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2.1 Introduction

During the past few decades, our understanding ofwhy, where, and when allergic contact dermatitis(ACD) might develop has rapidly increased. Criticaldiscoveries include the identification of T-cells as

mediators of cell-mediated immunity, their thymicorigin and recirculation patterns, and the molecularbasis of their specificity to just one or a few allergensout of the thousands of allergens known. Progresshas also resulted from the identification of genes thatdetermine T-cell function, and the development ofmonoclonal antibodies that recognize their prod-ucts. Moreover, the bio-industrial production of largeamounts of these products, e.g.,cytokines and chem-okines, and the breeding of mice with disruptions indistinct genes (knock-out mice) or provided with ad-ditional genes of interest (transgenic mice), have al-

lowed in-depth analysis of skin-inflammatory pro-cesses,such as those taking place in ACD.

Although humoral antibody-mediated reactionscan be a factor,ACD depends primarily on the activa-tion of allergen-specific T-cells [1],and is regarded asa prototype of delayed hypersensitivity, as classifiedby Turk [2] and Gell and Coombs (type IV hypersen-sitivity) [3]. Evolutionarily, cell-mediated immunityhas developed in vertebrates to facilitate eradicationof microorganisms and toxins. Elicitation of ACD byusually nontoxic doses of small-molecular-weight al-lergens indicates that the T-cell repertoire is often

slightly broader than one might wish. Thus,ACD canbe considered to reflect an untoward side-effect of awell-functioning immune system.

Subtle differences can be noted in macroscopicappearance,time course,and histopathology of aller-gic contact reactions in various vertebrates, includ-ing rodents and humans [4].Nevertheless, essentiallyall basic features are shared. Since both mouse andguinea pig models, next to clinical studies, havegreatly contributed to our present knowledge ofACD,both data sets provide the basis for this chapter.

In ACD, a distinction should be made betweeninduction (sensitization) and effector (elicitation)

Chapter 2

Mechanisms in Allergic ContactDermatitis

Thomas Rustemeyer, Ingrid M.W. van Hoogstraten,B. Mary E. von Blomberg, Rik J. Scheper

2

Contents

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . 11

2.2 Binding of Contact Allergens to Skin Components 132.2.1 Chemical Nature of Contact Allergens . . . . . . . 132.2.2 Hapten Presentation by LC . . . . . . . . . . . . . 132.2.3 Prohaptens . . . . . . . . . . . . . . . . . . . . . . 13

2.3 Hapten-Induced Activationof Allergen-Presenting Cells . . . . . . . . . . . . 142.3.1 Physiology of Langerhans Cells . . . . . . . . . . 142.3.2 Hapten-Induced LC Activation . . . . . . . . . . . 15

2.4 Recognition of Allergen-ModifiedLangerhans Cells by Specific T-Cells . . . . . . . . 17

2.4.1 Homing of Naive T-Cells into Lymph Nodes . . . 172.4.2 Activation of Hapten-Specific T-Cells . . . . . . . 17

2.5 Proliferation and Differentiation of Specific T-Cells 192.5.1 T-Cell Proliferation . . . . . . . . . . . . . . . . . 192.5.2 T-Cell Differentiation . . . . . . . . . . . . . . . . 192.5.3 Cytokine Environment . . . . . . . . . . . . . . . 202.5.4 Nature of the Allergen . . . . . . . . . . . . . . . . 212.5.5 Neuroendocrine Factors . . . . . . . . . . . . . . 21

2.6 Systemic Propagation of the Specific T-Cell Progeny 212.6.1 T-Cell Recirculation . . . . . . . . . . . . . . . . . 212.6.2 Different Homing Patterns . . . . . . . . . . . . . 222.6.3 Allergen-Specific T-Cell Recirculation:

Options for In Vitro Testing . . . . . . . . . . . . 23

2.7 The Effector Phase of Allergic Contact Dermatitis 242.7.1 Elicitation of ACD . . . . . . . . . . . . . . . . . . 242.7.2 Irritant Properties of Allergens . . . . . . . . . . . 242.7.3 Early Phase Reactivity . . . . . . . . . . . . . . . . 262.7.4 T-Cell Patrol and Specificity of T-Cell Infiltrates . 262.7.5 Effector T-Cell Phenotypes . . . . . . . . . . . . . 272.7.6 Downregulatory Processes . . . . . . . . . . . . . 28

2.8 Flare-up and Retest Reactivity . . . . . . . . . . . 282.8.1 Flare-up Phenomena . . . . . . . . . . . . . . . . 282.8.2 Local Skin Memory . . . . . . . . . . . . . . . . . 29

2.9 Hyporeactivity: Tolerance and Desensitization . . 302.9.1 Regulation of Immune Responses . . . . . . . . . 302.9.2 Cellular Basis of Active Tolerance . . . . . . . . . 312.9.3 Regulatory Mechanisms of the Effector Phase . . 322.9.4 Redundancy of Tolerance Mechanisms . . . . . . 322.9.5 Induction of Lasting Tolerance Only

in Naive Individuals . . . . . . . . . . . . . . . . . 322.9.6 Transient Desensitization in Primed Individuals . 32

2.10 Summary and Conclusions . . . . . . . . . . . . . 33

Suggested Reading . . . . . . . . . . . . . . . . . . 33

References . . . . . . . . . . . . . . . . . . . . . . 33

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phases [5] (Fig. 1). The induction phase includes theevents following a first contact with the allergen andis complete when the individual is sensitized and ca-pable of giving a positive ACD reaction. The effectorphase begins upon elicitation (challenge) and resultsin clinical manifestation of ACD. The entire processof the induction phase requires at least 3 days to sev-eral weeks,whereas the effector phase reaction is ful-ly developed within 12 days.Main episodes in the in-duction phase (steps 15) and effector phase (step 6)are:

Binding of allergen to skin components. Theallergen penetrating the skin readily asso-ciates with all kinds of skin components, in-cluding major histocompatibility complex(MHC) proteins. These molecules, in humansencoded for by histocompatibility antigen(HLA) genes, are abundantly present on epi-dermal Langerhans cells (LC).

Hapten-induced activation of allergen-present-ing cells. Allergen-carrying LC become acti-vated and travel via the afferent lymphatics tothe regional lymph nodes, where they settle asso-called interdigitating cells (IDC) in the par-acortical T-cell areas.

Recognition of allergen-modified LC by specificT-cells. In nonsensitized individuals the

frequency of T-cells with certain specificitiesis usually far below 1 per million. Within theparacortical areas, conditions are optimalfor allergen-carrying IDC to encounter naiveT-cells that specifically recognize the aller-genMHC molecule complexes. The dendriticmorphology of these allergen-presenting cellsstrongly facilitates multiple cell contacts, lead-ing to binding and activation of allergen-spe-cific T-cells.

Proliferation of specific T-cells in draininglymph nodes. Supported by interleukin-1

Thomas Rustemeyer et al.12

Fig. 1. Immunological events in allergic contact dermatitis(ACD). During the induction phase (left), skin contact with ahapten triggers migration of epidermal Langerhans cells (LC)via the afferent lymphatic vessels to the skin-draining lymphnodes. Haptenized LC home into the T-cell-rich paracorticalareas. Here, conditions are optimal for encountering naive Tcells that specifically recognize allergenMHC molecule com-plexes. Hapten-specific T-cells now expand abundantly andgenerate effector and memory cells, which are released via theefferent lymphatics into the circulation. With their newly ac-

quired homing receptors, these cells can easily extravasate pe-ripheral tissues.Renewed allergen contact sparks off the effec-tor phase (right). Due to their lowered activation threshold,hapten-specific effector T-cells are triggered by various hap-tenized cells, including LCand keratinocytes (KC), to produceproinflammatory cytokines and chemokines. Thereby, moreinflammatory cells are recruited further amplifying local in-flammatory mediator release. This leads to a gradually devel-oping eczematous reaction, reaching a maximum within1848 h, after which reactivity successively declines

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(IL-1), released by the allergen-presentingcells, activated T-cells start producing severalgrowth factors, including IL-2. A partly auto-crine cascade follows since at the same timereceptors for IL-2 are up-regulated in these

cells, resulting in vigorous blast formation andproliferation within a few days. Systemic propagation of the specific T-cell

progeny. The expanded progeny is subse-quently released via the efferent lymphaticsinto the blood flow and begins to recirculate.Thus, the frequency of specific effector T-cellsin the blood may rise to as high as 1 in 1000,whereas most of these cells display receptormolecules facilitating their migration into pe-ripheral tissues. In the absence of further al-lergen contacts, their frequency gradually de-

creases in subsequent weeks or months, butdoes not return to the low levels found innaive individuals.

Effector phase. By renewed allergen contact,the effector phase is initiated, which dependsnot only on the increased frequency of specif-ic T-cells, and their altered migratory capac-ities, but also on their low activation thresh-old. Thus, within the skin, allergen-presentingcells and specific T-cells can meet, and lead toplentiful local cytokine and chemokine re-lease. The release of these mediators, many of

which have a pro-inflammatory action, causesthe arrival of more T-cells, thus further ampli-fying local mediator release. This leads to agradually developing eczematous reaction thatreaches its maximum after 1848 h and thendeclines.

In the following sections, we will discuss these sixmain episodes of the ACD reaction in more detail.Furthermore, we will discuss local hyper-reactivity,such as flare-up and retest reactivity, and hyporeac-

tivity, i.e., upon desensitization or tolerance induc-tion.

2.2 Binding of Contact Allergensto Skin Components

2.2.1 Chemical Nature of Contact Allergens

Most contact allergens are small, chemically reactivemolecules with a molecular weight less than 500 Da[6]. Since these molecules are too small to be anti-

genic themselves,contact sensitizers are generally re-ferred to as haptens. Upon penetration through the

epidermal horny layer, haptens readily conjugate toepidermal and dermal molecules. Sensitizing organ-ic compounds may covalently bind to protein nucle-ophilic groups, such as thiol, amino, and hydroxylgroups, as is the case with poison oak/ivy allergens(reviewed in [7, 8]).Metal ions,e.g.,nickel cations, in-

stead form stable metalprotein chelate complexesby co-ordination bonds [9].

2.2.2 Hapten Presentation by LC

Sensitization is critically dependent on direct associ-ation of haptens with epidermal LC-bound MHCmolecules, or peptides present in the groove of thesemolecules. Both MHC class I and class II moleculesmay be altered this way,and thus give rise to allergen-specific CD8+ and CD4+ T-cells, respectively. Distinct

differences between allergens can, however, arisefrom differences in chemical reactivity and lipophi-licity (Fig. 2), since association with MHC moleculesmay also result from internalization of the haptens,followed by their intracellular processing as free hap-ten molecules or haptencarrier complexes. Lipo-philic haptens can directly penetrate LC, conjugatewith cytoplasmic proteins and be processed alongthe endogenous processing route, thus favoring as-sociation with MHC class I molecules [10]. In con-trast, hydrophilic allergens such as nickel ions may,after conjugation with skin proteins, be processed

along the exogenous route of antigen processingand thus favor the generation of altered MHC class IImolecules. Thus, the chemical nature of the haptenscan determine the extent to which allergen-specificCD8+ and/or CD4+ T-cells will be activated [1113].

2.2.3 Prohaptens

Whereas most allergens can form haptencarriercomplexes spontaneously, some act as prohaptensand may need activation,e.g., by light- or enzyme-in-

duced metabolic conversion, or oxidation [14].A pro-totype prohapten is p-phenylenediamine, whichneeds to be oxidized to a reactive metabolite, knownas Bandrowskis base [15, 16]. Tetrachlorosalicylani-lide is a typical photoallergen,which undergoes pho-tochemical dechlorination with UV irradiation, ulti-mately leading to photoadducts with skin proteins[17]. Reduced enzyme activity in certain individuals,related to genetic enzyme polymorphisms, explainsthe reduced risk of sensitization to prohaptens thatneed enzymatic activation [18]. Subsequent chaptersof this book will present in extensive detail the nu-

merous groups of molecules that have earned disre-pute for causing ACD [19].

Chapter 2Mechanism in Allergic Contact Dermatitis 13

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Allergenicity depends on several factorsdetermined by the very physicochemicalnature of the molecules themselves, i.e.,their capacity to penetrate the horny layer,lipophilicity, and chemical reactivity. Thesensitizing property of the majority of con-tact allergens can be predicted from thesecharacteristics. Two other factors, however,further contribute to the allergenicity ofchemicals, namely their pro-inflammatoryactivity and capacity to induce maturation

of LC.

2.3 Hapten-Induced Activationof Allergen-Presenting Cells

2.3.1 Physiology of Langerhans Cells

LC are professional antigen-presenting dendriticcells (DC) in the skin [20]. They form a contiguous

network within the epidermis and represent 2% to

5% of the total epidermal cell population [21]. Theirprincipal functions are internalization, processing,

transport, and presentation of skin-encounteredantigens [2223].As such,LC play a pivotal role in theinduction of cutaneous immune responses to infec-tious agents as well as to contact sensitizers [2426].LC originate from CD34+ bone marrow progenitors,entering the epidermis via the blood stream [27].Their continuous presence in the epidermis is alsoassured by local proliferation [28, 29]. They reside asrelatively immature DC, characterized by a high ca-pacity to gather antigens by macropinocytosis,whereas their capacity to stimulate naive T-cells isstill underdeveloped at this stage [30]. Their promi-

nent dendritic morphology and the presence of dis-tinctive Birbeck granules were observed long ago[3133]. In the last decade, their pivotal function inthe induction of skin immune responses was ex-plained by high expression of molecules mediatingantigen presentation (e.g., MHC class I and II, CD1),as well as of cellular adhesion and costimulatorymolecules [e.g., CD54, CD80, CD86, and cutaneouslymphocyte antigen (CLA)] [3436].


Fig. 2. Hapten presentation by epidermal Langerhans cells(LC). Allergen penetrating the epidermis readily associateswith all kinds of skin components, including major histocom-patibility complex (MHC) proteins,abundantly present on epi-

dermal LC. Both MHC class I and class II molecules may be al-tered directly or via intracellular hapten processing and, sub-sequently, be recognized by allergen-specific CD8+ and CD4+

T cells

Core Message

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2.3.2 Hapten-Induced LC Activation

Upon topical exposure to contact sensitizers, or oth-er appropriate stimuli (e.g., trauma, irradiation), upto 40% of the local LC become activated [37, 38], leavethe epidermis, and migrate, via afferent lymphatic

vessels, to the draining lymph nodes [39] (Fig. 3).This process of LC migration results from several fac-tors, including contact allergen-induced productionof cytokines favoring LC survival [4042] and loos-ening from surrounding keratinocytes [4345]. Thus,within 15 min after exposure to a contact sensitizer,production of IL-1 mRNA and release of IL-1 pro-tein from LC are induced [46, 47]. In turn,IL-1 stim-ulates release of tumor necrosis factor- (TNF-)and granulocyte-macrophage colony-stimulatingfactor (GM-CSF) from keratinocytes [47, 48]. Togeth-er, these three cytokines facilitate migration of LC

from the epidermis towards the lymph nodes [49].IL-1 and TNF- downregulate membrane-bound E-cadherin expression and thus cause disentanglementof LC from surrounding keratinocytes (Fig.3) [45,50,51]. Simultaneously, adhesion molecules are increas-ingly expressed that promote LC migration by medi-

ating interactions with the extracellular matrix anddermal cells, such as CD54,6 integrin, and CD44variants [5256]. Also, production of the epidermalbasement membrane degrading enzyme metallopro-teinase-9 is upregulated in activated LC [57].

Next, LC migration is directed by hapten-inducedalterations in chemokine receptor levels [58]. Uponmaturation,LC downregulate expression of receptorsfor inflammatory chemokines (e.g., CCR1, 2,5,and 6),whereas others (including CCR4, 7, and CXCR4) areupregulated (Fig. 3) (reviewed by [59] and [6062]).Notably, CCR7 may guide maturing LC into the


Fig.3ad.Hapten-induced migration of Langerhans cells (LC).a In a resting state, epidermal Langerhans cells (LC) reside insuprabasal cell layers, tightly bound to surrounding keratinoc-

ytes (KC), e.g., by E-cadherin.b Early after epidermal haptenexposure, LC produce IL-1, which induces the release of tu-

mor necrosis factor (TNF-) and granulocyte-macrophagecolony-stimulating factor (GM-CSF) from keratinocytes. To-gether, these three cytokines facilitate migration of LC from

the epidermis towards the lymph nodes.

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draining lymphatics and the lymph node paracorti-cal areas, since one of its ligands (secondary lym-phoid tissue chemokine, SLC) is produced by bothlymphatic and high endothelial cells [63, 64]. Not-ably, the same receptorligand interactions causenaive T-cells, which also express CCR7, to accumulatewithin the paracortical areas [65]. Migratory respon-

siveness of both cell types to CCR7 ligands is promot-ed by leukotriene C4, released from these cells via thetransmembrane transporter molecule Abcc1 (previ-ously called MRP1) [58, 66, 67]. Interestingly, Abcc1belongs to the same superfamily as the transporterassociated with antigen-processing TAP, known tomediate intracellular peptide transport in the en-dogenous route which favors peptide associationwith MHC class I molecules. Final positioning of theLC within the paracortical T-cell areas may be due toanother CCR7 ligand, EBI1-ligand chemokine (ELC),produced by resident mature DC [68]. Along withtheir migration and settling within the draininglymph nodes,haptenized LC further mature, as char-

acterized by their increased expression of costimula-tory and antigen-presentation molecules [69, 70]. Inaddition, they adopt a strongly veiled, interdigitatingappearance, thus maximizing the chances of produc-tive encounters with naive T lymphocytes, recogniz-ing altered self [48, 71, 72].

Professional antigen-presenting cells of theepidermis, called Langerhans cells, take uppenetrated allergens and present them inthe context of MHC molecules. Thereby,they are activated and emigrate from theepidermis via afferent lymphatics to thedraining lymph nodes, where they cancome into contact with naive T lympho-

cytes.


Fig.3ad.Hapten-induced migration of Langerhans cells (LC).c Emigration of LC starts with cytokine-induced disentangle-ment from surrounding keratinocytes (e.g., by downregula-tion of E-cadherin) and production of factors facilitating pen-etration of the basal membrane (e.g.,matrix metalloproteinas-es) and interactions with extracellular matrix and dermal cells(e.g., integrins and integrin ligands).d Once in the dermis, LC

migration is directed towards the draining afferent lymphaticvessels,guided by local production of chemokines (e.g., secon-dary lymphoid tissue chemokine, SLC) acting on newly ex-pressed chemokine receptors, such as CCR7, on activated LC.Along their journey, haptenized LC further mature as charac-terized by their increased dendritic morphology and expres-sion of costimulatory and antigen-presentation molecules

Core Message

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2.4 Recognition of Allergen-ModifiedLangerhans Cells by Specific T-Cells

2.4.1 Homing of Naive T-Cellsinto Lymph Nodes

More than 90% of naive lymphocytes present withinthe paracortical T-cell areas have entered the lymphnodes by high endothelial venules (HEV) [73]. Thesecells are characterized not only by CCR7 but also bythe presence of a high molecular weight isoform ofCD45 (CD45RA) [73, 74]. Entering the lymph nodesvia HEV is established by the lymphocyte adhesionmolecule L-selectin (CD62L), which allows rollinginteraction along the vessel walls by binding to pe-ripheral node addressins (PNAd), such as GlyCAM-1or CD34 [7577]. Next, firm adhesion is mediated by

the interaction of CD11a/CD18 with endothelialCD54, resulting in subsequent endothelial transmi-gration. Extravasation and migration of naive T-cellsto the paracortical T-cell areas is supported by chem-okines such as DC-CK-1, SLC, and ELC produced lo-cally by HEV and by hapten-loaded and resident DC[66,7880].In nonsensitized individuals,frequenciesof contact-allergen-specific T-cells are very low, andestimates vary from 1 per 109 to maximally 1 per 106[73, 81]. Nevertheless, the preferential homing ofnaive T-cells into the lymph node paracortical areas,and the large surface area of interdigitating cells

make allergen-specific T-cell activation likely withonly few dendritic cells exposing adequate densitiesof haptenized-MHC molecules [82, 83].

2.4.2 Activation of Hapten-Specific T-Cells

As outlined in Sect. 2.2,Binding of Contact Allergensto Skin Components,the chemical nature of the hap-ten determines its eventual cytoplasmic routing inantigen-presenting cells (APC), and thus whetherpresentation will be predominantly in context of

MHC class I or II molecules (Fig. 2). T cells, express-ing CD8 or CD4 molecules,can recognize the hapten-MHC class I or II complex, which in turn stabilizesMHC membrane expression [84,85].Chances of pro-ductive interactions with T-cells are high since eachMHCallergen complex can trigger a high number ofT-cell receptor (TCR) molecules (serial triggering)[86]. Moreover, after contacting specific CD4+ T-cells, hapten-presenting DC may reach a stable su-per-activated state,allowing for efficient activation ofsubsequently encountered specific CD8+ T-cells [87].The actual T-cell activation is executed by TCR-

chain-mediated signal transduction, followed by an

intracellular cascade of biochemical events, includ-ing protein phosphorylation, inositol phospholipidhydrolysis, increase in cytosolic Ca2+ [88, 89], and ac-tivation of transcription factors, ultimately leading togene activation (Fig. 4) [90].

For activation and proliferation, TCR triggering

(signal 1) is insufficient, but hapten-presentingAPC also provide the required costimulation (signal2; Fig. 4) [91, 92]. The costimulatory signals may in-volve secreted molecules, such as cytokines (IL-1), orsets of cellular adhesion molecules (CAMs) and theircounter-structures present on the outer cellularmembranes of APC and T-cells (summarized inFig. 5). Expression levels of most of these CAMs varywith their activational status, and thus can providepositive stimulatory feedback loops. For example, asmentioned above, after specific TCR binding and li-gation of CD40L (CD154) on T-cells with CD40 mole-

cules, APC reach a super-activated state, character-ized by over-expression of several CAMs, includingCD80 and CD86 (Fig. 4) [93, 94]. In turn, these mole-cules bind to and increase expression of CD28 on T-cells. This interaction stabilizes CD154 expression,causing amplified CD154CD40 signaling [94, 95].

The activational cascade is, as illustrated above,characterized by mutual activation of both hapten-presenting APC and hapten-reactive T-cells.Whereasthis activation protects the APC from apoptotic deathand prolongs their life to increase the chance of acti-vating their cognate T-cells, only the latter capitalize

on these interactions by giving rise to progeny. Asdiscussed below, to promote T-cell growth, cellularadhesion stimuli need to be complimented by a brothof cytokines, many of which are released by the sameAPC. Together, elevated expression levels of (co-)stimulatory molecules on APC and local abundanceof cytokines overcome the relatively high activationthreshold of naive T-cells [96].

The intricate structure of lymph node paracorti-cal areas, the differential expression of chemokinesand their receptors, the characteristic membrane ruf-fling of IDC, and the predominant circulation of

naive T lymphocytes through these lymph node are-as provide optimal conditions for TCR binding, i.e.,the first signal for induction of T-cell activation [97].Intimate DCT-cell contacts are further strength-ened by secondary signals, provided by sets of cellu-lar adhesion molecules,and growth-promoting cyto-kines (reviewed in [98, 99]).


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Fig. 4. Activation of hapten-specific T-cells. T-cell receptor(TCR) triggering by hapten-major histocompatibility complex(MHC) complexes (signal 1) is insufficient for T-cell activa-tion. But professionalantigen-presenting cells (APC),such asLangerhans cells, can provide the required costimulation(signal 2), involving secreted molecules such as cytokines,orsets of cellular adhesion molecules present on the outer cellu-lar membranes of APC and T-cells. T-cells, stimulated in thisway, activate nuclear responder elements (e.g., CD28RE). To-gether with nuclear transcription factors (NF), produced uponTCR triggering, these nuclear responder elements enable tran-scription of T-cell growth factors, e.g., IL-2.APCT-cell inter-

action gives rise to mutual activation (amplification): onAPC, ligation of CD40 with CD154 molecules on T-cells induc-es overexpression of several costimulatory molecules, includ-ing CD80 and CD86. In turn, these molecules bind to and in-crease expression of CD28 on T-cells. This interaction stabiliz-es CD154 expression, causing amplified CD154CD40 signal-ing, and preserves strong IL-2 production, finally resulting inabundant T-cell expansion. (DAG Diacylglycerol,IP3 inositol1,4,5-trisphosphate,PI phosphatidylinositol,PIP2 phosphati-dylinositol 4,5-bisphosphate, PKCprotein kinase C, PLCphos-pholipase C)

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2.5 Proliferation and Differentiationof Specific T-Cells

2.5.1 T-Cell Proliferation

When activated, naive allergen-specific T-cells startproducing several cytokines, including IL-2, which isa highly potent T-cell growth factor [100102].With-in 30 min after stimulation, IL-2 mRNA can alreadybe detected [100,103]. In particular, ligation of T-cell-bound CD28 receptors augments and prolongs IL-2production for several days [104]. Simultaneously,the IL-2 receptor -chain is upregulated,allowing forthe assembly of up to approximately 104 high-affinity

IL-2 receptor molecules per T-cell after 36 days[102]. This allows appropriately stimulated T-cells tostart proliferating abundantly. This process can bevisible as an impressive, sometimes painful lymphnode swelling.

2.5.2 T-Cell Differentiation

Whereas their allergen specificity remains strictlyconserved along with their proliferation, the T-cellprogeny differentiates within a few days into effectorcells with distinct cytokine profiles [105, 106]. While

naive T-cells release only small amounts of a limited

number of cytokines, e.g., IL-2, activated T-cells se-crete a broad array of cytokines which, besides IL-2,include IL-4, IL-10, interferon- (IFN-), and TNF-

(type-0 cytokine profile) [107109]. Within a fewdays, however, T-cell cytokine production can polar-ize towards one of the three major cytokine profiles,referred to as type 1 (characterized by a predomi-nant release of IFN- and TNF-), type 2 (IL-4and/or IL-10), or type 3 [transforming growth fac-tor- (TGF-); Fig. 6] [110, 111]. Evolutionarily, basedon requirements for combating different exogenousmicrobial infections, these polarized cytokine pro-files promote inflammation and cytotoxic effectorcell functions (type 1), antibody production (type 2),or anti-inflammatory activities in conjunction with

production of IgA (type 3) [112, 113].The latter excre-tory antibody excludes microbial entry, e.g., alongmucosal surfaces [114].As outlined above,both CD4+

and CD8+ allergen-specific T-cells may become in-volved in contact sensitization, and it is now clearthat both subsets can display these polarized cyto-kine profiles and, thereby, play distinct effector andregulatory roles in ACD [115117].

Polarization of cytokine production depends onseveral factors, including: (1) the site and cytokineenvironment of first allergenic contact, (2) the mo-lecular nature and concentrations of the allergen,

and (3) the neuroendocrine factors.


Fig. 5. Antigen-presenting cell and T-cell interaction mole-cules. On the outer cellular membranes of antigen-presentingcells (APC) and T-cells, respectively, sets of interaction mole-cules are expressed. They include antigen presentation (such

as MHC class I and II) and recognition (such as T-cell receptor,TCR/CD8, and CD4 complexes, respectively) and various ad-hesion molecules

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2.5.3 Cytokine Environment

In the skin-draining lymph nodes,allergen-activatedLC and macrophages rapidly produce large amountsof IL-12, switching off IL-4 gene expression,thus pro-moting the differentiation of type-1 T-cells [107, 118,119]. Notably, this process is reversible, and type-1 T-cells retain high IL-4R expression throughout, leav-ing these sensitive for IL-4 as a growth factor [120].On the other hand, functional IL-12R expression re-mains restricted to type-0 and type-1 cells [121].Type-2 T-cells, e.g., developing in mucosa-draininglymph nodes, lose the genes encoding the IL-12-R 2

chain and thus, type-2 differentiation is irreversible[121]. Early differentiation of type-1 T-cells is co-pro-moted by IL-12-induced secondary cytokines, e.g.,IFN-, released by nonspecific bystander lympho-cytes, including natural killer (NK) cells, within thelymph nodes [122, 123]. Next, cell-contact-mediatedsignals provided by APC during priming of naive T-cells constitute a critically important factor in skew-ing T-cell differentiation [124]: type-1 differentiationof T-cells is strongly stimulated by CD154 triggeringthrough CD40 on APC [125]. In contrast, ligation ofCD134L (gp 34; on APC) by CD134 (OX40; on T-cells)

promotes the differentiation of type-2 T-cells [126].

Also, CD86 expression on APC contributes to prefe-

rential differentiation of naive T-cells towards a type-2 cytokine profile [127130].

After a few days type-1, but not type-2, T-cells losefunctional IFN-R expression [131, 132] and thus be-come refractory to the growth inhibitory effects ofIFN- [133]. Once established,the type-1-differentiat-ed T-cells produce IFN- and IL-18, thereby furthersuppressing development of type-2 T-cells [134].Thus, considering that contact allergens will mainlyenter via the skin, type-1 pro-inflammatory T-cellsare thought to represent the primary effector cells inACD. Nevertheless, in sensitized individuals, type-2

T-cells also play a role, as shown by both IL-4 produc-tion and allergen-specific type-2 T-cells in the bloodand at ACD reaction sites (see Sect. 2.7,The EffectorPhase of Allergic Contact Dermatitis) [135137].Their role may increase along with the longevity ofsensitization,since several factors contribute to shift-ing type-1 to type-2 responses, including reversibilityof the former and not of the latter T-cells, as men-tioned above [138].

After mucosal contacts with contact allergens,type-2 T-cell responses are most prominent. In themucosal (cytokine) environment, DC release only

small quantities of IL-12, whereas IL-4 and IL-6 pro-


Fig.6. Generation and cross-regulation of different typesof T-cells. Depending on the immunological microenvi-ronment, activated naive T cells, which only release lowamounts of few cytokines (e.g., IL-2), can differentiateinto type-0 cells,secreting a broad array of cytokines, orthe more polarized T-cell types 1, 2, or 3, with their char-acteristic cytokine profiles. By secreting mutually inhib-itory cytokines, the latter cell types can interactively reg-ulate their activation and,thereby, control the type of im-mune response. (IFNInterferon, IL interleukin,LTlym-photoxin, TGFtransforming growth factor)

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duction by cells of the mast cell/basophil lineages,macrophages and NK(T) cells is relatively high[139141], abundantly present within the mucosallayers. Moreover, these tissues, as compared to theskin,contain high frequencies of B-cells,which, whenpresenting antigen, favor type-2 responses through

the abundant release of IL-10 [142, 143]. IL-10 isknown to inhibit type-1 differentiation, just as IFN-and IL-18 interfere with type-2 T-cell differentiation[106, 144, 145]. Along the mucosal surfaces, T-cellsmay also develop, exhibiting the third type-3T-cell-cytokine profile, characterized by TGF- production(reviewed by [146]).Since these cells play critical reg-ulatory roles in ACD,they will be described further inSect. 2.9,Hyporeactivity: Tolerance and Desensitiza-tion.

2.5.4 Nature of the Allergen

A second factor in determining T-cell cytokine-pro-duction profiles, although still poorly understood, isthe molecular character of the contact allergen itself,and the resulting extent of TCR triggering [106, 147,148]. For both protein and peptide antigens, highdoses of antigen might favor type-2 responses,whereas intermediate/low doses would induce type-1T-cell responses [106, 149]. To what extent this trans-lates to contact allergens is still unclear.Certainly,en-dogenous capacities of contact allergens to induce

IL-12 by LC, versus IL-4 by mast cells, basophils, orNK(T) cells, will affect the outcome. In this respect,some contact allergens are notorious for inducingtype-2 responses, even if their primary contact is bythe skin route, e.g., trimellitic acid, which is alsoknown as a respiratory sensitizer [150].

2.5.5 Neuroendocrine Factors

Diverse neuroendocrine factors co-determine T-celldifferentiation [151153]. An important link has been

established between nutritional deprivation and de-creased T-cell-mediated allergic contact reactions[154]. Apparently, adipocyte-derived leptin, a hor-mone released by adequately nourished and func-tioning fat cells, is required for type-1 T-cell differen-tiation. Administration of leptin to mice restoredACD reactivity during starvation [154]. Also, andro-gen hormones and adrenal cortex-derived steroidhormones, e.g., dehydroepiandrosterone (DHEA),promote type-1 T-cell and ACD reactivity. DHEA, liketestosterone, may favor differentiation of type-1 T-cells by promoting IFN- and suppressing IL-4 re-

lease [155, 156]. In contrast, the female sex hormone

progesterone furthers the development of type-2CD4+ T-cells and even induces, at least transiently,IL-4 production and CD30 expression in establishedtype-1 T-cells [157, 158]. Type-2 T-cell polarization isalso facilitated by adrenocorticotrophic hormone(ACTH) and glucocorticosteroids [159], and by pros-

taglandin (PG) E2 [160]. PGE2, released from mono-nuclear phagocytes, augments intracellular cAMPlevels, resulting in inhibition of pro-inflammatorycytokine, such as IFN- and TNF-, production[161164] and thus can influence the development ofeffector T-cells in ACD.

In healthy individuals, primary skin contacts withmost contact allergens lead to differentiation and ex-pansion of allergen-specific effector T-cells display-ing the type-1 cytokine profile. The same allergens, ifencountered along mucosal surfaces, favor the devel-opment of type-2 and/or type-3 effector T-cells. Fac-

tors skewing towards the latter profile remain un-known, despite their critical importance for under-standing mucosal tolerance induction (see Sect. 2.9,Hyporeactivity: Tolerance and Desensitization).For most, if not all, allergens prolonged allergeniccontacts, also along the skin route, ultimately lead toa predominance of type-2 allergen-specific T-cells,which may take over the role of type-1 T-cells in caus-ing contact allergic hypersensitivity.

2.6 Systemic Propagation

of the Specific T-Cell Progeny

2.6.1 T-Cell Recirculation

From the skin-draining lymphoid tissue,the progenyof primed T-cells are released via the efferent lym-phatic vessels and the thoracic duct into the bloodwhere they circulate for several minutes, up to 1 h(Fig. 7) [165, 166]. Like their naive precursors, theseeffector/memory T-cells can still enter lymphoid tis-sues upon adhering to HEV within the paracorticalareas, because they continue to express L-selectin

molecules (see Sect. 2.3, Recognition of Allergen-Modified Langerhans Cells by Specific T Cells) [167,168]. However, their lymph node entry via the affer-ent lymphatics increases as a consequence of theirhigher capacity to enter peripheral tissues [169, 170].The latter capacity relates to higher surface densitiesof adhesion molecules, such as VLA-4, facilitatingmigration through nonactivated, flat endothelia, e.g.,in the skin. Notably, vascular adhesion within pe-ripheral tissues is strongly augmented when expres-sion of vascular adhesion molecules, such as vascularcell adhesion molecule (VCAM), is upregulated, e.g.,

through cytokines released at inflammatory sites.


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Similarly, other ligandcounter structure pairs con-tribute to migration into peripheral tissues. Cutane-ous lymphocyte-associated antigen and the P-selec-tin glycoprotein ligand (PSGL-1; CD162) are overex-pressed on effector/memory T-cells, and mediatebinding to venules in the upper dermis through thesugar-binding counter structures CD62 E (E-selec-

tin) and CD62P (P-selectin) [171, 172]. Vascular ex-pression of the latter molecules is also greatly in-creased by local inflammatory reactions [173175].Notably, expression of the lymphocyte-bound li-gands is highest only for short periods after activa-tion, thus endowing recently activated T-cells withunique capacities to enter skin sites and exert effec-tor functions.

Upon repeated allergenic contacts, therefore, inparticular within a few weeks after sensitization, re-cently activated effector T-cells will give rise to aller-gic hypersensitivity reactions, as outlined below.However, within lymph nodes draining inflamed

skin areas, they can also contribute to further expan-sion of the allergen-specific T-cell pool.

2.6.2 Different Homing Patterns

Effector/memory T-cells show different recirculation

patterns depending on their sites of original prim-ing, e.g., within skin- or mucosa-draining lymphoidtissues [176, 177]. These differences are mediated bydistinct vascular adhesion molecules and by the in-volvement of different chemokinereceptor pairs.First, mucosal lymphoid tissue venules express yetanother L-selectin binding molecule, the mucosal ad-dressin MAdCAM-1. The latter molecule mediatespreferential binding of lymphoid cells generatedwithin the mucosal lymphoid tissues, showing over-expression of47, a MAdCAM-1 binding integrin[178]. Thus,along the gut, Peyers patches and laminapropria attract T lymphocyte progeny generated


Fig. 7. Systemic propagation of hapten-specific T-cells. Fromthe skin-draining lymphoid tissue, the progeny of primed T-cells is released via the efferent lymphatic vessels and the tho-

racic duct (DT

) into the blood and becomes part of the circu-lation. Like their naive precursors, these effector/memory T-cells can still enter lymphoid tissues by binding to peripheral

node addressins (PNAd). But increased expression of skin-homing molecules, e.g., cutaneous lymphocyte antigen (CLA),facilitates their migration in the skin.Via the afferent lymphat-

ic vessels, cells re-enter draining nodes and the recirculatinglymphocyte pool

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within other mucosal tissues, rather than contactallergen-specific cells derived from skin-draininglymph nodes. As outlined above, the latter are char-acterized by their high expression of CLA,facilitatingpreferential homing to the skin through its ligandCD62E [179, 180]. Second, T-cells biased towards pro-

duction of type-1 cytokines may show a higher pro-pensity to enter skin sites, as compared to mucosaltissues. In mice, the early influx of type-1 T-cells intodelayed-type hypersensitivity (DTH) reactions wasfound to be more efficient than that of type-2 T-cells,although both cell types expressed CLA.Here,CD162,highly expressed by type-1 T-cells, was found to beimportant for this preferential homing [173, 181, 182].Moreover, type-1 T-cells express distinct chemokinereceptors, notably CCR5 and CXCR3, contributing toskin entry [60, 183, 184]. In contrast, recirculationthrough mucosal tissues preferentially involves CCR3

and CCR4 [67, 185]. The latter chemokine receptorsare not only overexpressed on type-2 cytokine-pro-ducing T-cells, but also on basophils and eosinophils.Together, these cells contribute strongly to local im-mediate allergic hyper-responsiveness. Results ob-tained thus far favor the view that type-1 T-cells enterskin sites most readily [181, 186]. Their primary func-tion may be in the early control of antigenic pressure,e.g., through amplification of macrophage effectorfunctions. However, subset recirculation patterns arenot rigid, and, given the fact that type-1 cells can shiftcytokine production towards a type-2 profile,allergic

contact skin inflammatory lesions may rapidly bedominated by type-2 allergen-specific T-cells (seeSect. 2.4,Proliferation and Differentiation of Specif-ic T-Cells).

2.6.3 Allergen-Specific T-Cell Recirculation:Options for In Vitro Testing

The dissemination and recirculation of primed,aller-gen-specific T-cells throughout the body suggeststhat blood represents a most useful and accessible

source for T-cell-based in vitro assays for ACD.A ma-jor advantage of in vitro testing would be the non-interference with the patients immune system, thuseliminating any potential risk of primary sensitiza-tion by in vivo skin testing. Although such tests havefound several applications in fundamental research,e.g., on recognition of restriction elements, cross-re-activities and cytokine profile analyses, their use forroutine diagnostic purposes is limited. Even in high-ly sensitized individuals, frequencies of contact aller-gen-specific memory/effector cells may still be below1 per 103 [117, 187]. Given the relatively small samples

of blood obtainable by venepuncture (at only one or

a few time points), numbers of specific T-cells in anyculture well used for subsequent in vitro testingwould typically be below 100 cells/well.For compari-son, in vivo skin test reactions recruit at least 1000 ti-mes more specific T-cells from circulating lympho-cytes passing by for the period of testing, i.e., at least

24 h [165].The sensitivities required,therefore, for di-rect in vitro read-out assays, e.g., allergen-inducedproliferation or cytokine production, may often ex-ceed the lowest detection limits. However, the obser-vation that in vivo signal amplification may allow forthe detection of a single memory/effector T-cell[188190] suggests that it may be possible to solvesensitivity problems [190].

Appropriate allergen presentation, however, is amajor hurdle for in vitro testing, with a broad rangeof requirements for different allergens with uniquesolubilities, toxicities, and reactivity profiles. More-

over, in the absence of LC, monocytes are the majorsource of APC, though their numbers in peripheralblood may vary substantially within and between do-nors. Of note, optimal APC function is particularlycritical for recirculating resting/memory T-cells torespond. In the absence of repeated allergenic con-tacts, most CD45RO memory cells may finally revertto the naive CD45RA phenotype, with a higherthreshold for triggering [191, 192]. Supplementing invitro test cultures with an appropriate mix of cyto-kines may, however, compensate for this effect [187,190].

After antigenic activation the progeny of primedT-cells, i.e., effector/memory cells, are released viathe efferent lymphatics into the blood stream. Liketheir naive precursors, they can again leave the circu-lation and go into lymphoid organs anywhere in thebody, thus rapidly ensuring systemic memory. Theydiffer, however, from naive T-cells in many ways, in-cluding increased surface exposure of ligands facili-tating entry into the peripheral tissues, such as theskin. On the vascular side,distinct exit patterns fromthe circulation are determined by tissue-dependentexpression of vascular addressins and other adhe-

sion molecules,and locally released chemoattractantmolecules, i.e., chemokines. Once inside the tissues,these chemokines and stromal adhesion moleculesdetermine the transit times before recirculating T-cells eventually re-enter the blood stream. Thus, pe-ripheral blood provides a good source for in vitrostudies in ACD but, besides budgetary and logisticalreasons, theoretical considerations argue againstwide-scale applicability of in vitro assays for routinediagnostic purposes.


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In the paracortical areas of peripherallymph nodes mature antigen-presentingcells can activate antigen-specific naive T-cells. This results in the generation of effec-

tor and memory T-cell populations, whichare mainly released into the blood flow.Upon allergen contact these primed T-cellscan elicit an allergic contact dermatitis re-action.

2.7 The Effector Phaseof Allergic Contact Dermatitis

2.7.1 Elicitation of ACD

Once sensitized, individuals can develop ACD uponre-exposure to the contact allergen. Positive patchtest reactions mimic this process of allergen-specificskin hyper-reactivity. Thus, skin contacts induce aninflammatory reaction that, in general, is maximalwithin 23 days and, without further allergen supply,declines thereafter (Fig. 8). Looked at superficially,the mechanism of this type of skin hyper-reactivity

is straightforward: allergen elicitation or challengeleads to the (epi)dermal accumulation of contactallergen-specific memory/effector T lymphocyteswhich, upon encountering allergen-presenting cells,are reactivated to release pro-inflammatory cyto-kines. These, in turn, spark the inflammatory pro-

cess, resulting in macroscopically detectable erythe-ma and induration. As compared to immediate aller-gic reactions, developing within a few minutes aftermast cell degranulation, ACD reactions show a de-layed time course, since both the migration of aller-gen-specific T-cells from the dermal vessels and localcytokine production need several hours to becomefully effective. Still, the picture of the rise and fall ofACD reactions is far from clear. Some persistent is-sues are discussed below, notably: (1) irritant proper-ties of allergens, (2) role of early-phase reactivity,(3) T-cell patrol and specificity, (4) effector T-cell

phenotypes, and (5) downregulatory processes.

2.7.2 Irritant Properties of Allergens

Within a few hours after allergenic skin contact, im-munohistopathological changes can be observed, in-cluding vasodilatation, upregulation of endothelialadhesion molecules [193, 194], mast-cell degranula-tion [195, 196],keratinocyte cytokine and chemokineproduction [197], influx of leucocytes [198, 199], and


Core Message

Fig. 8a,b.

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Fig. 8af. The effector phase of allergic contact dermatitis.a 0 h In resting skin relatively few randomly patrolling, skin-homing CLA+ T-cells are present.b 04 h Re-exposure of thecontact allergen, binding to (epi)dermal molecules and cells,induces release of proinflammatory cytokines. The effectorphase of allergic contact dermatitis.c 26 h Influenced by in-flammatory mediators, activated epidermal Langerhans cells(LC) start migrating towards the basal membrane and endo-thelial cells express increased numbers of adhesion molecules.Endothelial-cell-bound hapten causes preferential extravasa-tion of hapten-specific T-cells, which are further guided by in-flammatory chemokines.d 48 h Hapten-activated T-cells re-

lease increasing amounts of inflammatory mediators,amplify-ing further cellular infiltration. e 1248 h The inflammatory re-action reaching its maximum, characterized by (epi)dermalinfiltrates, edema, and spongiosis.f48120 h Gradually, down-regulatory mechanisms take over,leading to decreased inflam-mation and disappearance of the cellular infiltrate.Finally,pri-mordial conditions are reconstituted except for a few residualhapten-specific T-cells causing the local skin memory. (DCDendritic cell,GM-CSFgranulocyte-macrophage colony-stim-ulating factor, IL interleukin, IFN interferon,KCkeratinocyte,PG prostaglandin, TGF transforming growth factor, TNF tu-mor necrosis factor)

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LC migration towards the dermis [53,200,201].Thesepro-inflammatory phenomena, which are also ob-served in nonsensitized individuals [202] and in T-cell-deficient nude mice [203], strongly contribute toallergenicity [5]. Clearly most, if not all, of these ef-fects can also be caused by irritants and, therefore, do

not unambiguously discriminate between irritantsand contact allergens [204206]. Probably, true dif-ferences between these types of compounds dependon whether or not allergen-specific T-cells becomeinvolved. Thus, only after specific T-cell triggeringmight distinctive features be observed, e.g., local re-lease of certain chemokines, such as CXCL10 (IP-10)and CXCL11 (I-TAC/IP-9) [207]. The latter chemo-kines are produced by IFN--activated keratinocytesand T lymphocytes [208].

Certainly, pro-inflammatory effects of contact al-lergens increase, in many ways, the chance of aller-

gen-specific T-cells meeting their targets. The firstcells affected by skin contact, i.e., keratinocytes andLC, are thought to represent major sources of pivotalmediators such as IL-1 and TNF- [46,209].First,asdescribed in Sect. 2.3,Hapten-Induced Activation ofAllergen-Presenting Cells, these cytokines causehapten-bearing LC to mature and migrate towardsthe dermis [34, 48]. But, these cytokines also cause(over)expression of adhesion molecules on dermalpostcapillary endothelial cells, and loosen intercellu-lar junctions. Thereby, extravasation of leucocytes,including allergen-specific T-cells, is strongly pro-

moted [209212]. Moreover, haptens can stimulatenitric oxide (NO) production of the inducible NO-synthase (iNOS) of LC and keratinocytes [213215],which contributes to local edema,vasodilatation,andcell extravasation [213,215].

Histopathological analyses support the view thatthe major causative events take place in the papillarydermis, close to the site of entry of allergen-specificT-cells, for instance at hair follicles, where haptenseasily penetrate and blood capillaries are nearby[216]. Here, perivascular mononuclear cell infiltratesdevelop, giving the highest chance of encounters

between allergen-presenting cells and specific T-cells. Once triggered,extravasated T-cells will readilyenter the lower epidermal layers,in which haptenizedkeratinocytes produce lymphocyte-attracting chem-okines, such as CXCL10 (IP-10) [207]. Subsequently,since memory T-cells can also be triggered by non-professional APC, including KC, fibroblasts, and in-filtrating mononuclear cells,ACD reactivity is ampli-fied in the epidermis [96, 98, 202]. Together, theseevents result in the characteristic epidermal damageseen in ACD, such as spongiosis and hyperplasia.Notably,in ongoing ACD reactions, the production of

chemokines attracting lymphocytes and monocy-

tes/macrophages,in addition to the production of cy-tokines, adds to the nonspecific recruitment and ac-tivation of leucocytes [60,217, 218].Thus, like the veryearly events in the effector phase reaction, the finalresponse to a contact allergen is antigen-nonspecific.It is therefore not surprising that allergic and irritant

reactions are histologically alike.

2.7.3 Early Phase Reactivity

The role of an antibody-mediated early phase reac-tion in the development of ACD is still unclear in hu-mans, although Askenase and his colleagues havegenerated robust data to support this view in murinemodels [219222]. Hapten-specific IgM, producedupon immunization by distant hapten-activated B-1cells [223, 224], can bind antigen early after challenge

[223, 225] and activate complement [226]. The result-ing C5a causes the release of serotonin and TNF-from local mast cells and platelets, leading to vascu-lar dilatation and permeabilization, detectable as anearly ear swelling peaking at 2 h [222, 227, 228]. Fur-thermore, C5a and TNF- induce the upregulation ofadhesion molecules on local endothelial cells [229,230], thereby contributing to the recruitment of T-cells in hapten challenge sites [222, 230]. In addition,human T-cells were recently found to express the C5areceptor and are chemoattracted to endothelium-bound C5a [231]. However, antibodies against most

contact allergens, including nickel, are only occasion-ally detectable in humans, arguing against humoralmechanisms playing more than a minor role in clini-cal ACD [232, 233]. Interestingly in mice, immuno-globulin light chains, which have long been consid-ered as the meaningless remnants of a spillover in theregular immunoglobulin production by B cells, wererecently discovered to mediate very early hypersensi-tivity reactions [234]. In addition to an auxiliary roleof humoral immunity, similar effects may be mediat-ed by allergen-specific T-cells with an unusual phe-notype (CD3CD4CD8Thy1+), which recognize the

hapten and, within 2 h of hapten application, werefound to elicit an early phase response [221].Also,-T-cells might contribute in a non-antigen-specific,probably non-MHC-restricted manner, to (early)elicitation responses [235237].

2.7.4 T-Cell Patrol and Specificityof T-Cell Infiltrates

Whereas early nonspecific skin reactivity to contactallergens is pivotal for both sensitization and elicita-

tion, full-scale development of ACD, of course, de-


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pends on allergen-specific T-cells within the(epi)dermal infiltrates. In healthy skin there is a con-stant flow of memory T-cells from the dermis to-wards the draining lymph nodes: about 200 T-cellsh1 cm2 skin [56]. Since just one single antigen-spe-cific T-cell can trigger visible skin inflammation

[190], randomly skin-patrolling memory/effector T-cells might account for the initiation of the allergen-specific effector phase.However, since frequencies ofhapten-specific T-cells in sensitized individuals maystill remain below 1 in 1000, this does not seem to bea realistic scenario. Thus, augmented random and/orspecific T-cell infiltration accompanies the develop-ment of ACD. Apparently, local chemokine release ispivotal in this respect [238]. The question concerningthe specificity of ACD T-cell infiltrates has so far re-ceived little attention. In a guinea pig model, prefe-rential entry of dinitrochlorobenzene (DNCB)-spe-

cific T-cells was observed within 18 h after elicitationof skin tests with DNCB, as compared to nonrelatedcompounds [239]. Probably, extravasation of hapten-specific T-cells benefits from T-cell receptor-mediat-ed interactions with endothelial MHC molecules,presenting hapten penetrated from the skin. Withinminutes after epicutaneous application, hapten canindeed be found in dermal tissues and on endothelialcells [193, 240, 241]. Interestingly, whereas preferen-tial entry may already contribute to extraordinarilyhigh frequencies of allergen-specific T-cells (within48 h up to 10%) [136, 188], at later stages, when the

ACD reaction fades away, the local frequency of aller-gen-specific T-cells may increase even further,due toallergen-induced proliferation and rescue fromapoptosis. Thus, at former skin reaction sites thesecells can generate local skin memory (see Sect. 2.8,Flare-up and Retest Reactivity).

2.7.5 Effector T-Cell Phenotypes

The debate on phenotypes of effector T-cells in ACDis ongoing, although recent studies have shed light on

longstanding issues [242]. This certainly holds truefor expression of membrane molecules determininglymphocyte-migration patterns. Once released fromreactive skin-draining lymph nodes to the blood, ef-fector T-cells express increased levels of moleculesmediating adhesion to peripheral vascular endothe-lia, e.g., the cutaneous lymphocyte antigen CLA [243,244]. Notably,the same molecule is used by precursorLC to find their way to the skin [245]. To what extentother cellular adhesion molecules associated with T-cell differentiation and maturation, in particular thelow-molecular-weight CD45 isoforms, contribute to

migration into skin-inflammatory foci is still unclear

[246, 247]. Tissue-bound ligand molecules clearly in-volved in lymphocyte extravasation and extra vascu-lar migration in the skin are fibronectin and colla-gens [248251].

Since cutaneous infiltrates show a clear prepon-derance of CD4+ T-cells, it is not surprising that these

cells have most often been held responsible for medi-ating ACD. Nevertheless, as discussed in Sect. 2.3,Recognition of Allergen-Modified Langerhans Cellsby Specific T Cells, infiltrates contain both allergen-specific CD4+ and CD8+ T-cells [252, 253]. The lattermight mediate skin inflammation through killing ofhapten-bearing target cells. Indeed, it has becomeclear that both CD4+ and CD8+ T-cells can act as ef-fector cells in DTH and ACD reactions [254257].Thus,neither of these subsets can be regarded simplyas regulatory or suppressor cells, although both ofthese subsets may,depending on the allergen models

and read-out assays,play such roles [116, 258].An essentially similar conclusion holds true for T-

cell subsets (whether CD4+ or CD8+), releasing type-1 or type-2 cytokines, or both (type 0) [190].Whereastype-1 cytokines,in particular IFN-, display well-es-tablished pro-inflammatory effects [133, 259], IL-4, ahallmark type-2 cytokine, can cause erythema andinduration when released in the skin [260, 261]. In-deed, blockage of IL-4 can interfere with ACD [261].Furthermore, analyses of skin test biopsy samplesdemonstrate the presence of not only type-1 T-cells,but also allergen-specific type-2 and type-0 T-cells

[117, 135,136].Entry of type-1 T-cells into skin-inflam-matory sites is facilitated by their expression ofCCR1, 5, and CXCR3 receptors for IFN--inducedchemokines such as MIP-1, MIP-1, and IP-10 [60,262, 263]. Type-2 T-cells overexpress a partially dif-ferent set of chemokine receptors, including, similarto eosinophils and basophils, CCR3,4, and 8 [67, 264].This would explain why local release of mediatorscommonly associated with immediate allergic reac-tions, such as eotaxins, preferentially involves type-2T-cells. Thus, a picture emerges in which ACD reac-tions can be caused both by allergen-specific type-1

or type-2 T-cells [117, 190, 265]. In retrospect, thedownregulatory effects of IL-4 on ACD reactions ob-served earlier in some mouse models [266] might beascribed to accelerated allergen clearance rather thanto blunt suppression.Still, both with time and repeat-ed allergen pressure, type-2 responsiveness may rap-idly take over [267]. Allergen-specific T-cells isolatedfrom skin test sites of sensitized individuals, as com-pared to blood, showed a strong bias towards type-2cytokine profiles [135].Additional local IFN- releaseseems,however,indispensable, since for a broad pan-el of contact allergens, clinical ACD reactions were

characterized by increased expression of mRNA en-

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coding IFN--inducible chemokines [207]. In addi-tion, transgenic mice expressing IFN- in the epider-mis showed strongly increased ACD reactivity [268].

2.7.6 Downregulatory Processes

Resolution of ACD reactions and risk factors for thedevelopment of chronicity are not yet fully under-stood. Of course, if the allergen source is limited, aswith skin testing, local concentrations of allergenusually rapidly decrease, thus taking away the criticaltrigger of the ACD reaction cascade. Since even ACDreactions due to chronic exposure to allergen seldomresult in permanent tissue destruction and scarifica-tion, immunoregulatory factors most likely contrib-ute to prevention of excessive cytotoxicity and fataldestruction of the basal membrane. Both IL-1 and he-

parinase, secreted from activated keratinocytes andT-cells, protect keratinocytes from TNF--inducedapoptosis [269, 270]. Moreover, activated effector T-cells can undergo activation-induced cell death(AICD) during the resolution phase [271]. Notably,pro-inflammatory type-1 T-cells,expressing high lev-els of Fas-ligand (CD95L) and low amounts of apop-tosis-protecting FAP-1 protein, are more susceptiblethan type-2 cells to AICD [272]. This may partly ex-plain the shift towards type-2 reactivity that is ob-served upon prolonged allergen exposure [267].Moreover, during the late phase of ACD, keratinocy-

tes, infiltrated macrophages, and T-cells start pro-ducing IL-10 [273275], which has many anti-inflam-matory activities, including suppression of antigen-presenting cell and macrophage functions [111, 276].In addition, the release of factors such as PGE2 andTGF-, derived from activated keratinocytes and in-filtrated leucocytes, e.g., type-3 T-cells, contributes todampening of the immune response [277, 278]. Re-lease of PGE

2, on the one hand, inhibits production of

pro-inflammatory cytokines [164, 279] and, on theother hand,activates basophils [280].These may con-stitute up to 515% of infiltrating cells in late-phase

ACD reactions [281] and are also believed to contrib-ute to downregulation of the inflammatory response[282, 283]. TGF- silences activated T-cells and inhib-its further infiltration by downregulating the expres-sion of adhesion molecules on both endothelial andskin cells [110]. Regulatory cells producing these sup-pressive mediators might even predominate in skinsites frequently exposed to the same allergen, andwhich are known to show local (allergen-specific)hypo-responsiveness [284].

ACD reactions can certainly be mediated by clas-sical effector cells, i.e., allergen-specific CD4+ type-1T-cells which, upon triggering by allergen-presenting

cells, produce IFN- to activate nonspecific inflam-matory cells such as macrophages. However, CD8+ T-cells, and other cytokines including IL-4, can alsoplay major roles in ACD. The conspicuous differencewith DTH reactions induced by intradermal admin-istration of protein antigens, i.e., the epidermal infil-

trate, can largely be attributed to hapten-inducedchemokine release by keratinocytes.

In sensitized individuals, allergen-specificT-cells migrate to allergen contact sites andrelease pro-inflammatory mediators,which, subsequently, attract various inflam-matory cells. This results in the elicitationof an allergic contact dermatitis reaction

within 2472 h.

2.8 Flare-up and Retest Reactivity

2.8.1 Flare-up Phenomena

Flare-up reactivity of former ACD and patch-test re-action sites is sometimes observed [285287]. Fromthe basic mechanisms of ACD, it can be inferred that

allergen-specific flare-up reactions depend either onlocal allergen or on T-cell retention at these skinsites. Flare-up reactions due to locally persisting al-lergen can readily be observed in humans, when,from about 1 week after primary sensitization, suffi-cient effector T-cells have entered the circulation toreact with residual allergen at the sensitization site[288]. This was most likely also the case when a pa-tient was patch tested with different penicillin deriv-atives,one of which released formaldehyde,(H. Neer-ing, personal communication). Pre-existing allergicreactivity and, thus, positive reactivity to formalde-

hyde apparently potentiated primary sensitization topenicillin, causing the other, previously negative,penicillin patch test sites to flare up from about1 week after skin testing. Local allergen retention,however, is usually of short duration only. In experi-mental guinea pig studies using DNCB, chromium,and penicillin allergens for sensitization, and skintesting at different days before or after sensitization,we never observed allergen retention in the skin tomediate flare-up reactions for periods exceeding2 weeks (R.J. Scheper et al., unpublished results).


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2.8.2 Local Skin Memory

In contrast, allergen-specific T-cells may persist forat least several months in the skin (Fig. 9) [289, 290].Thus, locally increased allergen-specific hyper-reac-

tivity, detectable through either accelerated retestreactivity (after repeated allergenic contacts at thesame skin site) or flare-up reactivity (after repeatedallergen entry from the circulation,e.g., derived fromfood), may be observed for long periods of time atformer skin reaction sites [291293].Typically,the er-ythematous reactions peak between 2 h and 6 h aftercontact with the allergen. Histological examinationof such previous skin reaction sites shows that themajority of remaining T-cells are CD4+ CCR10+

[290].The remarkable flare-up reactivity at such sitescan be understood by considering that just one spe-

cific effector T-cell can be sufficient to generate mac-roscopic reactivity [188]. Moreover, a very high fre-quency of the residual T-cells may be specific for theallergen, as discussed in Sect.2.7,The Effector Phaseof Allergic Contact Dermatitis. Notably, with higherallergen doses, in highly sensitized individuals, unre-lated skin test sites may show flare-up reactions [289]and even generalized erythematous macular erup-tions can be observed [294]. The latter reactivitiesare probably a corollary of the fact that recently acti-vated T-cells show strong expression of adhesion and

homing molecules, e.g., CLA, and chemokine recep-tors,such as CCR5, facilitating migration into periph-eral tissues and thus allergen-specific T-cell patrol inthe skin [244, 263, 295]. Upon allergen entry from thecirculation, these allergen-specific T-cells could me-diate generalized erythematous reactions [286, 296].

Recently, we have explored the possibilities of ex-ploiting the specific retest/skin memory phenome-non in both guinea pig models and humans, for dif-ferentiating between concomitant sensitization andcross-reactivity [297299]. We hypothesized that,with preferential local retention of T-cells reactive tothe first allergen used for skin testing, no acceleratedretest reactivity would be observed with a second,non-cross-reactive allergen, even when the individu-al would also be allergic to the latter allergen. But, ifretests were made with a second allergen, cross-reac-tive with the same T-cells, again an accelerated ery-

thematous reaction would be observed. Indeed, thishypothesis was confirmed for several different com-binations of contact allergens, in both guinea pigsand humans. Thus, retesting guinea pigs previouslysensitized to both methyl methacrylate (MMA) andDNCB, and skin tested with both allergens, showedaccelerated retest reactivities with four different me-thacrylate congeners on the former MMA, but notDNCB, patch test sites [297]. This retest model canalso be readily applied in clinical practice to dis-criminate between cross-reactivity and concomitant

Fig. 9. Local skin memory. In former allergic contact derma-titis sites a few hapten-specific T-cells can remain, mainlyclose to dermal dendritic cells (DC). Retest reaction: renewed

hapten contact can induce the rapid onset of an erythematousreaction, sparked off by the residual hapten-specific T-cells.(KCKeratinocyte, LCLangerhans cell)

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Fig. 10. Induction of oral tolerance. Hapten ingestion, prior to potential sensitizing skin contact(s), can induce hapten-specifictolerance

sensitization. Matura et al. [298] confirmed positivecross-retest reactions for cloprednol and tixocortolpivalate, both belonging to group A corticosteroids,and budesonide, amcinonide, and triamcinolone,all belonging to group B corticosteroids (see also[296]).

At skin sites of allergic contact dermatitisreactions, few but allergen-specific T-cellscan reside. Upon renewed allergen contact,these cells can cause an accelerated flare-up reaction peaking within few hours.

2.9 Hyporeactivity:Tolerance and Desensitization

Of course,uncontrolled development and expressionof T-cell-mediated immune function would be detri-mental to the host. During evolution, several mecha-nisms developed to curtail lymph node hyperplasiaor to prevent excessive skin damage upon persistingantigen exposure.

2.9.1 Regulation of Immune Responses

First, allergen contacts, e.g., by oral or intravenousadministration, may lead to large-scale presentationof allergen by cells other than skin DC (Fig.10).In theabsence of appropriate co-stimulatory signals (as de-

scribed in Sect. 2.3, Recognition of Allergen-Modi-fied Langerhans Cells) naive T-cells may be aner-gized, i.e., turned into an unresponsive state, eventu-ally leading to their death by apoptosis (Fig. 11)[300303]. With increasing density of MHCantigencomplexes on the surface of APC,multiple levels of T-cell tolerance might be induced,with the characteris-tic stages called ignorance, anergy, and deletion[304306]. Unresponsiveness of T-cells induced byallergenic contacts at skin sites where LC/DC func-tions have been damaged, e.g., by UV irradiation, orare naturally absent,e.g., in the tail skin of mice, maybe ascribed to T-cell anergy, frequently associatedwith TCR/CD4 or CD8 downregulation [307, 308].Whereas such anergy reflects passive unrespon-siveness, tolerance by active suppression may alsobe induced under similar circumstances [309]. Actu-ally, even regular epicutaneous allergenic contactsnot only induce effector T-cells but also lymphocytesregulating T-cell proliferation (afferently acting reg-ulatory cells) or, with frequent skin contacts, causingdecreased skin reactivity (regulatory cells of effector

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phase). Apparently, allergic contact hypersensitivityis the result of a delicate balance between effectorand regulatory mechanisms [284,310].

2.9.2 Cellular Basis of Active Tolerance

Upon preferential stimulation of regulatory cells,e.g., by feeding nonprimed, naive individuals withcontact allergens, strong and stable allergen-specific,

active tolerance may develop [311314]. The conceptof active regulatory (suppressor) cells controllingACD is based on the fact that, in experimental animalmodels, such allergen-specific tolerance can betransferred by lymphoid cells from tolerant to naiveanimals [237, 315]. Active suppression, as revealed bythese adoptive cell transfers, is a critical event in reg-ulating T-cell responses to contact sensitizers, and toall possible peptide/protein antigens, including bac-terial, autoimmune, and graft rejection antigens[316318].

Like effector T-cells in ACD, regulatory cells arenot a single subpopulation of cells.As outlined above,

depending among other things on the nature of theallergen and route of exposure,ACD can be mediatedby both CD4+ and CD8+ T-cells,either or both releas-ing type-1 or type-2 cytokines. Probably, given a pre-dominant effector phenotype for a particular aller-gen, each of the other phenotypes can act as regula-tory cells [319]. Nevertheless, earlier data suggestedthat type-2 cytokine-producing cells may be themost prominent regulatory cells in ACD, since aller-gic contact hypersensitivity was found to be en-

hanced, and tolerance reversed, by appropriatelytimed treatment with cytostatic drugs, including cy-clophosphamide [320322], preferentially affectingtype-2 T-cells [323]. Interferons and IL-12, both im-pairing type-2 and -3 cells, were also shown to inhib-it regulatory cells and to stimulate effector-cell func-tions in mouse models [324326]. On the other hand,in particular after mucosal allergen contact stimula-tion, T-cells predominantly producing TGF- (type-3cytokine profile) may act as regulatory cells [327,328]. These T-cells promote anti-inflammatory im-munity, e.g., by switching antibody production toIgA, which mediates secretory immunity and thus


Fig.11. The character of the APCT-cell interaction determinesthe immunological outcome. Sensitization: naive T-cells, acti-vated by antigen-presenting cells (APC) providing both hap-ten-specific (signal 1) and appropriate costimulatory (sig-nal 2) signals, develop into effector T-cells, characterized by

type-0, -1, and -2 cytokine secretion profiles. Tolerance: in theabsence of appropriate costimulatory signals, immunologicaltolerance may develop. With increasing density of MHChap-ten complexes on the surface of APC activating signal 1 T-cellpathways, multiple levels of T-cell tolerance might be induced

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contributes to antigen exclusion in the lumen, e.g., ofthe gastro-intestinal tract [329]. Of note, TGF-strongly suppresses development of both type-1 and -2 effector T-cells, and can silence T-cells in a semi-naive state [110]. Whether these type-3 T-cells, ortheir precursors, are more sensitive to cytostatic

drugs is not known. Another population of T-cells in-volved in tolerogenic processes is the group ofCD4+CD25+ T-cells [330].

2.9.3 Regulatory Mechanismsof the Effector Phase

A critical feature of the regulatory principles involv-ing mutual regulation of T-cell subpopulations bytype-1 and -2 cytokines, and both of these in turn byTGF--producing T-cells, is that their function is ob-

served foremost in primary immune responses(Fig. 6). Regulation may also pertain to the actualACD reactions, i.e., the effector phase. Several sup-pressive pathways could lead to decreased allergicskin reactivity, including hapten removal by in-creased blood flow and metabolism by cells of the in-flammatory infiltrate. Other regulatory mechanismscan also be involved, such as CD8+ T-cells, acting ei-ther as suppressor (CD28-CD11b+) or cytotoxic(CD28+CD11b) T-cells [331, 332], which may down-regulate skin reactivity by focusing on allergen-pre-senting DC as their targets [332].

2.9.4 Redundancyof Tolerance Mechanisms

Besides these types of regulatory T-cells producingdifferent cytokines, or exerting distinct cytotoxic-ities, other mechanisms may also contribute to im-mune regulation and tolerance. Apparently, the riskof excessive immune reactivity should be very low.These mechanisms involve allergen-specific T-cellsshedding truncated T-cell receptors, acting as antag-

onists and blocking allergen presentation [333], andhigh-dose allergen-induced anergic T-cells [307].Possibly, the latter cells, by actively suppressing DCfunctions, can function as active suppressor cells[307, 334]. Interestingly, DC, becoming suppressive bythis mechanism [307] or by suppressive cytokinessuch as IL-10 and PGE2 [164,335, 336],can, in turn,actthemselves as suppressor cells by conferring antigen-specific anergy to subsequently encountered T-cells[337339]. Although, at present, consensus has beenreached about a critical role of regulatory/suppressorcells in the development and expression of ACD, the

relative contributions of each of the various mecha-

nisms are still far from clear. Potential therapeuticapplications of regulatory cells in various disorders,such as allergic contact dermatitis and autoimmunediseases, are currently under investigation.

2.9.5 Induction of Lasting Tolerance Onlyin Naive Individuals

Both clinical and experimental findings indicate thatfull and persistent tolerance can only be inducedprior to any sensitizing allergen contacts [312, 340,341]. Upon primary allergenic contacts, naive T-cellsdifferentiate to produce polarized cytokine profiles(Fig. 6). Once polarized, however, T-cell profiles areirreversible, due to loss of cytokine (receptor) genes,or are at least very stable, due to the mutually sup-pressive activities of T-cell cytokines. An important

corollary of the latter concept of active suppression isthe bystander effect, in which the response to anyantigen can be downregulated by immunosuppres-sive cytokines acting at a very local tolerogenic mi-croenvironment [342]. The latter was observed forboth protein antigens [343, 344] and methacrylatecontact allergens [315]. The concept may also explainwhy even nonsensitizing doses of nickel applied tothe skin prevented subsequent tolerance inductionby feeding the metal allergen [345]. This may havecontributed to incomplete tolerance induction inearlier clinical studies when feeding with poison ivy-

/oak-derived allergens [346].Apparently, the progenyof naive allergen-specific cells, once on the stage,have been triggered to a subclinical degree towardseffector cells and become refractory to regulatory cellaction. Indeed,to our knowledge, permanent reversalof existing ACD in healthy individuals has, as yet,never been achieved. Nevertheless, as describedabove, effector cells still seem susceptible, thoughtransiently, to the downregulation of allergen reac-tivity, as was observed in desensitization procedures[345,347].

2.9.6 Transient Desensitizationin Primed Individuals

For dermatologists, methods by which patientsmight be desensitized for existing ACD would be awelcome addition to the currently prevailing symp-tomatic therapies, and investigators have made awide variety of attempts to achieve this goal. Unfor-tunately, therapeutic protocols involving ingestion ofpoison ivy allergen, penicillin, or nickel sulfate wereof only transient benefit to the patients [346350].

Similarly, in animal models, only a limited and tran-


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sient degree of hyposensitization was obtained byChase [351] when feeding DNCB-contact-sensitizedguinea pigs with the allergen, whereas, to achievepersistent chromium-unresponsiveness in presensi-tized animals, Polak and Turk [352] needed a rigor-ous protocol involving up to lethal doses of the aller-

gen. As outlined above, mechanisms underlying spe-cific desensitization in ACD probably depend on di-rect interference of allergen with effector T-cell func-tion, by blocking or downregulating T-cell receptors,leading to anergy [353]. As the onset of desensitiza-tion is immediate, no suppressor mechanisms mayinitially be involved.Apparently in the absence of LC,MHC class II-positive keratinocytes can serve as APCand are very effective in rendering allergen-specificeffector cells anergic [354]. Moreover, at later stages,active suppression may come into play resulting fromsecondary inactivation of DC function by anergized

T-cells [307]. Nevertheless, major problems with invivo desensitization procedures relate to the refrac-toriness of effector T-cells to regulatory cell func-tions, and the rapid replacement of anergized effec-tor cells by naive T-cells from relatively protected pe-ripheral lymphoid tissues, which can be the source ofa new generation of effector cells upon sensitizing al-lergen contacts. The same conclusions can be drawnfrom attempts to achieve local desensitization. It wasfound that local desensitization by repeatedly apply-ing allergen at the same skin site did not result fromlocal skin hardening or LC inactivation,as local reac-

tivity to an unrelated allergen at the site was unim-paired [284]. Persistence of cellular infiltrates, in theabsence of erythematous reactivity, at a desensitizedskin site could reflect local anergy, but also locally ac-tive regulatory cells. Upon discontinuation of aller-gen exposure, however, local unresponsiveness wasrapidly (within 1 week) lost. Collectively, these dataillustrate the problems encountered in attempting toeradicate established effector-T-cell function, not on-ly in ACD but also in autoimmune diseases [316].

2.10 Summary and Conclusions

Extensive research has led to a better understandingof the mechanisms of ACD. The basic immunology ofACD is now well defined, including T-cell migratorypatterns, recognition of distinct allergens, interac-tions with other inflammatory cells to generate in-flammation,and cytokine profiles.But new complex-ities have emerged. For instance, in contrast to earli-er belief, many of the currently known T-cell sub-populations can act either or both as effector andregulatory cells, depending on the nature of the aller-

gen, the route of entry, frequency of exposure, and

many other, still ill-defined factors. In particular, thepoor understanding of regulatory mechanisms inACD still hampers further therapeutic progress. Sofar, no methods of permanent desensitization havebeen devised.

Nevertheless, recently defined cellular interaction

molecules and mediators provide promising targetsfor anti-inflammatory drugs, some of which have al-ready entered clinical trials. Clearly, drugs found tobe effective in preventing severe T-cell-mediatedconditions, e.g., rejection of a vital organ graft,should be very safe before their use in ACD wouldseem appropriate. To date, prudence favors alterna-tive measures to prevent ACD, be it through legal ac-tion to outlaw the use of certain materials or throughavoiding personal contact with these materials. Inthe meantime, for difficult-to-avoid allergens, fur-ther studies on the potential value of tolerogenic

treatments prior to possible sensitization seem war-ranted.

Suggested Reading

Janeway CA, Travers P, Walport M, Shlomchik M (2001)Immunobiology, 5th edn. Garland, New York

Roitt I, Delves PJ (2001) Roitts essential immunology, 10thedn. Blackwell, London

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