Keywords

1 Introduction

Allergic contact dermatitis (ACD), such as metal allergy or plant allergy, is one of the most common skin diseases [1]. ACD is a kind of delayed-type hypersensitivity response, an essential immune response that eliminates the pathogens from the host. The antigens in ACD are usually not pathogens and not harmful to our body. However, once the antigen enters into skin and is recognized as harmful (sensitization phase), our host-defense system tries to eliminate them and provokes inflammation in skin, which is called the elicitation phase. In the sensitization phase, antigen-specific effector T cells are induced in the draining lymph nodes (LNs). In elicitation phase, the effector T cells infiltrate to the skin area where the antigens enter, and are activated by their specific antigen, and provoke inflammation in ACD.

The contact hypersensitivity model (CHS ) is a classic but easy and appropriate animal model for ACD [2]. By using CHS as a model of ACD, a number of studies have been performed and our knowledge of the immunological mechanisms of ACD has significantly expanded. In the past decade, several kinds of new types of immune cells have been discovered, such as regulatory T cells (Tregs) or langerin positive dermal dendritic cells (DCs). The roles of such new types of cells in ACD have been investigated using CHS, and some of the key dogma in ACD has been changing. In this section, we integrate the recent advancement of immunological mechanisms of both the sensitization and elicitation phases of CHS into the classic view, and discuss updated mechanisms on its development.

2 General Protocols of CHS

As this section discusses the mechanisms of ACD mainly based on mouse studies (CHS), we first briefly explain the experimental systems of CHS. For the induction of CHS, dinitrofluorobenzen (DNFB), oxazolone, Trinitrochlorobenzene (TNCB), and fluorescein isothiocyanate (FITC) are generally used as haptens [2]. Although there exist some variations in the protocol of CHS, the basic procedure is as follows. On day 0 (the first day of experiment), mice are painted with hapten (ex. 0.5 % DNFB) on their shaved abdominal skin or ear skin. This is the day of sensitization. Five or 7 days later, the same hapten of less concentration (ex. 0.3 % DNFB) is applied on the ear skin (elicitation), and ear thickness change is evaluated as a parameter of skin inflammation, which peaks at 24–48 h later. Histologically, severe edema and inflammatory cell infiltration are detected in dermis 24–48 h after elicitation, which correspond well with the ear thickness change.

As described above, the experimental system of CHS is quite simple, but well reflects many characteristic features of ACD.

3 Mechanisms of Sensitization Phase

3.1 Haptens and Keratinocytes

Most of the chemicals that induce ACD are small compounds called haptens , which typically have a molecular mass of less than 500. Haptens need to have chemical interactions with proteins to elicit adaptive immune responses [3]. Upon hapten application, keratinocytes are activated and produce various chemical mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and prostaglandin (PG) E2, which promote the migration and maturation of skin DCs [4–6]. Keratinocytes are activated by haptens through innate immune systems, such as Toll-like receptors (TLRs) and cytosolic NOD-like receptors (NLRs). Among the NLR family, NACHT-, LRR-, and pyrin (NALP) 3 control the production of pro-inflammatory cytokines through activation of caspase-1. Without NALP3 or its adaptor protein ASC, the production of IL-1β and IL-18 from keratinocytes was inhibited, which resulted in impaired DC migration and T cell priming, and led to impaired CHS [7–10]. Innate immune activation is also important for dendritic cell activations.

3.2 Roles of Cutaneous DC Subsets in Sensitization

There are at least three subsets of DCs in mouse skin: Langerhans cells in epidermis, langerin+ dermal DCs , and langerin–dermal DCs in dermis. After haptens enter into skin, they are captured by cutaneous DCs, which become mature and migrate to draining lymph nodes (dLNs), and present the antigen to naïve T cells to induce effector T cells. For the activation of DCs in the sensitization phase, both TLR and NOD pathways are required [11]. For example, among TLRs, TLR2 and TLR4 play critical roles for DCs maturation in CHS induced with trinitrochlorobenzene (TNCB), oxazolone (Ox), and fluorescein isothiocyanate (FITC) [12]. Among cutaneous DCs, LCs have long been considered to be central cells for antigen presentation in the sensitization phase of CHS. However, novel depletion systems of LCs (langerin–diphtheria toxin receptor [DTR] knockin mice) have revealed that langerin+ dermal DCs, but not LCs, may play a crucial role in sensitization, because depletion of langerin+ dermal DCs reduced CHS response whereas depletion of LCs did not [13–16]. Furthermore, it has also been reported that LCs play a regulatory role, rather than a stimulatory role, during sensitization in CHS by producing IL-10 [17, 18] or inducing ICOS+Foxp3+Tregs in dLNs [19]. We and others have, however, recently reported that these two populations work in a compensatory manner to initiate sensitization in CHS [20, 21]. Consistently, Batf3-deficient mice that lack langerin+ dDCs exhibited a normal CHS phenotype, suggesting the compensation of its function by other DCs [22].

Thus, although the function of LCs in sensitization remains controversial, langerin+ dDCs probably exert stimulatory effects during sensitization. As for the function of langerin- dDCs in sensitization, it has not yet been investigated intensively. However, they also appear to have stimulatory functions in sensitization, because ablation of both LCs and langerin+ dDCs prior to sensitization impairs CHS response but is unable to abrogate it completely [13, 20].

3.3 Metal Allergy and Innate Immune Activation

Nickel (Ni2+) is one of the most frequent causes of ACD, although it rarely occurs in mice. Earlier studies have reported that coadministration of adjuvant, such as complete Freund’s adjuvant or lipopolysaccharide, efficiently induced Ni2+ allergy in mice, suggesting the important role of TLR4 signaling in efficient sensitization with Ni2+. Very recently, Schmidt et al. reported that Ni2+ directly activates human TLR4 but not mouse TLR4, and that the transgenic expression of human TLR4 in TLR4-deficient mice resulted in efficient sensitization and elicitation to Ni2+ [23]. These reports indicate the crucial roles of TLR4 in Ni2+ allergy in both mice and humans.

Together, these hapten-induced irritant effects through innate immune systems are essential to the activation of cutaneous APCs and thereby determine the allergenic potential of a hapten during the sensitization phase.

3.4 Role of Mast Cells in Sensitization Phase

As the other authors describe this topic in the chapter, “Mast Cells and Basophils in Cutaneous Immune Response,” in this book, we just briefly explain this here. The role of mast cells in CHS has been controversial, because conflicting results have been reported in the CHS model using mice lacking mast cells constitutively [24–27]. However, a novel mast cell ablation system was recently established in two independent groups [28, 29]. In these mice, mast cells can be conditionally depleted through the administration of DT. Both groups reported that mice depleted of mast cells exhibited reduced CHS. It has also been revealed that mast cells stimulated DCs via intercellular adhesion molecule (ICAM)-1 or lymphocyte function-associated antigen (LFA)-1 interaction and by membrane-bound TNF- α on mast cells, and stimulated migration/maturation and subsequent T cell priming in the sensitization phase [28].

Overall, it would be very likely that mast cells play stimulatory roles both in the sensitization and elicitation phases in CHS.

4 Mechanisms of Elicitation Phase

4.1 Antigen Nonspecific Inflammation: Keratinocytes, Mast Cells, and Neutrophil Activation

The mechanism of inflammatory cell infiltration in the elicitation phase has been explained using two different inflammatory signals: antigen nonspecific and antigen specific [2, 30, 31]. Keratinocytes, neutrophils, and mast cells are the main players to create the antigen nonspecific inflammation at elicitation. First, haptens stimulate keratinocytes to produce pro-inflammatory cytokines such as IL-1β and TNF-α in an NLR-dependent manner [7, 10]. Those cytokines then activate vascular endothelial cells to express adhesion molecules such as ICAM-1 and P/E-selectins, which guide T cells in the blood to transmigrate to tissues. Haptens also increase vascular permeability through mast cell-derived histamin [29], which help neutrophils infiltrate the skin. In addition, haptens activate mast cells and keratinocytes to produce neutrophil-recruiting chemokines, such as CXCL1 and CXCL2, which further contribute to the neutrophil recruitment [25]. The initial neutrophil recruitment is proposed to be essential for subsequent T cell infiltration, because the depletion of neutrophils reduces the CD8+ T-cell infiltration and leads to impaired CHS [32, 33]. The aforementioned mechanism creates the first round of antigen nonspecific inflammation, which is an important step for subsequent antigen specific inflammation (Fig. 20.1a).

Fig. 20.1
figure 1

Representative image of neutrophil infiltration and T cells–DCs interaction in skin during elicitation phase. (a) Accumulation of neutrophils in dermis 24 h after elicitation (Green: neutrophils in Lysm-eGFP mice. Red: transferred CD4/CD8 T cells). (b) The interaction between T cells and dendritic cells in skin 24 h after elicitation (Green: Langerin positive cells in Langerin-GFP mice. Red: transferred CD4/CD8 T cells)

Full size image

4.2 Antigen Specific Inflammation: T-Cell Activation, CD4+ T (Th1/17) Cells, and CD8+ T (Tc1/Tc17) Cells

Following the antigen nonspecific inflammation, T-cell–mediated antigen-specific inflammation is initiated. When T cells infiltrate the skin, they are activated by cutaneous APCs, and produce cytokines such as IFN-γ and IL-17. In fact, stable interaction between skin DCs and T cells was observed in live imaging analysis [34] (Fig. 20.1b), and inhibition of CD86 expression by siRNA resulted in reduced inflammation [35], suggesting that effector T cells are activated locally by hapten-carrying APCs. Intriguingly, it has been reported that depletion of skin DCs in hapten-sensitized mice enhanced the effector phase of CHS [36], suggesting the existence of some DC subsets that play a regulatory function in the elicitation phase.

Cytokines produced by activated T cells then stimulate skin resident cells, which lead to further recruitment of T cells and amplify the inflammation. Each T-cell subset (i.e., Th1/Tc1 , Th2, Th17 /Tc17) activates the skin resident cells differently and forms their specific type of inflammation.

CHS was first considered to be a CD4+ T cell-mediated response as a representative of delayed-type hypersensitivity, but it is now recognized that both CD4+ and CD8+ T cells are important in the elicitation of CHS. CD8+ T cells mainly have pro-inflammatory effector functions, whereas CD4+ T cells have both pro- and anti-inflammatory functions that are dependent on their cytokine production pattern or subset.

CD4+ T helper (Th) cells and CD8+ T cytotoxic (Tc) cells can be subdivided into at least three subsets that are relevant for cutaneous immune responses: Th1/Tc1, Th2/Tc2, and Th17/Tc17 cells, respectively. Th1/Tc1 cells are characterized by the secretion of interferon (IFN)-γ; Th2/Tc2 cells by IL-4, IL-5, and IL-13; and Th17/Tc17 cells by IL-17A and IL-22 production. Although there exists some controversy regarding roles of each cytokine in CHS, the general trend is that IFN-γ from Tc1 is the major effector cytokine that provokes inflammation [37], and IFN-γ from Th1 and IL-17 from Th17/Tc17 also contribute for the full development of CHS [38–40]. It remains unknown whether Th2 cells contribute to the development of CHS, but several reports suggest that Th2 cells may also play important roles for the development of CHS in certain situations, depending on mouse strain or haptens [41].

IFN-γ or IL-17 produced by activated T cells induces various chemokine production from keratinocytes. Keratinocytes are an important source of chemokines in skin, and produce multiple chemokines, such as CXCL1, CXCL2, CXCL9, CXCL10, CCL8, CCL17, and CCL27. CXCL10 is a ligand for CXCR3, which are strongly expressed on Th1 cells and regulates their infiltration into skin [42, 43]. The CCL27-CCR10 and CCL17/22-CCR4 axes are another important mechanism for T-cell recruitment to skin [44, 45]. Neutrophil-recruiting chemokines also play important roles, because a blockade of CXCL1 or a deficiency of its receptor (CXCR2) leads to reduced CHS [32, 46].

5 Regulation of Inflammation: Regulatory T Cell (Treg)s

Evidence has accumulated regarding the regulatory mechanisms of Tregs in CHS [47]. Transfer of Tregs before elicitation suppresses the ear-swelling response [48], and depletion of endogenous Tregs before sensitization or elicitation enhances CHS response [49–51], indicating that Tregs play essential roles not only for the resolution of inflammation but also the initiation of T-cell priming. Studies of nickel allergy illustrated the existence of antigen-specific Tregs in healthy control individuals [52], suggesting the important roles of Tregs for the tolerance to allergens. As for the Tregs suppression mechanisms in CHS, several mechanisms such as IL-10 or CD39/73 dependent pathways are proposed [48, 53]. Table 20.1 summarizes the recent reports of Tregs function in CHS. Moreover, it has been revealed that Tregs display their inhibitory function by recirculating from skin to the draining [50]. As skin is an organ full of Tregs, such inhibitory mechanisms may also work in other skin diseases.

Table 20.1 An overview of recently published papers about Tregs and CHS
Full size table

6 Conclusion

Taken together, the broad view of CHS development during the sensitization and elicitation phases is summarized in Fig. 20.2 and Table 20.2.

Fig. 20.2
figure 2

A schematic view of the development of CHS. Step 1: Haptens activate keratinocytes (KCs) and mast cells directly or indirectly through innate immune systems. The activated KCs and mast cells produce various chemical mediators, which activate cutaneous DCs. Step 2: The activated DCs capture antigens and start maturation and migrate to the dLNs via afferent lymphatics. Step 3: Migrated DCs present antigen to naïve T cells in dLNs. Antigen-specific clones differentiate and proliferate into effector T cells. Tregs affect DCs function and play a suppressive role in effector T cell generation. Step 4: Upon re-exposure to haptens, KCs and mast cells are activated and produce various chemical mediators, which activate endothelial cells and cause inflammatory cell infiltration, including antigen-specific T cells. Step 5: Infiltrated antigen-specific effector T cells are activated and produce pro-inflammatory cytokines and chemokines, which activate KCs and cause further inflammatory cell infiltration. Step 6: In addition to effector T cells, Tregs infiltrate inflammatory sites and exert a suppressive function. Some infiltrated Tregs return to dLNs and may contribute to the resolution of inflammation

Full size image
Table 20.2 Summary of the functions of immune cells in CHS
Full size table

The CHS model has provided us with valuable lessons on the mechanisms of ACD as discussed above. However, there still remains a compelling need to reveal whether such findings in CHS are relevant to human ACD. In addition, recent reports suggest that innate immune cells, such as innate lymphoid cells, macrophages, natural killer T cells, or gamma-delta T cells contribute to the development of many more diseases than previously thought. Investigation of the roles of such cells in CHS would also be important for the understanding of ACD, which may lead to an innovative therapy for allergic skin inflammation.