Effector and regulatory mechanisms in allergic contact dermatitis

Authors


Marc Vocanson
INSERM U851
IFR 128 BioSciences Lyon-Gerland
21 av Tony Garnier
69365 Lyon
France

Abstract

Allergic contact dermatitis (ACD), one of the commonest occupational diseases, is a T-cell-mediated skin inflammation caused by repeated skin exposure to contact allergens, i.e. nonprotein chemicals called haptens. Allergic contact dermatitis, also referred to as contact hypersensitivity, is mediated by CD8+ T cells, which are primed in lymphoid organs during the sensitization phase and are recruited in the skin upon re-exposure to the hapten. Subsets of CD4+ T cells endowed with suppressive activity are responsible for both the down-regulation of eczema in allergic patients and the prevention of priming to haptens in nonallergic individuals. Therefore, ACD should be considered as a breakdown of the skin immune tolerance to haptens. Recent advances in the pathophysiology of ACD have demonstrated the important role of skin innate immunity in the sensitization process and have revisited the dogma that Langerhans cells are mandatory for CD8+ T-cell priming. They have also introduced mast cells as a pivotal actor in the magnitude of the inflammatory reaction. Finally, the most recent studies address the nature, the mode and the site of action of the regulatory T cells that control the skin inflammation with the aim of developing new strategies of tolerance induction in allergic patients.

Abbreviations:
ACD

allergic contact dermatitis

Ag

antigen

BAC

bacterial artificial chromosome

CHS

contact hypersensitivity

CLA

cutaneous lymphocyte antigen

CTL

cytotoxic T lymphocyte

DC

dendritic cell

dDC

dermal DC

dLN

draining lymph node

DNFB

2,4-dinitro-1-fluorobenzene

DT

diphtheria toxin

DTH

delayed-type hypersensitivity

EGFP

enhanced green fluorescent protein

FITC

fluorescein isothiocyanate

ICAM

intracellular adhesion molecule

IFN

interferon

Ig

immunoglobulin

IL

interleukin

iNKT

invariant NKTs

KI

knock-in

LC

Langerhans cell

LN

lymph node

LZT

low-zone tolerance

mAb

monoclonal antibody

MHC

major histocompatibility complex

ROS

reactive oxygen species

TARC

thymus- and activation-regulated chemokine

TCM

central memory T cell

TCR

T-cell receptor

TEFF

effector T cell

TEM

effector memory T cell

TGF

transforming growth factor

Th

T helper

TNCB

trinitrochlorobenzene

TNF

tumour necrosis factor

TNP

trinitrophenyl

Tregs

regulatory T cells

UV

ultraviolet

VCAM

vascular cell adhesion molecule

Contact dermatitis is a common inflammatory skin disease in industrialized countries with a great socioeconomic impact. It is one of the most common occupational diseases (1, 2). As the outermost barrier of the human body, the skin is the first to encounter chemical and physical factors from the environment. Two main types of contact dermatitis may be distinguished, according to the pathophysiological mechanisms involved: (i) irritant contact dermatitis is the expression of the proinflammatory and toxic effects of xenobiotics able to activate the skin’s innate immune system and (ii) allergic contact dermatitis (ACD) requires the activation of antigen (Ag)-specific acquired immunity, leading to the development of effector T cells (TEFF), which mediate the skin inflammation. ACD occurs at the site of contact with an allergen called a hapten, in sensitized individuals, and it is characterized by redness, papules and vesicles, followed by scaling and dry skin. Systemic administration of hapten to sensitized patients may possibly result in systemic ACD (3, 4).

Haptens

The origin and nature of the compounds able to induce an ACD reaction are very diverse, and comprise a limited number of strong contact sensitizers used in animal models of ACD and thousands of weak haptens responsible for human ACD (5, 6). Contact allergens are low molecular weight chemicals that behave as haptens as they are not immunogenic by themselves and need to bind to epidermal proteins to generate new antigenic determinants. Most haptens bear lipophilic residues, which enable them to cross through the corneal barrier, and electrophilic residues, which account for covalent bonds to the nucleophilic residues of cutaneous proteins (7).

Hapten–protein interaction

Hapten–protein conjugates generated in ACD are formed via the covalent binding of the hapten to specific amino acids of protein carriers. The binding is the result of classical nucleophilic–electrophilic reactions between electrophilic residues of the chemical and nucleophilic residues of some amino acids, such as cysteine [the one most often involved in hapten–protein interactions (8)], lysine, methionine, tyrosine or histidine (7, 9). Recent observations demonstrate that covalent binding is also produced via free radical reactions (10). Of note, sensitizing metal ions (nickel, chrome, cobalt, etc.) react somewhat differently in that they generate noncovalent coordination protein–metal chelate complexes. Nevertheless, those complexes are sufficiently stable to stimulate acquired immunity (11). Finally, it was proposed recently that noncovalent drug-major histocompatibility complex (MHC) interactions could constitute a novel type of hapten–epitope (12).

Prehaptens and prohaptens

The majority of contact allergens are not electrophilic molecules that can bind directly to self-proteins. However, upon transformation via their environment (oxidation), or via the enzymes they encounter in the skin, these chemicals are transformed into highly reactive metabolites. Such molecules are referred to as prehaptens and prohaptens respectively (13, 14).

Prehaptens.  It has recently been shown that the sensitizing properties of several terpene fragrances such as d-limonene, linalool, geraniol or β-carophyllene are borne by their air oxidation products and not by the pure compound (15–17). Photosensitizers are also prehaptens that are transformed by ultraviolet (UV) radiation (18).

Prohaptens.  Besides being a physical barrier, the skin exerts the role of a biochemical barrier and participates actively in the detoxification of chemicals through a large panel of detoxifying enzymes (cytochrome P450 isoenzymes, alcohol and aldehyde dehydrogenases, sulphotransferases, UDP glucuronosyl transferase, glutathione-S-transferase, etc.). These enzymes modify the structure of chemicals to lead to the formation of more hydrophilic derivatives that can be better eliminated from the body (14, 19). However, if a highly reactive intermediate is formed and cannot be easily detoxified, it may remain in the skin for long enough to form hapten-peptide conjugates. Numerous prohaptens have been identified, and include natural products (urushiol, a terpinen), dyes (paraphenylene-diamine, disperse blue), fragrances (eugenol, cinnamaldehyde), drugs (sulphamethoxazole, hydrocortisone) and industrially used chemicals (styrene, ethylenediamine) (10).

Pathophysiology of ACD

Knowledge of the pathophysiology of ACD is derived chiefly from animal models referred to as contact sensitivity or contact hypersensitivity (CHS), in which the skin inflammation is induced by painting a hapten onto the skin. ACD and CHS are thus considered synonymous and define a hapten-specific T-cell-mediated skin inflammation. The vast majority of available data on ACD have been obtained with strong experimental contact sensitizers [2,4-dinitro-1-fluorobenzene (DNFB), dinitrochlorobenzene (DNCB), trinitrochlorobenzene (TNCB), oxazolone], which are not present in our daily environment and are endowed with unique chemical and immunological properties, notably potent proinflammatory properties. Importantly, recent studies focusing on mouse ACD to common weak haptens demonstrated effector and regulatory mechanisms similar to those induced by experimental contact allergens. That the pathophysiology of human ACD is comparable to that derived from preclinical animal ACD is now under study.

Two temporally and spatially dissociated phases are usually necessary to achieve optimal ACD reactions: the sensitization and the elicitation phase. However, ACD also comprises another phase that still remains obscure: the regulation/resolution phase of inflammatory response (Fig. 1).

Figure 1.

 Schematic representation of the pathophysiology of allergic contact dermatitis/contact hypersensitivity. Sensitization phase: hapten penetration through the skin induces an inflammatory reaction with the following consequences: (1) activation of skin innate immunity including haptenization of skin proteins (1A), release of inflammatory mediators (IL-18, IL-1β, TNF-α, ATP, PGE2, LTB4, ROS, histamine) by resident-skin cells (1B) and release of neuromediators (noradrenaline) by sympathic nerves (1C). (2) Rapid recruitment of leucocytes from blood to skin including CCR2+ monocytes which differentiate into dendritic cells (DCs). (3) Activation/migration/maturation of haptenized dendritic cells (resident dermal and epidermal DC and recruited DC precursors) to the dLNs via afferent lymphatics. In the dLNs: (4) Activation of hapten-specific effector CD8+ T cells (producing IFN-γ and/or IL-17) by mature dendritic cells (CD86++, MHC class II++, CD54+, CD58+, IL-12+, IL-6+, TNF-α+) which differentiate into TEM and TCM. (5) Control of the CD8+ T-cell activation by Treg cells (CD4+ CD25+ T cells). The relative contribution of epidermal and dermal DC to the activation of CD8+ and CD4+ remains disputed. (6) Migration of CD8+ and CD4+ T cells from the LNs to the blood and recirculation of hapten-specific T cells. In the liver/in the spleen: (7) Some inflammatory events occurring at sites distant from the sensitization site are important for an optimal immunization including: (i) activation in the liver of IL-4 producing NKT cells by hapten or hapten-induced inflammatory mediators in the minutes following skin hapten exposure (7A) and (ii) NKT cell activation allows the recruitment and activation in the spleen of B-1 cells producing hapten-specific IgM, as early as day 1 after sensitization (7B). Challenge phase: re-exposure to hapten is followed by activation of skin innate immunity as depicted above, as well as secondary responses because of the existence of specific IgM and T cells (8): haptenization of skin proteins (8A); hapten-specific IgM antibodies fix complement components to generate C5a that activates mast cells to produce TNF-α and serotonin (8B). Release of inflammatory mediators (IL-18, IL-1β, TNF-α, ATP, PGE2, histamine, CCL2) by resident-skin cells (8C). (9) Recruitment of blood leucocytes including hapten-specific effector T cells (E/P-selectin ligands+) as early as 6–12 h. (10) Activation of hapten-specific effector CD8+ T cells (producing IFN-γ, TNF-α, IL-17) by MHC class I+ skin cells presenting haptenated peptides. CD8+ T cells induce keratinocyte apoptosis via Fas/Fas-L and perforin/granzyme cytotoxic activities. (11) Release of new inflammatory mediators (CXCL9, CXCL10) by resident-skin cells, particularly by mast cells (TNF-α, CXCL-8), that induce the influx of a second wave of leucocytes comprising neutrophils (12), NK cells (13) and Treg cells. 70% of recruited T cells are CXCR3+. (14) Appearance of eczematous lesion (24–48 h). Resolution/regulation phase: (15) Progressive resolution of eczematous lesion upon activation of CD4+ regulatory T cells (IL-10-producing Tr1, Th2 or CD4+ CD25+ FoxP3+ T cells). Potential participation of others cells, γδT cells, B cells in the down-regulation process. (16) Role of IL-10-producing mast cells activated by hapten-specific IgG.

Sensitization phase

The sensitization phase of ACD, also referred to as the afferent phase or induction phase, occurs at the first contact of skin with a strong experimental hapten and leads to the generation of hapten-specific T cells in the lymph nodes (LNs) and their migration back to the skin. The ability of a hapten to induce sensitization relies on two distinct properties. Through their proinflammatory properties, haptens activate the skin’s innate immunity and deliver signals that are able to induce the recruitment, migration and maturation of cutaneous dendritic cells (DC) (20–23). Through their binding to amino acid residues they modify self-proteins and allow the expression in the skin of new antigenic determinants (7). Haptens or haptenated proteins are then engulfed and processed within cutaneous DC and are expressed as haptenated peptides in the groove of MHC classes I and II molecules at the cell surface (24). Hapten-bearing DCs migrate from the skin to the regional LNs where specific CD8+ and CD4+ T lymphocytes are primed in the para-cortical area (25). T cells proliferate and emigrate out of the LNs to the blood where they re-circulate between the lymphoid organs and the skin.

The sensitization step lasts 10–15 days in man, and 5–7 days in the mouse. This first step has no clinical consequence in the majority of cases, but may induce a primary ACD characterized by a hapten-specific skin inflammation 5–15 days after the initial skin contact (26) (see Primary allergic contact dermatitis).

Elicitation phase

Re-exposure of sensitized individuals with the same hapten leads to the appearance of ACD  within 24/72 h. This phase is known as the elicitation (or efferent or challenge) phase of ACD. Haptens diffuse into the skin and are taken up by skin cells which express MHC classes I and/or II/haptenated peptide complexes. Specific T lymphocytes are activated in the dermis and the epidermis, and trigger the inflammatory process responsible for the cutaneous lesions. Recent studies have demonstrated that CD8+ cytotoxic T lymphocytes are the main TEFF of ACD to strong and weak haptens, and that they are recruited early after challenge. Their activation in the skin induces the recruitment of leucocytes, which participate in the development of the clinical eczematous lesion and contain the down-regulatory cells of ACD, found in the CD4+ T-cell subset (27, 28).

The efferent phase of ACD   takes 72 h in man, and 24–48 h in the mouse. The inflammatory reaction persists for only a few days and rapidly decreases following down-regulatory mechanisms (29).

Regulation/resolution phase of the inflammatory response

The resolution of ACD involves different mechanisms including the clearance of the hapten from the skin and the activation of CD4+ regulatory T cells (Tregs). Experimental evidence supports the hypothesis that CD4+ Treg cells control both the priming/expansion of specific CD8+ T cells in lymphoid organs and the activation of CD8+ T cells in the skin (30, 31). However, the precise phenotype of Treg cells involved in the down-regulation of ACD as well as their site and mode of action still remain unknown.

Primary allergic contact dermatitis

Although the development of ACD has been postulated to require two spatially and temporally dissociated phases, clinical evidence has demonstrated that ACD can develop after a single skin contact with a strong hapten in previously unsensitized patients. This phenomenon has been referred to as ‘primary ACD’. We have recently demonstrated, in a murine model, that the pathophysiology of this primary (one-step ACD) is identical to the classical (two-step) ACD reaction (26). The afferent and efferent phases of primary ACD can be induced after a single skin contact with haptens because of the persistence of the hapten in the skin for a long period of time, allowing for the recruitment and activation in the skin of CD8+ T cells, which have been primed in the lymphoid organs a few days before.

ACD to weak haptens

Weak haptens are the most frequently encountered haptens in humans (5, 6, 32). Most of them are devoid of skin toxicity, display minor proinflammatory activity only, and cannot activate innate immunity. Therefore, weak haptens induce ACD in a small proportion of individuals in contact with the chemical and mostly those who frequently manipulate them (occupational disease) (33). We recently developed a preclinical model of ACD to weak haptens (fragrance allergens) and showed that the pathophysiology of fragrance allergy is similar to that of ACD to strong haptens and involves CD8+ TEFF and CD4+ regulatory cells (34). However, there was one major difference between the two types of ACD. In ACD to strong haptens, the CD4+ Treg cells do not prevent priming but participate in the resolution of skin inflammation, whereas in ACD to weak haptens the presence of CD4+ Treg cells totally abrogates the CD8+ T-cell priming. Indeed, although ACD to three weak fragrance allergens could not be obtained in normal mice, animals acutely depleted in CD4+ T cells developed a typical ACD reaction. Hapten-specific CD8+ TEFF were primed in the draining LNs in the days following sensitization and were recruited into the skin upon challenge (34).

The central role of cutaneous dendritic cells

The skin hosts a dense network of DC, skilled to detect exogenous substances (pathogens, allergens) that penetrate the epithelial barrier, and to convey this information to T cells in the draining LNs. In the steady state, the skin contains at least two phenotypically distinct DC subsets: Langerhans cells (LCs) which pave the squamous epidermal layer of the skin and dermal interstitial DCs (dDCs). Epidermal resident LCs are usually characterized by their immature phenotype (low level of MHC class II and co-stimulatory molecules) and the presence of intracellular organelle known as Birbeck granules, whose expression is dependent of the C-lectin endocytic receptor Langerin (CD207). Conversely to LCs, the phenotypic characterization of dDCs is more complex and diverges between rodents and humans (35, 36).

The proinflammatory properties of haptens condition the ACD response

Upon hapten exposure, the skin DC network is profoundly modified with both emigration of resident DC from skin to LNs and recruitment of DC precursors and monocytes from blood to skin. This phenomenon is the consequence of early inflammatory events resulting from potent activation of the skin innate immunity by contact allergens. Along with their capacity to form a hapten–carrier complex, haptens deliver toxic/danger signals to skin cells leading to their activation and the production of a large panel of inflammatory mediators including primary and secondary cytokines, chemokines, nucleotides, eicosanoids, reactive oxygenated species (ROS), proteases and heat-shock proteins. Any component of this inflammatory cascade impacts on the recruitment, mobilization and activation of skin DCs and other leucocytes that transport haptenated peptides towards the draining lymph nodes (dLNs), and the subsequent activation of T-cell immunity. Hence, Bonneville et al. (37) have shown that hapten-induced skin inflammation conditions the development and severity of ACD responses. Recently, several excellent review articles have documented in detail the molecular and cellular events responsible for the mobilization, migration and maturation of haptenized-DCs en route towards the T-cell-rich para-cortical area of skin dLNs (21, 22, 38–42).

The sensitization phase of ACD was previously regarded as a unidirectional event in which resident-skin DCs migrate towards the dLNs after uptake of hapten–peptide conjugates. However, it is now clear that DC precursors, recruited from the blood to the skin in the hours following skin exposure to haptens, play a major role in sensitization, in as much as blocking DC recruitment leads to diminished ACD responses (43, 44).

Which DC subsets are responsible for sensitization?

Langerhans cells have been considered for decades as the prominent DC population, responsible for initiating the response to cutaneous Ags, notably haptens. However, recent studies have highlighted the essential role of dDCs in initiating hapten-induced T-cell responses (45–49). To date, the precise function of LCs is still an enigma, with contradictory observations describing their ability in inducing immunity as well as tolerance to skin Ags. Several basic experiments in CHS illustrated this LC paradigm (39).

Role of LC in sensitization.  Previous observations have hypothesized a role for LCs in sensitization: (i) animals painted with a hapten on cutaneous sites naturally or artificially depleted in LCs were unable to mount a ACD response (50, 51) and (ii) sensitization of naive mice can be achieved by injection of in vitro haptenized total epidermal cells, purified LCs, whereas injection of total epidermal cells depleted in LCs before haptenization revealed inefficiency in inducing sensitization (52–54). On the other hand, other observations suggested that LCs were not involved in sensitization but could rather trigger tolerance. In this respect, removal of LCs with corticosteroids or tape stripping during the effector phase of mouse ACD was found to increase the magnitude of the inflammatory response (55).

In the last 3 years, the role of LCs in the development of ACD has been revisited using genetically engineered knock-in mice (KI), in which diphtheria toxin receptor (DTR), fused to the enhanced green fluorescent protein (EGFP), was inserted to the langerin locus. In these mice, Langerin+ cells can be easily tracked (GFP) and conditionally ablated after diphtheria toxin (DT) administration. Confirming previous conflicting results, two groups obtained unmodified or slightly diminished ACD responses in similar LC-depleted KI animals (56, 57). However, opposite results were derived from experiments performed on bacterial artificial chromosome (BAC) transgenic mice, generated by inserting the subunit A of DT itself into the Langerin gene of human genomic BAC DNA, in which LCs are constitutively absent. In these mice, ACD appeared to be deregulated resulting in increased ACD response (58).

These inconsistent observations rely mostly on differences into the genetic constructions, the haptens and the technical procedures used by the different investigators, and the fact that Langerin is definitely not a specific marker for LCs. Indeed, utilizing Langerin–EGFP KI mice, it became apparent that various DC subsets express Langerin, and notably a particular dermal DC subset. These cells originate from a developmental pathway independent from that of epidermal LCs and must not be confused with LCs en route to the dLNs, which patrol the dermis in the steady state (45, 46, 47).

Role of dermal DC in sensitization.  All the Langerin+ populations are depleted following DT administration in Langerin–DTR–EGFP mice. However, unlike epidermal LCs, which do not repopulate the epidermis until several weeks after DT administration, the dermis is rapidly replenished by Langerin+ dDCs (≥50% by day 13). Bursch et al. demonstrated that in absence of both Langerin+ epidermal LCs and dDCs (hapten sensitization, 1 day after DT administration), mouse ACD responses were significantly reduced, but were unaffected in the absence of only epidermal LCs (hapten sensitization, 13 days after DT administration). These data suggest that Langerin+ dDCs can mediate sensitization in the absence of epidermal LCs.

Role of other DC subsets.  Of note, results obtained by Bursch et al. revealed that ACD response was not completely abrogated despite full ablation of all Langerin+ cells, indicating that several types of skin DCs are involved in T-cell priming. Recently, it was proposed that Langerin negative–dDC could also play an important role in inducing ACD responses (49). Therefore, it is possible that, depending on the dose and on the vehicle in which the hapten penetrates the skin, the type or the number of DC subsets initiating the ACD response may vary. Moreover, it is still unclear whether migratory skin DCs directly present haptenated peptides to naïve-specific T-cell precursors. Recent data obtained in a model of herpes simplex virus skin infection suggest that migratory DCs transfer Ags to LN-resident DC populations for efficient T-cell priming (59).

Hapten-specific T-cell activation

Allergic contact dermatitis reactions are dependent on the priming of T cells during the sensitization phase. Adoptive transfer of T cells from sensitized mice into naive recipients results in the transfer of sensitization. Moreover, T-cell depletion of sensitized mice abrogates the ACD reaction. T-cell activation requires the combination of three distinct signals. The first signal involves the interaction of T-cell receptor (TCR) and the MHC/haptenated peptide complex. The two additional signals require co-stimulatory molecules (signal 2) and the secretion of cytokines (signal 3) by DC. The absence of any of those signals may lead to anergy or to the death of the T cell, which has already engaged its TCR.

Hapten determinants for T cells provide the first signal

T lymphocytes usually recognize hapten-modified peptides in the groove of MHC molecules (24). Most of the results were obtained with the strong hapten trinitrophenyl (TNP) in mouse models. In vitro experiments have shown that for both MHC classes I (60, 61) and II-restricted determinants (62, 63), T cells react to MHC-associated TNP-peptides and not to covalently TNP-modified MHC molecules. TCR interacted mainly with the hapten TNP and parts of the MHC molecule, rather indifferently concerning the amino acid sequences of the modified MHC anchoring carrier peptide, but with high specificity for the position of the TNP-carrying lysine within this sequence (62, 64, 65).

T-cell recognition of metals may be different from the general scheme described above for nonmetallic chemicals. Different modes of T-cell activation have been suggested to explain the activation of metal-reactive T cells (66). Similar to TCRs binding classical haptens, metal-specific TCRs react to determinants formed by complexing of metal ions with MHC-anchored self-peptides. Some TCRs also recognize complexes formed by metal and amino acid residues of the MHC molecule itself and of MHC-anchored self-peptides. Further, Weltzien et al. have shown that nickel may behave like a superantigen and could directly link TCRs and MHC outside the groove of the MHC molecule, in a peptide-independent manner (66).

Of note, Pichler et al. (12) recently proposed a new concept (p-i concept) in which certain haptens (such as the drug sulphamethoxazole) would be presented to T cells in a noncovalent hapten–MHC–peptide association.

Finally, it was reported that haptens/metals can affect the processing of self-Ags, resulting in the generation of T cells reactive to cryptic self-peptides not containing haptens as part of the antigenic epitopes (14, 67).

Co-stimulatory molecules deliver the second signal

Important co-stimulatory molecules expressed by DCs are those of the B7 family, i.e. CD80 (B7-1) and CD86 (B7-2) (68) as well as CD40. CD86 and CD80 interact with the T-cell molecules CD28 and CTLA-4 (CD152). CD28 is constitutively expressed on T cells, particularly on naïve T cells. During their maturation, DCs up-regulate the expression of CD80 and CD86 (69, 70). CD28 ligation is mandatory for the development of ACD as mice deficient in the CD28 molecule have a strong reduction in skin inflammation (71). CD86 seems to be the principal CD28 ligand involved in the second signal (72). Indeed, injection of anti-CD86 blocking antibodies inhibits the activation of both CD4+ and CD8+ T cells and the development of ACD.

Other pairs of co-stimulatory molecules from the tumour necrosis factor (TNF)/TNF receptor family, such as CD40/CD40-L or RANK/RANK-L also participate in T-cell activation. Their interactions lead to the up-regulation of OX40-L on DC membranes. Interaction between OX40-L and OX40, expressed on activated T cells, induces an overexpression of CD80 and CD86 and thus a better T-cell activation, which is of relevance for ACD because mice deficient in OX40-L display both a dramatic decrease in ACD to oxazolone, DNFB and fluorescein isothiocyanate (FITC), and a diminished T-cell proliferation induced by DC from OX40-L-deficient mice (73).

To avoid excessive T-cell activation, critical negative signals are also delivered during DC–T cell cross-talk. Negative signals involve couples of the B7 family (CD80-CD86/CTLA-4; PD-L1-PD-L2/PD1, B7-H3/?, B7-H4/?), and their engagement allows a fine tuning of the T-cell response. CD80-CD86/CTLA-4 interaction is the predominant inhibitory pathway (68). CTLA-4 has synergistic, nonredundant functions with PD-1, B7-H3 or B7-H4, to attenuate T-cell responses and to promote T-cell tolerance (68, 74, 75). Thus, administration during sensitization of anti-PD-1 monoclonal antibody (mAb), anti-PD-L1 mAb, but not anti-PD-L2 mAb, significantly enhanced and prolonged ACD responses (76).

DCs produce cytokines (the third signal) that drive T-cell polarization

Differentiation of primed CD4+ and CD8+ T-cell precursors is mainly dependent on the type of cytokines secreted during DC–T cell interactions (77). Hence, interleukin (IL)-12, IL-4 or a cocktail of IL-6/IL-21/IL-23+/-transforming growth factor (TGF)-β1 or IL-1 drive the differentiation of naïve T cells into type 1 cells [producing interferon (IFN)-γ, IL-2 and TNF-α], type 2 cells (producing IL-4, IL-10, IL-13 and IL-5) or type-17 cells (producing IL-17A, IL-17F, IL-21 and IL-22) respectively. However, the mechanisms responsible for the polarization of T cells following T cell–DC dialogue in vivo are still poorly understood and depend on several factors including the type of DC subset, the nature of Ags and factors released by the tissular microenvironment [thymic stromal lymphopoietin (TSLP), histamine, kinins, complement anaphylatoxin C5, etc.] (41, 78, 79). It appears clear that in the absence of this third signal conveyed by cytokines, polarization of T cells fails, despite the existence of phenotypically mature DC (80).

Hapten-painting in mice results within 5 days in full activation and proliferation in the dLNs of both CD4+ and CD8+ T cells. Upon in vitro restimulation of dLNs, T cells with haptenated APCs, CD8+ T cells produce mainly type-1 cytokines and mostly IFN-γ. An unusually high frequency of about 50–100 CD8+ T-cell precursors/105 LN cells was detected in C57BL/6 mice sensitized for 5 days with the hapten TNCB (81). In contrast, CD4+ T cells produce type-2 cytokines, including IL-4 or IL-10 (25), but at much lower frequency (82). Recently, the priming of IL-17-secreting T cells was also described both in mice and humans (83, 84).

Polarization of type 1 T cells is crucial to the development of specific effector T-cell populations and optimal ACD reactions. For the quasi totality of haptens tested in experimental models, it was reported that the inflammatory skin response is mediated by CD8+ Tc1 cells. Type 1 polarization by injection of IL-12 at time of sensitization favours CD8+ T cell differentiation and increases the ACD reaction (85). It was also shown that IL-12 injection converted the tolerogenic hapten dinitrothiocyanobenzene (DNTB) in to an immunogen (86). On the contrary, type 2 polarization using IL-4 (or anti-IFN-γ mAbs) leads to a diminished ACD response associated with an altered CD8+ T-cell priming (85).

Hapten-specific CD8+ responses develop in the absence of CD4+ T cell help

Allergic contact dermatitis to strong haptens is mediated by CD8+ T cells and regulated by CD4+ T cells. More importantly, CD8+ effectors can develop in the absence of CD4+ T cells. This has been demonstrated by different sets of experiments: (i) mice depleted in CD8+ T cells or deficient in CD8+ T cells (MHC class I-KO mice) cannot develop ACD responses; (ii) mice depleted in CD4+ T cells or deficient in CD4+ T cells (MHC class II-KO mice) develop an enhanced ACD response and (iii) DC recovered from MHC class I-deficient mice cannot sensitize for ACD in transfer experiments whereas DC recovered from MHC class II-deficient mice are able to induce a normal ACD reactions (25, 29, 34, 65, 87–90). That CD4+ T cell help is not necessary for the development of ACD is in keeping with recent studies showing that Ag-specific cytotoxic lymphocyte (CTL) responses can be induced in the absence of help provided that (i) the immunogen has intrinsic proinflammatory properties (e.g. endotoxins and pathogenic microorganisms) able to generate a danger signal to the DC (91); (ii) the affinity of TCRs for MHC/peptide complexes is high and (iii) the frequency of specific CD8+ T-cell precursors is high (81). Contact-sensitizing haptens have two important properties that may explain their immunogenicity in the absence of CD4+ help. First, they are proinflammatory xenobiotics through induction of chemokine and cytokine production by skin cells (92). Secondly, covalent binding of haptens, such as DNFB or TNCB, on amino acid residues of proteins generates a high number of haptenated peptides allowing activation of high numbers of CTL precursors (81).

Migration of specific T cells in the blood and in the skin – generation of effector and memory T cells

Once activated, T cells emigrate from the LNs through the efferent lymphatic vessels and then circulate in the blood. Emigration of T cells outside the LNs is associated with modifications in the expression of chemokine and adressin receptors.

Different subsets of T cells are generated during an immune response. Two subsets of activated specific T cells down-regulate the expression of CCR7 and then lose the ability to re-circulate into the LNs. The CCR7-T cell subsets comprise TEFF and effector memory T cells (TEM) that are able to enter peripheral tissues and especially the skin. CCR7+ T cells constitute the other memory subset called central memory T cells (TCM), which maintain the ability to re-circulate from the blood to LN, but which cannot be recruited in peripheral tissues (93). Upon a subsequent Ag challenge, hapten-specific TEM may act as innate cells in respect to their quick and strong release of IFN-γ and CCL5, which confer an increased efficiency in the T-cell response (94). The TCM have a role in the preservation of the relatively high frequency of hapten-specific T cells.

Murine TEFF and TEM found in the skin express the chemokine receptors CCR4 and/or CCR10, the α4β1 integrin and cutaneous lymphocyte antigen (CLA), which are crucial for efficient T-cell homing into the skin (95). Skin-selective homing of primed T cells depends on the tissue microenvironment and more specifically on skin DCs (96, 97). Migration of T cells from the blood to the skin occurs at the site of postcapillary high endothelium venules through interactions of CLA and CCR4 with their respective ligands, E- or P-selectin and CCL17 [thymus- and activation-regulated chemokine (TARC)] constitutively expressed on endothelial cells (98–101). The passage of T cells in the dermis requires the sequential interaction of VLA-4 and LFA-1 receptors on T cells with vascular cell adhesion molecule (VCAM)-1 and intracellular adhesion molecule (ICAM)-1 on endothelial cells (102). Cutaneous T-cell-attracting chemokine CTACK (CCL27) that is constitutively expressed by noninflamed endothelium and preferentially by epidermal keratinocytes also participates in this process (103).

Thus, at the end of the afferent phase of ACD, specific T cells that have been activated by hapten-bearing DCs are found in the LNs (central memory cells), in the blood and in the skin (peripheral effector and memory cells). The skin has a normal-looking appearance. Specific T cells will be activated directly in the skin and massively recruited upon a subsequent skin contact with the same hapten.

CD8+ cytotoxic T cells initiate the development of ACD

Hapten skin-painting in sensitized individuals induces the ACD skin inflammatory reaction, which occurs in three steps. In the first step, early recruitment of CD8+ T effector cells is initiated via the activation of the endothelium upon hapten-induced skin inflammation. The second step follows the activation of hapten-specific T cells recruited upon hapten-presentation. This leads to activation of skin-resident cells through cytotoxicity and cytokine/chemokine production and is followed by the production of new mediators of the inflammatory reaction. The third step corresponds to the recruitment of leucocytes, especially neutrophils, macrophages and T cells, which progressively induce the morphological and clinical changes characteristic of ACD.

T-cell recruitment

Over the last decade, Askenase et al. have revealed a complex multi-step process in which innate and B-cell immune systems subtly collaborate to initiate the recruitment of hapten-specific T cells at the site of challenge. First, the recruitment process is instigated in the liver during the sensitization phase via the activation of IL-4-producing invariant NKTs (iNKTs) in the hours after epicutaneous painting. Invariant NKTs support the migration of a B1-like B-cell subset to lymphoid organs and their production of hapten-specific immunoglobulin M (IgM) antibodies as soon as 1 day after hapten sensitization (104–106). Upon challenge, circulating hapten-specific kappa chain light IgMs bind hapten Ag, in a complex that locally activates complement, to generate C5a (107). C5a stimulates local mast cells and platelets via their C5a receptors to release vasoactive serotonin (108, 109) and TNF-α (110) leading to local vascular activation and subsequent recruitment of hapten-primed TEFF, within a  2-h window that follows challenge. Thus, the activation of B-cell immunity during the sensitization phase has a great impact on the expression and the magnitude of the mouse ACD reaction, stimulating mast cells, which appear as the main amplificatory cells of this reaction.

Recruitment of TEFF into the skin is orchestrated by a coordinated and sequential release of chemokines by resident or newly immigrated skin cells. First, upon hapten-induced local inflammation and TNF-α/IL-1β release, TEFF are extravasated from the blood via the up-regulation of constitutively expressed E-/P-selectins and VCAM-1/ICAM-1 on endothelial cells. Effector T cells are then directed to the dermis and the epidermis notably through the up-regulation of CCL2, CCL5, CCL20, CCL22 or CCL27, which are detected  as early as 6–12 h after hapten challenge with a concomitant infiltration of mononuclear cells. Then, TCR-engagement on newly recruited effector cells induces the release of type 1 cytokines such as IFN-γ and TNF-α, as well as IL-4 and IL-17, which in turn stimulate the secretion of CXCL10 (IP-10), CXCL11 (I-TAC) and CXCL9 (Mig), the ligands of CXCR3, CXCL8 (MIP-2, the murin homologue of IL-8 in humans), CCL17 (TARC), CCL18 (PARC) or of IL-1, IL-6, TNF-α, GM-CSF by keratinocytes, mast cells or other skin cells (111). Of note, neither CXCL9 nor CXCL10 are detected into the challenged skin of naïve animals, indicating that those chemokines are selectively produced during hapten-specific immune responses (112). This complex cytokine and chemokine production then shapes and amplifies the inflammatory response initiated by the hapten exposure, and is responsible for a second wave of leucocyte infiltration. Recruited cells comprise polynuclear neutrophils, T cells (70% of infiltrating lymphocytes are CXCR3+) and inflammatory monocytes able to differentiate into macrophages and DCs. Of note, the amplification loop involves a previously unappreciated cross-talk between Sema7A+ activated T cells and α1β1 integrin+ monocytes/macrophages, secreting inflammatory cytokines and perhaps other mediators, at the site of inflammation (113).

The late and massive recruitment of polynuclear neutrophils is characteristic of inflammatory skin lesions such as ACD or psoriasis. The intensity of neutrophil infiltration correlates with the number of Ag-primed CD8+ T cells recruited into cutaneous Ag challenge sites. Hence, in their absence, ACD is reduced (114, 115). It has been proposed that resident mast cells are responsible for the influx of neutrophils (that express constitutively CXCR1 and 2), via the production of TNF-α and subsequently of CXCL-8 (116), confirming that mast cells play a pivotal role in the amplification of the ACD response. Mechanisms for mast cell activation are currently poorly understood, and could involve close interactions with activated type-1 cytokine-producing T cells. Interestingly, IL-4 and T helper (Th)2 cells down-regulate CXCL8 expression, limits neutrophil infiltration and reduces the severity of ACD (117).

CD8+ T cells are the main antigen-specific effector cells in ACD to strong and weak haptens

During the 1980s, studies from Cher and Mosmann (118) clearly established that classical delayed-type hypersensitivity (DTH) to nominal protein Ags was mediated by CD4+ T cells. The over-representation of CD4+ T cells in established ACD lesions and the presence of hapten-specific CD4+ T cells in the blood of sensitized patients (118, 119), have led to the hypothesis that ACD was mediated by CD4+ T cells. Experimental models have contributed to elucidating the nature of the effector T-cell population in ACD. Mice deficient in CD8+ T cells, following the invalidation of the MHC class I β2 microglobulin gene, or mice acutely depleted of CD8+ T cells by in vivo mAb treatment, are unable to develop ACD to experimental haptens (29). However, lack of CD8+ T cells in these mice does not affect the classical DTH to proteins as shown by Bour et al. (29). Conversely, mice deficient in CD4+ T cells following the invalidation of the MHC class II Aβ gene, or mice depleted of CD4+ T cells, develop a stronger and sustained ACD reaction, suggesting a regulatory function for the CD4+ T-cell compartment (29). Thus, ACD appears very different from classical DTH reactions in terms of T-cell subsets involved in effector proinflammatory functions.

Many investigators have confirmed that hapten-specific CD8+ T cells are the main effector cells of CHS, for several molecules tested in murine experimental models (34, 65, 120, 121). Moreover, studies on nickel ACD have suggested that the pathophysiology of ACD in humans is similar to that of ACD in mice and also involves CD8+ TEFF (122). Martin et al. (123) documented the reason for the dominance of hapten-specific CD8+ T cells by showing that they inhibit CD4+ T-cell priming via a Fas-mediated mechanism. However, the exact nature of hapten-specific effector CD8+ T cells, their mode of action and the role played by other potential effector cells is still unclear.

Role of IFN-γ and IL-17-secreting CD8+ T cells

As mentioned above, on presentation of haptenated peptides by resident or newly recruited skin cells, activated CD8+ T cells release IFN-γ and TNF-α. Both cytokines are potent activators of keratinocytes, and promote up-regulation of ICAM-1 and MHC class II and release of CXCL-9, CXCL-10 and CXCL-11 (111, 124). However, along with IFN-γ producers, a substantial number of IL-17-producing T-cell clones have also been detected in allergic patients (124, 125). Interleukin-17 also contributes actively and synergistically with IFN-γ and TNF-α to keratinocyte activation by augmenting ICAM-1 and CXCL-8 expression (124). The role of this cytokine has been confirmed in IL-17-deficient mice, which display an impaired ACD reaction (126). That IL-17-secreting CD8+ T cells play a crucial role in mediating ACD was suggested by the observation that neutralization of IL-17 suppresses the elicitation of mouse ACD (84). Nevertheless, a significant reduction in ACD responses was also recorded in mice deficient in IFN-γ or in IFN-γ receptors (127). Consequently, further investigations addressing the relative contribution of type-1 and/or type-17 CD8+ T cells, as well as the respective role of each cytokines, in the development of the inflammatory reaction will be necessary.

CD8+ T-cell cytotoxicity is mandatory for the expression of ACD

Whatever the role of cytokines (IFN-γ, IL-17, TNF-α) in the pathogenesis of ACD, CD8+ T-cell cytotoxicity appears to be mandatory for the development of CHS responses as mice deficient in the two main cytotoxic pathways, i.e. Fas/Fas-L and perforin, were unable to develop an ACD reaction despite the presence of IFN-γ-producing CD8+ T cells at the site of hapten challenge (28). Moreover, the two CD8+ CTL pathways are redundant as abrogation of ACD occurred only when the two pathways were inactivated in the same animal. Thus, the CD8+ T cells are effectors of ACD mostly through cytotoxicity. Keratinocytes are the main targets of the cytotoxic effect of hapten-specific CD8+ T cells (128) because keratinocyte apoptosis coincides with CD8+ T-cell arrival in the epidermis and increases proportionally with the number of infiltrating CD8+ T cells (27). This epidermal damage facilitates the penetration of haptens present on the skin, which may increase the inflammation (129). Whether type-1 and type-17 T cells feature diverse cytotoxic properties remains to be established.

CD4+ T cells and other cells may eventually mediate antigen-specific ACD

Although most recent studies have emphasized the major effector role of CD8+ T cells in ACD, it cannot be concluded that CD4+ T cells and other cells are unable to act as ACD effectors. Hapten-specific CD4+ T cells could participate appreciably in the inflammation, in support of effector CD8+ T cells. Cavani et al. observed that like CD8+ T cells, which kill resting nickel-modified keratinocytes in vitro, CD4+ Th1 clones were also able to kill keratinocytes, but exclusively after MHC class II induction by IFN-γ. They may thus cooperate with CD8+ T cells at a later time point to cause tissue damage (128).

It is important to stress that in some instances, CD4+ T cells can mediate mouse ACD to strong haptens. Martin et al. observed that, whereas CD8+ T cells are effector cells of ACD to TNCB in normal mice, CD4+ T cells can provoke the specific skin inflammation in mice deficient in CD8+ T cells. Moreover, for unique chemicals, such as FITC that provoke preferential type 2 cytokine production by CD4+ T cells in the dLNs, ACD reactions can be mediated by cells other than CD8+ T cells (64, 130). In MHC class II−/− × CD8−/− mice, inflammation depends on MHC class II-independent CD4+, probably CD4+ iNKTs (131). Therefore, it will be interesting to determine whether similar cells mediate the unusual ACD response recorded for TNCB in MHC class I−/− mice (123).

Two other lymphoid subsets have been described as potential effectors of ACD to haptens. Gamma delta T cells were reported to assist αβ T cells in the elicitation of ACD to the hapten paraphenylene-diamine (132). More striking is the contribution of NK cells to the response recorded in mice devoid of T and B cells (Rag−/− animals). Hapten-specific memory NK cells have been postulated to mediate the reaction in these lymphopenic animals (133). Nevertheless, NK cells seem to play a minor role in the standard ACD response to DNFB, as shown by the important inhibition of the ACD inflammation (about 80%) once specific CD8+ T-cell recruitment is blocked in the challenged ear skin (134).

Regulation of contact sensitivity

Down-regulation of ACD was initially attributed to the rapid clearance of the hapten from the skin in the few days following hapten-painting. However, recent studies have shown that experimental haptens, such as FITC, could stay in the epidermis for as long as 2 weeks after a single application to the skin (26). Furthermore, clinical observations of UV-induced ACD to ketoprofene suggest that haptens could stay in the skin for months or even years. Today, it appears clear that numerous regulatory mechanisms suppress the inflammation to avoid tissue damage. Regulatory feed-back mechanisms involve: (i) the elimination of Ag-loaded DC by effector CD8+ T cells (135); (ii) engagement on tissue cells of regulatory non MHC ligands (such as cadherins, PD-L1, RANK-L, etc.) for inhibitory immune receptors (74, 136–138); (iii) release of anti-inflammatory cytokines by skin cells and (iv) activation of T cells endowed with down-regulatory properties (Treg cells).

Skin-derived regulatory pathways

In ACD, in the hours following skin-painting and release of inflammatory molecules, immunosuppressive cytokines (IL-10, TGF-β) are produced by keratinocytes and possibly other skin cells (139–141). The immunosuppressive effect of IL-10 occurs through the inhibition of production of inflammatory mediators (IFN-γ, IL-6, IL-1, TNF-α), the modulation of DC maturation, and subsequently the inhibition of effector T-cell activity (142). Recently, Galli et al. documented that mast cells represent a quantitatively important source of skin IL-10 produced during the days following hapten challenge, and may therefore significantly limit the magnitude of the skin inflammation by regulating the numbers of leucocytes at this site (143). Interestingly, hapten-specific IgG1 antibody production would modulate in part the FcγR-dependent inhibitory effect of mast cells (143).

The large quantities of IFN-γ released during inflammation increase MHC class II expression on keratinocytes, stimulating their hapten-presenting capacities. Nevertheless, in the absence of positive co-stimulatory signals, they may induce anergy or death of the T cells that have already engaged their TCRs (144). It has also been reported that, in presence of IFN-γ, endothelial cells down-regulate the expression of E- and P-selectins, thereby limiting the arrival of new infiltrating leucocytes in the dermis (145).

However, although these diverse regulatory feed-back mechanisms are certainly very important in controlling inflammation, experiments performed in CD4+ T-cell-deficient animals, in which ACD is dramatically increased and lasts for several days, have demonstrated that the down-regulation of ACD is preferentially an active immune phenomenon mediated by a subset of suppressor/regulatory CD4+ T cells (25, 29). Thus, exaggerated responses recorded in mast cell-deficient mice are also progressively regulated (143).

CD4+ CD25+ regulatory T cells

Several CD4+ T-cell subsets with down-regulatory activities have been described in murine models and in human diseases, among which are CD4+ CD25+ FoxP3+ cells, Tr1 cells, Th3 cells, Th2 cells and NKT cells (31, 146). So far, little is known about the relative contribution of these different cell subsets in the control and the resolution of ACD.

The regulation of ACD can be divided into two phases, a central and a peripheral phase (147). The central regulatory phase controls the expansion and differentiation of CD8+ TEFF in the LNs while the peripheral phase limits the inflammatory process generated in the skin.

CD4+ CD25+ FoxP3+ T cells originating in the thymus are involved in tolerance to self-Ags as well as in the control of immune responses against pathogens, alloantigens and allergens (148). CD4+ CD25+ FoxP3+ T cells could also be generated at the periphery in tolerogenic (subimmunogenic) conditions of Ag presentation (149). In ACD, CD4+ CD25+ T cells were reported to exert the first line of control. Indeed, depletion of CD4+ CD25+ T cells by in vivo treatment of mice with an anti-CD25 mAb at the time of sensitization led to an increased CD8+ T-cell priming and an enhanced ACD reaction [B. Dubois and D. Kaiserlian, unpublished data and (30, 150)]. Moreover, mice treated with an IL-2-IgG2b fusion protein, showed a decreased CHS reaction associated with an increase in the CD4+ CD25+ T-cell subset (151). It was proposed that IL-2, secreted essentially by hapten-specific CD8+ T cells in the dLNs during the sensitization phase, would be required for the CD4+ CD25+ T-cell regulation of the effector T-cell priming (30). However, numerous questions about CD4 Treg cells in CHS remain unanswered: (i) What is their origin? (ii) What is their mode of action? (iii) Are they Ag-specific? (iv) What is their site of action? (v) How do strong hapten-specific CD8+ T cells succeed in overcoming the regulation exerted by CD4+ CD25+ Tregs in LNs at the time of sensitization? Recently, Ring et al. (152) reported an original mechanism by which CD4+ CD25+ Tregs suppressed ACD reactions by blocking the influx of TEFF into inflamed tissue. Moreover, Maeda et al. (153) reported the possible induction of Ag-specific cells CD4+ CD25+ T cells in a model of photoimmunosuppression to DNFB.

In humans, it has been shown that CD4+ CD25+ T cells isolated from nickel-allergic subjects showed a limited capacity to regulate nickel-specific T cells, when compared with Tregs isolated from healthy individuals (154), suggesting that ACD may develop upon defective Treg functions.

IL-10-producing CD4+ T cells

In human ACD, IL-10 releasing nickel-specific Tr1 are enriched in the peripheral blood of nonallergic individuals (154). Tr1 cells are essentially produced in vitro and in vivo in tolerizing conditions (antigenic stimulation in the presence of anti-inflammatory drugs, cytokines, etc.). That Tr1 or other IL-10-producing Th1 cells (155) are produced in the course of ACD remains to be established, as well as their respective roles/inter-relations with CD4+ CD25+ FoxP3+ T cells.

CD8+ T cells are suppressor cells in low-dose tolerance to haptens

Treg cells other than CD4+ CD25+ FoxP3+ T cells may participate in the control of ACD responses, depending on the concentration of hapten and the number of exposure. Repeated application onto the skin of a nonsensitizing amount of a strong sensitizer (DNFB or oxazolone) induces a tolerance window, during which any further skin application of the hapten at an optimal sensitization dose prevents the priming of effector cells and the development of ACD. This phenomenon is referred to as low-zone (low-dose) tolerance (LZT), and is regarded as the most common mechanism of tolerance to haptens in humans. Development of LZT is mediated by IL-4/IL-10-secreting CD8+ Tregs that are generated in presence of Tr1 CD4+ T cells (156).

Other regulatory T cells in CHS

The actual picture of regulatory populations involved in the control of ACD would not be complete without mentioning the potential role of hapten-activated spleen B cells, able to normalize exaggerated ACD responses recorded in CD19-deficient mice (157). Skin-resident Vγ5+ intraepithelial γδ T cells (referred to as dendritic epidermal T cells) present in mice would also provide local and non redundant regulation of hapten cutaneous inflammation (158).

Conclusion

Allergic contact dermatitis, as other inflammatory and autoimmune skin diseases, is mediated by cytotoxic CD8+ T cells. Recent studies have documented the dynamics and the complexity of the cellular and molecular inflammatory networks that lead to the priming of hapten-specific CD8+ T cells, their recruitment into the skin and the expression of ACD. Any of the inflammatory signalling pathways, cellular components or mediators that are activated upon epicutaneous exposure to sensitizing chemicals represents a potential target for the development of new drugs that treat this pathology. Nevertheless, more valuable avenues for the treatment of eczema will involve strategies designed to re-induce tolerance to haptens in ACD patients (159). Their success will necessitate a better comprehension of the regulatory mechanisms that limit and resolve the inflammatory reaction to strong haptens, or prevent ACD to clinically relevant weak allergens.

Ancillary