**3.1 Classification of CCL17 as an inflammatory chemokine**

The β-chemokine CCL17 formerly known as Thymus- and Activation Regulated Chemokine (TARC), was rst identied as a T cell chemoattractant by the group of O. Yoshie in human thymus, and phytohemagglutin stimulated peripheral blood mononuclear cells (Imai et al., 1996). Later on, the murine homologue was identified in murine bone marrow derived DC (BMDC) (Lieberam & Förster, 1999) and anti-CD40 stimulated splenic B cells (Schaniel et al., 1999). CCL17 shares the highest homology (32% amino acid identity) with CCL22 (macrophage-derived chemokine (MDC) and both chemokines signal through CCR4. CCR4 is expressed on T helper (Th)-1 and Th2 cells (D'Ambrosio et al., 1998; Sallusto et al., 1998a) but also on Th17 cells (Acosta-Rodriguez et al., 2007; Annunziato et al., 2007), CD8+ T cells (Kondo & Takiguchi, 2009; Semmling et al., 2010), regulatory T cells (Treg) (Iellem et al.,

Expression and Function of CCL17 in Atopic Dermatitis 85

which is able to strongly induce CCL17 in splenic DC, is the synthetic glycolipid α−galactosylceramide, a well known CD1d-dependent inducer of NKT cell activation (Semmling et al., 2010). After licensing by activated NKT cells, cross-priming CD8+ DC produce CCL17 and attract naïve cytotoxic T cells expressing CCR4. Based on the analysis of CCL17/EGFP reporter mice, expression of CCL17 is fairly restricted to DC and has not been observed in keratinocytes, B cells, endothelial or epithelial cells. Dermal DC (dDC) and LC in normal, untreated skin of mice are also CCL17-negative but turn on CCL17 expression

Although healthy human skin is devoid of CCL17 expression as well, human keratinocytes were shown to express CCL17 in inflamed skin, such as lesional skin of AD patients (Kakinuma et al., 2001; Vestergaard et al., 2000). *In vitro*, CCL17 expression is also found constitutively in HaCaT cells and is further inducible by cytokine stimulation (Vestergaard et al., 2001), whereas normal human keratinocytes did not express CCL17 protein *in vitro*, even after cytokine stimulation (Tsuda et al., 2003; Saeki & Tamaki, 2006). In contrast, human LC, Inflammatory Dendritic Epidermal Cells (IDEC) and dDC strongly express CCL17 (and CCL22) in inflammatory environments, particularly in lesional skin of AD patients, as do LPS stimulated human monocyte-derived DC (D'Ambrosio et al., 2002; Fujita et al., 2011; Kang et al., 2010; Soumelis et al., 2002). In addition, non-haematopoietic cells, such as bronchial epithelial cells, endothelial cells, broblasts, as well as smooth muscle cells can be a source of inducible CCL17 in humans (D'Ambrosio et al., 2002; Faffe et al., 2003; Sekiya et al., 2003; Yu et al., 2002). Taken together, under inflammatory conditions CCL17 appears much more widely expressed in different cell-types in human as opposed to mice, where CCL17 expression is quite restricted to DC subsets. However, detection of CCL17 protein expression by immunohistology as it has frequently been performed in human studies, may also pick up CCL17 that is passively absorbed to the cell membrane, for example by adhesion to surface glycosaminoglycans, and may not necessarily reflect

CCL17 interacts with two chemokine decoy receptors, the Duffy antigen (DARC) and the D6 receptor. The Duffy antigen is found on red blood cells and binds both CC and CXC chemokines. In contrast, D6 is expressed on lymphatic endothelial cells in skin, gut and lung and interacts with inammatory CC chemokines only. Although D6 is structurally similar to other chemokine receptors, it is most homologous to CCR4 and CCR5. Ligand binding induces rapid internalization of the ligand-receptor complexes, which facilitates regulation of inammatory immune responses and enables the final return to homeostatic levels. Interestingly, the homeostatic chemokines CCL19 and CCL21 cannot efficiently bind D6 (Bonecchi et al., 2004). Instead, CCL19, CCL21 and CCL25 are able to bind with high affinity to another hepta-helical surface protein, termed CCX-CKR, which also acts as a chemokine

AD is one of the most frequent chronic inflammatory skin diseases in children and adults and is characterized by pruritic skin lesions in typical body areas such as the flexural folds on dry skin (Bieber, 2008). Various exogenous and endogenous factors, including allergens

following irritation or injury of the skin (Stutte et al., 2010).

transcription of the *ccl17* gene in the same cell-type.

scavenger (Comerford et al., 2006; Gosling et al., 2000).

**4. CCL17 in atopic dermatitis** 

**3.3 Clearance of CCL17** 

2001), natural killer T (NKT) cells (Kim et al., 2002), NK cells (Inngjerdingen et al., 2000), platelets (Clemetson et al., 2000), eosinophils and monocytes (Bochner et al., 1999). Despite the broad expression pattern of CCR4, CCL17 has been mainly associated with Th2 type immune reactions and is implicated in the pathogenesis of several Th2-mediated diseases like atopic dermatitis (Sallusto et al., 1998a; Imai et al., 1999; Saeki & Tamaki, 2006). Besides attraction of Th2 cells, however, CCL17 may also induce chemotaxis of memory T cells, Treg and Th1 cells (Iellem et al., 2001; Lieberam & Förster, 1999). In addition, other CCR4 expressing cell types like CD8+ T cells, NK cells, basophils, eosinophils and DC may also respond to CCL17. As an inflammatory chemokine CCL17 is strongly upregulated in immature DC after stimulation with TLR ligands (Alferink et al., 2003; Lieberam & Förster, 1999). In addition, it can be upregulated following stimulation with various proinflammatory cytokines, as detailed below.
