Monocytes respond to activating signals, for example, chemokines, through chemokine receptors, setting in motion a series of adhesion and migration events associated with diapedesis.88 They play a direct role in sepsis and in more poorly defined changes associated with intravascular coagulation and platelet activation. Their phagocytic potential is mainly expressed after adherence to the vascular endothelium. Monocytes are relatively resistant to virus infection, compared with more differentiated macrophages. These cells selectively adhere to lipid- and platelet-activated endothelium, a precursor to atherogenesis.89 Although metabolic, microbial, or environmental stimuli are normally required to induce monocyte activation, once activated monocytes express a greater potential for cytotoxicity and antimicrobial functions than resident tissue macrophages.
Figure 67–11 schematically shows select surface receptors related to monocyte function. These include chemokine recognition, adhesion, and immunoregulatory molecules. Receptors involved in microbial recognition and innate immunity (e.g., cluster of differentiation [CD]14),90 phagocytosis (e.g., FcR, CR), secretory, and killing mechanisms are described, as are cytokine production and responses. Intracellular granule contents of monocytes include myeloperoxidase (MPO) and lysozyme, although these are less studied than in neutrophils.
MOTILITY OF MONOCYTES AND MACROPHAGES
An effective monocyte response to infection is predicated upon the ability to migrate and accumulate at sites of inflammation and infection. Monocytes are capable of both random and directed movement. Random migration is nondirected movement that occurs in the absence of attracting substances. Directed movement, as a result of chemotaxis, refers to monocyte migration that occurs in response to soluble factors or stimuli and that is mediated by different types of receptors on phagocyte cell surfaces. A number of different methods have been used to study macrophage movement both in vivo91 and in vitro.92
Monocytes and macrophages are unusual among hematopoietic cells in that they are motile (ameboid type), migratory, yet capable of sessile, “fixed” life in tissues as resident and more newly recruited cells. Although not as motile as neutrophils, and more difficult to study in physiologically relevant assays in vitro, they display lineage-specific, as well as shared, yet distinct properties with DCs, which can be considered as more motile, less-adherent cells specialized for antigen capture and delivery to naïve and primed lymphocytes.93 They also share receptors and cytoskeletal properties with fibroblasts. Apart from diapedesis in response to endothelial and extravascular signals, monocytes and their progeny display polarization and specialized adhesion structures, most evident in the tight seal of osteoclasts to bone surfaces, so as to localize secretion of powerful catabolic products.
Adhesion is a defining event in the differentiation of monocytes, profoundly influencing the organization of the cell, its plasma membrane, cytoplasm, and nuclear transcription machinery, as well as regulating posttranslational modification of the proteome. Monocytes express diverse integrins, implicated in outside-in as well as inside-out signaling.94 Particularly important are the β2-integrin heterodimers, restricted to myeloid cells, as opposed to β1 and β3 integrins shared with mesenchymal and other cells. The β2 integrins, lymphocyte function–associated antigen (LFA)-1 (CD11a/CD18), CR3 (CD11b/CD18), and CD11c/CD18, have been of great value in studies of monocyte/macrophage adhesion. Inhibitory and stimulatory monoclonal antibodies have been generated, and rare inborn errors of metabolism, such as the leukocyte adhesion deficiency syndrome, caused by a genetic deficiency of the common β2 chain, result in defective myeloid cell recruitment to inflammatory stimuli.
The well-known sequence paradigm of rolling (mediated by L-selectin), more stable adhesion (mediated by β2 integrins), and diapedesis has been extensively studied in neutrophils (Chap. 19), and is thought to be similar for monocyte recruitment in response to chemokines, as described in Chap. 68. Monocyte-specific and constitutive migration through different tissue compartments (marrow, blood, tissues) are still poorly understood. An unresolved question is whether circulating monocytes are already “bar coded” for entry to special tissues, such as the CNS, or whether cells enter tissues stochastically from blood.
The control of monocyte motility in relation to chemotaxis continues to be studied.95 In particular, the energetics and role of mitochondria in aerobic and hypoxic conditions deserve further study. Mitochondria are prominent in DCs and play a wider role than anticipated in innate resistance to viral infection and in cytosolic stress. Several well-known G-protein–coupled receptors (GPCRs), including the array of selective, shared, even redundant chemokine receptors, β-adrenergic receptors, and others contribute to the regulation of directed migration and other cellular functions (Table 67–5).96,97 In addition, a newly defined family of GPCR with large extracellular domains, includes myeloid-restricted members of the epidermal growth factor–seven transmembrane (EGF-TM7) subfamily with multiple EGF (epidermal growth factor) repeats. EMR2 (epidermal growth factor–like module containing mucin-like hormone receptor–like 2) and CD97, structurally related to the F4/80 antigen marker discussed in Chap. 68, likely support additional important monocyte functions.97 Their ligands include complement regulatory molecules (CD55, associated with paroxysmal nocturnal hemoglobinuria; Chap. 40) and chondroitin sulphate B, a matrix component. EMR2 expression on myeloid cells is upregulated by septic shock, its ligation on neutrophils potentiates a range of cellular responses.
Table 67–5.Selected G-Protein–Coupled Receptors Implicated in Functions of Monocytes and Macrophages ||Download (.pdf) Table 67–5. Selected G-Protein–Coupled Receptors Implicated in Functions of Monocytes and Macrophages
|Chemotaxis ||Adhesion/Cell–Cell Contact ||Activation and Resolution of Inflammation ||Alternative Activation ||Survival |
|Chemokine receptors ||EGF-TM7 receptors ||BAI-1 ||Purinergic receptors GPR86, GPR105, P2Y8, P2Y11, and P2Y12 ||Sphingosine-1-phosphate receptors |
|C5a receptor ||Sphingosine-1-phosphate receptors || || || |
|Leukotriene B4 receptor || ||Formyl peptide receptors ||Chemokine receptors || |
|Formyl peptide receptors ||CX3CR1 ||Chemokine receptors || || |
|Platelet-activating factor receptor || ||C5a receptor || || |
| || ||EMR2 || || |
|EMR2 || ||Protease-activated receptors || || |
|Neuropeptide Y receptor || ||Platelet-activating factor receptor || || |
| || ||Leukotriene B4 receptor || || |
| || ||Neurokinin receptors || || |
| || ||Neuropeptide Y receptor || || |
| || ||Vasoactive intestine peptide receptor || || |
| || ||Prostaglandin receptors || || |
| || ||Resolvin || || |
The roles of phosphoinositide metabolism, diacylglycerol generation, calcium fluxes, and phosphorylation/dephosphorylation in regulating actin assembly have been studied in human and mouse cells, using mainly neutrophils as a prototype.95 Genetic models of value for macrophage studies include src kinase knockout animals and the Wiskott-Aldrich syndrome. Small guanosine triphosphatases (GTPases; rac, rho, cdc42) have been implicated in diverse myeloid functions, including cell spreading and membrane ruffling. Specialized adhesion structures that deserve further study in macrophages include focal adhesion, podocyte formation (particularly prominent in osteoclasts) and possible participation in tight junctions; hemiconnexons have been reported in macrophages in marrow stroma. CR3 contributes to divalent cation-dependent adhesion of monocytes and macrophages to artificial, serum-coated substrates, such as bacteriologic plastic and the class A SR and MARCO (see “Non–Toll-Like, Nonopsonic Receptors” above), which mediate divalent cation-independent adhesion to serum-coated tissue culture plastic in vitro. However, the basis of the remarkable, even unique, protease-resistant adhesion of macrophages to foreign materials remains mysterious. Improved imaging studies, combined with genetic manipulations, will bring further insights into the regulation of monocyte/macrophage adhesion and migration in vivo.
INTERACTION WITH COAGULATION CASCADE
Monocytes and resident macrophages line the sinusoids of liver (Kupffer cells) and spleen and readily recognize activated platelets, binding them for clearance and destruction. In addition, monocytes produce potent procoagulants, such as tissue factor, initiating a clotting cascade which, if dysregulated, can lead to diffuse intravascular coagulation during septic shock. Following injury and inflammation, monocytes/macrophages produce urokinase, to generate plasmin, in concert with endothelial cell-derived tissue plasminogen activator.98 Macrophage production of urokinase is regulated by phagocytic and other stimuli, and the active enzyme can bind to receptors (urokinase plasminogen activator receptor) on the cell surface in a complex interaction with protease–antiprotease complexes, thus localizing fibrinolysis, which is important in wound repair.
The nature and source of the lipid tissue factor produced by monocytes is not well characterized. The cells also produce a complex mix of lipid metabolites, consisting of labile prostaglandins, leukotrienes, and thromboxanes, by utilization of arachidonate-derived precursors and substrates for phospholipase and cyclooxygenase-processing enzymes, among others.
RECOGNITION AND CLEARANCE OVERVIEW
Resident macrophages of the liver and marrow, as well as in lung and other nonhematopoietic tissues, play a major role in the recognition, phagocytosis, and endocytosis of foreign particles and macromolecules, as well as of modified host components. Clearance can be silent, even suppressing inflammation, mediated by transforming growth factor (TGF)-β generation, as observed after the uptake of apoptotic cells by macrophages.99 Production of hematopoietic cells is balanced by their programmed senescence and increased destruction, which can be enhanced in response to microbial and other toxic substances. Macrophages initiate and perpetuate inflammation, both acute and chronic, as a result of their biosynthetic and secretory responses to injurious particles. Uptake and vacuole formation sequester the membrane-enclosed contents for digestion and possible antigen processing and presentation, a specialized property of DCs after their further differentiation from active endocytic to APCs.33 Specialized studies show that blood-derived monocytes have unique functions. For example, in the human disorder multiple sclerosis and the model experimental autoimmune encephalitis, monocyte-derived macrophages initiate demyelination at nodes of Ranvier; whereas, microglia derived from yolk-sac progenitors during embryogenesis are relatively inert at disease onset.31 To illustrate the role of macrophages in the recognition and clearance of foreign substances, images of macrophage spreading and engulfment of erythrocytes can be visualized by scanning electron microscopy, and the sequence of engulfment by phase-contrast optics (see video talk on macrophage phagocytosis at http://hstalks.com/?t=BL1473311).
In addition, interest has grown explosively in cytosolic recognition systems, designed to protect the cell from various infectious and lytic agents.100,101,102 The process of autophagy shares aspects with both membrane-bound and cytoplasmic organelle injury, and has become of great current interest because of its contribution to pathogenesis of infectious, malignant, and inflammatory syndromes.103
Macrophages take up large numbers of naturally dying cells, hematopoietic and others, through a complex mechanism involving multiple, often redundant nonopsonic receptors.47,99 A possible role for complement has also been proposed. Figure 67–12 illustrates receptors and ligands that have been implicated. Apart from the SRs already discussed, they include receptors for opsonins and for milk-fat globulin, as well as for the vitronectin receptor. Phosphatidylserine (PS) expressed on the outer leaflet of apoptotic cells, contributes to apoptotic cell recognition, but its role is probably more complex as apparently healthy cells can express patches of PS on their surface and PS recognition plays a role in CD36-dependent macrophage–macrophage fusion.104 The recognition mechanisms for uptake of necrotic cells and enucleated erythroblast nuclei by macrophages are not clear (Chap. 15).
Phagocytic receptors for apoptotic cell phagocytosis. Macrophages and immature myeloid dendritic cells (DCs) are the main immune cells involved in the clearance of apoptotic cells. They express broadly similar multiple receptors that can bind directly or via opsonic-soluble proteins, for example, mannose-binding lectins (MBLs) to ligands. Phosphatidylserine (PS) becomes exposed on the outer surface of the apoptotic cell and a receptor for this ligand has been long sought. A new receptor (TIM4, and related TIM1) was discovered on resident mф, with specificity for PS. Other mф populations utilize MFGE8 (a milk-fat globulin protein secreted by mф) as an opsonin. Discrimination of non-self and altered self may involve combinations of different phagocyte receptors. Apoptotic cell uptake results in an antiinflammatory response by mф (e.g., release of transforming growth factor [TGF]-β and prostaglandin E2), but has also been implicated in cross-presentation by DCs. For further details see Ref. 47. (Reproduced with permission of Savill J, Dransfield I, Gregory C, et al: A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2002 Dec;2(12):965-975.)
ENDOCYTOSIS, PHAGOCYTOSIS, AND KILLING
Apart from the above ligands, macrophages express receptors for endocytosis of growth factors, cytokines, peptides, and lipids. Macrophages express a functional folate receptor that is induced during activation and can be used to target drugs or tracers to macrophages in situ.105 Hemoglobin–haptoglobin complexes are internalized by CD163, a glucocorticoid-regulated receptor with a remarkable SR-cysteine extracellular domain structure.106 CD163 is also upregulated by substance P.107
The cell biology of endocytosis and of phagocytosis is illustrated in Figs. 67–13 and 67–14. Apart from size and resultant involvement of the cytoskeleton, they have much in common; vesicle/phagosome formation, falling pH and initial digestion, fusion with secretory vesicles derived from the Golgi, and maturation to form secondary lysosomes/phagolysosomes with a more acidic pH, and further digestion.108,109 Apart from selective fusion with intracellular vesicles, there is extensive membrane flow, recycling, and fusion. Small GTPases play an important role in the control of membrane traffic.110 Early estimates revealed that a substantial fraction of surface membrane is internalized constitutively by endocytosis.
Phagocytosis and endocytosis pathways. Particulates are taken up by actin-dependent sequential maturation processes, involving membrane fusion and fission, which intersect with the endocytic pathway at several stages. Cytosolic small guanosine triphosphatases (rabs) determine organelle-specific interactions. Membrane is recycled to the plasma membrane, with processed antigen. Progressive acidification and delivery of lysosomal hydrolases result in terminal degradation. Compartment membranes express marker proteins such as lysosomal-associated membrane protein (LAMP)-1; the pan-macrophage CD68 antigen is associated with late endosomes and lysosomes.
A model for FcγR-mediated phagocytosis. A. Signaling upstream and downstream of Rho guanosine triphosphatases during FcγR-mediated phagocytosis. Immunoglobulin (Ig) G bound to antigen on the particle binds to FcγRI receptors at the surface of the mф and induces their aggregation (shown in red). This activates a Src family tyrosine kinase (probably Lyn). Lyn phosphorylates the receptor γ chain (phosphotyrosine residues in the γ chains are depicted as red diamonds) and Syk. Syk is activated and recruited to the phosphotyrosine residues of the γ chain through its two SH2 (Src homology 2) domains. Cdc42 activation by an unknown guanine–nucleotide exchange factor (GEF) allows the recruitment of WASP (Wiskott-Aldrich syndrome protein). In turn, WASP activates the Arp2/3 complex that triggers actin polymerization to generate the protrusive force for pseudopod extension (red arrowheads). Activation of a Rac1 GEF, possibly Vav, by tyrosine phosphorylation in conjunction with PI3 kinase products (PIP3) promotes GDP/GTP (guanosine diphosphate/guanosine triphosphate) exchange on Rac1. GTP-bound Rac1 interacts with and activates the serine/threonine kinase Pak1, which may induce the actinomyosin contractility involved in phagosome closure. B. In the next step, FcγRI is rapidly down-modulated and returned to an inactive state (shown in blue), resulting in actin filament disassembly. According to this model, actin assembly proceeds as a wave at the distal rim of the pseudopodia, while actin depolymerization occurs rearward. Polyphosphoinositide phosphatases such as the SH2 domain-containing SHIP, which selectively hydrolyze PIP3, may contribute to down-modulation. Modulation of FcγRI activation may also involve tyrosine phosphatases such as SHP-1, which associates with FcγRIIb, a member of the FcγR family that may be coligated with FcγRI. In addition, PEST family phosphotyrosine phosphatases (PTPases) may contribute to dephosphorylation by interacting with PSPIP, a cytoskeletal protein that interacts with WASP. GAPs may also contribute to down-modulation by returning Cdc42/Rac1 to the inactive, GDP-bound state. Eventually, cytoskeletal proteins are shed from the ingestion site to leave the phagosome free in the cytosol (not shown here). (Reproduced with permission from Chimini G, Chavrier P: Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nat Cell Biol 2000 Oct;2(10):E191-E196.)
Studies that used opsonic receptors to examine the uptake mechanism of antibody-coated erythrocytes via opsonic receptors gave rise to the zipper hypothesis: local segmental engagement of FcR, and circumferential flow of macrophage pseudopodia around the particle, followed by fusion at the tip, closure, and ingestion. Subsequent studies by several groups documented the role of phosphatidylinositol 3-kinase (PI3K) and phosphoinositides in the initial fusion and subsequent associations between the actin cytoskeleton and cellular membranes.111 Latex has provided a useful test particle to isolate latex-containing phagolysosomes by flotation. Proteomic analyses112 demonstrated the protein composition of phagosomes and drew attention to functional constituents in the phagolysosomal membrane.
These observations have provided the basis for numerous investigations regarding the interactions of diverse microorganisms with the vacuolar system, which are often necessary for pathogen survival and establishment of intracellular infection (Fig. 67–15). Organisms can inhibit acidification and fusion (Mycobacterium),101,113 multiply within secondary lysosomes (Leishmania),114 escape free into the cytosol (Listeria),115 or translocate their genomes into the cytoplasm by fusion (enveloped viruses); other organisms induce variations on this theme; for example, Brucella seeks out the endoplasmic reticulum after entry and Legionella can enter macrophages by inducing a phagosome membrane of unusual composition.116 Nonpathogenic organisms or pathogens taken up via opsonic receptors or after IFN-γ activation undergo a different fate, with killing and destruction.
Selected pathogens evade distinct phagocytic mechanisms. Pathogens have developed several mechanisms to enter and survive inside macrophages. Legionella pneumophila resides and multiplies in a vacuole studded with ribosomes as a result of interaction with the rough endoplasmic reticulum. The organism secretes effector molecules via its type IV secretion system into the cell, which inhibit phagosome/lysosome fusion. The Francisella tularensis phagosome acquires the early endosome markers EEA1 and Rab5 and then matures into a late endosome defined by the presence of the markers Lamp1, Lamp2, and Rab7. The late endosome does not acidify and the phagosomal membrane is disrupted, releasing the bacteria into the cytosol. The Mycobacterium tuberculosis phagosome acquires the early endosome marker Rab5 but excludes the late endosomal Lamps and Rab7. This organism also produces molecules that block fusion with the lysosome and resides and replicates in this early endosome. Acidification of the Listeria monocytogenes phagosome is essential for the perforation of the phagosomal membrane and escape of the bacteria into the cytosol. Here they mobilize the actin polymerization machinery to move within the cell and then from cell to cell. Candida albicans undergoes a conversion from a unicellular form to a multicellular hyphal form, which allows this fungus to escape the macrophage. The Leishmania mexicana phagosome develops into an acidic phagolysosome containing Rab7 where the parasite is able to survive and replicate. Viruses such as the influenza virus are able to inhibit the activation of antiviral mechanisms, such as the activation of IFN regulatory function proteins that induce IFN production upon viral infection, and enter the nucleus. Cytomegalovirus (not shown) incapacitates a range of major histocompatibility complex-antigen presenting pathways. (Used with permission of S. Seif, GraphisMedica, 2014.)
The zipper mechanism, with tight apposition of membrane to the particle’s surface ligands, does not apply to all forms of ingestion. For example, complement opsonized particles seem to sink into the cytoplasm, and other phagosomes can be spacious. A number of key methods of visualization109 illustrate the dynamic nature of phagocytosis. Figure 67–14 illustrates some of the signaling pathways that control the cytoskeleton.
Macrophages are rich in lysosomal digestive enzymes,33 activated by a falling pH of approximately 6.5 within the mature vacuole. Unless captured as peptides by MHC molecules, a feature of antigen processing by DCs, macromolecular substrates can be degraded to their constituent amino acids, sugars, or nucleic acid bases. Early studies117 probed the permeability of the lysosomal vacuolar membrane. If the content cannot be fully degraded because of its nature (e.g., sucrose), overload (e.g., lipid), or owing to a genetic deficiency in a catabolic enzyme (lysosomal storage diseases), it accumulates within residual lysosomes, altering macrophage gene expression and secretory output, thus mediating chronic inflammation or metabolic forms of modified inflammation, such as atherosclerosis, foam cell formation and Gaucher disease. Figure 67–16A illustrates the uptake of senescent erythrocytes, the breakdown of heme and storage of Fe2+.118 Figure 67–16B shows how phagocytosis by DCs can bring about processing and cross-presentation of exogenous antigens.119 By comparison (Fig. 67–16C), autophagy is the envelopment of damaged intracellular organelles and cytoplasm by cytoplasmic membrane, and sequestration within a digestive vacuole, resembling heterophagy (Chap. 15).116 Its biochemical and cellular basis has become of interest because of its apparent relevance to cancer, infections such as tuberculosis and Legionnaire disease, and inflammatory syndromes such as inflammatory bowel disease (IBD).
A. Macrophages have an important role in iron metabolism by processing effete erythrocytes, internalized by phagocytosis, and returning iron to the blood (through ferritin) for reuse. Dissociation of iron linked to heme on erythrocytes requires the action of heme oxygenase (HO), an enzyme present in the endoplasmic reticulum (ER). The process allowing the transfer of heme oxygenase from the ER to the phagosome lumen is so far unknown. B. Presentation of antigens from intracellular pathogens is mainly carried out by major histocompatibility complex (MHC) class II molecules loaded in phagosomes. Presentation of some pathogen antigens could also involve MHC class I molecules. Current models indicate that antigens generated by hydrolases in the phagosome lumen could use SEC61 for translocation to the cytoplasm. After processing by the proteasome, antigens could be translocated to the phagosome lumen through the transporter for antigen processing (TAP) complex where loading onto MHC class I or MHC class II molecules would occur. Transport to the cell surface from the phagosome lumen could take place by using the existing membrane recycling machinery, involving the small guanosine triphosphatases Rab4 and Rab11. C. Autophagy is a conserved membrane traffic pathway that equips eukaryotic cells to capture cytoplasmic components within a double-membrane vacuole, or autophagosome, for delivery to lysosomes. Although best known as a mechanism to survive starvation, autophagy is now recognized as a mechanism to combat infection by a variety of intracellular microbes.
Although the phagocytic mechanism has been investigated in depth, we do not understand fully how the process of internalization is controlled. For example, ingestion can be thwarted by attempts to ingest too large a particle or foreign surface, or by close apposition of plasma membrane to noninternalizable immune complexes. This results in redirecting secretory vesicles to the surface, reminiscent of osteoclast adhesion. In other circumstances, as in response to foreign bodies, and especially mycobacteria, and in the presence of the Th2 cytokines IL-4 and/or IL-13, individual macrophages can fuse to form giant cells, with a common cytoplasm and multinucleation. Several fusogenic surface molecules have been identified and DNAX-activating protein (DAP) 12 expression and signaling is important in generating a fusogenic differentiation phenotype in macrophages.120
The recognition of the multiprotein inflammasome complex101 has stimulated intense interest in the recognition by cytosolic proteins of foreign nucleic acid, uric acid-induced injury, and breakdown products of microbial walls, for example, muramyl dipeptide. More complex peptidoglycan structures can also be recognized by surface receptors in Drosophila. Several reviews chart the rapid growth in our knowledge of inflammasome function in health and disease.100,102,121,122 Figure 67–17 illustrates selected nucleotide-binding oligomerization domain (NOD)-like and related receptors (NLRs) with nucleotide oligomerization and other characteristic domains. Mutations in NLR have been implicated in IBD, in periodic familial Mediterranean fever, and in a range of autohyperinflammatory syndromes.123 More specifically, NOD-2 has been implicated in Crohn disease.124,125 Excessive caspase activation and IL-1β release can be countered therapeutically with IL-1 receptor antagonists. Figure 67–18 illustrates the role of inflammasome activation in intracellular infection. Antiviral production of IFN-α and -β involves retinoid-inducible gene (RIG)-I–like helicases, indicating a role for mitochondria in cytosolic sensing.
Nucleotide-binding and oligomerization domain (NOD)–leucine-rich repeat (LRR) and inflammasome structures. NOD-like receptors (NLRs) have three structural domains: The LRR domain at the C-terminus, the NACHT (domain present in NAIP, CIITA, AHD, HET-E, TP-1) domain, and the N-terminal domain that can be a pyrin domain (PYD), a caspase activation and recruitment domain (CARD), or a baculovirus inhibitor-of-apoptosis protein repeat domain (BIR). The LRR domain is considered as the ligand-sensing motif, thus involved in the interaction with pathogen-associated molecular patterns (PAMPs), in analogy to toll-like receptors (TLRs). The NACHT domain is responsible for the oligomerization and activation of NLRs. The PYD or CARD domain of NLR is the link to downstream adaptors (such as apoptosis-associated speck-like protein containing a CARD [ASC]) or effectors (such as caspase-1). The BIR domain is proposed to act as caspase inhibitor. During NACHT LRR protein (NALP) and NALP1 inflammasome activation, NALP3 or NALP1 interact through PYD–PYD homotypic interactions with ASC, resulting in its activation. Subsequently, the CARD domain of ASC interacts with the CARD domain of caspase-1 and mediates its activation. NALP1 may also activate directly the caspase-5 through its C-terminal CARD domain. In contrast, NALP3 does not simultaneously activate caspase-5, but NALP3 can recruit a second capsase-1 through the CARD domain of CARD inhibitor of nuclear factor-κB–activating ligand (CARDINAL), a component of the NALP3 inflammasome. Interleukin-1β–converting enzyme (ICE)-protease activating factor (IPAF), that can on its own sense PAMPs, possesses a CARD domain at the N-terminal and thus may directly activate caspase-1 without ASC recruitment (“IPAF inflammasome”). (Reproduced with permission of Sidiropoulos PI, Goulielmos G, Voloudakis GK, et al: Inflammasomes and rheumatic diseases: evolving concepts. Ann Rheum Dis 2008 Oct;67(10):1382-1389.)
Knockout studies show that IPAF (interleukin-1β–converting enzyme-protease activating factor) is essential for the activation of caspase-1 by Salmonella typhimurium, Shigella flexneri, and Legionella pneumophila in order to induce the release of interleukin (IL)-1β, IL-18, and macrophage cell death. Sensing intracellular S. typhimurium seems to be mediated by the detection of monomeric flagellin that is secreted by the bacterial type III secretion system (and is dependent on the protein SipB from S. typhimurium) by IPAF. The type III secretion system protein IpaB is involved in sensing S. flexneri. Sensing intracellular L. pneumophila seems to be mediated by the detection of monomeric flagellin that is secreted by the type IV secretion system by NAIP5 (neuronal apoptosis inhibitor protein 5), which, in conjunction with IPAF, induces caspase-1 activation and restricts the growth of these pathogens in macrophages. Although a specific NLR (nucleotide-binding oligomerization domain-like receptor) protein that detects cytosolic Francisella tularensis has not yet been identified, the adaptor molecule ASC (apoptosis-associated speck-like protein containing a CARD) seems to be essential for counteracting infections with F. tularensis. CARD, caspase activation and recruitment domain; LRR, leucine-rich repeat; NACHT, domain present in NAIP, CIITA, HET-E, and TP-1; PYD, pyrin domain. (Reproduced with permission of Mariathasan S, Monack DM: Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation. Nat Rev Immunol 2007 Jan;7(1):31-40.)
GENE EXPRESSION, SYNTHESIS, AND SECRETION
The development of microarray technology has had a dramatic impact on the analysis of macrophage gene expression in response to a wide range of stimuli, including microbial ligands, cytokines, and immunomodulators. Macrophages are able to express a large number of genes and are extremely versatile in their responses to environmental cues. It has been possible to discern signatures of particular agonists, for example, IFN-α and -β and IL-4, but many caveats remain in the interpretation of such data. Heterogeneity of cellular origin, differentiation stage, and populations from diverse origins, as well as substantial species differences, make it difficult to compare results within and among experiments. Validation of more quantitative messenger RNA analysis of protein synthesis and modification is difficult, although proteomic analysis is gaining ground. The study of macrophage chromatin organization in relation to gene expression is in its infancy.
There is extensive crosstalk between the secretory and endocytic pathways.126 Table 67–6 is a selected list of secretory products. This includes lysozyme, a major myelomonocytic product that is constitutively expressed in vitro, but upregulated in granulomata in vivo. The secretion pathway of lysozyme in monocytes and macrophages has not been defined. The well-known pro- and antiinflammatory cytokines are better characterized, both in terms of regulation and the secretion pathway.109 The response to IL-6 and TNF-α secretion in model systems shows a more complex pathway than previously recognized.127,128 In addition to these and other important growth and differentiation factors that regulate angiogenesis, for example, macrophages are able to produce and secrete enzymes and proenzymes for a range of activities, as well as their inhibitors, for example, proteinases and antiproteinases. Although the amounts of complement proteins produced, for example, are relatively small, they can be significantly concentrated in a local microenvironment. In addition, macrophages can produce a range of antimicrobial peptides and lytic agents, but their most important killing mechanisms depend on oxygen129 and nitrogen metabolites,109,114 which are illustrated in Figs. 67–19 and 67–20. Regulation of the nicotinamide adenine dinucleotide phosphate oxidase and of inducible nitric oxide synthase has been studied extensively in mice and humans through biochemical and genetic approaches. Apart from their antimicrobial activity, nitrogen metabolites contribute to signaling pathways.130 IFN-α and -β play an important role in macrophage antiviral activities131 and perhaps in the cellular response to bacteria.132 These cytokines also contribute significantly to immune and inflammatory pathways, as well as cancer immunoediting133 and autoimmunity.134
Table 67–6.Selected Secretion Products of Macrophages ||Download (.pdf) Table 67–6. Selected Secretion Products of Macrophages
|Proteins ||Product ||Comment |
|Enzymes ||Lysozyme ||Bulk product |
| ||Urokinase-type plasminogen activator ||Regulated by inflammation |
| ||Collagenase ||Regulated by inflammation |
| ||Elastase ||Regulated by inflammation |
| ||Metalloproteinases ||Also inhibitors |
| ||Complement ||All components and regulators |
| ||Arginase ||Alternative activation |
| ||Angiotensin-converting enzyme ||Induced glucocorticoids, granulomas |
| ||Chitotriosidase ||Gaucher disease, lysosomal storage |
|Inhibitors ||Acid hydrolases ||All classes (mainly intracellular) |
| ||TIMP || |
|Chemokines ||Many C-C, C-X-C, CX3C; e.g., MCP, RANTES, IL-8 ||Initiates acute and chronic recruitment of myeloid and lymphoid cells |
|Cytokines ||IL-1β, TNF-α ||Pro- and antiinflammatory |
| ||IL-6, IL-10, IL-12, IL-17, IL-18, IL-23 ||Also antagonists, e.g., IL-1Ra |
| ||Type I IFN ||Autocrine and paracrine amplification |
|Apolipoproteins ||Apolipoprotein E ||Local source, marrow origin after adoptive transfer |
|Growth/differentiation factors ||TGF-β ||Also other family members (activins), myeloid growth and differentiation |
| ||M-CSF || |
| ||GM-CSF || |
| ||FGF ||Fibrosis |
| ||PDGF ||Repair |
| ||VEGF ||Angiogenesis |
|Opsonins ||Fibronectin, pentraxin (PTX3) ||Also uncharacterized receptor on Mф |
|Soluble receptors ||Mannose receptor ||Soluble mannose receptor |
|Cationic peptides ||Defensins ||Subpopulations and species variation |
|Lipids ||Procoagulant ||Initiation clotting |
| ||Arachidonate metabolites: ||Pro- and antiinflammatory mediators |
| ||Prostaglandins || |
| ||Leukotrienes || |
| ||Thromboxanes || |
| ||Resolvins || |
|Metabolites ||Reactive oxygen intermediates || |
| ||Reactive nitrogen intermediates || |
| ||Haem breakdown (bile pigments) || |
| ||Iron, B12-binding protein || |
| ||Vitamin D metabolites || |
The respiratory burst in a phagocyte is triggered when a bacterium is phagocytosed. During the phagocytosis of bacteria by macrophages and neutrophils, the phagosome membrane pinches off and the microbe is endocytosed along with a small volume of extracellular fluid. The mechanisms discussed here are based on studies in neutrophils and are still controversial.112 Electrons are removed from nicotinamide adenine dinucleotide phosphate (NADPH) in the cytoplasm and transferred through the gp91phox component (which includes flavin adenine dinucleotide and two hemes) across the membrane, where they reduce extracellular (or intraphagosomal) O2 to O2–. Protons left behind in the cell are extruded through voltage-gated proton channels (red). Some of the reactive oxygen species (ROS) derived from O2– are indicated. Spontaneous or superoxide dismutase–catalyzed disproportionation of O2– produces hydrogen peroxide (H2O2), which may be converted to HOCl (hypochlorous acid, or household bleach) by myeloperoxidase (MPO). A. Traditional view of the respiratory burst with charge compensation by proton channels. A perfect match of one proton per electron results in no change in membrane potential, intracellular pH (pHi), or external pH (pHo) and little change in ionic strength. Because proton channels are separate molecules and for the most part operate independently of NADPH oxidase, perfect 1:1 stoichiometry is not obligatory. The large depolarization that occurs during the respiratory burst in intact neutrophils and eosinophils is likely the most important factor that causes proton channels to open, although both pHi and pHo tend to change in a direction that causes proton channels to open. That depolarization occurs demonstrates unequivocally that proton efflux initially lags behind electron efflux. B. If any fraction of the total charge compensation were mediated by K+ efflux, pHi would fall, pHo (or phagosomal pH) would increase, and the osmolality of the phagosomal contents would increase. In this model, the elevated pH and osmolality of the phagosomal contents are crucial to activating proteolytic enzymes that actually kill bacteria, as opposed to ROS, which are said to be inert. C. Respiratory burst reactions. During phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is formed. Cytochrome b588, which was part of the specific granule, combines with the plasma membrane NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the NADPH. The result is the production of superoxide anion. Some of the superoxide anion is converted to H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion can react with H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The result of all of these reactions is the production of the toxic oxygen compounds superoxide anion (O2–), H2O2, singlet oxygen (1O2) and hydroxyl radical (OH•). As the azurophilic granules fuse with the phagosome, myeloperoxidase is released into the phagolysosome. Myeloperoxidase uses H2O2 and halide ions (usually Cl–) to produce hypochlorite, a highly toxic substance. Some of the hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions is the production of toxic hypochlorite (Ocl–) and singlet oxygen (1O2). (A and B, modified with permission from Decoursey TE: Voltage-gated proton channels and other proton transfer pathways, Physiol Rev 2003 Apr;83(2):475-579.)
The role of nitrogen metabolism in mф function. Interferon-γ (IFN-γ) enhances the activity of nitric oxide synthase 2 (NOS2) to generate nitric oxide, and inhibits arginase. Interleukin (IL)-4 and IL-13 promote arginase-dependent formation of L-ornithine and, ultimately, fibroblast proliferation and collagen production. GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF, tumor necrosis factor. (Adapted with permission from Hesse M1, Modolell M, La Flamme AC, et al: Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism. J Immunol 2001 Dec 1;167(11):6533-6544.)
Macrophages may be able to produce IFN-γ, for example, under particular circumstances, but in vivo most of the cytokine derives from other sources. IFN-γ has a major impact on macrophage function (the initial name of IFN-γ was macrophage activating factor), including priming of biosynthetic and functional responses associated with cytotoxicity and inflammation in cell-mediated immunity (Fig. 67–21).135 Table 67–7 summarizes the markers and functions associated with various forms of macrophage activation and deactivation, as described in Chap. 68.136 Intracellular GTPases have been implicated in cell activation by IFN-γ, for example, and in relation to IBD.121,124,125 Similarly, the Th2 cytokines IL-4 and IL-13 induce characteristic changes in macrophage phenotype, which are associated with an alternative activation pathway. The cellular biology of alternatively activated macrophages is modified extensively (Fig. 67–22).137 Macrophages also express a range of inhibitory proteins, such as members of the suppressor of cytokine signaling family, that suppress cytokine production, in addition to IL-10138 and TGF-β. Lipid metabolites, mainly derived from arachidonate and other lipid precursors, provide another potent source of inflammatory and immunomodulatory products.139 The suppressive functions of monocytes and macrophages in chronic infections and experimental tumors require further study, including the development of new phenotypic markers in mice and humans.
Table 67–7.Immunomodulation of Macrophage Phenotype ||Download (.pdf) Table 67–7. Immunomodulation of Macrophage Phenotype
|Stimulus ||Category ||Markers ||Function |
|Microbial (bacterial) ||Innate activation ||Induction of MARCO ||Enhanced phagocytosis |
| || ||Costimulatory molecules ||Antigen presentation |
| || ||CD200 ||Inhibition (CD200R) |
|IFN-γ ||Classical activation ||Induction MHC II ||Cell-mediated immunity/delayed-type hypersensitivity |
| || ||Potentiation innate markers || |
| || ||- TNF-α ||Proinflammatory |
| || ||- iNOS induction ||Antimicrobial (NO) signaling |
| || ||- NADPH, respiratory burst ||Host defense, inflammation |
| || ||LGP47 induction ||Association with phagosome/intracellular pathogen killing |
| || ||Downregulation of MR ||Unknown |
| || ||Modulation of FcR expression || |
| || ||Proteasomal composition ||Antigen presentation |
|IL-4/IL-13 ||Alternative activation ||Enhanced MR ||Endocytosis |
| || ||Induction arginase ||Humoral immunity |
| || ||Induction YM1, FIZZ1 (mouse) ||Th2-responses, allergy, antiparasitic |
| || ||Induction CCL17 (MDC) and CCL22 (TARC) ||Immunity, repair/fibrosis |
| || ||Fusion, giant cell formation || |
| ||Upregulation ||CD23 (FcRε) || |
|Immune complexes ||Modified activation ||Selective IL-12 downregulation, IL-10 induction || |
|IL-10 ||Deactivation ||Downregulation MHC II || |
|TGF-β ||Deactivation ||Downregulation of proinflammatory NO and ROI || |
|Glucocorticoids ||Deactivation ||CD163 induction, monocyte recruitment downregulated, ACE induction, Stabilin induction ||Antiinflammatory |
| || || ||Homeostatic clearance of hemoglobin/haptoglobin complexes |
Signaling pathways induced by type I and type II interferon (IFN). The type I IFNs (IFN-α and IFN-β) bind a receptor that consists of the subunits IFN-α receptor (IFN-αR)-1 and IFN-αR2, which are constitutively associated with tyrosine kinase 2 (TYK2) and Janus kinase (JAK) 1, respectively. Type I IFN-induced JAK-STAT (signal transducer and activator of transcription) signaling is propagated similarly to IFN-γ–induced JAK-STAT signaling (below). Activated TYK2 and JAK1 phosphorylate STAT1 or STAT2. Type I IFN-induced signaling then induces homodimerization of STAT1 and heterodimerization of STAT1 and STAT2. STAT1 and STAT2 associate with the cytosolic transcription factor IFN-regulatory factor 9 (IRF9), forming a trimeric complex known as IFN-stimulated gene factor 3 (ISGF3). On entering the nucleus, ISGF3 binds IFN-stimulated response elements (ISREs). Studies of gene-targeted mice have shown that JAK1, STAT1, STAT2, and IRF9 are required for signaling through the type I IFN receptor. TYK2 is required for optimal type I IFN-induced signaling. IFN-γ signaling: IFN-γ induces reorganization of the IFN-γR subunits, IFN-γR1 and IFN-γR2, activating the Janus kinases JAK1 and JAK2, which are constitutively associated with each subunit, respectively. The JAKs phosphorylate a crucial tyrosine residue of IFN-γR1, forming a STAT1-binding site; they then tyrosine phosphorylate receptor-bound STAT1, which homodimerizes through Src homology 2 (SH2) domain–phosphotyrosine interactions and is fully activated by serine phosphorylation. STAT1 homodimers enter the nucleus and bind promoters at IFN-γ–activated sites (GASs) and induce gene transcription in conjunction with coactivators, such as CBP (cyclic adenosine monophosphate-responsive–element-binding protein [CREB]), p300, and minichromosome maintenance-deficient 5 (MCM5). IFN-γ–mediated signaling is controlled by several mechanisms: by dephosphorylation of IFN-γR1, JAK1, and STAT1 (mediated by SH2 domain-containing protein tyrosine phosphatase 2 [SHP2]); by inhibition of the JAKs (mediated by suppressor of cytokine signaling 1 [SOCS1]); by proteasomal degradation of the JAKs; and by inhibition of STAT1 (mediated by protein inhibitor of activated STAT1 [PIAS1]). (Reproduced with permission from Platanias LC: Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol 2005 May;5(5):375-386.)
Schematic cross-section of “activated” macrophage, showing ruffling of cell membrane and cellular organelles (also see Fig. 67–15). (Used with permission of S. Seif, GraphisMedica, 2014.)
In addition to cytokine and other soluble afferent and efferent responses, macrophages are able to directly interact among themselves, with all other cell types in the body, both viable and injured, as well as with all kinds of microorganisms. Their interactions are reciprocal and regulated, contributing to homeostasis and to pathogenesis, both acutely and following persistent injury, to chronic inflammation. Storage of poorly degraded materials in lysosomes, for example, results in sustained production of degradation products, whereas massive, acute responses have a profound impact on the systemic circulation, endocrine and nervous systems, and on metabolic pathways. Short-range interactions include giant cell formation during granulomatous inflammation, and also contact-dependent immunoregulation by surface molecules such as CD200/CD200R and SIRPα/CD47.140 Matrix and other surface interactions regulate the induction or suppression of adaptive immune responses, as well as of other functions. The availability of oxygen plays an important role in macrophage interactions with a range of other cells, both normally and in a range of pathologies inducing inflammation, repair, and malignancy (Fig. 67–23).
Hypoxia induces marked changes in the phenotype of macrophages. Macrophages upregulate hypoxia-inducible transcription factor (HIF)-1 and HIF-2 in hypoxia, which translocate to the nucleus to induce the expression of a wide array of target genes. Several important cell-surface receptors are upregulated in hypoxia, including the glucose receptor GLUT-1 (for increased glucose uptake as the cell switches to anaerobic glycolysis to make ATP in the absence of oxygen), the chemokine stromal cell-derived factor-1 (SDF-1) receptor CXCR4, and the angiopoietin receptor Tie-2. Hypoxia also stimulates the expression of a wide array of other protumor cytokines, enzymes, and receptors, grouped here according to their known function in tumors. Downregulation of a factor or tumor-associated macrophage function is indicated by an arrow. Ag, antigen; COX, cyclooxygenase; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; MIF, macrophage migration inhibitory factor; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; PGE2, prostaglandin E2; TF, tissue factor; uPA/R, urokinase-type plasminogen activator receptor; VEGF, vascular endothelial growth factor. (Modified with permission from Lewis CE, Hughes R: Inflammation and breast cancer. Microenvironmental factors regulating macrophage function in breast tumours: hypoxia and angiopoietin-2. Breast Cancer Res 2007;9(3):209.)
RELEVANCE TO HEMATOPOIETIC FUNCTIONS AND DISORDERS
In addition to their essential role in host defense (innate and acquired immunity), inflammation, and repair, macrophages contribute to hematopoiesis, as well as to the turnover of hematopoietic cells and their products. Macrophages can be induced to take up folate, sense and respond to oxygen levels, and promote vascular growth, regulating the integrity of the hematopoietic microenvironment. However, they also play a central effector role in pathogenesis. Their surface expression and secretion of TNF-α, other proinflammatory cytokines, enzymes, and metabolites contribute to vascular injury and increased permeability of the microvasculature, as well as to local and systemic catabolic effects associated with chronic inflammation. In this regard, anti–TNF-α therapy is of considerable value in selected inflammatory conditions and has been extended to the treatment of cancer and rheumatologic conditions.141,142,143,144 Stromal and other resident macrophage populations provide a niche for acute and persistent infections in marrow and elsewhere, and these macrophages also contribute to trophic support of hematopoietic malignancies, such as multiple myeloma. The macrophage, therefore, provides an important target cell for selective therapeutic intervention, without undue enhancement of vulnerability to infection. Additional molecular targets are needed, based on more detailed analysis of macrophage functions within their native hematopoietic tissue environment. A deeper understanding of macrophage physiologic functions and of their role in a broad range of diseases should lead to the development of fresh insights into the pathogenesis and management of hematologic disorders.