As with the granulocyte population, monocytes also represent downstream cells of myeloid cell progenitors but have a single indented nucleus, and are generally much larger. Out of the total leukocytes in human peripheral blood, approximately 5-10% are monocytes (Gordon and Taylor, 2005), which can be subdivided into three main cell types; the precursor monocytes found only in the blood, and two distinct monocyte- derived lineages; DCs and macrophages. The differentiation of these cells involves a complex pathway originating from myeloid progenitor cells. Initially a general precursor for monocytes is produced before subsequent formation of three independent groups which form two different arms to monocyte differentiation; common DC precursors, and two monocyte precursors (MPs) differentiated by their expression of Ly-6C. Along the first arm, both of the MPs enter the circulation from the bone marrow. Ly-6C- MPs
differentiate into lung resident, alveolar macrophages that function to maintain homeostasis. In contrast, Ly-6C+ MPs form a subset of DCs defined by the expression of
25 form both inflammatory macrophages and a subset of DCs known as TipDCs which produce nitric oxide and TNF. Additionally, Ly-6C+ cells can lead to the generation of
myeloid-derived suppressor cells which are known to target tumours. On the second arm, common DC precursors form both pre-classical and plasmacytoid DCs. If deployed in lymphoid tissues, pre-classical DCs fully differentiate into classical DCs, but upon migration to non-lymphoid tissues, they form a subset of DCs defined by the expression of CD103. In contrast, plasmacytoid DCs simply enter the blood and tissues and begin patrolling for antigen (see (Geissmann et al., 2010) for a detailed review of monocyte differentiation).
The differentiation of monocytes into either macrophages or DCs is dependent on the local cytokine milieu. Macrophage colony-stimulating factor (M-CSF) is a particularly important cytokine for macrophage survival. Indeed, IL-6 stimulates expression of the M-CSF receptor which promotes macrophage differentiation, whilst TNF promotes DC generation by limiting M-CSF cell surface expression. The overall function of these cells is the efficient phagocytosis of not only microorganisms but also in clearing the remnants of dead cells during steady-state homeostasis. Monocyte migration into tissues during infection is due to their expression of a range of chemokine and adhesion receptors (Chomarat et al., 2003).
1.1.3.1 Macrophages.
Macrophages, due to their tissue-resident nature, form a major component of steady- state immune homeostasis. They are often the first cell to detect a threat and signal through the release of cytokines to other cells, including neutrophils, that a response is necessary. Specifically, macrophages aid tissue homeostasis by producing growth factors
26 and clearing dead or dying cells, including tumour cells. Macrophages have a range of receptors e.g. TLRs, which allow them to detect molecular patterns produced by pathogenic organisms and damaged cells (Parham, 2009).
Macrophages can be divided in to two groups, M1 and M2, which are pro- and anti- inflammatory, respectively. M1 macrophages are characterised by a IL-12high, IL-23high, Il-
10low phenotype, and are activated in response to microbial products and inflammatory
cytokines such as IFN-γ. In direct contrast, the general phenotype of an M2 macrophage is IL-12low, IL-23low, IL-10high, and these cells can be further subdivided into three groups.
Those induced by IL-4 or IL-13 are defined as M2a, macrophages activated by IL-1R or TLR agonists as well as immune complexes are M2b, whilst M2c are activated by glucocorticoid hormones or IL-10. The typical effector mechanisms employed by M1 macrophages involve the production of inflammatory cytokines such as TNF, IL-6, and IL- 1β, but also the production of reactive oxygen species (ROS). This promotes a Th1 response. Opposing this, M2 Macrophages promote tissue repair and remodelling but primarily aid Th2 type responses leading to parasite phagocytosis (Mantovani et al., 2005; Mantovani et al., 2004).
1.1.3.2 Dendritic cells.
DCs represent another differentiation pathway for monocytes. Morphologically DCs can be separated from other monocytes in their mature state due to their long dendrites which give them their name. DCs express both CD11c and high MHC class II, whilst lacking haematopoietic lineage markers (Hashimoto et al., 2011). Similar to macrophages, DCs reside in the tissues, however their primary function is to present antigens for naïve T- cell priming. When DCs recognise an antigen they further upregulate their expression of
27 CD80/CD86 co-stimulatory receptor and MHC molecule surface expression is greatly increased. CCR7 is also expressed in order for the DCs to migrate to the lymph nodes, and lysosomal pathways are activated to process antigens into distinct peptides which are then presented at the cell surface bound to the MHC (Jiang et al., 2007). Mature DCs migrate to the draining lymph nodes where they present antigens to passing T-cells to promote the induction of an adaptive immune response (Parham, 2009). In fact, DCs are professional APCs and the only cell capable of naïve T-cell priming and thus are the driving force behind the establishment of immunological memory. For this reason, DCs are often considered to be of high importance in the balance between antigen clearance versus antigen tolerance as immature DC will promote tolerance due to a lack of co- stimulatory signalling, but mature DCs with the correct co-stimulatory signalling will lead to an immune response.
Another DC specialist task is antigen cross-presentation. This refers to the ability of MHC class I on the surface of DCs, which is normally used for the presentation of intracellular antigens, to present extracellular antigens. This enables a cytotoxic CD8+ T-cell to mount
a response against modified or infected cells that display modified-self proteins or foreign antigens, and is described in further detail in section 1.5.2. This process is aided by the highly phagocytic capabilities of immature DC (Joffre et al., 2012).
There are two main types of DC: classical and plasmacytoid. Classical DCs migrate between tissues and lymphoid organs but are short lived. Plasmacytoid DCs however are longer lived, and are found in all peripheral organs and the bone marrow, with activation associated with a much larger secretion of IFN type I than classical DCs, especially for those involved in reactions to viral infections (Geissmann et al., 2010). The activation of DCs leads to a plethora of antigen-specific responses including the activation of NK cells, non-antigen-specific macrophages, B-cells, and cytotoxic CD8+ T-cells (Banchereau et al.,
28 2000). It is also clear that DCs do not just stimulate a T-cell response but also direct it. A combination of IL-23 and IL-1β secretion from DC has been reported to aid Th17 differentiation, while IL-12 release polarises T-cells towards a Th1 phenotype, and IL-10 towards a Th2 phenotype (Liu et al., 1998; Macatonia et al., 1995; Sutton et al., 2009). While the primary function of such professional APCs is to present harmful antigens to passing T-cells, similar responses may occur in response to antigens that pose no threat such as drugs. Responses of this nature are categorised among the generalised term of adverse drug reactions (ADRs).