Growth and Differentiation: Life History and Turnover
In contrast to T- and B-lymphocytes, monocytes from blood give rise to terminally differentiated Mø
that cannot recirculate or reinitiate DNA replication except in a limited way. Unlike other myeloid granulocytic cells, Mø
can be long lived and retain the ability to synthesize RNA and protein to a marked extent, even when in a relatively quiescent state as “resident” cells. These are distributed throughout the tissues of the body and constitute a possible alarm-response system, but they also mediate homeostatic and poorly understood trophic functions. Following inflammatory and immune stimuli, many more monocytes can be recruited to local sites and give rise to “elicited” or “immunologically activated” Mø
with altered surface, secretory, and cytotoxic properties. The origins of Mø
from precursors are well known: from yolk sac (and possibly earlier paraaortic progenitors), migrating to fetal liver, then spleen and bone marrow, before and after birth.16
Yolk sac precursor cells may contribute to the establishment of selected tissue macrophages such as Langerhans cells in the adult.17
In the fetus, mature Mø
proliferate actively during tissue remodeling in developing organs. In the normal adult, tissue Mø
do not self-renew extensively except in specialized microenvironments such as epidermis,18
nervous system, or lung; after TH2-type parasitic infection, there can be considerable further replication at local sites of inflammation.19
Growth and differentiation are tightly regulated by specific growth factors and their receptors (eg, IL-3, CSF-1, granulocyte-macrophage [GM]-CSF/IL-34, IL-4, IL-13) and inhibitors (eg, IFN&agr;
, transforming growth factor [TGF]-&bgr;
, leukemia inhibitory factor), which vary considerably in their potency and selectivity. These processes are modulated by interactions with adjacent stromal and other cells (eg, through c-kit/ligand and Flt-3/ligand interactions). The growth-response of the target cell to an extrinsic stimulus decreases progressively and markedly (from 108
or more to 100
) during differentiation from stem cell to committed precursor to monoblast, monocyte, and Mø
, yet even the most terminally differentiated Mø
such as microglial cells can be “reactivated” to a limited extent by local stimuli. Elicited/activated Mø
respond more vigorously than resident Mø
to growth stimuli in vivo and in vitro, but the molecular basis for their enhanced proliferation is unknown.
FIG. 19.1. Differentiation of mononuclear phagocytes based on antigen markers FA-11 (macrosialin, murine CD68) and F4/80. See text for detailed discussion.
Although this general picture of blood monocyte-totissue Mø
differentiation has been accepted for some time as a result of parabiosis, adoptive transfer, and irradiation-reconstitution experiments, recent studies in mouse and man have demonstrated monocyte heterogeneity and distinct properties,20,21,22,23
with a subpopulation remaining within the vasculature, to perform a patrolling function. Our understanding of DCs and osteoclast differentiation is still compatible with a relatively simple model (Fig. 19.1)
in which major Mø
populations in mouse tissues can be characterized by selected antigen markers such as F4/80 (Emrl,
a member of a family of EGF-TM7 molecules) and macrosialin (CD68), a pan-Mø
endosomal glycoprotein related to the lysosome-associated membrane protein (LAMP) family. The DCs of myeloid origin (see elsewhere in this volume) share many properties with monocyte/macrophages,24
but are specialized to capture, process, and present antigens to naïve lymphocytes. Circulating precursors of DCs and and macrophages are normally present in the mononuclear fraction of blood in small numbers25
; studies in the mouse may not reflect the origin and differentiation of precursor cells in humans.26
Monocytes that have crossed the endothelium may be induced to “reverse migrate” into the circulation by selected stimuli in tissues.27
Finally, the mouse spleen has been shown to serve as a reservoir of monocyte/Mø
for recruitment to sites of inflammation.28
Circulating mononuclear precursors for osteoclasts are less defined and differentiate into mononucleate cells, recruited in response to sphingosine-1-phosphate to bone, for example,29
where they fuse to form multinucleate bone-resorbing osteoclasts.30
Local stromal cells, growth factors such as CSF-1, steroids (vitamin D metabolites), and hormones (eg, calcitonin, for which osteoclasts express receptors) all contribute to local maturation. Osteoprotegerin, a naturally occurring secreted protein with homology to members of the TNF-receptor family, interacts with TRANCE, a TNF-related protein, to regulate osteoclast differentiation and activation in vitro and in vivo.
Use of antigen markers such as CD34 on progenitors, CD14 and CD16 on monocytes, and chemokine receptors and multichannel fluorescein-activated cell sorter analysis have made it possible to isolate leukocyte subpopulations and study their progeny and differential responses in different mouse tissues and models of disease.31
The mononuclear fraction of blood may contain precursors of other tissue cells, including mesenchymal stem cells able to synthesize matrix proteins such as collagen, and some endothelial cells. Perhaps the mysterious follicular dendritic cells (FDCs) with mixed hemopoietic and mesenchymal properties fall in this category.
The large-scale production of immature and mature DClike cells from bulk monocytes in cytokine-supplemented culture systems (IL-4, GM-CSF, TNF&agr;) has revolutionized the study of these specialized APCs. Individually, the same cytokines give rise to Mø-like cells, and early during in vitro differentiation, the cellular phenotype is reversible. Later, when mature DCs with high MHC II, APC function, and other characteristic markers are formed, differentiation is irreversible. This process is independent of cell division, although earlier progenitors in bone marrow and GM-CSF-mobilized blood mononuclear cells can be stimulated to multiply, as well as differentiate, in vitro. These examples of terminal differentiation observed with DCs and osteoclasts may extend to other specialized, more obvious Mø-like cells. Mature Mø can be derived by growth and differentiation in steroid-supplemented media in Dexter-type long-term bone marrow cultures that contain stromal fibroblasts and hemopoietic elements. These Mø express adhesion molecules responsible for divalent cation-dependent cluster formation with erythroblasts (EbR). This receptor, possibly related to V-CAM, cannot be induced on terminally differentiated peritoneal Mø if these are placed in the same culture system. This contrasts sharply with the ready adaptation of many tissue Mø to conventional cell culture conditions, when the cells often adopt a common, standard phenotype. Irreversible stages of Mø differentiation may therefore occur in specialized microenvironments in vitro or in vivo.
Little is known about determinants of Mø longevity and turnover. Growth factors such as CSF-1 enhance Mø survival and prevent induction of an apoptotic program. The expression of Fas-L and Fas on Mø has been less studied than on lymphocytes; they and other members of the TNF and its receptor family may play a major role in determining Mø survival, especially in induced populations, where cell turnover is markedly enhanced. Tissue Mø vary greatly in their lifespan, from days to months. Apart from inflammatory and microbial stimuli, local and systemic environmental factors such as salt loading and hormones, including estrogen, are known to influence Mø turnover.
Tissue Distribution and Phenotypic Heterogeneity of Resident Macrophages in Lymphoid and Nonlymphoid Organs
The use of the F4/80 plasma membrane antigen made it possible to detect mature Mø
in developing and adult murine tissues and define their anatomic relationship to other cells in endothelium, epithelium, and connective tissue, as well as the nervous system.32,33
Subsequently, other membrane antigens,34
macrosialin, sialoadhesin, and others were identified as useful markers for Mø
in situ (Table 19.1)
subpopulations in different tissues display considerable heterogeneity in expressing these and selected receptor antigens (eg, complement receptor [CR]3 and class A scavenger receptor [SR-A]), drawing attention to unknown mechanisms of homing, emigration, and local adaptation to particular microenvironments. From the viewpoint of immune responses, a few aspects deserve comment.
Fetal Liver and Bone Marrow
form an integral part of the hemopoietic microenvironment and play a key role in the production, differentiation, and destruction of all hemopoietic cells. The fetal liver is a major site of definitive erythropoiesis from midgestation.16
The bone marrow becomes active in the production of hemopoietic cells from shortly before birth, and Mø
are a prominent component of the hemopoietic stroma throughout adult life. Mature “stromal” Mø
in fetal liver and adult bone marrow express nonphagocytic adhesion molecules such as sialoadhesin (Sn), an immunoglobulin (Ig)-superfamily sialic acid-binding lectin (Table 19.1)
, and the EbR referred to previously, which is also involved in adhesion of developing myeloid and possibly lymphoid cells (Fig. 19.2)
. VLA-4 has been implicated as a ligand for EbR. Ligands for Sn include CD43 on developing granulocytes and on lymphocyte subpopulations. Sn clusters at sites of contact between stromal Mø
and myeloid but not erythroid cells. Chemokines are able to induce polarized expression of adhesion molecules such as intercellular adhesion molecules and CD43 in leukocytes, but the significance
of altered ligand distribution for interactions between Mø
and bound hemopoietic cells is unknown. Adhesion of immature cells to stromal Mø
may play a role in regulating their intermediate stages of development before release into the bloodstream, whereas fibroblasts in the stroma associate with earlier progenitors, as well as with Mø
. Discarded nuclei of mammalian erythroid cells are rapidly engulfed by stromal Mø
, but the receptors involved in their binding and phagocytosis are unknown. Mø
also phagocytose apoptotic hemopoietic cells generated in bone marrow, including large numbers of myeloid and B cells. We know little about the plasma membrane molecules and cytokine signals operating within this complex milieu, but it is clear that stromal Mø
constitute a neglected constituent within the hemopoietic microenvironment.
TABLE 19.1 Selected Differentiation Antigens Used to Study Murine Macrophage Heterogeneity
Mature Mø, absent T areas
Useful marker development, CNS
Glycoforms regulated by inflammation and phagocytosis
CR3 (CD11b, CD18)
Monocytes, microglia, PMN, NK cells
Important in inflammatory recruitment, PMN apoptosis
SR-A (I, II)
Collagenous, type II glycoprotein
Polyanions, LTA, LPS, bacterial proteins
Protects host against LPS-induced shock
Isoforms differ, cysteine-rich domain
Modified proteins &bgr;-amyloid apolipoprotein A, E
Mø, sinusoidal endothelium
Phagocytosis of apoptotic cells and bacteria
Sialyl glycoconjugates (eg, CD43)
Subsets tissue Mø
Marginal zone metallophils in spleen and subcapsular sinus of lymph nodes
CNS, central nervous system; DC, dendritic cell; ICAM, intercellular adhesion molecule; Ig, immunoglobulin; LAMP, lysosome-associated membrane protein; LPS, lipopolysaccharide; LTA, lipoteichoic acid; Mø, macrophages; NK, natural killer; OX-LDL, oxidised low density lipoprotein; PMN, polymorphonuclear neutrophil; Sn, sialoadhesin; SR-A, type A scavenger receptor.
FIG. 19.2. Associations of tissue macrophages with other hemopoietic cells to illustrate variations on a common theme. See text for details.
Apart from their remarkable capacity to remove apoptotic thymocytes, the possible role of Mø
in positive and negative selection of thymocytes has been almost totally overlooked; more attention has been given to local DCs and their specialized properties. Mature Mø
with unusual features are also present in cortex and medulla. Clusters of viable thymocytes and Mø
can be isolated from the thymus of young animals by collagenase digestion and adherence to a substratum (see Fig. 19.2
). The nonphagocytic adhesion receptors responsible for cluster formation are more highly expressed by thymic than other Mø
, but their nature is unknown (N. Platt, unpublished observations). These Mø
also express MHC class II antigens and other receptors such as the SR-A (see subsequent discussion), which contributes to phagocytosis of apoptotic thymocytes in vitro, but is redundant in vivo; other markers, such as the F4/80 antigen, are poorly expressed in situ but can be readily detected after cell isolation. A striking difference between thymic and several other tissue Mø
subpopulations is their independence of CSF-1; the CSF-1-deficient op/op
mouse lacks osteoclasts and some Mø
populations, including monocytes, peritoneal cells, and Kupffer cells, but contains normal numbers of thymic Mø
, as well as DCs and selected Mø
in other sites. A second ligand for the CSF-1 receptor Fms, IL-34, may account for CSF-1 independence.35
Factors involved in constitutive recruitment of thymic Mø
are unknown; following death of thymocytes induced by ionizing radiation or glucocorticoids, intensely phagocytic Mø
appear in large numbers; it is not known what proportion arises locally and by recruitment.
From the viewpoint of the Mø
, the spleen is perhaps the most complex organ in the body.36,37
It contributes to hemopoiesis, which persists postnatally in some species or can be induced by increased demand, can serve as a reservoir as noted previously, and contributes to the turnover of all blood elements at the end of their natural lifespan. The spleen filters a substantial proportion of total cardiac output, captures particulate and other antigenic materials from the bloodstream, and plays an important role in natural and acquired humoral and cellular immunity. The organ is rich in subpopulations of Mø
that differ in microanatomic localization, phenotype, life history, and functions (Fig. 19.3)
are central to antigen capture, degradation, transport, and presentation to T- and B-lymphocytes, and contribute substantially to antimicrobial resistance. Recent work has unveiled an unexpected role in facilitating activation of other lymphocyte subsets, such as invariant natural killer T cells37
; CD 169+ macrophages also activate CD8 T cells in response to dead cell-associated antigens in lymph nodes and by transferring antigen to DCs in the spleen. Because other hemopoietic and secondary lymphoid organs can replace many of these functions after maturation of the immune system, the unique properties of the spleen have been mainly recognized in the immature host and in immune responses to complex polysaccharides. Splenectomy in the adult renders the host susceptible to infection by pathogenic bacteria such as pneumococci that contain saccharide-rich capsular antigens; the marginal zone of the spleen in particular may play an essential role in this aspect of host resistance.
The properties of Mø in the unstimulated mature mouse spleen are different according to their localization in red or white pulp and the marginal zone. Mø are intimately associated with the specialized vasculature. Species differences in splenic anatomy and phenotype are well recognized, although Mø display broadly common features in humans and rodents. Subpopulations of Mø, DCs, and cells with mixed phenotypes have been characterized by in situ analysis by antigen markers, liposome or diphtheria toxin-depletion studies, various immunization and infection protocols, and cytokine and receptor gene ko models in the mouse. The results raise questions about the dynamics and molecular basis of cell production, recruitment, differentiation, emigration, and death within each distinct splenic compartment. Cell isolation methods are still primitive in correlating in vitro properties with those of Mø subpopulations in vivo and remain an important challenge. Detailed aspects of splenic architecture, DC origin and function, and T- and B-lymphocyte induction and differentiation are described elsewhere in this volume. Here, some features of Mø in the normal and immunoreactive organ are highlighted.
Marginal Zone Macrophages
The marginal zone of spleen consists of a complex mixture of resident cells (reticular and other fibroblasts, endothelium), Mø
, DCs, and lymphoid cells, including subpopulations of B-lymphocytes. It constitutes an important interface with the circulation that delivers cells, particulates, or soluble molecules directly into the marginal sinus or via the red pulp. Resident Mø
are present as specialized metallophilic cells in the inner marginal zone, and other Mø
are found in the outer zone; the latter may be more phagocytic. Sn is strongly expressed by the marginal metallophils, compared with only weak expression in red pulp and virtual absence in the white pulp. Sn+ cells appear in this zone 2 to 4 weeks postnatally in the mouse as the white pulp forms. Liposomes containing clodronate, a cytotoxic drug, can be delivered systemically and deplete Sn+ cells and other Mø
; regeneration of different Mø
subpopulations in spleen occurs at different times, and this procedure has been used to correlate their reappearance with distinct immunologic functions. Marginal
lack F4/80 but may express an undefined ligand for F4/80 on circulating activated DCs, which mediates peripheral tolerance to anterior chamber or gut-derived antigens.38
Marginal zone Mø
express phagocytic receptors, such as SR-A, which is more widely present on tissue Mø
, as well as MARCO, a distinct collagenous scavenger receptor, which is almost exclusively present on these Mø
in the normal mouse. The structures and possible role of these pattern recognition receptors in uptake of microbes are discussed subsequently. In vivo studies have shown that an Mø
lectin, the MR, may be involved in transfer of mannosylated ligands to the site of an immune response in the white pulp.39
The MR contains a highly conserved cysteine-rich domain, not involved in mannosyl recognition, that reacts strongly with ligands on a subset of marginal metallophilic Mø
, sulfated glycoforms of Sn, and CD45, among others; this has been demonstrated with a chimeric probe of the cysteine-rich domain of the MR and human Fc (CR-Fc) and by immunochemical analysis of tissue sections and affinity chromatography of spleen ligands. After immunization, this probe additionally labels undefined cells in the FDC network of germinal centers, as well as tingible body Mø
. It is possible that marginal zone Mø
can be induced to migrate into white pulp as described after LPS injection; alternatively, they may shed complexes of soluble MR-glycoprotein ligand for transfer to other CR-Fc+ cells, which may be resident or newly recruited mononuclear cells. Finally, the marginal metallophilic Mø
population depends on CSF-1 for its appearance and on members of the TNF receptor family, as shown with op/op
and experimentally produced ko mice.
FIG. 19.3. Microheterogeneity of macrophages in spleen, resting, and antigen-stimulated lymph nodes. See text for markers and details.
White Pulp Macrophages
The F4/80 antigen is strikingly absent on murine white pulp Mø, which do express FA-11 (macrosialin), the murine homolog of CD68. Actively phagocytic Mø express this intracellular glycoprotein in abundance compared with DCs. After uptake of a foreign particle (eg, sheep erythrocytes or an infectious agent, such as BCG or Plasmodium yoellii), white pulp Mø become more prominent, although it is not known whether there is migration of cells into the white pulp or transfer of phagocytosed material and reactivation of previous resident Mø. Tingible body Mø appear to be involved in uptake and digestion of apoptotic B-lymphocytes.
Red Pulp Macrophages
These express F4/80 antigen and MR strongly and in the mouse include stromal-type Mø
involved in hemopoiesis. Extensive phagocytosis of senescent erythrocytes results in accumulation of bile pigments and ferritin, and play an important role in iron turnover40
The role of various phagocytic receptors in clearance of host cells and pathogens by red pulp Mø
requires further study.
There is no evidence that Mø, other than interdigitating DCs, associate directly with CD4+ T-lymphocytes in the normal spleen. Following infection by BCG, for example, or by other microorganisms such as Salmonella, there is massive recruitment and local production of Mø, many of which associate with T-lymphocytes. Newly formed granulomata often appear first in the marginal zone (focal accumulations of activated Mø and activated T cells). As infections spread into the white and red pulp, the granulomata become confluent and less localized, obscuring and/or disrupting the underlying architecture of the spleen. The possible role of activated Mø in T-cell apoptosis and clearance in spleen has not been defined.
F4/80 antigen is relatively poorly expressed in lymph node (see Fig. 19.3
), but many macrosialin (CD68)+ cells are present. The subcapsular sinus is analogous to the marginal zone and contains strongly Sn+ cells; this is the site where afferent lymph enters, containing antigen and migrating DCs derived from skin and mucosal surfaces. The medulla contains Sn+, CD68+ Mø
, which also express high levels of SR-A. As in the spleen marginal zone, subcapsular sinus Mø
are strongly labeled by the CR-Fc probe. Following primary or secondary immunization, the staining pattern moves deeper into the cortex and eventually becomes concentrated in germinal centers. The kinetics of this process strongly suggests a transport process by Mø
-related cells resembling antigen transport cells described previously. CR-Fc+ cells can be isolated by digestion of lymph nodes and form clusters with CR-Fc- lymphocytes. Adoptive transfer has shown that fluorescein-activated cell sorter-isolated CR-Fc+ cells resemble DCs in their ability to home to T-cell areas and to present antigen to naive T and B cells. Overall, there is considerable heterogeneity in the population of migratory APCs involved in antigen capture, transport, and delivery to T and B cells, and it may turn out that specialized tissue Mø
as well as myeloid-type DCs can migrate in response to immunologic stimuli, especially TLR ligands.42
Although less studied, the Mø
in Peyer patch resemble the CD68+, F4/80- cells described in spleen and white pulp and in other T-cell-rich areas. They are well placed to interact with gut-derived antigens and pathogens taken up via specialized epithelial M cells in the dome, and deliver antigens to afferent lymphatics, as myeloid DCs. These cells are distinct from abundant F4/80+ cells in the lamina propria found all the way down the gastrointestinal tract and may play a role in the induction of mucosal immunity. Recent studies have described heterogeneous populations of resident and recruited macrophages and DC in the mouse intestine.43
The role of the microbiome44
has received a great deal of attention in regard to innate cell phenotype and epithelial integrity in the gut.
Regional F4/80+ and CD68+ Mø
are well described in liver (Kupffer cells), dermis, neuroendocrine and reproductive organs, and serosal cavities, where they are able to react to systemic and local stimuli. In the lung, alveolar Mø
are strongly CD68+ but only weakly F4/80+ and are distinct from interstitial Mø
and intraepithelial DCs. In the lamina propria of the intestine, Mø
display a downregulated phenotype, ascribed to TGF&bgr;
of local origin.45
In addition, resident Mø
are found throughout connective tissue and within the interstitium of organs, including heart, kidney, and pancreas. These cells vary greatly depending on their local microenvironment; for example, in the central nervous system, microglia within the neuropil differ strikingly from Mø
in the meninges or choroid plexus.46
in the brain can be distinguished from resident microglia by their expression of endocytic receptors (eg, the SR-A and MR, and of MHC I and II antigens). Microglia are highly ramified, terminally differentiated cells of monocytic origin; many Mø
markers are downregulated. Their phenotype is influenced by the blood-brain barrier, normally absent in circumventricular organs, and disrupted by inflammatory stimuli. Microglia can be reactivated by local LPS and neurocytotoxins; they are then difficult to distinguish from newly recruited monocytes, which acquire microglial features once they enter the parenchyma of the brain. Resting microglia are unusual among many tissue Mø
in that they constitutively express high levels of CR3 and respond to CR3 ligands, such as mAb, by induced DNA synthesis and apoptosis. In other sites, such as lung and liver, CR3 expression is a feature of recent myeloid recruitment, including monocytes. Resident Kupffer cells lack constitutive CR3 but express a novel CR implicated in clearance function.
Resident tissue macrophages in human tissues express CD68 antigen, but their phenotypic diversity and microheterogeneity in different organs remain poorly defined. Access to skin biopsies, bronchoalveolar lavage, and placenta, for example, provides material for further analysis.
Enhanced Recruitment of Monocytes by Inflammatory and Immune Stimuli: Activation in Vivo
In response to local tissue and vascular changes, partly induced by resident Mø
during (re)activation by inflammatory, infectious, and immunologic stimuli, monocytes are recruited from marrow pools and blood in increased numbers; they diapedese and differentiate into Mø
with altered effector functions as they enter the tissues.47
are classified as “elicited” when cells are generated in the absence of IFN&ggr;
and as “immunologically activated” after exposure to IFN&ggr;
. Enhanced recruitment can also involve that of other myeloid or lymphoid cells; selectivity of the cellular response depends on the nature of the evoking stimulus (immunogenic or not), the chemokines produced,
and the receptors expressed by different leukocytes. Mø
and other cells produce a range of different chemokines and express multiple seven-transmembrane, G protein-coupled chemokine receptors. The chemokines can also act in the marrow compartment, especially if anchored to matrix and glycosaminoglycans, may display other growth regulatory functions, and can control egress. Locally bound or soluble chemokines induce the surface expression and activity of adhesion molecules on circulating white cells, as well as directing their migration through and beyond endothelium. Feedback mechanisms from periphery to central stores and within the marrow stroma may depend on cytokines and growth factors such as macrophage inflammatory protein-1&agr;
and GM-CSF, which inhibit or enhance monocyte production, respectively. The adhesion molecules involved in recruitment of monocytes, originally defined by studies in humans with inborn errors and by use of inhibitory antibodies in experimental animal models,48
overlap with those of polymorphonuclear neutrophils and lymphocytes and include L-selectin, &bgr;
2-integrins, especially CR3, CD31, an Ig-superfamily molecule, and CD99; additional monocyte adhesion molecules for activated endothelium include CD44, vascular cell adhesion molecules, &bgr;1
-integrins, and newly described receptors such as EMR2 and CD97, members of the EGF-TM7 family.33
The mechanisms of constitutive entry of monocytes into developing and adult tissues, in the absence of an inflammatory stimulus, are unknown.
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