Introductory Remarks

The haematopoietic system produces a wide variety of new functional cells as they are needed, including monocytes, macrophages and myeloid dendritic cells. Macrophages and dendritic cells are spiised for phagocytosis, tissue remodelling and antigen processing and presentation, as we will discuss later. They are derived from primitive precursor cells that can be grown in vitro or in vivo, and which replenish the entire haematopoietic compartment throughout life.

This chapter reviews our current knowledge of the role of cytokines and their receptors in the proliferation and differentiation of pluri-potent stem cells (PPSC) as they develop into cells of the monocyte/macrophage/dendritic cell lineage. Detailed discussion is therefore limited to the roles of CSF-1, SCF, FL, GM-CSF, IL-3, TNF, IFN-gamma, and IL-4, and to a lesser extent IL-6 and TPO. We have concentrated on experiments that use gene manipulation technology to unravel pathways of activation of cellular function and proliferation.

Much of our knowledge of haematopoiesis comes from mouse models. Till and McCulloch^1-3 were the first to show that individual bone marrow cells could form colonies in the spleens of irradiated recipient mice, and that these colonies also contained transplantable colony forming cells as well as all the other cellular elements of blood. In spite of this early observation most of what we know about pluri-potent stem cells of blood is inferred from analysis of their progeny, since the PPSC is present in very small numbers and until recently was difficult to maintain in vitro.⁴

Mouse PPSC are capable of the long-term (greater than six months) repopulation of both myeloid and lymphoid systems in lethally irradiated recipient mice. PPSC represent only 10-5 of the nucleated bone marrow cell pool, and in the steady state they either do not cycle or they cycle very slowly.^5,6 During times of demand, and under the control of a number of cytokines, PPSC proliferate and differentiate to give rise to common myeloid⁷ and common lymphoid⁸ progenitor cells that become increasingly more committed to a particular lineage until they can only form one mature type of blood cell.⁹ Progenitor cells can be detected in in vitro assays where they form colonies in a soft agar matrix under the influence of a particular group of cytokines called colony-stimulating factors. Initially the ‘colony assay’ was used to detect macrophage and granulocyte colonies.^10,11 It was shown that colony numbers were linearly related to the number of cells cultured and, at constant cell numbers, the number of colonies generated was proportional to the concentration of cytokine present. This permitted the use of in vitro colony assays in the purification of cytokines that stimulated colony formation,^12-14 and in the molecular cloning of the genes that encoded them.

Sources of Cytokines and Regulation of Their Production

Haematopoietic cytokines are glycoproteins that are normally present in the circulation (CSF-1, SCF, FL, G-CSF, EPO and TPO) in very low (pico-molar) doses, or are induced to appear in response to infection or inflammation (GM-CSF, IL-3, IL-5, IL-6, and IL-11). Some are induced in response to particular stimuli e.g., IL-3 is induced almost entirely by antigen stimulation of T cells. Cytokines contain carbohydrate moieties that are not required for activity but which seem to affect their half-life in the circulation and their localisation in vivo. The crystal structures of many cytokines have been determined including those of CSF-1,¹⁵ GM-CSF,¹⁶ G-CSF,¹⁷ IL-2^18,19 IL-3²⁰ and IL-6.²¹ Surprisingly the tertiary structures of these very disparate primary amino acid chains are rather similar. In general, like growth hormone,²² they contain four α-helical bundles and an antiparallel β ribbon. Some are homo-dimers (SCF, CSF-1, FL and IL-5) but others are monomeric. SCF, CSF-1 and FL are expressed as membrane spanning forms that are active at the cell surface and are involved in local regulation.

Signalling

Cytokines exert their action through high-affinity receptors on the cell surface that are linked to pathways of cellular activation, survival, proliferation and differentiation. Cross-linking of receptor subunits on the outside of the cell wall leads to abutting of kinases associated with the intracellular receptor tails, either as intrinsic activities, or because of pre-association of secondary kinase molecules. This intracellular association of signalling molecules results in phosphorylation of tyrosine residues in the receptor tail and binding of further signalling molecules that have phospho-tyrosine-binding domains. Several aspects of the downstream intracellular pathways of cytokines are similar, because different activated receptor cytoplasmic domains often bind a common signalling molecule or family of signalling molecules. Thus stoichiometry and rate of reaction are important regulatory influences that allow discrimination between signalling processes with different outcomes.

Cooperative Interaction of Cytokines in Proliferation and Differentiation

For efficient in vitro proliferation and differentiation, PPSCs and multi-potent progenitor cells require a combination of cytokines. (eg SCF, IL-1, IL-3, IL-6, GM-CSF, and CSF-1).^23,24 As might be expected from this observation, immature haematopoietic cells have been shown to co-express a number of different lineage specific receptors at low levels.²⁵ As these immature cells develop, they lose receptors for some cytokines e.g., SCF and IL-3, while retaining receptors for later acting cytokines such as CSF-1. Eventually at least some of the immature cells reach the stage of committed progenitor cell, where their further proliferation and differentiation are along one particular lineage, dictated by the relevant lineage-restricted cytokine. It may be important in lineage commitment for the level of receptors for the lineage-restricted cytokine to increase as differentiation proceeds.^26-28 Single lineage cytokines (such as CSF-1) often regulate the survival of their target cells including fully differentiated end cells^29,30 where they may prime the cells to receive secondary activation signals from elements such as bacterial lipo-poly-saccharide or immune complexes. Despite the apparent overlap in target cell specificity of several cytokines their functions are largely non-redundant as indicated by the distinct haematopoietic phenotypes of cytokine or cytokine-receptor deficient mice.

Synergy occurs between some late-acting lineage restricted cytokines such as CSF-1, EPO and G-CSF, with early acting cytokines such as SCF and IL-3, in stimulating the proliferation and differentiation of multi-potent cells. This provides a mechanism by which the tightly regulated changes in the level of a late acting cytokine, can be coupled to the channelling of multi-potent progenitor cells into a lineage to satisfy the demand for differentiated cells. The underlying mechanism of synergy may lie at the level of either the receptors or at the level of post receptor signalling pathways. There is little evidence that one cytokine directly influences levels of receptor for a synergistic cytokine, although it has been shown that IL-3 induces increased expression of c-fms mRNA in macrophages and bone marrow cells of A/J mice.^27,31 It is not clear whether the apparent increase in mRNA is due to de novo synthesis or to stabilisation of existing messenger RNA. The signalling is unique in that it almost certainly occurs as the result of binding of IL-3 to the mouse unique IL-3 specific beta sub-unit of the IL-3 receptor.

In an interesting experiment to determine the influence of cytokines on haematopoiesis, it was shown that common lymphoid progenitors can be redirected to the myeloid lineage by stimulation through exogenously expressed IL-2 and GM-CSF receptors. Furthermore it was shown that granulocyte and monocyte differentiation signals can be regulated by different domains of the IL-2 receptor.³²

The Role of Transcription Factors

Experiments using gene manipulation have shown that nuclear transcription factors are essential for stem cell lineage commitment.GATA-1³³ reprograms avian myelomonocytic cells from a myoblast fate to eosinophil, thromboblast and erythroblast differentiation. PU.1 on the other hand induces myeloid lineage commitment in multi-potent haematopoietic progenitors.³⁴ Pre B cells in Pax 5 negative mice fail to develop into B cells, instead becoming progenitors of macrophages, osteoclasts, granulocytes, NK cells and T cells.³⁵ This suggests that transcription factors may act as both positive and negative regulators of differentiation

Tyrosine Kinase Receptors

CSF-1, SCF and FL are homodimeric cytokines that share some sequence homology and structurally are similar to one another.^36-40 All three cytokines have complex patterns of expression due to alternative mRNA splicing. This allows them to be expressed as either membrane-spanning, cell-surface or secreted glycoproteins. All three growth factors are widely expressed in all tissues and at least two of them, SCF and CSF-1, affect non-haematopoietic as well as haematopoietic cells.

The receptors for the above cytokines are members of the PDGF receptor family.^39-42 Each receptor possesses an extra-cellular domain comprising five Ig-like repeats which are heavily glycosylated with N-linked sugars, a trans-membrane domain, and intra-cellular domains containing a juxta-membrane region, a src-related tyrosine kinase domain that is interrupted by a kinase insert domain, and a carboxy-terminal tail. The three amino terminal Ig-like domains incorporate the ligand binding domains of the SCF and CSF-1 receptors.

Binding of this type of dimeric receptor to its cognate ligand stabilises the non-covalent association between the two chains of the receptor at the cell surface and permits the trans-phosphorylation of the intra-cellular domain of one chain by the other.

Tyrosine phosphorylation in response to cytokine binding is not restricted to those proteins that are stably associated with the receptor. Regions containing tyrosines that are phosphorylated as a consequence of receptor activation act as docking sites for src homology region 2 (SH2) domains of signalling and adaptor proteins. These proteins in turn may interact with plasma membrane associated proteins. An example is the association of recruited Grb2/Sos with Ras, which leads to their activation. Or the associated proteins may themselves become tyrosine phosphorylated.

Many of the signalling pathways activated by SCF, CSF-1 and FL receptors, including the Ras/Raf-mitogen activated protein kinase cascade, the Janus kinase (JAK) signal transducers and activation of transcription (STAT) pathway, Src family members and phosphatidylinositol-3-kinase (PI3K), are shared.⁴³ All three receptors are likely to exhibit ligand induced, Cbl-mediated decreases in receptor expression.⁴⁴

The SCF and CSF-1 receptors are encoded by the proto-oncogenes c-kit and c-fms respectively. ⁴⁵ The oncogenes derived from these two proto-oncogenes are present in mutated forms in retroviruses that cause sarcoma in cats. The mutations in the receptor genes cause constitutive activation of the receptors in the absence of cytokines.⁴⁵ thus contributing to unregulated cell proliferation.

Cytokines and Receptors with a Significant Effect on Cells of the Monocyte Macrophage Lineage

CSF-1

CSF-1 regulates the survival, function, proliferation and differentiation of cells of the mononuclear phagocyte series, as well as the function of cells of the female reproductive tract.^40,52-54 The 150 amino terminal amino acids of CSF-1 are sufficient for biological activity. This section contains the four alpha helical bundle-anti-parallel beta sheet structure.¹⁵ and is expressed as an end to end disulfide-linked dimer. It is synthesised in the endoplasmic reticulum as a disulphide linked homo-dimeric, membrane-spanning precursor containing 522 amino acids. In secretory vesicles the mature proteoglycan (120 — 160 kD) and glycoprotein (100kD) are cleaved from this precursor and secreted.⁵⁵ A smaller CSF-1 glycoprotein precursor of 224 amino acids from which the proteolytic cleavage and glycosaminoglycan sites have been due to alternative splicing is expressed on the cell surface when the secretory vesicle fuses with the cell membrane.

The 58 kb gene encoding the CSF-1 R is known as c-fms and maps to the long arm of chromosome 5⁴⁵ near to the genes encoding GM-CSF and IL-3. When ligand binds the CSF-1 receptor dimerization occurs and the cytoplasmic tails of the molecule trans-phosphorylate on tyrosine. This results in a cascade by which several cytoplasmic proteins, some of which have been shown to play a role in signal transduction, are phosphorylated.^55,56 After activation and signalling the receptor ligand complexes are internalised and destroyed within lysosomes.^44,56-58

CSF-1 is synthesised by many cell types, including fibroblasts, endothelial cells, bone marrow stromal cells, osteoblasts, keratinocytes, astrocytes, myoblasts, and uterine epithelial cells in pregnancy.⁴⁰ Circulating CSF-1 is probably made by endothelial cells of small blood vessel walls. Ninety five percent of circulating CSF-1 is cleared by sinusoidal macrophages such as Kupffer cells, through CSF-1R-mediated endocytosis and intra-lysosomal destruction. The number of sinusoidal macrophages serves as a simple feed-back control on the circulating level of CSF-1.^58,59

The half-life of circulating CSF-1 at physiological concentrations is about ten minutes but at saturating pharmacological concentrations the half-life can extend to 1.6 hours.⁵⁷ Bacterial endotoxin, viruses, and parasitic infections raise the levels of circulating CSF-1.^40,52-54 Repeated injection of CSF-1 into mice can cause a tenfold increase in circulating monocytes and an increase in macrophages in the periphery.⁶⁰

Osteopetrotic (Csf1^op/Csf1^op) mice have an inactivating mutation in the CSF-1 gene.^61,62 These mice display impaired bone resorption associated with a paucity of osteoclasts, the production of which requires both osteoprotergerin ligand and CSF-1. They have no incisors, poor fertility, a lower body weight, absence of evoked auditory and visual responses, a thinner than normal dermis, a deficiency in blood monocytes and in macrophages in certain tissues and a shorter life expectancy.^63,64

Restoration of circulating CSF-1 in Csf-1op/CSF-1op mutant mice cured their osteopetrosis and monocytopenia and caused increases in some but not all tissue macrophage populations. This is consistent with local as well as humoral regulation by CSF-1.^63-65 There was complete rescue of these phenotypes by normal tissue specific and developmental expression of a transgene encoding all three forms of CSF-1 but only partial rescue by a transgene encoding the cell surface iso-form.^66,67 The two secreted forms of CSF-1 are both found in the circulation. Both the cell surface and secreted proteoglycan CSF-1 variants are thought to play a role in local regulation in part because the proteoglycan form is sequestered by specific extracellular matrices.^40,45

Studies with CSF-1op/CSF-1op and Csf1r^-/Csf1r^- mice clearly indicate that CSF-1 is the primary regulator of the monocyte macrophage series, including osteoclasts. The pleiotropic phenotype of Csf1^op /Csf1^op mice, and the restricted distribution of CSF-1R to cells of the mononuclear phagocyte system and female reproductive tract, suggest that the major role of CSF-1 is to generate and maintain macrophages that have trophic, as well as scavenger functions that are important in organogenesis and tissue turnover.⁶⁵ Csf1op/Csf1op mice exhibit normal T- and B-cell-dependent responses to simple and corpuscular antigens.^68,69 However, CSF-1 has a well-documented priming role in macrophage activation and it plays an important role in innate immune responses.⁷⁶ It appears that the development of macrophages involved in inflammatory and immunologic functions depends on other cytokines that regulate the activities of macrophages generated by CSF-1.^70,71

The CSF-1 R is expressed at low levels on early multi-potential cells. The level of CSF-1R increases tenfold as these cells give rise to committed macrophage precursors.^26,72 There is some evidence that this increase can be mediated by IL-3.²⁷ In vitro experiments have shown that in contrast to committed progenitors the multi-potent cells are unable to respond to CSF-1 alone, but CSF-1 can synergise with IL-1, SCF, IL-3 and IL-6 to stimulate multi-potential cell proliferation and differentiation to committed macrophage progenitor cells.^{23,24,26,71-73}

As well as its role in macrophage development, CSF-1 is involved in bone metabolism through its role in the proliferation and differentiation of osteoclast precursors. CSF-1 is synthesised locally by osteoblasts and synergizes with osteoprotergerin ligand in the maturation of osteoclasts from small mononuclear cell precursors that proliferate in response to CSF-1 alone.⁷⁴ CSF-1 also plays and important role in both male and female reproductive systems⁷⁵ and the action of CSF-1 on the trophoblast is critical for embryonic resistance to certain infections. ⁷⁶ Under control of the female endocrine system, CSF-1 is synthesised in the oviduct and the uterine epithelium during pregnancy and can influence several cell populations including maternal macrophages and decidual cells, as well as early embryonic cells and various trophoblast cell types.^40,60 Circulating levels of CSF-1 are elevated in patients with myeloid and lymphoid malignancies or with carcinomas of the ovary, breast and endometrium. In ovarian cancer elevated levels of circulating CSF-1 are associated with a poor prognosis^77,78 IL-3 and GM-CSF.

IL-3, GM-CSF and IL-5 are cytokines that signal through receptors that contain a common beta sub-unit.⁷⁹ Although their amino acid sequences share little homology, it appears that IL-3, IL-5 and GM-CSF share a common ancestral gene. All three genes map to in close proximity on the long arm of chromosome 5 and have similar structure. The tertiary protein structure for all three is similar, with four alpha helical bundles and antiparallel beta ribbons except that IL-5 is a covalently disulphide linked dimer. All three cytokines signal through a receptor that contains an alpha chain that exhibits low affinity for the cytokine and a common beta chain. When cytokine-specific alpha chain and beta chains join, a high affinity receptor complex capable of signalling is formed. In mice there is a second form of the beta chain which appears to be IL-3 specific and which binds IL-3 with low affinity. This variant beta chain may be able to signal in the absence of alpha chain because in some strains of mice the alpha chain is missing and yet gene expression but not proliferation can be induced in bone marrow cells of these mice by IL-3.²⁷ The affinity of binding of the IL-3 specific beta chain is contentious as is the availability of complementary alpha chain in mutant mice.

Ligand-induced disulphide bonding between alpha and beta sub units as well as dimerization of beta subunits are required for signalling (REF). In strains in which the alpha chain is missing, possibly beta chain dimerization is sufficient. The high affinity cytokine-receptor complex resembles the IL-6/IL-6R high affinity complex, consisting of two receptor alpha chains, two beta chains and two ligand molecules.⁷⁹ Because of the shared beta sub-unit, IL-3, IL-5 and GM-CSF compete with each other for binding to their high affinity receptors,⁸⁰ though in mice IL-3 can to some extent circumvent this because of the IL-3 unique variant of the beta chain. The biological significance of the competition between IL-3, IL-5 and GM-CSF for the shared beta sub-unit is not understood. However, it is clear that the cytoplasmic domains for both alpha and beta receptor subunits can be involved in signalling.⁸⁰

The 120kD beta sub-unit contains the conserved cytokine motif found in the alpha sub-unit. It has a much longer cytoplasmic tail which is required for proliferative signalling and it has box I and box 2 motifs that contain docking sites required for the recruitment of signalling molecules by the activated receptor. Activation of the receptor complex results in tyrosine phosphorylation of the receptor and recruited molecules.

Interleukin-3

Interleukin-3 is a secreted monomeric 133 amino acid 25-30kD glycoprotein containing an intramolecular disulphide bridge that stabilizes the loop containing the binding site. The IL-3R comprises a low affinity alpha sub-unit and a beta sub-unit in the human but, as indicated above, in the mouse there is also a low affinity IL-3 specific beta sub-unit which interacts in a productive manner only with the IL-3 specific alpha chain.^79-83

IL-3 is synthesised almost exclusively by T cells in response to antigenic stimulation.⁸² It is a pleiotropic cytokine supporting the proliferation and differentiation of multi-potent progenitor cells and committed progenitors.^83-84

IL-3 alone stimulates multi-potent stem cells to form colonies containing neutrophils, basophils, monocytes, eosinophils and megakayocytes. Yet more primitive precursors are stimulated by IL-3 in the presence of CSF-1⁸³ in populations enriched for haematopoietic stem cells. IL-3 nullizygous mice have diminished DTH but no obvious steady state haematopoietic phenotype. ⁸² This suggests that IL-3 may be involved only when there is an unusual demand for increased haematopoiesis such as eventuates in an immune response.

Granulocyte-Macrophage Colony Stimulating Factor

GM-CSF is a glycoprotein of between 18 and 32 kD.⁸⁵ It has a receptor similar to that of IL-3. The alpha sub-unit of the receptor is a membrane spanning protein of 80 kD containing the extracellular cytokine-binding domain. There are three alternative transcripts, one for the main form just described, one encoding a soluble form that has the region encoding the trans-membrane and cytoplasmic region spliced out, and a third species encoding an alternative membrane spanning alpha chain with a slightly longer c-terminal end.⁸⁶ All bind ligand but their physiological significance is poorly understood.

GM-CSF is constitutively synthesised by macrophages, endothelial cells and fibroblasts and its expression is inducible in T cells.⁸⁷ The cytokine acts as survival molecule and as a mitogen on progenitor cells of many lineages.⁸⁸ including all the cells of the neutrophil, macrophage and eosinophil lineages. At most stages the cells of these lineages also require more lineage specific factors such as G-CSF, CSF-1 and IL-5. GM-CSF synergises with EPO and TPO to generate erythroid and megakaryocyte progeny. In vivo GM-CSF acts to increase the number of circulating neutrophils, monocytes and eosinophils and the number of fixed tissue macrophages. GM-CSF-deficient mice have pulmonary alveolar proteinosis caused by a deficiency of alveolar macrophages.⁸⁹ However these mice have normal granulocytes and macrophage numbers in the steady state, thus GM-CSF apparently does not play an indispensable role in blood cell production.

Interleukin-4

Is a glycoprotein of 15 to 19 kD. There is a splice variant lacking amino acids 22 to 37 that is expressed more strongly in thymocytes. This IL- 4d2 inhibits IL-4 induced T cell proliferation. The IL-4 R is expressed on a wide range of cell types including B and T lymphocytes, monocytes, granulocytes, fibroblasts, endothelial and epithelial cells and expression is induced by IL-4.⁹⁰

Two forms of IL-4 R are known, one comprising the IL-4Rβ and γ_c subunits requires JAK3 for STAT activation is predominantly expressed in haematopoietic cells. The other comprising the IL-4β and IL-13Rα subunits requires JAK2 for STAT 6 activation and is predominantly expressed in non hematopoietic cells.⁹⁰ A soluble form of IL-4 receptor binds IL-4 with high affinity.⁹¹

IL-4 is expressed in TH2 cells. It has complex biological actions many of which may be indirect through the modulation of cytokine production.^90-92 IL-4 enhances antigen-presenting activity of B cells to T cells. It enhances antigen receptor and CD40 triggering of B cell proliferation and differentiation, but it antagonises IL-2-induced co-stimulation, possibly because it sequesters the γ_c chain, shared with the IL-2 receptor. Normal B cell development is seen IL-4 nullizygous mice suggesting that some of its in vitro effects can be subsumed by other factors in vivo.⁹² IL-4 stimulates the generation of TH2 cells and the proliferation and maturation of thymocytes, while in contrast it inhibits early T cell development in foetal thymus organ culture. IL-4-nullizygous mice have a TH2 cell deficiency and diminished IgG1 and IgE responses consistent with a role for IL-4 in regulation of T helper cell development and in Ig switching.^90-94

IL-4 inhibits CSF-1 induced macrophage and megakaryocyte colony formation, probably by accelerating terminal cell differentiation, but enhances G-CSF induced colony formation and, with IL-3, the generation of basophils and eosinophils.

Interferon-γ

A non-covalent homo-dimeric glycoprotein of 50 kD is produced by activated or cytokine-stimulated T lymphocytes and NK cells. Interferon-γ has a receptor composed of two or three subunits. The primary receptor contains a ubiquitously expressed alpha chain, an inducible beta chain, and in some circumstances, a third sub-unit is required for signalling.⁹⁵ IFN-γ deficient mice have defects in the immune system that become apparent following infection suggesting that these mice fail to deal with infectious agents effectively at the phagocytic level. IFN augments the immune response by activating macrophages toward phagocytosis, and enhancing antigen recognition by increasing the expression of class one and class two antigens on antigen processing DCs. IFN is also a general inhibitor of cell proliferation by a variety of mechanisms.⁹⁶

Tumour Necrosis Factor

Tumour necrosis factor alpha and lympho-toxin alpha (TNF-beta) are products of activated macrophages and T cells. Receptors for these mediators are ubiquitous, serving important functions in sepsis, granulomatous disease, autoimmunity, graft versus host disease and other inflammatory events particularly involving the activation of macrophages or neutrophils. TNF-α deficient mice have a disorganised splenic architecture and peripheral lymph node deficiencies, and have a reduced capacity to fight infection.^97,98 The TNFs induce production of myeloid CSFs, IFN-γ, IL-6 and chemokines by various cell types and they inhibit production of TGF-β. The actions of the TNFs can therefore be quite complex, particularly in the regulation of antigen processing cells (APC's).

Antigen Processing Cells

Recently, renewed effort has been directed towards establishing the role of APC's in immune regulation.⁹⁹ The best understood APC's are macrophages, Langerhan's cells (LC), myeloid dendritic cells (MDC) and lymphoid dendritic cells (LDC).¹⁰⁰

Macrophages are M-CSF (CSF-1) responsive, strongly adherent, highly phagocytic and express cell-surface F4/80,¹⁰¹ Mac-1 and cell surface Fms.¹⁰² They are derived from blood monocytes and reside in discrete sites as tissue macrophages.⁶⁵ As monocytes they are highly mobile and can migrate readily into sites of inflammation. Macrophages are subject to regulation by a wide variety of stimuli that affect their maturation and function. They are important in tissue remodelling and removal of debris including opsonised micro-organisms. The role of the classical macrophage in immune induction is contentious. Macrophages present antigen poorly and in fact may be immuno-suppressive.¹⁰³ It is generally thought that only cells which express an intact antigen processing and presentation pathway, including an active TAP system and appropriate MHC class II antigens, as well as other required co-stimulatory molecules, are effective in antigen presentation.^70-71 These are properties not generally seen in macrophages, though activation of some phagocytes by IFN-γ for example, induces expression of some of these molecules.⁹⁴ The phagocytes so induced may be more closely related to DC than to classical macrophages as we shall discuss later.

MDC and LC take up antigen in the periphery by pinocytosis or phagocytosis then migrate to T-cell areas of lymphoid organs such as spleen, lymph nodes and Peyer's patches. LC ingest antigens in the dermal region then migrate via afferent lymphatics to local lymph nodes.¹⁰⁵ Studies of mouse.¹⁰⁶ and human^107,108 LC and MDC have shown that both cell types pass through an immature stage where they express receptors that enhance antigen capture and adsorption such as the macrophage mannose receptor¹⁰⁷ and Fcγ and Fcε receptors.¹⁰⁸ At this stage MDC and LC lack the cell surface molecules CD40, CD54 and CD86,¹⁰⁰ which are important for the delivery of activation signals to naive T cells. After DC have taken up antigen, it is transported to a particular type of late endosomal compartment which is rich in MHC Class II.^108-111 In this compartment the antigen is digested into peptides and loaded onto MHC class II molecules. When microbial or inflammatory products stimulate DC to mature, the DC migrate to T cell areas of lymphoid tissue and the antigen-loaded MHC class II molecules are passed to the cell surface for presentation to a re-circulating pool of T cells.^111,112 T cell clones with particular antigen specificities are then activated and expanded to achieve effector or helper cell status.

LDC are different from MDC in that they secrete much higher levels of IL-12, and they are located in the T cell areas of the spleen whereas MDC are found in the marginal zone.¹¹³ In addition, LDC are long lived cells whose MHC molecules tend to carry peptides derived from self antigens.¹⁰⁰ It has been suggested that unlike MDC which specialise in triggering T cell immune responses against foreign antigens such as those of pathogenic micro-organisms. LDC may also be required for maintaining self tolerance.^114,115

Classification and Sources of Antigen Processing Cells

MDC can be isolated from blood, lymph nodes, liver, bone marrow, cord blood and thymus either as mature DC, monocytes, or CD34+ myeloid precursor cells. DC progenitor cells can then be stimulated to differentiate into DC by the addition of various combinations of cytokines and growth factors,¹¹⁶ Some of these stimuli in mouse systems include GM-CSF,¹¹⁷ GM-CSF and IL-1,¹¹⁸ GM-CSF, SCF and TNF-α,¹¹⁹ and in human systems include IL-4 and IFN-γ,¹²⁰ IL-4 and GM-CSF,^107,120-122 IL-4, GM-CSF and IFN-γ,123-124 TNF-α and GM-CSF,¹²⁵ TNF-α, GM-CSF and CD40L.¹²⁶

MDC are derived from the same GM-CSF-responsive progenitor cell pool in bone marrow that gives rise to macrophages and LC's.^99,127-132 Human CD34+ blood cells also contain a macrophage, DC and LC progenitor cell.^130-133 The point at which the LC and DC diverge during maturation in vitro appears to be after CD34+ progenitor cells are exposed to GM-CSF and TNF-α. This results in two populations of cells, one of which is CD1a+ and the other CD14+.¹⁰⁰ The CD1a+ cells differentiate into LC in response to further culture in the presence of GM-CSF and TNF-α.¹³⁴ The CD14+ cells are bi-potent maturing into DC in response to GM-CSF and TNF-α (or other cytokine combinations described above), or macrophages in response to M-CSF.

Currently MDC are characterized by the expression of CD1a, CD40, CD80, CD86 and MHC class II cell surface antigens, they endocytose FITC dextran, and stimulate T cells in a mixed lymphocyte reaction.^70,71 LC are distinguished from MDC by expression of CLA,¹³⁵ Birbeck granules, a granule-associated antigen called LAG-1¹³⁶ and E-cadherin.¹³⁷

LDC progenitors can be isolated from the thymus and express low levels of CD4 whereas the mature LDC expresses CD8 and lack myeloid antigens such as CD11b/CR3 receptor, CD13 and CD33.¹³⁸ LDC share most properties of MDC, but unlike the MDC which is CD11_c+, the LDC is CD11_c-, and lacks the myeloid markers CD13/CD33 and CD45RO. LDC are claimed to express high levels of the alpha chain of the IL-3 receptor but not alpha chains for the GM-CSF receptor and mature in response to IL-3 treatment.¹³⁹ LDC also express high levels of self peptides and fas-ligand capable of inducing CD4+ T-cell death, but they do not endocytose FITC-dextran.¹³⁹

Origin of APC

Studies performed on the differentiation of both mouse and human DC suggest the existence of a bi-potent myeloid progenitor cell that can develop, via blood monocytes, into either a dendritic cell or a macrophage.^134,140-147 The resulting dendritic cells and macrophages are closely related and in certain circumstances seem to inter-convert.^132,148-150 En route to their final location committed progenitors probably receive the various combinations of signals that modulate their ability to home and acquire their final set of functional characteristics. This may account for the apparent heterogeneity in sorted DC populations and hence the notion of “Subsets of DC”.

The developmental pathways of MDC's and macrophages are complex. At one extreme, driven by M-CSF, is the classical tissue macrophage, certain trophic functions which we would suggest is involved largely in tissue re-modelling and general phagocytosis and destruction of unwanted debris, including opsonised bacteria, viruses and tumour cells, as well as some aspects of innate immunity.^70,71,76 Next are the cells that are regulated by a combination of IL-3 and CSF-1, the so called high proliferative potential cells that give rise to very large numbers of progeny in response to the synergistic mitogenic effect of these two cytokines.⁷³ These cells may give rise to a variety of other types including those with increased ability to respond to GM-CSF. Switching the primary mitogenic signal in some of these cells to GM-CSF seems to select for a population with increased ability to acquire dendritic cell properties following exposure to TNF-α and IFN-γ.

It is likely that all DC and ‘immune’ macrophages arise from the same myeloid progenitor population and that the plurality of ‘end’ cells seen is a reflection of different final maturation pathways induced by the signalling molecules indicated (and probably by others). It may even be that many supposed end cells in this system can undergo a form of trans-differentiation as we have been able to show for TNF-α/IFN-γ induced DC, which in the presence of IL-4 rapidly acquire the characteristic morphology of macrophages (Hapel et al unpublished))

Whether the LDC is derived from this same myeloid pool awaits further investigation. Studies thus far suggest that the LDC is derived from a pool of progenitor cells that can also give rise to T lymphocytes.^151-152

In Vitro Model Systems for Studying the Differentiation of Dendritic Cells

There are significant difficulties in precisely defining DC and their progenitors because cell populations isolated ex vivo are somewhat heterogeneous with respect to cell surface phenotype, morphology and tissue origin. By setting parameters carefully, FACS selection can provide a relatively homogeneous population of cells. But results generated by this sort of experimental approach must be treated with caution because FACS sorting provides a selected, not necessarily homogeneous population. The recent notion that there may be subsets of DC that preferentially stimulate different subsets of T cells, e.g., Th1 and Th2, further complicates analysis of FACS sorted populations of DC and their progenitors.

The majority of in vitro studies on the development of DC have relied on FACS sorted populations of CD34+ human peripheral blood monocytes. These cells are cultured in the presence of GM-CSF and IL-4 for between 7 and 11 days after which time dendritic cells appear in the cultures.^153,154 Treatment of these cultures with TNF-α, CD40L or LPS is required to stimulate full antigen-presenting function. It is difficult, if not impossible, to precisely analyze the differentiation steps that are required to produce DC from this heterogeneous cell population over the 7-11 day culture period. Neither the starting cell nor the final mature DC have been identified. What one studies is a population effect.

In a more recently developed system¹⁵⁵ co-culture of a FACS sorted CD34+ progenitor population with umbilical vein endothelial cells was reported to yield functional DC in 2 days. This suggests that DC can be generated quite rapidly from progenitor cells given a suitable environment and adequate differentiation signals. The DC maturation system described by Randolph¹⁵⁵ may resemble more closely the in vivo maturation of DC but it still does not resolve the problem of using an innately heterogeneous FACS sorted progenitor cell population to study the DC lineage. A preferred system would be clonally derived progenitor cell lines that retain the ability to respond to differentiation-inducing cytokines, and other stimuli, in a homogenous manner. In such a system more precise determination of DC-specific cell markers can be made and correlated with different stages of functional maturation.

Conclusion

Macrophages and dendritic cells appear to be overlapping populations of cells that are specialised for tissue remodelling, innate immunity, and removal of cellular debris on one hand (macrophages) and antigen procesing and presentation to the immune response on the other (dendritic cells). Classical macrophages are induced by CSF-1 while the dendritic lineage relies for its maintenance on GM-CSF and other “immune” cytokines. Both cell lineages display phagocytosis but dendritic cells are far more potent at antigen processing and presentation. In some circumstances the two lineages may appear to be indistinguishable. Further experimentation with cytokine and receptor gene knock out mice is required to fully elucidate the precise function of these two lineages in immune regulation.

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