Inflammatory stimuli (e.g. local infection) enhance the recruitment of monocytes (and often other myeloid cells) from blood, and ultimately from bone marrow stores.
Monocytes adhere to activated endothelium through a series of well-described interactions involving selectins, β2integrins, and CD31, as well as chemotactic molecules
acting on their transmembrane G protein-coupled recep- tors (Fig. 9.8, see also Chapter 6). When ligands bind to these receptors it causes the recruitment of intracellular proteins to the area of membrane activation of integrins and polarization of the cells (Fig. 9.9).
Diapedesis results in local tissue interactions and accu- mulation of macrophages with enhanced turnover and an altered phenotype (e.g. the upregulation of endocytic receptors and production of proinflammatory mediators). Phagocytosis by recently recruited monocytes pro- foundly alters their differentiation into:
• migratory dendritic cells; or • more sessile macrophages.
Recent studies have confirmed the potential of monocytes to differentiate into either of these cell types in vivo, as they do in cell culture.
It is convenient to distinguish the macrophages ‘elicited’ by a non-specific inflammatory stimulus from ‘immunologically activated’ macrophages. The latter cells: • respond to IFNγ by acquiring enhanced antimicrobial
properties; and
Differentiation and distribution of macrophages
Fig. 9.5 Blood monocytes are derived from bone marrow in the adult host and enter tissues initially as sinus-lining or extravascular mature cells. Interstitial or intraepithelial adherent macrophages and serosal macrophages can enter afferent lymphatics. Monocyte-derived dendritic cells can arise from tissue macrophages by reverse migration across endothelium. Stromal macrophages in bone marrow may also derive from the circulating monocyte population. stromal macrophage blood monocyte perivascular macrophage epithelium endo- thelium tissue macrophage pluripotent stem cell pro-monocyte interstitial fixed macrophages
sinus-lining macrophages free macrophages bone
marrow blood tissue
afferent
lymph epithelium
serous cavity
• express enhanced MHC class II molecules, whereas other genes (e.g. the mannose receptor) are downregulated.
Q. What will be the functional effect of the changes in cell surface molecules described above?
A. The increase in MHC class II molecules will enhance the
ability to present antigen to CD4+T cells, while the reduction in
the MR may mean a reduced ability to take up some microbes by non-immune mechanisms.
Cytokines produced by NK cells, lymphocytes, and various antigen-presenting cells (APCs) influence the pattern of gene expression by macrophages. Work in
vitro, and some studies in vivo, especially with cytokine/ receptor knockout mouse strains, have made it possible to classify macrophages differing in their functional state (Fig. 9.10).
The initial and further interactions with endocytic and phagocytic stimuli give rise to further heterogeneity in phenotype. It should be emphasized that, although the evidence for individual molecules as mediators of acti- vation is good, we have little insight into the complex interactions that pertain in vivo.
Fig. 9.11 illustrates a mycobacterium-induced granu- loma, rich in macrophages. Cytokines, for example TNFα, and receptors such as CR3 play an important role in granuloma formation.
RESIDENT AND RECRUITED MACROPHAGES RESPOND TO INJURY AND IMMUNE STIMULI
Macrophages in secondary lymphoid tissues
Fig. 9.6 Heterogeneity of macrophages in secondary lymphoid organs of mouse. (1) Red pulp of spleen stained for F4/80. Macrophages stain strongly positive. (Courtesy of Dr DA Hume) (2) Mouse spleen stained with antibody to sialoadhesin. The marginal metallophils of spleen are strongly sialoadhesin positive. (Courtesy of Dr PR Crocker) (3) The marginal zone contains inner metallophilic and outer zones. In the diagram of the marginal zone of the spleen, the central arteriole (CA) branches into small capillaries, which either pass through the marginal zone and end in the red pulp (RP) or open into the marginal sinus (MS). The marginal zone is composed of
reticular cells (RCs). Within this reticular framework, the large marginal zone macrophages (MZMs), dendritic cells, and marginal zone B cells (MZBs) are localized. The marginal metallophilic macrophages (MMM) are situated at the inner border of the marginal sinus and the white pulp (WP) (Courtesy of Dr G Kraal) (4) Lymph node contains subcapsular sinus macrophages, which are sialoadhesin positive and bind CR-Fc, a chimeric probe of the mannose receptor cysteine-rich domain, and human Fc (left); medullary macrophages express only sialoadhesin (right). (Courtesy of Dr L Martinez-Pomares)
1 2 3 4 RC MZM MS CA WP MMM RP MZB Ly CR-Fc α-Sn
Environmental influences on macrophages
Fig. 9.7 Some of the mediators that act on macrophages.
Macrophage 7tm G protein-coupled receptors for chemotactic stimuli
Fig. 9.8 Macrophages express a variety of receptors for chemotactic agents. Fractalkine (CX3CL1) is unique as a transmembrane ligand – its receptor, CX3CR1, and CCR2 may be differentially expressed by monocytes, giving rise to inflammatory and resident macrophages.
stimulus example/receptors response
M-CSF, GM-CSF IFNγ TNFα IL-4, IL-13 IL-10 CCL2 (macrophage chemotactic protein-1, MCP-1) fibronectin / β1-integrin
vasoactive peptide (VIP) prostaglandin E2 (PGE2) leukotriene B4 (LTB4) fibronectin / β1- integrin accessory molecules (CD80, CD86) LPS (CD14) growth factors cytokines chemokines extracellular matrix peptides eicosanoids cellular interactions: endothelium T cells microbial interactions
proteases neutral proteinases (e.g. plasmin)
growth, differentiation, survival activation activation alternate activation deactivation migration adhesion, phagocytosis modulation of various functions modulation of various functions recruitment antigen presentation secretion, activation adhesion, secretion
M-CSF, macrophage colony stimulating factor; GM-CSF, granulocyte—macrophage colony stimulating factor; IFNγ, interferon-γ; IL, interleukin; LPS, lipopolysaccharide; TNFα, tumor necrosis factor-α
receptors ligands sources
fMLP fMet-Leu peptides prokaryote protein synthesis
C5a receptor complement C5a lytic activation
LTB4 receptor leukotriene B4 mast cells, macrophages
PAF receptor platelet-activating factor platelets, neutrophils, macrophages
CCR1, CCR2, CCR5 chemokines CCL2, 3, 4, 5, 7, 8
leukocytes, tissue cells
CXCR1, CXCR2 CXCL8 (IL-8) endothelium, lymphocytes
CXCR3 CXCL9, 10, 11 endothelium, tissue cells
CX3CR1 CX3CL1 (fractalkine) endothelium, epithelium, neurons,
smooth muscle cells
7tm, seven-transmembrane segments; CCL, CC chemokine ligand; CXCL, CXC chemokine ligand; CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; fMLP, f.Met-Leu-Phe; PAF, platelet-activating factor
Immunologically activated macrophages express the capacity to produce antimicrobial products including lysozyme and reactive oxygen and nitrogen species (see below). However, characteristic morphologic features such as epithelioid cell and giant cell formation, hallmarks of mycobacterial granulomas, remain mysterious in origin.
Cytokines modulate the phenotype of macrophages
A further level of heterogeneity derives from the distinct effects of TH1- and TH2-type cytokines on monocyte/
macrophage differentiation (Fig. 9.12).
In-vitro studies with macrophages treated with differ- ent cytokines have indicated that there is a spectrum of gene expression induced by different cytokines (Fig. 9.13): • IFNγ characteristically enhances proinflammatory and
antimicrobial activities (‘immune activation’);
• IL-10 efficiently counteracts proinflammatory and antimicrobial activities (‘deactivation’);
• IL-4 and IL-13 exert distinctive effects on MHC class II and MR expression, which we have termed ‘alter- native activation’, rather than focusing on the modest inhibition of proinflammatory products.
Broadly speaking:
• ‘activated’ macrophages mediate cellular immunity; • ‘alternatively activated’ macrophages may promote
humoral immunity, including repair processes.
It is well established that macrophages and myeloid dendritic cells produce IL-12 and IL-18, which enhance the production of IFNγ by NK cells and T cells. There is, as yet, no comparable IL-4/IL-13-inducing activity that can be ascribed to the APCs, though subsets of dendritic cells have been implicated in TH2 differentiation, which
has also been postulated to constitute a ‘default’ pathway.
RESIDENT AND RECRUITED MACROPHAGES RESPOND TO INJURY AND IMMUNE STIMULI
Intracellular signaling via chemotactic agents such as f-met-leu-phe (fMLP)
Fig. 9.9 fMLP binds to its receptor and causes guanosine triphosphate (GTP) to displace guanosine diphosphate (GDP) on the heterotrimeric G protein. This causes theβ and γ chains to dissociate from the α chain and they in turn recruit phosphatidyl inositol (PI)-3 kinase to the membrane. The kinase phosphorylates phosphatidyl inositol diphosphate (PIP2) to the
triphosphate PIP3. A number of proteins
that have pleckstrin homology (PH) domains are then recruited to the membrane at this point, causing focal adhesion, activation of integrins, and polarization of the cell, in preparation for migration. chemotactic agent eg FMLP GPCR α γ γ β β PIP2 PIP3 PI-3 kinase pleckstrin-homology domain containing proteins P GTP GDP Activation of macrophages
Fig. 9.10 Elicited and immunologically primed macrophages differ from resident macrophages. Monocyte recruitment is enhanced and yields macrophages with proinflammatory and cytotoxic properties. Activation by cytokines enhances expression of MHC class II molecules and the complement receptor CR3. Phagocytosis and the production of proinflammatory mediators and cytotoxic products are increased. By contrast, resident
macrophages (e.g. in bone marrow) lack inflammatory functions, but participate in trophic reactions (e.g. with developing hematopoietic cells) as well as performing endocytosis. Microbial stimuli can act directly on resident macrophages to induce distinct surface and secretory properties.
MHC class II CR3 cytotoxic molecules phagocytosis endocytic receptors hemagglutinins monocyte elicited/primed macrophage stromal resident macrophage cytokines
The links between innate and acquired immune responses are therefore understood only in part.
MACROPHAGES CLEAR APOPTOTIC CELLS