**The Role of Prostanoids in Atopic Dermatitis**

Tetsuya Honda and Kenji Kabashima

*Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto Japan* 

#### **1. Introduction**

64 Atopic Dermatitis – Disease Etiology and Clinical Management

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Atopic dermatitis (AD) is a common pruritic and chronic inflammatory skin disease, with a prevalence of up to 3% among adults and up to 25% among children (Bieber; Guttman-Yassky et al.; Odhiambo et al., 2009). The clinical features of AD are varied, with patients generally having dry skin, but wet eczematous lesions in the acute stage and lichenification lesions in the chronic stage (Guttman-Yassky et al., 2011). In terms of histology, an increased number of lymphocytes, eosinophils, and mast cells in the dermis are detected. A barrier defect with decreased cornification and epidermal hyperplasia are also characteristic features of AD (Elias and Schmuth, 2009; Guttman-Yassky et al., 2009).

AD is a multi-factorial disease that arises from complex interaction between genetic and environmental factors. As for its pathogenesis, two models have been proposed: the outside-in model and the inside-out model (Bieber, 2008). In the outside-in model, the decreased skin barrier function caused by genetic defects, such as mutations in filaggrin, allows for the penetration of large immunogenic proteins, which subsequently cause T helper type 2 (Th2) deviated immune activation (Elias et al., 2008; Elias and Schmuth, 2009). In the inside-out model, activation of Th2 cells results in reactive epidermal hyperplasia (Nograles et al.; Ong and Leung, 2006). It has been proposed that the lack of environmental antigens during childhood lead to reduced T helper type 1 (Th1) cellmediated immunity and increased activation of Th2 cells (hygiene hypothesis). In recent reports, involvements of T helper type 17 (Th17) cells and T helper type 22 (Th22) cells have also been proposed (Koga et al., 2008; Nograles et al., 2009).

As for the treatment of AD, various therapies have been employed, and the use of topical steroids plays a major role in therapies (Guttman-Yassky et al.). Although the use of topical corticosteroids is the first-line therapy and provides rapid relief of symptoms, prolonged use can cause severe side effects such as skin atrophy. Therefore, alternative therapies with fewer and less extreme side effects are needed.

#### **2. Characteristics of prostanoids**

When tissues are exposed to diverse pathophysiological stimuli, arachidonic acid (AA) is released from membrane phospholipids and converted to lipid mediators, such as prostanoids, leukotrienes (LTs) and hydroxy-eicosatetraenoic acids (HETEs). Prostanoids, including prostaglandins (PG) and thromboxane (TX), are formed by the cyclooxygenase (COX) pathway, whereas LTs and HETEs are formed by the 5-, 12- and 15-lipoxygenase (LO) pathways. COX has two isoforms, COX-1 and COX-2. While COX-1 is constitutively

The Role of Prostanoids in Atopic Dermatitis 67

receptors as agonists or antagonists (Narumiya and FitzGerald, 2001). These genetic and pharmacological approaches have revealed new roles for prostanoids and their receptors in

In this chapter, we will discuss the recent findings regarding the role of prostanoids in skin immunity, and discuss the possible involvement of prostanoids in the pathogenesis of AD, and also the clinical potential of receptor-selective drugs as a new therapeutic target for AD.

Human bodies are exposed to external stimuli continuously. The skin plays an important role in self-defense during exposure to foreign antigens and consists of a vast array of immune cells, such as keratinocytes (KCs), T cells, B cells, mast cells, eosinophils, fibroblasts, and two types of cutaneous dendritic cells (DCs) including epidermal Langerhans cells (LCs) and dermal DCs (dDCs). In the normal human skin, immunohistochemical examinations have revealed that COX-1 is observed throughout the epidermis, whereas COX-2 exists in more differentiated suprabasilar KCs and outer root sheath cells of hair follicles (Leong et al., 1996; Torii et al., 2002). Among prostanoids, PGE2 is the main COX product in human epidermal homogenates (Hammarstrom et al., 1979). PGD2 has been detected in human skin (Hammarstrom et al., 1979), and PGD synthase is present predominantly in LCs, dDCs, dermal macrophages and mast cells, but not in KCs (Ruzicka and Aubock, 1987; Ujihara et al., 1988). Among these, mast cells have been found to be one of the major cellular sources of PGD2. TX synthase activity has been found in keratinocytes (Andoh et al., 2007) and high levels of TXB2, as a metabolite of TXA2, were detected in the cultured supernatant of LCs and DCs (Kabashima et al., 2003a). PGI2 was detected in the skin of the murine AD model (Sugimoto et al., 2006). PGF2a, was observed in skin exudates of nickel allergy patients (Lerche et al., 1989). In biopsy specimens from patients with AD, PGE2 has been determined in biologically active amounts in both lesional and perilesional skin (Fogh et al., 1989). In contrast, normal levels of eicosanoids were found in the uninvolved skin of these patients. The

above findings on the synthesis of prostanoids are summarized in Table 1.

Adult human KCs express mRNA for all subtypes of PGE2 receptors (Konger et al., 1998; Tober et al., 2006) and the expression of all PGE2 receptors has been detected in mouse KCs by immunohistochemistry (Tober et al., 2007). Mouse LCs and DCs express DP (Angeli et al., 2001), EP1, 2, 3, 4 (Kabashima et al., 2003b), and IP (Huang et al., 2001), and T cells express EP1, 2, 3, 4 (Tilley et al., 2001), IP (Takahashi et al., 2002) and TP (Nataraj et al., 2001). PGE2 suppresses T cell proliferation and differentiation in the thymus, and interleukin (IL)-1 production by acting at EP2 and EP4 *in vitro* (Nataraj et al., 2001). Mast cells express EP1, 2, 3, 4, DP, and IP (Fedyk and Phipps, 1996; Tilley et al., 2001), and PGE2 acts at EP3 to suppress their degranulation (Kunikata et al., 2005). Human eosinophils express EP2, EP4, DP, CRTH2 and TP (Nguyen et al., 2002; Schratl et al., 2007), and PGE2 seems to prolong eosinophil survival (Mita et al., 2002; Peacock et al., 1999). PGE2 suppresses TNF-a production and enhances IL-6 production from neutrophils stimulated by lipopolysaccharide (LPS) through EP2 and EP4 (Nguyen et al., 2002; Yamane et al., 2000). As summarized in Table 1, prostanoids and their receptors are produced and expressed by a wide variety of cells in the skin. This varied expression pattern of prostanoids maintains the homeostasis of the human body.

**4. Prostanoid receptor expression in skin** 

allergic and immune diseases (Honda et al., 2010).

**3. Production of prostanoids in skin** 

expressed in cells, COX-2 requires specific stimulation by substances such as acetone and the phorbol ester TPA (Narumiya et al., 1999; Scholz et al., 1995). This reaction results in the formation of an unstable endoperoxide intermediate PGH2, which, in turn, is metabolized to PGD2, PGE2, PGF2a, PGI2, and thromboxane TXA2 by specific synthases.

Prostanoids are released from cells immediately after their formation. Because they are chemically and metabolically unstable, they usually function only locally through their specific membrane receptors on target cells (Narumiya et al., 1999). Nine types and subtypes of membrane prostanoid receptors are conserved in mammals from mouse to human: two subtypes of the PGD receptor (DP (DP1) and the chemoattractant receptor homologousmolecule expressed on Th2 cells, CRTH2 (DP2)), four subtypes of the PGE receptor (EP1, EP2, EP3, and EP4), the PGF receptor (FP), the PGI receptor (IP), and the TXA receptor (TP) (Figure 1). All are G protein-coupled rhodopsin-type receptors with seven transmembrane domains (Figure 1). The main signal transduction mechanisms of these prostanoid receptors are through the regulation of intracellular cyclic adenosine monophosphate (cAMP) concentration and intracellular free calcium concentration. DP, EP2, EP4 and IP are Gscoupled receptors and elevate intracellular cAMP concentration, while EP3 and CRTH2 are Gi-coupled receptors and decrease intracellular cAMP. EP1, FP and TP are Gq and other G protein-coupled receptors, which increase intracellular calcium concentration (Narumiya et al., 1999). However, most prostanoid receptors may bind with more than one G proteincoupled receptors via their specific signaling pathway. Recently, individual prostanoid receptor gene-deficient mice have been used as models to dissect out the respective roles of each receptor in combination with the use of compounds that selectively bind to prostanoid

Fig. 1. Biosynthetic pathways of prostanoids. The formation of PGD2, PGE2, PGF2a, PGG2, PGH2, and PGI2, and TXA2 from arachidonic acid is shown. The first two steps of the pathway (i.e., conversion of arachidonic acid to PGG2 and then to PGH2) are catalyzed by COX, and the subsequent conversion of PGH2 to each prostanoid is catalyzed by the respective synthase as shown. All are G protein-coupled rhodopsin-type receptors.

expressed in cells, COX-2 requires specific stimulation by substances such as acetone and the phorbol ester TPA (Narumiya et al., 1999; Scholz et al., 1995). This reaction results in the formation of an unstable endoperoxide intermediate PGH2, which, in turn, is metabolized to

Prostanoids are released from cells immediately after their formation. Because they are chemically and metabolically unstable, they usually function only locally through their specific membrane receptors on target cells (Narumiya et al., 1999). Nine types and subtypes of membrane prostanoid receptors are conserved in mammals from mouse to human: two subtypes of the PGD receptor (DP (DP1) and the chemoattractant receptor homologousmolecule expressed on Th2 cells, CRTH2 (DP2)), four subtypes of the PGE receptor (EP1, EP2, EP3, and EP4), the PGF receptor (FP), the PGI receptor (IP), and the TXA receptor (TP) (Figure 1). All are G protein-coupled rhodopsin-type receptors with seven transmembrane domains (Figure 1). The main signal transduction mechanisms of these prostanoid receptors are through the regulation of intracellular cyclic adenosine monophosphate (cAMP) concentration and intracellular free calcium concentration. DP, EP2, EP4 and IP are Gscoupled receptors and elevate intracellular cAMP concentration, while EP3 and CRTH2 are Gi-coupled receptors and decrease intracellular cAMP. EP1, FP and TP are Gq and other G protein-coupled receptors, which increase intracellular calcium concentration (Narumiya et al., 1999). However, most prostanoid receptors may bind with more than one G proteincoupled receptors via their specific signaling pathway. Recently, individual prostanoid receptor gene-deficient mice have been used as models to dissect out the respective roles of each receptor in combination with the use of compounds that selectively bind to prostanoid

Fig. 1. Biosynthetic pathways of prostanoids. The formation of PGD2, PGE2, PGF2a, PGG2, PGH2, and PGI2, and TXA2 from arachidonic acid is shown. The first two steps of the pathway (i.e., conversion of arachidonic acid to PGG2 and then to PGH2) are catalyzed by COX, and the subsequent conversion of PGH2 to each prostanoid is catalyzed by the respective synthase as shown. All are G protein-coupled rhodopsin-type receptors.

PGD2, PGE2, PGF2a, PGI2, and thromboxane TXA2 by specific synthases.

receptors as agonists or antagonists (Narumiya and FitzGerald, 2001). These genetic and pharmacological approaches have revealed new roles for prostanoids and their receptors in allergic and immune diseases (Honda et al., 2010).

In this chapter, we will discuss the recent findings regarding the role of prostanoids in skin immunity, and discuss the possible involvement of prostanoids in the pathogenesis of AD, and also the clinical potential of receptor-selective drugs as a new therapeutic target for AD.

#### **3. Production of prostanoids in skin**

Human bodies are exposed to external stimuli continuously. The skin plays an important role in self-defense during exposure to foreign antigens and consists of a vast array of immune cells, such as keratinocytes (KCs), T cells, B cells, mast cells, eosinophils, fibroblasts, and two types of cutaneous dendritic cells (DCs) including epidermal Langerhans cells (LCs) and dermal DCs (dDCs). In the normal human skin, immunohistochemical examinations have revealed that COX-1 is observed throughout the epidermis, whereas COX-2 exists in more differentiated suprabasilar KCs and outer root sheath cells of hair follicles (Leong et al., 1996; Torii et al., 2002). Among prostanoids, PGE2 is the main COX product in human epidermal homogenates (Hammarstrom et al., 1979). PGD2 has been detected in human skin (Hammarstrom et al., 1979), and PGD synthase is present predominantly in LCs, dDCs, dermal macrophages and mast cells, but not in KCs (Ruzicka and Aubock, 1987; Ujihara et al., 1988). Among these, mast cells have been found to be one of the major cellular sources of PGD2. TX synthase activity has been found in keratinocytes (Andoh et al., 2007) and high levels of TXB2, as a metabolite of TXA2, were detected in the cultured supernatant of LCs and DCs (Kabashima et al., 2003a). PGI2 was detected in the skin of the murine AD model (Sugimoto et al., 2006). PGF2a, was observed in skin exudates of nickel allergy patients (Lerche et al., 1989). In biopsy specimens from patients with AD, PGE2 has been determined in biologically active amounts in both lesional and perilesional skin (Fogh et al., 1989). In contrast, normal levels of eicosanoids were found in the uninvolved skin of these patients. The above findings on the synthesis of prostanoids are summarized in Table 1.

#### **4. Prostanoid receptor expression in skin**

Adult human KCs express mRNA for all subtypes of PGE2 receptors (Konger et al., 1998; Tober et al., 2006) and the expression of all PGE2 receptors has been detected in mouse KCs by immunohistochemistry (Tober et al., 2007). Mouse LCs and DCs express DP (Angeli et al., 2001), EP1, 2, 3, 4 (Kabashima et al., 2003b), and IP (Huang et al., 2001), and T cells express EP1, 2, 3, 4 (Tilley et al., 2001), IP (Takahashi et al., 2002) and TP (Nataraj et al., 2001). PGE2 suppresses T cell proliferation and differentiation in the thymus, and interleukin (IL)-1 production by acting at EP2 and EP4 *in vitro* (Nataraj et al., 2001). Mast cells express EP1, 2, 3, 4, DP, and IP (Fedyk and Phipps, 1996; Tilley et al., 2001), and PGE2 acts at EP3 to suppress their degranulation (Kunikata et al., 2005). Human eosinophils express EP2, EP4, DP, CRTH2 and TP (Nguyen et al., 2002; Schratl et al., 2007), and PGE2 seems to prolong eosinophil survival (Mita et al., 2002; Peacock et al., 1999). PGE2 suppresses TNF-a production and enhances IL-6 production from neutrophils stimulated by lipopolysaccharide (LPS) through EP2 and EP4 (Nguyen et al., 2002; Yamane et al., 2000). As summarized in Table 1, prostanoids and their receptors are produced and expressed by a wide variety of cells in the skin. This varied expression pattern of prostanoids maintains the homeostasis of the human body.

The Role of Prostanoids in Atopic Dermatitis 69

For example, PGE2-EP1 signaling has been reported to facilitate Th1 differentiation in the sensitization process through the skin (Nagamachi et al., 2007). PGE2 produced by DCs in draining lymph nodes (dLNs) stimulates EP1 receptors on naïve CD4+ and CD8+ T cells and promotes Th1 and Tc1 differentiation (Nagamachi et al., 2007). PGI2-IP signaling promotes Th1 and Tc1 differentiation through a cAMP dependent mechanism (Nakajima et al., 2010). Intriguingly, IP deficient mice showed enhanced Th2 response such as elevated IgE concentration in serum in the mouse OVA-induced asthma model (Nagao et al., 2003), suggesting that lack of PGI2-IP signaling might result in a Th2 biased immune response

The regulatory mechanism of prostanoid signaling on Th differentiation is complex, because it depends on the context of immune system. For example, PGE2-EP2/EP4 signaling regulates Th1 and Th17 differentiation (Yao et al., 2009). In a weaker co-stimulation signaling through CD28, PGE2 suppresses the Th1 differentiation via EP2 and EP4 receptors. In the case of strong co-stimulation signaling, however, stimulation of EP2 and EP4 signaling conversely facilitates the Th1 differentiation through a PI3-kinase-dependent mechanism (Yao et al., 2009). These results suggest that the action of prostanoid receptor signaling can be changed in a context-dependent manner. EP2 and EP4 signaling also regulates the Th17 differentiation. Th17 is a recently identified Th subset, and can be detected in a number of diseases, including AD (Guttman-Yassky et al., 2011; Koga et al., 2008). *In vitro*, Th17 differentiation is induced from naïve T cells in the presence of IL-6 and TGF-b. In this condition, PGE2 acts on naïve T cells through EP2/EP4 signaling and suppress the Th17 differentiation in a cAMP-dependent manner. However, PGE2-EP2/EP4 signaling also acts on DCs and increases the IL-23 production from the DCs. Thus, PGE2 facilitates the expansion of Th17 (Yao et al., 2009). The blockade of EP4 signaling consistently ameliorated the disease progression in a CHS model and an EAE model, which are mediated by Th1 and Th17 cells, respectively (Yao et al., 2009). These results clearly indicate the importance of prostanoid signaling in Th differentiation *in vivo.* The facilitation

effect of PGE2 on Th17 is also reported in human T cells (Boniface et al., 2009).

In the initial step of sensitization in AD, allergens which enter the skin are captured by skin DCs and presented to the naïve T cells in the dLNs. Prostanoids can regulate this step by affecting the migration ability or antigen presentation ability of the skin DCs (Figure 2). PGE2, which is produced by KCs, acts on EP4 on LCs, and stimulates the migration of LCs (Kabashima et al., 2003b). Conversely, stimulation of DP on DCs inhibits the migration of skin DCs. Topical administration of DP inhibits the migration of DCs to dLNs and significantly suppresses the development of the mouse AD model (Angeli et al., 2001; Angeli et al., 2004). Prostanoids also regulate DC-T cell interaction in the priming of naïve T cells (Kabashima et al., 2003a). Cutaneous DCs produce abundant TXA2, which acts on naïve T cells and increases the motility of T cells, which impairs the stable DC-T cell interaction (Kabashima et al., 2003a). TP-deficient mice or wild-type mice treated with a TP antagonist during the sensitization period show enhanced CHS responses, indicating that TP signaling

Although the role of IgE in AD is still controversial (Guttman-Yassky et al., 2011), high serum IgE is one of the hallmarks of AD. Compared to the analysis of T cells and DCs, the reports about the role of prostanoids on B cells are relatively scarce. From the *in vivo* data using COX-2 deficient mice or IP deficient mice, which show increased IgE production in OVA sensitization

**7. Prostanoids as regulatory factors of DC function** 

negatively regulates the priming of T cells *in vivo*.

through the inhibition of Th1 differentiation.


PG; prostaglandin, S; synthase, m; mouse, h; human Modified from the reference by Tilley et al.

Table 1. Expression of prostanoid synthases and prostanoid receptors in the skin
