Monocytes, Macrophages, and Dendritic Cells

Matthew Collin

Derralynn A. Hughes

Annette Plüddemann

Siamon Gordon

INTRODUCTION

Monocytes, macrophages, and dendritic cells (DCs) constitute a group of myeloid cells which share common hematopoietic origins and express related functions in host homeostasis and innate and acquired immunity. They develop in hematopoietic organs, enter the circulation, and are widely distributed throughout almost all tissues. They express a variable capacity for migration, phagocytosis, antigen presentation, and secretion.

Study of this family of cells, initially known as the reticuloen-dothelial system (RES), and later as the mononuclear phagocyte system (MPS), is undergoing rapid change, with recent emphasis on cellular heterogeneity and differentiation, compounded by complex modulation of their phenotype within tissues. As a result of their specialized functions, the fields of macrophage and DC immunobiology have diverged to a great extent. Our goal in this chapter is to re-integrate these divisions, to emphasize their related properties and variations on a common theme. While much remains unknown concerning their life history and properties in situ, there is a considerable body of detail now available from studies in cell culture and in experimental animals, especially in the mouse, which may not necessarily reflect their behavior in humans. In this chapter, we summarize their cellular and molecular properties and emphasize studies in humans, drawing attention where possible to clinically relevant functions and pathogenic mechanisms. While primarily aimed at hematologists, this chapter highlights the major populations present in the
tissues, both normally and in a range of inflammatory, infectious, metabolic, and neoplastic diseases.

A BRIEF HISTORY¹^,²^,³ and ⁴^,⁵^,⁶^,⁷

The award of the Nobel Prize in 1908 to Elie Metchnikoff, shared with Paul Ehrlich, established the macrophage as a specialized phagocyte, distinct from the microphage (neutrophil) and an important part of the inflammatory cellular response to foreign bodies and infection. There was still considerable confusion between monocytes and lymphocytes, based on morphology and histology alone. Electron microscopy in the 1950s (such as the work of Marchesi and Florey) characterized the ultrastructure of monocytes, their diapedesis and the pleomorphic appearance of macrophages in tissues. The precursor-product studies of Ebert and Florey and the migration studies of Volkman and Gowans helped to clarify the distinctions between these mononuclear cells. Recent reviews by Cavaillon²^,³ cover the origins, the first half of the 20th century, and the emergence of the RES in detail. During this time the importance of mycobacterial infection of monocytes and macrophages was a popular research topic: for example, in the significant work of Florence Sabin.

In the 1960s Zanvil Cohn and James Hirsch turned from their studies on granulocytes and defined the endocytic and cellular properties in culture of mouse macrophages that had been isolated from the peritoneal cavity. Together with Ralph van Furth and a group of investigators, they established the classification of macrophage-related cells as members of the MPS. A series of influential international Leiden conferences reviewed and published progress of research in the field from the late 1960s to the early 1990s.

During the 1950s and 1960s George Mackaness described the antigen-dependent but nonspecific activation of macrophages by cell-mediated protective immune responses to intracellular pathogens such as BCG and Listeria monocytogenes. This association of macrophages with T lymphocytes had profound implications for acquired immunity. The search for accessory cells responsible for antigen specific activation of T helper lymphocytes led to the discovery of DC, by Ralph Steinman and Zanvil Cohn⁸ in 1973. The unique role of DCs in antigen presentation, later shown by others to depend on the Major Histocompatibility Antigen complex, forms a bridge between innate and adaptive immunity and was recognized by part of the Nobel Prize for Medicine and Physiology in 2011.

Macrophages and DCs are both considered to be components of the MPS, although significant debate remains concerning their exact relationship and derivation in the steady state and inflammation. Derivatives of macrophages and DCs form an abundant component of the organized structures known as granulomata, as shown by Walter Spector. Although they are a prominent feature of important chronic infectious diseases such as tuberculosis the nature of their interactions with T lymphocytes and their precise functions are still unclear.

Other milestones of macrophage research include the discovery and exploitation of growth factors for macrophages and other hematopoietic cells, notably by Donald Metcalf; the identification of cytokines such as tumor necrosis factor (TNF) by Anthony Cerami and Lloyd Old, that play an important role and provide a therapeutic target in diseases such as rheumatoid arthritis; and the recognition by Russell Ross of the central role of monocytes that adhere to the walls of large arteries and give rise to lipid-laden macrophages in atherosclerosis. In addition, Marcel Bessis recognized the hematopoietic importance of macrophages in erythroblastic islands, although the full implications of his discovery remain to be explored.

The DC field has gained increasing momentum since the 1970s. The discovery of toll-like receptors (TLRs), C-type lectins, and other pathogen-associated molecular pattern recognition systems were key events in understanding how DCs and macrophages distinguish self from nonself. The contributions of Jules Hoffman and Bruce Beutler to this area were also recognized in the 2011 Nobel Prize. Another important breakthrough was the isolation of distinct precursors of DCs and macrophages through the work of several groups including Frederic Geissmann, Marcus Manz, Ken Shortman, Miriam Merad, and Michel Nussenzweig. More recently, the genetic control and specialized functions of distinct DC subsets have been documented in both mouse and humans. Unique pathways of ontogeny have also been described for special members of the MPS including osteoclasts, epidermal Langerhans cells, and microglia of the brain.

FIGURE 10.1. Summary of the main pathways of monocyte, macrophage, and dendritic cell (DC) development in human tissue during embryogenesis, fetal development, adult steady state, and inflammation. A: Embryonic. Yolk sac macrophages are the first hematopoietic cells to appear. Studies in mice indicate that these cells migrate through the mesoderm prior to vasculogenesis, proliferating and colonizing all the tissues of the embryo. Lineage tracing indicates that 100% of microglia arise from the yolk sac, becoming isolated in the developing brain by closure of the blood-brain barrier. They are maintained in adult life by local proliferation. B: Fetal. In models derived from lineage tracing in mice, hematopoiesis in the liver gives rise to the first monocytes, which circulate widely and dilute the contribution of yolk sac macrophages in almost all tissue sites. Residual yolk sac contribution of approximately 10% is detectable in the Langerhans cells of the epidermis; the other 90% are fetal liver-derived and become locally self-renewing in adult life. Tissue macrophages and specialized populations of macrophages: alveolar, Kupffer, and osteoclasts, may also derive from fetal liver monocytes, at least initially. C: Adult steady-state hematopoiesis, arising from multi-lymphoid progenitors (MLP) capable of differentiating into monocytes, DCs, lymphoid cells, and granulocyte-macrophage progenitors (GMP), contributes a wide variety of cells with macrophage and DC potential into the blood. These include circulating CD34+ MLP, classical CD14+ and nonclassical CD16+ monocytes, CD1c+ myeloid DCs, CD141+ myeloid DCs, and plasmacytoid DCs. The contribution of these steady state blood components to macrophage homeostasis in a wide range of tissues, in addition to those illustrated, has not been rigorously defined. CD34+ precursors and both subsets of monocytes potentially contribute to tissue macrophages in interstitial and specialized sites. CD14+ monocytes and CD16+ monocytes are closely related to CD14+ interstitial DCs, while CD1c+ and CD141+ blood DCs both have phenotypically similar interstitial DC counterparts. CD34+ precursors also have the potential to form tissue DCs. Plasmacytoid DCs do not migrate into the tissues in the steady state. D: Inflammation. Upregulation of myelopoiesis under conditions of stress is likely to involve expansion of GMP production of monocytes, myeloid DCs, and plasmacytoid DCs. Multiple inflammatory signals potentially recruit all peripheral blood populations to the tissues. Classical CD14+ monocytes are the most significant component giving rise to inflammatory activated “M1” macrophages and TNF/i-NOS-producing DCs (Tip-DCs) in a range of tissues. This may include the CNS (pathway not shown for simplicity here). Langerhans cells are also replaced by inflammatory classical monocytes in desquamating epidermal inflammation. Myeloid DCs may be directly recruited to give inflammatory DCs, and infiltration with plasmacytoid DCs is well documented.

In the following section, we summarize general and specialized features of monocytes, macrophages, and DCs, collectively termed mononuclear phagocytes (MPs). Figure 10.1 illustrates a compre-hensive overview of human MP differentiation and distribution, to be discussed in detail in subsequent sections.

FUNCTIONAL PROPERTIES

Cellular Morphology⁹^,¹⁰

Aspects of mononuclear cell differentiation and morphology in situ and ex vivo are illustrated in Figures 10.1, 10.2, 10.3 and 10.4 and summarized in Table 10.1.

FIGURE 10.2. Ultrastructure of promonocyte and monocyte. A: Electron micrograph of a promonocyte from human bone marrow stained for peroxidase. The nucleus (n), situated at one end of the cell, exhibits an irregular outline and deep indentation. The cytoplasm contains a number of cytoplasmic organelles. Peroxidase reactivity is demonstrable throughout the rough endoplasmic reticulum (er), Golgi complex (G), and all cytoplasmic granules (g⁺₁, g⁺₂, g⁺₃). Apparently, all granules mature from the earliest forms, which are spherical and dense (g⁺₁), with a homogenous matrix, to more condensed and elongated forms (g⁺₂), and then to dumbbell forms (g⁺₃). The Golgi complex (G) is composed of several stacks of cisternae and occupies a large area adjacent to the nucleus. Bundles of filaments (f) are prominent in the cytoplasm and are believed to be useful in characterizing the cell as a monocyte form. Several mitochondria (m) are also seen ×18,000. B: Electron micrograph of a normal human monocyte examined for peroxidase. In the nucleus (n), the chromatin is more condensed than in earlier forms, is mainly peripheral in distribution, and is interrupted at the nuclear pores. The voluminous cytoplasm (c) contains a full complement of organelles associated with protein synthesis and export of secretory granules. Peroxidase is present in only some of the granules (g⁺), but others (g:), as well as the endoplasmic reticulum (er) and Golgi complex (G), now lack the reaction product. At this stage, the two kinds of granules are approximately equal in number and similar in size and shape, ranging from 90 to 450 nm in length and from spherical or rodlike to dumbbell in shape. Microtubules (mt) radiate from the cell center, where a centriole can be seen adjacent to the Golgi complex (G). The moderately abundant endoplasmic reticulum has a more peripheral distribution than in the promonocyte, and modest numbers of mitochondria (m) are present. Numerous pseudopodia (ps) extend from the cell surface. The peripheral lacunae (l) represent a tangential section through surface irregularities ×16,200. From Nicholls BA, Bainton DF: Differentiation of human monocytes in bone marrow and blood. Sequential formation of two granule populations. Lab Invest 1973;29:27-40, with permission.

FIGURE 10.3. Morphology of mouse mononuclear phagocytes. A: Mouse peritoneal macrophages in culture on cover glasses undergo cell fusion shortly after addition of UV-irradiated Sendai virus. Note cytoplasmic bridge formation in nascent homokaryon. Phase contrast microscopy reveals characteristic nuclear morphology, rudimentary nucleoli, and extensive phase-lucent and phase-dense vesicles. Plasma membrane shows spreading of organelle-poor cell periphery with ruffling. From Gordon S, Cohn Z. Macrophage-melanocyte heterokaryons. I. Preparation and properties. J Exp Med 1970:131:981-1003, with permission. B,C: Phase contrast micrograph of dendritic cell isolated by Steinman and Cohn from mouse spleen. B: Note extensive dendrites, euchromatin nucleus, and mitochondria-rich cytoplasm, a feature of their high motility. C: Note lack of resetting of DCs with antibody-coated sheep erythrocytes, compared with macrophages in the same preparation, one of the features which allowed separation and their distinction. From Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 1973:1142-1162, with permission. D: Scanning electron microscopy of human alveolar macrophages cultured on cellulose membrane in a biphasic cell culture system. Note characteristic rounded appearance with extensive ruffles. From Wallaert B, Fahy O, Tsicopoulos A, Gosset P, Tonnel AB. Experimental systems for mechanistic studies of toxicant induced lung inflammation. Toxicol Lett 2000;112-113:157-163, with permission.

FIGURE 10.4. Mononuclear phagocytes in human skin and thymus. A: Epidermis: CD1a⁺ Langerhans cells. B: Dermis/interstitium Lyve-1+ macrophages and CD31+ endothelium. C: Epidermis: Langerhans cells express langerin and MHCII, which co-localize. D: Electron micrograph of Birbeck granules. E: Dermis: Triple labeling shows CD11c (DCs, green), XIIIa (macrophages, red) and CD3 (T cells, blue). F: Thymic macrophages and DCs express EMR2. Photomicrograph D from Romani N, Clausen BE, Stoitzner P. Langerhans cells and more: langerin expressing cell subsets in the skin. Immunol Rev 2010;234(1):120-141, with permission. Other images from M. Collin, N. Romani and T. Marafioti.

TABLE 10.1 SELECTED PROPERTIES OF HUMAN MONONUCLEAR PHAGOCYTES

Properties		Functions
A. Monocytes
Low level in blood (˜1-4%)
*Subpopulations:*
	Classical (˜90%) CD14^hi CD16^var	Phagocytosis, secretion, inflammation
	Nonclassical (˜10%) CD14^mod/dim CD16^hi	Patrolling, intravascular
*Migration/adhesion:*		Recruitment by inflammation, infection, immune stimulation
	CCR2⁺ (classical)
	CX₃CR₁, variable
	CD11b⁺
	CD11c⁺
	LFA-1 (nonclassical)
*Recognition, endocytosis, phagocytosis:*		Clearance
	Opsonic receptors, e.g., FcR, CR	Humoral immunity
	Nonopsonic receptors, e.g., TLR, CD36, Lectins	Innate immunity, homeostasis
	Cytosolic sensors	Inflammasome activation
*Biosynthesis: varied secretion:*		Link to acquired immunity
	Myeloperoxidase (MPO), Lysozyme	Antimicrobial
	Respiratory burst (RB)	Microbial killing
	Cytokines	Cell activation, regulation
	Leukotrienes	Inflammation
	Tissue factor	Procoagulant
*Differentiation/modulation:*
	Precursor for macrophages and DCs	Inflammation, T cell activation
B. Macrophages
	Widely distributed: Lymphohemopoietic tissues as resident and recruited populations	Local and systemic homeostasis Trophic functions—inflammation and repair
	Variable life span
	Heterogeneous phenotype
	Sessile (mainly)
*Endocytosis and phagocytosis:*		Clearance, digestion, host defense
	Opsonic (Fc, CR)	Humoral immunity
	Nonopsonic recognition (SR, Lectins)	Defense and homeostasis
	CD163, CD71, folate receptors	Uptake hemoglobin-haptoglobin, transferrin, B12
	Sensing (TLR, cytosolic)	Innate immunity
	Regulatory receptors, e.g., TREMs, EMR2, GPCR	Potentiate responses
*Biosynthesis and secretion:*		Trophic and cytocidal
	Pro- and anti-inflammatory cytokines	Inflammation, repair
	E.g., IL-1, TNF, IL-6, IL-12, IL-18, IL-10, TGF-β	Innate immunity
	Neutral proteinases, enzymes	Tissue catabolism
	Low-molecular-weight mediators: RB, nitric oxide, prostanoids	Defense and inflammation
*Modulation:*
Classical (M1) and alternative activation (M2)		Regulation of cellular immunity
*Immune interactions:*		Activation/suppression of primed T cells
	Expression of MHCII and costimulatory molecules	Adaptive immune regulation
C. Myeloid Dendritic cells (mDC)
	Minor population in blood, lymph tissues	Sentinel cells
	Short to medium-lived	Bridge innate to adaptive immunity or tolerance
	Subsets described, varied antigen markers
	Motile	Migration
	Chemokine receptors, e.g., CCR7
	Transient adhesion
	Endocytosis and phagocytosis	Antigen capture
*Two-stage maturation/activation:*		Induced by LPS, other ligands, e.g., TLR
	Loss endocytosis, macropinocytosis, phagocytosis	Processing, direct and cross-presentation to naive lymphocytes
	Intracellular MHCII redistributed to surface
	Limited digestion, capture peptides DM
	Specialized endosomal compartment (multivesicular bodies, Birbeck granules)
	CD1d expression	Lipid antigen presentation
	Cytosolic sensors	Nucleic acid recognition
	Costimulatory molecules	Activation and inhibition of APC functions
	TLR, PRR-dependent cytokine production, e.g., IL-12, IL-18	Activation, regulation of tissue activation
D. Plasmacytoid Dendritic Cells (pDCs)
	Minor, blood, tissue
	Short-lived, labile
	Mixed phenotype markers, e.g., CD123 (IL-3R); TLR3, 7, 9; Siglec H (mouse), RLR, helicases
*Biosynthesis, secretion:*
Type I interferon+		Antiviral responses
IL-1, interleukin-1; MPO, Myeloperoxidase; RLRs, RIG-I-like receptor; PRRs, pattern recognition receptors; TGF-β, tumor growth factor-β; TNF, tumor necrosis factor; TLR, Toll-like receptors; LFA-1, Lymphocyte function-associated antigen 1; APC, antigen presenting cell; MHCII, major histocompatibility complex Class II.

Monocytes are rounded cells, 10 to 15 µm in size, with oval, kidney-shaped, or indented nuclei, a rim of heterochromatin, and mostly euchromatic nucleoplasm. Their cytoplasm is relatively abundant, compared with nonactivated lymphocytes, containing myeloperoxidase+ and rudimentary lysozyme+ granules, nonspecific esterases, and lysosomes. Scanning electron microscopy reveals extensive surface folds. They adhere readily to native and artificial substrates by a range of adhesion receptors, flattening out and spreading in characteristic fried egg or stellate shape with fine plasma membrane processes. They respond slowly to chemotactic gradients in vitro, compared with neutrophils.

Macrophages are larger, rounded (e.g., in alveolar space, peritoneal cavity), or stellate, with two or more processes, sticky and sluggish though motile, and with extensive, dynamic plasma membrane processes and filopodia. Podosomes have been observed. The cytoplasm of cultured cells has a well-developed centrosome, with an organized cytoskeleton, and is rich in synthetic organelles and endocytic vesicles, often containing debris and residues of phagocytosis in abundant lysosomes. They lose their peroxidase granules as monocytes mature in culture. Macrophages love lining up along surface irregularities on culture substrata and usually keep their distance from one another in culture; inflammatory stimuli cause aggregation. Occasional binucleate cells arise by failure of cytokinesis; macrophage fusion can be observed “spontaneously,” especially on selected substrates, or after exposure to serum lipids or cytokines such as interleukin (IL)-4.

DCs in the blood are smaller than monocytes and may be found in the “lymphoid” gates on flow cytometry analyses. In the tissues, they are smaller than macrophages and less extensively arborized with multiple short processes. During migration through the afferent lymph they mature into “veiled cells” with extensive macropinocytic processes. In lymph nodes they are highly motile and transit rapidly between T cells until a cognate antigen interaction is detected by the T cell. In vitro they are lightly or nonadherent compared with monocytes or macrophages, a property that was exploited in their original isolation from mouse spleen.

FIGURE 10.5. (A) Recruitment. Stages of monocyte adherence to endothelium and diapedesis, induced by inflammatory stimuli. The model is mainly based on the recruitment of neutrophils, with which it shares many features, although monocyte- specific chemokines, receptors, and adhesion ligands exist, especially in constitutive and noninfectious, metabolic forms of inflammation. Figure provided by S. Yona. (B) Monocyte diapedesis. Electron micrograph from collection of H. Florey.

Chemotaxis, Migration, and Adhesion

See Figures 10.5 and 10.6A. Selected antigen markers and receptors are listed in Tables 10.2 and 10.3.¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹^,²⁰^,²¹ and ²²^,²³^,²⁴^,²⁵

Chemotaxis of MPs to inflammatory sites is stimulated by factors such as complement component C5a, N-formylated oligopeptides, fragments of fibronectin, elastin, and collagen; and by secreted proteins called chemokines. Stimulation by chemotactic factors results in increased integrin affinity and therefore binding to the endothelium at the same time as formation of lamellipodia and actin polymerization, resulting in cell movement. Chemokines are divided into subclasses on the basis of the spacing of the N-terminal cysteine residues. CCL#, CXCL#, CL#, and CX3CL# refer to four families of chemokine ligands, in which # denotes the identifying number, C denotes a cysteine, and X denotes a noncysteine amino acid. CC chemokines are responsible for attracting monocytes and lymphocytes, directing traffic of leukocytes under steady state conditions; whereas inflammatory chemokines are expressed by circulating leukocytes and other cells only upon activation. In the mouse two subsets, classical (Ly6C+ analogous to human CD14⁺⁺CD16⁻) and nonclassical monocytes (Ly6C^{lo, human} CD14^dimCD16⁺) express different chemokine receptors, especially CCR1, CCR2, and CX3CR1 (fractalkine receptor). Under conditions of inflammation, monocytes are recruited to tissues by CCL2 (MCP-1), CCL3 (MIP1a), and CCL5 (RANTES) acting through CCR1, CCR2, and CCR5. These inflammatory monocytes are able to differentiate into macrophages or DCs that appear to play important roles in the initiation of immune responses. The signals that drive steady state recruitment have not been solved but are independent of CCR1, CCR2, and CCR5.

Monocytes and macrophages adhere to other cell types, including lymphocytes and vascular endothelial cells, and to extracellular matrix components, e.g., fibronectin and laminin, using specific cell surface adhesion receptors. Inflammatory macrophages and DCs originate from the migration and differentiation of circulating monocytes into virtually all tissues, contributing to

the pathophysiology of many human diseases including atherogenesis and other inflammatory diseases. The origin of steady state tissue macrophages and DCs is much less certain, as discussed below. It is likely that blood monocytes and blood DCs contribute, but circulating hematopoietic progenitor cells may also give rise to tissue leukocytes.

FIGURE 10.6. Selected macrophage plasma membrane receptors. (A) Growth, differentiation, adhesion and migration. (B) Recognition and sensing.

FIGURE 10.6. Selected macrophage plasma membrane receptors. (C) Potentiation and negative regulation.

Three families of cell surface glycoproteins mediate most cell adhesion: integrins, immunoglobulin-related molecules, and selectins. Integrins are large heterodimeric glycoproteins classified in subfamilies according to the common beta subunit; beta 1 (CD49/cd29) or VLA, beta 2 (CD11/CD18), beta 3 (cytoadhesion), and beta 7 are the most relevant. Integrins recognize Ig-related molecules such as intercellular adhesion molecule-1 (ICAM-1), ICAM-2, and vascular cell adhesion molecule (VCAM). Selectins (L-selectin, E-selectin, and P-selectin) recognize oligosaccharides, e.g., Sialyl Le^x. CD44 and CD36 are structurally unrelated but important adhesion molecules which bind to hyaluronic acid and thrombospondin. Macrophage entry into tissues is a multistep process involving both adhesion and transendothelial migration. The process involves monocyte tethering and rolling on the surface of activated endothelial cells mediated mainly by activated selectins; firm adhesion is mediated by VCAM-1 and ICAM-1 on the endothelium, binding to β1 and β2 integrins expressed on leukocytes. Subsequently, monocytes transmigrate the endothelium.

The dynamic complexity of adhesion receptor utilization in chronic inflammatory processes such as atherosclerosis is exemplified by the following studies. Analysis of integrin, immunoglobulin-related, and selectin expression on blood monocytes, in vitro differentiated macrophages, and alveolar macrophages reveals monocyte expression of β1 (CD29), α4, α5, α6, β2 (CD18), CD11a, CD11b, and CD11c subunits, but not αV (CD51). Some expression of CD41b (IIb) and CD61 (β3) has been detected. The Ig-related molecules CD54 (ICAM-1), ICAM-2, and CD58 (LFA-3) are expressed, as well as L-selectin and the carbohydrate ligands Le^x (CD15) and sialyl Le^x. CD44 and CD36 are strongly positive. Alveolar macrophages exhibit lower expression of α4, α6, β2, CD11a, CD11b, L-selectin, Le^x, and sialyl Le^x. ICAM-2 and CD36 are absent, whereas expression of α3, but not of CD11c, is higher. Similar results were obtained with in vitro differentiated macrophages. The profile of adhesion receptors expressed as monocytes differentiate into macrophages varies according to tissue location and the disease state. For example, endothelium overlying human atherosclerotic lesions and fatty streaks expresses high levels of E-and P-selectins, ICAM-1, and VCAM-1. Selectin deficiency reduces atherosclerosis, and genetic mutation of ICAM-1 or VCAM-1 reduces atherosclerosis in mice. Oxidized LDL leads to endothelial dysfunction, leading to expression of adhesion molecules and recruitment of monocytes into the subendothelial space. Ox-LDL is taken up by macrophages via scavenger receptors such as SR-A1, SR-A2, and LOX-1, which are themselves implicated in local macrophage adhesion. CD36, a multifunctional membrane receptor present on MPs and other cells, functions as a scavenger receptor for oxidized phospholipids. On macrophages, CD36 interaction with oxidized LDL triggers a pro-inflammatory and pro-atherogenic response involving activation of src-family kinases, MAP kinases, and Vav family guanine nucleotide exchange factors and results in ligand internalization, foam cell formation, and inhibition of migration.

Macrophage adhesion molecules also have an important role in homotypic cell fusion, forming osteoclasts and multinucleated giant cells associated with chronic inflammation. Progress has recently been made in identifying molecules involved in macrophage fusion. Signaling processes mediated by DAP12 and STAT6 induce a fusion-competent status. Chemotaxis through CCL2, cell-cell adhesion mediated by E-cadherin, exposure of phosphatidylserine, lipid recognition by CD36, purinergic receptors, and cytoskeletal rearrangement dependent on RAC1 are prerequisites for successful macrophage fusion.

TABLE 10.2 SELECTED ANTIGEN MARKERS EXPRESSED BY HUMAN MONONUCLEAR PHAGOCYTES

	Antigen	Ligand/Comment
Monocytes:	CD14	LPS, TLR
	CD16^+/−	FcRγ
	CD115	CSF-1
	CD45	Leukocyte common, phosphatase
	CD11b	Complement receptor
	CD11c^+/−	Complement receptor
	CD11a	LFA-1—patrolling monocytes
	CD163	Haptoglobin-hemoglobin complex
	CD36	OxLDL
	SLAN^+/−	6-SulfoLacNAc
	CX₃CR₁	Fractalkine
	MHCII	Peptides
	CD4
Macrophages	CD115	CSF-1
	CD68	Endosomal pan-macrophage, DC osteoclasts
	CD312 (EMR2)	Myeloid adhesion GPCR (also monocytes, DCs, neutrophils)
	CD169	Siglec-1 (sialoadhesin), macrophage subsets, e.g., marginal zone, subcapsular sinus
	CD206	MMR, alternative activation
	fXIIIa
mDCs:	CD115	CSF-1, IL-34
	CD116	GM-CSF
	CD135	Flt-3L
	CD11c	Not unique to DCs
	CD1a, c (BDCA-1), d	Major subset
	MHCII^hi
	CD83, 80, 86	Costimulatory molecules
	CD205 (Dec205)	Lectin-like
	CD206	MMR
	CD209 (DC-SIGN)	Interstitial DCs, not unique
	CD141 (BDCA-3, thrombomodulin)	Minor subset—cross presenting (widely expressed)
	Langerin	Mannosylated and sulfated glycans
	CD103 (α_Eβ₇ Integrin)	Gut DCs tolerogenic
pDCs:	CD123 (IL-3R)
	CD303 (BDCA-2)	Type II C-type lectin
	CD304 (Neuropilin-1; BDCA-4)
	(Siglec H-member mouse CD33 family)
	CD2	Lymphoid antigen
	CD7	Lymphoid antigen
CSF-1, colony stimulating factor-1; GM-CSF, granulocyte-macrophage colony stimulating factor-1; TLR, Toll-like receptors; LPS, lipopolysaccharide; MMR, macrophage mannose receptor; OxLDL, oxidized low-density lipoprotein; BDCA, blood dendritic cell antigen.

The regulation of DC function is intimately linked to migration. At sites of antigen contact, myeloid or “classical” DCs are found in immature forms with high expression of pattern recognition receptors (PRRs) and endocytic activity, but low surface expression of MHC and costimulatory molecules. Migration and maturation are believed to occur in the steady state, but are massively increased by local inflammatory stimuli mediated by TLR agonists, cytokines, and other danger signals. DC migration from tissue sites to afferent lymphatics is integrin-independent and driven by chemokine gradients of CCL19 and CCL21 acting through CCR7. Recent studies show that these chemokines decorate the endothelium of afferent lymphatics. The passive drainage of fluid through afferent lymphatics may also play a part in the migration of DCs towards lymph nodes. Within the node, similar chemokine gradients position DCs in the T cell areas, where they upregulate antigen-bearing MHC molecules and costimulatory antigens. Antigen uptake declines and morphological changes ensue, including the extension of dendrites. Myeloid and plasmacytoid DCs also enter the lymph nodes directly from the bloodstream, where they undergo a parallel process of maturation. In the later stages of immune activation, monocyte-derived DCs also reach the lymph nodes by direct recruitment from the blood or via inflamed tissues.

TABLE 10.3 SELECTED PLASMA MEMBRANE PATTERN RECOGNITION RECEPTORS (PRRs) EXPRESSED BY HUMAN MONONUCLEAR PHAGOCYTES

Receptor	Ligand	Comment
FcR	Immunoglobulins	Phagocytosis, ADCC
CD11b/18	Complement, other	Phagocytosis, myeloid adhesion
CD163	Haptoglobin-hemoglobin complex
CD14	LPS	TLR complex
TLR (various)	Microbial, endogenous
Siglec-1 (CD169)	Sialylated glycoconjugates	Subset macrophages
DC-SIGN (CD209)	Mannose, fucose, GlcNAc
Dectin-1 CLEC-1	β-glucan	Fungal infection
Dectin-2	High mannose	Th17 activation
Langerin	Mannosyl and sulfated glycans	Abundant in Birbeck granules
Mincle	C-type lectin-like	Binds mycobacterial cord factor
CLEC-9A (DNGR-1)	Actin	Cross-presentation
TAM (Tyro3, Axl, MER)	Gas6, ProtS	Apoptotic cell uptake
SR-A	Polyanions, ApoA1, LPS, LTA Neisseria proteins	Endocytosis, adhesion
MARCO	LPS, Neisseria proteins	Innate immunity, phagocytosis, adhesion
CD36	OxLDL, phosphatidylserine	Adhesion, apoptotic cell clearance, fusion of macrophages
TIM(1-4)	Phosphatidylserine	Apoptotic cell uptake
CD300a	Phosphatidylserine	Apoptotic cell uptake
EMR2	Chondroitin sulfate B	Adhesion GPCR
CD97	CD55	Adhesion GPCR
TLR, toll-like receptor; ADCC, antibody-dependent cell-mediated cytotoxicity; GPCR, G protein-coupled receptor; LPS, lipopolysaccharide; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; OxLDL, oxidized low-density lipoprotein; LTA, lipoteichoic acid; TIM, T-cell immunoglobulin mucin; EMR2, epidermal growth factor-like module-containing mucin-like hormone receptor 2.

Recognition: Plasma Membrane Antigen Markers, Sensors, and Regulators

See Figures 10.6, 10.7, 10.8, 10.9, 10.10, 10.11 and 10.12 and Tables 10.1, 10.2 and 10.3.²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹ and ³²^,³³^,³⁴^,³⁵^,³⁶^,³⁷

Monocytes and macrophages express a range of opsonic and nonopsonic plasma membrane and endosomal receptors to recognize foreign and modified self-ligands. These include activatory (ITAM cytoplasmic motif) and inhibitory (ITIM motif) Fc receptors, receptors for complement-derived ligands, TLRs, lectins, and scavenger receptors, as well as receptors for other humoral ligands, for example, collectins and pentraxins. Monocytes also express CD14, a GPI-linked receptor for LPS, which is downregulated upon cell maturation in culture. Resting monocytes do not express class A scavenger receptor I/II, which is upregulated on macrophages during maturation, but do express MARCO,
a related SR, and CD36, another promiscuous adhesion molecule also present on platelets and endothelial cells. The lectins include Dectin-1, the receptor for fungal β-glucan, expressed by monocytes, macrophages, neutrophils, and DCs; the mannose receptor, a multilectin with distinct carbohydrate recognition domains for GlcNac, mannosyl, and fucosyl ligands on microbes and selected host molecules; and a cysteine-rich domain which recognizes endogenous sulfated sugars. Other mannose binding lectins include DC-SIGN and Dectin-2; galectin is a receptor for galactosyl recognition. Additional plasma membrane receptors include CD163, an SR-like molecule that recognizes haptoglobin – hemoglobin complexes, and which is induced by glucocorticoids. Additional SR, not shown, are present on endothelial cells, which also express SR-A. Apart from the scavenger receptors, several other receptors can recognize apoptotic cells, e.g., the Tyro3, Axl, and Mer (TAM) receptors, which recognize Gas 6 and Protein S ligands. Several TIM molecules and CD300A bind phosphatidyl serine, another ligand for apoptotic cell recognition.

FIGURE 10.7. A: Fc receptors of human macrophages show diversity for Ig class ligands and signaling. See references for further details. B: Macrophage receptors for complement recognition and regulation.

FIGURE 10.8. Cooperation of Toll-like receptor signals and Dectin-1 (with cytosolic inflammasome activation and type I interferon) in innate immune defense. From Trinichieri G, Sher A. Cooperation of Toll-like receptor signals in innate immune defense. Nat Immunol Rev 2007;7:179-190, with permission.

In common with monocytes and macrophages, DCs are equipped with an array of PRRs, which can detect evolutionary-conserved pathogen-associated molecular patterns (PAMPs), including proteins, lipids, carbohydrates, and nucleic acids. The PRRs encompass families of membrane-bound TLRs and C-type lectin receptors (CLRs), such as Langerin, MMR, DC-205, DC-LAMP, DC-SIGN, Dectin-1, and DCIR. The PRRs also include cytosolic NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and helicase nucleic acid receptors, as discussed further below. Human DCs are equipped with multiple CD1 antigens for the recognition of bacterial lipid antigens (notably CD1a and CD1c) and a range of receptors for complement (CD11b and CD11c) and coagulation proteins (CD141: thrombomodulin).

The SIGLEC family of leukocyte plasma membrane molecules interacts with sialylated glycoconjugates, and members are variably expressed on myeloid cells of different species, including macrophages and plasmacytoid DCs. Siglec-1 (sialoadhesin, CD169) has been implicated in binding of hematopoietic cells, including lymphocytes.

The murine macrophage plasma membrane antigen marker F4/80 is part of a small family of seven transmembrane adhesion GPCR which includes CD97 and EMR2, more broadly expressed on myeloid cells. EMR2 is a useful immunocytochemical marker for human tissue macrophages, as well as neutrophils, in which it potentiates a range of cellular functions. The TREM family also modulates myeloid cell responses; TREM-2, for example, associates with DAP-12 as one of several signaling partners. Counterreceptors include the negative receptor pair CD200/CD200R.

Genetic polymorphisms have been described for a range of the above surface molecules; for example, TLR mediating pro-inflammatory signaling and Fc receptors, which mediate important functions such as phagocytosis, antibody-dependent cytotoxicity, and immunomodulation.

Cytosolic Recognition and Inflammasome Activation

See Figures 10.8 and 10.9.³⁸^,³⁹^,⁴⁰^,⁴¹

This topic has benefited enormously from the study of auto-inflammatory syndromes associated with rare genetic
disorders, and their dramatic amelioration by IL-1 antagonists. Inflammasomes are protein complex platforms which are required for the activation of inflammatory caspases and the maturation of their pro-inflammatory cytokines including IL-1β and IL-18. They are constructed around several proteins, including NLRP3, NLRC4, AIM2, and NLRP6, and recognize inflammatory signals arising from receptors for PAMPs, damage-associated molecules (DAMPs), and sterile particulates, such as uric acid crystals. Recognition of immune signals by one of several families of receptors results in direct activation of caspase-1 and caspase-5, secretion of potent pro-inflammatory cytokines, and a form of cell death called pyroptosis. PRRs of innate immune cells can be classified into phagocytic and sensor PRRs and, in addition to the plasma membrane receptors described above, include intracellular TLRs, retinoic acid-inducible gene I-like receptors (RLRs), and nucleotide-binding oligomerization domain-like receptors (NLRs). Inflammasome-mediated processes are important during microbial infections and also in regulating both metabolic processes and mucosal immune responses. For example, the NLRP3 inflammasome has
been demonstrated to be involved in antibacterial, viral, fungal, and parasitic immune responses. On infection with influenza A, endosomal TLR7 recognizes viral RNA and induces transcription of the NLRP3 inflammasome components. Selected bacteria have been shown to allow the cytoplasmic entry of flagellin, the NLRC4 ligand, leading to activation of the NLRC4 pathway. Some inflammasome activators are exogenous particles, e.g., silica and asbestos, whose uptake by pulmonary macrophages activates NLRP3 inflammasome-dependent caspase activation, cytokine, and cellular reactive oxygen species release, contributing to silicosis or asbestosis. Inflammasomes have also been implicated in metabolic pathologies with activation of caspase-1 by NLRP3 in adipose tissue, resulting in inhibition of insulin signaling, expression of TNF-α, and induction of CD4 T helper cells. However, a protective role for activated inflammasomes in age-related macular degeneration has also been proposed, since lack of NLRP3 or IL-18 exacerbates choroidal neovascularization.

FIGURE 10.9. Models of inflammasome structure. A: A Toll-like receptor (TLR). TLRs are integral membrane glycoproteins with an N-terminal ectodomain and a single transmembrane domain. The ectodomain of a TLR7, TLR8, and TLR9 family member is depicted, with the leucine-rich repeat (LRR) solenoid shown with a gray molecular surface, and the N- and C-terminal flanking regions shown in green and purple, respectively. An undefined region present in TLR7, TLR8, and TLR9, but not in the other TLRs, is shown as a light blue string. Insertions within LRRs at position 10 are indicated in red and might contribute to the formation of the pathogen-associated molecular pattern (PAMP) binding site. An insert at position 15 is indicated in yellow. Also shown is a cartoon of the transmembrane domain (presumed to be a single α-helix), followed by a molecular surface representation of the TLR1 Toll-IL-1 and IL-18 receptor (TIR) domain. From Bell JY, Mullen GE, Leifer CA, Mazzoni A, Davies DR, Segal DM. Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol 2003;24:528-533, with permission. B: Activators of the inflammasome. Sterile activators include host- and environment-derived molecules, and pathogen-associated activators include PAMPs derived from bacteria, viruses, fungi, and protozoa. Assembly of the NLRs, ASC, and caspase-1 leads to the formation of a pentad- or heptamer structure: the inflammasome. Activation of the inflammasome leads to maturation and secretion of IL-1β and IL-18, as well as inflammatory cell death, by either pyroptosis or pyronecrosis. ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; CPPD, calcium pyrophosphate dihydrate; MDP, muramyl dipeptide; MSU, monosodium urate; NLR, nucleotide-binding domain, leucine-rich repeat containing; PAMP, pathogen-associated molecular patterns. From Davis BK, Wen H, Ting JP. The inflammasome NLRs in immunity, inflammation, and associated diseases. Annu Rev Immunol 2011;29:707-735. C: Electrostatic attraction underlies innate dsDNA recognition by the HIN domains. Both oligonucleotide/oligosaccharide binding folds and the linker between them engage the dsDNA backbone. An autoinhibited state of AIM2 is activated by DNA that liberates the PYD domain. DNA serves as an oligomerization platform for the inflammasome assembly. From Jin T, Perry A, Jiang J, et al. Structures of the HIN domain: DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor. Immunity 2012;36(4):561-571, with permission.

FIGURE 10.10. A: 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 LAMP1; the pan-macrophage CD68 antigen is associated with late endosomes and lysosomes. B: Schematic representation of FcR-mediated uptake of a bacterium by a zipper-like mechanism, initiating actin assembly. Modified from Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature 2003;422(6927):37-44. C: Dectin-1 transfected fibroblast taking up unopsonized zymosan (yeast wall particle) through β-glucan recognition, to initiate a phagocytic cup. (Courtesy of G. Brown.) D: Uptake of Pneumocystis carinii via Dectin-1. Contact zone resembles phagocytic synapse described by Goodridge et al.³⁰

Intracellular detection of nucleic acids by DCs is very sophisticated. TLR3, TLR7, and TLR9 cooperate with a large family of helicases, RLRs, and DNA sensors in the recognition of single- and double-stranded nucleic acids. Ligation of these receptors leads to rapid phenotypic maturation, activation of antigen-presenting capacity, and cytokine release; plasmacytoid DCs, for example, release abundant type 1 interferon in response to viral infection.

Endocytosis and Phagocytosis

See Figures 10.10 and 10.11.⁴²^,⁴³^,⁴⁴^,⁴⁵ and ⁴⁶^,⁴⁷ and ⁴⁸

Monocytes and macrophages are able to internalize and ingest soluble and particulate ligands, including apoptotic cells and micro-organisms, with variable efficiency. Surface molecules which mediate receptor-mediated endocytosis, apart from those listed above, include transferrin (CD71) and folate receptors. Macrophages are able to internalize the equivalent of their surface in 20 minutes; and a series of fusions and fissions, assisted by small GTP-ases, result in membrane retrieval, receptor recycling to the cell surface, or cargo delivery from endosomes to lysosomes for digestion. Phagosome formation and fusion result in formation of phagolysosomes. Fc receptor-mediated uptake depends on circumferential engagement of the target ligands by pseudopodia, a zipper-like process, and ingestion depends on the actin cytoskeleton. Complement-mediated uptake proceeds by a distinct “sinking” mechanism. Underwood and colleagues have described formation of a phagocytic synapse in the uptake of yeast particles
by Dectin-1, which has a hemi-ITAM in its cytoplasmic domain, by a syk- and CARD9-dependent pathway. Uptake is associated with acidification, to promote digestion of all macromolecules.

FIGURE 10.11. A: Phagocytic receptors for apoptotic cell phagocytosis. Macrophages and immature myeloid dendritic cells (mDCs) express multiple receptors for direct or opsonic binding, e.g., mannose binding lectin (MBL) or milk fat globulin (mFGE8). Phosphatidylserine (PS) exposed on the outer surface of the apoptotic cell can be recognized by a range of receptors, including TIM4, TIM1 and CD300A (not shown). Discrimination of nonself and altered-self may involve combinations of different phagocyte receptors. Apoptotic cell uptake results in an antiinflammatory response (e.g., release of transforming growth factor-β and prostaglandin E2), but has also been implicated in cross-presentation by DCs. From Savill J, Dransfield I, Gregory C, Haslett C. A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2002;2:965-975, with permission. B: Thymic macrophage with apoptotic thymocyte in phagocytic vacuole. The vast majority of thymocytes never leave the thymus, undergo apoptosis, and are rapidly removed by macrophages (×4,500).

FIGURE 10.12. Plasma membrane remodeling, cytoskeletal assembly, and cytoplasmic signaling associated with Fc receptor-mediated particle ingestion. From Greenberg S. Diversity in phagocytic signalling. J Cell Sci 2001;114(Pt 6):1039-1040, with permission.⁴⁴

Endocytosis by DCs follows special pathways that preserve antigens for presentation on MHC molecules. Most DCs are competent to present externally acquired antigen to maturing MHC class II dimers in the late endosomes. A complex pathway involving invariant chain and HLA-DM maintains MHC class II in an open configuration for peptide binding. A subset of DCs is competent at cross-presenting externally acquired antigen to Class I MHC. These
DCs are characterized by high expression of CD141 in humans and CD8 in mice. They also possess a unique chemokine receptor XCR1 and lectin CLEC9A (DNGR-1) with actin as ligand; and they express a high level of TLR3. Specialized biochemical machinery allows these cells to transfer exogenous antigen to the endogenously loaded MHC class I presentation pathway.

Autophagy is an analogous process by which macrophages and DCs surround damaged cytoplasmic organelles with membrane for delivery to lysosomes, especially after microbial infection. Several components involved in this process have been identified and some have been implicated in genetic predisposition to inflammatory bowel disease. This is also thought to be a key process in antigen presentation of cytosolic antigens to MHC class I.

Signaling and Gene Expression

See Figures 10.8, 10.9, and 10.12.⁴⁹^,⁵⁰^,⁵¹ and ⁵²

A range of signaling pathways have been characterized in myeloid cells following on receptor ligation and collaboration, which differ depending on the cell type involved. These include sequential phosphorylation/dephosphorylation by kinases and phosphatases, the assembly and disassembly of protein complexes, and redistribution of molecules including phosphoinositides and lipid metabolites from cytoplasm to the plasma membrane as well as to the nucleus. The adaptor proteins for TLR-signaling include MyD-88, TRIF, and TIRAP/MAL. Signaling pathways that are particularly well characterized include MAP kinase activation, classical and alternative NFkB activation, and GPCR activation, e.g., by leukotrienes. Actin assembly and disassembly play a prominent role in cell migration and phagocytosis, as well as secretion. Gene expression depends on dynamic changes in chromatin conformation, binding and release of transcription complexes, and epigenetic modifications of DNA as well as histones by methylation and acetylation. Recent studies have begun to compare transcription by monocytes, macrophages, and DCs in considerable detail, including microRNA analysis. Translational modifications include glycosylation, prenylation and other lipid modifications, and ubiquitination, which determine protein turnover and export, as well as biosynthetic quality control. Current high throughput sequencing and microarray and proteomic analysis is set to transform our knowledge of cell functions and differences as well as of similarities among monocytes, macrophages, and DCs. We include a few recent references of useful resources in a fast-moving field.

FIGURE 10.13. Maturation/activation of dendritic cells (DCs) leads to acquisition of APC function. A: DC maturation ex vivo. Langerhans cells upon explantation from mouse skin undergo “spontaneous” maturation with redistribution of MHCII from an intracellular compartment to the cell surface. (Courtesy of Ira Mellman and Ralph Steinman.) B: One of the first systems to produce large numbers of human dendritic cells from blood monocytes; the added cytokines and other maturation stimuli influence the function of the DCs. Based on studies by Lanzavecchia, Sallusto, Romani, Schuler, Bhardwaj, and their colleagues.

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