on microbes are listed in the table.

antimicrobials, is essential for designing appro- from larger proteins, and in addition to their
priate in vitro conditions to probe mechanisms antimicrobial activity, they may potentiate
of action. DNA activation of dendritic cells (DCs) (50).
The neutrophil cationic antimicrobial Neutrophils also contain a number of
peptides include defensins and cathelicidins. full-length cationic antimicrobial proteins,
Neutrophils mostly produce α-defensins, a including BPI and histones. BPI is cationic
protein family whose members possess multi- and binds LPS avidly, much like its structural
ple disulfide bonds and whose structures may cousin the LPS binding protein. BPI binding to
change under physiological conditions and LPS results in increased bacterial permeability
increase their activity (48). A surprising num- and hydrolysis of bacterial phospholipids; cell
ber of functions are assigned to defensins, but death then follows (51). Interestingly, histones
none have been validated in vivo. Interestingly, are extremely effective antimicrobials and
inhibition of bacterial cell wall synthesis (49) were one of the first antimicrobials described
was recently shown at low concentrations that (52). The significance of histones (and of the
may be more similar to those present at inflam- peptides derived from them) as microbials
matory sites. Cathelicidins, including the well- remains to be demonstrated in vivo (53).
studied LL-37, are proteolytically processed Given their dual role as an architectural

468 Amulic et al.


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1 neutrófilos - primeira apresentação.pdf