Phagocyte partnership during the onset and resolution of inflammation

REVIEWS

Phagocyte partnership during the onset and resolution of inflammation Oliver Soehnlein*‡ and Lennart Lindbom*

Abstract | Neutrophils, monocytes and macrophages are closely related phagocytic cells that cooperate during the onset, progression and resolution of inflammation. This Review highlights the mechanisms involved in the intimate partnership of phagocytes during each progressive phase of the inflammatory response. We describe how tissue-resident macrophages recognize tissue damage to promote the recruitment of neutrophils and the mechanisms by which infiltrating neutrophils can then promote monocyte recruitment. Furthermore, we discuss the phagocyte-derived signals that abrogate neutrophil recruitment and how the uptake of apoptotic neutrophils by macrophages leads to termination of the inflammatory response. Finally, we highlight the potential therapeutic relevance of these interactions.

*Department of Physiology and Pharmacology, Karolinska Institutet, S‑171 77 Stockholm, Sweden. ‡ Institute for Molecular Cardiovascular Research (IMCAR), University of Aachen, 52074 Aachen, Germany. Correspondence to O.S. e‑mail: osoehnlein@ ukaachen.de doi:10.1038/nri2779

Inflammation is a tightly regulated process initiated following tissue injury or infection. The main function of inflammation is to eliminate the pathogenic insult and to remove damaged tissue, with the aim of restoring tissue homeostasis. The concerted action of professional phagocytes — neutrophils, monocytes and macrophages — is crucial to the effective elimination of intruders and cell debris. Neutrophils are short-lived cells that are recruited early in the inflammatory response1. They use a large array of inflammatory mechanisms, some of which are potentially harmful to the host, and so neutrophil activation has to be tightly controlled to avoid excessive tissue damage. Monocytes can be classified into at least two subpopulations with distinct phenotypical and functional characteristics: classical human CD14+CD16– or mouse GR1+ monocytes and nonclassical human CD14lowCD16+ or mouse GR1– monocytes (BOX 1). Gene expression profiles of monocyte subsets are well conserved between humans and mice2. The physiological roles of the respective monocyte subsets in vivo are not fully defined, but the two subsets might have different roles during homeostasis and inflammation. Studies suggest that classical monocytes initiate inflammatory activities whereas non-classical monocytes promote wound healing and angiogenesis3–6. However, it has also been suggested that classical monocytes produce lower levels of pro-inflammatory cytokines and are less potent in presenting antigens compared with non-classical monocytes7,8. To enter the site of inflammation, each monocyte subset uses a different set of

adhesion molecules and chemokine receptors. Classical monocytes use P-selectin glycoprotein ligand 1 (PSGL1), CC-chemokine receptor 2 (CCR2) and CCR6 to migrate from the blood; however, the extravasation of nonclassical monocytes depends on CX3C-chemokine receptor 1 (CX3CR1; also known as the fractalkine receptor)9–11. Following extravasation, monocytes can develop into dendritic cells (DCs) and tissue-resident macrophages, or they can give rise to inflammatory macrophages12,13. However, the fate and differentiation of the monocyte subsets after entering the tissue is still not fully defined (BOX 1). As far back as a century ago, neutrophils, monocytes and macrophages were appreciated for their phagocytic and microbicidal capacity 14. However, the abilities of these phagocytes go far beyond eating and killing. In fact, these cells are indispensible for host defence; they effectively regulate innate and adaptive immune responses and can clear inflammatory sites without the contribution of the adaptive immune system. The effectiveness of these phagocytes crucially depends on the close partnerships they maintain, and they have complementary roles in reconstituting tissue integrity following injury or infection. The literature on neutrophils, monocytes and macrophages is so large that few authors have attempted to cover their inflammatory roles comprehensively 15,16. This Review makes no such effort; in fact, large elements of phagocyte biology are omitted here, including myelopoiesis, classical adhesion and emigration cascades, signal transduction and antimicrobial mechanisms.

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REVIEWS Box 1 | Heterogeneity of monocytes In humans, classical monocytes are defined as CD14+CD16−, whereas non-classical monocytes are CD14lowCD16+ (REFS 3,13). Mouse monocytes can be divided into either classical ‘inflammatory’ GR1+ monocytes that express high levels of CC-chemokine receptor 2 (CCR2), CD62 ligand (CD62L) and low levels of CX3C-chemokine receptor 1 (CX3CR1), or non-classical ‘resident’ GR1– monocytes that are characterized by high expression of CX3CR1 but do not express CCR2 or CD62L2,12. Current dogma suggests that the differential expression of chemokine receptors and cell adhesion molecules between these mouse monocyte subsets, which is similar to that seen in human monocyte populations, is crucial for their extravasation behaviour. Functionally, classical CD14+CD16– monocytes produce lower levels of pro-inflammatory cytokines but contribute more effectively to bacterial clearance compared with non-classical monocytes7,8. By contrast, non-classical CD14lowCD16+ monocytes are more potent at presenting antigens and also account for more vigorous production of pro-inflammatory cytokines7,8. Mouse GR1+ monocytes are thought to correspond to human classical monocytes, whereas the GR1– subset is believed to correspond to the human non-classical monocytes. This is, however, confounded by functional features, as well as by relative numbers in peripheral blood. Whereas human CD14hiCD16– monocytes produce the immunoregulatory cytokine interleukin-10 (IL-10) in response to lipopolysaccharide (LPS), mouse GR1+ monocytes efficiently produce pro-inflammatory cytokines, such as tumour necrosis factor and IL-1. In addition, CD14hiCD16– monocytes account for about 90% of monocytes in human peripheral blood, whereas the suggested mouse correlate account for only 50% of the total blood monocytes. The fate and differentiation of monocyte subsets towards dendritic cells or macrophages is largely unclear and remains an issue of debate. Diversity and plasticity of macrophages is also an important feature of the mononuclear phagocyte system137. For example, in response to bacterial moieties (such as LPS) and interferon-γ, macrophages undergo classical activation to become M1 macrophages, and are characterized by the production of reactive nitrogen and oxygen species, expression of co-stimulatory molecules and the release of pro-inflammatory cytokines. By contrast, M2 macrophages, developing following exposure to IL-4 and/or IL-13, express high levels of scavenger, mannose and galactose receptors137.

Instead, we discuss selected studies that provide evidence for the partnership of phagocyte subsets during various phases of the inflammatory response (FIG. 1). we discuss the role of resident phagocytes in recognizing tissue damage and subsequently inducing neutrophil recruitment (phase I), the mechanisms underlying neutrophil-mediated monocyte recruitment (phase II), the phagocyte-derived signals that abrogate neutrophil recruitment (phase III) and, finally, the termination of the inflammatory response by macrophages that adopt an anti-inflammatory signature following ingestion of apoptotic neutrophils (phase Iv).

Common origin but functional heterogeneity The ability of neutrophils, monocytes and macrophages to phagocytose microorganisms and cell debris was appreciated by Élie Metchnikoff a century ago14. However, only in recent years have we started to understand the relationship between these cells. Their partnership begins in the bone marrow, where they arise from a common precursor 17. This close origin is the basis for many of the functional characteristics that they share, such as powerful phagocytic capacity and elaborate intracellular killing machineries, similar transcriptional profiles and common phenotypical markers. Nevertheless, functional differences between the cell types do exist and these explain why the interplay between these subpopulations is crucial for the resolution of inflammation and a return to tissue homoeostasis. For example, mobilization of

phagocyte subsets from the bone marrow is mediated by different chemokine–chemokine receptor axes. while the CXC-chemokine ligand 12 (CXCL12)–CXCR4 axis is crucial for neutrophil homeostasis, the CCL2–CCR2 axis is crucial for the bone marrow mobilization of inflammatory monocytes18–21. The pool of circulating monocytes can be further increased with inflammatory monocytes mobilized from the spleen; this is a recently identified monocyte reservoir in mice22 that enables instant dispatch of monocytes in case of emergency. In addition, the phagocyte subsets have potent antimicrobial killing abilities, but use distinct mechanisms to achieve this. Neutrophils are equipped with a large range of antimicrobial polypeptides that are readily stored in granules and can also rapidly produce oxygen radicals1,23 (BOX 2). However, this potent killing and relative lack of specificity can lead to damage to self tissues, making neutrophils less suitable as sentinels in tissues and body cavities. By contrast, the less microbicidal and less cytotoxic resident macrophages are long-lived and more specific in their recognition and subsequent downstream activity, and are therefore well suited to guard body compartments16. examples such as these show that despite their kinship in origin and function, neutrophils and mononuclear phagocytes are not redundant; instead, they complement each other in the inflammatory process.

Phase I: macrophages fuel neutrophil egress The initiation of inflammatory responses must be tightly regulated to avoid considerable collateral damage. Molecular alarm signals generated in response to tissue damage and/or microbial invasion are recognized by antigen-presenting cells (APCs) such as resident macrophages or DCs. owing to their strategic location in close proximity to the site of injury, tissue-resident cells are the primary inducers of an inflammatory reaction (FIG. 2). For example, in contrast to cells undergoing apoptosis, necrotic cell death resulting from infection or injury leads to the disruption of cell membranes and the release of cytoplasmic and nuclear components24 that contain damage-associated molecular patterns (DAMPs). DAMPs (such as DNA) are recognized by pattern recognition receptors (PRRs; such as Toll-like receptor 9 (TLR9)) expressed by leukocytes and promote the production of pro-inflammatory cytokines, such as interleukin-1 (IL-1). In addition, proteases and hydrolases released from dead cells modify extracellular components to generate mediators (such as complement fragments) or other DAMPs (such as fragments of extracellular matrix) that can activate leukocytes. Pathogenassociated molecular patterns (PAMPs) can also initiate immune responses by activating PRRs on APCs25. A typical example is activation of TLR4 by bacterial lipopolysaccharide (LPS), which promotes the production and release of pro-inflammatory cytokines such as tumour necrosis factor (TNF)25. Sentinel monocytes initiate inflammatory responses. Following the discovery of two distinct subsets of blood monocytes12,26, researchers tried to address their respective functions. Most emphasis was placed on classical

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REVIEWS a

b

Phase I

Phase II

Neutrophil recruitment

Patrolling monocyte

c

d

Phase III

Phase IV

Macrophages and apoptotic neutrophils prevent further neutrophil infiltration

Inflammatory monocyte recruitment

Blood

Endothelium

Tissue Injury leads to release of DAMPs that alert patrolling monocytes and macrophages

Macrophage

Production of cytokines and chemokines

LL-37 and azurocidin

Neutrophil granule contents promote monocyte extravasation

Macrophage growth factors promote repair

Lipoxin A4 and resolvins

‘Find me’ signals attract scavengers

IL-1, G-CSF, GM-CSF and TNF

Macrophage Macrophages control neutrophil life-span

Apoptotic neutrophil

‘Find me’ signals attract scavengers

TGFβ, IL-10 and PGE2 Clearance of apoptotic neutrophils promotes ‘anti-inflammatory’ macrophage response

Figure 1 | Phagocyte interactions in inflammation. a | Phase I: patrolling non-classical monocytes and resident Nature Reviews | Immunology macrophages are among the first cells to sense a disturbance in tissue homeostasis. They rapidly produce cytokines and chemokines to alert the immune system and to recruit neutrophils. b | Phase II: shortly after the alarm has gone off, neutrophils invade the site of injury and release granule contents that promote the extravasation of inflammatory monocytes. c | Phase III: the life-span of emigrated neutrophils is rather short and is subject to modification by pro- or anti-apoptotic signals, some of which are produced by macrophages. Macrophages and apoptotic neutrophils prevent further infiltration of neutrophils, but signals from apoptotic neutrophils promote continued monocyte influx. d | Phase IV: the clearance of apoptotic neutrophils promotes an anti-inflammatory programme in macrophages, which leads, ultimately, to the reconstitution of tissue homeostasis. G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–monocyte colony-stimulating factor; IL, interleukin; PGE2, prostaglandin E2; TGFβ, transforming growth factor-β;TNF, tumour necrosis factor.

monocytes, and non-classical monocytes were somewhat neglected. However, recent intravital microscopy studies have shown that in the steady-state non-classical monocytes crawl along the luminal side of the endothelium thereby patrolling healthy tissues4. In response to tissue damage or infection, these monocytes rapidly migrate out of the circulation and invade the damaged site. Such emigration was reported for both septic (Listeria monocytogenes) and aseptic (dibutylphtalate acetone or micropipette-induced) tissue injury, indicating that distinct alarm signals can trigger the rapid efflux of non-classical monocytes. This early efflux may be relevant for the subsequent inflammatory cascade as recently emigrated non-classical monocytes transcribe genes that encode antimicrobial proteins, PRRs, scavenger receptors, cytokines and chemokines, as well as genes associated with antigen presentation27. However, the precise role of sentinel monocytes in initiating immune responses remains unclear and requires further investigation. The recruitment of patrolling non-classical monocytes is transient and these cells are rapidly superseded by recruited neutrophils and, subsequently, by classical monocytes27,28.

Resident macrophages recruit neutrophils. In the past decade, there have been important advances in our understanding of how the inflammatory response to pathogens is initiated. The host senses the presence of microorganisms using receptors that are specialized for the recognition of microbial components, which are chemically distinct from the host’s endogenous molecules. Although the molecular identification of these PRRs, such as the TLRs, has resulted in a detailed understanding of how pathogens trigger inflammatory immune responses25, it is less well understood how stimuli derived from dying cells are recognized. However, several recent studies have addressed this issue and we are beginning to appreciate the complexity of this process. we focus here on the importance of DAMPs for the early interaction of resident macrophages and neutrophils, but it should be mentioned that DAMPs are also sensed by other cells, such as DCs, and induce the recruitment of cells other than neutrophils (for example, monocytes) to the site of inflammation. Damaged cells spill cytoplasmic and nuclear components into the extracellular milieu and these ‘alarm signals’ activate tissue-resident cells, such as macrophages24.



REVIEWS Box 2 | The neutrophil’s armour • Granule proteins. Granule proteins are synthesized at different stages of myelopoiesis and targeted to granule subsets49. Different granule subsets show distinct propensities for release. Secretory vesicles are mobilized following initial neutrophil–endothelial cell contact and tertiary granules released during subsequent neutrophil transendothelial migration. Primary and secondary granules are discharged once the neutrophil has entered the tissue. Secretory vesicles are rich in membrane-bound receptors, but granules released at later stages mainly contain proteases and antimicrobial polypeptides50. • Reactive oxygen species (ROS). During neutrophil activation, cytoplasmic and membrane components assemble to form a functional multi-component electron-transfer system, which catalyses the reduction of molecular O2 at the expense of cytoplasmic NADPH23. ROS are associated with antimicrobial activity but also damage host tissue. • Lipid mediators. Activated neutrophils produce leukotrienes in a reaction that involves oxygenation of arachidonic acid and further processing of the intermediate metabolite leukotriene A4 into the potent chemoattractant leukotriene B4 (REF. 46). Leukotriene B4 released by neutrophils can amplify the immune response and promote further recruitment of the phagocytic cells in an autocrine or paracrine manner48. • Neutrophil extracellular traps (NETs). Neutrophils activated through Toll-like receptors, Fc receptors or chemokine and cytokine receptors release nuclear content such as chromatin. This forms a scaffold for the extracellular exposure of antimicrobial proteins and histones110,111.To date, NETs have been mainly linked to bacterial trapping and killing but they may also be involved in the activation of antigen-presenting cells. • De novo synthesis programme. Following extravasation, neutrophils initiate a second transcriptional burst resulting in the production of various cytokines, chemokines and growth factors75.

Recently, CLeC4e (also known as MINCLe), a transmembrane C-type lectin receptor (CLR), was shown to be involved in initiating the early inflammatory response after necrotic cell death29. Activation of CLeC4e by spliceosome-associated protein 130 (SAP130; also known as SF3B3), a nuclear protein released from necrotic cells, initiates an intracellular signalling cascade involving spleen tyrosine kinase (SYK) and caspase-recruitment domain 9 (CARD9)30. This ultimately leads to the production of pro-inflammatory cytokines and chemokines, including TNF, interleukin-6 (IL-6), CXCL1 and CXCL2, which stimulate the recruitment of neutrophils to the injured tissue. one of the best known endogenous alarm signals is high-mobility group box 1 protein (HMGB1), which is a nuclear protein that modulates chromatin accessibility and is released from cells undergoing necrosis31. early reports indicated that extracellular HMGB1 induces macrophage synthesis of pro-inflammatory cytokines, such as TNF and IL-6, as well as phagocyte-attracting chemokines, such as CXCL8 (also known as IL-8), CCL3 and CCL4, thereby promoting the recruitment of neutrophils. It later became apparent that HMGB1 required a partner to initiate inflammation32. we now know that complexes between HMGB1 and DAMPs (such as single-stranded DNA, LPS or nucleosomes) interact with the canonical HMGB1 receptor RAGe (receptor for advanced glycation end products) as well as TLR2, TLR4, TLR9 and IL-1R to induce cytokine production33,34. Importantly, HMGB1 activates nuclear factor-κB (NF-κB) through a TLR–myeloid differentiation primary-response protein 88 (MYD88)-dependent pathway, which is similar to pattern recognition associated with infection25,32. So, trauma and pathogens might engage the same immune cell receptors. However, the outcomes of trauma and infection are different in many ways; therefore the signalling pathways are not identical. Interestingly, it has recently been shown that HMGB1 can interact with CD24, a glycosylphosphatidylinositol

(GPI)-anchored membrane protein, which in turn binds sialic acid-binding immunoglobulin-like lectin G (SIGLeCG)35. As CD24 does not contain a cytoplasmic domain, it signals through SIGLeCG, which has an immunoreceptor tyrosine-based inhibitory motif (ITIM). ITIM activation decreases the activation of NF-κB and the subsequent secretion of cytokines and chemokines. So this pathway limits the APC response to DAMPs but is not activated by PAMPs and might therefore help to avoid unnecessary collateral damage in the absence of pathogens36. The production of neutrophil-recruiting chemokines by monocytes and macrophages is therefore a common response following the recognition of danger signals (TABLE 1). The importance of macrophage-derived chemokines in promoting neutrophil egress from the vasculature has repeatedly been shown in experimental models of inflammation and also in clinically relevant models. Typically, researchers have studied the effects of macrophage-derived chemokines in promoting neutrophil infiltration into the pleural cavity and the peritoneum, as well as to the lung 37–41. The induction of chemokine synthesis is not, however, restricted to receptor ligation, as indicated by chemokine production by macrophages following exposure to reactive oxygen species (RoS)42. RoS are not only antimicrobial and tissue destructive but also function as signal-transduction molecules contributing to the expression of various inflammatory cytokines and chemokines. In monocytic cells, RoS activate transient receptor potential protein M2 (TRPM2) Ca2+ channels42. Downstream signalling involves extracellular signalregulated kinase (eRK) activation and NF-κB translocation into the nucleus, ultimately promoting the induction of a neutrophil-recruiting chemokine milieu42. Cleavage and conversion initiate inflammation. The potency of chemokines to attract neutrophils can be further increased by the enzymatic cleavage of chemokines by matrix metalloproteinases (MMPs). In this context,

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REVIEWS Blood

Activated macrophages recruit neutrophils by producing inflammatory cytokines and chemokines

Endothelium

Tissue

CXCL1, CXCL2, TNF and IL-6

SAP130

CLEC4E

SYK FcR γ-chain

TLR9 Nucleus

RAGE Necrotic cells

HMGB1–DNA complex

ERK TRPM2 Resident macrophage

ROS Necrotic cells release DAMPs and activate tissue macrophages

Figure 2 | Resident macrophages promote neutrophil invasion. Antigen-presenting cells such as macrophages sense tissue damage through damage-associated molecular pattern (DAMP) receptors and produce a neutrophil-recruiting chemokine Necrotic Nature Reviewsmilieu. | Immunology cells release spliceosome-associated protein 130 (SAP130), which is recognized by CLEC4E (also known as MINCLE), initiating a signalling cascade through the Fc receptor γ-chain (FcR γ-chain) and spleen tyrosine kinase (SYK). High-mobility group box 1 protein (HMGB1) forms complexes with DNA that are recognized by receptor for advanced glycation end products (RAGE) and Toll-like receptor 9 (TLR9), leading to cell activation. Under stress conditions, inflammatory cells produce and release vast amounts of reactive oxygen species (ROS). ROS induce Ca2+ influx through transient receptor potential protein M2 (TRPM2) Ca2+ channels, which leads to downstream signalling and chemokine production. CXCL, CXC-chemokine ligand; ERK, extracellular signal-regulated kinase; IL-interleukin; TNF, tumour necrosis factor.

Secretagogue A substance that causes another substance to be secreted.

it was shown that MMP9, a protease common to neutrophils, macrophages and endothelial cells, cleaves and increases the chemotactic activity of CXCL1 and CXCL8, thereby amplifying neutrophil tissue infiltration43. In addition, the secretion of MMP8 by macrophages and neutrophils is a non-redundant mechanism for cleaving CXCL5 and CXCL8, both of which are key ligands for the neutrophil receptor CXCR2 (REF.44). Consequently, chemokine modification by proteases provides a feedforward mechanism to promote the initial neutrophil extravasation. Lipid mediators, such as prostaglandins and leukotrienes, are widely appreciated for their pro-inflammatory activities45. using either cyclooxygenases or lipoxygenases, monocytes and macrophages rapidly synthesize these lipid mediators from membrane-derived arachidonic acid within seconds to minutes of acute inflammatory challenge45. Sequential actions of cytoplasmic 5-lipoxygenase and leukotriene A4 hydrolase yield leukotriene B4, which has long been known as a potent chemoattractant for myeloid cells46 and, more recently,

also for certain T cell subsets47. In addition to its chemotactic effects, leukotriene B4 is a strong secretagogue of neutrophil granule proteins and a powerful inducer of RoS production48. with regard to its chemoattractant activity, leukotriene B4 has substantial functional overlap with certain chemokine peptides and it exerts its effect through activation of G protein-coupled transmembrane receptors, although it has no structural similarity to the chemokines. Two such receptors have been identified on neutrophils, known as BLT1 (also known as LTB4R) and BLT2 (also known as LTB4R2) with high and low affinity for leukotriene B4, respectively 48. All the main effects of leukotriene B4 on neutrophils can be achieved through the activation of BLT1, whereas little is known about the contribution of BLT2 to neutrophil responses. In addition to being activated by leukotriene B4 released from tissue-resident macrophages, neutrophils can produce leukotriene B4 themselves48, triggering activation of BLT1 in an autocrine manner. Therefore, neutrophils that have already entered the site of inflammation can in this way amplify the chemotactic signal and call for the further recruitment of other inflammatory cells.

Phase II: neutrophils recruit monocytes Following the activation of tissue-resident macrophages and formation of chemotactic signals, neutrophils rapidly emigrate into the tissue as an advance guard for subsequent inflammatory, classical monocytes (FIG. 3). Prepacked cargo attracts classical inflammatory monocytes. Following extravasation, neutrophils sequentially release preformed granule proteins. The earliest granule compartments to be released during the neutrophil’s journey to the site of injury or infection are secretory vesicles. These are rich in membrane-bound receptors49,50. Therefore, the mobilization of this granule subset and fusion with the cell membrane transforms the neutrophil to a highly perceptive cell. Although secretory vesicles are generally devoid of soluble mediators49,50, it has been shown that neutrophils adhering to the vessel wall release a soluble component that induces endothelial cell activation and subsequent plasma leakage51. Later research showed that neutrophil-derived azurocidin is responsible for inducing permeability changes in vessel walls and also lung oedema formation 52–54. Azurocidin is cationic in nature and this favours its deposition on the endothelium55,56, where it is presented to rolling monocytes and promotes their firm adhesion; this is complemented by the capacity of azurocidin to increase the expression of endothelial cell adhesion molecules57. During the passage of neutrophils through the endothelium and their subsequent extravascular migration, granules are discharged58. Many of the liberated proteins have antimicrobial and matrix-degrading activity 49,50. Clinical evidence from patients with specific granule deficiencies has indicated an important role for neutrophil granule proteins in the extravasation of monocytes59. These patients lack, among other proteins, 18 kDa cationic antimicrobial protein (CAP18), which is the pro-form of the cathelicidin LL-37, and



REVIEWS Table 1 | Chemokines and chemokine receptors involved in phagocyte interplay during the onset or resolution of inflammation Chemokine Source

Receptor

Target

mechanism leading to production or activation

Role in phagocyte interplay

CXCL1

CXCR2

Neutrophils

Onset: sensing necrotic cell death (by CLEC4E, ROS and HMGB1)

Onset: neutrophil recruitment

Macrophages

Refs 29, 33, 34, 37–42

Onset: increased activity after cleavage by MMP9

CXCL2

Macrophages

CXCR2

Neutrophils

43

Resolution: cleavage by MMP12

Resolution: termination of neutrophil efflux

Onset: sensing necrotic cell death (by CLEC4E, ROS and HMGB1)


Resolution: cleavage by MMP12

Resolution: termination of neutrophil efflux

CXCL5

Macrophages, eosinophils and epithelial cells

CXCR2

Neutrophils

Onset: increased activity following cleavage by MMP8


CXCL8

Macrophages

CXCR2

Neutrophils

Onset: sensing stress (by ROS)


Onset: increased activity after cleavage by MMP8 or MMP9

CCL2

CCL3

102 29, 33, 34, 37–42 102 44

42 43,44

Neutrophils

Monocytes

Onset: transcriptional activation

Onset: monocyte recruitment

65

Macrophages

Neutrophils

Resolution: lipoxin A4 blocks transcription

Resolution: termination of neutrophil influx

91

Inflammatory monocytes

Onset: IL-6 trans-signalling

Onset: inflammatory monocyte recruitment

Endothelial cells and macrophages

Endothelial cells, monocytes and macrophages

CCR2

CCR1


Onset: synthesis following exposure to neutrophil granule proteins Resolution: cleavage by MMP1, MMP3 and MMP12

Resolution: termination of leukocyte influx

Onset: synthesis following exposure to neutrophil granule proteins



68–70 28

102 28

77

Neutrophils

CCR5

Apoptotic neutrophils

Resolution: chemokine sequestration


101

CCL4

Neutrophils

CCR1




CCL5

Macrophages

CCR5

Apoptotic neutrophils

Resolution: chemokine sequestration


CCL6

Neutrophils and macrophages

CCR1


Onset: increased activity following truncation by neutrophil proteases


CCL7

Macrophages

CCR2


Resolution: cleavage by MMP2 and Resolution: termination of MMP12 leukocyte efflux

102

CCL8

Neutrophils and monocytes

CCR1 and CCR5


Resolution: cleavage by MMP1, MMP3 and MMP12

Resolution: termination of leukocyte efflux

102

CCL9

Macrophages and epithelial cells

CCR1




74

CCL15

Macrophages and epithelial cells

CCR1




74

CCL20

Neutrophils, macrophages and lymphocytes

CCR6




31,77,78

CCL23

Macrophages

CCR1




74

CX3CL1

Apoptotic cells, endothelial cells and macrophages

CX3CR1

Monocytes

Resolution: released by apoptotic cells

Resolution: monocyte recruitment

118

28 101 74

CC, CC-chemokine; CX3C, CX3C-chemokine; CXC, CXC-chemokine; HMGB1, high-mobility group box 1 protein; L, ligand; MMP, matrix metalloproteinase; R, receptor; ROS, reactive oxygen species.

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REVIEWS Pertussis toxin Pertussis toxin blocks Gαi-coupled receptor signalling (including chemokine-receptor signalling) by catalysing ADP ribosylation of the Gαi subunit.

α-defensins, all of which have been shown to have direct monocyte-attracting activity 60,61. In addition, cathepsin G and azurocidin attract monocytes62. The sensitivity of these cellular responses to pertussis toxin indicated the involvement of G protein-coupled receptors, and the monocyte-attracting activity of cathepsin G, LL-37 and azurocidin was shown to involve activation of formylpeptide receptors (FPRs), a family of evolutionarily conserved heptahelical G protein-coupled receptors. In humans, three FPRs have been identified, all of which are expressed by monocytes63. Cathepsin G and LL-37 activate FPR164 and FPR260, respectively, but it remains unknown which specific FPR azurocidin acts through65. In addition, the receptor for α-defensins on monocytes is yet to be identified61. It is important to note that different monocyte subsets are recruited by distinct mechanisms. In this respect, it has recently been shown in mice that neutrophil depletion specifically decreases the recruitment of classical inflammatory monocytes28,65. Interestingly, this deficiency could be rescued, almost completely, by the local application of supernatant from activated neutrophils65. Subsequent experiments identified the FPR ligands LL-37 and azurocidin as the principal mediators of this effect. Therefore the immediate availability of neutrophil-derived granule proteins may enable the rapid recruitment of classical monocytes to the site of inflammation65,66.

Blood

Neutrophil granule proteins Monocyte rolling

Granule proteins anchor on endothelial proteoglycans

Tissue

Endothelial activation transmits neutrophil-triggered monocyte recruitment. Although the recruitment cascade for neutrophils and monocytes is similar, both populations differ in their use of cell adhesion molecules and chemokines. Adhesion and extravasation of neutrophils depends largely on the expression of P-selectin (also known as CD62P), intercellular adhesion molecule 1 (ICAM1, also known as CD54) and ICAM2 (also known as CD102) by the endothelium; ICAM1 and ICAM2 are recognized by the β2 integrins CD11b–CD18, which are expressed by neutrophils67. Neutrophil recruitment is promoted by chemotactic agents, such as complement component C5a, leukotriene B4, platelet activating factor (PAF) and CXCL8, many of which are derived from tissue-resident cells or infiltrating leukocyte subsets. By contrast, monocytes also express β1 integrins that promote their recruitment through interactions with endothelial cell-expressed e-selectin (also known as CD62e) and vascular cell adhesion molecule 1 (vCAM1; also known as CD106). Furthermore, monocytes are recruited in response to CCL2 (REF. 67). The chemotactic mediators involved in neutrophil recruitment tend to be pre-stored or synthesized through enzymatic cleavage, which leads to their rapid mobilization. However, monocyte chemoattractants tend to require de novo synthesis. In the transition from neutrophil to monocyte tissue influx, IL-6 and its soluble receptor sIL-6Rα have an important role68,69. Activation

Soluble IL-6–IL6R complexes activate endothelial cells

Extravasation

Neutrophil products promotes chemokine synthesis by endothelial cells gp130

VCAM1 CCL2

Endothelium

Azurocidin, LL-37 and cathepsin G Neutrophil derived proteases modify chemokine pro-forms Proteases Neutrophil granule proteins promote chemokine production by macrophages

Chemokines

Figure 3 | neutrophils promote classical inflammatory monocyte influx. Granule proteins discharged from| activated Nature Reviews Immunology neutrophils anchor on endothelial proteoglycans. In this location, the granule proteins are recognized by monocytes rolling along the endothelium and promote firm adhesion of these cells. Azurocidin, LL-37, and cathepsin G released from recruited neutrophils activate formyl peptide receptors on classical inflammatory monocytes and promote their extravasation. Neutrophils produce soluble complexes of interleukin-6 (IL-6) and IL-6 receptor (IL-6R), which activate endothelial cells through gp130 to express CC-chemokine ligand 2 (CCL2) and vascular cell-adhesion molecule 1 (VCAM1). Neutrophil granule proteins promote the de novo synthesis of monocyte-attracting chemokines by neighbouring endothelial cells and macrophages. Finally, neutrophil-derived proteases cleave chemokine pro-forms, altering their activity. NATuRe RevIewS | Immunology


REVIEWS of neutrophils by chemoattractants promotes the shedding of IL-6Rα; sIL-6Rα then binds IL-6 released by cells at the inflammatory site, such as macrophages and endothelial cells. IL-6–IL-6Rα complexes bind to gp130 on endothelial cells and trigger a cellular response known as IL-6 trans-signalling. As a consequence, endothelial cells upregulate the expression of CCL2 and vCAM1, which trigger the adhesion and emigration of classical monocytes68,70. Shedding of IL-6Rα from neutrophils is also induced by apoptosis, thereby linking signals from apoptotic neutrophils with subsequent monocyte infiltration71. By contrast, the recruitment of neutrophils is not promoted by this mechanism71. Neutrophils alter chemokine networks to attract classical inflammatory monocytes. Secreted neutrophil granule proteins, such as proteinase 3, directly activate cells in close proximity to secrete chemokines, thereby creating a milieu that favours the recruitment of classical monocytes.

Apoptotic neutrophils promote monocyte recruitment Blood

Lipid mediators decrease neutrophil recruitment Neutrophil

Monocyte

Endothelium

Tissue

Lipoxins and resolvins

Lactoferrin from apoptotic neutrophils decreases neutrophil infiltration Lactoferrin, LPC, nucleotides and CX3CL1

Macrophage

CCR5

Apoptotic neutrophil CCR5 on apoptotic neutrophils depletes CCL3 and CCL5, decreasing neutrophil infiltration

Uptake of apoptotic neutrophils promotes a pro-resolution programme in macrophages, increasing TGFβ and IL-10

Figure 4 | Phagocyte interplay during the resolution of inflammation. The restoration of tissue homeostasis is a regulated programme,Nature in which the abrogation of Reviews | Immunology neutrophil infiltration and the clearance of dead neutrophils are key events. Lipid-mediators, such as lipoxins or resolvins, turn off the influx of neutrophils, while the influx of monocytes is further promoted. Lactoferrin from apoptotic neutrophils abrogates neutrophil infiltration but promotes the recruitment of monocytes. Lysophosphatidlycholine (LPC) is released from apoptotic cells in a caspase-3-dependent manner and activates the G protein-coupled G2A receptor on monocytes, which induces their recruitment. The release of nucleotides and CX3C-chemokine ligand 1 (CX3CL1) from apoptotic neutrophils induces monocyte recruitment through P2Y2 and CX3C-chemokine receptor 1 (CX3CR1), respectively. Resolvins and protectins increase the expression of CC-chemokine receptor 5 (CCR5) on apoptotic neutrophils: this depletes CCR5-binding chemokines (for example, CCL3 and CCL5) and prevents further influx of neutrophils. Finally, the clearance of apoptotic neutrophils by macrophages launches a pro-resolution transcriptional programme with release of transforming growth factor-β (TGFβ) and interleukin-10 (IL-10).

In this context, proteinase 3 was shown to induce the production of CCL2 from endothelial cells72, whereas azurocidin enhanced the expression of CCL3 from monocytes and macrophages73. CCL3 is a major ligand for CCR1, which is mainly expressed by classical monocytes. In addition, CCL6, CCL9, CCL15 and CCL23, all of which are primarily macrophage derived (TABLE 1), are weak ligands for CCR1. However, following exposure to neutrophil serine proteases these chemokines undergo amino-terminal modification that results in an up to 1,000-fold increase in their ability to activate CCR1 and induce the chemotaxis of monocytes74. Recruited neutrophils also undergo a transcriptional burst75, thereby providing another mechanism to alter the chemokine milieu. In terms of chemokine production, the main chemokine produced by neutrophils is CXCL8, which activates neutrophils through CXCR2 in an autocrine loop. CXCL8 also binds to CXCR2 expressed by monocytes. Therefore, it is not surprising that CXCL8 also mediates adhesion of human monocytes to the vascular endothelium76. Appropriately stimulated neutrophils can also produce CC-chemokines, such as CCL3 and CCL4 (REF. 77) . Both chemokines mainly bind to CCR1, thereby acting on classical monocytes. It has also been described that neutrophils can express CCL20 when exposed to pro-inflammatory stimuli such as TNF78,79. CCL20 potently attracts inflammatory monocytes to the inflamed dermis through CCR6 (REFS 78,80). Collectively, these data indicate that neutrophils both produce and modify chemokines to preferentially recruit inflammatory monocytes. Similar mechanisms may also apply to non-classical chemokines, such as chemerin. Chemerin has been identified in inflammatory exudates and recruits monocytes, macrophages and plasmacytoid DCs through activation of ChemR23. Pro-chemerin, the secreted pro-form of chemerin, is released by a broad range of cells. Bone marrow-derived cells however, do not express prochemerin81 but produce serine proteases (such as neutrophil elastase and cathepsin G) that cleave pro-chemerin to produce active chemerin82.

Phase III: phagocyte signals stop neutrophil influx once neutrophils, classical inflammatory monocytes and macrophages have entered the site of injury or infection, they collaborate to remove foreign entities (for example, bacteria). The mechanisms involved in this interplay have recently been described83 and reviewed84,85 and are not further discussed here. After the inflammatory stimulus has been eliminated, the ongoing inflammatory response must be resolved to avoid excessive tissue damage and to initiate the healing process. During the resolution of inflammation a set of ‘brakes’ prevent further infiltration of leukocytes and promote the removal of debris from the inflamed site, thereby restoring tissue homeostasis (FIG. 4). Change in local lipids limits neutrophil infiltration. The process of resolution is not a mere termination of the inflammatory reaction, but it is an active process requiring signals that turn off neutrophil infiltration

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ELR motif A conserved glutamatic acid– leucine–arginine (ELR) sequence found in CXC-chemokines immediately preceding the first cysteine residue near the amino-terminal end. The motif is crucial for receptor ligation and the chemotactic activity of neutrophils.

and, at the same time, promote the uptake and clearance of apoptotic cells. Lipid mediators seem to have a key role in this process 86, and the resolution of inflammation is accompanied by an active switch in the types of lipid mediator found at the inflamed site. During the initial inflammatory response, prostaglandins and leukotrienes that amplify inflammation are generated by various cell types, including endothelial cells, neutrophils, monocytes and macrophages45. Following this, prostaglandin e 2 (PGe 2) and PGD 2 gradually promote the synthesis of mediators with anti-inflammatory and pro-resolving activity, such as the lipoxins86. This process is known as lipid mediator class-switch87. Neutrophils can interact with other cells in their immediate vicinity, such as platelets and epithelial cells, to produce lipoxins86. other resident cells can also produce lipoxins in response to specific cytokines or growth factors. In the case of macrophages, the uptake of apoptotic neutrophils promotes lipoxin production88. Lipoxin A4 inhibits neutrophil entry to the site of inflammation, but promotes monocyte migration89. This differential response can be explained by the celltype specific signalling pathways that are triggered following interaction of lipoxin A4 with its receptor 90. In neutrophils, lipoxin A4 induces changes in the phosphorylation of cytoskeletal proteins resulting in cell arrest 90; by contrast, lipoxin A4 induces mobilization of intracellular Ca2+ in macrophages, which promotes chemotaxis90. Lipoxin A4 generally decreases neutrophil activity, characterized by lower levels of CD11b–CD18 expression, RoS formation and NF-κB activity, as well as decreased synthesis of pro-inflammatory chemokines and cytokines90. In monocytes and macrophages, lipoxin A4 not only stimulates migration but also promotes the non-phlogistic phagocytosis of apoptotic neutrophils and inhibits CXCL8 release91. Resolvins are another class of pro-resolving lipid mediators that were initially identified in exudates from mouse air-pouch models during the spontaneous resolution of inflammation92. They are derived either from eicosapentaenoic acid (ePA) or docosahexaenoic acid (DHA) and termed e-resolvin and D-resolvin, respectively. e-resolvins are produced by neutrophil-derived 5-lipoxygenase following the conversion of lipid precursors by endothelial cells or bacteria86. Resolvin e1 binds ChemR23 on monocytes, macrophages and DCs and attenuates TNF-mediated NF-κB activation, thus forming an anti-inflammatory signalling pathway 93. Ligation of the pro-inflammatory leukotriene B4 receptor BLT1 on neutrophils by resolvin e1 has antagonizing effects93,94. D-resolvins are potent inhibitors of neutrophil transendothelial migration86. owing to the abundance of DHA in neuronal cells, D-resolvins might have an important physiological role in neuronal host defence. The same is true for the related class of DHA-derived lipid mediators known as protectins86. Recently, another anti-inflammatory lipid mediator known as macrophage mediator in resolving inflammation 1 (maresin 1) was identified95. DHA was found to be converted by resident peritoneal macrophages to maresin 1 in a process

involving 12,15-lipoxygenase. Similarly to resolvin e1, in a mouse model of peritonitis maresin 1 was found to decrease neutrophil accumulation but to promote the recruitment of macrophages. Furthermore, maresin 1 induces the uptake of zymosan particles by macrophages and might promote macrophage uptake of dead neutrophils95. Annexin A1 and chemerin-derived peptides provide pro-resolution signals. In resting conditions, neutrophils, monocytes and macrophages contain high levels of annexin A1. Following cell activation, annexin A1 is rapidly discharged96. In macrophages this requires involvement of an ABC transporter system, whereas neutrophil-derived annexin A1 is released from tertiary granules following activation; for example, by chemoattractants. Following release, annexin A1 dampens the accumulation of neutrophils in the tissue by several mechanisms, including by downregulation of transendothelial migration97. Furthermore, annexin A1 promotes neutrophil apoptosis96. Finally, annexin A1 released from apoptotic neutrophils acts on macrophages to promote the phagocytosis and removal of dead neutrophils98. Similar activities have recently been ascribed to chemerin-derived peptides. Although chemerin mainly attracts APCs, carboxy-terminal peptides released following the cleavage of chemerin by cysteine proteases have opposing effects — they block neutrophil and monocyte tissue infiltration and prevent the release of pro-inflammatory mediators from classically activated macrophages99. In addition, chemerin-derived peptides promote the clearance of dead neutrophils by macrophages100. Chemokine inactivation dampens neutrophil recruitment. In addition to inhibiting neutrophil recruitment and promoting the non-phlogistic uptake of dead neutrophils by macrophages, another inflammationresolving pathway has been described for resolvin e1 and protectin D1. Both increase the surface expression of CCR5 on apoptotic neutrophils101 and this leads to the clearance of pro-inflammatory chemoattractants, such as CCL3 and CCL5, from the site of inflammation and termination of inflammatory cell influx (TABLE 1). Recently, it was found that macrophage-specific MMP12 dampens the influx of neutrophils in vivo in a mouse model102, suggesting that MMPs might terminate neutrophil recruitment (TABLE 1). MMP12 specifically cleaves CXC-chemokines in the ELR motif, which is crucial for receptor binding. Chemokines modified in this way lose their neutrophil-recruiting activity. MMP-dependent chemokine cleavage also affects the expression of CC-chemokines that preferentially attract classical monocytes. CCL7 is a physiological substrate for MMP2 (REF. 103); cleaved CCL7 continues to bind chemokine receptors CCR1, CCR2 and CCR3, but fails to induce downstream signalling and chemotaxis103,104. Instead, modified CCL7 acts as a general chemokine antagonist and dampens inflammation103,104. A similar function was recently shown for CCL2, CCL8, and CCL13 following cleavage by MMP1 and MMP3 (REF. 104).



REVIEWS Macrophages send live and die signals to neutrophils. Neutrophils are short-lived cells, but migration towards the site of injury or infection exposes neutrophils to survival signals that increase their life-span. IL-1β, granulocyte colony-stimulating factor (G-CSF) and granulocyte–monocyte colony-stimulating factor (GM-CSF) are released by macrophages and can delay neutrophil apoptosis 16. However, macrophage-derived factors do not always extend neutrophil lifespan. In inflamed sites macrophages are the main source of TNF, and this cytokine shows divergent, dose-dependent effects on neutrophil survival. Although low concentrations of TNF prevent neutrophils from dying, higher concentrations promote neutrophil apoptosis105. Monocytes and macrophages also display membrane-bound TNF, which can induce neutrophil death and therefore promote resolution of inflammation106,107.

‘Find me’ signal A signal emitted by dying cells to promote the recruitment of scavenger cells that clear the apoptotic cell body.

‘Eat me’ signal A signal emitted by dying cells to facilitate their recognition and phagocytosis.

Electrotaxis Movement of an organism or a cell in response to stimulation by electric charges.

Apoptotic neutrophils operate negative feedback loops. Apoptotic neutrophils can themselves produce signals that abrogate further neutrophil infiltration. one pathway initiated here involves the release of annexin A1 as detailed above. Lactoferrin released by apoptotic cells is another negative regulator of neutrophil recruitment108. It also acts as a chemoattractant for cells that phagocytose apoptotic neutrophils109 underlining its inflammationresolving properties. Neutrophils store lactoferrin in secondary granules, but lactoferrin can also be synthesized de novo following neutrophil emigration75. Lactoferrin is released during neutrophil extravasation or when the cell undergoes apoptosis and may localize in neutrophil extracellular traps (NeTs)110,111 (BOX 2). Complementary to the inhibition of neutrophil influx, a second feedback loop that might be more powerful in abolishing continued extravasation is the regulation of granulopoiesis under inflammatory conditions. Several years ago, it was shown that IL-17 levels are severely increased in mice deficient in adhesion molecules necessary for neutrophil influx, such as CD18 (REF. 112). The resulting neutrophilia was therefore associated with high levels of IL-17, which is known to stimulate granulopoiesis through G-CSF112. IL-17 synthesis by CD4+ T cells, γδ T cells, and natural killer T cells is partially controlled by IL-23, which is produced by macrophages and DCs113. Increased IL-23 production in CD18-deficient mice was shown to be the underlying reason for augmented IL-17 levels. These findings suggest a model of granulopoiesis driven by a cytokine cascade, which starts with macrophage IL-23 secretion. IL-23 promotes IL-17 production by T cells and this leads to increased G-CSF levels and neutrophil recruitment. Recruited neutrophils undergoing apoptosis are phagocytosed by macrophages, resulting in downregulation of macrophage IL-23 secretion. This then leads to decreased downstream production of IL-17 and G-CSF production, which curbs granulopoiesis112. Dying neutrophils attract their scavengers. In recent years, several apoptotic cell-derived ‘find me’ signals have been identified. Among these, are S19 ribosomal protein dimer, split tyrosyl-tRNA synthetase, thrombospondin 1

and lysophosphatidylcholine (which has received much attention114). In an in vitro migration assay, apoptotic cells secreted lysophosphatidylcholine and attracted monocytes and macrophages in a caspase 3-dependent manner 114. In this context, it should also be noted that lysophosphatidylcholine has been identified as an ‘eat me’ signal on the apoptotic cell surface, recognized by naturally occurring IgM antibodies115. The model of monocyte recruitment in response to lysophosphatidylcholine from apoptotic cells suggests that a calcium-independent phospholipase A2 (iPLA2) is activated in apoptotic neutrophils in a caspase 3-dependent manner 114. Activated iPLA2 subsequently hydrolyses phosphatidylcholine, yielding arachidonic acid and lysophosphatidylcholine. In turn, lysophosphatidylcholine is externalized and secreted by an unknown mechanism114,115. Two putative receptors for lysophosphatidylcholine have been identified on monocytes, the G protein-coupled receptors G2A and GPR4. The chemotactic activity of lysophosphatidylcholine is mediated through activation of G2A, but not through the related receptor GPR4 (REF. 116). Lipid mediators are not the only type of chemoattractant molecule to be released from dying cells; proteins and nucleotides also attract scavenger cells. It was recently shown that the release of the nucleotides ATP and uTP from dying cells could induce the recruitment of monocytes and macrophages117. ATP and uTP are recognized by P2Y2, a G protein-coupled nucleotide receptor that mediates, among other functions, cell migration. In addition, dying cells release CX3CL1 and this chemokine attracts macrophages to sites of cell death118. The receptor for CX3CL1, CX3CR1, is expressed by both classical and non-classical monocyte subsets, but has been shown to be particularly important for the extravasation of non-classical monocytes. In conclusion, the data suggest that several classes of molecules are involved in recruiting scavenger cells. The relative importance of these molecules is yet to be explored. More recently, it has been shown that changes in the membrane composition of apoptotic cells (specifically, negative surface charges) are signals that attract scavenging cells119. Although such a mechanism has only been established for epithelial cells, it could be anticipated that apoptotic neutrophils might also generate such electric potential leading to electrotaxis of scavengers. Taken together, neutrophils undergoing apoptosis release lipid mediators, proteins and nucleotides that attract macrophages, the scavengers of dead neutrophils.

Phase IV: macrophages restore homeostasis uptake of apoptotic neutrophils can stimulate macrophages to release mediators that suppress the inflammatory response. early studies showed that incubation of LPS-activated monocytes with apoptotic lymphocytes inhibited monocyte release of TNF, but their production of the anti-inflammatory cytokines transforming growth factor-β (TGFβ) and IL-10 was increased120. Apoptotic neutrophils were found to have a similar effect on monocytes and macrophages121,122. Although the molecular mechanisms involved in TGFβ expression

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REVIEWS remain poorly understood, they seem to be tightly linked to events induced by apoptotic cell contact with macrophages and the subsequent transcription and translation of TGFβ. Phagocyte recognition of apoptotic cells involves a complex molecular array, sometimes referred to as the ‘engulfment synapse’123. Apoptotic cells display eat-me signals by exposure of apoptotic cell-associated molecular patterns such as phosphatidylserine, which is the best-studied marker of apoptosis123. Phagocytes express various receptors that might bind either directly to the exposed phosphatidylserine or indirectly though bridging molecules. These receptors include a proposed phosphatidylserine receptor, a tyrosine kinase receptor MeR, scavenger receptors, integrins and complement receptors123–128. Thrombospondin, collectins, C1q and annexin A1 serve as bridging molecules128. Lipoxin A4 is another mediator produced during the resolution and repair stage of inflammation that can induce TGFβ in resolving exudates and is therefore involved in promoting resolution of inflammation and tissue fibrosis. Phagocytosis of apoptotic cells not only prevents activated macrophages from killing tissue-resident cells but also triggers the release of vascular endothelial growth factor (veGF) and other growth factors that are crucial for repair. However, although the release of TGFβ, IL-10 and PGe2 can contribute to restoration of homeostasis after tissue injury, these anti-inflammatory mediators also dampen antibacterial mechanisms. In this context, it has been shown that macrophage-mediated clearance of apoptotic cells promotes intracellular parasite persistence, indicative of an immunosuppressive effect 129. Recent data provide a potential mechanism underlying this effect: ingestion of apoptotic cells by alveolar macrophages suppresses their ability to phagocytose and kill bacteria in a pathway involving macrophage cyclooxygenases130. Therefore, the timing of the initiation of the repair process is crucial for the outcome: resolving the inflammatory response too early might prolong microbial infection.

Conclusion The coordinated interplay between macrophages, neutrophils and monocytes is crucial for the effective elimination of noxious agents and the restoration of tissue homeostasis after injury or infection. Initial phagocyte interactions

1. 2. 3. 4.

5.

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promote mutual recruitment and antimicrobial activities, but later cross-talk between phagocyte populations dampens pro-inflammatory responses and promotes resolution of the inflammatory response. The mechanisms detailed here might stimulate the development of new anti-inflammatory and pro-resolving therapeutic interventions aimed at modulating phagocyte function. Targeting individual neutrophil granule proteins could be an elegant approach to control the degree of inflammation. In this context, insight into neutrophil– macrophage interactions has recently led to the synthesis of innate defence regulator 1 (IDR1), a peptide that has similar activities to defensins and the cathelicidin LL-37 (REF. 131). Despite a lack of direct antimicrobial effects, this peptide activates antimicrobial function in macrophages131. A contrasting approach is to inhibit neutrophil degranulation in acute inflammatory processes, thereby preventing over-zealous immune activation such as that seen during sepsis. So far, there have been no attempts to specifically antagonize LL-37, azurocidin or human neutrophil defensin peptides. This is somewhat surprising, as there has recently been a great deal of interest in targeting the endothelial deposition of platelet chemokines that promote monocyte adhesion132,133. In terms of resolution, apoptosis of neutrophils holds a central position as it brings sustained neutrophil recruitment to an end while the phagocytic clearance of apoptotic neutrophils reprogrammes macrophages to an anti-inflammatory phenotype. Therefore, the induction of neutrophil apoptosis might be an interesting strategy with which to terminate inflammation. Inhibition of cyclin-dependent kinases overrides signals from neutrophil survival factors such as GM-CSF, thereby promoting resolution of inflammation134. In a complementary approach, it has repeatedly been shown that pro-resolving lipid mediators such as lipoxins, resolvins, protectins and maresins promote the return to tissue homeostasis135,136. An in-depth understanding of the phagocyte circuits that control initiation, progression and resolution of inflammation will be important for therapeutic interventions. Decoding the complex interactions between phagocytes will allow us to create specific and custom-made therapies with limited side effects. As such, research addressing the complex and finely balanced nature of the phagocytes of the innate immune system warrants more attention.

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Acknowledgements

The authors’ research is supported by the Deutsche Forschungsgemeinschaft (SO876/3-1, SO876/4-1 and FOR809), the German Heart Foundation and German Foundation of Heart Research, the START programme within the Faculty of Medicine at the RWTH Aachen University, the Swedish Research Council, the Swedish Heart-Lung Foundation, Swedish Foundation for Strategic Research and Karolinska Institutet.

Competing interests statement

The authors declare no competing financial interests.

DATABASES UniProtKB: http://www.uniprot.org annexin A1 | azurocidin | BLT1 | BLT2 | CAP18 | cathepsin G | CCL2 | CCL3 | CCL4 | CCR2 | CCR6 | CD24 | CX3CR1 | CXCL8 | E-selectin | FPR1 | FPR2 | G-CSF | GM-CSF | HMGB1 | ICAM1 | ICAM2 | IL-1β | MMP8 | MMP9 | MMP12 | P-selectin | PSGL1 | RAGE | SAP130 | TLR2 | TLR4 | TLR9 | VCAM1

FURTHER INFORMATION Oliver Soehnlein’s homepage: http://www.imcar.rwthaachen.de/team/oliver-soehnlein/ Lennart Lindbom’s homepage: http://ki.se/ki/jsp/polopoly. jsp?d=2625&l=en All lInkS ARe ACTIve In The onlIne Pdf
