**Keratinocytes, Innate Immunity and Allergic Contact Dermatitis - Opportunities for the Development of** *In Vitro* **Assays to Predict the Sensitizing Potential of Chemicals**

Jochem W. van der Veen1,2, Rob J. Vandebriel2, Henk van Loveren1,2 and Janine Ezendam2 *1Maastricht University, Department of Toxicogenomics, The Netherlands 2National Institute for Public Health and the Environment, The Netherlands* 

#### **1. Introduction**

38 Contact Dermatitis

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> Allergic contact dermatitis (ACD) is the most prevalent form of immunotoxicity in humans characterized by clinical manifestations such as red rashes, itchy skin and blisters. The disease is caused by skin sensitizers which are allergenic low-molecular weight chemicals. ACD is an important occupational disease that gives problems at different workplaces, including hair dressers, metal workers, construction workers, and cleaners. In addition, ACD can develop in the general population as well, since several consumer products contain skin sensitizers. Important skin sensitizers are metals (nickel, chromium), fragrances, hair dye ingredients and preservatives (Kimber et al., 2002a; Vandebriel & van Loveren, 2010).

> ACD is a typical type IV (delayed-type) hypersensitivity response that develops in two phases, the initiation phase in which the immune system is sensitized and the elicitation phase in which the clinical symptoms develop. At initiation, the low-molecular weight and polarity of skin sensitizers allow for penetration of the stratum corneum of the skin. In addition, protein reactivity is a hallmark of skin sensitizers. After binding to proteins in the skin, hapten-carrier complexes are formed (Kimber et al., 2002a; Berard et al., 2003; Vandebriel & van Loveren, 2010). The formation of these hapten-protein complexes is crucial, since the chemicals themselves are not immunogenic and priming of T cells can only occur after formation of these complexes. After taking up these hapten-carrier complexes, immature Langerhans cells and dendritic cells start to migrate to the draining lymph node and become potent T cell activators through the upregulation of costimulatory molecules (Gaspari, 1997; Kimber et al., 2002a; Berard et al., 2003; Vocanson et al., 2009; Vandebriel & van Loveren, 2010). Hapten specific T cells are activated through a combination of haptenized peptide presentation on major histocompatibility complex (MHC) molecules and costimulatory molecules, such as CD54, CD80 and CD86. Activated T cells undergo clonal expansion, thereby generating skin homing CD8+ Tc1/Tc17 and CD4+ Th1/Th17 effector T

Keratinocytes, Innate Immunity and Allergic Contact Dermatitis - Opportunities

**2. Innate immune responses: Toll-like receptors and other pattern** 

The induction of innate signaling pathways by skin sensitizers in keratinocytes is believed to be a crucial factor in skin sensitization and a requirement for activation of Langerhans cells and dendritic cells and subsequent T cell priming (Martin et al., 2011). Studies have demonstrated that human primary keratinocytes express mRNA for Toll-like receptors (TLRs), such as TLR 1, 2, 3, 4, 5 and 9 (Kollisch et al., 2005; Son et al., 2006), which are important receptors of the innate immune system. Upon TLR activation, keratinocytes are able to produce a range of cytokines and chemokines, which allows for attraction of other

TLRs are the first family to be identified of the germ-line encoded pattern recognition receptors (PRR). These receptors are involved in the first line of defense against pathogens. To date, 10 different TLRs have been identified in humans (Boehme & Compton, 2004). The TLRs are known to recognize various pathogen associated molecular patterns (PAMPs), which are conserved and essential molecules of pathogens. Some well-known examples are lipopolysaccharide (LPS) that is recognized by TLR4, double-stranded RNA recognized by TLR3 and lipopeptides recognized by TLR2 (Hosogi et al., 2004; Lebre et al., 2007; Kumar et al., 2009). The primary function of TLRs is the initial recognition of pathogenic microorganisms and subsequent activation of the innate immune response. Most pathogens express multiple PAMPs and are thus recognized by multiple TLRs. Activated TLRs will promote the phagocytosis of pathogens in innate immune cells such as macrophages and induce a respiratory burst, production of ROS and RNS, to neutralize pathogens. Combined, this promotes the presentation of pathogen specific peptides to cells of the adaptive immune system. Furthermore, the secreted reactive oxygen species can act as signaling molecules

Fig. 1. Keratinocyte responses to sensitizers

immune cells, such as dendritic cells (Lebre et al., 2007).

**recognition receptors** 

for the Development of *In Vitro* Assays to Predict the Sensitizing Potential of Chemicals 41

In the development of *in vitro* assays, human keratinocyte cell lines, primary keratinocytes, and 3D skin models are used. In order to base read-outs of these assays on toxicological relevant pathways it is important to understand the underlying mechanisms of skin sensitization. In this chapter, an overview of current knowledge on the role of innate immune and oxidative stress pathways in skin sensitization will be provided together with the relevance of these pathways for the development of *in vitro* assays using keratinocytes.

cells that enter the blood circulation (Kimber et al., 2002a; Freudenberg et al., 2009 ; Vocanson et al., 2009; Vandebriel & van Loveren, 2010). After being sensitized, subsequent skin contact with the hapten will elicit skin symptoms within 48 hours caused by the hapten specific T cells that migrate into the skin. Here they recognize the protein/hapten complex presented by either dendritic cells or keratinocytes. Upon recognition, the T cells become activated and start to produce typical Th1 and Th17 response cytokines, such as IFN-γ, IL-12, IL-17, and IL-23 (Kimber & Dearman, 2002; Zhao et al., 2009).

The identification of chemicals with skin sensitizing capacity is of great importance to ensure the safety of industrial chemicals and cosmetic ingredients. The current testing methods for skin sensitization are animal tests, either using guinea pigs (Guinea Pig Maximization Test or the Buehler Test) or mice (the murine Local Lymph Node Assay) (Kimber et al., 1994; Gerberick et al., 2007a). Recently, the pressure to develop non-animal testing strategies has increased due to public and political influence. The 7th amendment to the European Union (EU) Cosmetics Directive forbids the use of animal tests and the sensitizing potential of a great number of chemicals will have to be evaluated within the framework of Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH). Therefore, there is a great demand for in vitro alternative test methods that can replace the currently used animal assays. Knowledge on physical-chemical properties of haptens together with insight in the immunological mechanisms that lead to sensitization need to be applied in the alternative methods that are currently being developed or validated. The elucidation of pathways that play a role in the initiation phase of skin sensitization have long been subject of investigations. Several *in vitro* and *in vivo* studies have shown that pathways linked to innate immunity and oxidative stress are important in the first phase of skin sensitization (Natsch, 2009; Vandebriel et al., 2010; Martin et al., 2011). Activation of these pathways induces cell stress and damage, and production of pro-inflammatory cytokines and chemokines. In this way, 'danger' signals are produced in the skin, which are considered to be required for further development of an adaptive immune response (Kimber et al., 2002b; Martin et al., 2011).

In the skin, keratinocytes are abundantly present and these cells are the first to encounter haptens that penetrate through the skin. Keratinocytes are considered to be key players in the initiation phase of skin sensitization for several reasons (Figure 1). Keratinocytes contain enzymes with metabolic activity required for the conversion of prohaptens into biologically active haptens, thereby facilitating binding to proteins (Van Pelt et al., 1990; Gelardi et al., 2001). In addition, keratinocytes have been shown to express chemotactic factors upon exposure to sensitizers, including chemokines (CXCL8, CXCL9, CXCL10, CXCL11) and adhesion molecules (ICAM-1). These attract more immune cells to the exposed skin area, thereby strengthening the immune response (Gaspari, 1997; Albanesi, 2010). Keratinocytes are important in the elicitation phase of ACD as well, since they are able to present antigen to the surroundings through both MHC class I and MHC class II molecules (Albanesi et al., 2005; Nestle et al., 2009). In addition, after being targeted by IFN-γ, keratinocytes upregulate costimulatory molecules such as CD80 and are able to function as antigen presenting cells and facilitating activation of hapten specific T cells (Gaspari, 1997; Albanesi, 2010). Hence, keratinocytes are important in the sensitization and elicitation phase of ACD and for that reason these cells are often used for the development of *in vitro* assays for skin sensitization testing (Van Och et al., 2005; Corsini et al., 2009; Vandebriel et al., 2010; Galbiati et al., 2011).

In the development of *in vitro* assays, human keratinocyte cell lines, primary keratinocytes, and 3D skin models are used. In order to base read-outs of these assays on toxicological relevant pathways it is important to understand the underlying mechanisms of skin sensitization. In this chapter, an overview of current knowledge on the role of innate immune and oxidative stress pathways in skin sensitization will be provided together with the relevance of these pathways for the development of *in vitro* assays using keratinocytes.

Fig. 1. Keratinocyte responses to sensitizers

40 Contact Dermatitis

cells that enter the blood circulation (Kimber et al., 2002a; Freudenberg et al., 2009 ; Vocanson et al., 2009; Vandebriel & van Loveren, 2010). After being sensitized, subsequent skin contact with the hapten will elicit skin symptoms within 48 hours caused by the hapten specific T cells that migrate into the skin. Here they recognize the protein/hapten complex presented by either dendritic cells or keratinocytes. Upon recognition, the T cells become activated and start to produce typical Th1 and Th17 response cytokines, such as IFN-γ, IL-

The identification of chemicals with skin sensitizing capacity is of great importance to ensure the safety of industrial chemicals and cosmetic ingredients. The current testing methods for skin sensitization are animal tests, either using guinea pigs (Guinea Pig Maximization Test or the Buehler Test) or mice (the murine Local Lymph Node Assay) (Kimber et al., 1994; Gerberick et al., 2007a). Recently, the pressure to develop non-animal testing strategies has increased due to public and political influence. The 7th amendment to the European Union (EU) Cosmetics Directive forbids the use of animal tests and the sensitizing potential of a great number of chemicals will have to be evaluated within the framework of Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH). Therefore, there is a great demand for in vitro alternative test methods that can replace the currently used animal assays. Knowledge on physical-chemical properties of haptens together with insight in the immunological mechanisms that lead to sensitization need to be applied in the alternative methods that are currently being developed or validated. The elucidation of pathways that play a role in the initiation phase of skin sensitization have long been subject of investigations. Several *in vitro* and *in vivo* studies have shown that pathways linked to innate immunity and oxidative stress are important in the first phase of skin sensitization (Natsch, 2009; Vandebriel et al., 2010; Martin et al., 2011). Activation of these pathways induces cell stress and damage, and production of pro-inflammatory cytokines and chemokines. In this way, 'danger' signals are produced in the skin, which are considered to be required for further development of an

In the skin, keratinocytes are abundantly present and these cells are the first to encounter haptens that penetrate through the skin. Keratinocytes are considered to be key players in the initiation phase of skin sensitization for several reasons (Figure 1). Keratinocytes contain enzymes with metabolic activity required for the conversion of prohaptens into biologically active haptens, thereby facilitating binding to proteins (Van Pelt et al., 1990; Gelardi et al., 2001). In addition, keratinocytes have been shown to express chemotactic factors upon exposure to sensitizers, including chemokines (CXCL8, CXCL9, CXCL10, CXCL11) and adhesion molecules (ICAM-1). These attract more immune cells to the exposed skin area, thereby strengthening the immune response (Gaspari, 1997; Albanesi, 2010). Keratinocytes are important in the elicitation phase of ACD as well, since they are able to present antigen to the surroundings through both MHC class I and MHC class II molecules (Albanesi et al., 2005; Nestle et al., 2009). In addition, after being targeted by IFN-γ, keratinocytes upregulate costimulatory molecules such as CD80 and are able to function as antigen presenting cells and facilitating activation of hapten specific T cells (Gaspari, 1997; Albanesi, 2010). Hence, keratinocytes are important in the sensitization and elicitation phase of ACD and for that reason these cells are often used for the development of *in vitro* assays for skin sensitization testing (Van Och et al., 2005; Corsini et al., 2009; Vandebriel et al., 2010; Galbiati et al., 2011).

12, IL-17, and IL-23 (Kimber & Dearman, 2002; Zhao et al., 2009).

adaptive immune response (Kimber et al., 2002b; Martin et al., 2011).
