++
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.
++
++
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).
++
+++
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.
++
++
++
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.
++
++
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).
++
++
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.
++
++
+++
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
++
++
++
++
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.
++
++
++
+++
CELLULAR INTERACTIONS
++
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).
++
+++
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.