Gregory A. Taylor
J. Brice Weinberg
The mononuclear phagocyte system, also known as the reticuloendothelial system (RES), is composed of monocytes, macrophages, and their precursor cells (1). The cells composing this system (Table 12.1) possess common origins, morphologies, and functions, and they arise in the bone marrow from progenitors committed to mononuclear phagocyte production. Monocytes are released into the blood, and after a short time in the circulation, migrate into different tissues, either randomly or specifically in response to chemotactic stimuli. In the tissues, they differentiate in response to soluble stimuli to become tissue macrophages with characteristic morphologic and functional qualities. This process of differentiation (the acquisition of different, more specialized functions) has been termed “activation.” Figure 12.1 depicts the bidirectional, factor-influenced modulation of function of a monocyte or macrophage.
Cells of the mononuclear phagocyte system are phylogenetically very primitive. No animals can live without them. They participate in a wide variety of important functions in the body, including removal of dead, senescent, foreign, or altered cells or foreign particles; regulation of the function of other cells; processing and pre-sentation of antigens in immune reactions; participation in various inflammatory reactions; and destruction of microbes and tumor cells.
History
Ilya Metchnikov studied ameboid cells of invertebrate animals. He thought that these mobile cells, which were capable of ingesting solid particles, could serve in the defense of the organism against “noxious intruders.” In an experiment using the transparent starfish larva, he observed the cells migrating to and aggregating around a rose thorn introduced into the tissue (2,3). He coined the term macrophage for these phagocytic connective tissue cells (as opposed to the more common granular leukocytes or microphages). Based on his subsequent observations on phagocytosis in invertebrates and the prior observations of Cohnheim regarding the migration of leukocytes through vessel walls (4), he stated in 1882 that “Diapedesis and accumulation of white corpuscles in inflammatory diseases must be regarded as modes of defense of the organism against microorganisms, the leukocytes in this struggle devouring and destroying the parasites” (5).
In the late 1800s, Ehrlich introduced techniques for staining blood cells; these allowed a more exact identification of the white corpuscles. Using several different dyes, he divided the blood cells into lymphocytes, large mononuclear cells, large mononuclear cells with indented nuclei, and polymorphous nucleated cells with neutrophilic, acidophilic, or basophilic granules (6). The “large mononuclear cells with indented nuclei” were later called monocytes (3).
The term reticuloendothelial system was first used by Aschoff in 1924 to designate cells that have the ability to remove and store injected carbon particles or the dye neutral red from the blood and lymph (7). “Reticulo” refers to the property of forming reticular or lattice networks in various organs, and “endothelial” refers to the location of most of the phagocytic cells identified in these early studies. The microscopic techniques at that time could not discern that the vascular linings in organs such as the spleen, liver, and lymph nodes were composed of endothelial cells (essentially nonphagocytic cells) as well as phagocytic cells (macrophages). Modern methods permit the distinction to be made, and thus the term RES for the body’s system of monocytes and macrophages is a misnomer. During the early investigations, different components of the system were named descriptively, depending on their location, the nature of material engulfed by the cells, and the diseases affecting the cells, as well as their relationship to other structures. For example, macrophages of the tissues were called “histiocytes,” the histiocytes of loose connective tissue were referred to as “resting wandering cells” or “ameboid wandering cells” depending on their observed motility in the tissues, while the highly vacuolated histiocytes were called “rhagiocrine cells” because of their supposed secretory activity. The histiocytes of the omentum were named “clasmatocytes” because of the cytoplasmic budding interpreted by Ranvier as indicating that the cells served a nutritive function. The “littoral cells” were those histiocytes adjacent to the bloodstream (e.g., the von Kupffer cells of the liver, or the splenic macrophages) (8). In the early 1900s, several investigators demonstrated that blood monocytes, after long-term culture in vitro, developed into cells that were similar in appearance to tissue macrophages, inflammatory epithelioid cells, and multinucleated giant cells (9,10,11). Using meticulous observations with supravital dyes through the transparent rabbit ear, Ebert and Florey showed that blood monocytes emigrated into the tissue in vivo, and changed into cells with the appearance of inflammatory tissue macrophages (12).
In the 1950s and 1960s, work by George Mackaness et al. helped define mechanisms by which macrophages become activated to control the growth of intracellular organisms (13). Zanvil Cohn and et al. in the 1960s through the 1990s defined many aspects of the cell biology of mononuclear phagocytes (14). John Hibbs, Carl Nathan, and colleagues in the 1970s through 1990s further defined the processes of macrophage activation and the importance of nitric oxide as a macrophage effector molecule for antimicrobial and antitumor action (15,16). Studies beginning in the 1970s by Seymour Klebanoff, Bernard Babior, John Curnutte, and colleagues helped clarify mechanisms of oxidative microbial killing and molecularly define the phagocyte oxidase (17,18,19). Donald Metcalf, Richard Stanley, and coworkers in the 1970s through 1990s did detailed studies of hematopoiesis and growth factors demonstrating basic mechanisms of hematopoiesis and monocytopoiesis, the clinical uses of colony-stimulating factors, and the actions of macrophage colony-stimulating factor (M-CSF) in monocytopoiesis and mononuclear phagocyte function and embryogenesis (20,21). Molecular characterization of leukocyte receptors and ligands by a variety of investigators in the 1980s and 1990s gave new insights into mononuclear phagocyte interactions with other cells (e.g., lymphocytes, endothelium, and tumor cells) and microorganisms. In the 1990s, gene transfer or gene disruption techniques (22,23) gave new tools for the analysis of mononuclear phagocyte and cytokine biology. In the late 1990s and early 2000s, researchers identified mammalian analogues of the Drosophila toll receptors, and identified the microbial components that serve as their ligands; these included the toll-like receptor 4 (TLR4) and its ligand lipopolysaccharide (LPS) (24,25). The complex interplay of novel cytokines and receptors (osteoprotegerin, osteoprotegerin ligand, and receptor activator of NF-κB [RANK]) controlling osteoclast formation and differentiation was defined (26). Researchers further established the arginine/inducible nitric oxide synthase/ arginase pathway as a regulator of cell growth and toxicity, apoptosis, inflammation, and host resistance to tumors and microbes (16,27,28,29,30). New details of monocyte and macrophage heterogeneity revealed morphologic, immunologic, and functional differences in blood monocytes and tissue macrophages that dictate normal physiology as well as recruitment of monocytes into inflammatory sites (31). And finally, further particulars of the pathways of macrophage activation (classical, alternative, and type II) were dissected (32,33).
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Table 12.1 Cells of the Mononuclear Phagocyte System |
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Phylogeny and Ontogeny
Phylogenetically, mononuclear phagocytes are primitive cells. Invertebrates have phagocytic mononuclear cells that are known by various names (e.g., celomocytes, amebocytes, hemocytes) and are morphologically and functionally similar to the mononuclear phagocytes of mammals (34). These or comparable phagocytes are present in all the phyla of the animal kingdom (metazoan or higher). Whereas lymphocytes first appear in the cyclostome hagfish (vertebrate), immunoglobulins (Igs) and complement (C′) appear later in evolution. Amebocytes serve various functions in these primitive animals. First, they are important in nutrition. Different particles and microbes are digested and transported by the amebocytes, and the digested products are used by the host (35). Second, amebocytes serve as scavengers by surrounding and ingesting/digesting apoptotic, dead, or dying tissue. This is especially important in the physiologic process of metamorphosis in insects and amphibians (2,34,36) as well as in encapsulating and walling off foreign material (34,37). Third, amebocytes play an important role in protecting the animal against infections and possibly cancer. For example, cells of the earthworm, crayfish, starfish, oyster, cockroach, and sponge recognize bacteria as foreign, and phagocytize and destroy them. Cells from crayfish also can kill mammalian tumor cells in vitro by a nonspecific, nonphagocytic process comparable to that of mammalian macrophages (38). Invertebrates have soluble “recognition factors” that function as primitive opsonins for particles (34,38); these may have been forerunners of the Ig and C′ systems. As in mammalian macrophages, products from bacteria appear to be specifically chemotactic for amebocytes (39). Phagocytes mediate the rejection of foreign tissue grafts (34,40). Thus, phagocytes from evolutionarily primitive animals function very effectively in protecting the host in the absence of B or T lymphocytes, Ig, or C′. With evolution and the acquisition of lymphocytes, Ig, and C′, phagocytes developed receptors for these ligands, and their nutritive function decreased, but the important functions of phagocytosis, endocytosis, encapsulation, and recognition of foreign substances remained.
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Figure 12.1. Modulation of mononuclear phagocyte function. Monocytes and macrophages can produce and elaborate many different bioactive products, and their function can be modulated by different substances. The figure depicts the reversible “activation” and “deactivation” mediated by these substances. Also see Table 12.6 regarding different types of “activated” macrophages. CC, chemokine; CSF, colony-stimulating factor; D3, vitamin D3; GF, growth factor; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; ROS, reactive oxygen species; RNS, reactive nitrogen species; TGF, tumor growth factor; TNF, tumor necrosis factor. |
During ontogeny, pluripotent hemopoietic stem cells sequentially occupy the embryonic yolk sac, fetal liver, spleen, and adult bone marrow. Hematopoietic stem cells are derived from ventral mesodermal tissue. Stem cells migrate from the yolk sac and colonize receptive intraembryonic hemopoietic organs where they can then respond to stimuli for differentiation along certain cellular pathways (41). The embryonic macrophages play important roles in tissue metamorphosis by scavenging dying and dead cells, and possibly by mediating the destruction of cells in areas of tissue remodeling (2,36). Mononuclear phagocytes may also regulate embryonic growth by the secretion of various mitogens or growth factors (42,43).
Before day 9 in the mouse embryo, no cells identifiable as mononuclear phagocytes are seen anywhere (including the yolk sac), even though the yolk sac contains cells capable of forming macrophage colonies in appropriate assays (44). After day 10, glass-adherent macrophages with Fc receptors are seen in the yolk sac. After 12 days’ gestation, mononuclear phagocytes are found in the liver and then in other tissues (44). In fetal rats, macrophages with phagocytic capability can be detected in inflammatory lesions as early as 16 days. Work in transparent zebrafish embryos has shown that macrophages appear as early as erythroid cells, originating from the ventrolateral mesoderm (45). The macrophages migrate to the yolk sac, differentiate, and then join the blood or invade the head region. These macrophages can phagocytize apoptotic cells and also ingest and kill injected bacteria. In zebrafish hematopoiesis, macrophages apparently use a rapid differentiation pathway that goes from early hematopoietic cells directly to relatively mature, functional macrophages without use of the monocyte as an intermediary cell (45). Macrophages in zebrafish rather specifically express high levels of the gene for the actin-binding protein l-plastin and the gene for lysosome C (46).
The macrophage inflammatory response is seen at earlier times in gestation than is the neutrophil response (47). Research in transparent zebrafish has shown that infection with mycobacteria in the embryo at a stage before lymphocyte development results in macrophage aggregates with hallmarks of granuloma (48). At birth, mononuclear phagocyte function is not fully developed. Neonates are highly susceptible to infections by viruses and intracellular pathogenic bacteria and have a poorly developed inflammatory response. Studies in mice show that the abnormalities relate to abnormal immune-response–related antigen (Ia) expression that results in poor antigen presentation, macrophage-mediated cytotoxicity, and interleukin-1 (IL-1) production. These defects can be bypassed by treatment of macrophages with lymphokines (49,50). Prostaglandins of the E series and α-fetoprotein suppress the expression of major histocompatibility complex (MHC) class II antigen on macrophages; these substances may play a role in the low expression of MHC class II antigen in neonatal macrophages. The uptake and degradation of opsonized bacteria and the phagocytosis of complement-coated erythrocytes are normal in neonatal macrophages (49,50).
Morphology
Monoblasts
The monoblast normally is found only in the bone marrow. This cell is difficult to identify with certainty (51). However, the ability of single cells grown in agar culture to produce mixed granulocyte-macrophage or pure macrophage colonies suggests that there is a common precursor that develops into neutrophil (microphage) or macrophage cells (52,53). The monoblast is defined by its ability to form mature colonies of mononuclear phagocytes in in vitro cultures. It is nonmotile, is nonadherent to glass or plastic, and mea-sures about 14 μm in diameter. It is characterized by deeply basophilic cytoplasm, a large nucleus with little indentation, fine stringy chromatin, and one or two large nucleoli. Numerous fine, spherical, or slender, rodlike mitochondria can be seen in supravitally stained cells, but neutral red-containing vacuoles are absent or only a few very fine bodies are seen. These are presumably pinocytic vesicles. On electron microscopic examination, aggregated ribosomes and scattered strips of endoplasmic reticulum are seen in the cytoplasm, but the Golgi apparatus is small (54). Distinguishing the monoblast from the myeloblast on morphologic grounds is difficult and probably impossible even when using the electron microscope.
Promonocytes
The human promonocyte, essentially the earliest cell clearly recognizable morphologically in this lineage, is 11 to 13 μm in diameter and has a high nuclear:cytoplasmic ratio (51). The nucleus is indented with fine chromatin and may contain a nucleolus. The cytoplasm has considerable basophilia on Wright stain, and the cells stain for nonspecific esterase, peroxidase, and lysozyme (Table 12.2). These cells can phagocytize opsonized bacteria and IgG-coated erythrocytes but not IgM C′-coated erythrocytes. There is active pinocytosis as determined by uptake of dextran sulfate. A high proportion of these cells can incorporate tritiated thymidine, indicating that they are actively synthesizing DNA (55). On electron microscopy, rough endoplasmic reticulum (RER) is well developed, and free polyribosomes are scattered throughout the cytoplasm (56) (Fig. 12.2). The Golgi apparatus is well developed, and granules of varying size and shape surround it, together with small vesicles that seem to arise from the Golgi cisternae. The RER, Golgi cisternae, and early granules contain peroxidase, aryl sulfatase, and acid phosphatase (57). Numerous bundles of filaments are present in the cytoplasm around the nucleus, a feature of value in identifying these cells as monocytes (56,57,58,59). The cytoplasmic membrane of the promonocyte exhibits various processes and projections that presumably are related to its property of motility and active endocytosis (56,57). The promonocyte is distinguishable from the promyelocyte by the fact that promonocytes contain fewer and smaller granules, and their granules lack crystalloids; the cytoplasm characteristically contains bundles of filaments, and the nuclei are quite irregular and deeply indented (58).
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Figure 12.2. 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. (From Nichols BA, Bainton DF. Differentiation of human monocytes in bone marrow and blood. Lab Invest 1973;29:27, with permission.) |
Monocytes
Monocytes are 10 to 11 μm in diameter, being generally smaller than promonocytes but larger than other mature leukocytes. Monocytes seen in the bone marrow and the peripheral blood vary considerably in size and shape, but the “typical” monocyte is usually easily distinguishable from other leukocytes. The nucleus is large and oval or indented and centrally placed. By light microscopy, nucleoli usually are not seen. The nuclear chromatin is delicate, and the membrane is thin. The cytoplasm is abundant, is gray or light blue-gray in Wright-stained preparations, and contains numerous fine, clear or lilac vacuoles (Fig. 12.3). The granules resemble fine dust and give the bluish cytoplasm a ground-glass appearance. In certain circumstances (e.g., bacteremia) or with heavy staining, the granules appear more prominent. The delicate chromatin and the bluish color of the cytoplasm in the monocyte are most helpful in differentiating these cells from metamyelocytes or band forms of neutrophils. Granules in monocytes contain peroxidase, but are much smaller than those found in neutrophils.
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Figure 12.3. Monocyte ×1,000 (approximately). Wright stain. |
The cells attach avidly to plastic (polystyrene) or glass, are motile in their adherent state, and spread and project thin processes within 1 to 3 hours. On phase microscopy, phase-dense granules, which correspond to lysosomes, can be seen in the cytoplasm. The lysosomes are excluded from the actin-rich thin border area of cortical cytoplasm (hyaloplasm). Cell motion is ameboid in nature; large, filmy, irregular pseudopods extend slowly from the delicate cytoplasm as the cell moves randomly. In response to chemotactic signals, the movement is directed, and the cell assumes a δ shape with the slender, pointed edge trailing (60).
Electron microscopy shows that the mature monocyte contains a horseshoe-shaped nucleus, with dense, granular peripheral chromatin surrounding extensive, light-staining central nucleoplasm (Fig. 12.4). Nucleoli have been observed in as many as 50% of blood monocytes. The mitochondria are spherical or elongated and are usually located in the periphery of the abundant cytoplasm. The Golgi apparatus is well developed, and small vesicles are especially numerous in this region but may be found throughout the cytoplasm.
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Figure 12.4. 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 form 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 tangential section through surface irregularities. ×16,200. (From Nichols BA, Bainton DF. Differentiation of human monocytes in bone marrow and blood. Lab Invest 1973;29:27, with permission.) |
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Figure 12.5. Electron micrograph of a peroxidase-negative human macrophage that developed after 14 days in liquid culture. The eccentric nucleus contains a distinct nucleolus (nu) and the cytoplasm is filled with many organelles (i.e., mitochondria [m], rough endoplasmic reticulum [rer], a large Golgi complex [G], and numerous small vesicles of vacuoles [v]). No peroxidase can be detected at this late stage of maturation. In addition to the occasional clear vacuoles (v) are many inclusions, which are peroxidase-negative granules (p-g). Their content is unknown. ×14,500. e, filipodium. (From Bainton D, Golde DW. Differentiation of macrophages from normal human bone marrow in liquid culture. J Clin Invest 1978;61:1555, with permission.) |
At the monocyte stage, peroxidase production ceases, and the RER and Golgi complex no longer contain the enzyme, but peroxidase is present in storage granules (58). A second population of granules is produced that contain no peroxidase (58,59,61). In normal human monocytes, peroxidase activity can be detected in the Golgi area and in the rough endoplasmic reticulum after adherence to plastic or glass for 2 to 18 hours. Patients with hereditary deficiency of neutrophil peroxidase have monocytes that lack the enzyme in their storage granules (61). However, after adherence and in vitro culture, peroxidase activity appears as in normal monocytes, suggesting that the peroxidase stored in monocyte granules and the peroxidase that appears in the RER and Golgi after adherence are two distinct proteins (61). The peroxidase of the RER and Golgi is inhibited by aminotriazole and sodium azide, while that of the granules is not (59). Cytochemical staining at the light and electron microscope levels have demonstrated many other enzymes including acid phosphatase, sodium fluoride–resistant nonspecific esterase (α-naphthyl butyrate or acetate esterase), naphthylaminidase, lysozyme, β-glucuronidase, 5′ nucleotidase, galactosidases, and numerous other hydrolases and proteases (62,63,64,65,66).
Macrophages
Macrophages represent the tissue component of the mononuclear phagocyte system. This is a very heterogenous group of cells with many different phenotypes. In general, they are thought to arise from emigrated blood monocytes, and apparently differentiate in response to local conditions and factors (1). Macrophages are large, actively phagocytic cells measuring 15 to 80 μm in diameter. Their shape is irregular, and their motility comparable to that of blood monocytes. Bleblike and filiform pseudopods are seen frequently. The cytoplasm is abundant. The nucleus is egg shaped or may be indented or elongated. When stained with Wright stain, the chromatin appears “spongy,” and the nuclear membrane is distinct. The cytoplasm is sky-blue and contains coarse, azure granules and vacuoles. Electron microscopy of macrophages shows a spectrum of cell types ranging from those comparable to monocytes to much larger cells with more cytoplasm and more vacuolization and granulation (Fig. 12.5). Peroxidase is seen in the RER and Golgi, but in contradistinction to monocytes, is absent in the granules. Some “intermediate” cells are seen in which peroxidase is present in the RER, Golgi, and granules; these may be monocytes that have recently emigrated into the tissues (67).
Mononuclear Phagocyte Production and Kinetics
The major site of production of mononuclear phagocytes is the bone marrow. Primitive stem cells become committed to the mononuclear phagocyte lineage in a stochastic fashion, and then, under the influence of growth factors and cytokines, differentiate into monocytes. Myeloid cell growth is controlled by different glycoprotein growth factors that can cause the development in vitro of colonies composed of granulocytes only (G-CSF), macrophages only (M-CSF), both granulocytes and macrophages (GM-CSF), or granulocytes, macrophages, normoblasts, megakaryocytes, mast cells, and stem cells (IL-3) (52,68). The macrophage growth factors cause both proliferation and differentiation of primitive hematopoietic cells to monoblasts, promonocytes, and mature monocytes. They work through specific, high-affinity cell-surface receptors that initiate the cell proliferation (52,69). Receptors for M-CSF are restricted to cells of the mononuclear phagocyte system and are species specific. Mouse mononuclear phagocytes bind M-CSF specifically with high affinity (69). The receptor for the mononuclear phagocyte growth factor, M-CSF is closely related to the c-fms proto-oncogene product (70,71). The transcription factor PU.1 is necessary for the M-CSF–dependent proliferation of mononuclear phagocytes (72). Mononuclear phagocyte development during fetal and neonatal life is dependent on M-CSF (73). The critical importance of M-CSF versus GM-CSF in the development of mononuclear phagocytes is underscored by the absence of abnormalities in mononuclear phagocytes in mice lacking GM-CSF (those with targeted disruption of the GM-CSF gene) (74,75).
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Figure 12.6. Model of the production and kinetics of the monocyte-macrophage system in humans. The marrow and blood compartments are drawn to show their relative sizes. The compartment transit times are derived from 3HTdR and 3H-DFP labeling studies. A small number of young cells may enter the blood from the G1 phases of the promonocyte cell cycle (curved arrows). The box outlined by dashed lines indicates a large compartment of marrow promonocytes in the resting or G0 stage (see text). CMP, circulating monocyte pool; G’ cell generation time with the subcompartments G1 (pre-DNA synthesis gap), S (DNA synthesis period), and G2 (post-DNA synthesis gap); GI, gastrointestinal; MMP, marginal monocyte pool; T1/2 half life. |
Studies of human mononuclear phagocytopoiesis have shown that 3% of bone marrow cells are promonocytes (76). When marrow cells were incubated in vitro with 3HTdR, one group of investigators showed that about 79% of the promonocytes were labeled (77), while another group showed that 12% were labeled (76,78). Double-labeling studies (3HTdR and 14CTdR), as well as serial 3HTdR injections, indicated a DNA synthesis time of about 10 hours and a generation time of 29 hours for promonocytes. When 3HTdR was injected into normal subjects and the subsequent appearance of labeled monocytes in the blood was monitored, a lag period of only 5 to 7 hours was noted, thus demonstrating the absence of a significant storage compartment. Subsequently, several waves of labeled cells appeared, reflecting the flow into the blood of cells labeled during promonocyte proliferation in the marrow (78). From studies of the time course of labeled monocytes in the blood after the intravenous injection of 3HTdR, the transit time through the marrow proliferation pool was estimated to be 54 hours (78). This is about twice the promonocyte generation time of 29 hours and suggests that there are, on the average, two catenated promonocyte generations in the bone marrow of normal man (Fig. 12.6). The size of the marrow promonocyte pool in normal man has been calculated from data on total marrow cellularity to be about 60 × 106 cells/kg of body weight. From these data, the labeling index of 12% and DNA synthesis time of 10 hours, a production rate of 7 × 106 cells/kg/hour can be calculated (60 × 106 × 0.12/10 hours) (78). Other workers have demonstrated comparable values using different techniques (51,77). Similar numbers of cells are turning over through the blood as determined from blood monocyte kinetic measurements (79).
Monocyte Kinetics in Blood
Blood monocytes are a population of recently formed young cells on their way from the bone marrow to their ultimate sites of activity in the tissues. Therefore, monocyte production can be calculated from the turnover of blood monocytes. Blood monocyte kinetics have been evaluated in humans by in vivo and in vitro labeling of autologous blood with tritiated diisopropyl fluorophosphate (3H-DFP), reinjecting the labeled cells, and measuring the proportion of labeled cells present at later times by autoradiographic techniques (78,79). These studies have shown the marginal monocyte pool to be about 3.5 times the size of the circulating pool. Blood monocytes left the vascular system in an exponential manner with a half-time of 4.5 to 10.0 hours (mean of 8.4 hours) (78,79). This 8.4-hour half-life is shorter than the 71.0 hours calculated from experiments using in vivo labeling with 3HTdR (80). From the blood pool size and the half-time value, the blood monocyte turnover rate was calculated to be about 7 × 106 cells/kg/hour (78).
Alterations in monocyte kinetics have been measured after the acute or chronic administration of adrenal glucocorticoids to humans (81) and mice (82). Profound monocytopenia develops promptly in both species, and its duration and degree depend on the amount, solubility, and route of steroid administration. In man, the cellularity of induced exudates is decreased by steroid administration (81). In mice, the number of cells harvested from the peritoneum is only moderately decreased (about 30%), but the flow of 3HTdR-labeled monocytes from the blood into the peritoneum in response to an inflammatory stimulus is markedly reduced. The mechanism of the sudden monocytopenia is not fully understood, nor is it clear whether the reduced cellularity at the site of inflammation merely reflects the monocytopenia, is the result of other steroid effects on the monocytes or the vascular wall, or is due to yet other factors. In septicemia, monocytopoiesis is enhanced in the marrow, and the blood monocyte turnover rate is increased in humans (78). Administration of recombinant G-CSF, GM-CSF, or IL-3 to humans amplified bone marrow production of mononuclear phagocytes and release of monocytic cells into the blood (83). The peripheral half-life of blood monocytes in subjects receiving the growth factors was reduced (83). G-CSF administration to normal people in preparation for blood stem cell harvesting caused an eightfold increase in neutrophil and monocyte counts, and a slight decrease in platelet counts (84). The effects of recombinant growth factors on tissue macrophage numbers and function are not known.
Recent work with mouse cells has documented that blood monocytes consist generally of two subsets (85). One population corresponds to the human monocytes that express CD14, CD62L, and the CC-chemokine receptor 2 (CCR2). These “inflammatory monocytes” are recruited to inflamed tissues. The other population is comparable to human monocytes that are CD16+ and CCR2-. These cells are thought to be resident cells recruited to tissues in the absence of inflammation (85).
Tissue Macrophage Kinetics
Tissue macrophages arise primarily from emigrated blood monocytes that differentiate into macrophages. Chimera studies in experimental animals have shown that marked donor marrow or blood monocytes (radioactive label, abnormal or opposite sex chromosome marker, unique enzyme, or distinctive morphologic marker) eventually are found in tissues as macrophages. This has been shown in the case of peritoneal macrophages (86,87,88,89), liver Kupffer cells (90,91,92), alveolar macrophages (93,94,95,96), osteoclasts (97), type A synovial cells (98), and inflammatory tissue macrophages (99). Studies in humans after bone marrow or liver transplants from opposite sex donors have given evidence for the bone marrow or blood origin of liver Kupffer cells (100) and alveolar macrophages (101). Animals receiving total body irradiation with shielding of a small portion of bone marrow followed by in vivo 3HTdR injection have near-normal levels of labeled (bone marrow–derived) peritoneal (55), Kupffer (90), and alveolar macrophages (102), suggesting that these tissue macrophages are derived from the bone marrow. Furthermore, glucocorticosteroids, which inhibit the egress of blood monocytes into tissues but not the 3HTdR incorporation into mononuclear phagocyte precursors (82), decrease the labeling indices of peritoneal, Kupffer, and alveolar macrophages to near zero (77,90,102). However, most studies in experimental animals and in humans have shown a detectable, albeit small, 3HTdR labeling index in peritoneal, alveolar, Kupffer, and wound macrophages (51,77,90,102,103,104). Some have contended that local proliferation of tissue macrophages plays an important physiologic role in the maintenance of macrophage numbers there. For example, in mice made severely monocytopenic by the injection of strontium-89 (which localizes in bone marrow and dramatically reduces marrow mononuclear phagocyte production), the levels of peritoneal macrophages remain stable for months with 3HTdR labeling indices of 3 to 5% in the tissue macrophages (105). Local proliferation of tissue macrophages in physiologic conditions contributes to tissue macrophage renewal, but the precise extent of this is unknown.
Studies in mice have attempted to define the kinetics of tissue macrophages. Pulse labeling with 3HTdR has given information on blood half-times, the numbers of macrophages in the various compartments, and the number of monocytes that enter the tissues per unit time for peritoneum, liver, and lung (77,90,102). Approximately 8% of the blood monocytes go to the peritoneal cavity, and there are about 1 to 3 × 106resident peritoneal macrophages per mouse. The turnover rate for peritoneal macrophages is low (about 0.1% per hour), giving a turnover time of about 41 days. There are approximately 0.9 × 106 Kupffer cells per liver in the mouse (90), and the calculated mean turnover time is 21 days. Approximately half of the labeled blood monocytes migrate to the liver, where they reside as Kupffer cells. Approximately 15% of labeled blood monocytes migrate into the lung. The total mouse lung macrophage population is about 2 × 106 (102); of these, 93% are in alveoli while 7% are found in the interstitium. The calculated mean turnover time for lung macrophages is about 27 days. Data for other tissue mononuclear phagocytes are not available.
To maintain a steady number of tissue macrophages in the face of the noted steady influx of monocytes into the tissues, the tissue macrophages must have a steady local death rate and/or a steady efflux from the tissues. Some alveolar macrophages are removed by the mucociliary pathway and are expectorated or swallowed (102,106,107). There is evidence that interstitial lung macrophages migrate to lymph nodes via the lymphatics (108,109), where they may die. Macrophages from the small intestine and liver migrate to regional lymph nodes (110,111). Macrophages in granulomas (epithelioid cells and multinucleated giant cells) die in situ (112). There is no good information that tissue macrophages ever re-enter the blood. However, when researchers injected fluorescent spheres into the subcutaneous tissue of mice, the spheres were phago-cytized by exuded monocytes and then migrated to draining lymph nodes, where they had characteristics of dendritic cells (113). In monocyte-deficient osteopetrotic mice, the appearance of the labeled cells in lymph nodes was reduced by more than 85%. These data indicate that tissue macrophages can leave one tissue (subcutaneous tissue) and enter another (lymph node), where they may differentiate into another cell type (dendritic cell) (113).
Characteristics and Distribution of Tissue Macrophages
Cells of the mononuclear phagocyte system are widely distributed throughout the body (Table 12.1). The cells in the various tissues are quite heterogeneous, differing in numerous parameters. Because of the broad distribution and the difficulty of isolating those deeply embedded in some tissues, it has been difficult to quantitate the numbers in different compartments or the total number in the body. Studies using a monoclonal antibody that recognizes a specific mononuclear phagocyte antigen (F4/80) have provided some data (114). The macrophage antigen was found in a wide variety of tissues; the organs with the highest total F4/80 antigen content were the bowel, liver, bone marrow, spleen, lymph nodes, and kidney (114). Although levels of F4/80 may vary with the “activation” state of macrophages, these studies are helpful in assessing macrophage content.
Lung Macrophages
The interstitial and the alveolar macrophages constitute the lung macrophage pool, approximately 7% being interstitial (102). Interstitial cells are likely the immediate antecedents of the free alveolar macrophages (115,116). Pulmonary macrophages are essential components of the respiratory defense system, and because of their accessibility, alveolar macrophages have been studied most extensively. Alveolar macrophages are 15 to 50 μm in diameter. Their appearance and structure depend on the age of the cell and the nature and quantity of the material that has been endocytized. In Giemsa- or Wright-stained preparations, their cytoplasm is gray or light blue and contains numerous cytoplasmic granules. The nuclear:cytoplasmic ratio is about 1:3. Nucleoli are sometimes seen. There is abundant smooth and rough endoplasmic reticulum. A variable number are extremely vacuolated or “foamy.” Phagosomes may contain dust particles, bacteria, erythrocytes, and cellular debris. Some of the inclusions contain surfactant, the phospholipid synthesized by alveolar type II cells that is important in the maintenance of normal lung function. The normal alveolar macrophage removes and degrades surfactant material (117). It does not contain hemoglobin, and only trace amounts of iron are present. However, in situations of pulmonary hemorrhage (occult or overt), large quantities of hemoglobin and iron in the form of hemosiderin can be found in the cells (117). They contain α-naphthyl butyrate esterase (nonspecific esterase), lysozyme, and acid phosphatase, and stain positively with the periodic acid-Schiff (PAS) stain. There is little or no peroxidase activity in alveolar macrophages, but they do contain catalase. They have receptors for the Fc fragment of IgG and complement (C3b) (118,119,120). Ultrastructurally, the nuclei of alveolar macrophages are polymorphous and frequently have an eccentrically placed nucleus with a nucleolus (117). Multinucleated cells are rarely seen. There are numerous membrane-bound cytoplasmic vesicles or primary lysosomes that contain various enzymes typical of lysosomes, including cathepsins, lysozyme, β-glucuronidase, aminopeptidase, β-galactosidase, aryl sulfatase, acid ribonuclease, and phospholipases (117).
Alveolar macrophages exist at the tissue–air interphase, where they encounter inhaled pollutants and microorganisms. Despite encountering these microbes, these macrophages rarely display inflammatory properties and trigger adaptive immune responses. Researchers have shown that alveolar macrophages bind to alveolar epithelial cells and induce expression of αvβ6 integrin on the epithelial cells, causing transforming growth factor (TGF)-β expression. The latent TGF-β is activated by macrophage-elaborated matrix metalloproteinases, and the active TGF-β inhibits macrophage phagocytosis and cytokine production (121,122). Alveolar macrophages exist in an environment with a high partial pressure of oxygen as compared to the other mononuclear phagocytes of the body. As a consequence, alveolar macrophages have metabolic properties different than macrophages in other sites. Their basal glucose consumption and respiratory rate is greater than those of all other phagocytes studied (123). However, unlike phagocytes from other sites, they have a poor respiratory burst and little increase in hexose monophosphate shunt activity in response to soluble stimuli or phagocytosis (123). Rat alveolar macrophages have high levels of cytochrome oxidase and low levels of the glycolytic pathway enzymes pyruvate kinase and phosphofructokinase when compared to peritoneal macrophages. This particular enzyme phenotype in alveolar macrophages can be changed to that of peritoneal macrophages by incubating the cells in relatively anaerobic conditions in vitro (124).
Alveolar macrophages contain and secrete numerous enzymes typical of mononuclear phagocytes. Elastase and collagenase may have a role in the production of tissue destruction and the evolution of emphysema (125), especially in people with αl antitrypsin (αl antiprotease) deficiency. In addition to the various proteases, alveolar macrophages have been noted to contain the protease inhibitor αlantiprotease (126). The elastolytic enzyme activity of alveolar macrophages (possibly a cathepsin B) is poorly inhibited by the natural protease inhibitors of serum, while that of neutrophils is well inhibited (127). This suggests that macrophages of the lung may be more important in elastin degradation than are neutrophils.
Numerous environmental agents can alter alveolar macrophage function (128). Cigarette smoke is one of the most important toxins to which alveolar macrophages are exposed and contains many materials in the particulate and vapor phases. Smokers have increased numbers of alveolar macrophages (117,129), and cells from smokers show several abnormalities. They are slightly larger in diameter than those from nonsmokers and contain variable numbers of inclusions (117,130) such as electron-dense areas, lipid vacuoles, and “needlelike” and “fiberlike” structures. The needlelike structures may represent ingested kaolinite or aluminum silicate from the cigarettes (131). Cells from smokers spread more on glass and have more lamellipodia and filipodia than do those of nonsmokers (117). The smokers’ cells contain higher levels of elastase and lysosomal enzymes, and these enzymes are released more completely from smokers’ cells either spontaneously or in response to cigarette smoke (117). These enzymes are important in the pathogenesis of pulmonary emphysema. Alveolar macrophages from smokers also have impaired synthesis of RNA and protein (132), increased glucose utilization (133), increased hydrogen peroxide production, increased hexose monophosphate shunt activity, and reduced levels of glutathione peroxidase (117). In vitro cigarette smoke exposure in high amounts impairs various functions of macrophages (117). Also, alveolar macrophages contain the enzyme aryl hydrocarbon hydroxylase, and it is increased in macrophages from smokers (118,134). This enzyme transforms carcinogenic, polycyclic aromatic hydrocarbons into less dangerous hydrophilic compounds. There may be a relationship between the inducibility of this enzyme and the susceptibility to lung cancer (135).
Pulmonary macrophages are important in specific immunologic reactions because of their processing of inhaled antigens and interactions with lymphocytes (117). Alveolar macrophages can produce IL-1 (136), Interferon-α and -γ (137,138), chemotactic substances (139), and colony-stimulating factors (140). Interleukin-1, with its multiple effects, may be important in many different physiologic and pathologic conditions in the lungs (43). Interleukin-1 is a mitogen for fibroblasts. Alveolar macrophages interacting with silica or asbestos have been noted to contain or secrete a factor that enhances fibroblast proliferation and collagen synthesis (141,142), suggesting that interleukin-1 may play a role in the fibrosis seen in humans with silicosis and asbestosis. Chemokines produced by macrophages may also be important in the pathogenesis of fibrotic lung disease (143).
Splenic Macrophages
Splenic macrophages are present both in the red and the white pulp (114). The red pulp is composed almost exclusively of macrophages involved in the phagocytosis and destruction of blood cells. Although the precise routes and dynamics of cellular movement within and through the spleen are not fully understood, red pulp macrophages apparently emigrate there through the fenestrations of the basement membranes of the splenic sinuses, and occasionally can be seen extending their pseudopods through these fenestrations, possibly interacting with blood elements (144). The red pulp has the densest concentration of the macrophage antigen F4/80 found in the mouse (145). In the white pulp, the macrophages are much less frequent and are found in the germinal centers, where occasionally they are seen phago-cytizing lymphocytes (146). White pulp macrophages are presumably involved in antigen processing and generation of the immune response. Splenic macrophages measure 17 to 50 μm in diameter and contain more rough endoplasmic reticulum and Golgi zone than do blood monocytes (146). Red pulp macrophages contain numerous inclusions including phagocytized erythrocytes, neutrophils, eosinophils, and platelets (144,147). In hemolytic anemias and immune thrombocytopenias, an increased amount of phagocytized debris is present; occasionally the macrophages appear as “sea blue histiocytes” with the endocytized cellular membranes converted to ceroid (148). Splenic macrophages have receptors for the Fc portion of IgG and for complement (149), express antigens typical of mononuclear phagocytes at other sites (145), and contain ferritin and hemosiderin (144).
Bowel Macrophages
Using immunohistochemical techniques, researchers demonstrated numerous macrophages bearing the macrophage-specific antigen F4/80 in the lamina propria throughout the gastrointestinal tract (150). Many of the labeled cells are spread along the base of the epithelial cells, apparently underlying the basement membrane. In the small intestine, the macrophages extend to the epithelium of the crypts. Some of the macrophage antigen-positive cells are associated with capillaries or with numerous lymphocytes and plasma cells in the lamina propria (150). The cortical areas of the gut-associated lymphoid areas contain very few of the antigen-positive macrophages (145). There are very few of the macrophages in the Peyer patches, but there are macrophages in the dome epithelium of Peyer patches (145,151).
In addition to immunohistochemical means of detection, bowel macrophages are recognizable by observing ingestion of carbon particles, PAS staining, and cytochemical stains for acid phosphatase. By electron microscopy, bowel macrophages are often located adjacent to blood vessels or in close association with plasma cells in the lamina propria. They have large lysosomes, phagosomes, and electron-dense inclusions (152). Mitoses are rarely seen (151).
The role of intestinal macrophages in physiologic and pathologic conditions is not fully understood. Gut-associated macrophages probably play a role in the development of an immune response to absorbed antigens, in suppressing immune responses, or in developing immune tolerance (151). The cells may be important in phagocytizing bacteria and cell debris. Sequestration of waste material in macrophages and then shedding of these macrophages into the intestinal lumen may serve as a mechanism for eliminating accumulated or dead intestinal wall material (151). Likewise, iron-laden macrophages are shed from the villi into the gut lumen in patients with iron overload (153,154). In the obligate sanguivore vampire bat, the bowel apparently plays a large role in the elimination of excess body iron; iron-laden macrophages migrate there and desquamate into the stool (155). We do not know if this excretory system for iron is important in man.
Intestinal macrophages are involved in diseases affecting the bowel. Various investigators have postulated that macrophage-secreted proteases (including acid hydrolases, elastases, and collagenases) (156) may injure the bowel in conditions such as Crohn disease in humans, and in experimentally induced cecal inflammation in guinea pigs (151,157). In inflammatory bowel disease, there is an increase in the mucosal macrophage population derived from circulating monocytes. These macrophages contribute to inflammation by secreting cytokines such as IL-1, IL-6, IL-8, IL-12, IL-18, and tumor necrosis factor (TNF) (158). They also generate reactive oxygen and nitrogen species, molecules with proinflammatory properties. Elevated bowel lumen nitric oxide and increased expression of inducible nitric oxide synthase have been noted with various inflammatory bowel disorders (159). These include celiac disease (160,161,162), ulcerative colitis (163), and inflammatory bowel disease (164). IL-10 generally serves as a deactivator of macrophage function, and mice with genetically disrupted IL-10 gene have overactive bowel macrophages and an inflammation of the bowel comparable to that seen in humans with inflammatory bowel disease (165). Lysozyme, an enzyme found in highest concentrations in macrophages, is elevated in stool and intestinal mucus in humans with bowel inflammation (166). Serum lysozyme is elevated in patients with Crohn disease (167) and in other types of inflammatory bowel disease (168). Granulomas seen in Crohn disease and in intestinal tuberculosis are composed of monocytes/macrophages, together with lymphocytes and plasma cells (169,170,171). Fibrosis that accompanies these disorders may, in part, be caused by proliferation of local fibro-blasts stimulated by macrophage-derived fibroblast growth factors, such as interleukin-1 (43).
Using epidemiologic and linkage studies, a susceptibility locus for inflammatory bowel disease has mapped to chromosome 16. The gene at this location (caspase recruitment domain 15 [CARD15]/nucleotide-binding oligomerization domain 2 [Nod2]) (172,173,174) encodes a monocyte/macrophage-specific protein that is involved in activation of NF-κB and regulation of apoptosis (175). CARD15/Nod2 is mutated in about 50% of patients with Crohn disease versus 20% of healthy controls in Caucasian populations (172,173,174). However, susceptibility to ulcerative colitis is not influenced by CARD15/Nod2 mutations.
Whipple disease, a multisystem disease caused by an infection with an unusual and fastidious microorganism, is characterized by an accumulation of epithelioid macrophages in various organs, including prominently the bowel wall inside macrophages (176,177). The macrophages contain PAS-positive material. The causative organism, Tropheryma whippelii, has been identified by polymerase chain reaction studies of nucleic acid from involved tissues (178,179). The organism is very difficult to culture in vitro, but by incubating blood mononuclear cells with the monocyte “deactivating” cytokine IL-4, researchers are able to grow T. whippelii in blood monocytes in tissue culture (180,181). The organism can also be grown in vitro in fibroblasts under certain conditions. Practical laboratory diagnosis is based on identifying typical PAS-positive–containing macrophages in affected tissues, demonstration of T. whippelii nucleic acid in blood leukocytes or affected tissues using polymerase chain reaction amplification, and possibly by use of specific tests using antiorganism antibody or in situ hybridization using specific nucleic acid probes (176,177).
Skin Macrophages (Langerhans Cells)
The mononuclear phagocytes of the skin are called Langerhans cells (182). These cells arise from monocytes (183). Not easily seen in routine stains, these cells must be stained with metal salts such as gold chloride, with cytochemical stains, or with immunohistochemical stains (182,184). Langerhans cells have a relatively clear cytoplasm; a lobulated nucleus, without organelles characteristic of keratinocytes and melanocytes; and a unique cytoplasmic granule (Birbeck granule) (182,185) (Fig. 12.7). The tennis racket–shaped granules are seen only by electron microscopy and may measure 0.8 to 2 μm in length. The origin and function of these granules are unknown.
Langerhans cells make up 3 to 8% of the epidermal cells (186). They are of bone marrow origin (187) but can proliferate locally as well (188). Reticular dysgenesis is a rare inherited immunodeficiency characterized by lack of blood monocytes and neutrophils and low lymphocyte numbers (despite normal erythrocyte and platelet counts). These patients have normal skin macrophages but absent Langerhans cells (189). After bone marrow transplant therapy, the Langerhans cells return. Langerhans cells have receptors for the Fc portion of IgG and for C3 (190,191) and express the MHC class II antigens (immune associated [Ia] in mice and HLA-DR in the human) (182,192). The cells express a macrophage-related antigen recognized by monoclonal antibody F4/80 (150), and the CD1a antigen (193). CD1a is not expressed on normal blood monocytes, but it is found on intrathymic thymocytes. Langerhans cells contain no peroxidase, but ATPase activity is present. They adhere to glass and have some pinocytic and phagocytic activity, but much less than do macrophages from other areas (182).
The primary function of Langerhans cells is to serve as antigen-presenting cells (194). In this respect, they are closely related to dendritic cells, marginal zone macrophages, medullary macrophages, and interdigitating cells of the T-cell areas of lymph nodes (182). There is a strong correlation between the expression of MHC class II antigen and the ability to serve as an accessory cell. Langerhans cells synthesize and express large amounts of MHC class II antigen and are seen in apposition to lymphocytes at sites of contact allergic dermatitis, suggesting an important role in contact-delayed hypersensitivity (195,196). Langerhans cells can also acquire antigen in the skin and carry it to regional lymph nodes, where they are sometimes seen as “veiled” cells (182). Langerhans cells are probably involved also in rejection of skin allografts, and there is some evidence that they aid in the differentiation (keratinization) of epidermal cells by an unknown mechanism (197).
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Figure 12.7. Electron micrograph of a Langerhans cell from the skin of a normal human. The cell displays a lobulated structure and numerous cytoplasmic Langerhans (“Birbeck”) granules. The inset shows a higher-power view of several of the granules. ×25,000. (Photograph courtesy of Dr. John Shelburne.) |
Langerhans cells are involved in various disease states. As discussed above, they participate in allergic contact dermatitis reactions. In histiocytosis X (or so-called Langerhans cell granuloma), there is an accumulation of these cells in various areas of the body (198). Histologically, this does not appear to be a malignant condition (199). The cells in this condition have the Birbeck granules, express Fc and C3 receptors, and have more phagocytic capability than do normal Langerhans cells (198).
Liver Macrophages (Kupffer Cells)
A large portion of the body’s mononuclear phagocytes resides in the liver (90,114). Liver macrophages (or Kupffer cells) are the phagocytes of the liver sinusoidal walls (200). They lie on the endothelial cells or are embedded in the endothelial lining and may extend into the space of Disse. Kupffer cells are also in close contact with other liver cells including fat-storing cells and pit cells, as well as reticulin fibers. By scanning electron microscopy, they have microvilli and occasional lamellipodia. The cells have slightly irregular oval nuclei, small mitochondria, and numerous endocytic vacuoles. They contain “wormlike structures,” apparently unique internal structures consisting of internalized membrane with a thick coat of membrane material (201). By transmission electron microscopy, the external membrane has a thick, fuzzy coat. Kupffer cells contain peroxidase in the endoplasmic reticulum, whereas liver endothelial cells contain no peroxidase (200).
Kupffer cells play important roles in liver injury and in various disorders including viral hepatitis, steatohepatitis, alcoholic liver disease, intrahepatic cholestasis, liver fibrosis, and rejection of transplanted liver (202,203). In their critical position along the sinusoidal wall, they phagocytize different blood-borne particles. These include injected experimental particles such as carbon and latex, liposomes, bacteria (204), parasites (205), viruses (206), and altered or opsonized erythrocytes (147,207). They efficiently remove portal blood bacteria that come from the gut (204). Furthermore, they are important in clearing endotoxin from the blood passing through the liver (208). Kupffer cells can produce IL-1, TNF, arachidonic acid catabolites, reactive oxygen species, and reactive nitrogen species, and alter the functions of hepatocytes (203,209). These alterations range from subtle Kupffer cell–mediated changes to marked hepatocyte injury. Kupffer cells can regulate hepatocyte general protein and fibrinogen synthesis (210) and cytochrome P450 drug transformation reactions (211). Bacteria and endotoxin cause an increase in the membrane microvilli and an increase in the levels of certain lysosomal enzymes (212). Kupffer cells may play a role in eliminating fibrin and fibrin degradation products from blood; in disseminated intravascular coagulation, this capability is depressed (213).
Phagocytosis by the macrophages in the liver is mediated by Kupffer cell Fc or complement receptors (149,214,215,216). Antibody opsonization of various particles dramatically enhances their removal from the circulation by these liver cells (200). The Kupffer cells recognize erythrocytes that have been injured or aged in vitro (207) or that have been treated with neuraminidase (217). The cells have receptors for N-acetylglucosamine (218). Plasma fibronectin also acts as a particle opsonin recognized by Kupffer cells (219). Murine Kupffer cells synthesize and secrete fibronectin, complement factor B, and apolipoprotein E (220). They also display MHC class II antigen and the macrophage antigen F4/80, but they lack the Mac-1 antigen (220).
Kupffer cells can serve as accessory cells in presenting antigens to and stimulating DNA synthesis in T lymphocytes (221,222). This interaction, like that involving macrophages from other sites, is genetically restricted (222). Kupffer cells from normal animals are cytostatic for tumor cells (223), and those from animals injected with Corynebacterium parvum are cytolytic for tumor cells (224). In mice, unlike peritoneal macrophages, Kupffer cells produce hydrogen peroxide very poorly in response to phagocytosis or soluble stimuli (220). Interferon-γ (IFN-γ), which enhances capacity for peroxide production in peritoneal macrophages and blood monocytes, has no such effect on Kupffer cells (220). Kupffer cells are not very effective in in vitro killing of Toxoplasma gondii trophozoites and Leishmania donovani promastigotes and amastigotes, and IFN-γ will not enhance this cytotoxicity (220). IFN-γ will, however, increase the expression of MHC class II antigen in murine Kupffer cells. In experimental sublethal infection of mice with Listeria monocytogenes, there is an influx of immigrant macrophages into the liver (220). These newly immigrated liver macrophages, unlike the Kupffer cells, are able to produce substantial amounts of peroxide, and display significant antimicrobial effect (220).
Kupffer cells, like hepatocytes, can produce large amounts of nitric oxide (NO), and this can have profound effects on hepatocyte function and liver homeostasis in general (209). NO is responsible for many of the Kupffer cell–mediated effects on hepatocytes. These include altered hepatocyte total protein synthesis, decreased glycolysis, decreased glyceraldehyde-3-phosphate dehydrogenase activity, increased cellular cyclic guanosine monophosphate (cGMP) content, decreased cytochrome P450 activity, decreased mitochondrial respiration, and tumor cell cytostasis. In microbial sepsis and endotoxemia, there is an increase in hepatic production of IL-1 and TNF, as well as an increase in NO production. While the NO likely mediates the vasodilation and hypotension and has the potential for toxic effects on hepatocytes, experiments in sepsis models using mice have shown that inhibition of NO production by administration of L-arginine analogues causes an accentuation of hepatic necrosis (209,225). Part of this accentuated toxicity may be caused by worsening of thrombosis and coagulative necrosis.
Brain Macrophages (Microglial Cells)
Investigators in the early 1900s noted special-appearing cells in brain prepared by silver impregnation staining methods (226). These cells have been called microglial cells, gitter cells, and rod cells (227). Although early workers postulated that these microglial cells were a type of brain macrophage (226,227,228), only recently have studies given unambiguous evidence of this (229,230). Using immunohistochemical techniques, researchers have shown that cells with the typical appearance of microglial cells display the macrophage-specific antigen F4/80, the Fc receptor for IgG1/IgG2b, and the myelomonocytic antigen Mac-l (CD11b/CD18) (230). In the adult mouse, there are two distinct populations of brain cells bearing the macrophage antigens. Round monocytelike cells with short processes associated with the choroid plexus, ventricles, and leptomeninges measure approximately 10 μm in diameter and more typical microglial cells have little cytoplasm but long processes. The body of the latter cell measures about 5 to 7 μm in diameter, and the processes extend up to 40 μm from the body (230). Most of the microglial cells are in the gray matter. By electron microscopy, the nucleus is irregularly shaped with unevenly distributed nuclear chromatin. The cytoplasm has elongated cisternae of endoplasmic reticulum, and the long processes themselves contain few organelles. Mitochondria are present in moderate numbers. Lysosomes, endocytic vacuoles, and internalized lipid material are seen in the cells. The cells can internalize erythrocytes and carbon particles, and they contain nonspecific esterase. With injury or inflammation, there is a dramatic increase in the number of microglial cells (227,231,232,233). Dying neurons in the injured area likely act as a chemotactic stimulus for bone marrow–derived monocytes to migrate into the area and differentiate into microglial cells (229,230,234). Increased numbers of microglial cells in a lesion result from emigrated cells, proliferation of resident microglia, and migration of microglia from adjacent normal brain tissue (234). In developing mouse retina, degenerating neurons can be observed, and macrophages migrate from the blood vessels overlying the developing retina, phagocytize the dying neurons, and then develop into F4/80+ microglial cells (229). In the brain gray matter, the microglial cells are seen in highest concentrations about vessels and about degenerating, pyknotic cells (227,230). During development in the mouse, macrophages invade the brain and can be followed through a series of transitional forms as they differentiate to become microglial cells. Apparently, the programmed neuronal cell death that accompanies brain maturation acts as a stimulus to attract monocytes (230).
Brain macrophages are frequently infected with human immunodeficiency virus type 1 (HIV-1) in subjects with HIV-1 infection and/or acquired immunodeficiency syndrome (AIDS). Blood monocytes infected can emigrate into the brain carrying HIV-1 with them, and then differentiate and reside in the central nervous system (CNS). This “Trojan horse” introduction of the virus apparently is the primary method of infection (235). Also, brain macrophages and microglial cells express the HIV-1 receptors/coreceptors CD4, CCR3, CCR5, and CXCR4. Products of infected macrophages may directly injure and kill neurons; candidate neurotoxins from macrophages include NO, platelet-activating factor, kyneurine metabolites, and proinflammatory cytokines (235).
The function of brain mononuclear phagocytes is not fully known. They phagocytize dying neurons and their processes, debris, and foreign material (227,228). Although no experimental evidence has been presented, they probably also play a role in the induction of immune responses, in mediating antimicrobial and antitumor effects, and possibly in modulating neuronal cell function. Brain macrophages may function to prevent excessive inflammatory responses by controlling T-lymphocyte responses through the inhibition of T-lymphocyte proliferation, and in some cases by inducing T-lymphocyte anergy (236).
Peritoneal Macrophages
Much of the work with mononuclear phagocytes in experimental animals has been done with peritoneal macrophages because they can be conveniently obtained in adequate numbers and cultured. Peritoneal macrophages differ morphologically from their precursor blood monocytes. They measure 15 to 45 μm in diameter, adhere avidly to glass or plastic substrates, and frequently manifest vacuolated cytoplasm. Human studies have primarily employed cells from women undergoing elective laparoscopy or laparotomy (237,238,239,240), from patients with indwelling catheters immediately after operative placement or patients undergoing chronic dialysis with indwelling peritoneal catheters (241), or from patients with benign or malignant ascites (242). The cell population from normal women is composed of 85 to 95% macrophages, the remainder being lymphocytes (237,238,243). The macrophages are heterogenous, with some resembling blood monocytes morphologically, but most looking more like tissue macrophages (237). There are about 2 to 5 × 106 peritoneal macrophages in a normal woman (238,239,243). The number varies during the menstrual cycle, the highest numbers being near the time of menstruation; retrograde menstruation may cause this increase (243). Women with endometriosis have increased numbers of peritoneal macrophages (238,239,243,244). These peritoneal macrophages in endometriosis are activated as judged by a number of parameters, including increased expression of inducible nitric oxide synthase (NOS2) and increased ability to produce NO (245).
Peritoneal macrophages are peroxidase negative, have Fc and complement receptors, and are heterogeneous with respect to cytochemistry and phagocytic ability (238,240,241). Hydrogen peroxide and superoxide production in response to phorbol myristate acetate is comparable to that of blood monocytes (246). Peritoneal macrophages are capable of killing tumor cells and bacteria (241,242,246), and in general, are more effective in killing tumor cells than are blood monocytes (246). The number of peritoneal macrophages in patients with acute inflammatory peritonitis or with malignant ascites is elevated (242). However, the functional capabilities in comparison to resident normal peritoneal macrophages are not known.
Renal Macrophages
Although inconspicuous on routine hematoxylin and eosin–stained sections, kidneys contain a large number of macrophages (114,247). They are of bone marrow origin, and are phagocytic, bear Fc and complement receptors, express MHC class II antigen, stain positively for nonspecific esterase, and adhere to plastic or glass substrate. The highest concentration of macrophages is in the medullary interstitial cell population, where they cover much of the outer surfaces of the collecting tubules (145). Macrophages are also seen in close proximity to the macula densa and afferent and efferent arterioles of the glomerular capillary bed, as well as around the Bowman capsule (145). The glomerular mesangium of normal animals usually does not contain cells typical of mononuclear phagocytes (248,249).
In conditions of glomerular injury, however, there is an influx of monocytes into the glomerulus. This is especially prominent in disorders characterized by crescent formation where macrophages constitute up to 50% of the cells in the crescent (250). IL-1, TNF, and IL-6 may contribute to the inflammation noted in glomerulonephritis (250). Interleukin-1 secreted by the mononuclear phagocytes may play a role in the glomerular cell proliferation seen in these disorders (251). Mice that develop a spontaneous disease similar to human system lupus erythematosus (SLE) overexpress inducible nitric oxide synthase and overproduce nitric oxide. Blocking the nitric oxide synthase in vivo with an L-arginine analogue prevents development of arthritis and nephritis (252). In humans with lupus nephritis or IgA nephropathy, there is an increased expression of inducible nitric oxide synthase that correlates with the severity of disease (253).
Milk Macrophages
There are a large number of mononuclear phagocytes in human colostrum and milk, and these macrophages can be transferred from mother to nursing baby. There are from 0.2 to 21.0 × 106 cells/ml milk (254,255). About 40% of milk leukocytes are nonspecific esterase-positive macrophages capable of adhering to plastic or glass substrate (254). These cells have Fc and complement receptors, are able to mediate cytostasis and cytolysis of tumor cells (254,255,256), and can phagocytize and kill bacteria (257). They can also modulate the mitogenic activity of lymphocytes (258). Breast milk macrophages (unlike blood monocytes) spontaneously produce GM-CSF in in vitro culture, and differentiate to dendritic cells after treatment with IL-4 alone (259). Milk macrophages express high levels of CD86, CD40, and HLA-DR, but they do not express CD1a, a molecule characteristic of dendritic cells. CD83, a molecule usually found in dendritic cells, is expressed by milk macrophages but not blood monocytes (259).
Reproductive Tract Macrophages
The testis contains large numbers throughout the interstitium (260,261,262,263). The cells adhere to glass or plastic, bear Fc and complement receptors, and express MHC class II antigen and nonspecific esterase activity. Testicular macrophages display F4/80, CD11b, CD11c, CD13, CD14, and CD68. The cells can produce cytokines typical of mononuclear phagocytes (e.g., IL-1 and TNF). Also, they phagocytize sperm in the seminiferous tubules and vas deferens (264), and are thought to be important in eliminating unejaculated, dying sperm (265). They can phagocytize bacteria (266), participate in the destruction and elimination of invading organisms (261), and participate in the formation of “sperm granulomas” seen occasionally in men who have had vasectomies (267). Because of their location and the presence of MHC class II antigen on their surfaces, some have speculated that testicular macrophages are important in the induction of immunity against antigens (including sperm antigens) (267). In a group of men with infertility of various causes, testicular macrophages were more prominent in the tubular wall and tubular lumen (compared to those with other types of infertility and to fertile men), and in those with germ cell arrest and Sertoli-only causation, the numbers of macrophages were increased in number by twofold (268). This suggests that the testicular macrophages may adversely affect fertility in certain situations. Macrophage function can be modified by sex hormones, and conversely, macrophages can modulate sex hormone production (266,269,270). In particular, macrophages apparently can synthesize testosterone (270). Testicular macrophages express the HIV-1 receptors CD4, CCR5, and CXCR4 (but not CCR3) (271). This indicates that these macrophages can be infected with HIV-1 and possibly play a role in HIV-1 transmission.
The ovary contains macrophages that phagocytize degenerating luteal cells during normal estrus, postpartum luteolysis, and follicle atresia (262,272). These mononuclear phagocytes have the usual characteristics of tissue macrophages—Fc receptors, ability to ingest latex spheres, tight adherence to plastic substrate, and nonspecific esterase activity (272). Macrophages isolated from mouse corpora lutea enhance progesterone production by ovarian cells. It is likely that intraovarian macrophages play a role in maintaining progesterone secretion by luteal cells in vivo (272).
The number of macrophages in the body of the uterus is increased immediately following insemination (273), in the presence of intrauterine contraceptive devices (274,275), and during pregnancy (276,277). Intrauterine devices are seen to be coated with macrophages, and these inflammatory macrophages may play a part in damaging the gametes or zygote and preventing implantation (274,275,278). Uterine macrophages are frequently seen phagocytizing sperm (273). In mice, macrophages constitute 10 and 22% of uterine cells from normal and pregnant mice, respectively (277). These macrophages represent a large part of the uterine decidua during pregnancy (up to 10 to 20% of the total uterine cells). A large proportion of uterine macrophages in pregnancy are MHC class II antigen or CD54 positive, suggesting that they may be “activated” in some fashion (277). Uterine macrophages (many expressing NOS2) increase as pregnancy progresses, but there is a decline in endometrial macrophage numbers at least 1 day before onset of parturition (279,280). Since NOS2-derived NO exerts a relaxing effect of uterine smooth muscle and promotes uterine quiescence, some have suggested that the decrease in macrophages just before delivery results in a decrease in this general inhibitory effect on uterine contractility (279). Decidual macrophages produce nitric oxide; NO elaborated by activated decidual macrophages may be an effector molecule in mediating early embryo loss in spontaneous abortion (281).
Oviducts contain a few typical macrophages that, based on analysis of women with oviducts blocked either proximally or distally, arise from migration of peritoneal macrophages through the fimbrial openings (282). The numbers of macrophages in these sites reflect the numbers in the peritoneum. Oviductal macrophages may interfere with fertilization by injuring or phagocytizing normal or antibody-opsonized sperm (239,282,283).
Mice with a genetically disrupted M-CSF gene have reduced fertility (284). Homozygous M-CSF-/- mice have markedly reduced numbers of testicular and ovarian macrophages that accompanies their reduced fertility. Males have reduced numbers of sperm and decreased libido, and females have extended estrous cycles and poor ovulation rates. However, the major reproduction defect appears to be a defective feedback regulation of the hypothalamic-pituitary-gonadal axis, suggesting that defects in brain macrophages (microglial cells) contribute to the problem. Also, it is likely that there is abnormal macrophage-influenced function of steroidogenic cells such as Leydig cells in males and corpus luteal cells in females (284).
Bone Macrophages (Osteoclasts)
Osteoclasts are specialized multinucleated cells found in bone that mediate bone resorption (285). They form from circulating hematopoietic cells of bone marrow origin (286,287). Because of their morphologic similarity to inflammatory multinucleated giant cells, investigators have postulated that they are formed from emigrated blood monocytes (285,286). Blood monocytes and peritoneal macrophages can degrade bone in vitro (288,289). In murine systems, bone degrading ability relates to the degree of macrophage multinuclearity (288). In osteopetrosis, there is an inability to produce M-CSF with a resultant defect in osteoclast production and function (21).
Osteoclasts lack certain antigens usually noted on macrophages (e.g., Fc and C3 receptors), but they express high levels of tartrate-resistant acid phosphatase, the vitronectin receptor, and calcitonin receptors (26). Several factors have been discovered that enhance osteoclast formation in vitro. These include 1,25-dihydroxyvitamin D3, prostaglandin E2, IL-1, IL-11, TNF, and glucocorticoids. These factors work through induction of osteopro-tegerin ligand (OPGL, identical to TNF-related activation-induced cytokine [TRANCE]) in osteoblasts. OPGL in turn binds to the receptor activator of NF-κB (RANK) on osteoclast precursors, and (in the presence of M-CSF) the osteoclast precursors develop into mature osteoclasts. Osteoprotegerin (OPG) is a soluble decoy receptor for OGGL that serves to inhibit osteoclast formation and differentiation (285,290). TGF-β increases the proportion of precursors that become osteoclasts, and it is an essential costimulator of osteoclast formation. Research using mice with disruption of various genes has been very helpful in understanding the control of osteoclast formation and function and bone resorption. Mice with disruptions of M-CSF, C-src, C-fos, NF-κB, OPGL, and RANK have osteopetrosis, while those with disrupted OPG have osteoporosis (26).
Granuloma Macrophages
A granuloma is defined as “a compact, organized collection of mononuclear phagocytes (macrophages or epithelioid cells), which may or may not be accompanied by accessory features such as necrosis or the infiltration of other inflammatory leukocytes” (291). Granulomas are characteristic of host responses to many different living and nonliving agents. In general, they form as reactions to particulate or indigestible agents that persist in tissues for long periods of time. Cells of granulomas serve both protective and destructive functions for the host. They protect by killing microbial agents and possibly tumor cells, by processing antigen, and by interacting with lymphocytes. But they also cause destruction and fibrosis of adjacent normal tissues (112,291,292). Multinucleated giant macrophages are prominent features of these granulomas. Multinucleated macrophages form by a process of cellular fusion of uninuclear monocytes or macrophages (112,293,294). Cytokines, growth factors, dihydroxyvitamin D3, and fusion proteins of viruses (including HIV-1) are important in inducing this fusion process (112,293,294,295). IFN-γ, IL-3, IL-4, GM-CSF, TNF, and CD44 (the hyaluronate receptor) can induce the fusion of monocytes or macrophages to form cells in vitro resembling those seen in human granulomas (294,296,297,298,299,300,301,302).
Granulomatous inflammation in vivo is characterized by heterogenous responses controlled by inciting antigens and different cytokines, with Th1-type (e.g., IL-2 and IFN-γ) or Th2-type cytokines (e.g., IL-4 and IL-10) influencing patterns of responses. The specific cytokines involved in granulomatous inflammation vary with the inciting organisms/antigens. IFN-γ, IL-1, and TNF generally serve to enhance granulomatous inflammation associated with mycobacteria (Th1 type), while IL-4 and IL-10 enhance that associated with schistosomiasis (Th2 type) (303,304). In studies of mice with either Th1 (induced with mycobacterial purified protein derivative [PPD] on plastic beads) or Th2 (induced Schistosomamansoni egg antigens [SEA] on plastic beads) antigens, there are differing patterns of chemokine responses (305). Of 24 different chemokines analyzed, there were characteristic profiles for Th1- and Th2-type responses that appeared to be controlled by certain cytokines. Inducible nitric oxide synthase is noted in induced granulomas indicating that nitric oxide may play a role in the inflammation (306), and NOS2-derived NO regulates the size, quantity, and quality of granulomas in Mycobacterium avium– infected mice (307).
There is increased expression of osteopontin in granulomas, and osteopontin is absolutely required for granuloma formation (308). Osteopontin is a secreted soluble cytokine that influences migration of mononuclear phagocytes and T lymphocytes. Osteopontin deficiency in mice is associated with impaired resistance to herpes simplex virus, Listeria monocytogenes, and mycobacterial infection (308), and relatively low-level osteopontin in tissues in humans is associated with poorly formed granulomas, lack of multinucleated giant cells in granuloma, and impaired resistance to infection with Mycobacterium bovis (BCG) (308,309). Secreted lymphotoxin (LT-α3, also known as TNF-β) is also required for granuloma formation and for control of mycobacterial infection, while membrane-bound LT-β is not (310). While LT-β is required for lymph node formation, absence of LT-β does not in itself modify granuloma formation. Lung inflammatory lesions that develop in LT-α–deficient mice show absence of macrophages and multi-nucleated giant cells and increased numbers of neutrophils. Generally, however, LT-α–deficient mice have normal T-cell and macrophage function (310). In transparent zebrafish, infection with mycobacteria of the embryo at a stage before lymphocyte development results in macrophage aggregates with hallmarks of granuloma (48). These studies indicate that granuloma formation is possible in the context of “innate” immunity.
The functions of multinucleated macrophages are not precisely known. They differ from mononuclear phagocytes in several ways, namely, diminished ability to phagocytize particles and to reduce nitroblue tetrazolium, diminished expression of 5′-nucleotidase, and reduced expression of various mononuclear phagocyte antigens (311,312). Fusing macrophages express a unique surface molecule that may be involved in the fusion process (313). Multi-nucleated macrophages can also kill tumor cells (314,315). The lifespan of multinucleated macrophages is thought to be shorter than that of other tissue macrophages (112).
Synovial Macrophages
Synovial type A cells are macrophages. These cells are phagocytic, have Fc receptors, express MHC class II antigen and nonspecific esterase activity, are of bone marrow origin, and look like mononuclear phagocytes on light and electron microscopy (98). In inflammatory arthritides such as rheumatoid arthritis (RA), there are increased numbers of synovial tissue macrophages (316), and the numbers of synovial macrophages correlate well with the degree of articular destruction (317). Macrophages play a critical role in the initiation and propagation of arthritis; they participate in antigen presentation, regulation of T- and B-lymphocyte activity, secretion of proteases, production of proinflammatory cytokines and arachidonic acid derivatives, and generation of reactive oxygen and nitrogen species (316). Humans with RA have accelerated generation of mononuclear phagocytes by bone marrow cells (318). Elimination of macrophages in rat models of arthritis ameliorates arthritis (319). Evidence points toward proteases (316), reactive oxygen species (320), and reactive species such as nitric oxide and peroxynitrite (252,321,322) as the most important macrophage-derived effector molecules in arthritis.
Mononuclear Phagocyte Antigens and Receptors
The plasma membrane is a dynamic structure in a constant state of change. The mouse macrophage, through endocytosis, completely internalizes the equivalent of its entire surface membrane area every 33 minutes (323). Accompanying this and other kinds of endocytosis, various surface membrane components are internalized and diminished in activity. These include 5′-nucleotidase (CD73) (324) and the antigen F4/80 (325). This probably accounts for the reduced levels in “activated” macrophage cells that possess enhanced rates of endocytosis (326). The membrane lipids of macrophages influence membrane fluidity and can be altered by environmental conditions. For example, membrane cholesterol and phospholipids change after phagocytosis (327), and membrane fatty acid composition changes after incubation in medium with high levels of saturated fatty acids (328); these changes can alter the activities of the macrophages.
As noted above in describing mononuclear phagocytes in the diverse areas of bone marrow, blood, and various tissues, there is great heterogeneity in mononuclear phagocyte populations. The abilities to define expression of unique antigens and receptors (31) on these cells has made possible good determination of these blood monocyte and tissue macrophage subsets (31,329,330,331,332). The mononuclear phagocyte interacts with the environment through its receptors. Some ligands such as glucocorticoids diffuse through the membrane to interact with intracellular receptors, but most bind to plasma membrane receptors. Once a ligand binds to its receptor, the macrophage reacts in some way (e.g., by altering gene expression, inducing secretion, or changing shape). General classes of mononuclear phagocyte receptors include families for receptors of antibodies and complement; the pattern recognition receptors toll-like receptors such as TLR-4 and nucleotide-binding oligomerization domain receptors such as Nod2; scavenger receptors such as SR-A; glycosylphosphatidylinositol (GPI)-anchored receptors such as CD14; integrins such as CD18/CD11b; immunoglobulin superfamily receptors such as the Fc receptor; seven transmembrane spanning receptors such as chemokine receptor 2 (CCR2) and the C5a receptor; and others (333,334,335). Table 12.3 lists some of the mononuclear phagocyte receptors.
Although once thought to be exclusively a lymphocyte antigen, the CD4 (T4) molecule is also expressed on mononuclear phagocytes. CD4 is necessary for infection of these cells with HIV-1 (336). HIV-1 infection of lymphocytes generally leads to rapid death of the infected cells, but HIV-1–infected monocytes and macrophages persist in in vitro culture and in vivo, frequently forming multinucleated giant cells (syncytia) and serving as reservoirs for the virus (336,337). HIV-1 infection of cells is mediated by interaction of viral envelope components with cellular CD4 and one of two chemokine coreceptors (CCR5 and CXCR4) (338). Virus use or the CCR5, CXCR4, or CCR5 + CXCR4 regulates lymphocyte and macrophage entry and cell tropism for primary T lymphocytes, mononuclear phagocytes, and/or transformed T-cell lines. Monocytotropic strains are generally preferentially transmitted in new infections, despite an evolution of viruses to lymphocytotropic phenotype over the course of infection in individuals (339,340,341). The CCR5 molecule also serves as a receptor for RANTES, MIP-1α, and MIP-1β; these β chemokines can inhibit HIV-1 infection of cells (342). Some people resist infection with HIV-1 despite repeated challenges. It appears that these individuals are resistant because they have a defect in CCR5, which renders their cells noninfectible by primary strains of HIV-1 (343,344). Furthermore, infected individuals who are heterozygotic for the mutant CCR5 have a slow progression of disease and prolonged life after infection (345). Approximately 1% and 10 to 15% of western Europeans are homozygotic and heterozygotic for the mutant gene, respectively, while very few (essentially zero) Africans have the mutation (345). Dendritic cells (DCs), unique cells differentiated from monocytes, display the DC-specific intercellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN; CD209) antigen. DC-SIGN, which can also be expressed by some macrophages, is a C-type lectin that serves as an antigen receptor recognizing pathogens through carbohydrates (346). It regulates DC trafficking, and it also binds HIV-1 and protects it in early endosomes allowing HIV-1 transport by DC to lymphoid tissues, where it enhances infection of permissive target cells (346). DC-SIGN also serves as the receptor for Mycobacterium tuberculosis on DC, using lipoarabinomannan as the ligand for binding (347,348).
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Table 12.3 Mononuclear Phagocyte Receptors |
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Mononuclear phagocytes react with immunoglobulins through Fc receptors (FcRs). All FcRs are integral membrane proteins in the immunoglobulin superfamily, except for FcγRIIIB, which is linked to the membrane by a GPI anchor. The receptors interact with the Fc portions of immunoglobulins. Immunoglobulin interaction with cells through FcRs results in many responses, ranging from effector functions such as antibody-dependent cytotoxicity, particle phagocytosis, enzyme secretion, and reactive oxygen and nitrogen species generation (349,350,351,352). The best-characterized FcRs are those for IgG and IgE (FcγR and Fc∊R, respectively). FcγRI (CD64) is the high-affinity Fc receptor for IgG; it is expressed on numerous leukocytes including neutrophils, monocytes, and macrophages. IFN-γ can enhance FcγRI expression on mononuclear phagocytes by more than 20-fold. FcγRII (CD32) is found on essentially all cells that bear FcγRs except NK cells. FcγRII binds IgG with low affinity, and unlike FcγRI, it does not bind monomeric IgG. FcγRII is encoded by a minimum of three related genes, and isoforms of FcγRIIB are formed by alternative splicing. FcγRIIB is expressed by mononuclear phagocytes, lymphocytes, and some primitive hematopoietic cells, but not by NK cells or neutrophils. FcγRIIA and IIC are preferentially expressed in neutrophils, and IIB in lymphocytes; monocytes express all three. FcγRIII (CD16) is seen only in mononuclear phagocytes, NK cells, and myeloid precursor cells. FcγRIIIB noted in neutrophils is not an integral membrane protein; it is linked by the GPI anchor and is absent in patients with paroxysmal nocturnal hemoglobinuria. FcγRIIIA is a conventional integral membrane protein with an intracytoplasmic tail. FcγRIIIA is able to mediate antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis, while FcγRIIIB is not.
Fc∊RI, the high-affinity Fc receptor for IgE, is present only on mast cells and basophils; it is involved in allergic reactions. Fc∊RII (CD23), the low-affinity receptor, is present on B and T lymphocytes, mononuclear phagocytes, and eosinophils. This receptor does not belong to the Ig superfamily of receptors. It is somewhat homologous to the receptor for asialoglycoprotein. Fc∊RIIA is found in resting B cells, while Fc∊RIIB is noted in activated B cells, mononuclear phagocytes, and eosinophils, especially after stimulation with IL-4. Engaging Fc∊RII on mononuclear phagocytes induces a variety of cellular changes, many of which relate to the induction of nitric oxide synthesis (352).
IgA receptors (FcαR; CD89) have been detected on monocytes, polymorphonuclear neutrophils (PMNs), and eosinophils, and on phagocytic cells at mucosal sites (353). These receptors bind both secretory and serum forms of IgA and require the Ca2 region of the IgA molecule for ligand recognition. Monocytes and PMNs modulate their expression of the FcαR upon treatment with cytokines, such as GM-CSF, and lipopolysaccharide. Purified FcαRs appear as heavily glycosylated molecules with an average molecular weight of 60 kD, dropping to 32 and 36 kD upon treatment with N-glycanase. Ligation of FcαRs on phagocytic cells by multivalent IgA complexes induces a variety of responses, including superoxide generation, release of inflammatory mediators, phagocytosis, and killing of various pathogenic microorganisms. Thus, the apparent role of these receptors is to amplify the protective effects of the IgA antibody, a function of potential importance to mucosal defense (353).
Mononuclear phagocytes have different types of complement receptors (CRs) (354,355). CR1 (CD35) binds C3b/C4b and is found primarily on erythrocytes, but also on monocytes and neutrophils. The receptor is a 200-kD transmembrane glycoprotein. Expression of CR1 on human mononuclear phagocytes can be increased by treatment of the cells with agents such as chemotactic peptides, lymphokines, and fibronectin. CR2 (CD21), the receptor for C3d,g and the Epstein-Barr virus, is not found on mononuclear phagocytes. CR3 (CD11b/CD18, Mac 1) binds C3bi. It is found on mononuclear phagocytes as well as neutrophils (354,355). CR3 exists as a dimer (one α chain CD18, and one β chain CD11b) in the membrane. The α chains are shared by the antigens recognized by antibodies specific for lymphocyte function–associated antigen-1 (LFA-1), Mac-l/Mo1, and pl50,95, while the β chains are distinct for each antigen (356). CR3 appears to function as an adherence protein, and cells lacking these antigens (and CR3) show defective adherence, phagocytosis of C3bi-coated particles, and depressed production of reactive oxygen species (357,358,359). When normal human monocytes bind particles to their CR1 or CR3, these particles are generally not ingested. However, cultured blood monocytes as well as freshly isolated human peritoneal macrophages both bind and ingest these particles (240,354). In addition to binding C3bi, CR3 also binds ICAM-1 (CD54). Certain patients with recurrent bacterial infections have been described whose monocytes, granulocytes, and null cells lack these antigens (357,358,359). Cells from these patients manifest reduced phagocyte adherence to glass or plastic, phagocytosis of C3bi-coated particles, and reduced production of reactive oxygen species with phagocytosis of zymosan.
C5a anaphylatoxin, the amino terminal fragment of C5 produced after the enzymatic cleavage, causes a variety of effects on mononuclear phagocytes, most notably chemotaxis (360). C5a receptors (CD88) are members of the superfamily of G-protein–coupled receptors characterized by unique motifs including seven membrane-spanning hydrophobic domains. These receptors are closely related to receptors for chemotactic (fMLP) peptides (360,361). C5a and fMLP receptors are expressed on both neutrophils and mononuclear phagocytes.
Mononuclear phagocytes have specific α2-macroglobulin receptors (A2M) (CD91) that have been modified by interacting with proteases or certain ligands. A2M is synthesized by many different cells, including mononuclear phagocytes, hepatocytes, and fibro-blasts. A2M not only serves as a broad-spectrum protease inhibitor, but it also binds numerous growth factors, cytokines, hormones, antigens, and microbial proteins including IL-1, leukemia inhibitory factor, TNF, IL-6, TGF-β, IFN-γ, streptococcal cell wall proteins, and modified lipoproteins (362). Modified A2M (A2M that has interacted with protease or ligand [A2M*]) binds to mononuclear phagocyte A2M* receptors and initiates cellular changes. Expression of the receptor can be modulated by several factors including IFN-γ, endotoxin, and M-CSF. A2M* binding is accompanied by receptor-mediated internalization and modulation of cell function. Antigen bound to A2M is apparently targeted to mononuclear phagocytes for subsequent antigen presentation to lymphocytes and enhanced immune responses. A2M* may either antagonize or enhance cellular responses to bound growth factors and cytokines by blocking growth factor/cytokine receptor binding or by enhancing cellular uptake and processing of the ligand.
Macrophages also have specific receptors for glycoproteins with mannose, fucose, or N-acetylglucosamine terminal groups (363). These proteins are bound and internalized by the phagocytes, and the receptors are probably recycled to the surface. This system may represent a mechanism for clearing escaped or excess glycoprotein hydrolases in the macrophage’s environment. Macrophages from mice with chronic infection with bacillus Calmette-Guérin (BCG) (so-called “activated macrophages”) have reduced numbers of receptors for these glycoproteins, while receptors for other ligands such as α2-macroglobulin are not altered (364). The mannose-binding protein (CD206) can serve as a primitive opsonin for various microbes (32).
The mononuclear phagocyte diferric transferrin receptor (CD71) plays a role in the transport of iron into the cell (365). Diferric transferrin binds to a specific membrane receptor, is internalized from a coated pit into an acid-uncoated vesicle, and at the acid pH of the vesicle iron dissociates from the transferrin protein. Then the apotransferrin dissociates from the receptor, and the receptor is available for another round of ligand binding (366). The number of transferrin receptors per cell is reduced in mouse macrophages “activated” in vivo or in vitro (367). Mononuclear phagocytes also have specific cellular lactoferrin receptors (368). In episodes of acute endotoxemia or bacteremia, apolactoferrin is released from the specific granules by the action of interleukin-l. This apolactoferrin, after competing for and binding iron, is internalized by the mononuclear phagocyte receptors, thus possibly contributing to the hyposideremia seen in these conditions (43,362,369).
Mononuclear phagocyte lipoprotein receptors control the entry of these proteins and their associated lipids into the cells (370). Subendothelial vascular mononuclear phagocytes accumulate lipoproteins and cholesteryl esters and form “foam cells,” thus contributing to the generation of the atherosclerotic plaque (371,372). Certain receptors for low-density lipoprotein (LDL) and modified lipoproteins (“macrophage scavenger receptors,” or MSRs) are important in this accumulation (362). The LDL receptor recognizes apolipoproteins B-100 and E, which are constituents of LDL, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and catabolized chylomicrons (chylomicron remnants). Mouse peritoneal macrophages have very few receptors for LDL, but they do have many MSRs (371). Human blood monocytes have both LDL receptors and MSRs, and in the course of in vitro culture of monocytes, there is a dramatic increase of MSRs (333). Modified LDL with its cholesteryl esters binds to the MSR, and is internalized into the lysosomal compartment where an acid lipase produces free cholesterol. This cholesterol is re-esterified to cholesterol esters that are stored in the cytoplasm as lipid droplets. Unlike the LDL receptor, the levels of acetyl-LDL receptors are not regulated by the cellular level of cholesterol or cholesteryl esters, so the process can proceed unchecked and result in engorgement of the macrophages with lipid (371). Patients with homozygous familial hypercholesterolemia who lack the receptors for LDL have normal numbers of acetyl-LDL receptors (371). Modified LDL can bind to a variety of mononuclear phagocyte receptors; these include (a) MSR (CD204), (b) FcγRII-B2, (c) CD36 (thrombospondin), and (d) CD68 (macrosialin), a lysosomal-associated membrane protein (“lamp”) of macrophages (333,362).
Mononuclear phagocyte scavenger receptors (SRs) are a group of proteins that bind chemically or oxidatively modified lipoproteins, polyanions, and apoptotic cells (333,373,374). There are at least six classes of SRs (classes A through F). SR-AI and SR-AII bind acetylated and oxidized LDL, polyanions, and apoptotic cells. SR-B includes CD36 and SR-BI. CD36 binds acetylated and oxidized LDL, phosphatidyl serine, and apoptotic cells; interacts with the vitronectin receptor in binding apoptotic cells; and is a phagocytic receptor. SR-B-I is a receptor for high-density lipoprotein (HDL), the lipoprotein that can remove cholesterol from cells. SR-CI binds acetylated LDL and polyanions. The class D SR macrosialin (CD68) binds oxidized LDL, the class E SR LOX-1 binds oxidized LDL and polyanions, and the class F SR binds acetylated and oxidized LDL and polyanions. Humans with CD36 deficiency have reduced myocardial fatty uptake and an increased incidence of hypertrophic cardiomyopathy (333,374).
Expression of the SR can be regulated by various factors, including LPS, M-CSF, TGF-β, IFN-γ, retinoic acid, glucocorticoids, and vitamin D3. M-CSF stimulates SR expression, and in vivo M-CSF lowers cholesterol levels. Based on the large variety of ligands for these receptors, SR appears to play a role in several physiologic and pathologic processes (e.g., atherosclerosis, cell adhesion, host defense and innate immunity, cell interactions involving adhesion molecules, inflammatory responses to microbes [viz., bacterial LPS and lipoteichoic acid], and inanimate particulates [i.e., asbestos]) (333,362,374,375).
Cholesteryl esters are removed from mononuclear phagocytes only after de-esterification with the production of free cholesterol (371), which is then removed by an HDL. Mononuclear phagocytes produce and secrete apolipoprotein E in the form of phospholipid discs. These particles, along with cholesteryl ester formed by the action of lecithin-cholesterol acyl transferase, are assembled extracellularly into a large spherical particle containing a core of cholesteryl esters and a coat containing apolipoproteins A-l and E. This particle may be HDLc that transports the cholesterol to the liver, where it is metabolized.
The LDL receptor–related protein (also called the “remnant receptor”) binds chylomicrons and VLDL that have been modified by hydrolases (e.g., lipoprotein lipase) and by association and disassociation with apolipoproteins (362). Receptors for these “remnants” are expressed on hepatocytes and Kupffer cells. LDL receptor–related protein (LRP) has a C-terminal cytoplasmic portion, a single transmembrane domain, and an extracellular domain that has similarity to that of the LDL receptor. The receptor mediates the endocytosis of apoE-containing, remnantlike lipoproteins; lipoprotein lipase; chylomicron remnants; lactoferrin; A2M-ligand complexes; plasminogen activator–plasminogen activator inhibitor-I complexes; and certain toxic ligands such as Pseudomonas exotoxin A and possibly bacterial LPS. LRP expression is decreased by treatment of cells with IFN-γ, LPS, or prostaglandin E2 and is increased by M-CSF.
Mononuclear phagocytes express members of the TNF receptor family. This family of receptors includes the p55 (TNFR1; CD120a) and p75 (TNFR2; CD120b) components of the TNF receptor, CD40, CD27, CD30, and CD95 (Fas) (376). These receptors are type I membrane proteins containing homologous extracellular domains, with cytoplasmic domains of variable length without significant sequence homology. Ligands for these proteins are all type II membrane molecules, with extracellular carboxy-terminal regions and intracellular amino termini. Binding of ligand to receptor can induce cellular growth, differentiation, or apoptotic death. In the case of TNFR1 and Fas, engaging the receptor with TNF-α/β and Fas ligand, respectively, induces apoptotic death of the cell by activating a complex pathway leading to fragmentation of DNA by endonucleases, nuclear dissolution, and nonnecrotic death (377). This apoptotic cell death occurs in a variety of cell types under physiologic (“programmed cell death”) and pathologic conditions. It serves as an important means of immunologic cell selection of lymphocytes during development (negative selection or clonal deletion of immature T cells), tissue remodeling during embryogenesis and inflammation, and cell-mediated cytotoxicity. MRL-lpr/lpr mice have no or very little Fas (CD95) due to a retrotransposon insertion in the gene resulting in aberrant splicing and premature termination of transcription; they have defective apoptosis with consequent massive lymphadenopathy and autoimmune disease comparable to humans with rheumatoid arthritis and systemic lupus erythematosus. Mice with the gld defect have a point mutation in the gene for Fas ligand, defective apoptosis, lymphadenopathy, and autoimmunity (378,379). Humans with CD95 defects, impaired apoptosis, and aspects of autoimmunity have been identified (380,381).
Mouse macrophages and human monocytes have high-affinity specific interferon-γ receptors (IFN-γ-Rs; CD119) (382,383). The effects of IFN-γ in modulating the activities of mononuclear phagocytes apparently are mediated through these receptors. IFN-γ serves as a “macrophage-activating factor” in mouse (384) and human (294,385) mononuclear phagocytes. Individuals with mutations in IFN-γ-R receptor chains are susceptible to severe infections with bacillus Calmette-Guérin, nontuberculous mycobacteria, listeria, and salmonella (386,387). Monocytes also have receptors for interferon-α (IFN-α-Rs; CD118) (388). IFN-α administered in vivo or used in vitro with isolated cells can activate monocytes for expression of NOS2 and for nitric oxide production (389). IFN-γ-R signaling activates STAT1 homodimers that serve as transcription factors to trigger production of IFN-γ–activated genes; similarly, IFN-α-R activates STAT1-STAT2 heterodimers that drive transcription of IFN-α–activated genes. Consequently, genetic inactivation of STAT1 or STAT2 in humans and mice leads to loss of IFN-α/IFN-γ or IFN-α responsiveness, respectively (390). Macrophages have IL-12 receptors (CD212), and genetic deficiencies of this receptor predispose to severe infections with poorly virulent mycobacteria and salmonella, at least in part, because IL-12 is a major activator of IFN-γ production (383,391).
As noted earlier, mononuclear phagocytes have M-CSF receptors (M-CSF-Rs; CD115), the specific growth factor for the mononuclear phagocyte lineage (52,69,392). Mononuclear phagocytes also express integrin receptors. The integrins are a family at least 24 different widely expressed cell-surface receptors. Integrins are αβ heterodimers including the leukocyte-specific β2 in-tegrin LFA-2 (CD11a/CD18), Mac-1 (CR3, CD11b/CD18), and p150,p95 (CD11c/CD18) (393). Ligands for the various integrins include collagen, laminin, fibronectin, vascular cell adhesion molecule-1 (VCAM-1), vitronectin, ICAM-1 and -2, fibrinogen, factor X, and C3bi. Integrins are important in cell–cell adhesion, transmembrane signaling, fertility, leukocyte function, inflammation, hemostasis, bone remodeling, and angiogenesis (393).
Mononuclear phagocytes have specific high-affinity chemotactic peptides receptors (394) such as N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys and N-formyl-Met-Leu-Phe. The chemotactic peptide (“fMLP”) receptor is a member of the seven-transmembrane-spanning, G-protein–linked receptor family (361). There is sequence and structural similarity of the fMLP receptor to the C5a receptor, and the responses of neutrophils and mononuclear phagocytes (the major cell types expressing these receptors) are likewise quite similar (360,361). These peptides induce not only directed movement (chemotaxis) of the cells, but also lysosomal enzyme release and reactive oxygen species production.
Mononuclear phagocytes are capable of synthesizing coagulation factors (see below), and they also display coagulation factor receptors (395). Not only are coagulation processes and activated coagulation factors important mediators of blood coagulation and anticoagulation, but they also play roles in cell mitogenesis, inflammation, atherogenesis, tissue remodeling, cell adherence, and chemotaxis. Monocytes and macrophages have well-characterized receptors for factor VII/VIIa (tissue factor), fibrinogen (CD11b/CD18 and CD11c/CD18), factor X (CD11b/CD18), factor Xa (membrane-bound factor V and an “effector cell protease receptor type 1”), thrombin (the thrombin protease–activated receptor), and urokinase-type plasminogen activator (uPA) (uPA-R, CD87). Tissue factor (TF) is a membrane lipoprotein that initiates the extrinsic pathway of coagulation on mononuclear phagocytes and endothelial cells (396,397). TF acts as a high-affinity receptor and cofactor for factor VII/VIIa. Monocyte expression of TF is increased by treatment with LPS, TNF, immune complexes, and lymphocyte products. The thrombin receptor (or protease-activated receptor) is a G-protein–linked seven-transmembrane segment receptor expressed on several cell types including mononuclear phagocytes (398,399). Thrombin cleavage of the extracellular part of the receptor exposes a tethered ligand, which mediates functional changes in the receptor-bearing cell. The urokinase plasminogen activator receptor (CD87) (400) localizes uPA on the membrane and contributes to cell-mediated fibrinolysis, tissue remodeling, and cell invasiveness through tissues, cell adhesiveness, wound repair, and control of apoptosis (401). The receptor is a GPI-linked membrane protein. Its expression is increased by treatment of cells with IFN-γ and TNF.
The effects of several different hormones (e.g., insulin, glucagon, thyrotropin, somatomedin, prostaglandins of the E series, dexamethasone, dihydroalprenolol, 1,25-dihydroxyvitamin D3, and sex hormones) on mononuclear phagocytes are probably mediated by specific cellular receptors (266,269,402,403). Monocytes and macrophages display receptors for a variety of other cytokines and growth factors as noted inTable 12.3.
Receptors for Microbes and Microbial Products
Mononuclear phagocytes respond to picomolar to nanomolar amounts of microbial components in complex ways; TLRs are central to the mediation of these effects, and key to the innate immune system in animals (25,404). The toll protein was originally identified in Drosophila (the fruit fly) as an essential determinant of dorsoventral polarity in embryogenesis, and it was subsequently shown that a family of toll proteins and related proteins were important in antifungal and antimicrobial immunity in the flies (25). The cytoplasmic portion of the IL-1 receptor has homology to Drosophila toll. In vertebrates, the TLRs are a family of proteins (TLR1 through TLR10) (Table 12.4). Members of the TLR family are involved in the recognition of pathogen-associated molecular patterns, with various TLR-binding recognized microbial components. TLR2 binds bacterial peptidoglycan, LPS-associated protein, and bacterial laminoarabinomannan. TLR2 also cooperates with TLR6 to bind and mediate the effects of Mycoplasma lipoprotein. Mice with disrupted TLR2 have impaired production of TNF, IL-6, and NO in response to several Gram-positive cell walls (likely the peptidoglycan component). TLR2 may also interact with the LPS of Porphyromonas gingivalis and Leptospira interrogans, two unique LPSs. TLR2 also reacts with GPI membrane components of Trypanosoma cruzi.
Investigators noted in the mid-1900s that mice of the inbred strains C3H/HeJ and C57Bl/10ScCr were unresponsive to LPS from Gram-negative bacteria in vivo, and that LPS did not activate macrophages and B cells of these mice in vitro. Researchers cloned the mouse TLR4 gene and discovered that this LPS insensitivity is caused by mutations in the TLR4 gene in these strains—a point mutation in the cytoplasmic domain of TLR4 in C3H/HeJ mice, and a TLR4 null mutation in mice (24,405). Subsequently, investigators have demonstrated TLR4 mutations in humans who were unresponsive to inhaled LPS (406). TLR4 is expressed by mononuclear phagocytes, cardiomyocytes, airway epithelium, endothelial cells, and smooth muscle cells, and to a small extent in other cells. People with TLR4 mutations appear to be at increased risk for infection with Gram-negative organisms and for septic shock (407), but they have decreased risk of atherosclerosis (408). This protection from atherosclerosis may be due to diminished inflammatory responses to certain microbes (e.g., Chlamydia pneumoniae) thought to be important in the pathogenesis of atherosclerosis (408). Levels of TLR4, MD2, and MyD88 are increased in human mononuclear phagocytes by IFN-γ treatment, and these increases correlate with increased responsiveness to LPS (409).
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Table 12.4 Toll-Like Receptors |
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TLR3 is the receptor for double-stranded RNA and polyinosine/polycytosine, and TLR5 serves as the receptor for flagellin, the major protein of bacterial flagella. Microbial DNA is a potent immunostimulant and inducer of inflammation, and it is a potent adjuvant in vaccines (410,411,412). Microbial DNA (unlike DNA from vertebrates) has a large content of unmethylated cytosines adjacent to guanosines (CpG DNA). TLR9 is the receptor for CpG DNA, and it mediates the cellular effects of this immunostimulant (413,414). Mice with a disrupted gene for TLR9 have absent or diminished responses to CpG DNA.
Signaling through the TLR/IL-1R pathways proceeds through several common signaling pathways, leading to the nuclear translocation of NF-κB and activation of various kinases. For example, LPS binds a plasma protein (LPS-binding protein), which in turn can bind in a specific manner with membrane or soluble CD14 (415,416). Also, LPS alone (or the LPS/LPS-binding protein complex) binds to TLR4 that is complexed with protein MD-2. Signal transduction starts with the adapter protein MyD88 interacting with the TLR/IL-1R cytoplasmic tail and recruitment of the IL-1R–associated kinase (IRAK). Activated IRAK then binds and activates TNFR–associated factor 6 (TRAF6), which in turn stimulates the IκB kinase and MAP kinase. This leads to nuclear translocation of NFκB and transcription of proinflammatory cytokines and growth factors (25). MyD88 is absolutely critical to this complex pathway that mediates responses through TLR2, TLR4, TLR9, and the IL-1R pathways (25,417). MyD88-deficient mice lack cellular responses to LPS, peptidoglycan, CpG DNA, IL-1, and IL-18.
Endocytosis and Phagocytosis
Mononuclear phagocytes internalize substances by either pinocytosis (the uptake of solutes) or phagocytosis (the uptake of particulates >0.5 μm). Pinocytosis can proceed by receptor-mediated processes (discussed above) or by receptor-independent processes. Pinocytosis is facilitated at certain areas of the cell membrane that are coated on the inner surface with proteins such as clathrin, or that contain complex structures of lipid and protein called lipid rafts (418,419). Following internalization, pinocytic vesicles fuse with lysosomes, and their contents are then processed in the lysosome (420). Parts of the lysosome/vesicle membrane bud off and return to the plasma membrane, thus recycling the originally internalized membrane and providing a mechanism for maintaining membrane surface area without requiring new membrane synthesis (420,421).
Particles are phagocytized through either opsonin-independent or opsonin-dependent mechanisms (422). Polystyrene (latex) spheres and aldehyde-fixed erythrocytes are examples of particles avidly phagocytized by mononuclear phagocytes without the need for opsonins. Though the precise mechanism of opsonin-independent uptake is not known, it may involve surface hydrophobicity and/or charge of the particle. Additionally, it may occur through a lipid raft-mediated mechanism (419). Phagocytosis is facilitated by opsonization, which is essentially coating the particle with substances such as immunoglobulin, complement, and fibronectin. The ability of macrophages to phagocytize opsonized particles depends on the source and activation state of the macrophages, likely as a result of differential expression of surface receptors for the opsonins (240,354). Further, a particle must be surrounded circumferentially with the opsonin in order to be phagocytized; for instance, in the case of an IgG-coated particle, the macrophage membrane will extend around the particle only as far as the IgG coating (423,424).
Opsonin-dependent phagocytosis is a complex process that varies with the receptor that may trigger the process; however, some generalizations are true for most situations (418). Following engagement of the macrophage receptor, polymerization of actin occurs at the membrane, allowing the macrophage to send out pseudopods that surround the particle and draw it into the cell. The membrane that surrounds this nascent phagosome is thought to be derived from the plasma membrane, though a controversial alternative model suggests that the endoplasmic reticulum may contribute (425). The phagosome undergoes a series of sequential fusions with endosomes and lysosomes in a process termed phagosome maturation. The result of maturation is extensive remodeling of the lipid components of the membrane and the delivery of protein cargo, ultimately resulting in a phagosome that is fully mature, contains lysosomal hydrolases, and has a reduced pH. When the phagocytized particle is a microbe, completion of the maturation process results in elimination of the organism, though a pathogenic microbe is able to thwart the process through a variety of mechanisms.
Chemotaxis
Mononuclear phagocytes, like neutrophils, exhibit general movement (chemokinesis) as well as directed movement (chemotaxis) in response to various factors (85,426). Movement of monocytes and macrophages to and within sites of inflammation and tissue injury is influenced by chemotactic factors, which are a variety of substances derived from plasma, cells, bacteria, and connective tissue. These include complement components such as C5a (427); extracellular matrix fragments including those of fibronectin (428), elastin (429), and collagen (430); N-formylated oligopeptides (394,431); proteins derived from malignant cells (432,433,434); and a family of secreted proteins called chemokines (435,436).
Circulating monocytes are a heterogeneous group, some of which are recruited to inflamed tissue and some to noninflamed tissue. They express a complex array of chemokine and other receptors that lead to their different responses to chemotactic stimuli, and different propensities to migrate into distinct tissues. Inflammatory monocytes can be characterized as CD14hiCD16- in humans or CCR2+/CX3CR1lowLy6+ in mice, and noninflammatory/resident monocytes as CD14+/CD16+ in humans or CCR2-/ CX3CR1hiLy6- in mice (31).
The migration of monocytes from the vascular space into tissue requires highly coordinated interaction between receptors on the monocyte and the endothelial or extracellular matrix (85). Circulating monocytes maintain loose contact with the endothelium via low-affinity interaction of selectins on the surface of the monocytes with the endothelial surface. When monocytes are stimulated by chemotactic factors, integrin receptors on the monocytes are activated, resulting in much firmer binding to the endothelial cell. The monocytes then extravasate between endothelial cells to reach the tissue. Underlying this general scheme is a complex series of molecular events that can be briefly summarized. Following binding of chemotactic factors to G-protein–coupled or a tyrosine kinase receptor on the monocyte, phosphatidyl inositol-3 kinase is activated, which catalyzes formation of lipid second messengers including phosphatidylinositol 3,4,5-triphosphate (437). Subsequently, several small guanosine triphosphate (GTP)-binding proteins are activated. Rap1 and RAPL are involved in increasing integrin affinity (85), while RhoA, Rac1, and Cdc42 stimulate cell movement by weakening cell adhesions and promoting formation of cell extensions (lamellipodia) that drive movement forward (437). Underlying increased cell movement are changes in actin polymerization that occur in the actin-related (arp) 2/3 complex, a group of proteins that includes actin monomers, arp2, arp3, and the Wiskott-Aldrich syndrome protein (WASp) (438,439).
Products of Mononuclear Phagocytes
Mononuclear phagocytes are capable of producing many substances that can influence the host. Some of these substances are secreted constitutively, not requiring any particular stimulus to initiate the secretion. However, secretion of most macrophage products is influenced by stimuli acting on the cell. In general, the secretory capacity of macrophages parallels their state of “activation,” and correlates well with their ability to mediate bactericidal and tumoricidal activities. Some of the products can act on the phagocytes themselves, thus providing the possibility for autoregulation of cell function. Table 12.5 lists some of the secretory products of mononuclear phagocytes.
Enzymes
Lysozyme (also called muramidase) is a 14-kD protein that is secreted in a constitutive manner from cultured macrophages (440), but apparently in an inducible manner from populations of activated macrophages in vivo (441). It is a major secretory product, composing up to 2.5% of cellular protein produced per day (442). While neutrophils also secrete lysozyme, mononuclear phagocytes contain and secrete more. The main function of the enzyme is to lyse bacteria (especially Gram-positive organisms) that contain a certain glucosidic linkage. Animals or patients with an increased mononuclear phagocyte mass or with monocytic or myelomonocytic leukemia have high levels of plasma and/or urine lysozyme (443), which sometimes leads to tubular dysfunction and excess loss of potassium and refractory hypokalemia (444). Additionally, elevated lysozyme expression may be useful in the diagnosis of sarcoidosis (445).
The neutral proteases, including plasminogen activator, collagenase, and elastase, are enzymes that are active at neutral pH and can degrade connective tissue components. These enzymes may be used by the mononuclear phagocytes to degrade components of the extracellular matrix as they migrate through the tissues. Plasminogen activator secretion from macrophages is enhanced if the cells have been “activated” to a certain state and if they are phagocytizing particles (446,447). However, endotoxin, a stimulus known to produce highly “activated” or tumoricidal macrophages, dramatically reduces plasminogen activator secretion (448). Plasminogen activator not only generates plasmin from plasminogen, but also cleaves C1, C3, and activated Hageman factor. As noted earlier, elastase and collagenase secreted by alveolar macrophages may play a role in the genesis of emphysema (125). Elastase can degrade a large number of substrates other than elastin including αl-antiprotease (449), immunoglobulin (450), proteoglycans, fibronectin, fibrin, and fibrinogen (451,452,453).
A large number of acid hydrolases are present in macrophage lysosomes. The content of these enzymes is increased as mononuclear phagocytes are stimulated by microbial products, lymphokines, phagocytosis, and in vitro culture (62,65,454). The secretion of these enzymes can be stimulated by phagocytosis, by engaging Fc or complement receptors, or after treatment with lymphokines (454). In certain instances of tissue inflammation with low tissue pH (455), the acid hydrolases may be able to act on tissue components. Potential substrates include basement membranes, cartilage, collagen, complement components, proteoglycans, and immunoglobulin (454).
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Table 12.5 Products of Mononuclear Phagocytes |
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Mononuclear phagocytes are able to secrete nearly all of the complement factors of the classical and the alternate pathways, except C6 through C9 (454,456,457). Some of these factors can be acted upon by various proteases to form products that can alter the function of macrophages. In particular, complement opsonization of particles stimulates phagocytosis by macrophages (458).
Mononuclear phagocytes produce several coagulation factors. These include those enhancing coagulation, such as the membrane-bound tissue factor/procoagulant activity that binds and activates factor VII (397,459), as well as factor XIII, which cross-links fibrin (448,459,460,461). The procoagulant activity of macrophages and monocytes is dramatically enhanced by endotoxin or antigen–antibody complexes (462,463). Mononuclear phagocyte-associated procoagulant/anticoagulant activities are important in atherogenesis, inflammation, cellular growth control, tissue remodeling, cell migration, and invasiveness. Much of the fibrin deposition seen in delayed hypersensitivity reactions and in tumors likely results from mononuclear phagocyte-derived procoagulants (464,465). In inflammatory lesions such as those seen in rheumatoid arthritis, macrophages express a variety of procoagulant and anticoagulant molecules (466,467,468). Mononuclear phagocyte-associated procoagulants play important roles in disseminated intravascular coagulation seen in malignancy and infection, and in the prothrombotic state noted in association with malignancy (469,470).
Among the protease inhibitors produced by mononuclear phagocytes, α2-macroglobulin is very important because it affects many proteases, including plasminogen activator, plasmin, elastase, kallikrein, and thrombin (471). Mononuclear phagocytes also synthesize and secrete αl-antiprotease (126,472,473). As noted earlier, mononuclear phagocytes have receptors for α2-macroglobulin–protease complex, and this complex can modulate the function of these cells (474,475,476).
Reactive Oxygen Species
As mononuclear phagocytes ingest particles or as they are stimulated with certain surface active agents, they exhibit a respiratory burst, much like that seen in neutrophils (477). Oxygen is consumed at an increased rate, the hexose monophosphate shunt is stimulated, reduced glutathione and reduced nicotinamide adenine dinucleotide phosphate (NADPH) are generated, and oxygen is reduced to superoxide anion by an oxidase. This oxidase requires NADPH, a flavoprotein, cytochrome b245, and ubiquinone (477,478). The superoxide anion dismutates to hydrogen peroxide, and some hydroxyl radical may also form in the presence of iron (479). Some peroxide diffuses out of the cell, while some is changed to water and oxygen by the action of catalase. In addition to phagocytic stimuli and surface-active agents (e.g., arachidonic acid or phorbol myristate acetate), antigen–antibody complexes, C5a, ionophores, fatty acids, and lectins can also trigger the mononuclear phagocyte respiratory burst (480). If mononuclear phagocytes have been “activated” by in vivo infection or by in vitro treatment with lymphokines such as IFN-γ, their ability to secrete reactive oxygen species is greatly enhanced (294,385,480,481). These oxygen species have the ability to lyse nucleated and nonnucleated cells, to inactivate certain enzymes, and to alter lipids, nucleic acids, and proteins (482). They are particularly important for defense against intracellular bacteria and protozoa.
The NADPH oxidase enzyme complex (NOX)-2 (also known as phagocyte oxidase) is responsible for generating ROS in neutrophils and mononuclear phagocytes. Other NOX enzymes and related dual oxidase (DUOX) enzymes are expressed in nonphagocytic cells (478). NOX2 consists of membrane components (cytochrome b558, p22-phox, the GTP-binding protein Rac2, and flavin-adenine dinucleotide [FAD]) and cytoplasmic components (p47-phox and gp91-phox) (17,477). Humans with chronic granulomatous disease (CGD) have a defect in one of these subunits rendering their phagocytes unable to generate ROS. Mouse models of CGD created by genetically disrupting expression of gp91-phox (483) or p47-phox (484) are useful in analyzing CGD and in developing new treatments for the disease.
Reactive Nitrogen Species
The simple gas NO has multiple important physiologic and pathologic functions (485,486). These include roles in (to mention only a few) host resistance to tumors and microbes, regulation of blood pressure and vascular tone, neurotransmission, learning, neurotoxicity, carcinogenesis, and control of cellular growth and differentiation (485,486,487,488,489). In the presence of oxygen, NO rapidly (in seconds) is converted to nitrite and nitrate, substances that are generally not bioactive (490). NO binds with high affinity to iron in heme groups of proteins such as hemoglobin (Hb), myoglobin (Mb), and guanylyl cyclase; Hb and Mb are very effective quenchers of NO action. NO also nitrosylates the hemoglobin globin β chain cysteine at position 93 (491). This S-nitrosohemoglobin can subsequently release NO and participate in a cycle of NO/oxygen loading/unloading that is somewhat comparable to that of oxygen/carbon dioxide in the respiratory cycle (491,492).
NO also reacts with O2-, and superoxide dismutase (SOD) prolongs NO life by eliminating O2-. On reacting with O2-, NO may generate peroxynitrite (OONO–), a very toxic/reactive molecule that is a very important final effector toxic molecule when one thinks of NO toxicity in oxygenated systems (486). Peroxynitrite reacts with protein and nonprotein sulfhydryls, DNA, and membrane phospholipids. Peroxynitrite also nitrates free and protein-associated tyrosines (and other phenolics) via the metal-catalyzed formation of the nitronium ion to form nitrotyrosine. Nitration of proteins modifies their functions, and nitrated proteins serve as a relatively long-lived marker (a “track”) of NO and peroxynitrite action in tissues (486,493). Tyrosine nitration has been associated with inflammatory disorders (486,493,494).
There are three forms of the enzyme nitric oxide synthase encoded by three different genes. Neural NOS (nNOS or NOS1) and endothelial cell NOS (eNOS or NOS3) are constitutive enzymes, demonstrating low-level, constant transcription of messenger RNA (mRNA). The enzymatic expressions of NOS1 and NOS3 are modulated by regulation of cytoplasmic calcium levels, with agents inducing increases in calcium (e.g., calcium ionophores or ligands such as acetylcholine), with subsequent binding to calmodulin and activation of the enzyme. Inducible NOS (iNOS or NOS2) can be regulated at multiple levels, but induction of mRNA transcription by agents such as cytokines or LPS appears to be of major importance (495) (Fig. 12.8). Although NOS2 was described initially in mononuclear phagocytes, it also is found in synoviocytes, chondrocytes, endothelial cells, smooth muscle cells, hepatocytes, and others (485,489,496,497,498). NOS3 is myristoylated and membrane bound; while NOS1 and NOS2 are not myristoylated, they can exist in membrane-bound and cytoplasmic forms. In the mouse macrophage NOS2 promoter, there are two functionally important areas upstream of the 5′ end of the gene, which contain consensus sequences for several known DNA-binding proteins (495). The human NOS2 promoter differs from that in the mouse, extending at least 26 kb upstream from the transcription start site (499).
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Figure 12.8. Factors inhibiting or enhancing expression of nitric oxide (NO) synthase type 2 (NOS or NOS2) and production of NO. The transcription of NOS2 messenger RNA (mRNA) can be enhanced (+) or diminished (-) by various cytokines/growth factors and bacterial products. The enzyme NOS converts to L-arginine (L-arg) to nitric oxide and L-citruline, and it requires reduced nicotinamide adenine dinucleotide phosphate (NADPH), biopterin, flavin mononucleotide (FMN), flavin-adenine nucleotide (FAD), oxygen, and heme for activity. Depletion of L-arginine or biopterin diminishes activity. L-Arginine analogs, such as Ng-mono-methyl—arginine, block enzyme activity. NO can inhibit NOS activity by oxidizing the enzyme’s heme group. Superoxide and oxygen rapidly diminish NO activity by oxidizing it. Iron, heme, and cobalamins can blunt NO’s activity by binding NO to their iron or cobalt. CaM, calmodulin; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; TGF, transforming growth factor; TNF, tumor necrosis factor. |
Regulation of NOS2 can occur at multiple steps (495), including mRNA transcription, mRNA stability, and mRNA translation. At the protein level, NOS may be regulated by calmodulin binding, dimer formation (the functional enzyme exists as a dimer), substrate (L-arginine) depletion, substrate recycling to L-citrulline, tetrahydrobiopterin availability, end-product inhibition (NO interaction with NOS heme), phosphorylation, and subcellular localization. Important NOS cofactors include FAD, flavin mononucleotide (FMN), NADPH, tetrahydrobiopterin, and calmodulin-calcium (500,501,502,503). For NOS2, calmodulin is tightly bound to protein, making it relatively resistant to inhibition by calcium chelators.
Although many have shown high-level NO production by murine macrophages, there has been some difficulty showing that human mononuclear phagocytes produce NO in vitro. However, some studies have clearly documented that human mononuclear phagocytes from patients with a variety of illnesses express high levels of NOS2 and produce NO in vitro and in vivo (159). Some researchers have seen NOS2 antigen in human alveolar macrophages (504,505,506). Cancer patients treated with IL-2 overproduce NO via an NOS mechanism (507). Blood mononuclear cells from rheumatoid arthritis patients express higher levels of NOS2 than do normal individuals, and the cells are more responsive to IFN-γ and endotoxin stimulation for NO production in vitro, indicating that their cells are activated for NOS expression and NO production in vivo (322). Tanzanian children spontaneously express NOS2 antigen in their blood mononuclear cells, and this is increased in those with mild or asymptomatic malaria and markedly decreased in those with cerebral malaria (508). Patients with hepatitis C infection receiving IFN-α treatment in vivo have monocytes that express high levels of NOS2 mRNA and antigen, and have high-level NOS enzyme activity (389). Also, IFN-α activates normal blood monocytes in vitro for NOS2 expression and NO production (389). Finally, as noted above, peritoneal macrophages from women with endometriosis and infertility spontaneously express NOS2 antigen and produce NO in vivo and in vitro, and they are more responsive to IFN-α or IFN-γ and LPS in vitro (245).
NOS2 expression is controlled primarily by levels of mRNA transcription. Human NOS2 promoter polymorphisms have been identified that are associated with resistance to infection and NOS2 expression/NO production. The G-954C (a G to C change at 954 bases upstream from the transcription site) polymorphism is associated with protection from severe malaria in Gabonese children (509), and mononuclear cells from those with the G-954C genotype express more NOS activity and produce more NO in vitro (510). The polymorphic pentanucleotide repeat CCTTT (approximately 7 to 20 repeats) is at about position –2,500 in the NOS2 promoter. Gambian children with high numbers of repeats were found to have more severe malaria and to be more likely to die from malaria (511). Other researchers discovered the C-1173T NOS2 promoter polymorphism that was very highly significantly associated with protection from severe malaria in Tanzanian children (primarily cerebral malaria) and severe malaria in Kenyan children (primarily severe malaria anemia) (512). Those with the C-1173T polymorphism had increased levels of NO in vivo, suggesting that the polymorphism was functional. C-1173T was noted only in Africans, and its protective effects against malarial disease was independent of any other NOS2 polymorphism (512).
Bioactive Lipids
Much of the fatty acid in macrophages is arachidonic acid, which can be mobilized for the synthesis of cyclooxygenase and lipoxygenase products. These bioactive lipids are important for a variety of inflammatory processes. The ability of the macrophage to produce these substances is dramatically influenced by activation of the cells, engagement of the Fc receptors, phagocytosis of particles, or treatment with phorbol diesters or ionophores (440,513,514,515).
Peroxisome Proliferator-activated Receptor Ligands
Macrophages secrete ligands that bind the peroxisome proliferator-activated receptor-α (PPAR-α) and PPAR-γ nuclear receptors (516,517,518). These molecules are members of the nuclear hormone receptor family of ligand-induced transcription factors. PPARs are expressed in macrophages, and thus, they influence immune responses of macrophages. In particular, they are important in regulating inflammation, mainly eliciting anti-inflammatory responses, though this is dependent on the ligand and type of macrophage examined (517). A number of natural and synthetic ligands for PPARs have been identified. Endogenous ligands for PPAR-α include leukotriene B4, eicosanoids, and certain fatty acids, while synthetic ligands include fibrates. In contrast, endogenous ligands for PPAR-γ include oxidized LDLs and the prostaglandin D2 (PGD2) dehydration products PGJ2 and 15d-PGJ2, while the antidiabetic thiazolidinediones and nonsteroidal anti-inflammatory drugs also serve as ligands (517).
Cytokines and Chemokines
Macrophages secrete an array of polypeptide cytokines and chemokines that play divergent but important roles in innate and acquired immune responses. Macrophages are particularly important sources of TNF-α, IL-1, IL-6, IL-8, IL-12, TGF-β, and CSFs (440).
IL-1 is a major mediator of local and systemic inflammation. It is secreted in response to diverse stimuli including bacterial infection, viral infection, tissue trauma, and tumors. It can stimulate proliferation of T and B lymphocytes; cause hyperthermia by action through hypothalamic cells; alter synovial cell synthesis of prostaglandins, collagenase, and plasminogen activator; enhance fibroblast proliferation; enhance catabolic activities in muscle; cause specific granule release from neutrophils; and cause hepatocyte synthesis of acute-phase reactants (43,519,520).
TNF-α (also called cachectin) is the prototype for a large family of cytokines that have important roles in immunity (521,522,523). TNF-α is secreted by macrophages after exposure to endotoxin. It binds either a 55-kD or 75-kD receptor (524). Collectively, the TNF-α family members have multiple activities that include beneficial functions for organogenesis, inflammation, and host defense. However, overproduction of TNF-α can lead to cachexia, sepsis, and autoimmune disease. A number of TNF-α inhibitors are used to effectively treat rheumatoid arthritis, though suppression of host defense is a side effect with consequent activation of infections (particularly mycobacterial infections) and occurrence of B-cell lymphoma (525,526).
Antigen Presentation
Mononuclear phagocytes act as antigen-presenting cells to promote activation, antibody production, and immune responses from B and T lymphocytes (527,528,529). While macrophages are not as adept at antigen processing and presentation as dendritic cells, macrophages are present in high numbers at sites of infection, and therefore, are likely to play important roles as antigen-presenting cells in vivo (527). Within the macrophage, antigens are degraded to peptide fragments, which are then presented via either class I or class II MHC molecules, with class I molecules being involved with endogenously generated antigens and class II with exogenous antigens. Antigen peptides generated endogenously in the cytoplasm (e.g., those generated from an intracellular infection such as influenza virus) by the actions of the proteasome are actively transported across the rough endoplasmic reticulum and presented at the mononuclear phagocyte membrane in association with the class I MHC molecule. This antigen is then presented to CD4 T lymphocytes for the generation of sensitized, cytotoxic lymphocytes. In contrast, exogenous protein antigens presented at the membrane of mononuclear phagocytes are endocytized and processed in a lysosomal compartment. Class II α and β chains associate with invariant (Ii) chains and progress through the endoplasmic reticulum. Exogenous antigen peptide fragment then complexes with the αβ chains in an acidic endosome with dissociation of Ii from the complex. The antigen class II αβ complex is then transported to the cell membrane, where antigen can be presented to responding cells in the context of MHC class II molecule. The two pathways of antigen presentation are not entirely independent; cross-presentation is the process whereby exogenous antigens leave the endosomal/lyso-somal compartments to enter the cytosol and subsequently be processed through the endogenous antigen pathway (530).
Antigen-presenting cells must physically interact with responding lymphocytes. The process of MHC class I or II–associated antigen presentation to the T-cell receptor complex is facilitated by certain receptor–ligand couplings between mononuclear phagocytes and responding T lymphocytes; these pairs include CD2 with LFA-3, LFA-1 with ICAM-1 or 2, and CD28 with B7-1 (CD80) or B7-2 (CD86). Lymphokines and monokines elaborated by the interacting cells influence expression of these accessory molecules and act as costimulatory molecules for activation of lymphocytes. CD8 and CD4 have a low affinity for binding to MHC class I and II molecules, respectively, and they likely contribute to the interactions.
Antimicrobial Effects
Macrophages are major effectors of innate resistance to pathogens. This ability to provide host resistance to pathogens is enhanced when the macrophages are activated with cytokines. Somewhat paradoxically, in absence of the appropriate activation signals, macrophages are able to harbor pathogens, such as M. tuberculosis, providing safe niches in which to evade immune surveillance. Hence, an effective innate immune response hinges not only on the ability of macrophages to migrate to sites of infection and phagocytize pathogens, as discussed above, but also to be appropriately activated to stimulate intracellular pathways that drive pathogen killing.
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Table 12.6 Types of “Activated” Macrophages |
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At least three pathways of macrophage activation have been identified: (a) classical activation, (b) alternative activation, and (c) type II activation (32,33) (Table 12.6). Classical activation required two signals; the first is IFN-γ (382,384,385,481,531) that “primes” the macrophage, and the second is TNF-α or a substance that induces TNF-α production such as LPS. The hallmark of the activated macrophage is increased NO production and potent intracellular killing capacity, as discussed in more detail below. Antigen presentation is also enhanced in the activated macrophage as a consequence of increased MHC class II expression.
In contrast, alternative activation occurs when macrophages are exposed to glucocorticoids or cytokines produced by Th2 cells, notably IL-4 and IL-13, instead of Th1 cytokines like IFN-γ (532). Alternatively activated macrophages do not increase NO production; rather, the cells produce increased levels of arginase, an enzyme that metabolizes arginine and limits NO production through substrate depletion. Further, these cells are poor at presenting antigen to T cells, though they do have increased fibronectin and extracellular matrix synthesis. Thus, while alternatively activated macrophages display poor ability to kill intracellular pathogens, their functions are thought to lie in wound repair, limiting tissue damage resulting from inflammation, and resistance to parasites (533,534).
The third type of macrophage activation—type II activation—requires two signals, the first being engagement of the FcγR, and second, signaling through TLRs. The result is a cell that produces large amounts of IL-10 (535), which is thought to prevent the overexpression of inflammatory cytokines. Thus, in this context, the type II–activated macrophage functions as an anti-inflammatory cell, though it is clearly distinct from the alternatively activated macrophage in several ways, including the fact that arginase is not produced.
The antimicrobial pathways that are stimulated in classically activated macrophages have been studied in some detail. As mentioned above, when a pathogen is phagocytized by a macrophage, the nascent phagosome undergoes a maturation process in which the phagosomal environment is ultimately altered to decrease survival of the pathogen (536). Maturation involves sequential interaction of the phagosome with early endosomes, late endosomes, and lysosomes. It is not clear whether this interaction involves complete fusion of the phagosome with the endosomes/lysosomes to completely mix the contents of the two compartments, or whether only some of the endosomal/lysosomal contents are transferred to the phagosome through a “kiss-and-run” mechanism (537). Maturation also involves removal of phagosomal contents, which eventually appear in the endosomes of the macrophage (538,539). This membrane trafficking to and from the phagosome is mediated by the microtubule and actin cytoskeletons. Recent data also suggest that, at least in some cases, the contents of the nascent phagosome are transferred to an autophagic compartment called the autophagosome, which then interacts with lysosomes to become an autophagolysosome (Fig. 12.9). This process has been termed xenophagy, referring to a selective form of autophagy in which pathogen-containing phagosomes are selectively targeted, rather than the entirety of the cytosolic contents as is the case for autophagy (540). The general importance of xenophagy is not yet certain.
Among the key components that are transferred to the phagosome and are critical in creating an antimicrobial environment are the vacuole ATPase, NOS2, and phagocyte oxidase (phox). The vacuolar ATPase lowers the pH of the phagosome from a near-neutral pH to <5 (541,542). This negatively affects growth of the pathogen, and also enhances the activity of lysosomal hydrolases and the production of reactive oxygen intermediates. Additionally, lowering the phagosomal pH is thought to enhance endosomal/ lysosomal trafficking to the phagosome. Acidification, in fact, precedes and is necessary for lysosomal fusion (536,543).
Transfer of NOS2 to the phagosome is somewhat controversial, as it has been observed in some studies but not others (544,545). Nevertheless, whether NOS2 works directly in the phagosome or distally from another site within the cell, it is a major effector of induced innate immunity in the classically activated macrophage. As mentioned in more detail above, NOS2 is an enzyme that uses L-arginine as a nitrogenous donor to produce NO, which is, in itself, microbicidal. Production of NOS2 is stimulated at the transcriptional level by microbial products and cytokines, among them being IFN-γ (546). NO is required in the macrophage for its full killing capacity against many intracellular pathogens; likewise, in intact mice, absence of NOS2 leads to severely impaired resistance to many intracellular bacteria and protozoa (547). The role of human NOS2 in resistance to infection was initially more difficult to establish, but it is now generally accepted. Further, some polymorphisms in the human NOS2 have been correlated with altered resistance to infection. For instance, an NOS2 promoter polymorphism has been linked to increased NO production and increased resistance to malaria (512).
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Figure 12.9. Antimicrobial mechanisms in macrophages. Several pathways that lead to the elimination of internalized pathogens exist in macrophages. Phagosomal maturation: Nascent phagosomes that contain pathogens may interact with endosomal and lysosomal elements, leading to production of phagolysosomes. Multiple killing mechanisms are present in phagolysosomes. These include production of reactive oxygen intermediates (ROIs) by phagocyte oxidase, production of nitric oxide (NO) by inducible nitric oxide synthase (NOS2), a pH decrease due to vacuolar ATPase activity, and the presence of active lysosomal hydrolases. Alternatively, nascent phagosomes may enter an autophagic compartment, where they interact with lysosomes to become autophagolysosomes, in which pathogen killing also takes place. Nutrient deprivation: Nutrients required for pathogen growth (e.g., tryptophan or iron) are selectively removed or sequestered by the macrophage. Apoptosis: Macrophages undergo apoptosis, releasing pathogens for immune recognition and destruction. |
Phagocyte oxidase is a complex of at least five proteins, including membrane-bound gp91phox and p22phox, and cytosolic p40phox, p47phox, and p67phox. These proteins complex on membranes following an appropriate stimulation (548), and then catalyze the production of superoxide from molecular oxygen, using NADPH as a source of reducing potential. Subsequently, superoxide reacts spontaneously to produce reactive oxygen intermediates (ROIs), including hydrogen peroxide, hydroxyl radical, and hypochlorous acid. Phagocyte oxidase activity is activated by IFN-γ, which promotes transcription of certain phox components including gp47 and gp91 (385,549). ROIs have a profound impact on survival of pathogens in macrophages as well as neutrophils. Absence of phagocyte oxidase in mice (550,551) and humans leads to profoundly impaired resistance to intracellular pathogens, and leads to a condition in humans called chronic granulomatous disease.
Activated macrophages also possess several other antimicrobial mechanisms that do not directly involve phagosomal maturation. These include sequestration of essential nutrients required for pathogen growth and survival. For example, activated macrophages are able to limit free iron, by increasing production of transferrin that binds intracellular iron (512,552) and by decreasing transferrin receptors on their cell surface that normally mediate iron uptake (553). Similarly, activated macrophages reduce cellular tryptophan stores, in this case, through the actions of the enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan (548,554). In addition, metabolic products of tryptophan degradation, such as hydroxyanthranilic acid, are also toxic to pathogens.
A variety of factors and actions can reverse activation, or deactivate macrophages, though it is important to define activation when assessing these processes. The cytokines IL-10 and TGF-β reverse classical activation by decreasing MHC class II expression, NOS2 expression/NO production, and cyclooxygenase-2 (COX2) expression/prostaglandin production. IL-4 diminishes NOS2 expression and NO production, but it also increases arginase expression; so as mentioned above, IL-4–treated macrophages, though “deactivated” for some functions, are activated for others.
Antitumor Effects
Classically activated macrophages elicit antitumor effects that are potent and complex. These effects are immunologically nonspecific in that they act upon tumor cells irrespective of species or antigenic makeup, but they are selective in that tumor cells are affected while normal cells are relatively spared (555). Antitumor activity is both cytostatic and cytolytic, and occurs as a result of macrophage–target cell contact and/or through the secretion of cytokines by the macrophage. TNF-α is a major factor released by macrophages that may account for much of their antitumor activity (524,556), while release of reactive nitrogen intermediates is also important (557). Additional effector mechanisms of activated macrophages that have been linked to antitumor activity include reactive oxygen species (558), thymidine (559), arginase (560), and a cytolytic protease (561).
Although macrophages can elicit potent antitumor properties, it is paradoxic that in some cases, the presence of tumor-associated macrophages inversely correlates with a positive prognosis (562). The ultimate role of the macrophage in tumor immunoregulation is thought to be determined by whether the cell is classically activated to enhance antitumor properties or alternatively activated to promote tumor growth (563). Thus, the actual role of the tumor-associated macrophage is complex, and in many cases the balance may lean toward alternatively activated macrophages, in which case the macrophage would promote tumor progression, angiogenesis, and metastasis, largely through the secretion of chemokines that drive these processes (563,564,565).
Apoptosis
Apoptosis, or programmed cell death, is a natural, active process that can be envisioned as a way to rid the body of effete cells, excess cells, or cells that are potentially dangerous (566,567,568). Cells undergo apoptosis in response to many extracellular and intracellular signals. At least two biochemical pathways trigger the process: The cell death receptor pathway (initiated by a variety of ligands through TNF receptors and CD95) and the mitochondrial pathway (initiated by a variety of signals such as x-irradiation, UV irradiation, chemotherapy drugs, and p53/DNA damage). The biochemical events of these pathways have been defined in some detail, with both pathways ultimately leading to the activation of several cysteine-aspartate proteases (caspases) that cleave intracellular targets and initiate apoptosis. Additionally, caspase-independent pathways have been identified. Cells that undergo apoptosis are morphologically distinct, as the cells shrink, their cytoskeleton and nuclear envelope break down, and DNA is fragmented.
Apoptosis impinges on macrophage function in several respects. Macrophages themselves undergo apoptosis in certain situations, particularly after they have been infected with certain intracellular pathogens such as Mycobacteria, Salmonella, and Yersinia species (569,570,571). These pathogens actively stimulate apoptosis of macrophages through the actions of virulence factors that the pathogens introduce into the macrophage. This is thought to be beneficial to the pathogens and detrimental to the host in that it undermines the ability of the macrophages to effect pathogen killing, while causing release and consequently dissemination of the pathogens.
Macrophages also stimulate apoptosis of other cells. As mentioned above, classically activated macrophages are able to trigger apoptosis of tumor cells through production of TNF-α and reactive nitrogen intermediates, which may be important for immunosurveillance of cancerous cells. Additionally, macrophages are able to trigger apoptosis of specific cells during embryonic (572,573) and lymphocyte development (574) when highly coordinated patterns of cell destruction and elimination are required.
Macrophages are also critically important for the removal of apoptotic cells from tissues (575,576), a process that functions to reduce the release of toxic cell debris in local environment, and to diminish any proinflammatory effects of the debris. Additionally, when macrophages phagocytize apoptotic cells, they reduce inflammation by increasing production of the anti-inflammatory substances IL-10, TGF-β, and prostaglandin E2 and decreasing production of the proinflammatory substances TNF, IL-1, IL-8, GM-CSF, NO, leukotriene C4, and thromboxane B2(575,576). In contrast, phagocytosis of necrotic (as opposed to apoptotic) cells by macrophages generally results in production of proinflammatory mediators and a decrease in anti-inflammatory mediators. Recognition of apoptotic cells occurs primarily through phosphatidyl serine (PS) that is exposed on the outer leaflet of apoptotic cells, as well as through other surface molecules such as sugars on proteins and lipids, oxidized LDL-like proteins, ICAM-3, and a C1q-binding site (575,576). The macrophage receptors that recognize these substances include a macrophage PS receptor, lectins, the C1q-receptor, CR3, CR4, the vitronectin receptor, CD14, and scavenger receptors (SRA, CD36, CD68, and LOX-1) (575,576).
Erythrocyte Destruction and Iron Metabolism
A major function of the mononuclear phagocyte system is to acquire and maintain a storage pool of iron capable of meeting the needs of erythropoiesis (577). The predominant means through which macrophages acquire iron is by phagocytosis of damaged or senescent erythrocytes, particularly by macrophages in the liver, spleen, and bone marrow (147,578). The mechanism by which these erythrocytes are recognized is poorly understood, with various studies suggesting that the recognition signal may be peptidic, glucidic, and/or lipidic in nature (579,580). Once erythrocytes are internalized into macrophage phagosomes, their membrane is lysed, hemoglobin is oxidized to methemoglobin, heme and globin dissociate, and globin is degraded to amino acids. The heme is subsequently degraded by the heme oxygenase complex to carbon monoxide, biliverdin, and iron (577). Carbon monoxide (acquired exogenously or generated endogenously from heme oxygenase) modulates many different cellular functions (581); these include cell proliferation, vasorelaxation, neural function, and apoptosis. Likewise, biliverdin is bioactive, possessing free radical scavenging and anti-inflammatory activity (581). In addition to acquisition of iron from phagocytized erythrocytes, iron is also acquired by macrophages in the form of free hemoglobin, as it is estimated that 10 to 20% of erythrocytes are degraded intravascularly before they can be cleared through erythrophagocytosis (582). Free hemoglobin is taken in through the CD163 scavenger receptor on the macrophage surface (583). Finally, a third mechanism through which macrophages may take up iron is by internalization of transferrin-bound iron via transferrin receptors that are expressed in large numbers by macrophages (577). However, the extent to which this pathway contributes to iron stores in human macrophages in vivo is uncertain (584).
Iron that is derived from erythrophagocytosis is stored in ferritin within the mononuclear phagocyte (585,586). In contrast, iron derived from hemoglobin uptake may be stored in a separate pool that has not been clearly defined (587). When needed, ferritin iron is made available to transferrin and released from the macrophage for subsequent use in heme protein synthesis. Several macrophage proteins are important for iron release: Ceruloplasmin oxidizes iron for transfer to transferrin (588,589); ferroportin (also known as IRGE1 and MTP1) is an iron exporter protein (590,591,592); and Nramp1 is a transmembrane protein that mediates iron release from the phagosome (593). Additionally, the hepatically derived serum protein hepcidin is an important mediator of iron efflux from the macrophage, as it binds ferroportin and triggers its internalization and degradation, thus inhibiting iron export (594). It is notable that ferroportin (595), Nramp1 (596,597,598), and hepcidin have also been implicated in resistance to pathogens, indicating that iron homeostasis in the macrophage plays an important role in innate immunity, as well as in hemoglobin biosynthesis. For instance, hepcidin concentrations increase during inflammation. The increased hepcidin decreases iron efflux from macrophages, thus inhibiting growth of invading pathogens because of decreased iron availability for pathogenic growth. This additionally contributes to the development of the anemia of inflammation (599). Thus, the antimicrobial and antitumor effects of macrophages can be inhibited by iron overload (600,601).
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