Chapter 67: Structure, Receptors, and Functions of Monocytes and Macrophages

Modern study of mammalian phagocytes began with Metchnikoff in the 19th century. An understanding of the ontogeny, kinetics, and function of phagocytic cells in animals led to the concept of the mononuclear phagocyte system.^1,2 Kinetic studies indicate that marrow monoblasts and monocytes develop from the common myeloid progenitor, a derivative of the hematopoietic stem cell, and that tissue macrophages develop from monocytes that have migrated from the blood pool in response to chemotactic stimuli (Table 67-1 and Chap. 18). Tissue macrophages share many functional characteristics, such as phagocytic and microbial killing capabilities and adherence to glass or plastic surfaces in vitro. Vascular endothelium, reticular cells, and dendritic cells of lymphoid germinal centers usually are not included in the mononuclear phagocyte system, although the now obsolete term reticuloendothelial system³ denoted those cells as playing some complementary part with mononuclear phagocytes. In addition to developing the multitude of types of tissue macrophages, monocytes can differentiate into myeloid-derived dendritic cells.^4,5

The blood monocyte is a medium to large motile cell that can marginate along vessel walls and has a propensity for adherence to surfaces. Monocytes respond to inflammation and chemotactic stimuli by active diapedesis across vessel walls into inflammatory foci, where they can mature into macrophages, with greater phagocytic capacity and increased content of hydrolytic enzymes. Free macrophages also are present in mammary glands, alveolar spaces, pleura, peritoneum, and synovia. The somewhat less-motile fixed-tissue macrophages are found in different tissues and serous cavities. The functions of mononuclear phagocytes include phagocytosis, killing, and digestion of microorganisms, particulate material, or tissue debris; secretion of chemical mediators and regulators of the inflammatory response; interaction (as dendritic cells) with antigen and lymphocytes in the generation of the immune response; cytotoxicity, such as killing of some tumor cells; and other functions specific for macrophages of particular tissues.

The development of techniques to isolate monocytes from blood of adult human subjects led to the discovery that monocytes are heterogeneous with regard to cell volumes. Isolation of purified monocytes by adherence to glass substrates or to gelatin-coated flasks or by centrifugal elutriation reveals distinct populations of monocytes.^1,2 In addition to the usual 12- to 15-μm diameter (when measured on a dried blood film) monocyte, so-called regular monocytes, a somewhat smaller cell that is less active than its larger, more mature counterpart has been identified. This cell is referred to as a small immature monocyte, but its functional significance is not clear.

Monocytes continuously emigrate from the blood into tissue, with a half-life in the blood of approximately 1 day in mice.⁶ Nondividing monocytes can be induced to differentiate into dendritic like cells in vitro. However, this process requires culture of the cells for 7 to 10 days with exogenous cytokines, typically interleukin (IL)-4 and granulocyte-monocyte colony-stimulating factor (GM-CSF).⁷ The major lineage regulator of nearly all macrophages is monocyte/macrophage colony-stimulating factor (M-CSF; also termed CSF-1) and its receptor (M-CSF R). The M-CSF R is a class III transmembrane tyrosine kinase receptor, which is expressed on most mononuclear phagocytes.⁸ In the presence of endothelial cells grown on an extracellular matrix, monocytes differentiate along two distinct pathways: toward dendritic cells or macrophages. Monocytes that migrate across endothelium in an abluminal to luminal direction differentiate into dendritic cells. In contrast, monocytes that remain in the subendothelial matrix differentiate into macrophages.

MORPHOLOGY OF MONOCYTE PRECURSORS

Monoblasts and promonocytes are the precursors of monocytes, bearing finely dispersed nuclear chromatin and nucleoli when observed in the stained film of the marrow. The monoblast is a very-low-prevalence marrow cell, indistinguishable by light microscopy from the myeloblast. Promonocytes are 12 to 18 μm in diameter (as measured on dried blood films) and have characteristic deeply indented, irregularly shaped nuclei with condensed chromatin, and numerous cytoplasmic microfilaments.

In animal studies, a small percentage of marrow cells are phagocytic, synthesize DNA, adhere to glass surfaces, and contain nonspecific esterases.⁹ These cells have been referred to as promonocytes and are considered as intermediate between monoblasts and the monocytes of the blood.⁹ Cytochemical studies identify the promonocyte in normal human marrow. Promonocytes have deeply indented and irregularly shaped nuclei and bundled and scattered single filaments in the cytoplasm. These morphologic features distinguish the promonocyte from the progranulocyte.^10,11 Peroxidase is present throughout the cell secretory apparatus in all cisternae of the rough-surfaced endoplasmic reticulum, the Golgi complex, associated vesicles, and all immature and mature granules. Cytochemical reaction products for acid phosphatase and arylsulfatase also are deposited throughout the secretory apparatus of the promonocyte.

MORPHOLOGY OF MONOCYTES

Light Microscopy

The morphology of monocytes has been investigated by light and phase-contrast optics,¹² scanning and transmission electron microscopy, and freeze-fracture and freeze-etch procedures.¹³

On the stained blood film the monocyte has a diameter of 12 to 15 μm (Fig. 67–1). The monocyte nucleus occupies approximately half the area of the cell and usually is eccentrically placed. The nucleus most often is reniform, but may be round or irregular. It contains a characteristic chromatin net with fine strands bridging small chromatin clumps. Chromatin aggregates are arranged along the internal side of the nuclear membrane. The cytoplasm is spread out, stains grayish-blue with Wright stain, and contains a variable number of fine, pink-purple granules, which at times are sufficiently numerous to give the entire cytoplasm a pink hue. Clear cytoplasmic vacuoles and a variable number of larger azurophilic granulations often are encountered in these cells.

Phase Microscopy

The monocyte nucleus has a distinct chromatin pattern on a cloudy background when examined by phase-contrast microscopy. The cytoplasm is clear gray. Mitochondria are extremely fine and occasionally form a small, juxtanuclear rosette surrounding the centrosome. The phase-dense cytoplasmic granules, varying in number, are generally at the limit of resolution of light microscopy and appear as fine intracytoplasmic dust. Monocytes contain several types of cytoplasmic vacuoles. The reniform nucleus with a juxtanuclear depression filled by a centrosome and its active undulating movement similar to that of other leukocytes are characteristic of the monocyte. The locomotion of the monocyte has the same pattern of undulating cytoplasmic veils seen in macrophages. The monocyte generally assumes a triangular shape as it moves, with one point trailing behind and the other two points advancing before the cell. Blood monocytes undergo adherence and cytoplasmic spreading following attachment to glass surfaces.¹⁴ The extent of spreading increases in the presence of antigen–antibody complexes, certain divalent metals, and proteolytic enzymes.^14,15 The spread form of the monocyte reveals that the nucleus and granules are located centrally and the abundant hyaloplasm is in the periphery of the cell, terminating in a fringed border that displays undulating movement. The small monocyte may be difficult to distinguish from the large lymphocyte when examined by phase-contrast microscopy.

A striking feature on phase-contrast microscopy is the ruffled plasma membrane that forms prominent phase-dense folds at the cell surface and edges. Some cells have a dense thickening at the edge of the cytoplasm, with microextensions on the thickened edge.

Scanning Electron Microscopy

The monocyte surface has very prominent ruffles and small surface blebs.^16,17 Extensive ruffling on the monocyte plasma membrane is of functional significance. The monocyte is both motile and phagocytic, and these functions require physical contact with particles or cell surfaces. Reduction in the radius of curvature of the cell surface by formation of ruffles or microvilli may reduce repulsive forces when surface negative-charge groups on the cell approach and contact a negatively charged substratum or cell. In addition, redundancy of the cell membrane may provide reserve membrane required for locomotion and phagocytosis.

Transmission Electron Microscopy

The nucleus of the monocyte contains one or two small nucleoli surrounded by nucleolar-associated chromatin (Fig. 67–2).¹⁸ The cytoplasm contains a relatively small quantity of endoplasmic reticulum and a variable quantity of ribosomes and polysomes. The mitochondria are numerous, small, and elongated. The Golgi complex is well developed and is situated about the centrosome within the nuclear indentation. Centrioles and filamentous centriolar satellites are often visualized in this region. Microtubules are numerous, and microfibrils are found in bundles surrounding the nucleus. In cultured macrophages, collections of microfilaments are present underneath the plasma membrane near sites of cell attachment either to a substratum or to phagocytosable particles.¹⁹ The cell surface is characterized by numerous microvilli and vesicles of micropinocytosis. The cytoplasmic granules resemble the small granules found in the granulocytic series, measuring approximately 0.05 to 0.2 μm in diameter. They are dense and homogeneous and are surrounded by a limiting membrane. These granules, as with the lysosomal granules of other leukocytes, are packaged by the Golgi apparatus after their enzymatic content has been produced by the ribosomal complex of the cell.^10,11 These cytoplasmic granules contain acid phosphatase and arylsulfatase and, therefore, are primary lysosomes. After endocytosis, lysosomes fuse with the phagosome, forming secondary lysosomes. Some monocyte granules stain positive for peroxidase, whereas others are peroxidase negative.^10,11

Freeze-Fracture Microscopy

In this technique, a cell suspension is frozen, placed in a high-vacuum chamber, and struck with a blunt edge, thus producing a fracture that propagates through the frozen specimen. The utility of the procedure comes from the remarkable finding that when the fracture encounters a cell, the fracture tends to propagate along the interior of the plasma membrane and thus split the lipid bilayer into its two constituent layers. After fracture, the specimen is coated with platinum, which is electron dense when viewed with transmission electron microscopy. All cell types examined thus far by the freeze-fracture technique reveal intramembrane particles (IMPs) as the predominant topographic feature of the interior of the bilayer. Studies of the erythrocyte show that at least some particles contain intercalated membrane proteins, and this is assumed to be the case for nucleated cells as well. The distribution of IMPs is dramatically altered in a number of cell systems by physiologic stimuli, for example, hormonal stimulation.

Profound changes in the distribution of IMPs on mononuclear phagocytes occur following binding of antibody-coated erythrocytes.¹³ Because redistribution of IMPs also occurs in some nonphagocyte Fc receptor (FcR)–bearing cells¹³ and after exposure to aggregated immunoglobulin (Ig) G, this alteration in IMPs presumably reflects interaction with FcR. Freeze-etch electron micrographs of the monocyte show nuclear pores traversing both lamellae of the nuclear membrane and contours of cytoplasmic lysosomes and mitochondria (Fig. 67–3).

HISTOCHEMISTRY OF MONOCYTES

Table 67–2 compares the hydrolytic enzyme contents of monocytes, neutrophils, and lymphocytes. Monocytes also give a weak but positive periodic acid–Schiff reaction (for polysaccharides) and Sudan black B reaction (for lipids). Nonspecific esterase^20,21,22 is frequently used as a marker for monocytes. Monocyte esterases are inhibited by sodium fluoride, whereas the esterases of the granulocytic series are not. The nonspecific esterase reaction is positive in promyelocytes and myelocytes; therefore, analysis of fluoride inhibition is necessary to distinguish marrow monocytes from early myelocytes. Monocyte granules, although heterogeneous in size (0.3 to 0.6 μm), are not separable into populations by routine electron microscopic criteria. Identification of monocyte granule populations has depended on subcellular localization of monocyte enzymes by electron microscopic cytochemistry.¹⁰ Human marrow promonocytes and blood monocytes contain granules that comprise two functionally distinct populations.^10,11 One population contains the enzymes acid phosphatase, arylsulfatase, and peroxidase. These granules are modified primary lysosomes and are analogous to the azurophil granules of the neutrophil. The monocyte azurophil granule population is heterogeneous in cytochemical reactivity for peroxidase, acid phosphatase, and arylsulfatase.^23,24 Moreover, primary granules that are morphologically identical with other vesicles can be identified as lysosomes cytochemically. The other population of monocyte granules lacks alkaline phosphatase²³ and is not strictly analogous to the specific granules of neutrophils.

MORPHOLOGY OF MACROPHAGES

Macrophage characteristics are heralded by a significant increase in cell size, increase in the number of cytoplasmic granules, increase in the heterogeneity of cell size and shape, and increase in the number of cytoplasmic clear vacuoles in comparison to monocytes.

Light and Phase-Contrast Microscopy

In vitro culture of monocytes purified from adult human blood has provided an opportunity to observe the maturation of these cells into mature macrophages. The macrophages of the pulmonary alveoli, peritoneal and pleural cavities, and inflammatory exudates are hypermature cells that have undergone in vivo stimulation and maturation. This process results in enhanced bactericidal activity^1,2 because of augmentation of lysosome number and acid hydrolase content. Macrophages display attributes of morphologic specialization specific to their location and function. The fixed macrophages of the spleen (littoral cells) are involved in the sequestration and destruction of effete or abnormal red cells and exhibit stages of erythrophagocytosis and intracytoplasmic aggregates of ferritin (Chap. 6). The macrophages of the marrow, the “nurse cells” of the erythroblastic island, play a similar role in erythrophagocytosis and iron storage and transfer (Chaps. 5 and 31). Hepatic macrophages (Kupffer cells), found in liver sinusoids, also phagocytize red cells and other cellular elements and are important sites of iron storage. Macrophages of the pulmonary alveoli, the lamina propria of the gastrointestinal tract, and the peritoneal and pleural fluids reflect in their morphology a specific function of phagocytosis of microorganisms, cells, and cellular and noncellular debris, characteristic of the specific organ location.

Most macrophages are 25 to 50 μm in diameter on Wright or hematoxylin-and-eosin–stained films (Fig. 67–4). They have an eccentrically placed reniform or fusiform nucleus with one or two distinct nucleoli and finely dispersed, loosely stranded nuclear chromatin that tend to clump in the nuclear interior and along the internal aspect of the nuclear membrane (Fig. 67–5A). A juxtanuclear clear zone (Golgi complex) is well defined when the Wright stain is used. The cytoplasm shows fine granules and multiple pink-purple, large azurophil granules. The cytoplasmic borders are irregularly serrated. Cytoplasmic vacuoles are present near the cell periphery, reflecting the active pinocytosis in these cells.

The surface antigen CD68, also known as macrosialin, is commonly used as a macrophage marker. Figure 67–5B shows an immunohistochemistry micrograph of a macrophage in a lymph node. The cytoplasm of the macrophage is intensely positive for CD68, while the surrounding lymphocytes are negative.

On phase-contrast microscopy, living macrophages are large cells with a propensity to adhere to and spread on glass surfaces. Thus, the cell organelles are concentrated within the central portion of the cell and clear veils of hyaloplasm spread about the cell, with intense ruffling of the membrane borders. Vesicles and contractile vacuoles are seen about the cell periphery and in the cell interior. The juxtanuclear clear zone bearing the centrosome and the Golgi complex is particularly dynamic and displays an undulating motion.

Electron Microscopy

Scanning electron micrographs of macrophages adherent to glass surface show membrane ruffling and pseudopodia (Fig. 67–6). Transmission electron microscopy of monocyte-derived macrophages show a variable degree of differentiation, nuclear “maturity,” ribosomes, mitochondria, and lysosome content, and the nucleus varies in shape from horseshoe to fusiform (Fig. 67–7). Clear spaces between membrane-fixed chromatin aggregates mark the sites of nuclear pores that are relatively abundant on freeze-etch electron micrographs of macrophages and monocytes (see Fig. 67–3). Polyribosomes and scant smooth and rough endoplasmic reticulum are seen about the cell periphery. A well-developed Golgi complex is in a juxtanuclear location. It often is multicentric and contains a concentration of vesicles, some with dense inclusions that mark them as early lysosomes. A relatively constant feature of cells engaged in endocytosis is the large number of microvilli at the cell surface. The degree of development of this surface adaptation is related to the phagocytic activity of the cell and its rate of pinocytosis. The number and size of mitochondria vary with the phagocytic and hence metabolic activity of the cell. Mitochondria tend to be grouped about the region of the Golgi complex, although several usually are seen dispersed about the cell periphery, presumably supplying energy for the active endocytic processes occurring there.

The most constant and characteristic ultrastructural features of macrophages are the electron-dense membrane-bound lysosomes that often can be seen fusing with phagosomes to form secondary lysosomes. Within the secondary lysosomes, ingested cellular, bacterial, and noncellular material can be seen in various stages of degradation, often recognizable as degenerating mitochondria or nuclear material. These secondary lysosomes also contain partially degraded material from the late stages of the endocytic process, often appearing as multilamellar lipid bodies. Microtubules and microfilaments are prominent in macrophages. Actin- and myosin-like proteins have been isolated from monocytes and partially characterized. Resting macrophages have irregular cell borders and pseudopodia pushed out in all directions. Their cytoplasm has rough endoplasmic reticulum and Golgi complex in the perinuclear area. Lipid globules, primary lysosomes, and mitochondria are characteristically prominent. Activated monocytes/macrophages are motile cells that extend a leading pseudopod as they move forward.²⁵

MEMBRANE RECEPTORS AND OTHER SURFACE PROTEINS OF MONOCYTES AND MACROPHAGES

Monocyte/macrophage cells have surface receptors that have been characterized by their binding to specific monoclonal antibodies. These receptors (Fig. 67–8) are markers for origin, growth, differentiation,²⁶ activation, recognition, migration, and function of the monocyte/macrophage. Monocytes have been classified into distinct subtypes based on surface expression of CD14 and CD16, molecules that form part of the lipopolysaccharide (LPS) toll-like receptor (TLR) and one of the immunoglobulin FcRs, respectively. These include CD14+-bright/CD16– monocytes, CD14+-dim/CD16+ monocytes, and CD14-dim/CD16+ monocytes. Monocyte heterogeneity was initially divided into the CD14+-bright/CD16-negative cells, which comprise 90 to 95 percent of total circulating monocytes (classical monocyte)²⁷—CD14-bright or dim refer to the fluorescence magnitude of staining using a specific CD14 monoclonal antibody. The minor subset is CD14-dim, CD16-positive, and less phagocytic than the classical monocyte. The classical monocyte produces reactive oxygen species (ROS) and cytokines in response to TLR engagement. The minor subset selectively secretes tumor necrosis factor (TNF)-α, IL-13, and CCL2 in response to viruses and immune complexes containing nucleic acids via TLR-7, TLR-8, MyD88-MEK (myeloid differentiation factor 88–MAPK kinase), and AHD.²⁸ This minor subset, CD14-dim,²⁹ is competent in (SR [scavenger receptor]) function of vascular, intraluminal debris and uptake of immune complexes.³⁰ In addition, their phenotype is related to the ability to produce and secrete select cytokines.³¹

Macrophages are proficient at endocytosis (both fluid phase and receptor-mediated) and are highly professional phagocytes of particulates of all origin, organic (cellular, microbial) as well as inorganic foreign materials.³² In contrast, when dendritic cells (DCs) mature into antigen-presenting cells (APCs), they have reduced uptake capacity and induce an adaptive immune response or tolerance. Immature DCs display active macropinocytosis and capture exogenous materials for cross-presentation.³³ It is convenient to classify plasma membrane uptake receptors as opsonic and nonopsonic TLRs and non–TLR-dependent. The latter category includes a range of SRs^34,35 and a family of lectin-like, carbohydrate recognition molecules.^36,37 Given the complex ligands presented on the surface of microorganisms and damaged host cells, or generated within the vacuolar system after uptake, these receptors frequently cooperate with one another.

Fc Receptors

FcRs for IgG are expressed on the surface of mononuclear cells, macrophages, granulocytes, and platelets.^38,39 FcRs are divided into three distinct classes: FcRI, FcRII, and FcRIII (Fig. 67–9). These receptors have broad ranges of expression on different cells. The first IgG receptor, FcRI (CD64), is a receptor found on monocytes, macrophages, and activated neutrophils. This receptor binds monomeric IgG through the Fc portion of the molecule. This Ig receptor has increased expression on activated monocytes and macrophages. CD64 allows for receptor-mediated endocytosis of IgG–antigen complexes for presentation to T cells, can trigger the release of cytokines and ROS, and can play a role in granulocyte-mediated antibody-dependent cytotoxicity. The second IgG receptor, FcRII (CD32), is a widely distributed receptor present on many cell types, including monocytes, platelets, neutrophils, B cells, some T cells, and some capillary endothelium. This receptor can bind complexed IgG rather than monomeric IgG. This FcR regulates B-cell function when coengaged with the B-cell receptor for antigen, namely, surface Ig. It also can induce mediator release from myeloid cells and phagocytosis of Ig-coated particles in vitro. Finally, this FcR also can target antigen into presenting pathways. The third IgG receptor, FcRIII (CD16), is expressed by neutrophils, natural killer cells, and tissue macrophages.⁴⁰ This receptor can bind Ig in immune complexes and Ig bound to cell-surface membranes. It is the main FcR responsible for antibody-dependent cellular cytotoxicity. All three FcRs specifically bind the human IgG subclasses IgG₁ and IgG₃ (Chap. 75). The interaction of FcR on macrophages with immune complexes results in cell “activation,” with an increase in phagocytosis, superoxide production, and prostaglandin and leukotriene release.

Complement Receptors

Activation of the complement system results in liberation of numerous ligands that bind to specific receptors on mononuclear phagocytes. Four receptors that bind fragments of the complement component C3 have been identified (see Fig. 67–9).⁴¹ Complement receptor (CR) 1 (or CD35) binds dimeric C3bi and is found on both monocytes and macrophages. CR3 (or CD11b) binds the complement fragment C3b. CR3 is a heterodimeric glycoprotein that is composed of two noncovalently linked polypeptides. The α chain of the polypeptide has an Mr of 185,000, and the β subunit has an Mr of 95,000. This receptor and the leukocyte antigens lymphocyte function–associated antigen (CD11a) and alpha-X integrin chain (CD11c) compose a family of heterodimers that share a common β subunit (CD18).⁴² This family is designated the leukocyte integrin (β₂) subfamily.⁴³ These heterodimers are involved in cell–cell interactions, including leukocyte trafficking into the tissues, binding of opsonized particles and plasma proteins, and attachment to various substrates. They also may modulate intercellular adhesion. Elimination of the integrin β₂ subunit causes leukocyte adhesion deficiency.⁴⁴

The classical opsonins, which promote the uptake of particles, are antibody, IgG complexed with antigens, and complement, activated by the classical pathway (antibody-dependent IgM or IgG) or recognized directly via the lectin-carbohydrate–stimulated alternative pathway. Fc and CRs are heterogeneous in structure, expression, and function, activating or inhibiting macrophage responses,^45,46 as illustrated in Fig. 67–9. Other opsonins include fibronectin and milk-fat globulin.⁴⁷ Through their expression of various opsonic receptors, monocytes, macrophages, and DCs perform versatile roles in innate and adaptive immunity,⁴⁸ in antigen clearance and destruction, in autoimmunity, and in pathogenesis of a range of inflammatory and infectious disorders. Genetic polymorphisms influence the expression and functions of FcRs in homeostasis and disease. Although prominent in host protection, invading microorganisms may be able to exploit, even subvert these receptors to facilitate their entry and survival.⁴⁹ Opsonic receptors play an important role in clearance of hematopoietic cells, for example, antibody-coated platelets, giving rise to thrombocytopenia, and in therapeutic antibody treatment, for example, to facilitate engraftment. Antibody engineering has provided novel therapeutic agents to minimize undesirable consequences, such as cell activation. The initiation or avoidance of complement activation in particular controls an important effector pathway in tissue injury and repair.

Toll-Like Receptors

The family of TLRs, identified on macrophages in mammals, is a pattern-recognition receptor that bind structurally conserved molecules derived from microorganisms, including endotoxins (LPS) and viral nucleic acids. TLRs are now considered key molecules responsible for alerting the immune system to the presence of microbial infections. For example, TLR4 is part of a recognition couple for LPS. Pathogen recognition by TLRs activates the innate immune system through the signaling pathway and provokes inflammatory responses, such as cytokine production.⁵⁰ These are shown schematically in Fig. 67–10 to illustrate their diverse structures and signaling pathways.

The discovery of TLR has transformed the study of innate immunity, inflammation, and adjuvant actions on APC.^51,52,53 Receptor structures, heterogeneity of expression, microbial and endogenous ligands, and signaling have been defined, and knowledge of their regulation has begun to offer agents to manipulate TLR signaling in humans. The discovery of inborn errors, such as the interleukin receptor–associated kinase (IRAK)-4 deficiency,⁵⁴ and the role of toll-interleukin receptor adaptor protein (TIRAP) function in Plasmodium falciparum infection,⁵⁵ for example, have illustrated their role in human disease. Several concepts have emerged. From the original studies on LPS recognition and signaling by the multiprotein complex formed by CD14, LPS binding protein, and MD2, and the clarification of the distinct adaptor pathways (MyD88 [myeloid differentiation factor 88], TIRAP/MAL [MyD88 adaptor-like], TRIF [TIR domain-containing adaptor inducing interferon (IFN)-β], and TRAM [TRIF-related adaptor molecule]), the recognition and sensing of TLR ligands have become clear. The tertiary structure of TLR4 has been reported.⁵⁶ TLRs are expressed either on the plasma membrane of myeloid and other cells, or within the vacuole, especially in the case of TLRs 3, 7, and 9, which are implicated in viral nucleic acid recognition. Crosstalk among nuclear factor (NF) κB, IFN, and mitogen-activated protein kinase (MAPK) kinase pathways has also become apparent.⁵⁷ TLRs collaborate with other recognition receptors,⁵⁸ such as dectin-1. Furthermore, a role has been proposed for TLR signaling in nontranscriptional activities, such as the kinetics of phagosome maturation in macrophages.⁵⁹

Non–Toll-Like, Nonopsonic Receptors

The study of lectins and SRs has lagged behind that of the above receptors, but is gaining ground, documenting receptor expression and ligands, mainly in mouse models of inflammation and infection.^35,60,61 These receptors are present on macrophages and DCs, and variably on monocytes and neutrophils. They are implicated in the recognition and uptake of microbial and host ligands, and vary in their ability to activate host defense functions. Figure 67–11 and Table 67–3 illustrate the functional attributes of these receptor systems. The mannose receptor is mainly involved in endocytosis, with a predominant intracellular localization.^62,63 The multilectin mannose receptor displays dual functions, contributing to the clearance of mannose-terminal lysosomal hydrolases and of neutrophil granule glycoproteins such as MPO, as well as of hormones (e.g., thyroglobulin) and exocrine secretion products (e.g., amylase). It plays a role in the capture and transport of mannose-terminal glycoproteins to targets in spleen (marginal metallophilic macrophages) and in lymph nodes (subcapsular sinus macrophages) that express sulfated receptors for its cysteine-rich domain. The outcome of such targeting is either silent disposal or, if combined with TLR stimulation, induction of an immune response.⁶⁴ In common with several other nonopsonic receptors, it can play dual, even opposing actions in host protection or in pathogenesis, as shown by ongoing studies in mice.

Dectin-1 is a lectin-like receptor that is widely expressed on myeloid cells, with a single immunoreceptor tyrosine-based activation motif (ITAM)–like motif in its cytoplasmic tail.⁶⁵ It recognizes β glucans, abundant in fungal walls, including bioactive zymosan particles, and has been implicated in innate resistance to fungal infection. Dectin-1 activates syk and caspase activation and recruitment domain (CARD)-9, regulating various effector pathways such as TNF-α, leukotriene production, and T-helper (Th) 17 cell activation, with heterogeneity in responses by macrophages and DCs. Dectin-1 collaborates with TLR 2/6 in the response to zymosan. Other lectins expressed by macrophages include sialic acid recognition molecules, Siglec-1 (sialoadhesin),⁶⁶ an extended Ig superfamily plasma membrane protein implicated in cell–cell interactions (Chap. 68 discusses a possible role in the hematopoietic system).

SRs are a diverse family of structurally unrelated, promiscuous receptors, with a predilection for polyanionic ligands, expressed by diverse microorganisms, apoptotic cells, and modified host lipoproteins.³⁴ SR-A I/II and MARCO (macrophage receptor with collagenous structure) (class A SR) are collagenous transmembrane receptors that mediate endocytosis, phagocytosis, and cell adhesion. SR-A I/II is upregulated by M-CSF and MARCO by TLR and MyD88-dependent microbial ligands, triggers of innate immune activation.⁶⁷ A number of naturally occurring ligands for SR-A have been identified, including apolipoprotein A₁ and Neisserial outer-surface proteins,⁶⁸ as well as previously described lipid A, lipoteichoic acid, and modified (acetylated) low-density lipoproteins, among others. After initial interest primarily in its role in atherogenesis (Chap. 134), attention has also focused on innate immune functions in bacterial infection.

Class B SRs, such as CD36 and SR-BI, have distinct structures and have been implicated in mycobacterial recognition as well as in the uptake and exchange of lipids.^69,70 CD36, together with thrombospondin, plays a role in apoptotic cell uptake⁴⁷ and has been implicated in macrophage fusion. Other SRs, expressed on a variety of cells as well as macrophages, have similar roles in clearance.

Human Leukocyte Antigen Class II Receptors

Monocytes and macrophages serve an important function as APCs. They bear the class II glycoproteins of the major histocompatibility gene complex, human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ. Expression of major histocompatibility complex (MHC) class II antigens on macrophages from different tissues varies widely. Splenic macrophages contain a high percentage of HLA-DR–positive cells (50 percent), whereas peritoneal macrophages have relatively few (10 to 20 percent).⁷¹ The proportion of Ia-positive alveolar macrophages is only approximately 5 percent.⁷² Lymphokines, primarily IFN-γ, can induce macrophages to express higher levels of MHC class II antigens, whereas prostaglandin E, α-fetoprotein, and glucocorticoids downregulate HLA-DR antigen expression on macrophages.

CD11

CD11 defines a family of three accessory adhesion surface glycoproteins: CD11a, CD11b, and CD11c. These proteins are distinct α subunits for three heterodimeric surface glycoproteins, each sharing a common β subunit, designated CD18. The α subunits have different isoelectric points, molecular weights, and cell distribution (Chap. 15).⁷³ Whereas CD11a is expressed on all leukocytes, CD11b and CD11c are expressed predominantly on monocytes and macrophages, a minor subset of B lymphocytes, and most polymorphonuclear leukocytes. CD11b is expressed on more than 95 percent of fresh human monocytes and macrophages but declines rapidly on cells maintained in vitro. Antibodies specific for CD11b, such as OKM1 or Mo1, may block this CR’s ability to bind to CD3bi.⁷⁴ Accordingly, these antibodies strongly inhibit CR-mediated rosetting of erythrocyte–IgM antibody–complement complexes.

CD14 and CD16

The CD14 molecule is one of the most characteristic surface antigens of the monocyte lineage. It is a polypeptide of 356 amino acids that is anchored to the plasma membrane by a phosphoinositol linkage.⁷⁵ It is expressed strongly on the surface of monocytes and weakly on the surface of granulocytes and most tissue macrophages. It can be detected on some nonmyeloid cells (e.g., hepatocytes and some epithelial cells). CD14 functions as a receptor for endotoxin (LPS). LPS binds to a serum protein, LPS-binding protein, which facilitates the binding of LPS to CD14. The coreceptor MD2 and TLR4 also are vital in this process. When LPS binds to CD14/MD-2/TLR4 expressed by monocytes or neutrophils, the cells become activated and release cytokines such as TNF and upregulate cell surface molecules, including adhesion molecules. In vitro, soluble CD14 binds to LPS, and the complex stimulates cells that do not express CD14 to secrete cytokines and coregulate adhesion molecules.⁷⁶

A subset of human blood monocytes that express low levels of CD14 molecules and high levels of the Fcγ receptor III (FcγRIII) CD16 has been identified.^77,78 These CD14+CD16+ monocytes resemble alveolar but not peripheral macrophages. CD14+CD16+ monocytes represent 5 to 10 percent of blood monocytes in normal individuals and can be dramatically expanded in pathologic conditions, such as sepsis, HIV infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines.

CD4

T lymphocytes express several surface receptors. The surface antigen CD4 is expressed primarily in T-helper lymphocytes (Chap. 76). CD4 and its corresponding messenger RNA have been demonstrated on monocytes, macrophages, and the monocyte-like cell line U-937.^79,80 Although CD4 is present at low concentrations in blood monocytes, the proportion of cells that display this plasma membrane determinant ranges from less than 5 percent to 90 percent. The CD4 molecule is involved in induction of T-lymphocyte helper functions (T₄) and T proliferative responses to antigen stimulation; however, its role in the function of monocyte/macrophages has not been determined. An important aspect of the monocyte/macrophage phenotype is the presence of CD4 molecules on the surface of monocytes that can act as receptors for HIV type 1 (HIV-1). HIV-1 uses the CD4 receptors as an entry pathway for infection of monocyte/macrophages.^79,80

Chemokine Receptors

Chemokines mediate their activities by binding to target cell surface chemokine receptors that belong to a large family of G-protein–coupled, seven transmembrane domain receptors. Human monocytes/macrophages express several chemokine receptors (Table 67–4). The chemokine receptor CCR5 has been implicated in HIV-1 infection of monocytes/macrophages.^{81,82,83,84,85} CCR5 is a major coreceptor on monocytes/macrophages for M-tropic HIV-1 infection. At least one copy of a 32-nucleotide deletion within the CCR5 gene (CCR5Δ32) has been found in approximately 4 to 16 percent of individuals, depending on their background; when in the homozygous state, individuals are highly protected against acquisition of HIV.^86,87

To illustrate the dynamic interaction of macrophages and virus, a video showing an HIV-1 infected human macrophage sensing its environment was captured from a spinning disk confocal microscope using a 100× objective by Raphael Gaudin (see http://www.cellimagelibrary.org/images/41568#.VAR6eNcDfRo.email).

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

APOPTOSIS

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

INFLAMMASOME

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.