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  • 1. 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 R still occur before final publication S online and in print.) C E I N N 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.orgby 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
  • 2. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 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).Annu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org still enjoys frequent use and that is used inter-by Universidade Federal do Amazonas on 03/21/12. For personal use only. 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. Changes may still occur before final publication online and in print
  • 3. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 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 boneAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org comprehensive review of the subject matter. marrow intent on pursuing one simple, yetby Universidade Federal do Amazonas on 03/21/12. For personal use only. 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 Changes may still occur before final publication online and in print
  • 4. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 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 signalingAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org endothelial cells near the inflammatory site. bacterial products results in activation andby Universidade Federal do Amazonas on 03/21/12. For personal use only. 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. Changes may still occur before final publication online and in print
  • 5. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 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-Annu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org produced cytokines (e.g., IL-8) and, in parallel, tivators, IL-8. At low concentrations, IL-8by Universidade Federal do Amazonas on 03/21/12. For personal use only. 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 www.annualreviews.org • Neutrophil Functions 463 Changes may still occur before final publication online and in print
  • 6. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 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 THEAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org believe that neutrophil activation refers only toby Universidade Federal do Amazonas on 03/21/12. For personal use only. 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. Changes may still occur before final publication online and in print
  • 7. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 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 enzymeAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org of dangerous substances through the blood- critical in the oxidative burst (32, 33). Otherby Universidade Federal do Amazonas on 03/21/12. For personal use only. 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. www.annualreviews.org • Neutrophil Functions 465 Changes may still occur before final publication online and in print
  • 8. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 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 integrinAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org number of metalloproteases, such as gelatinase interaction with the endothelium. As they pro-by Universidade Federal do Amazonas on 03/21/12. For personal use only. 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. Changes may still occur before final publication online and in print
  • 9. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 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 fromAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org cruitment factors (see section on Neutrophils those of other species. Indeed, as already men-by Universidade Federal do Amazonas on 03/21/12. For personal use only. 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 www.annualreviews.org • Neutrophil Functions 467 Changes may still occur before final publication online and in print
  • 10. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 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 theAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.orgby Universidade Federal do Amazonas on 03/21/12. For personal use only. 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. Changes may still occur before final publication online and in print
  • 11. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 scaffold for DNA and as antimicrobials, their Reactive Oxygen Species in vivo significance is particularly difficult to Upon activation, neutrophils produce ROS in demonstrate. a process called the respiratory burst. It is mis- Chronic The second class of neutrophil antimi- leading to think of ROS as a single entity be- granulomatous crobials encompasses a broad assortment of cause they differ in their stability, reactivity, and disease (CGD): proteolytic enzymes that participate in microbe permeability to membranes (62). However, all caused by mutations destruction. Lysozyme destroys the bacterial rendering the ROS can modify and damage other molecules, NADPH oxidase wall, making it an obvious antimicrobial, as properties exploited by the host cell for signal- nonfunctional, shown in mice deficient in this enzyme (54). ing and antimicrobial action. characterized by Surprisingly, this occurred independently of its The NADPH oxidase complex assembles susceptibility to enzymatic activity (55). Neutrophils also con- on the phagosomal and plasma membranes infection and tain several serine proteases (including PR3, autoinflammation and begins the reactive oxygen cascade by CG, and NE, collectively known as the serpro- reducing molecular oxygen to superoxide. cidins) that exhibit differing specificities. They Downstream of superoxide, many potentialAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org are tightly regulated intra- and extracellularlyby Universidade Federal do Amazonas on 03/21/12. For personal use only. reactions can occur (for details, see References by serpins, indicating that their activity is 62–64). Superoxide, though not a strong deployed under specific conditions. NE cleaves oxidant, rapidly dismutates, forming hydrogen enterobacterial virulence factors with high peroxide. Superoxide can also react with nitric specificity (56), indicating the possibility of the oxide, which is produced at high levels at coevolution of microbial virulence factors and inflammatory sites, to form peroxynitrite, a antimicrobial effectors. Of further interest, NE strong oxidant. Upon degranulation into the mutations in humans, but not genetic ablation phagosome, MPO can react with hydrogen of this enzyme in mice, results in neutropenia. peroxide to produce various reactive species, This can be rescued by the administration of including hypohalous acids. Hypochlorous recombinant granulocyte macrophage colony- acid, thought to be the major product of MPO stimulating factor (GM-CSF); however, these in the phagosome, is more reactive than su- patients still exhibit significant susceptibility peroxide and is antimicrobial in vitro. Thus, it to infections. Mice deficient in NE or CG is assumed to have direct antimicrobial effects are highly susceptible to bacterial and fungal in the phagosome. However, a theoretical infections (57, 58). Another protein, azuro- model of the phagosome suggests that most of cidin, is a member of the same family but lacks the hypochlorous acid produced would react protease activity. Unexpectedly, it still kills with host proteins before reaching the bac- microbes, suggesting that these proteins may terium. This model predicts that chloramines, all have antimicrobial activity independent produced when hypochlorous acid reacts of proteolysis, perhaps as a result of their with amine groups, may be the most relevant cationicity. These serine proteases also play a antimicrobial actors in the phagosome (65). salient role in autoimmunity (see discussion in ROS are clearly important for neutrophil section on Autoimmunity, below) (59). antimicrobial activity: Neutrophils from The final class of neutrophil antimicrobials chronic granulomatous disease (CGD) patients consists of a number of proteins that chelate kill microbes poorly, making these patients essential metals from microbes and possibly susceptible to many infections. Interestingly, impact bacterial growth. Two of these chela- CGD patients can control catalase-negative tors are lactoferrin, first identified in milk, bacteria, which produce, but do not degrade, which binds preferentially to iron, and cal- their own hydrogen peroxide, thus providing protectin (also called S100A and many other a substrate for reactions downstream in the names), which sequesters zinc (60) and results in reactive oxygen cascade (66). NADPH ox- “nutritional immunity” (61). idase is also implicated in the regulation of www.annualreviews.org • Neutrophil Functions 469 Changes may still occur before final publication online and in print
  • 12. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 inflammation, which explains why CGD (73, 74). Furthermore, superoxide generation patients often suffer from autoinflammatory leads to an ionic influx into the phagosome to diseases (67). compensate for charge; this may activate gran- Paradoxically, although MPO is required ule proteases by releasing them from their pu- for neutrophil microbicidal activity in vitro, tative matrix (75). There is controversy around MPO-deficient individuals do not have striking which ions and which channel are responsible clinical manifestations (68, 69). Some MPO- for charge compensation, but this theory of deficient individuals suffer from frequent or se- protease activation is certainly intriguing (69). vere infections, especially with Candida species, Studies of ROS are hampered by various and a few have been mistaken for CGD patients. technical issues. Ideally, a probe for ROS However, most MPO-deficient individuals in should be specific, targetable to particular the developed world have apparently normal intracellular compartments, and capable of immunity. The mild effects of MPO deficiency being used in vivo. Traditional probes for suggest that MPO’s products are not essential ROS do not meet these specifications; inAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org for antimicrobial action. Indeed, in the absence addition, the probes often become radicalby Universidade Federal do Amazonas on 03/21/12. For personal use only. of MPO, other reactive species (e.g., superox- species (76). One promising new approach ide, hydrogen peroxide, hydroperoxyl radical, for ROS detection that meets these criteria is peroxynitrite) can still be produced in the the use of redox-sensitive fluorescent protein- neutrophil phagosome; hydroperoxyl radical is based probes, such as roGFP and HyPer predicted to be present at antimicrobial concen- (76). Other methods that can be used in vivo trations (65). However, there may be a broader include transcription profiling of superoxide reason for this discrepancy. Modern technolo- or hydrogen peroxide–sensitive genes as well gies can distinguish between individuals who as the detection of relatively stable products of are partially and completely MPO deficient, reactive oxygen using mass spectrometry (76). and partial MPO deficiency does not correlate with pathology (70). Residual activity of MPO may be sufficient for antimicrobial activity: In Phagocytosis the case of CGD, even 1% of normal NADPH Phagocytosis is the major mechanism to re- oxidase activity leads to an improved prognosis move pathogens and cell debris. It is an active, (71). Epidemiological studies distinguishing receptor-mediated process during which a par- the degrees of MPO deficiency and their ticle is internalized by the cell membrane into correlation with clinical manifestations may be a vacuole called the phagosome. As with other necessary to understand the function of MPO. phagocytes, the mechanistic details of internal- In addition to direct antimicrobial action, ization depend on the type of interaction be- ROS can modify host molecules. Because tween the neutrophil and the microorganism. these species are highly reactive, they are often Interaction can be direct, through recognition thought to be too nonspecific to be involved in of PAMPs by pattern-recognition receptors, or signaling. However, specificity can be achieved opsonin mediated. The latter mechanism is bet- on the submolecular level, by cellular redox ter characterized and includes two prototypical buffering systems and by limited diffusion of examples: FcγR-mediated phagocytosis, which ROS owing to their short half-lives (72). A relies on the formation of pseudopod extensions well-studied example of ROS in signaling is for engulfment of IgG-opsonized particles, and the reversible regulation of various targets complement receptor-mediated phagocytosis, (including phosphatases, metalloproteinases, which does not require membrane extensions and caspases) by direct oxidation of cysteine or pseudopods (77). residues. In addition, neutrophil granule After engulfment, the nascent phagosome proteases can be regulated by oxidative inacti- is relatively benign to microorganisms, acquir- vation of their inhibitors or by direct oxidation ing its lethal properties only after a drastic 470 Amulic et al. Changes may still occur before final publication online and in print
  • 13. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 maturation process. Our understanding of importance of phagocytosis in the innate this process is largely based on studies in immune defense. macrophages, and although these are certainly Autophagy: a process instructive, essential differences exist in neu- in which cellular trophils. Macrophage phagocytosis follows an Neutrophil Extracellular Traps contents are degraded endocytic maturation pathway: In neutrophils, Upon stimulation, neutrophils can undergo in lysosomes, phagosome maturation happens upon fusion of NETosis, an active form of cell death that especially in conditions of nutrient granules to the phagosome, whereby delivery leads to release of decondensed chromatin into scarcity and infection of antimicrobial molecules into the phagoso- the extracellular space (86, 87). The fibrous mal lumen occurs. Simultaneously, assembly structures termed NETs contain histones as of the NADPH oxidase on the phagosomal well as antimicrobial granular and cytoplasmic membrane allows ROS production, and jointly, proteins (88). NETs trap many types of mi- these two mechanisms create an environment crobes ex vivo and have been found in various toxic to most pathogens. Neutrophil phago- disease models in vivo; they are thought toAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org somal pH regulation also differs significantly kill microbes by exposing them to high localby Universidade Federal do Amazonas on 03/21/12. For personal use only. from that observed in macrophages. While the concentrations of antimicrobials (89). macrophage phagosome gradually acidifies, The mechanism of NET formation is not neutrophil phagosomal pH is initially alkaline completely understood. The reactive oxygen (78) and remains neutral for prolonged periods pathway is involved, as NADPH oxidase and of time (79). The maintenance of this alkaline MPO are required for NET formation in re- pH is essential for the activation of the major sponse to chemical and biological stimuli (87, serine proteases NE and CG, and it is sustained 90, 91). Nitric oxide donors can induce NETs via NADPH oxidase activity, despite contin- via a mechanism that also requires ROS (90), a uing fusion of acidic granules. Key events of finding that awaits genetic confirmation. All ac- the maturation process are described in more tivators of NET formation tested so far require detail in Reference 80. ROS production. S. aureus may be an exception, Not all pathogens succumb to the hostile although those experiments were done using environment of the phagosome. In fact, some pharmacological inhibitors, not cells deficient have evolved strategies to survive inside neu- in ROS production (92). Upstream of NADPH trophils. These strategies include interfering oxidase, the Raf-MEK-ERK pathway is impli- with engulfment, modulating phagosome cated in NET formation (93), but further along maturation, and creating a more hospitable in the process, NE translocates from the gran- intraphagosomal environment. The polysac- ules to the nucleus and degrades histones, lead- charide capsule expressed by Staphylococcus ing to chromatin decondensation (94). Histone aureus confers antiphagocytic properties (81). citrullination may also play a role in NET for- Helicobacter pylori can disrupt targeting of mation, although this has not been confirmed NADPH oxidase to the phagosome so that in primary human neutrophils (95–97). Au- superoxide anions accumulate extracellularly tophagy is also thought to be required for NET rather than in the phagosome (82). Francisella formation, but this has so far been shown only tularensis prevents triggering of the oxidative using a nonspecific inhibitor of autophagy (98). burst and also inhibits ROS production in The majority of research on NETs has been response to other stimuli (83). Finally, other conducted ex vivo. Ideally, to test the relevance pathogens, such as Salmonella typhimurium and of NETs, a “NETs knockout” organism should Streptococcus pyogenes, can efficiently block gran- be generated to investigate its response to ule fusion with the phagosome (84, 85). The pathogens. Unfortunately, it is not possible to variety of mechanisms evolved by intracellular eliminate the main components of NETs— pathogens to resist killing and enable survival DNA and histones—from an infection model. within the phagosome further emphasizes the Moreover, the factors that are important for www.annualreviews.org • Neutrophil Functions 471 Changes may still occur before final publication online and in print
  • 14. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 NET formation, such as NADPH oxidase, establishing the correct environmental condi- MPO, and NE, are also critical for other an- tions to launch the adaptive immune response. timicrobial neutrophil functions. For now, the The cytokines released by PMNs are often Cystic fibrosis: caused by defects in evidence for the relevance of NETs is indirect. synthesized de novo. Although neutrophils the CFTR ion On the one hand, bacteria that express DNases transcribe little after leaving the bone marrow, transporter, as virulence factors disseminate more efficiently once activated, these cells undergo a tran- characterized by thick, in the host, which may point to evolutionary scriptional burst that results in the synthesis sticky mucus and pressure to avoid entrapment by NETs (99, of signaling molecules (110, 111). Compared decreases in lung and digestive function 100). In addition, a persistent Aspergillus with other immune cells (e.g., macrophages), infection in a CGD patient was cleared after neutrophils typically produce lower amounts gene therapy, which restored NADPH oxidase of cytokines per cell, but they are so abundant activity, NET formation, and NET-mediated at inflammatory sites that their contribution but not phagocytosis-mediated killing by the to total cytokine levels is significant (4). Fur- patient’s neutrophils ex vivo (101). On the other thermore, neutrophil-secreted proteases canAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org hand, the immune system has redundant mech- modulate signaling networks in vivo throughby Universidade Federal do Amazonas on 03/21/12. For personal use only. anisms to fight infection, and it may be that cytokine processing (112). NETs are especially important under certain The initial neutrophil cytokine response is conditions, such as during infections with large an appeal for immunological reinforcement. pathogens that are not readily phagocytosed. The most abundantly produced cytokine, IL-8, NETs can also have detrimental effects on primarily serves to recruit other neutrophils the host. Because NETs expose self molecules (113). Similarly, neutrophil-derived proinflam- extracellularly, they lead to autoimmunity: matory IL-1β and TNF-α induce other cells NETs have been implicated in systemic to produce neutrophil chemoattractants (114, lupus erythematosus (SLE), an autoimmune 115) (for a comprehensive list of cytokines disease characterized by the formation of produced by neutrophils, please see References autoantibodies, often against chromatin and 115, 116). In addition to cytokines, neutrophils neutrophil components (102–106) (see section release other signaling mediators, including on Autoimmunity, below). Platelet-induced granule contents (117), lipids (118), and ROS NETs, formed during sepsis, are associated such as hydrogen peroxide (119). They also with hepatotoxicity due to tissue damage communicate via cell-cell contact (120). Here (107). Platelets also bind to NETs, raising the we provide examples of how neutrophils possibility that NETs nucleate blood clots in interact with other cells to shape the immune the context of deep vein thrombosis (108). response (see Figure 3). NETs have also been observed in the airway fluids of cystic fibrosis patients, where they may increase the viscosity of the sputum and Monocytes and Macrophages decrease lung function (109). As they respond to infection or injury, neutrophils and their relatives in the mono- cyte/macrophage lineage coordinate their NEUTROPHILS IN IMMUNE activities, leading to alternating waves of re- CELL CROSS TALK cruitment of these two cell types. Macrophages Neutrophils participate in the communica- and patrolling monocytes are among the initial tion networks that form the foundations of detectors of PAMPs and endogenous activators, immunity, issuing instructions to practically the danger-associated molecular patterns (121), all other immune cells. As one of the first cell and these cells work to summon large numbers types to arrive at sites of infection, neutrophils of neutrophils to the inflammatory locus. The secrete cytokines and chemokines critical in the influx of neutrophils is followed closely by the unfolding of the inflammatory response and in arrival of monocytes, suggesting a causal link 472 Amulic et al. Changes may still occur before final publication online and in print
  • 15. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 Tissue T cell Activation and CD8+ Lymph node IFN-γ differentiation DC T cell Crosspriming ROS? T cell Arginase? Neutrophil IFN-γ Activation DC DC NK cell IL-12 DC Antigen presentation CD4+ Macrophage Neutrophil T cell Th1 Activation Neutrophil BacteriaAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.orgby Universidade Federal do Amazonas on 03/21/12. For personal use only. Neutrophil Monocyte DC Blood Figure 3 Neutrophil communication with other immune cells. Neutrophils interact with a variety of cell types. They are important both for recruitment of monocytes and dendritic cells (DCs) to infected tissues and for enhancement of macrophage and DC activity. In contrast, in the lymph nodes, neutrophils impede DC function by inhibiting antigen presentation to CD4+ cells. Neutrophils also interact with the adaptive arm of the immune system: They can act as antigen-presenting cells by cross-presenting antigen to CD8+ T cells; they also secrete IL-12, which activates T cells. T cells, in turn, activate neutrophils by secreting IFN-γ. Finally, neutrophils, DCs and natural killer (NK) cells colocalize and enhance each other’s activity via receptor-receptor interactions and soluble mediators. behind these temporal dynamics. Indeed, neu- microbicidal activity (129). The circuitous trophils recruit monocytes via several different nature of the cross talk of these two cell types mechanisms. They express classical monocyte becomes obvious during inflammation abate- chemoattractants such as CCL2 (MCP-1) ment: Monocytes, recruited by neutrophils (122), CCL3 (MIP-1α) (123), CCL20 (MIP- and differentiated into macrophages, repress 3α), and CCL19 (MIP-3β) (124). Additionally, further neutrophil chemotaxis and ensure and perhaps more unexpectedly, neutrophils the appropriate removal of their postmortem use granule proteins to induce extravasation remains (see section on Neutrophils and of monocytes in vivo, as shown for LL-37, Resolution of Inflammation, below). azurocidin (HBP/CAP37), and CG (125–127). Monocyte recruitment is also affected indirectly by neutrophils: via upregulation of endothelial Dendritic Cells adhesion factors, increase of transendothelial Neutrophils can also recruit and activate permeability, enhancement of production of DCs in vivo. This was recently illustrated chemoattractants by other cell types, and mod- in a mouse model of Leishmaniasis, where ulation of the activities of these chemokines subcutaneous inoculation of Leishmania major via proteolytic processing (reviewed in 128). triggered a massive and rapid infiltration of In addition to recruitment, neutrophils mod- neutrophils (130). These cells secrete the ulate monocyte and macrophage cytokine chemokine CCL3, recruiting DCs to the production (128), directly enhancing their site of inoculation and initiating a protective www.annualreviews.org • Neutrophil Functions 473 Changes may still occur before final publication online and in print
  • 16. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 Th1 response (131). Interestingly, activated performed in vitro, and their interpretation is neutrophils can induce the maturation of DCs frustratingly difficult owing to the question- in vitro through specific receptor-receptor able purity of cell preparations. Recently, it DC-SIGN: dendritic cell–specific interactions between Mac-1 and DC-SIGN, was shown that neutrophils, NK cells, and DCs intercellular adhesion leading to local secretion of TNF-α (120). interact in a m´ nage a trois involving both e ` molecule-3-grabbing In this case, the reduced levels of cytokine cytokine signaling and direct cell-cell contact nonintegrin production foster specificity, as only proximal (137, 138). In one report, infection of mice Granulocyte DCs receive the maturation signal. A similar with Legionella pneumophila triggered produc- receptor 1 (Gr1): activation model was earlier proposed for Tox- tion of IFN-γ by NK cells; this was dependent the anti-Gr1 antibody oplasma gondii (132). Neutrophil-activated DCs on both PMN-derived IL-18 and DC-derived RB6-8C5 reacts with both Ly6G (specific produce the proinflammatory cytokine IL-12 IL-12 (137). Similarly, human neutrophils, NK for neutrophils) and and induce proliferation of T cells (120, 132). cells, and DCs colocalize at inflammatory sites, Ly6C (present on However, some of these experiments should and a positive feedback loop has been proposed many immune cell be interpreted cautiously because they are on the basis of in vitro data. In this scheme, neu- types)Annu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org based on the injection of the anti-Gr1 antibody trophils interact with a specific subset of DCs,by Universidade Federal do Amazonas on 03/21/12. For personal use only. Th17 cells: subset of (RB6), which depletes neutrophils but may also (via CD18-ICAM-1 interactions), prompting T helper cells that result in depletion of many other cell types in the DCs to produce IL-12p70, which in turn produce IL-17, important in mice. The anti-Ly6G monoclonal antibody is stimulates IFN-γ production by NK cells and inflammation and more specific and hence a better reagent for this further activates neutrophils. Simultaneously, implicated in type of experiment (133). The crucial role of neutrophils also activate NK cells by direct con- autoimmunity neutrophils in DC activation was recently con- tact (139). Additional in vitro interactions be- firmed using anti-Ly6G antibody depletion: In tween neutrophils and NK cells are extensively Mycobacterium tuberculosis infection, timely traf- reviewed in Reference 138. ficking of DCs to lymph nodes and activation of CD4+ T cells were both dependent on PMNs. Furthermore, this study demonstrated that Lymphocytes DCs presented bacterial antigens when they A surprising finding in recent years is the exten- ingested infected neutrophils just as efficiently sive cross talk between cells located at opposite as they did via direct uptake of Mycobacterium ends of the immune spectrum. Previously (134). In sharp contrast to the above findings, thought to belong to isolated compartments, a separate study using an immunization model neutrophils and T cells shape and impact showed that neutrophils recruited to lymph each other’s functions, both qualitatively and nodes compete for antigen with DCs and quantitatively (140). Neutrophils affect T cell macrophages and that these neutrophils inhibit function indirectly via DCs, as outlined above, their interactions with T cells (135). It is possi- but can also influence T cell function directly. ble that neutrophils have site-specific effects on PMNs secrete IL-12, which may be crucial for DCs and can be stimulatory at peripheral sites Th1 cell differentiation (141, 142). They also and inhibitory in the lymph nodes. Neutrophils express several T cell chemoattractants (116) exhibit fascinating and somewhat enigmatic be- as well as B cell development and maturation havior in the lymph nodes, where they engage factors (143, 144). Cytokine communication in swarming activity in response to parasitic occurs in both directions: For instance, IFN-γ, infection (136). The functions and mechanistic which is secreted by T cells, prolongs neu- details of these swarms are unknown and trophil life span, induces gene expression, and represent questions of immense interest. increases phagocytic capacity (145). The T helper 17 (Th17) cell subset secretes IL-17, Natural Killer Cells a key cytokine in the control of neutrophil Studies of interactions between neutrophil and dynamics, which acts by upregulating expres- natural killer (NK) cells have historically been sion of CXCL8 (IL-8), G-CSF, and TNF-α 474 Amulic et al. Changes may still occur before final publication online and in print
  • 17. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 by epithelial, endothelial, and stromal cells Although some collateral damage to host (146). Collectively, these Th17-associated tissues is inevitable during infection, neu- cytokines increase granulopoeisis as well as the trophils must be removed before they have Ulcerative colitis: a recruitment and life span of neutrophils. serious, detrimental effects on inflamed tissues. type of inflammatory Neutrophils potentially have suppressive ef- Resolution of inflammation is an active process bowel disease fects on T cells via two proposed mechanisms: that limits further leukocyte infiltration and characterized by ulcers (a) L-arginine depletion by release of arginase, removes apoptotic cells from inflamed sites. and tissue erosion in the colon and rectum which inhibits T cell responses in vitro (147), This process is essential for maintenance of and (b) hydrogen peroxide–mediated suppres- tissue homeostasis and, if impeded, leads to sion, as proposed in a cancer model (119) (see “nonresolving inflammation,” a problematic section on Cancer, below). Direct evidence of condition that contributes to many diseases. such interactions in vivo is still missing. Interestingly, neutrophils influence CD8+ T cell responses by cross-presenting exogenous Apoptosis and ClearanceAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org antigens in vivo. Using mice in which profes-by Universidade Federal do Amazonas on 03/21/12. For personal use only. sional antigen-presenting cells do not express Apoptosis is a central aspect of inflammation functional MHC class I, Beauvillain et al. (148) resolution. Once neutrophils have executed showed that antigen-pulsed neutrophils can their antimicrobial agenda, they die via a built- induce differentiation of cytotoxic T cells. in cell-death program. However, not only does These striking findings imply that neutrophils apoptosis reduce the number of neutrophils have characteristics of antigen-presenting cells. present, it also produces signals that abro- Neutrophils also appear capable of expressing gate further neutrophil recruitment. Phagocy- MHC class II and costimulatory molecules tosis of apoptotic neutrophils also reprograms under inflammatory conditions (149–151), macrophages to adopt an anti-inflammatory and they can present antigen to CD4+ T cells phenotype. in vitro (152–154). However, the functional Neutrophil death is influenced by inflamma- significance for protective immunity remains tory mediators such as GM-CSF and LPS and unclear, especially in light of the finding that by environmental conditions such as hypoxia, mouse neutrophils that migrate to the lymph all of which prolong neutrophil survival. The node have a negative effect on CD4 responses signaling networks that regulate survival have in an immunization system (135). In humans, also been well characterized. These networks there are large variations in the ability of also control the expression of known antiapop- donors to express MHC class II (149, 151), totic (Mcl-1 and A1) or proapoptotic proteins suggesting concomitant variations in the ability (Bad, Bax, Bak, and Bid), and they also activate to activate T cells, a finding that could have caspases (for an extensive review, see Reference implications for susceptibility to autoimmune 155). Given that neutrophils are terminally diseases. Therefore, neutrophil modulation of differentiated, it is unexpected that molecules adaptive immunity seems to be highly complex controlling cell proliferation regulate survival. and is only now starting to be unraveled. Proposed to have prosurvival effects, one such protein is survivin. It is expressed more highly in immature neutrophils than in mature ones, NEUTROPHILS AND but its expression can be restored in mature RESOLUTION OF cells by inflammatory signals such as G-CSF or INFLAMMATION GM-CSF. In line with these findings, survivin The lethal cargo of neutrophils is not only is also highly expressed in neutrophils at sites destructive toward invading microbes, but of inflammation, such as cystic fibrosis sputum, also harmful to host cells. Thus, neutrophil appendix infiltrates, and intestines of patients deployment must be tightly controlled. with ulcerative colitis (156). www.annualreviews.org • Neutrophil Functions 475 Changes may still occur before final publication online and in print
  • 18. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 Similarly, cyclin-dependent kinases func- their vicinity (epithelial cells, endothelial cells, tion as prosurvival factors in neutrophils. fibroblasts, platelets, and leukocytes) and par- Pharmacological inhibition of these cell cycle ticipate in the transcellular biosynthesis of lipid Wegener’s granulomatosis: regulators induce caspase-dependent apoptosis mediators with anti-inflammatory and prore- vasculitis affecting the and block life-span extension by survival factors solving activities, such as lipoxins, resolvins, and lungs, nose, and (157). More recently, prosurvival effects were protectins. A major lipid mediator class switch kidneys; inflammation also attributed to proliferating cell nuclear thus exists, governed by temporally regulated leads to reduced blood antigen (PCNA). This factor usually resides expression of different lipoxygenases and the flow, tissue destruction, and in the nucleus, where it is involved in DNA mobilization of different fatty acid substrates. damage of vital organs replication, but in neutrophils, it associates The different biosynthesis pathways of prore- Prostaglandins and with procaspases in the cytosol and is thought solving lipid mediators have been reviewed in leukotrienes: lipids to prevent their activation. During apoptosis, detail elsewhere (118). Interestingly, microor- synthesized by PCNA is targeted for proteosomal degradation, ganisms are also a source of lipid precursors cyclooxygenases and which correlates with an increase in caspase-3 that can be used by neutrophils for resolvin 5-lipoxygenase,Annu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org and caspase-8 activities. This mechanism is rel- synthesis. Thus, microbes also likely participateby Universidade Federal do Amazonas on 03/21/12. For personal use only. respectively, in the arachidonic acid evant in Wegener’s granulomatosis and sepsis, in synthesis of mediators with proresolving pathway; have where stabilization of PCNA is associated with functions at the site of infection (159, 160). proinflammatory resistance of neutrophils to apoptosis (158). How do lipid mediators contribute to functions including Equally important for the resolution of in- the termination of inflammation? Lipoxins, leukocyte recruitment flammation is the proper removal of apoptotic resolvins, and protectins exert cell-type specific cells. This relies on the release of “find-me” effects, promoting monocyte/macrophage signals at early stages of cell death, which at- recruitment and activation while inhibiting tract phagocytes. Likewise, distinct “eat me” neutrophil functions. The inhibitory effect signals are required for specific recognition of extends to all essential steps of neutrophil apoptotic cells. Ingestion of apoptotic cells by responses: migration, adhesion, and activation. macrophages drives the production of the anti- All three lipid mediators reduce neutrophil inflammatory cytokines tumor growth factor recruitment, a process that involves the lipoxin- (TGF)-β and IL-10 (155). Failure to clear these A4 receptor and the leukotriene B4 receptor apoptotic cells, by contrast, results in secondary (BLT1) (161–167). Ariel et al. (168) also pro- necrosis and release of products that generate posed an interesting mechanism of action for proinflammatory signals (Figure 4). lipoxins, resolvins, and protectins in clearing in- flammatory sites. They showed that neutrophil exposure to these lipids increases expression Lipid Mediator Class Switch of CCR5 on the surface of late apoptotic neu- Soluble mediators play a crucial role in the trophils, leading to efficient sequestration of the resolution of inflammation. In neutrophils, chemoattractants CCL3 and CCL5. The se- a particularly prominent role is assumed by questration of these chemokines means they are lipid mediators. The successful progression unavailable to recruit neutrophils to inflamed of inflammation appears to hinge on a shift sites (168) (Figure 4). This mechanism com- in the composition of secreted lipids. At early plements other anti-inflammatory processes stages of inflammation, neutrophils synthesize in which chemokines are inactivated by neu- proinflammatory lipid mediators, such as trophil proteases. Of these lipids, lipoxins are prostaglandins and leukotrienes. These are the most completely understood. In addition to derived from arachidonate precursor molecules neutrophil recruitment, lipoxins can inhibit the and are synthesized through the cyclooxy- shedding of L-selectin and the upregulation of genase and lipoxygenase pathways. During β2 integrins in response to proinflammatory the later stages of the inflammatory response, stimuli, thereby reducing adhesion of neu- neutrophils interact with various cell types in trophils to endothelial cells (169, 170). Finally, 476 Amulic et al. Changes may still occur before final publication online and in print
  • 19. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 Initiation Resolution of inflammation Leukotrienes Prostaglandins TNF-α Lipoxins Resolvins Protectins IL-10 TGF-β of inflammation Platelets Monocyte Lipoxins Neutrophil Lipoxin Resolvins Protectins Macrophage ApoptoticAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.orgby Universidade Federal do Amazonas on 03/21/12. For personal use only. neutrophil Chemokines CCR5 IL-10 Leukotrienes TGF-β Prostaglandins PGE-2 Chemokines Chemokine clearance Microorganisms ? TNF-α NETotic IL-6 neutrophil Macrophage Figure 4 From inflammation to homeostasis: neutrophil apoptosis and lipid mediator class switching in the resolution of inflammation. At the site of infection, resident macrophages initiate an inflammatory response, secreting proinflammatory cytokines and chemokines that alert the immune system and promote neutrophil recruitment. In the early stages of inflammation, microbes trigger the production of proinflammatory lipid mediators, such as leukotrienes and prostaglandins, which also recruit neutrophils. As inflammation progresses, a switch occurs, and anti-inflammatory lipid mediators such as lipoxins, resolvins, and protectins are produced. Notably, interaction of neutrophils with platelets induces the production of lipoxins. Anti-inflammatory lipid mediators initiate the resolution of inflammation by blocking neutrophil and promoting monocyte recruitment. Monocytes differentiated into macrophages ingest apoptotic neutrophils, driving the production of the anti-inflammatory cytokines tumor growth factor (TGF)-β and IL-10 and prostaglandin-E2 (PGE-2), which drive the lipid mediator class switch. Pro-resolving lipid mediators also promote the expression of CCR5 on the surface of apoptotic neutrophils, providing a means of scavenging chemokines. Chemokine clearance upon phagocytosis of apoptotic neutrophils by macrophages further contributes to the reduction of neutrophil infiltration and the return to tissue homeostasis. The contribution of macrophages to the clearance of NETotic neutrophils, and how this could impact inflammation resolution, is currently unknown. A timeline of the inflammation process from initiation to resolution is summarized in the upper part of the figure. lipoxins also impact neutrophil activation by Disorders Associated with inhibiting ROS and peroxynitrite production, Nonresolved Inflammation NF-κB activation, and IL-8 expression (170). The failure of neutrophils to apoptose or mal- In addition to directly impacting neu- functions in the removal of their apoptotic re- trophil functions, lipid mediators promote mains result in chronic inflammation. These nonphlogistic (noninflammatory) phagocyto- conditions lead to the accumulation of cyto- sis of apoptotic neutrophils by monocytes toxic substances and are associated with severe Chronic obstructive and macrophages. In the presence of anti- pathologies, including cystic fibrosis, chronic pulmonary disease inflammatory lipids, engulfment of apoptotic (COPD): lung disease obstructive pulmonary disease (COPD), and neutrophils is not accompanied by the release of caused by noxious rheumatoid arthritis (RA). The severity of in- proinflammatory mediators, as typically occurs particles or gas, e.g., flammation often directly correlates with poor during macrophage activation. Instead, produc- tobacco smoking; clinical outcome. inflammation leads to tion of the anti-inflammatory cytokines TGF-β COPD is a major cause of death in indus- lung obstruction and IL-10 is increased (163, 171). trialized nations, where smoking is a prime www.annualreviews.org • Neutrophil Functions 477 Changes may still occur before final publication online and in print
  • 20. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 instigator of this disease. A chronic neutrophil the previous section. It is, however, unknown infiltration in the lungs of COPD patients whether all neutrophils are capable of adapting promotes tissue damage and organ dysfunc- to the changing chemoattractant environment Rheumatoid arthritis (RA): chronic tion. One of the key molecules controlling or if different subsets of neutrophils are suc- inflammatory disease the inflammatory response in the lung is cessively involved. The relevance of this model that affects many leukotriene A4 hydrolase (LTA4H). This in human disease remains to be established, tissues and organs but enzyme has two opposing activities. First, its although the clinical similarities between this primarily synovial hydrolase activity converts leukotriene A4 into mouse model and human RA are encouraging. joints; severe inflammation causes leukotriene B4, a potent neutrophil chemoat- deformity tractant and proinflammatory agent. Second, LTA4H is an aminopeptidase that inactivates NEUTROPHILS IN DISEASE a specific neutrophil chemoattractant, the Neutrophils are prominent players in the innate proline-glycine-proline tripeptide (PGP), thus immune response and the clearance of infec- conferring the enzyme with anti-inflammatory tion, a subject addressed in several prominentAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org properties. Interestingly, tobacco smoke selec- reviews. However, neutrophil action can alsoby Universidade Federal do Amazonas on 03/21/12. For personal use only. tively inhibits only the aminopeptidase activity support disease progression in other illnesses. of LTA4H, promoting the accumulation of A host of autoimmune disorders belong to this both leukotriene B4 and PGP. This in turn category. In addition, certain malignant cancers promotes neutrophil recruitment and fuels are also prime examples of illnesses in which chronic lung inflammation (172). neutrophils play a salient role. Another prime example of a disease linked to nonresolving inflammation is RA. Neutrophils are the most abundant leukocytes present in the Cancer synovial fluid of RA patients, and their role in The link between cancer and inflammation pathogenesis has been demonstrated in several was noted as early as 1863 by Rudolf Virchow animal models. These models primarily used (177). Since then, it has been proposed that neutrophil depletion or adoptive transfer of neutrophil-derived ROS have the potential to wild-type neutrophils in leukotriene-deficient initiate tumor formation by genotoxic stress mice (173–175). In one model, synthesis and induction of genomic instability. Although of leukotriene B4 by neutrophils in joints this has been demonstrated in vitro (178, 179), is essential for disease development (174). convincing evidence for PMN-mediated DNA Leukotriene B4 can act in an autocrine manner mutagenesis in vivo is still lacking. Neutrophils via the neutrophil receptor BLT1 to promote do, however, impact cancer progression. the recruitment of a first wave of neutrophils They are abundant in tumors and influence into the joint. Later, the recruitment of a tumor development through several secreted second wave of neutrophils is independent of mediators, including cytokines, ROS, and this leukotriene B4–BLT1 pathway. At this matrix-degrading proteases (reviewed in Ref- stage, immune complexes are essential for erence 180). The majority of findings support stimulating infiltrating neutrophils to deliver a “protumor” and “antihost” effect of these IL-1β into the joint. This in turn induces the cells; clinical studies indicate that neutrophil production of chemokines by synovial tissue infiltration of tumors is associated with poorer cells and sustains neutrophil recruitment (175, prognosis (181, 182). Indeed, some cancers 176). These studies exemplify the complex seem to actively recruit neutrophils through regulation cascades involving lipids, cytokines, production of IL-8 (183). In agreement with and chemokines that orchestrate neutrophil this, antibody depletion of neutrophils reduces recruitment in chronic inflammation. They tumor growth (184). The protumor function also demonstrate the cross talk between neu- of neutrophils operates at multiple levels, trophils and other immune cells discussed in including production of angiogenic factors 478 Amulic et al. Changes may still occur before final publication online and in print
  • 21. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 (185), enhancement of metastasis (186), and of monocytic and granulocytic cells (189). In suppression of the antitumor immune response human renal cell carcinoma, MDSCs have (119, 187). Using the anti-Ly6G antibody, identical morphology and express the same sur- Acute-phase Fridlender and colleagues (187) depleted neu- face markers as do activated neutrophils (190, proteins: secreted by trophils and confirmed their tumorigenic role. 191). MDSCs inhibit T cell proliferation by liver, concentration in Moreover, the study showed that neutrophils in limiting L-arginine availability via arginase and plasma changes by the tumor microenvironment could, under cer- NOS activities, both of which use this amino 25% or more during inflammation tain circumstances, be induced to target their acid as a substrate (189, 191, 192). Furthermore, cytotoxic arsenal at tumor cells, whose growth MDSCs are strong producers of ROS, which they usually help to fuel. Pharmacological suppresses T cell responses (119, 192). Inter- inhibition of TGF-β signaling led tumor- fering with the release of MDSCs or using drug associated neutrophils to assume a heightened interventions to polarize neutrophil responses proinflammatory state, causing a reduction in in the tumor microenvironment could repre- tumor growth. These alternatively activated sent novel therapeutic strategies against cancer.Annu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org neutrophils underwent a complete reversal inby Universidade Federal do Amazonas on 03/21/12. For personal use only. their effect on CD8+ T cells, serving to activate rather than suppress these cells. Differential Autoimmunity neutrophil responses were also demonstrated in Deregulated neutrophil cell death and/or a melanoma study. In this instance, increased clearance often accompanies autoimmune syn- systemic levels of the acute-phase protein dromes (193–195) and may play a major role serum amyloid A (SAA-1) induced neutrophils in disease pathogenesis, given that release of to secrete the anti-inflammatory cytokine IL- proteolytic and cytotoxic molecules from neu- 10, which also inhibited T cell responses. Cross trophils can trigger organ damage. Neutrophil talk with invariant NKT cells could counter products act as both targets and mediators of this response, restoring a proinflammatory autoimmunity. MPO and PR3 are the main tar- activation status (188). Thus, investigation of gets of antineutrophil cytoplasmic antibodies neutrophils in cancer has revealed considerable (ANCA), autoantibodies directed against anti- plasticity in their responses. Although little gens present in the cytoplasm of neutrophils. evidence currently supports the existence of Wegener’s granulomatosis is consistently as- different populations, it is likely that neutrophil sociated with the presence of ANCA. Further- responses are more flexible and less stereotyped more, the extent of organ damage in patients than previously thought. with Wegener’s granulomatosis correlates with Another major mechanism of tumor escape the PMN infiltrate rather than with traditional from immune control has recently been autoimmunity parameters such as T cell acti- attributed to a heterogeneous category of im- vation or autoantibody titers (196). Likewise, mature myeloid cells, called myeloid-derived ANCA bind MPO and PR3 expressed on the suppressor cells (MDSCs) (189). In healthy surface of activated neutrophils, promoting individuals, MDSCs are found in the bone degranulation and release of chemoattractants marrow, where they differentiate into mature and ROS, which together lead to a vicious neutrophils and monocytes. In cancer and cycle of tissue damage and inflammation. Early some autoimmune and infectious diseases, reports also suggest that, in an inflammatory en- differentiation is partially blocked, leading to vironment, ANCA accelerate ROS-dependent accumulation of these precursors, which act as neutrophil apoptosis, suggesting a feed-forward powerful suppressors of T cell functions. MD- cycle culminating in organ damage (194, 195). SCs have characteristics of neutrophils, and in SLE is another chronic autoimmune disease mice, they are typically detected using the neu- affecting multiple tissues and organs. Autoan- trophil surface markers CD11b+ and Gr-1+ , tibodies produced in SLE are predominantly although they consist of variable proportions either ANCA or directed against chromatin. www.annualreviews.org • Neutrophil Functions 479 Changes may still occur before final publication online and in print
  • 22. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 Although neutrophils had long been suspected to produce IFN-α, a central cytokine in SLE to be causative agents, their role in SLE patho- pathogenesis (103, 104). However, it remains to genesis remained elusive. The recent discovery be determined if DCs can present NET com- Vasculitis: inflammation of blood of a link between SLE and NET formation ponents or if they contribute to autoreactive B vessels has helped to shed light on this quandary. cell activation. It is also possible that NETs are It was proposed that TNF-α and IFN-α involved in other autoimmune diseases. Should prime cells for NET formation in response to this prove to be the case, understanding the anti-PR3, antiribonucleoprotein, anti-HNP, role of NETs may provide critical insights into or anti-LL-37 autoantibodies (103, 104, 106). the role of microbial infections as a trigger of Thus, high levels of inflammatory cytokines in autoimmunity. autoimmune patients are believed to sensitize neutrophils to NETosis, whereas autoantibod- ies may trigger a switch from apoptosis to NE- CONCLUDING REMARKS Tosis. Additional evidence suggesting a role for Neutrophils are specialized phagocytes thatAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.org NETs in autoimmune pathology was obtained arose as an evolutionary adaptation in verte-by Universidade Federal do Amazonas on 03/21/12. For personal use only. when NETs were identified in renal and/or brates to coordinate and execute one of the most skin biopsies from patients with SLE and small fundamental physiological responses: inflam- vessel vasculitis (103–106). Several studies have mation. They are endowed with antimicrobial reported the presence of a particular subset of mechanisms that make them the preeminent neutrophils in PBMC preparations from pedi- microbe exterminators of the immune system. atric and adult SLE patients. These low-density In addition to this important role, PMNs also granulocytes display phenotypic characteristics network with many other immune cells and of immature neutrophils with nonsegmented help regulate the initiation of specific T and nuclei and higher expression of MPO, NE, B cell immunity. However, neutrophils do not and defensin-3, and they may be related to the always act in ways beneficial to the host: Uncon- MDSCs discussed previously (see section on trolled neutrophil responses can exacerbate and Cancer, above) (197, 198). An increased capac- even cause autoimmune and inflammatory dis- ity to form NETs and a heightened cytotoxicity eases. Many challenges remain in understand- toward endothelial cells could bestow them ing neutrophil function: Is there specialization with pathogenic properties in lupus (105). among PMNs? Are they more plastic than we Because NETs appear to be formed during suspect? How do they make decisions before autoimmune disease, their timely removal may deploying their armamentaria? How do they be an essential mechanism for maintaining kill microbes? How specific are their instruc- tissue homeostasis. Human serum contains the tions to other cells? Answering these questions nuclease DNase I, which degrades NETs in will better define neutrophils’ role in defense vitro. Notably, a familial form of SLE is linked and disease and will provide a rational path for to a mutation in DNase I (199). Furthermore, pursuing new therapies. Moreover, neutrophils in a cohort of SLE patients, 36% exhibited can potentially provide insights into several either elevated titers of autoantibodies directed unique aspects of basic cell biology. Their strik- against NET components or inhibitors of ingly short life spans make them excellent mod- DNase I, both of which may protect NETs els for investigating cell death, whereas their from degradation. Most notably, impaired reliance on ROS as biochemical effectors may NET degradation correlates with development reveal novel ways for relaying intracellular of lupus nephritis, one of the most severe signals. The uniquely lobulated neutrophil manifestations of SLE (102). nucleus is a feat of higher-order nuclear Can it be that NETs play a general role architecture that is just beginning to yield in modulation of autoimmune responses? We its secrets. In short, exciting times await the know that NETs induce plasmacytoid DCs humble neutrophil. 480 Amulic et al. Changes may still occur before final publication online and in print
  • 23. IY30CH19-Zychlinsky ARI 27 December 2011 13:33 DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. ACKNOWLEDGMENTS We thank Diane Schad for assistance with graphic design and Cornelia Heinz for administrative help. G.H. is an Alexander von Humboldt Foundation Scholar, and B.A. is supported by an EMBO Long-Term Fellowship. LITERATURE CITED 1. Ehrlich P. 1880. Methodologische Beitr¨ ge zur Physiologie und Pathologie der verschiedenen Formen a der Leukocyten. Z. Klin. Med. 1:553–60 2. Waller A. 1846. Microscopic observation on the perforation of capillaries by the corpuscles of blood andAnnu. Rev. Immunol. 2012.30. Downloaded from www.annualreviews.orgby Universidade Federal do Amazonas on 03/21/12. For personal use only. the origin of mucus and pus globules. Lond. Edinburgh Philos. Mag. J. Sci. 2:271–80 3. Metchnikoff E. 1893. Lecon sur la pathologie comparee de inflammation. Ann. Inst. Pasteur 7:348–57 4. Nathan C. 2006. Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 6:173–82 5. Borregaard N. 2010. Neutrophils, from marrow to microbes. Immunity 33:657–70 6. Borregaard N, Cowland JB. 1997. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89:3503–21 7. Finger EB, Puri KD, Alon R, Lawrence MB, von Andrian UH, Springer TA. 1996. Adhesion through L-selectin requires a threshold hydrodynamic shear. Nature 379:266–69 8. Lawrence MB, Kansas GS, Kunkel EJ, Ley K. 1997. Threshold levels of fluid shear promote leukocyte adhesion through selectins (CD62L,P,E). J. Cell Biol. 136:717–27 9. Kansas GS. 1996. Selectins and their ligands: current concepts and controversies. Blood 88:3259–87 10. McEver RP, Cummings RD. 1997. Role of PSGL-1 binding to selectins in leukocyte recruitment. J. Clin. Investig. 100:S97–103 11. Yago T, Shao B, Miner JJ, Yao L, Klopocki AG, et al. 2010. E-selectin engages PSGL-1 and CD44 through a common signaling pathway to induce integrin αLβ2-mediated slow leukocyte rolling. Blood 116:485–94 12. Mueller H, Stadtmann A, Van Aken H, Hirsch E, Wang D, et al. 2010. Tyrosine kinase Btk regulates E-selectin-mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) γ2 and PI3Kγ pathways. Blood 115:3118–27 13. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. 2007. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7:678–89 14. Campbell JJ, Hedrick J, Zlotnik A, Siani MA, Thompson DA, Butcher EC. 1998. Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279:381–84 15. Constantin G, Majeed M, Giagulli C, Piccio L, Kim JY, et al. 2000. Chemokines trigger immediate β2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow. Immunity 13:759–69 16. Liu S, Kiosses WB, Rose DM, Slepak M, Salgia R, et al. 2002. A fragment of paxillin binds the α4 integrin cytoplasmic domain (tail) and selectively inhibits α4-mediated cell migration. J. Biol. Chem. 277:20887–94 17. Goldfinger LE, Han J, Kiosses WB, Howe AK, Ginsberg MH. 2003. Spatial restriction of α4 inte- grin phosphorylation regulates lamellipodial stability and α4β1-dependent cell migration. J. Cell Biol. 162:731–41 18. Xu J, Wang F, Van Keymeulen A, Herzmark P, Straight A, et al. 2003. Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell 114:201–14 19. Schenkel AR, Chew TW, Muller WA. 2004. Platelet endothelial cell adhesion molecule deficiency or blockade significantly reduces leukocyte emigration in a majority of mouse strains. J. Immunol. 173:6403– 8 www.annualreviews.org • Neutrophil Functions 481 Changes may still occur before final publication online and in print
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