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Chemokines are the largest family of cytokines identified. They are secreted proteins, with the noted exceptions of CX3CL1 and CXCL16 which have membrane anchored forms allowing them to also act as adhesion molecules [Bazan et al., 1997; Matloubian et al., 2000]. Mature chemokines are 8-14 kDa, basic proteins which even in cases of low overall sequence identity adopt a similar tertiary folding as determined by X-ray crystallography and/or NMR [Allen et al., 2007]. This tertiary structure is maintained by the presence of disulphide bonds between four conserved cysteine residues. (figure 7.1). The spacing between the first and second of these cysteine residues is used to classify the mammalian chemokines into four groups: CXC (a.k.a. α), CC (a.k.a. β), XC (a.k.a. γ) and CX3C (a.k.a. δ); with each chemokine being assigned an 'L' as signifier for 'ligand' and an identifying arabic number. Currently there are 17 α-chemokines designated CXCL1-CXCL17; 28 β-

chemokines designated CCL1-CCL28; 2 γ-chemokines designated XCL1-XCL2 and a single δ-chemokine CX3CL1, formally recognised [Zlotnik & Yoshie, 2000; Bacon et al., 2002; Zlotnik & Yoshie, 2012]. However, the existence of isoforms and alternatively spliced variants plus the possibility of post-translational modifications can be considered to greatly increase the number of functional chemokines.

Figure 7.1 Schematic representation of the structural motifs which retain chemokine tertiary structure and define the four chemokine subfamilies. Beta sheet structures are depicted as arrows.

Disulphide bonds between the conserved cysteine residues are represented by dashed lines. The CX3C chemokine is depicted with a transmembrane domain [Figure from Frederick & Clayman,

2001].

In addition to these structural criteria, chemokines may also be classified into two groups upon the basis of function: homeostatic and inflammatory chemokines (figure 1.6). The homeostatic chemokines are typically constitutively expressed in organs and tissues in the absence of inflammatory stimuli, resulting in leukocyte trafficking important during

normally occurring processes such as immune cell maturation or immune surveillance. While inflammatory chemokines are predominantly only expressed by monocytic, epithelial, endothelial or fibroblastic cells in response to pro-inflammatory stimuli, to initiate the migration of leukocytes to inflammatory sites as well as activating other mediators of immune responses and wound healing. Typically, chemokines belonging to the homeostatic category are considered to bind a single receptor to exert their

function(s), while the inflammatory chemokines display a more promiscuous receptor binding profile. However, as with many aspects of the chemokine system, the division of chemokines into these groups is not absolute, with a number falling into both categories [Allen et al., 2007].

While the interaction of chemokines with their respective receptors is the major interaction defining chemokine activity it is not the only interaction important for

chemokine function. In vivo, predominantly on luminal surface of endothelial cells, certain chemokines also undergo essential lower-affinity interactions with glycosaminoglycan (GAG) moieties of proteoglycans; although the affinities and GAG specificities can vary widely, representing another mechanisms for the control of their site specific expression [Proudfoot et al., 2003; Witt & Lander, 1994; Kuschert et al., 1999]. GAGs are highly

charged, highly sulfated and heterogeneous polysaccharides which, while expressed by nearly all mammalian cells, possess expression patterns which can be varied, for example, upon a different pathological state [Mortier et al., 2012]. It is an electrostatic interaction between the negative charge on GAGs and the predominantly basic chemokine protein which is thought to be primarily be responsible for binding [Hileman et al., 1998;

Proudfoot, 2006]. While these GAG interactions are essential for the activity of certain chemokines in vivo, the exact mechanism still remains uncertain. It is believed that in vivo the secretion of chemokines alone, especially in presence of shear forces of blood flow, is not sufficient for the establishment and maintenance of the gradient required for

leukocyte chemotaxis. As such the chemokine-GAG interaction permits a mechanism for localisation through establishment of the chemokine gradient [Handel et al., 2005]. However, the possibility that chemokine-GAG interactions may contribute to other important chemotactic processes, such as leukocyte arrest, protection from proteolysis and transcytosis of chemokines across the endothelium, still remains [Middleton et al., 2002; Salanga & Handel 2011].

In addition to the interaction with GAGs, many chemokines also possess the ability to form both homo- and hetero-oligomers both in solution at high concentrations and in the presence of GAGs [Handel et al., 2005; Proudfoot, 2006; Salanga & Handel 2011]. It should be noted that a number of chemokines exist in naturally occurring obligate monomeric forms, such as CCL1, CCL7 and CCL11[Proudfoot et al., 2003]. While there is some

evidence suggesting that these higher order oligomers may play a role in vivo, such as by increasing the affinity for GAGs or potentially modulating chemokine function, the true role of such structures remains unclear [Salanga & Handel 2011]. However, despite the existence of oligomeric forms, it is now generally accepted that the interaction of the chemokine with the receptor responsible for receptor activation and chemotaxis, occurs via the monomeric form. This was elucidated through the generation of obligate

mononmeric mutants of a number of chemokines, CCL2, CCL3, CCL4, CCL5 and CXCL8. These obligate monomeric mutants failed to induce chemotaxis in in vivo migration assays however, they retained wild-type receptor binding affinities and chemotactic activity in vitro [Paavola et al., 1998; Handel et al., 2008; Avalos et al., 1994; Laurence et al., 2000; Proudfoot et al., 2003; Rajarathnam et al., 1994]. Thus it seems while oligomeric forms may to play a role in chemotactic responses in vivo, at least in the case of some

chemokines, the actual chemokine-chemokine receptor interaction which leads to receptor activation and induction of cellular movement occurs via the monomeric chemokine form.