Within tissues the mononuclear phagocytes undergo maturation, adapt to their local microenvironment, and differentiate into various cell types (Fig. 7.1). Distinctive populations of resident macrophages are found in most tissues of the body; they differ in their life span, morphology, and phenotype, for example the microglial cells in the brain appear quite unlike mononuclear phagocytes in other tissues (Fig. 7.2). Resident cells have usually ceased to proliferate, but may remain as relatively long-lived cells, with low turnover, unlike neutrophils.

Fig. 7.1 Differentiation of mononuclear phagocytes

Mononuclear phagocytes and some dendritic cells differentiate from a precursor in the bone marrow. Differentiation into monocytes and tissue macrophages is driven by M-CSF. Inflammatory dendritic cells and Langerhans’ cells also appear to belong to this lineage although Langerhans’ cells may be directly bone-marrow derived. They are effective in presenting antigen to naive T cells. Some of the populations, indicated by a circular arrow, have some capacity for self renewal.

Fig. 7.2 Microglia in mouse brain stained for F4/80

Microglia are widely distributed in non-overlapping fields and show a dendritic morphology – mouse brain stained with antibody to F4/80.

(Courtesy of Dr Payam Rezaie.)

M-CSF is required for macrophage differentiation

Macrophage colony stimulating factor (M-CSF) is a major growth, differentiation, and survival factor, selective for monocytes and macrophages. It is produced constitutively by fibroblasts, stromal cells, endothelial cells, macrophages and smooth muscle. Most members of the mononuclear phagocyte family express the receptor for M-CSF, CD115 and mice deficient in M-CSF have major defects in different macrophage populations, including osteoclasts.

In contrast, granulocyte-macrophage colony stimulating factor (GM-CSF) primarily regulates myeloid cell production. It is only produced after cell activation and mice deficient in GM-CSF have no major defects in macrophage populations, with only the maturation of alveolar macrophage being altered leading to pulmonary alveolar proteinosis.

Macrophage populations have distinctive phenotypes

Some of the key features of resident macrophage populations in tissues are shown in Figure 7.w1. The study of the heterogeneity and distribution of mononuclear phagocytes has been made possible through the use of antibodies against differentiation markers. These markers can be located at the plasma membrane or endosomal compartments. In the mouse the F4/80 antigen and macrosialin (CD68) have proved useful in defining the distribution of mature macrophages in many (but not all) tissues. Differentiation antigens such as sialoadhesin, a lectin-like receptor for sialylated glycoconjugates, are particularly strongly present on populations of macrophages in lymphoid organs that do not express F4/80 or CD68. Macrophages expressing sialoadhesin are exposed to the blood and lymph and are thought to be involved in antigen delivery. In humans, the CD68 antigen, the human homolog of macrosialin, is widely expressed in macrophages while the F4/80 homolog EMR2 labels subsets of macrophages. Anatomical differences between mouse and human spleen correlate with different distribution of macrophage differentiation markers. The mannose receptor is expressed by macrophages in the red pulp of mouse spleen but is absent from these cells in human spleen.

Fig. 7.w1 Resident tissue macrophage populations

Resident tissue macrophage populations.

Comparing the differentiation markers expressed by macrophages in tissues with those expressed by macrophages differentially stimulated in vitro, allows researchers to propose hypotheses regarding the role of different macrophage populations in health and disease. For instance, alternatively activated macrophages were first characterized in vitro by studying macrophages treated with IL-4 in culture; the markers identified in IL-4/IL-13 treated cells together with additional markers discovered in the case of parasite infection are being used to investigate the acquisition by macrophages of an alternative activation profile in tissues. Interestingly, macrophages with characteristics of alternative activation are being detected during allergic responses, nematode infection and during wound healing. Differences in activation markers between human and mouse macrophages need to be taken into consideration when translating results obtained with preclinical models into the clinical setting.

Some of the important stimuli that modulate macrophage phenotype are listed in Figure 7.w2 and some of their characteristic cell surface receptors in Figure 7.w3.

Fig. 7.w2 Environmental influences on macrophages

Mediators that act on macrophages.

Fig. 7.w3 Macrophage 7tm G protein-coupled receptors for chemotactic stimuli

Macrophages express a variety of receptors for chemotactic agents. Fractalkine (CX3CL1) is unique as a transmembrane ligand – its receptor, CX3CR1, and CCR2 may be differentially expressed by monocytes, giving rise to inflammatory and resident macrophages.

The tissue environment controls differentiation of resident macrophages

It is possible to reconstruct a constitutive migration pathway in which monocytes become endothelial-like and line vascular sinusoids, as in the liver (Kupffer cells, see Fig. 2.3), or penetrate between endothelial cells. They underlie endothelia or epithelia or enter the interstitial space or serosal cavities (Fig. 7.3). The molecular mechanisms of constitutive macrophage distribution and induced migration are beginning to be defined, and involve cellular adhesion molecules, cytokines, and growth factors, as well as chemokines and chemokine receptors.

Fig. 7.3 Differentiation and distribution of macrophages

Blood monocytes are derived from bone marrow and enter tissues initially as extravascular macrophages or tissue macrophages which may subsequently migrate into the serous cavities. At sites of inflammation monocytes may develop into elicited inflammatory macrophages or dendritic cells which can migrate through afferent lymph to the local lymph nodes.White arrows indicate recruitment during steady state conditions. Red arrows denote recruitment during inflammation.

Mature macrophages are themselves part of the stromal microenvironment in bone marrow. They associate with developing hematopoietic cells to perform poorly defined non-phagocytic trophic functions, as well as removing effete cells and erythroid nuclei. In bone, osteoclasts, highly specialized multinucleated cells of monocytic origin, mediate bone resorption and their deficency leads to osteopetrosis.

Secondary lymphoid organs contain several distinct types of macrophages. These macrophages have been better characterized in the mouse (Fig. 7.4) and subsets involved in the clearance of apoptotic lymphocytes (tingible body macrophages) or presentation of naive antigens to B cells (subcapsular sinus macrophages) have been identified. Anatomical differences between human and mouse spleen, such as the absence of a well defined marginal sinus, correlates with phenotypical differences in splenic macrophages (see Fig. 7.4)

Fig. 7.4 Macrophages in secondary lymphoid tissues

Heterogeneity of macrophages in secondary lymphoid organs of mouse and human. (1) Red pulp of spleen stained for F4/80. Macrophages stain strongly positive. (2) Mouse spleen stained with antibody to sialoadhesin. The marginal metallophils of spleen are strongly sialoadhesin (CD169) positive. (3) Human spleen stained for CD68 and (4) immunofluorescence staining with anti-CD68 (green), and for the mannose receptor (red) indicates staining along sinusoids but most CD68+ macrophages lack the mannose receptor. Nuclei are stained blue.

((1) Courtesy of Dr DA Hume. (2) Courtesy of Dr PR Crocker.)

Macrophages can act as antigen-presenting cells

Although macrophages are often regarded as sessile cells, they readily migrate to draining lymph nodes after an inflammatory stimulus and become arrested there. They are therefore absent from efferent lymph and do not, as a rule, re-enter the circulation.

Q. Why is there no advantage for the immune system in the recirculation of macrophages, whereas recirculation of lymphocytes is a central element in immune defense?

A. Macrophages do not develop an immunological memory, and do not undergo the selective clonal expansion of antigen-stimulated lymphocytes that occurs in lymphoid tissues. Therefore there is no advantage in having macrophages return to the circulation from lymphoid tissues.

Macrophages, like dendritic cells, have all the machinery required for antigen processing and presentation of exogenous peptides and endogenous peptides on MHC class II and class I, respectively. Cross-presentation, a process by which peptides of exogenous origin are presented on MHC class I, also takes place in macrophages. While dendritic cells are uniquely suited for stimulating naive T cells in secondary lymphoid organs, macrophages present antigen in the periphery to activated (already primed) T cells. This interaction makes macrophages important effector cells during adaptive immunity (Fig. 7.5). The specialization of dendritic cells for antigen presentation correlates with a reduced degradative capacity that facilitates the generation of MHC-peptide complexes.

Fig. 7.5 Comparison of the functions of macrophages and dendritic cells

Comparison of the functions of macrophages and dendritic cells.

Macrophages act as sentinels within the tissues

Macrophages react to a wide range of environmental influences that help to fulfill their role as sentinels of the innate immune system (Fig. 7.6). The presence of cells within tissues with the potential to initiate inflammation through the release of cytokines and chemokines and to cause tissue damage through the production of reactive oxygen species requires control systems capable of downmodulating macrophage activation. One of these systems involves the molecule CD200L, which is an inhibitory receptor expressed by myeloid cells. CD200L inhibitory signaling is triggered through interaction with CD200 expressed by non-hematopoietic cells as well as macrophages. The CD200–CD200L interaction is important for the control of macrophage activation by other cells present in tissues.

Fig. 7.6 Macrophage phenotype and function

Macrophage phenotype and function are modulated by the cytokines shown left. And they can sense their environment via opsonic receptors for antibody (FcR) and complement C3b (CR3) and via pattern-recognition receptors. They are also negatively regulated via CD200 and SIRPα (signal regulatory protein-alpha). Macrophages secrete cytokines and eicosanoids (prostaglandins and leukotrienes) and may release reactive oxygen and nitrogen intermediates (ROIs, RNIs) at sites of inflammation.

Phagocytosis and endocytosis

Soluble compounds are internalized by endocytosis

Macrophages play a key role in the clearance of altered forms of lipoproteins (e.g. acetylated lipoproteins) or glycoproteins (e.g. asialylated mannosylated proteins or proteins bearing advanced glycan products), damaged or apoptotic cells, pollutant particles and microbes. For this purpose, macrophages have a highly developed endocytic compartment that mediates the uptake of a wide range of stimuli and targets them for degradation in lysosomes. Soluble compounds are internalized through fluid phase or receptor-mediated endocytosis (see below) leading to the generation of vesicles called endosomes. Endosomes mature by fusing with different endocytic vesicles from the early and late endocytic compartments and finally the endocytosed material is targeted to lysosomes. This endosomal trafficking is also characterized by a reduction in the luminal pH which facilitates the action of acid hydrolases and degradation of the endosomal content.

Large particles are internalized by phagocytosis

Phagocytosis involves the uptake of particulate material (>0.5 μm) after recognition by opsonic or non-opsonic receptors (see Fig 7.6), its engulfment through the generation of pseudopodia and the formation of phagosomes. Phagosomes follow a similar maturation process to endosomes through the fusion with components of the early and late endocytic compartments so that maturing phagosomes sequentially adopt characteristics of early and late endosomes; this process culminates in the fusion of phagosomes to lysosomes to form phagolysosomes (Fig. 7.7). Phagosomal maturation is accompanied by acidification of the lumen (from 6.1–6.5 in early phagosomes to 4.5 in phagolysosomes), which controls membrane traffic and has a direct effect on microbial growth. Other microbicidal mechanisms associated with phagosome maturation are the generation of reactive oxygen and nitrogen species and the presence of antimicrobial proteins and peptides.

Fig. 7.7 Phagocytosis mediated by opsonic receptors

(1) Pathogens, such as bacteria or fungi, are taken up by binding to opsonic receptors including the Fc receptor, complement receptors, and receptors for carbohydrate e.g. mannose receptor (MR) or dectin-1. (2) The particle is engulfed and the phagosome forms (3). Acidification of the phagosome follows as toxic molecules (reactive oxygen and nitrogen intermediates) are pumped into the phagosome. The marker CD68 is located in the phagosome membrane. (4) Lysosomes fuse with the phagosome, releasing proteolytic enzymes into the phagolysosome, which digest the pathogen. (5) On completion the membrane of the phagolysosome is disrupted. Antigenic fragments may become diverted to the acidic endosome compartment for interaction with MHC class II molecules and antigen presentation. The process induces secretion of toxic molecules and cytokines.

Host cells control the phagocytic activity of macrophages by displaying the ‘don’t eat me’ signal CD47. CD47 engages a receptor in macrophages called SIRPα that through its immunoreceptor tyrosine-based inhibitory motif (ITIM) motif inhibits the uptake process. CD47 expression by tumor cells has been proposed as an immunosurveillance escape mechanism.

Macrophages sample their environment through opsonic and non-opsonic receptors

Macrophages are endowed with a wide range of receptors that act as sensors of the physiological status of organs, including the presence of infection. These receptors can be categorized as opsonic or non-opsonic depending on their capacity to interact directly with the stimuli or their need for a bridging molecule such as antibody or fragments of the complement component C3, which act as opsonins.

Opsonic receptors require antibody or complement to recognize the target

Bacteria opsonized by C3 fragments or antibody engage complement receptors (CR) or Fc receptors (FcR). CR-dependent phagocytosis is not an automatic process but requires additional stimulation such as inflammation. Monocytes and macrophages express a range of receptors (CR1, CR3, CR4) for C3 cleavage products that may become bound to pathogens, immune complexes or other complement activators. The role of CR3 role in regulated phagocytosis has been well studied, and the mechanism of CR3-mediated ingestion differs strikingly from that mediated by Fc receptors.

Q. What other function does CR3 have, unrelated to its role as an opsonic receptor?

A. CR3 is an adhesion molecule, which also has a role in cell migration by binding to ICAM-1 on endothelium or fibrinogen in the extracellular matrix (see Fig. 6.13).

FcRs belong to the immunoglobulin superfamily (see Fig. 3.17). The best characterized FcR is CD64 (FcγRI), the high affinity receptor for IgG which signals through the common γ chain that contains an immunoreceptor tyrosine based activation motif (ITAM). The common γ chain is also used by some non-opsonic receptors that bind carbohydrates (see below) and it signals through the key kinase Syk. In humans other activating receptors for IgG are low affinity FcγRIIa (CD32) and FcγRIII (CD16), which require the recognition of immune complexes for inducing internalisation. IgG-opsonized material is readily internalized by macrophages and leads to the production of reactive oxygen species and cellular activation. The activating effect of ITAM-associated FcγRs is regulated by the presence of the inhibitory form of CD32 (FcγRIIb), which bears an ITIM.

The mechanism for ingestion of antibody-coated particles is distinct from that mediated by CR3 (Fig. 7.8). FcR-mediated uptake proceeds by a zipper-like process where sequential attachment between receptors and ligands guides pseudopod flow around the circumference of the particle. In contrast, CR3 contact sites are discontinuous for complement-coated particles which ‘sink’ into the macrophage cytoplasm. Small GTPases play distinct roles in actin cytoskeleton engagement by each receptor-mediated process.

Fig. 7.8 Zipper model of phagocytosis

(1) During phagocytosis, receptor–ligand interactions guide the extension of tightly apposed pseudopods around the particle’s total circumference until a fusion of the plasma membrane occurs at the tip. This is known as the zipper mechanism. Alternative ‘trigger’ mechanisms, in which spacious phagosomes result from flipping over of ruffles back onto the plasma membrane, have also been described. The cytoskeleton of phagocytes plays a key role in engulfment, during which there is extensive remodeling of actin filaments. Some microorganisms and intracellular parasites induce novel mechanisms to recruit cell membranes during entry into phagocytes. (2 and 3) Electron micrographs of ingestion of antibody (IgG)-coated sheep erythrocytes by peritoneal macrophage by the zipper mechanism.

(Scanning electron micrograph (2), courtesy of Dr GG MacPherson. Transmission electron micrograph (3), courtesy of Dr SC Silverstein.)

The best characterized non-opsonic receptors are the Toll-like receptors (TLRs)

Non-opsonic receptors or pattern recognition receptors (PRRs) recognize unusual features characteristic of damaged, malfunctioning or infected tissues and their general characteristics are described in Chapter 6.

TLRs are membrane glycoproteins with an extracellular region responsible for ligand binding and a cytoplasmic domain responsible for triggering an intracellular signaling cascade. They can form hetero- or homo-dimers with each other or complex with other receptors in order to detect a wide range of microbial components. They are located at the cell surface or within endosomes. In humans there are 10 of these receptors and together they are able to recognize a wide range of microbes including Gram-positive bacteria and mycobacteria (see Fig. 6.20). For example, TLR4 detects Gram-negative bacteria because of its ability to recognize endotoxin. It then signals to the cell using similar systems to those mediated by IL-1 (Fig. 7.9). It can also activate the macrophage by a second pathway that is initiated by Trif, which leads to a secondary production of IFNβ and autocrine activation of additional macrophage genes.

Fig. 7.9 Activation of macrophages by lipopolysaccharide and IL-1

LPS binds leukocyte CD14 (which is GPI-anchored to the membrane) through LPS binding protein (LPS-BP) and interacts with transmembrane TLRs, by transferring LPS to MD-2 to initiate signal transduction. Signaling pathways of the receptor share elements with the IL-1R pathway (e.g. IRAK – IL-1R-associated kinase). Cell activation proceeds via both the mitogen-activated protein (MAP) kinase pathways and the induction of NF-κB. (IL-1R, IL-1 receptor.)

CD14 is a GPI-linked membrane protein that facilitates the recognition of LPS by TLR4 so that it increases LPS sensitivity (see Fig. 7.9). Recently CD14 has also been shown to facilitate recognition of ligands by TLR2 and TLR3, which opens the possibility of CD14 acting as a multifunctional adaptor protein.

TLR4 also recognizes degraded extracellular matrix and the nuclear protein high mobility group 1 protein (HMGB1) which can be released by necrotic cells, an example of damage-associated molecules.

TLRs activate macrophages through several different pathways

The key adaptor molecules that mediate TLR-signalling are MyD88 (Fig. 7.w4) and Trif, both of which interact with the TIR cytoplasmic domain of TLRs. Most TLRs signal either through MyD88 or Trif, but TLR4 is unique in its ability to signal through both. The range of responses elicited by TLRs in macrophages is vast and includes the activation of the NFκB signaling pathway (see Fig. 7.w4) and MAP kinases, responsible for the production of proinflammatory cytokines and induction of microbicidal mechanisms. Signaling through the IL-1 and IL-18 receptors is also mediated by MyD88.

Fig. 7.w4 The nuclear factor kappa B activation pathway

Activation of NF-κB can be induced by ligation of a number of cell surface receptors including the Toll-like receptors (TLRs) illustrated. Apart from MyD88, a small number of other adapters transmit signals from the TLRs to cause NF-κB activation. An intracellular signaling cascade leads to the activation of inhibitor of kappa B kinase (IKK). NF-κB is normally retained in the cytosol by interaction with an inhibitor (I-κB). Phosphorylation of I-κB by IKK leads to dissociation and degradation of the inhibitor and NF-κB translocates to the nucleus to act as a transcription factor that activates a set of genes involved in inflammatory reactions.

The Trif pathway induces IRF transcription factors that will lead to the production of type 1 interferons, which can then stimulate the macrophage to cause a second, delayed wave of gene activation (Fig. 7.w5).

Fig. 7.w5 Activation of the IFNβ gene by Toll-like receptors

TLRs can activate the IFNβ gene via a pathway that is independent of MyD88, but involves activation of the transcription factor interferon regulatory factor-3 (IRF-3). Released IFNβ acts on type 1 IFN receptors causing the aggregation of the two subunits of the receptor. This leads to activation and phosphorylation of two Jak kinases, Jak1 and Tyk2. These two transcription factors form a complex with a DNA-binding protein, P48. The complex moves to the nucleus and induces transcription of genes bearing an IFN-specific response element (ISRE), such as the chemokine CXCL10.

Lectin and scavenger receptors are non-opsonic receptors that recognize carbohydrates and modified proteins directly

In the plasma membrane, members of the scavenger receptor and lectin families mediate the recognition of modified lipoproteins and carbohydrates. Most of these receptors have signaling and internalisation motifs in their cytoplasmic region and are capable of mediating endocytosis and phagocytosis in isolation but their role is largely confined to the fine-tuning of TLR signaling.

Scavenger receptors (SR) (Fig. 7.10) such as SR-A are involved in LPS clearance, and may serve to downregulate responses induced via the TLR4-CD14 pathway (see Fig. 7.9) and therefore limit the systemic release of TNFα and resultant septic shock. SR-A has also been involved in bacterial uptake. Another member of this family, CD36, collaborates with TLR2 in the recognition of S. aureus and M. tuberculosis.

Fig. 7.10 Class A and related scavenger receptors

Selected scavenger receptors are shown. Scavenger receptors of macrophages are responsible for the uptake of apoptotic cells, modified lipoproteins, and other polyanionic ligands (e.g. LPS and lipoteichoic acids [LTA]), as well as selected bacteria such as Neisseria spp. CD163 is involved in endocytosis of hemoglobin–haptoglobin complexes.

Dectin-1 (Fig. 7.11), a lectin with a single lectin-like domain and an intracellular ITAM-like motif, is highly specific for the fungi-derived compound β-glucan (Fig. 7.12). Dectin-1-mediated effects are largely mediated by the kinase, Syk and Card 9 Dectin-1 mediates phagocytosis of β-glucan particles and synergizes with TLRs to boost immune responses. It also has a role in Th cell differentiation and β-glucan treated dendritic cells promote the development of TH17 cells. Humans deficient in dectin-1 are more susceptible to mucosal candidiasis.

Fig. 7.11 Lectin receptors

The mannose receptor contains eight C-type lectin domains involved in binding to mannosylated carbohydrates and related glycoconjugates. It is related to a more broadly expressed endocytic receptor, ENDO 180. A distinct lectin domain located in the distal (C terminal) segment binds sulfated glycoconjugates. The β-glucan receptor (dectin-1) contains a single lectin domain and an intracellular immunoreceptor tyrosine-based activation motif (ITAM). Both dectin-2 and MINCLE recognize mannose and are associated with the common γ-chain that is associated with Fc receptors. DC-SIGN recognizes mannose and fucose through four associated lectin domains. ‘+’ indicates charged amino acid residues.

Fig. 7.12 Zymosan particles phagocytosed by a macrophage

The micrograph shows zymosan (yeast) particles phagocytosed by a macrophage, a process dependent on dectin-1. Truncation of the cytoplasmic tail prevents phagocytosis.

The mannose receptor (MR) may play a unique role in tissue homeostasis as well as host defense (see Fig. 7.11). Endogenous ligands include lysosomal hydrolases and myeloperoxidase. The N terminal cysteine-rich domain of the MR is a distinct lectin for sulfated glycoconjugates highly expressed in secondary lymphoid organs. The cysteine-rich domain also contributes to the clearance of hormones such as lutropin. MR can internalize collagen, which is recognized through the fibronectin type II domain and recent evidence suggests that MR promotes TH2 responses, which correlates with its capacity to interact with multiple glycosylated allergens and secreted helminth products.

DC-SIGN, another mannose-binding C-type lectin (see Fig. 7.11), is expressed on some macrophages. It forms tetramers and lacks obvious signaling motifs at its cytoplasmic region. It has been implicated in interactions between APCs and T cells and in microbial recognition. DC-SIGN has been shown to modulate TLR signaling to promote transcription of various cytokine genes, particularly IL-10 and IL-8.

Other lectin receptors are langerin and dectin-2, which have mannose specificity, and Mincle, which recognizes ligands expressed by necrotic cells in addition to fungal pathogens. Dectin-2 and Mincle (see Fig. 7.11) signal through the common γ-chain that also mediates signaling by the FcR CD64.

DCIR is the only single lectin receptor bearing an ITIM. Animals deficient in DCIR have altered DC numbers and increased susceptibility to autoimmune diseases.

Cytosolic receptors recognize intracellular pathogens

Cytosolic receptors include two families of molecules that recognize intracellular bacteria and viruses:

• the nucleotide binding and oligomerization domain (NOD)-like receptors (NLR) recognize, among others, bacterial compounds such as peptidoglycan; and

• the retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) recognize nucleic acids such as dsRNA which are produced during viral replication (Fig. 7.13).

Fig. 7.13 Intracellular PRRs

Viral nucleic acids are recognized by RIG-1 and MDA-5, which assemble with the adapter protein IPS-1 onto the mitochondrial outer membrane and activate the transcription factors IRF3 and NFκB, which induce genes involved in inflammation. Bacterial peptidoglycans are detected by NOD-1 and NOD-2, which signal through RICK to activate NFκB and the MAP-kinase pathway. Molecules of this type form inflammasomes that can lead to the induction of caspases and production of IL-1β and, IL-18.

Some of the NLRs form part of a multi-protein complex, the inflammasome, which is assembled in the cytoplasm and triggers inflammatory cell death (pyroptosis) of the infected cell. Pyroptosis causes release of cell contents and induces inflammation. Caspase I also processes the precursors of IL-1 and IL-18 to produce the active inflammatory cytokines. The composition of the inflammasome varies depending on the initiating stimulus as the NLRs responsible for the formation of the inflammasome complex are activated by different agents.

The two RLH proteins RIG-1 and MDA5 both recognize RNA viruses, but they have different specificity. For example RIG-1 is important in recognition of influenza virus, whereas MDA5 recognizes polio virus. Both, RIG-1 and MDA-5 are involved in the recognition of Dengue virus. There are also cytosolic receptors able to recognize DNA.

Q. What other non-opsonic receptors can recognize products of viral replication?

A. The Toll-like receptors TLR3, TLR7 and TLR8 also recognize RNA molecules (see Fig. 6.20).

Mechanism of action of NLRs

All NLR proteins have a similar domain structure:

• an N-terminal region that is involved in the recruitment of adaptor molecules that link ligand recognition to signal transduction;

• a central nucleotide binding and oligomerization domain (NACHT/NOD) that mediates oligomerization; and

• a C-terminal leucine-rich repeat (LRR) that mediates recognition of PAMPs or DAMPs.

Two key members of the NLR family are NOD-1 and NOD-2, which recognize fragments of peptidoglycan generated during the division of Gram-negative and Gram-positive bacteria (Fig. 7.w6). Detection of peptidoglycan through NOD receptors leads to the recruitment of the adaptor RICK, activation of NFkB and promotion of inflammation (see Fig. 7-w6).

Fig. 7.w6 NOD-1 signal transduction

NOD-1 is an intracellular pattern recognition molecule. Peptidoglycan from, for example, Shigella flexneri and some strains of Escherichia coli binds to the protein. A domain (RICK) then activates IKK (see Fig. 7.w4) leading to the transcription of proteins dependent on NF-κB.

NOD-1 polymorphisms are associated with the development of atopic eczema, asthma, and increased serum IgE concentrations. Three common mutations of amino acid residues near or within the NOD-2 LRR region are genetic risk factors for the development of Crohn’s disease. NOD-2 deficiency leads to increased susceptibility to bacterial infection via the oral route, implying that it may be required for expression of antimicrobial peptides.

Other NLR proteins such as NLRP3, NLRP1 and IPAF, are components of multiprotein complexes called inflammasomes that lead to the activation of inflammatory caspases. Inflammatory caspases mediate the processing of pro-IL-1β and pro-IL-18 into their active counterparts and can lead to inflammation (see Fig. 7.13). Inflammatory caspases also lead to the induction of inflammatory cell death or pyroptosis in which the affected cell dies alerting the immune system of the presence of DAMPs by releasing its contents.

In inflammasomes NLR proteins act as scaffold proteins that trigger caspase activation. The assembly of inflammasomes can be induced by pathogens as well as by host derived molecules, signs of metabolic stress and particles such as uric acid crystals and asbestos.

Mechanisms of action of RLH receptors

The RLH receptors include RIG-1 and MDA-5. These proteins have an RNA helicase domain with ATPase activity which is activated by ligand binding and is necessary for signaling. After binding, RIG-I and MDA5 interact with the signaling-adaptor molecule, IPS-1 (IFN-β promoter stimulator-1), present in the outer membrane of the mitochondria. This unit causes the activation of transcription factors, leading to the expression of IFN-β, IRF3-target genes, and NFκB target genes. RNA recognition by RIG-I requires the presence of a free 5′-triphosphate structure, which allows for differential recognition of non-self/viral RNA versus self-RNAs, as host RNA is either capped or post-translationally modified to remove the 5′-triphosphate.

The composition of the RNA molecule also has a role:

• MDA5 preferentially binds long dsRNAs (greater than 1 kb in length) and poly I:C;

• RIG-I is responsible for binding shorter dsRNA fragments.

Cytosolic DNA is capable of eliciting proinflammatory responses and several pathways can mediate this effect:

• engagement of a DNA receptor;

• detection by RIG-1 of an intermediate RNA molecule produced through the action of RNA polIII;

• activation of the AIM-2 inflammasome. AIM-2 is an IFN-inducible protein and the first example of a non-NLR family member forming an inflammasome scaffold.

Functions of phagocytic cells

Clearance of apoptotic cells by macrophages produces anti-inflammatory signals

To maintain appropriate cell numbers during development, normal tissue homeostasis and pathological responses, cells die naturally by apoptosis, which involves activation of non-inflammatory caspases.

Q. Give an example in the immune system where macrophages phagocytose apoptotic cells

A. Thymocytes that fail the processes of positive and negative selection die by apoptosis and are phagocytosed by thymic macrophages (Chapter 2). Also B cells that die within lymphoid follicles are taken up by tingible body macrophages as described in Chapter 9.

Cellular and biochemical pathways resulting in apoptosis are conserved in evolution and apoptotic cells are rapidly and efficiently cleared by macrophages (Fig. 7.14), although they can also be engulfed by non-professional phagocytes. The appearance of phosphatidylserine (PS) in the outer leaflet of the plasma membrane is characteristic of apoptotic cells. PS recognition by PS-binding proteins stimulates the uptake of apoptotic cells and the production of anti-inflammatory mediators, especially TGFβ, which inhibit production of proinflammatory chemokines and cytokines.

Fig. 7.14 Phagocytosis of apoptotic thymocyte by thymic macrophage

Thymic macrophages phagocytose the large numbers of thymocytes that die by apoptosis during T cell development. The arrow indicates the nucleus of a phagocytosed thymocyte.

There is redundancy in the receptors involved in apoptotic cell recognition. These include a range of scavenger receptors (SR-AI, CD36), T-cell immunoglobulin receptors (Tim) 3 and 4 and stabilin-2, and the complement component C1q which directly recognize the ‘eat-me’ signals displayed by the apoptotic cells.

Inefficient clearance of apoptotic cells may also contribute to autoimmune disorders such as systemic lupus erythematosus, and may explain their association with genetic deficiencies of complement components.

Macrophages coordinate the inflammatory response

Recognition of necrotic cells and microbial compounds by macrophages initiates inflammation

In contrast to the recognition of apoptotic cells, uptake of microbial products and necrotic cells by resident macrophages promotes cellular activation leading to the production of secreted molecules (Fig. 7.15). Recognition of PAMPS and molecules from damaged cells through PRRs leads to the activation of the NFκB pathway (see Fig. 7.w4) and the production of cytokines and chemokines. The activation of cytosolic phospholipase A2 causes the release of arachidonic acid, the precursor of prostaglandins and leukotrienes, through the actions of the cycloxygenase and lipoxygenase pathways, respectively. Proinflammatory prostaglandins control blood flow and vascular dilation and permeability at sites of inflammation. Trafficking of lymphocytes and neutrophils into tissues is induced by leukotriene-B4 (see Figs. 6.17 & 6.18).

Fig. 7.15 Secretory products of macrophages

Macrophages produce a wide range of secreted molecules.

Resident macrophages recruit neutrophils to inflammatory sites

Neutrophils are the first cells recruited to the site of inflammation and play a key role in elimination of the inflammatory insult (Fig. 7.16). Appropriate neutrophil recruitment is essential for successful resolution of the inflammatory response as deficiencies in the clearance of the triggering stimuli will result in chronic inflammation and tissue dysfunction (Fig. 7.17). Although macrophages and neutrophils share the capacity to mediate phagocytosis and intracellular killing, there are key differences between them:

• neutrophils are short lived, store a wide range of antimicrobial polypeptides in intracellular granules and readily produce oxygen radicals;

• resident macrophages are long lived, less microbicidal and cytotoxic, and display a higher degree of specificity which makes them better suited as sentinels of the immune system.

Fig. 7.16 The role of mononuclear phagocytes in inflammation

(1) Tissue-resident macrophages respond to injury by the release of IL-1, TNFα and IL-6, which attract neutrophils from the blood. (2) As the inflammatory reaction develops the release of leukotrienes, prostaglandins and chemokines attracts monocytes and lymphocytes. (3) As the inflammation resolves dead neutrophils are phagocytosed by mononuclear phagocytes and the profile of cytokines switches towards production of IL-10 and TGFβ. (4) The production of lipoxins, protectins and resolvins is associated with restoration of normal function, and termination of the inflammatory response.

Fig. 7.17 Crohn’s disease

A section of the gut wall from a patient with Crohn’s disease, showing the intense inflammation of the tissue (1). The histological section shows a granulomatous reaction with lymphocyte and macrophage infiltration and the formation of giant cells (2).

Activated tissue macrophages produce chemokines CXCL5 and CXCL8 that promote neutrophil recruitment (see Fig. 7.16) and neutrophil extravasation is also promoted through proteolytic cleavage of chemokines by metalloproteases (MMP8 and MMP9), which enhance their chemotactic activity. The primary role of neutrophils in a wound is to eliminate invading pathogens. To aid this goal recruited neutrophils that fail to encounter bacteria in a short period of time will soon release their microbicidal compounds leading to the liquefaction of tissue and the formation of pus. Tissue destruction facilitates bacterial clearance by eliminating collagen fibrils that limit cellular movement.

Monocyte recruitment to sites of inflammation is promoted by activated neutrophils

There is a transition from neutrophil recruitment to monocyte influx that is prompted by the shedding of IL6Rα from the surface of neutrophils after activation. sIL6Rα complexed with IL6, produced by macrophages and endothelial cells, is recognized by gp130 on endothelial cells leading to the expression of VCAM-1 (a ligand for the integrin VLA-4 expressed by monocytes) and CCL2.

Q. What effect will the expression of VCAM-1 and CCL2 have at a site of inflammation?

A. VCAM-1 is an adhesion molecule that binds to the integrin VLA-4 expressed on some monocytes and lymphocytes. CCL2 is a chemokine that attracts monocytes by its interaction with CCR2. Their expression will therefore enhance monocyte and lymphocyte transendothelial migration.

Shedding of IL6Rα by neutrophils is also induced by apoptosis. Additionally, serine proteases released by neutrophils are capable of modifying chemokines to increase their affinity for CCR1 and promote the recruitment of inflammatory monocytes.

Macrophages and neutrophils have complementary microbicidal actions

Macrophages and neutrophils complement each other in the clearance of pathogens. Following extravasation neutrophils release preformed proteins stored in granules, in three phases:

• secretory vesicles loaded with membrane-bound receptors are mobilized to the plasma membrane;

• secondary and tertiary granules containing lactoferrin, lipocalin, lysozyme and LL37 are released. These granules also contain matrix metalloproteases MMP-8, -9 and -25, that digest extracellular matrix and facilitate tissue destruction;

• primary or azurophilic granules containing defensins (see below) and myeloperoxidase are released –myeloperoxidase converts H₂O₂ to hypochlorous acid which reacts with amines to produce anti-bacterial chloramines (Fig. 7.18).

Fig. 7.18 Oxygen-dependent microbicidal activity

(1) An enzyme (NADPH oxidase) in the phagosome membrane reduces oxygen to the superoxide anion (). This can give rise to hydroxyl radicals (^•OH), singlet oxygen (Δg’O₂), and hydrogen peroxide (H₂O₂), all of which are potentially toxic. Lysosome fusion is not required for these parts of the pathway, and the reaction takes place spontaneously following formation of the phagosome. (2) If lysosome fusion occurs, myeloperoxidase (or, under some circumstances, catalase from peroxisomes) acts on peroxides in the presence of halides (preferably iodide). Then additional toxic oxidants, such as hypohalites (HIO, HClO), are generated.

The defensins are a group of highly cationic polypeptides which contribute to antibacterial activities. The defensins:

• are small peptides (30–33 amino acids) found in some macrophages of many species and specifically in human neutrophils, where they comprise up to 50% of the granule proteins;

• form ion-permeable channels in lipid bilayers and probably act before acidification of the phagolysosome;

• are able to kill a range of pathogens, including bacteria (S. aureus, Pseudomonas aeruginosa, E. coli), fungi (Cryptococcus neoformans), and enveloped viruses (Herpes simplex).

Q. What other molecule can generate ion-permeable channels in lipid bilayers?

A. The final component of the lytic complement pathway, C9 (see Chapter 4).

The primary granules also contain BPI (LPS-binding bactericidal permeability increasing protein) and serprocidins that include 3 serine proteases which in addition to their microbicidal activity, cause tissue destruction.

Cytosolic and nuclear components of neutrophils can also contribute to antimicrobial activity – chromatin from neutrophils forms extracellular nets that associate with proteases from the azurophil granules.

Phagocytes kill pathogens with reactive oxygen and nitrogen intermediates

Phagocytes mediate microbial killing through a wide range of mechanisms including acidification of the phagosome, through the formation of a H⁺ ion gradient by V-ATPase. Acidification has direct microbicidal activity and facilitates the action of enzymes that have acidic pH optima. Additionally the H⁺ ion gradient facilitates the extrusion of nutrients needed by the microbes.

Macrophages and neutrophils can also kill pathogens by secreting highly toxic reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) into the phagosome (see Fig. 7.18). The NOX2 NADPH oxidase located at the phagosomal membrane generates ROIs and this property is most prominent in neutrophils. This oxidase transfers electrons from cytosolic NADPH to molecular oxygen releasing O⁻₂ into the lumen. ROIs can interact with macromolecules (for instance through sulfur groups) rendering them inactive. Patients with chronic granulomatous disease lacking essential oxidase components suffer from repeated bacterial infections.

One major difference between resting and activated macrophages is the ability to generate hydrogen peroxide (H₂O₂), and other metabolites generated by the respiratory burst. Whereas neutrophils are readily endowed with microbicidal properties, macrophages require activation through the engagement of activating PRRs, reaching maximal microbicidal activity in the presence of IFNγ, which triggers classical activation (see below). Failure of macrophage activation in AIDS contributes to opportunistic pathogen infections and persistence of HIV, as well as reactivation of latent tuberculosis.

Q. Some people have a defect in the type-2 IFN receptor – the condition is very rare. What effect do you think it would have on their resistance to different types of pathogen?

A. It makes them very susceptible to infection by intracellular bacteria.

Macrophages can also be activated by IFNγ to express high levels of inducible nitric oxide synthase (i-NOS, NOS2), which catalyzes the production of nitric oxide (NO) from arginine (Fig. 7.19). ROIs and RNIs can interact to produce peroxynitrites and these reactive species all act within the phagosome to cause toxic effects on intraphagosomal pathogens; they interact with thiols, metal centers and tyrosines, damaging nucleic acids and converting lipids through oxidative damage. In this way bacterial metabolism and replication are impaired.

Fig. 7.19 The nitric oxide pathway

IFNγ and other inflammatory cytokines cause production of inducible nitric oxide synthase (i-NOS, also known as NOS2) which combines oxygen with guanidino nitrogen of L-arginine to give nitric oxide (NO^•), which is toxic for bacteria and tumor cells. Toxicity may be increased by interactions with products of the oxygen reduction pathway, leading to the formation of peroxynitrites. Cell activation by TH2-type cytokines promotes breakdown of arginine to ornithine and urea. Polyamines are products of ornithine which promote collagen synthesis and cell proliferation.

Q. Describe four roles of IFNγ in the development of the immune response, each modulating a different group of cells

A. IFNγ induces MHC molecules and co-stimulatory molecules on APCs, and induces adhesion molecules and chemokines on endothelium. It also activates macrophage microbicidal activity, modulates class switching by B cells (see Chapter 9), and regulates the balance between TH1 and TH2 populations of T cells. It has some capacity to induce production of anti-viral proteins.

Another mechanism that limits bacterial growth involves the sequestration of key nutrients by lactoferrin (Fe²⁺) or NRAMP1, which extrudes Fe²⁺, Zn²⁺ and Mn²⁺ from the lumen. Additionally, phagosomes contain endopeptidases, exopeptidases, and hydrolases, which break down the pathogens. The proteases are delivered by different granules at different stages during the maturation of the phagosome.

Some pathogens avoid phagocytosis or escape damage

Some pathogens have developed elaborate mechanisms to evade killing within phagosomes. These include the escape of the bacteria from the phagosome (Listeria monocytogenes), promotion of fusion to the endoplasmic reticulum (Legionella) and inhibition (M. tuberculosis) or delay (C. burnetii) of phagosome maturation. This will depend on the mechanism used for internalization; while direct recognition of the pathogen through non-opsonic receptors has limited activating capacity and enables the pathogen to exploit particular escape mechanisms, internalization of antibody-coated pathogens leads to enhanced cellular activation and triggering of microbicidal mechanisms. Macrophages once activated through PRRs such as TLRs induce autophagy which mediates degradation of vacuolar M. tuberculosis and Toxoplasma gondii. Autophagy is also used by macrophages to sequester and degrade cytosolic pathogens such as Francisella tularensis and Salmonella enterica.

Functions of secreted molecules

Following encounters with microorganisms and antigens, resident macrophages can enhance their transcription and translation of a wide range of gene products, including secreted molecules, which often act locally close to the cell surface.

Secretory products include:

• eicosanoids involved in control of inflammation;

• cytokines;

• complement proteins;

• antibacterial peptides and enzymes, especially lysozyme.

Lysozyme acts directly on the bacterial cell-wall proteoglycans, present especially in the exposed cell wall of Gram-positive bacteria (Fig.7.w7). The cell walls of Gram-negative bacteria may also become exposed to lysozyme if they have been damaged by complement membrane attack complexes. Lysozyme is constitutively produced by macrophages.

Fig. 7.w7 Action of lysozyme on the cell wall of Staphylococcus aureus

The structure of the cell wall of Gram-positive bacteria such as Staphylococcus aureus includes a backbone of N-acetylglucosamine (GlcNac) alternating with N-acetylmuramic acid (MurNac) cross-linked by amino acid side chains (yellow) and bridges of five glycine residues (orange). Lysozyme splits the molecules at the places indicated.

Cytotoxic products and powerful neutral proteinases such as elastase, collagenase, and urokinase (generating plasmin) (Fig.7.w8) are able to induce tissue injury and contribute to destructive chronic inflammation in joints and lung. Monocyte-derived procoagulant/tissue factor can also induce vascular occlusion and tissue damage.

Fig. 7.w8 Actions of macrophage neutral proteinases

Macrophage-derived neutral proteinases interact with plasma and tissue enzymes and their inhibitors (which can also be generated by macrophages) to regulate fibrinolysis and matrix catabolism. (PA, plasminogen activator; TIMP, tissue inhibitor of matrix metalloproteinase.)

In addition to their local actions, IL-6 and IL-1 act as circulating mediators of the acute phase response. Distant targets of these macrophage-derived cytokines include thermoregulatory centers in the central nervous system, muscle and fat stores, liver and the neuroendocrine system.

Resolution of inflammation by macrophages is an active process

Complete removal of the inflammatory trigger initiates the resolution of inflammation, in which neutrophil infiltration stops and apoptotic neutrophils are phagocytozed. A key event in this process is ‘lipid mediator class-switch’ in which prostaglandin and leukotriene synthesis is replaced by the synthesis of lipoxins, resolvins and protectins (see Fig. 7.16). Intriguingly, the signaling pathways leading to the proinflammatory prostaglandins E2 and D2, synthesized early during inflammation, also lead to the transcription of the enzyme responsible for lipoxin synthesis. Lipoxin synthesis by neutrophils occurs through their interaction with platelets and epithelial cells that provide metabolic precursors through a process called transcellular biosynthesis. In the case of macrophages, lipoxin synthesis is triggered by uptake of apoptotic cells. Lipoxin A4 reduces neutrophil activity, increases the migration of monocytes, favors uptake of apoptotic neutrophils and inhibits the synthesis of CXCL8. Resolvin E1 and protectin D1 increase the expression of CXCR5 on the surface of apoptotic neutrophils which facilitates clearance of CXCL3 and CXCL5 (neutrophil chemoattractants) from the inflammatory site. Apoptotic neutrophils produce ‘find me’ signals (e.g. lysophosphatidylcholine) that attract macrophages. Uptake of these neutrophils by macrophages inhibits production of IL-23, a cytokine involved in promoting granulopoiesis. As described above, recognition of apoptotic cells by macrophages also leads to the production of the anti-inflammatory cytokines IL-10 and TGFβ that together with factors such as vascular endothelial growth factor promote tissue repair.

Different pathways of macrophage activation

Previous sections have hinted at the capacity of macrophages to adapt to their surroundings (i.e. macrophages in different anatomical compartments display distinct phenotypes and resident macrophages have lower microbicidal activity compared to recruited macrophages, even after activation). Study of the phenotype of macrophages exposed in culture to different combinations of cytokines further illustrated the plasticity of these cells. Broadly, macrophages can follow classical (M1) or alternative (M2) activation profiles though it is possible to encounter intermediate phenotypes:

• M1 activation refers to macrophages with enhanced microbicidal activity that can be generated in culture by treating with selected TLR ligands or pathogens in the presence (classical activation) or absence (innate activation) of IFNγ;

• M2 activation generally refers to macrophages exposed to IL-4 or IL-13 although within the spectrum of M2 activation, macrophages with a regulatory phenotype can also be obtained in response to immunocomplexes and TLR engagement (Fig. 7.20).

Fig. 7.20 Modulation of macrophage activation

Signals from microbial products, phagocytosis, and cytokines result in changes to the surface and secretory properties of macrophages, which can be classified as activated, deactivated, or alternatively activated. (NO, nitric oxide; TGFβ, transforming growth factor-β.)

Major attempts are being made to correlate the phenotypes observed in vitro with patterns of differentiation in vivo through the analysis of signature markers, some of them having been identified during parasite infection in mice (Fig. 7.21). Classical activation appears to be consistent with the role of macrophages as effector cells during the course of cell-mediated immune responses where IFNγ produced by TH1 T cells will enable the elimination of intracellular pathogens by macrophages (see above). Additionally M1 macrophages promote TH1 responses by producing IL-12, and secreting CXCL9 and CXCL10 which selectively recruit TH1 T cells.