Ann. N.Y. Acad. Sci. ISSN 0077-8923

A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: The Year in Immunology

Regulation of immune responses by neutrophils Jing Wang1,2,3 and Hisashi Arase3,4,5 1 Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada. 2 Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada. 3 Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. 4 Laboratory of Immunochemistry, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan. 5 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama, Japan

Address for correspondence: Jing Wang, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada; and Hisashi Arase, Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, 3-1 YamadaOka, Suita, 565-0871 Osaka, Japan. [email protected]; [email protected]

Neutrophils, the most abundant circulating cells in humans, are major pathogen-killing immune cells. For many years, these cells were considered to be simple killers at the “bottom” of immune responses. However, recent studies have revealed more sophisticated mechanisms associated with neutrophilic cytotoxic functions, and neutrophils have been shown to contribute to various infectious and inflammatory diseases. In this review, we discuss the key features of neutrophils during inflammatory responses, from their release from the bone marrow to their death in inflammatory loci. We also discuss the expanding roles of neutrophils that have been identified in the context of several inflammatory diseases. We further focus on the mechanisms that regulate neutrophil recruitment to inflamed tissues and neutrophil cytotoxic activities against both pathogens and host tissues. Keywords: neutrophils; inflammation; recruitment; chronic diseases

Introduction As the most abundant circulating white blood cells in humans, neutrophils provide the first line of host defense against a wide range of infectious pathogens. For many years, neutrophils were considered as simple nonspecific pathogen killers.1 Despite their discovery more than a century ago, functional studies of neutrophils have encountered many difficulties associated with the handling of these cells. Although neutrophils can be easily isolated and enriched from blood and other tissues, the short lives of neutrophils make them difficult to study in cultures. Isolated neutrophils are viable and easily activated, and neutrophil-like cell lines barely reflect the functional diversity of neutrophils.2 Therefore, in vitro studies using these cells should be carefully interpreted. In addition, in vitro studies can never reflect the complex biological realities and complexities in tissues or circulation. In the last two decades, emerging technologies such as intravital microscopy have enabled researchers to perform real-time analyses of neutrophil dynamics in vivo.3 Since then,

novel functions of neutrophils have been studied intensively. However, in vivo studies of neutrophil function may also feature some problems. For example, it remains difficult to label neutrophils, specifically in vivo. The LysM-GFP mice, which are widely used to study neutrophils in vivo, express GFP in both monocytes and neutrophils; therefore, neutrophil-specific antibodies must be used to detect neutrophils even though they may exert some effects on neutrophil function.4–6 In addition, a recent paper also raised concerns about in vivo studies of neutrophil function in mice because mouse models differ from human systems in certain important aspects.7 Although not restricted to neutrophils, the study demonstrated a low correlation between the changes in gene expression in humans and mice by comparing the gene response patterns associated with serious human injuries to those in the corresponding mouse models. In fact, there are differences in the biology of neutrophils between mouse and human. For example, human blood contains 50–70% neutrophils, whereas mouse blood contains 10–25% neutrophils.

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Human and mouse neutrophils express different surface and intracellular molecules that are important for immune responses.8,9 Despite the difficulties associated with neutrophil functional studies in vivo and in vitro, comprehensive immune responses cannot be understood without an understanding of the role of neutrophils, as these cells appear in all scenarios of inflammatory responses, from innate to adaptive immune responses. It turns out that the neutrophils are more sophisticated than previously expected. These cells are not only simple suicide killers but provide protection against intracellular bacteria or viruses.10–13 There are different subsets of neutrophils with functional heterogeneity.14 In addition to antimicrobial agents, neutrophils produce numerous cytokines, chemokines, and growth factors.15 Neutrophils can direct adaptive immune responses by communicating with various other cell types, including B cells, dendritic cells, and T cells.16–22 Neutrophils may act as decision-makers in immune responses through their unique capacity to both help and destroy host tissues in the body. During the last decade, a tremendous amount of data was generated regarding neutrophils. In this review, we will focus on the most crucial aspects and molecular mechanisms of neutrophil functions in immune responses (Fig. 1). Because detailed discussions on neutrophils in adaptive immunity and interaction between neutrophils and other types of immune cells in adaptive immune responses can be found elsewhere (e.g., Refs. 15 and 23), this review will mainly focus on neutrophils in innate immune responses. The beginnings of neutrophils In steady state, neutrophils are generated in the bone marrow at a rate of 109 cells per kilogram of body weight per day.24 Under the instruction of growth factors and cytokines, hematopoietic cells can differentiate into myeloblasts, promyelocytes, myelocytes, metamyelocytes, band cells, and finally, granulocytes.25 During neutrophil maturation, several proteins are synthesized and sorted into different granules, including azurophilic, specific, and gelatinase granules. Azurophilic granules contain myeloperoxidase (MPO), an enzyme critical to the oxidative burst.26,27 Specific granules contain lactoferrin and are formed after the generation of azurophilic granules. Gelatinase granules 2

contain few antimicrobial agents and instead store numerous metalloproteases, such as gelatinase and leukolysin. These granules are not only repository organelles for dangerous substances but also indispensable for nearly all neutrophilic activities during inflammation.28 Unlike macrophages or other myeloid cells, neutrophils are terminally differentiated before being released into the bloodstream, and they exhibit a short life span of approximately 6–8 h in circulation.29 In the absence of inflammatory signals, neutrophils die by spontaneous apoptosis, with the clearance of dead neutrophils occurring in the liver, spleen, and bone marrow.30 However, several recent studies have challenged some of the above ideas about neutrophils. For example, two studies demonstrated that neutrophils recruited into inflammatory lesions can further differentiate into neutrophils-dendritic cells hybrids. The hybrid cells exhibit several properties of both neutrophils and dendritic cells.31,32 A recent study that incorporated a novel labeling method suggested that the life spans of neutrophils may be as long as 5 d in humans and 90 h in mice under homeostatic conditions.33 While these findings hold tremendous interest, the validity of the labeling method and mathematical modeling used in the study remain controversial.34,35 It is unclear why neutrophils have such a short life span and require daily clearance. An interesting study suggested that the daily cycle of neutrophil elimination triggers signals that modulate the hematopoietic niche and promotes subsequent cycles of hematopoietic stem/progenitor cell release.36 Neutrophil turnover can be delayed or accelerated during inflammatory responses, indicating that a longer or shorter neutrophil life span may be important to determine whether an inflammatory response will be extended or terminated.37,38 Neutrophil retention and release from the bone marrow is tightly regulated by the balance between the levels of two chemokine receptors that are expressed on neutrophils, CXCR4 and CXCR2, and their respective ligands, stromal derived factor 1 (SDF-1) and KC (or Gro␤).39 CXCR4 is crucial for neutrophil maintenance in the bone marrow; CXCR4 deletion causes a shift in the pool of mature neutrophils from the bone marrow to the circulation. SDF-1, which is expressed by bone marrow endothelial cells and osteoblasts, binds to CXCR4, and the CXCR4/SDF-1 signal axis is necessary to maintain a neutrophil bone marrow

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Figure 1. Neutrophil dynamics: from inflammation to homeostasis. Neutrophil retention and release in the bone marrow is determined by the balance between CXCR4 (retention) and CXCR2 (release). During an inflammatory response, circulating neutrophils in the blood are recruited to the site of inflammation through sequential steps. After migrating to the site of inflammation, neutrophils phagocytose and digest microorganisms or cell debris and release antimicrobial agents through degranulation. Next, neutrophils undergo either apoptosis or NETosis. The uptake of apoptotic neutrophils by macrophages drives production of the anti-inflammatory cytokines tumor growth factor (TGF)-␤, IL-10, and prostaglandin-E2 (PGE-2).

pool that is ready for immediate release.40,41 Transient blockade of CXCR4 signaling induces neutrophilia, which is thought to result from increased neutrophil migration from the BM. However, a recent study has challenged this idea.42 In this elegant study using intravital multiphoton microscopy, inhibition of CXCR4 signaling by a small antagonist increased peripheral neutrophils that are independent of mobilization from bone marrow. Instead, the release of neutrophils from the marginated pool in the lung had a significant impact on neutrophilia induced by CXCR4 inhibition, suggesting the presence of alternative reservoirs for circulating neutrophils. Signaling through CXCR2 affects neutrophil release. CXCR2 depletion causes a myelokathexis phenotype, in which mature neutrophils are retained in the bone marrow. Depletion of both CXCR2 and CXCR4 leads to constitutive mobilization of neutrophils, suggesting a dominant role for CXCR4 in neutrophil trafficking.39 Neutrophil release can also be stimulated by G-CSF, which has been shown to tilt the CXCR4 and CXCR2 signaling balance in favor of CXCR2 by downreg-

ulating SDF-1 expression and upregulating CXCL1 and CXCL2 expression in bone marrow endothelial cells.43 Neutrophil recruitment At sites of tissue inflammation or infection, circulating neutrophils are trapped by activated endothelial cells and further activated during passage into the tissues. Neutrophil exit from the blood is a sequential process, also referred to as the neutrophil recruitment cascade, and includes the steps of tethering, rolling, adhesion, crawling, and transmigration.44–46 During the initial stage of an inflammatory response, pathogen or host-derived inflammatory signals stimulate endothelial cells near inflammatory sites and promote the expression of adhesion molecules such as P and E selectins. Neutrophils constitutively express glycosylated ligands for these selectins such as the P-selectin glycoprotein ligand 1 (PSGL-1). These molecules engage P and E selectins on endothelial cells, leading to the tethering of circulating neutrophils to the vessel wall. Tethering is followed by the rolling of neutrophils along the

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vessel in the direction of blood flow. Neutrophils are unique in their ability to roll along the endothelium under shear stress of 1–10 dyn/cm2 , whereas most leukocytes roll along postcapillary venules at shear stress of 0–3 dyn/cm2 .47 Neutrophils display resistance to high shear forces by flattening out over the endothelium in order to engage higher numbers of adhesive molecules and to form membrane tethers that can extend for several cell diameters behind the rolling neutrophil to reduce speed. Shear-resistant rolling also involves the formation of long membrane tethers at the rear of the cell that shift to the front of the rolling cell and facilitate resistance to high fluid pressures. When neutrophil rolls forward, these tethers, or slings, wrap around the rolling cells, and the patchy PSGL-1 distribution along each sling provides a unique adhesive substrate.48 The engagement of PSGL-1 on neutrophils also activates various kinases, including the Src family kinases, Syk, phosphoinositide 3-kinase (PI3K), and p38 mitogen-activated protein kinase, which set the stage for integrin activation and firm adhesion.49,50 Rolling neutrophils also evaluate the local microenviroment for inflammatory mediators such as chemokines or chemoattractants.51 As neutrophils roll along the vessel wall, interactions with these molecules lead to conformational changes (insideout activation) and ␤2-integrin clustering.52 Conformational changes in ␤2-integrins expose their binding domain to their ligand intracellular adhesion molecule 1 (ICAM-1), which is expressed on the inflamed endothelium. Integrin clustering also increases the affinity of these molecules for ICAM1.53 Integrin activation and clustering enable neutrophils to stop rolling and promote firm adhesion. This inside-out integrin activation is known to be mediated by intracellular molecules such as kindlin-3, Talin, and guanine exchange factors for small GTPases.54 Patients who are deficient in these molecules are known to have congenial leukocyte adhesion deficiencies, which are characterized by life-threatening, recurrent bacterial infections.55 However, excessive integrin activation can also lead to unexpected cell adhesion and aggregation that may have profound pathological consequences. Several studies have identified endogenous inhibitors that counteract ␤2-integrin activation, including growth differentiation factor 15 (GDF-15) and developmentally regulated endothelial cell locus 1 protein (DEL-1).56–58 A study published by our 4

group also identified a key transmembrane receptor, the paired immunoglobulin-like type 2 receptor ␣ (PILR␣), on neutrophils that can modulate integrin inside-out activation and protect against excessive activation.59 Pentraxin 3 (PTX3), which is stored in neutrophil-specific granules and is released after neutrophil degranulation, can block neutrophil recruitment by competitive binding to P-selectin in vivo.60 After adhesion to the endothelium, neutrophils must navigate a path through the endothelium into the underlying tissue. Neutrophils can migrate through the endothelial cell barrier by two pathways, the paracellular (between endothelial cell junctions) and transcellular (through endothelial cells) pathways.61 Both routes depend on hemophilic interactions between adhesion molecules such as PECAM-1 and CD99, which are expressed on both the endothelium and leukocytes.62–64 Other molecules, such as junctional adhesion molecules (JAM-A, JAM-B, and JAM-C), also play essential roles in transmigration through interactions with the integrin Mac-1.65 Once through the endothelium, neutrophils must cross the basement membrane, a protein mesh composed of laminins and collagens. The preferred neutrophil transmigration route is dictated by the basement membrane composition, thus allowing an easier exit from the vessel. Within the interstitial space, neutrophils migrate toward the site of inflammation. Recent studies have shown that venular pericytes, which lie between endothelial cells and the basement membrane and ensheath the vessel itself, can support neutrophil crawling during their egress from the vessel lumen.66 NG2-expressing arteriolar and capillary pericytes attract and interact with neutrophils after their extravasation from postcapillary venules by instructing neutrophils with pattern recognition and motility programs.67 The above information describes the general cascade of neutrophil recruitment in an inflammatory setting. This cascade is based on early observations of cultured endothelial cells, as well as numerous organs, including the mesentery, peritoneum, and skeletal muscles, in vivo. The development of intravital imaging techniques to visualize more organs and deeper tissues has facilitated our understanding that neutrophil recruitment mechanisms vary substantially in different organs, such as the lung, liver, and kidney.68 These

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tissue-specific neutrophil recruitment mechanisms can be observed because of the presence of unique cell populations and structural organization in each organ. For example, in the liver, neutrophil recruitment into the sinusoidal capillaries does not require the rolling step; instead, neutrophils adhere directly to the hepatic endothelium.69 The integrin–ICAM-1 interaction is required for neutrophil adherence to sites of sterile inflammation, whereas in the infected liver, integrin-independent interactions occur between endothelial cell-expressed hyaluronan and neutrophil-expressed CD44.70 The lung is another exception to the usual neutrophil recruitment cascade. Neutrophil extravasation occurs mainly in the small capillaries surrounding the alveoli but not in postcapillary venules. It has been speculated that neutrophils must alter their shape in order to pass through the thin vasculature in the lung.71 Recent studies that incorporated multiphoton imaging of the intact kidney suggested that both neutrophils and monocytes constitutively patrol the capillaries of the glomeruli.72 In antibody-induced inflammation, these cells exhibit Mac-1–dependent activation, an increased dwelling time, and increased ROS generation.72,73 These studies demonstrate the ability of state-of-the-art confocal multiphotonfluorescence microscopy to provide new insights into neutrophil recruitment under different inflammatory states. Neutrophil activation in sites of inflammation Once neutrophils pass through the endothelial barrier, they move into a very different environment that contains abundant chemoattractants and inflammatory stimulants, which will act as the primary directors of neutrophil behavior. As neutrophils follow chemotactic gradients in the interstitial space, the chemoattractants bind to their respective neutrophil receptors, thus activating downstream molecules and promoting the oxidative burst. The oxidative burst can also be induced by direct microbial activation through patternrecognition receptors, such as TLR4, TLR2, and TLR9.74–76 Neutrophils can kill pathogens through several different mechanisms, including phagocytosis, the production of antimicrobial peptides and proteins, and the release of ROS during a respiratory burst.28 It is worth mentioning that while many of the an-

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timicrobial functions of neutrophils were identified through in vitro studies, it remains challenging to demonstrate in vivo relevance. A detailed discussion on microbial elimination by neutrophils can be found in References 1 and 77. In addition to possessing antimicrobial weapons, neutrophils can also to expel their nuclear contents (DNA and histone) in a complex with antimicrobial granules and cytoplasmic proteins into the extracellular space.78 These web-like structures, or neutrophil extracellular traps (NETs), can trap different types of microbes, including viruses; the process of NET formation is termed NETosis. DNase-mediated perturbation of NETs increases susceptibility to infections, suggesting that NETosis is an active process in neutrophil host defense.79 Neutrophils undergoing NETosis are distinguished from those undergoing apoptosis by the lack of “eat me” signals on the cell surfaces.80 NETs are cleared mainly by nucleases derived from either the host or the pathogen. Interestingly, a recent study showed that Staphylococcus aureus could generate deoxyadenosine from NETs by using two of its enzymes, nuclease and adenosine synthase. Deoxyadenosine can trigger the caspase3–mediated apoptosis of immune cells, specifically macrophages in this case.81 Therefore, S. aureus has evolved to escape host defense by reusing NETs to destroy the immune system. That study also suggested that the existence of NETs presents an evolutionary pressure on bacteria to avoid killing. Various inflammatory stimuli can induce NET formation, including cytokines, chemokines, and PAMPs. The mechanism of NET formation is not completely understood and differs according to the stimulus. NADPH oxidase and MPO are required for NET formation in response to chemical and biological stimuli.82,83 Neutrophils from patients with chronic granulomatous disease (CGD), which is characterized by mutations that render NADPH oxidase nonfunctional, cannot form NETs.84 However, NET formation can also occur independently of oxidase activity and MPO is not required for bacterial induction of NETs.85 Gene knockout mice lacking peptidylarginine deiminase 4 (PAD4), an enzyme required for histone deamination, exhibit impaired NETosis, suggesting that chromatin disassembly allows full NET dispersion.86 In addition, emerging evidence has shown that platelets play important roles in inducing NET formation during infection and injury.87–89

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Similar to other neutrophilic antimicrobial agents, NETs can also exert detrimental effects against the host.90 Excessive NET formation has been linked to various neutrophil-mediated pathologies, including autoimmune diseases, such as systemic lupus erythematous, sepsis, vasculitis, and thrombosis.91–94 In vivo studies using imaging techniques also demonstrate NET formation and the relevance of NETs in several pathological situations.12,88,95 Inflammation that occurs in the absence of microorganisms is termed sterile inflammation, and sterile inflammation is also marked by neutrophil recruitment.96 In contrast to an important role in killing pathogens, neutrophils appear to be more harmful during sterile inflammation. Even small amounts of dead cells trigger a huge recruitment of neutrophils, and neutrophils are redundant to cell types such as monocytes and macrophages. 97,98 On the other hand, the production of ROS, proteases, and growth factors by neutrophils results in tissue destruction, as well as fibroblast proliferation, aberrant collagen accumulation, and fibrosis. Neutrophil activation during sterile inflammation is involved in many physiological and pathological processes, such as autoimmune diseases, cancer, and atherosclerosis, and will be later described in detail. Although neutrophils are generally considered to be more detrimental in sterile inflammation, a recent study identified a proangiogenic subset of neutrophils that are recruited to transplanted pancreatic islets and are required for islet revascularizaton. This study suggested that neutrophils themselves may also have the potential to promote tissue and wound repair.99 Neutrophil clearance Regardless of whether inflammation is microbial or sterile, the lethal cargo carried by neutrophils is not only destructive against invading microbes or dead cells, but is also harmful to host tissues. Therefore, the disposal of recruited neutrophils at inflammatory sites is an important step in the resolution of inflammation.100 Since Metchnikoff discovered that neutrophils were ingested by tissue macrophages more than 100 years ago, it has been believed that the active clearance of inflammatory infiltrates by phagocytes, such as macrophages and monocytes, is responsible for neutrophil clearance and promotion of a return to homeostasis.101 6

Neutrophils that have accumulated at inflammatory sites can undergo apoptosis and express “find-me” signals to attract phagocytes such as monocytes and macrophages. Recently, several find-me signals expressed by apoptotic cells have been identified, including the S19 ribosomal protein dimer, split tyrosyl-tRNA synthetase, thrombospondin 1, lysophosphatidylcholine, and the nucleotides ATP and UTP.102–104 The roles of such find-me signals in orchestrating neutrophil removal have not been thoroughly investigated, and it may be argued that monocytes and macrophages are recruited to inflammation sites independent of these signals. Apoptotic neutrophils must be ingested rapidly before they progress to necrosis.37 Inflammation can thus be prolonged by the failure of neutrophils to undergo timely apoptosis or the failure of macrophages to clear apoptotic neutrophils. Therefore, neutrophil elimination is an active process that prevents further leukocyte infiltration and removes apoptotic cells from inflamed tissues. After accomplishing their primary mission in the inflamed sites, neutrophils undergo apoptosis. Although neutrophils possess a built-in celldeath program and can die spontaneously, survival signals such as IL-1␤, G-CSF, and GM-CSF, which are released by macrophages, can delay neutrophil apoptosis.105 Interestingly, hypoxia inducible factor-2␣ (HIF-2␣), whose expression is upregulated in neutrophils during acute and chronic inflammation, reduces neutrophil apoptosis. HIF2␣–deficient animals show increased neutrophils apoptosis in vivo and reduced tissue injury.106 On the other hand, macrophages can also produce TNF-␣, which can promote neutrophil apoptosis at high concentrations and thus promote the resolution of inflammation.107 Apoptotic neutrophils can also produce signals to prevent further neutrophil recruitment. Lactoferrin and annexin A1, which are released from apoptotic cells, are two negative regulators of neutrophil recruitment.108,109 Equally important regulators of resolution of inflammation include signals that promote the uptake and clearance of apoptotic cells. Lipid mediators play a crucial role in this process. During the initial inflammatory response, proinflammatory lipid mediators, such as prostaglandins and leukotrienes, which can amplify inflammation, are generated by various cell types, including endothelial cells, neutrophils, monocytes, and macrophages.110 Prostaglandins

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and leukotrienes are derived from arachidonate precursors and synthesized by the cyclooxygenase and lipoxygenase pathways.111 During the later stage of inflammation, neutrophils can interact with various cell types in their vicinities, including epithelial cells, endothelial cells, fibroblasts, and platelets. Following these interactions, neutrophils can produce lipid mediators with anti-inflammatory and proresolution activities, such as lipoxins, resolvins, and protectins. Macrophages that ingest apoptotic neutrophils can also produce lipoxin. Lipoxin A4 inhibits neutrophil recruitment by inhibiting L-selectin shedding and ␤2-integrin upregulation in response to proinflammatory stimulation, thereby reducing the firm adhesion of neutrophils to endothelial cells.112 Lipoxin can also promote monocyte migration by inducing intracellular Ca2+ mobilization in these cells and, consequently, promoting chemotaxis. Lipoxins also impact neutrophil activation by inhibiting ROS and IL-8 production.113 Resolvins are another class of lipid mediators induced during the resolution stage of inflammation. Resolvins are synthesized from the omega-3 fatty acids eicosapentaenoic acid (E-resolvin) and docosahexaenoic acid (D-resolvin).114 E-resolvins are produced by neutrophil-derived 5-lipoxygenase, following the conversion of lipid precursors by endothelial cells or bacteria. E-resolvins can attenuate TNF-␣–mediated NF-␬B activation in monocytes, macrophages, and DCs, thereby inducing an antiinflammatory signaling pathway.115 Resolvins can also increase CC-chemokine receptor 5 (CCR5) expression on apoptotic neutrophils, leading to the clearance of CCR5-binding chemokines such as CCL3 and CCL5 and preventing further neutrophil influx.116 In a mouse model of aspiration pneumonia, resolvin E1 treatment reduced neutrophil accumulation in the lung by 55% and the levels of cytokines and chemokines.117 The phagocytosis of apoptotic neutrophils by macrophages triggers production of the antiinflammatory cytokines TGF-␤ and IL-10.118 TGF-␤ is a critical tissue repair mediator that induces mesenchymal cells to express NADPH oxidase 4 (NOX4), which mediates myofibroblast differentiation and extracellular matrix production and promotes tissue homeostasis.119 Although resolution of inflammation is an active process requiring the coordinated regulation of different cell types and mediators, it is certain that neutrophil apoptosis occupies

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a central position during the resolution response. Therefore, the timely induction of neutrophil apoptosis may be an interesting target for terminating inflammation. Neutrophils in diseases Previously, intensive studies of neutrophils have focused on their prominent roles in innate immune responses against pathogens. However, recent studies have suggested that neutrophils are also major contributors to the progression of many diseases, such as autoimmune disorders, cancer, and atherosclerosis. Here, we provide examples of how neutrophils are involved in these diseases (Fig. 2).

Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) is a major cause of death in industrialized countries and is most commonly caused by smoking. Chronic neutrophilic infiltration in the lungs of COPD patients promotes tissue damage and organ dysfunction. Epithelial cells are key regulators of neutrophil trafficking into the airways, neutrophils being attracted by chemotactic factors such as IL-8 and leukotriene B4 (LTB4). Leukotriene B4 is converted from leukotriene A4 by leukotriene A4 hydrolase (LTA4H). LTA4H is also an aminopeptidase that inactivates a specific neutrophil chemoattractant, the proline-glycineproline tripeptide (PGP), thus conferring antiinflammatory properties upon the enzyme. Interestingly, cigarette smoke selectively inhibits the aminopeptidase activity of LTA4H, thereby promoting the accumulation of both leukotriene B4 and PGP. This subsequently promotes neutrophil recruitment and fuels chronic lung inflammation. Neutrophil removal by the mucociliary escalator results in the release of proteases and proinflammatory mediators. Increased levels of GM-CSF and LTB4 delay neutrophil apoptosis and lead to the retention of neutrophils, further increasing inflammation.120 Autoimmune disease Rheumatoid arthritis (RA) is a prototypical autoimmune disease that affects approximately 1% of the human population.121 RA is characterized by chronic autoimmune polyarthritis with synovial hyperplasia and joint destruction, which leads to pain, loss of joint function, and concomitant reductions in the quality of life. Neutrophils are by far the most abundant immune cells present in

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Figure 2. Neutrophils in various diseases. Neutrophils play a significant role in both initiating and shaping the immune response during inflammatory diseases and cancer. Dashed lines indicate signals that recruit neutrophils; solid lines indicate effectors that are released by neutrophils. Detailed information is discussed in the text.

synovial fluid from the joints of RA patients and are also abundant at the pannus/cartilage interface, the site of active tissue damage.122 Neutrophils isolated from RA joints exhibit an activated (primed) phenotype, indicating that the cells have undergone an activation process and likely have released important inflammatory mediators. After entering the joint tissues, neutrophils can be activated by immunoglobulin aggregates (rheumatoid factor, RF) that can engage Fc␥ receptors to trigger degranulation and ROS production.123,124 Oxygen radicals induce DNA damage and may induce mutations to immunoglobulins that lead to the formation of RF.125,126 Granules released from neutrophils are implicated in the damage to the host tissues, particularly the destruction of the collagen matrix within cartilage.127 The availability of several animal models of inflammatory arthritis provides the opportunity to 8

study the role of neutrophils in vivo. Serum transfer from K/B×N mice can induce arthritis in other mice and initiate rapid infiltration of neutrophils into the synovial cavity and the onset of arthritis. Neutrophil depletion completely protects mice from K/B×N serum transfer arthritis.128 In Gfi-deficient mice, which exhibit impaired neutrophil development, K/B×N serum transfer also failed to induce arthritis development in mice.129 In the K/B×N serum-transfer mouse model, neutrophil-derived lipid chemoattractant LTB4 has been identified as a key mediator of the inflammatory response. LTB4, together with cytokine IL-1, CCR1 and CXCR2 chemokine ligands, drives neutrophil recruitment into the joint.130 Blocking of the LTB4 receptor BLT1 has been shown to reverse K/B×N seruminduced disease.130,131 Other molecules such as Vav or phospholipase C␥ , which are associated with downstream integrin and Fc␥ receptor signaling,

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have also been shown to contribute to neutrophilmediated arthritis.132 Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is a group of diseases characterized by the systemic chronic inflammation of small blood vessels; these diseases occur at a frequency of 1 per 50,000 people.133 ANCA activates primed neutrophils by binding to certain antigens expressed on the neutrophil surface in specific inflammatory microenvironments.134 ANCAs act directly against cytoplasmic neutrophil products such as MPO, proteinase 3 (PR3), and Lamp-2 (lysosome-associated membrane protein 2). However, the mechanism by which neutrophil granular proteins become antigenic remains unclear.135 A previous study showed that a LAMP-2 epitope recognized by ANCA shares 100% homology with the bacterial adhesion molecule FimH, suggesting that autoantibody production may result from cross-reactivity triggered by infectious agents.136 ANCAs can induce human PMNs to produce superoxide, release intracellular granules, and secrete proinflammatory cytokines in vitro. The ANCA-activated neutrophils can also activate alternative complement pathway and the generation of chemoattractant C5a, further amplifying the recruitment of neutrophils.137,138 The role of neutrophils in disease pathogenesis was indicated by neutrophil depletion studies in which neutrophil-depleted mice were protected from anti-MPO IgG–induced ANCA, thus supporting the conclusion that neutrophils are the key effector cells in vasculitis.139 In addition, it was proposed that NETs may participate in vasculitis pathogenesis, as NETs are produced by ANCA-stimulated neutrophils and have been identified in glomeruli and the interstitium of kidney biopsies from patients with vasculitis;139 these findings suggest that NETs may be involved in glomerular capillary damage,93 a cytotoxic effect that could be due to the presence of histones in NETs.140 Neutrophil activation can be induced by both the Fab and Fc portions of ANCA, leading to degranulation, cytokine release, and ROS production. Interestingly, human neutrophils exhibit variable expression of CD177, a glycosylphosphatidylinositol-anchored surface receptor. CD177 associates with the ANCA antigen PR3 and anchors PR3 to the surfaces of CD177+ neutrophils. Therefore, only CD177expressing neutrophils are activated by ANCA, and

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the proportion of this neutrophil subset was found to be increased in Wegener’s granulomatosis.141 In addition, neutrophils are also implicated in disease pathogenesis of systemic lupus erythematosus (SLE).91,92 Two recent studies reported existence of autoantibodies in SLE patients that are against selfDNA and antimicrobial peptides in NETs. NETs can also activate plasmacytoid DCs to produce type I interferon in a TLR9-dependent way, therefore providing a new therapeutic target for the treatment of SLE.

Cancer Neutrophils comprise a significant portion of the inflammatory cell infiltrates observed in a wide variety of murine models and human cancers.142 Neutrophil recruitment to sites of tumorigenesis is mediated by chemotactic factors secreted by many cell types, including tumor cells. Studies by Sparmann and Houghton showed that tumor cells could release the chemokines IL-8, KC (IL-8 homologue in mouse), and MIP-2.143,144 Some studies have suggested that neutrophils attracted by tumors may elicit protumor responses. For instance, GM-CSF, when produced by breast cancer cells, has been shown to induce the production of oncostatinM, an IL-6–like cytokine by neutrophils.145 This IL-6–like cytokine can subsequently stimulate VEGF production by cancer cells and promote invasiveness. Elastase is another effector protein secreted by neutrophils. In lung tumors, elastase enters tumor cells through clathrin-coated pits and is trafficked through the early endosomes. IRS-1, an adaptor protein known to bind the p85 subunit of PI3K, is a substrate for elastase. Elastase-mediated IRS-1 degradation was shown to increase the interaction between PI3K and the potent mitogen platelet–derived growth factor receptor (PDGFR), thereby skewing the PI3K signaling axis toward tumor proliferation.143 NETs derived from neutrophils during sepsis have also been shown to trap circulating tumor cells and promote the adhesion of tumor cells to distant organ sites.95 Increasing clinical evidence also indicates that the presence of neutrophils in tumor is associated with poor prognosis.146–148 Although many studies have supported a protumor role of neutrophils, several early studies also claimed that under certain circumstances, neutrophils within the tumor microenvironment could be induced to target their

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cytotoxic arsenals against tumor cells. The inhibition of TGF-␤ signaling led to the generation of “antitumor” neutrophils that could produce high levels of proinflammatory cytokines, which are cytotoxic to tumor cells. These studies suggested that neutrophils in cancer settings exhibit considerable plasticity in their responses, and that the antitumor/protumor paradigms that exist for macrophage (M1/M2) polarization may also exist for neutrophils.149 However, these studies are reported only in the mouse. Evidence for neutrophil plasticity in human remains to be obtained.

Atherosclerosis Cardiovascular diseases are among the most prevalent human diseases and the leading causes of death in developed countries; atherosclerosis is the primary cause of mortality and morbidity consequent to cardiovascular disease.150 Because atherosclerotic lesions are rarely detected, the role of neutrophils in the pathophysiology of atherosclerosis has been long-neglected. However, several recent studies have suggested that neutrophils are crucial players in atherogenesis.151,152 It has long been appreciated that atherosclerosis results from chronic inflammation of the vascular wall. In previous years, use of refined staining techniques has allowed the sensitive and specific detection of neutrophils present in unique areas of atherosclerotic plaques in both humans and mice.153,154 Also, the presence of neutrophils has been correlated with both the incidence and severity of coronary artery disease.155 In early murine lesions, neutrophils have been identified in subendothelial and intimal locations, whereas in more advanced plaques, neutrophils have been found within the plaque shoulder and in the adventitia.151 Importantly, neutrophil depletion strongly reduced the atherosclerotic lesion areas in apolipoprotein E– deficient (Apoe−/− ) mice fed a high-fat diet, suggesting an important role for neutrophils in atherosclerotic plaque formation.156 A recent study provided additional support for the role of neutrophils in atherosclerosis by showing that a genetic deficiency in CRAMP, a member of the cathelicidin family of antimicrobial peptides that is expressed in neutrophils, led to reduced atherosclerotic lesion sizes in Apoe−/− mice fed a high-fat diet.157 CRAMP and the homologous human cathelicidin peptide LL37 (the

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C-terminal portion of its precursor hCAP-18) promoted monocyte recruitment and macrophage accumulation in early atherosclerotic lesions. CRAMP is deposited in activated neutrophils located on inflamed endothelium in large arteries, but not in monocytes or macrophages. Neutrophil depletion was shown to specifically decrease classical monocyte recruitment in mice, which could be rescued by LL-37 and azurocidin. Neutrophil recruitment to the inflamed mouse arterial wall is mediated by the CCL5 receptors CCR1 and CCR5, in comparison to the restricted use of CCR2 and CXCR2 for peripheral venous recruitment.158 CCL5 release and deposition appear to depend on Pselectin expression on platelets. Platelets were previously suggested to participate in atherosclerotic plaques, and platelet-derived CCL5 was shown to promote monocyte adhesion to the luminal surfaces of early atherosclerotic lesions in the inflamed arterial walls of mice.159,160 Aspirin-mediated platelet inhibition has proven to be an enduring pillar of secondary prevention in patients with acute coronary syndrome.161 Mice lacking P-selectin, CD18, or ICAM-1 were previously shown to exhibit reduced atherosclerotic plaque formation.162–164 In the vascular wall, oxidized low-density lipoprotein (LDL) can induce neutrophil transmigration and degranulation, which triggers further intimal recruitment of classical monocytes by the cathelicidin-induced activation of formyl-peptide receptors. These monocytes differentiate into macrophages and internalize native and modified LDL, leading to foam cell formation. Collectively, atherosclerotic plaque formation likely involves the sequential recruitment of platelets, neutrophils, and monocytes.152 The aforementioned studies suggest potentially important roles for neutrophils in atherosclerosis and related vascular diseases. However, additional work will be required to investigate the exact spatiotemporal control mechanisms of neutrophils that participate in plaque formation. For example, are neutrophils activated inside or outside of the vasculature? Also, how do intravascular events promote plaque formation below the endothelium? However, intravital imaging around the heart is extremely difficult, compared to that of other organs. A recent study of leukocyte imaging in a beating heart may provide a novel technique for studying atherosclerotic plaque behavior.165

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Regulation of immune responses by neutrophils

Table 1. Potential therapeutic neutrophil targets

Target

Function

Associated diseases

Elastase

Degradation of ECM (extracellular matrix) components Bactericidal properties Cleavage of inflammatory mediators Cleavage of receptors Cytokine and chemokine induction Induction of airway submucosal gland secretion

Papillon–Lef`evre syndrome166 Hereditary neutropenia167 Atherosclerosis168 Cystic fibrosis169

Proteinase 3

ECM degradation Bactericidal properties Induction of endothelial cell apoptosis Regulation of granulopoiesis

Papillon–Lef`evre syndrome166 Wegener’s granulomatosis 170,171

Cathepsin G

ECM degradation Bactericidal properties Platelet activation Induction of airway submucosal gland secretion

Papillon–Lef`evre syndrome166

␤2 integrin

Neutrophil adhesion and migration Generation of respiratory burst

Atherosclerosis172 Vasculitis173

Gelatinase (MMP-9)

Contribute to tumor proliferation Degradation of serine protease inhibitor ␣1-antityrpsin Cleavage of interferon-␤

Cancer142 COPD174 Asthma175 Multiple sclerosis176

Oncostatin M

IL–6–like cytokine Stimulation of VEGF production from breast cancer cells

Cancer142

Reactive oxygen species

Carcinogen Induction of tissue damage Induction of apoptosis

Pulmonary hypertension177 Cancer142 Atherosclerosis178

Summary Recent studies have significantly advanced our understanding of novel neutrophil functions within immune responses. Studies published in recent decades have illustrated the contributions of neutrophils to multiple components of both innate and adaptive immune responses. Neutrophils are no longer considered simply destructive bacteria killing cells, but are instead known to play more diverse roles in the immune system. The functions of neutrophils in immune defense and their roles in various diseases suggest that these cells could be potential targets in clinical medicine (Table 1). Despite the difficulties associated with manipulating these short-lived cells, our knowledge about neutrophils

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Regulation of immune responses by neutrophils.

Neutrophils, the most abundant circulating cells in humans, are major pathogen-killing immune cells. For many years, these cells were considered to be...
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