Mature Mø 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.

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). Mø 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.

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
zone Mø 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.

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.

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 and tolerance.⁴¹ 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 Perivascular Mø 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.