**1. Introduction**

80 Atopic Dermatitis – Disease Etiology and Clinical Management

Yoshimura-Uchiyama, C., Iikura, M., Yamaguchi, M., Nagase, H., Ishii, A., Matsushima,

*15*, 633-640.

Clin Exp Allergy *34*, 1283-1290.

inflammation through Th1 cell differentiation and Th17 cell expansion. Nat Med

K., Yamamoto, K., Shichijo, M., Bacon, K.B., and Hirai, K. (2004). Differential modulation of human basophil functions through prostaglandin D2 receptors DP and chemoattractant receptor-homologous molecule expressed on Th2 cells/DP2.

> Chemokines are a superfamily of potent leukocyte chemoattractant cytokines with a molecular weight of 8-12 kDa. Historically, many chemokines had more than one name until the 1999 Keystone Symposium on Chemokines, when a new nomenclature was introduced (Zlotnik & Yoshie, 2000). Chemokines have been subdivided into four subfamilies on the basis of the position of either one or two cysteine residues located near the N-terminus of the protein and an L (ligand) was added (CXCL, CCL, CL and CXXXCL) to designate all chemokines as ligands of their respective receptors (R). The chemokine network comprises about 50 chemokines, as well as 20 classical (10 CCRs, 7 CXCRs, two XCR and a single CX3CR) and 3 atypical chemokine receptors (Duffy antigen receptor for chemokines = DARC, CC-X-chemokine receptor (CCX-CKR), and the D6 molecule) (Cyster, 2005; Comerford & McColl, 2011; Hansell & Nibbs, 2007; Ransohoff, 2009; Sallusto & Baggiolini, 2008). Many ligands bind multiple receptors, although each of them bind in a slightly different way, thereby inducing distinct downstream responses. The timing and venue of specific ligand-receptor interactions determines the nature of various biological processes. Although their best known function is the regulation of leukocyte migration, chemokines also enhance cell adhesion or costimulation, and stimulate myelopoiesis, tumor growth or angiogenesis (Ransohoff, 2009; Sallusto & Baggiolini, 2008; Viola & Luster, 2008). In addition, chemokines participate in the organization of the microenvironmental architecture of primary and secondary lymphoid organs during physiological and pathological conditions (Cyster, 2005). Importantly, a defined subset of chemokines and their receptors drive certain inflammatory immune responses to protect the body against microbial and environmental pathogens. Dysregulation of such chemokines may contribute to the pathogenesis of inflammatory diseases, like acute respiratory distress syndrome, multiple sclerosis, inflammatory bowel diseases, atherosclerosis, or rheumatoid arthritis (Charo & Ransohoff, 2006). Furthermore, chemokines produced in barrier organs are known to

Expression and Function of CCL17 in Atopic Dermatitis 83

a disturbance of the epidermal barrier and subsequent activation of keratinocytes by penetrating microbial and environmental pathogens or allergens. Release of proinflammatory cytokines, such as thymic stromal lymphopoetin (TSLP), IL-25 and IL-33 initially leads to the activation and/or attraction of innate immune cells, including cutaneous DC, and induction of a Th2-biased immune response (Carmi-Levy et al., 2011). During this phase a panel of homeostatic and inflammatory chemokines are upregulated in the affected skin areas, promoting the attraction of pro-allergic effector cells, like mast cells, eosinophils, inflammatory DC and cutaneous lymphocyte antigen (CLA)+CCR4+ skinhoming Th cells. Chemokines associated with an AD phenotype comprise CCL1, CCL2, CCL3, CCL5, CCL11, CCL13, CCL17, CCL18, CCL20, CCL22, CCL26, CCL27 and CX3CL1, and serum levels of CCL11, CCL17, CCL22, CCL26, CCL27 and CX3CL1 correlate with

CCL1, CCL11 and CCL26 have been shown to interact with CCR8 and CCR3 on endothelial cells in AD, thereby inducing angiogenesis and tissue remodelling (Owczarek et al., 2010; Salcedo et al., 2001; Yawalkar et al., 1999). CCL1-CCR8 interactions also lead to recruitment of T cells and LC-like DC to the inflamed skin (Gombert et al., 2005), and have been

CCL27 is already expressed under homeostatic conditions and is further induced under inflammatory conditions in epidermal keratinocytes. In addition, CCL27 binds to the extracellular matrix and is displayed on endothelial cells in inflamed skin (Homey et al., 2002). CCR10, the receptor of CCL27, is preferentially expressed by CLA+CD4+ or CD8+ memory T cells (Hudak et al., 2002). Neutralization of CCL27 significantly inhibited inflammatory skin responses in mouse models that mimic allergic contact dermatitis and AD (Homey et al., 2002; Hudak et al., 2002). In addition to CCR10, the skin-homing CLA+ memory T cells express CCR4 on their cell surface. As further discussed below, CCR4 and CCR10 ligands cooperate in the recruitment of memory T cells to sites of skin inflammation

The human chemokine CCL18 is one of the most highly expressed chemokines produced by DC in lesional skin of AD patients but not in psoriasis (Fujita et al., 2011; Gros et al., 2009; Pivarcsi et al., 2004). Pivarcsi et al. showed that allergen exposure, as well as staphylococcal products induced its expression *in vitro* and *in vivo*. Although the receptor of CCL18 is still unknown, this chemokine has been shown to attract CLA+ memory T

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.,

associated with emigration of LC to the draining LN (Qu et al., 2004).

disease activity (Homey et al., 2006).

(Mirshahpanah et al., 2008; Reiss et al., 2001).

**3. Function and expression of CCL17** 

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

cells (Günther et al., 2005).

substantially contribute to the pathogenesis of atopic diseases, like asthma, rhinitis and atopic dermatitis (D'Ambrosio, 2005; Homey et al., 2006; Pease, 2011).

### **1.1 Chemokine signaling**

Chemokine receptors are pertussis toxin-sensitive heterotrimeric (αβγ) G-protein coupled receptors (GPCR) with seven helical membrane-spanning regions connected by extramembranous loops. Ligand binding induces a series of intracellular signalling pathways, leading to changes in actin cytoskeleton, activation of integrins, cell migration and alterations of the cellular activation status. For detailed information on chemokine receptor signaling pathways we refer the reader to other review articles covering this topic specifically (Randolph et al., 2008; Thelen & Stein, 2008; Wu, 2005). Briefly, chemokine ligand binding induces activation of the G proteins associated with the chemokine receptor causing the dissociation of Gα-GTP from the receptor and from the Gβγ heterodimer. Whereas the Gα subunit inhibits adenylyl cyclases, the Gβγ subunit is able to activate several effectors, including phosphatidylinositol-3-OH-kinase (PI3K) and members of the phospholipase C family. In addition, chemokine receptor signaling leads to activation of small GTPases of the Rho and Ras families.

#### **1.2 Regulation of chemokine receptor expression**

Many chemokine receptor genes are constitutively expressed and their cell surface expression ranges from as few as around 1000/cell in the case of CXCR4 to around 40,000/cell in the case of CXCR2 on neutrophils (Holmes et al., 1991; Loetscher et al., 1994). Chemokine receptor expression can be regulated by two major mechanisms: enhanced/reduced gene expression and/or desensitization. Altered gene expression of chemokine receptors is evident in naïve T lymphocytes expressing high levels of homeostatic receptors that mediate circulation through secondary lymphoid organs. Once activated, homeostatic receptors are down-regulated, and inflammatory chemokine receptors are up-regulated on effector cells ( Ebert & McColl, 2002; Sallusto et al., 1998a). This allows effector cells to migrate into tissues where the ligands for the inflammatory receptors are being expressed. This mechanism also regulates DC trafficking into tissues, within tissues and from tissues into draining LN. Chemokine receptors may also undergo transient homologous or heterologous desensitization (Aragay et al., 1998; Mashikian et al., 1999). Binding of the ligand leads to phosphorylation-dependent internalization of the receptor and abolishes further chemokine stimulation. This is called homologous desensitization. In contrast, heterologous desensitization happens when molecules other than those that bind directly can desensitize chemokine receptors, for example by utilization of common intracellular signalling pathways, or by alterations in the phosphorylation status of the receptor.

#### **2. Chemokine and chemokine receptor expression in AD**

AD represents a chronic relapsing skin disease induced by epidermal barrier dysfunctions, sensitization to environmental allergens, microbial stimulation, and genetic predisposition (Bieber, 2008). The lesional skin contains many signs of leukocytic inflammation, resulting from enhanced production of proinflammatory cytokines and chemokines (Gros et al., 2009; Homey et al., 2006; Pastore et al., 2004). One of the initial events in the pathogenesis of AD is

substantially contribute to the pathogenesis of atopic diseases, like asthma, rhinitis and

Chemokine receptors are pertussis toxin-sensitive heterotrimeric (αβγ) G-protein coupled receptors (GPCR) with seven helical membrane-spanning regions connected by extramembranous loops. Ligand binding induces a series of intracellular signalling pathways, leading to changes in actin cytoskeleton, activation of integrins, cell migration and alterations of the cellular activation status. For detailed information on chemokine receptor signaling pathways we refer the reader to other review articles covering this topic specifically (Randolph et al., 2008; Thelen & Stein, 2008; Wu, 2005). Briefly, chemokine ligand binding induces activation of the G proteins associated with the chemokine receptor causing the dissociation of Gα-GTP from the receptor and from the Gβγ heterodimer. Whereas the Gα subunit inhibits adenylyl cyclases, the Gβγ subunit is able to activate several effectors, including phosphatidylinositol-3-OH-kinase (PI3K) and members of the phospholipase C family. In addition, chemokine receptor signaling leads to activation of

Many chemokine receptor genes are constitutively expressed and their cell surface expression ranges from as few as around 1000/cell in the case of CXCR4 to around 40,000/cell in the case of CXCR2 on neutrophils (Holmes et al., 1991; Loetscher et al., 1994). Chemokine receptor expression can be regulated by two major mechanisms: enhanced/reduced gene expression and/or desensitization. Altered gene expression of chemokine receptors is evident in naïve T lymphocytes expressing high levels of homeostatic receptors that mediate circulation through secondary lymphoid organs. Once activated, homeostatic receptors are down-regulated, and inflammatory chemokine receptors are up-regulated on effector cells ( Ebert & McColl, 2002; Sallusto et al., 1998a). This allows effector cells to migrate into tissues where the ligands for the inflammatory receptors are being expressed. This mechanism also regulates DC trafficking into tissues, within tissues and from tissues into draining LN. Chemokine receptors may also undergo transient homologous or heterologous desensitization (Aragay et al., 1998; Mashikian et al., 1999). Binding of the ligand leads to phosphorylation-dependent internalization of the receptor and abolishes further chemokine stimulation. This is called homologous desensitization. In contrast, heterologous desensitization happens when molecules other than those that bind directly can desensitize chemokine receptors, for example by utilization of common intracellular signalling pathways, or by alterations in the

AD represents a chronic relapsing skin disease induced by epidermal barrier dysfunctions, sensitization to environmental allergens, microbial stimulation, and genetic predisposition (Bieber, 2008). The lesional skin contains many signs of leukocytic inflammation, resulting from enhanced production of proinflammatory cytokines and chemokines (Gros et al., 2009; Homey et al., 2006; Pastore et al., 2004). One of the initial events in the pathogenesis of AD is

atopic dermatitis (D'Ambrosio, 2005; Homey et al., 2006; Pease, 2011).

**1.1 Chemokine signaling** 

small GTPases of the Rho and Ras families.

phosphorylation status of the receptor.

**2. Chemokine and chemokine receptor expression in AD** 

**1.2 Regulation of chemokine receptor expression** 

a disturbance of the epidermal barrier and subsequent activation of keratinocytes by penetrating microbial and environmental pathogens or allergens. Release of proinflammatory cytokines, such as thymic stromal lymphopoetin (TSLP), IL-25 and IL-33 initially leads to the activation and/or attraction of innate immune cells, including cutaneous DC, and induction of a Th2-biased immune response (Carmi-Levy et al., 2011). During this phase a panel of homeostatic and inflammatory chemokines are upregulated in the affected skin areas, promoting the attraction of pro-allergic effector cells, like mast cells, eosinophils, inflammatory DC and cutaneous lymphocyte antigen (CLA)+CCR4+ skinhoming Th cells. Chemokines associated with an AD phenotype comprise CCL1, CCL2, CCL3, CCL5, CCL11, CCL13, CCL17, CCL18, CCL20, CCL22, CCL26, CCL27 and CX3CL1, and serum levels of CCL11, CCL17, CCL22, CCL26, CCL27 and CX3CL1 correlate with disease activity (Homey et al., 2006).

CCL1, CCL11 and CCL26 have been shown to interact with CCR8 and CCR3 on endothelial cells in AD, thereby inducing angiogenesis and tissue remodelling (Owczarek et al., 2010; Salcedo et al., 2001; Yawalkar et al., 1999). CCL1-CCR8 interactions also lead to recruitment of T cells and LC-like DC to the inflamed skin (Gombert et al., 2005), and have been associated with emigration of LC to the draining LN (Qu et al., 2004).

CCL27 is already expressed under homeostatic conditions and is further induced under inflammatory conditions in epidermal keratinocytes. In addition, CCL27 binds to the extracellular matrix and is displayed on endothelial cells in inflamed skin (Homey et al., 2002). CCR10, the receptor of CCL27, is preferentially expressed by CLA+CD4+ or CD8+ memory T cells (Hudak et al., 2002). Neutralization of CCL27 significantly inhibited inflammatory skin responses in mouse models that mimic allergic contact dermatitis and AD (Homey et al., 2002; Hudak et al., 2002). In addition to CCR10, the skin-homing CLA+ memory T cells express CCR4 on their cell surface. As further discussed below, CCR4 and CCR10 ligands cooperate in the recruitment of memory T cells to sites of skin inflammation (Mirshahpanah et al., 2008; Reiss et al., 2001).

The human chemokine CCL18 is one of the most highly expressed chemokines produced by DC in lesional skin of AD patients but not in psoriasis (Fujita et al., 2011; Gros et al., 2009; Pivarcsi et al., 2004). Pivarcsi et al. showed that allergen exposure, as well as staphylococcal products induced its expression *in vitro* and *in vivo*. Although the receptor of CCL18 is still unknown, this chemokine has been shown to attract CLA+ memory T cells (Günther et al., 2005).
