4 Mechanisms of Irritant ContactDermatitis

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4.1 Introduction

Irritant contact dermatitis is an eczematous reaction in the skin of external origin. In contrast to allergic contact dermatitis, no eliciting allergens can be identified. The spectrum of irritant reactions includes subjective irritant response, acute irritant contact dermatitis, chronic irritant contact dermatitis, and chemical burns (Table 1). Irritant contact dermatitis is in its acute form characterized by erythema, infil-

tration, and vesiculation. In its more chronic form, dryness, fissuring, and hyperkeratosis are more pronounced. It is thus clear that the clinical reaction pat- tern of mild to moderate irritant contact dermatitis is often indistinguishable from the allergic contact dermatitis reaction. Thus, differentiation between these two reaction types is often based solely on pa- tient history and skin allergy tests. Despite the common hallmarks of irritant contact dermatitis, the clinical manifestation of the skin lesions developing following contact with different irritants varies. Fac- tors that may influence the outcome of skin contact with irritants can be divided as follows:

쐽 Exogenous: such as structural and chemical properties of the irritant, exposure to other irritants, and environmental conditions, e.g., temperature and humidity.

쐽 Endogenous: such as body region that is exposed (the scrotum is much more sensitive than, e.g., the upper back), age [1], race [2], and pre-existing skin disease.

Moreover, in addition to the capacity of different irritants to induce clinically different reactions, it has been reported that marked interindividual variation in the threshold for eliciting clinical irritant reaction in skin is present [3].

In the past, the pathogenesis of irritant contact dermatitis was thought to be nonimmunological.

However, today it is generally accepted that the immune system plays a key role in eliciting irritant reactions. This has been underscored by human and animal studies demonstrating the importance of sig-

Chapter 4

Mechanisms of Irritant Contact Dermatitis

Steen Lisby, Ole Baadsgaard

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Table 1.Type of irritant reactions Subjective irritant reaction (stinging) Acute irritant contact dermatitis Chronic irritant contact dermatitis Chemical burn

Contents

4.1 Introduction . . . . 69 4.2 Clinical Spectrum of Irritant Skin Reactions 70 4.2.1 Subjective Irritant Reaction . . . . 70 4.2.2 Acute Irritant Contact Dermatitis . . . . 70 4.2.3 Chronic (Cumulative) Irritant Contact

Dermatitis . . . . 70 4.2.4 Chemical Burn . . . . 71 4.3 Skin – the Outpost of the Immune System . 71 4.3.1 Immunocompetent Cells of the Skin . . . . . 71 4.3.2 Skin Infiltrating T Lymphocytes . . . . 72 4.4 Pathogenesis of Acute Irritant Contact

Dermatitis . . . . 73 4.4.1 Skin Barrier Perturbation . . . . 73 4.4.2 Cellular Immunological Changes

in Irritant Contact Dermatitis . . . . 74 4.4.3 Epidermal Cytokines Involved

in Irritant Contact Dermatitis . . . . 75 4.5 Irritant-induced Interleukin-1 . . . . 76 4.6 Irritant-induced TNF-α . . . . 76 4.7 Mechanisms of Irritant-induced TNF-α

in Keratinocytes . . . . 77 4.7.1 Regulation of the Inflammatory Milieu

Locally in Inflamed Skin . . . . 78 4.8 Hypothesis of the Immunological Events

Leading to Irritant Contact Dermatitis . . . . 79 Suggested Reading . . . . 80 References . . . . 80

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nal molecules, e.g., cytokines, in eliciting the irritant reaction.

쐽

Irritant contact dermatitis is an eczematous reaction in the skin caused by exposure to external agents/chemicals. Clinical- ly the reaction manifests similar to the allergic contact dermatitis reaction.

4.2 Clinical Spectrum of Irritant Skin Reactions

The spectrum of the clinical appearances of irritant contact dermatitis is extremely broad. It is therefore widely accepted that no single mechanism underly- ing the development of this disease entity exists. In this chapter, we briefly outline the different clinical reaction types. For more extensive description, the reader is referred to Chap. 15.

쐽

Irritant contact dermatitis can be divided into different reaction types, including stinging, acute irritant reaction, chronic irritant reaction, and chemical burn.

4.2.1 Subjective Irritant Reaction

The hallmark of this type of irritation is the lack of clinical manifestation. Subjective registration of a burning or stinging feeling following contact with certain chemicals is diagnostic (Table 2). Despite no clinical manifestation, the reaction can be repro- duced. Typically, symptoms occur rapidly following exposure (i.e., within seconds to minutes). There seem to be interindividual differences in eliciting this type of reaction, and several studies have classed individuals as sensitive (stingers) and nonsensitive (nonstingers) [4]. One example of immediate stinging is the appliance of a mixture of chloroform and methanol to the skin. In stingers, even when applied to intact skin, a sharp pain develops within seconds to minutes following exposure to the chloroform/

methanol mixture [5].

4.2.2 Acute Irritant Contact Dermatitis This type of reaction is often the result of a single exposure to an irritant. The clinical appearance is very variable and often indistinguishable from the allergic contact dermatitis reactions. The manifestation may vary from a little dryness and redness to severe reactions with edema, inflammation, and vesiculation.

Often the clinical reactions are located to areas of exposure and the skin manifestations often disappear within days to weeks.

4.2.3 Chronic (Cumulative) Irritant Contact Dermatitis

This type of reaction develops as a result of cumulative exposures of the skin to irritants. Clinically, this type of reaction is characterized by dryness, redness, infiltration, scaling, fissuring, and vesiculation to only a minor degree. Often this type of irritant contact dermatitis is located on the hands. A hallmark of this type of reaction is its chronicity. Despite removal of irritant exposure, the clinical reaction may continue for several years. Several external factors are known to contribute to elicitation of chronic irritant eczema.

These agents include water, detergents, organic sol- vents, oils, alkalis, acids, oxidizing agents, heat, cold, friction, and multiple microtrauma.

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Table 2. Chemicals involved in subjective skin reactions (adapted from [4])

Immediate stinging potential Chloroform

Methanol Hydrochloric acid Retinoic acid

Delayed stinging potential Weak:

Aluminum chloride Benzene

Phenol Phosphoric acid Resorcinol Salicylic acid Moderate:

Propylene glycol Dimethylsulfoxide Benzoyl peroxide Severe:

Crude coal tar Lactic acid Hydrochloric acid Sodium hydroxide

Amyldimethyl-p-aminobenzoic acid

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4.2.4 Chemical Burn

Reactions are induced by highly alkaline or acid com- pounds. These agents can result in severe damage of the skin. The reaction often develops within minutes, and frequently manifests with the appearance of a painful erythema, followed by vesiculation, and the formation of necrotic scars. This type of reaction is often sharply demarcated and may lead to deep tissue destruction even after only a short exposure.

4.3 Skin – the Outpost of the Immune System

To understand the pathogenic mechanisms involved in irritant contact dermatitis, it is important to ad- dress the involvement of the different cell types constitutively present within the skin, and the cell types that can be recruited to the site of the irritant reaction as well as the proinflammatory and inflammatory mediators induced by the different cell popula- tions following irritant exposure.

4.3.1 Immunocompetent Cells of the Skin The outermost part of the skin is the epidermis. Epi- dermis is mainly composed of keratinocytes, Langer- hans cells, and melanocytes. Both keratinocytes and Langerhans cells are involved in immunological processes. In contrast, the immunological importance of the epidermal melanocyte, if any, is not known.

The involvement of the keratinocyte in the skin immune system was first indicated in 1981/1982 by Luger et al. and Sauder et al. who described a keratinocyte-derived cytokine, epidermal-derived thymo- cyte activating factor (ETAF) [6, 7]. The majority of ETAF activity was later confined to interleukin-1 (IL- 1). It has now been demonstrated that the keratinocyte is capable of producing a variety of immunological active cytokines/factors (Table 3), including IL-1, IL-6, IL-8, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β). The involvement of some of these factors in irritant contact dermatitis is reviewed later in this chapter. Beside cytokine expression, keratinocytes can be induced to express or increase expression of major histocompatibility complex (MHC) molecules [8, 9] and cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) [10, 11].

Expression of these molecules, in combination with the release of chemotactic cytokines, and factors involved in the upregulation of E-selectin and vascular

cell adhesion molecule-1 (VCAM-1) on dermal endothelial cells [12], makes the keratinocyte an important player in the induction and maintenance of inflammatory cells within the skin.

The epidermal Langerhans cell is the only cell type in normal epidermis that exhibits all accessory cell functions and thus acts as a complete antigen- presenting cell. The epidermal Langerhans cell was originally described in 1868 by Paul Langerhans [13]

and comprises 2–5% of the total epidermal cell popu- lation. It is constitutively present in the skin and is lo- calized to the suprabasal part of the epidermis. The Langerhans cell is a dendritic, bone marrow-derived cell characterized by surface expression of type-1a cluster of differentiation (CD1a) antigen, as well as MHC class I, and MHC class II (HLA-DR, -DP, -DQ) molecules. Ultrastructurally, the Langerhans cell contains characteristic intracytoplasmic Birbeck’s granules. Beside its capacity to present antigens to T- cells, the Langerhans cell is capable of secreting cytokines such as IL-1β, IL-6, IL-10, IL-12, and TNF-α [14].

The Langerhans cell has been implicated in the immune surveillance of the skin; it is also required for induction of primary immune responses in skin, and as such is suggested to be a key player in allergic contact dermatitis. In addition, recent research has associated this cell type with events occurring during the development of irritant contact dermatitis.

Several dermal antigen-presenting cell subsets have been described including macrophages and dendritic cells. Macrophages are bone marrow-de-

Table 3.Keratinocyte-derived cytokines Interleukin-1α

Interleukin-1β Interleukin-3 Interleukin-6 Interleukin-7 Interleukin-8 Interleukin-10 Interleukin-12 Interleukin-15 Interleukin-18

Tumor necrosis factor-α Transforming growth factor-α Transforming growth factor-β Granulocyte colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor Platelet-derived growth factor

Epidermal cell-derived lymphocyte differentiation inhibiting factor

Keratinocyte lymphocyte inhibitory factor

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rived cells with a broad range of functions, including antimicrobial activity, anti-tumor activity, regulation of B and T lymphocytes, release of cytokines and processing antigens – thereby functioning as antigen-presenting cells. These cells are characterized by surface expression of Fc-receptors, including CD16 and CDw32, and MHC class II molecules. Further- more, these cells express LFA-1 (CD11a) and when activated also CD11b.

In ultraviolet-irradiated skin, dermal and epidermal monocyte/macrophage-like cells expressing a HLA-DR⁺, CD11b⁺, CD36⁺phenotype have been observed [15]. These cells are involved in downregula- tion of the immune response, revealed by their capacity to preferentially activate CD4⁺suppressor-in- ducer T lymphocytes [16, 17]. In addition, these CD11b⁺, MHC class II⁺ cells were found to secrete large amounts of IL-10, in contrast to the residual epidermal Langerhans cells, which secrete mainly IL-12 [18]. Thus, different bone marrow-derived cells of the macrophage or dendritic cell lineage are differently involved in the ongoing immune regulation within the skin during an inflammatory reaction.

In skin diseases, such as mycosis fungoides and contact dermatitis, cells with a similar HLA-DR⁺, CD36⁺phenotype have been detected within the epidermis [19, 20]. Their functional role is underscored by observations that depletion of the epidermal Langerhans cells only partially inhibits an autologous epidermal lymphocyte reaction. Furthermore, when isolated from involved epidermis, HLA-DR⁺, CD36⁺ cells exhibit the capacity directly to stimulate autologous T lymphocytes in vitro [21]. In addition, HLA- DR⁺, CD36⁺cells have been observed in the irritant reaction [22]. However, their functional role in the development of an irritant reaction is still unknown.

쐽

Immunocompetence of normal epidermis is restricted to the epidermal Langerhans cell. In irritant contact dermatitis, other dendritic cells are present, and the keratinocytes develop immunoregulatory functions, including but not limited to MHC class II and ICAM-1 expression.

4.3.2 Skin Infiltrating T Lymphocytes It has been known for several years that many skin diseases are characterized by skin infiltration by T

lymphocytes. These T lymphocytes often express a CD3⁺, CD4⁺ phenotype, although CD8⁺ T lymphocytes are also present. While trafficking the skin, these T lymphocytes are capable of releasing a variety of cytokines, including IL-2, IL-4, IL-10, interferon- γ (IFN-γ) and TNF-α. Based on their cytokine secretion, T lymphocytes can be divided into T helper-1- like (Th1-like), Th2-like or Th0-like cells (Table 4).

This division was originally suggested in 1986 by Mosmann et al. based on investigation of murine T lymphocyte clones [23]. He distinguished two different subsets of T lymphocyte clones. The first was named Th1 and comprised clones preferentially producing IL-2 and IFN-γ, while the other group of clones was termed Th2 and produced large amounts of IL-4 and IL-5. Following this observation, several studies have included more cytokines in this subdivi- sion and furthermore suggested a similar division of human T lymphocytes. Many of the T lymphocyte- derived cytokines are involved in regulation of inflammatory processes. IL-2 is known as a T lymphocyte growth factor, another cytokine like IFN-γ is involved in the induction or upregulation of cell adhesion molecules [10], and IL-10 downregulates Th1- type cytokine secretion [24] and thus acts as an inhibitory molecule.

In humans, a disease such as atopic eczema is characterized by skin infiltration by T lymphocytes expressing a Th2 like profile in its acute phase whether the skin-infiltrating T lymphocytes in allergic contact dermatitis, psoriasis, and late-phase chronic atopic dermatitis express a Th1 like profile. In irritant contact dermatitis, studies investigating cytokine profiles in the acute reactions have mainly detected increased levels of IL-2 and IFN-γ, thereby indicating a Th1-cytokine profile, as discussed in this chapter.

Recent, it has been demonstrated that T lymphocytes entering the skin often are characterized by increased expression of a surface molecule – cutaneous lymphocyte-associated antigen (CLA) [25]. This molecule participates directly in transendothelial migration of T lymphocytes. The ligand for CLA is E-

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Table 4.T helper (Th) lymphocyte cytokine profiles: cytokines predominant in the different groups

Th1 Th2 Th0

IFN-γ IL-4 INF-γ

IL-2 IL-5 IL-2

TNF-α IL-6 IL-4

TNF-β IL-9 TGF-β

IL-10 IL-13

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selectin, which is found to be upregulated in various skin diseases, including contact dermatitis. Other receptor-ligand pairs, such as lymphocyte function-associated antigen (LFA)-1/ICAM-1 and very late antigen-4 (VLA-4)/VCAM-1, are also involved in this process [26]. The importance of CLA has been demonstrated by blocking CLA in vitro, which resulted in inhibition of transendothelial T lymphocyte migration [26]. Furthermore, studies on T lymphocytes from individuals with contact allergic dermatitis have revealed that preferentially CLA⁺cells are activated and recruited to the skin [27]. Thus, the importance of CLA as a selective skin homing receptor for T lymphocytes has been established and this molecule seems to play an important role in the recruit- ment of T lymphocytes to the local inflammatory reaction site in the skin. Despite these observations, the role of CLA expression in irritant contact dermatitis is still not clarified.

쐽

Inflammatory skin diseases, including irritant contact dermatitis, are characterized by influx of activated T lymphocytes. In general the skin-infiltrating T lymphocytes express CLA; however, their role in irritant contact dermatitis is unknown. In irritant contact dermatitis, studies investigating cytokine profiles are preferentially performed in the acute reactions and these investigations have detected increased levels of IL-2 and IFN-γ and thereby indicate a Th1-cytokine profile.

4.4 Pathogenesis of Acute Irritant Contact Dermatitis

Research within the field of irritant contact dermatitis has primary been focused on the development of the acute irritant reaction and only to a lesser degree the chronic irritant reaction. For many years re- searchers have tried to differentiate between the allergic and irritant skin reactions by the means of his- topathology or immunohistopathology [28, 29] – as described in Chapter 8. However, only minor differences have been revealed. Until recently, skin irritation was thought to be a nonimmunological reaction in the skin; however, recent work has indeed implicated the immune system in the development and maintenance of irritant-induced skin reactions. In

contrast to allergic skin reactions, no immunological memory seems to be involved in eliciting irritant contact dermatitis and the development of irritant skin reactions does not require prior sensitization.

Although chemical differences exist between different irritants, exposure of the skin to irritants often lead to skin barrier perturbation, skin infiltration by immunocompetent cells, and induction of inflammatory signal molecules.

4.4.1 Skin Barrier Perturbation

One major finding following exposure to skin irritants is perturbation of the skin barrier. The skin barrier is composed of the outermost layer of the epidermis – the stratum corneum. The stratum corneum consists of protein-rich cells, the corneocytes, which are embedded within a continuous lipid-rich matrix. Within the stratum corneum, the barrier function is mainly confined to the inner one-third – included within the compact part of the stratum corneum [30]. The dynamic process of damaging and re-normalization of the skin barrier can be quanti- fied using a noninvasive technique based on the measurement of transepidermal water loss (TEWL).

This method has today been accepted as a reliable marker of skin barrier disruption. Much research has been conducted using the anionic surfactant sodium lauryl sulfate (SLS).Application of SLS to human skin results in perturbation of the skin barrier and an increased TEWL measurement as compared to control values [31]. This effect is not only a transient phenomenon. Increased TEWL values have indeed been observed more than 6 days following exposure to SLS [32]. In addition, another study demonstrated that complete recovery of the skin barrier was first obtained more than 3 weeks after irritant challenge [33].

This was demonstrated by re-testing the irritant- treated skin area with the same irritant. Thus, long- lasting perturbation of the skin barrier is observed following SLS challenge of the skin in vivo.

The mechanisms behind the irritant-induced barrier perturbation are not fully understood; however, increased hydration [34] and disorganization of the lipid bilayers of the epidermis [35] have been reported. Although one could argue that disruption of the skin barrier is merely a mechanical change of the skin, several studies have demonstrated the importance of an intact stratum corneum. Disruption of the barrier could actually result in the induction of a danger signal. In support of this, it has been demonstrated that acetone treatment or impeachment of the skin barrier by tape stripping results in increased mitotic activity in the basal keratinocytes [36]. Fur-

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thermore, studies have indicated that, following disruption of the skin barrier, increased levels of immunological active signal molecules, in particular IL-1α, IL-1β, TNF-α and GM-CSF, are present within the skin [37]. Thus, taken together, perturbation of the skin barrier itself could actually initiate an immunological stress signal leading to the subsequent development of an inflammatory reaction locally in the skin.

Finally, an impaired skin barrier also facilitates skin penetration by the irritant itself, or by other external agents including allergens and bacteria. Thus, perturbation of the skin barrier is thereby implicated in many skin diseases and thought to be a major player in the induction of irritant contact dermatitis.

쐽

One hallmark of irritant exposure is perturbation of the skin barrier. This facilitates penetration by external agents and by itself induces inflammatory signals locally in challenged skin.

4.4.2 Cellular Immunological Changes in Irritant Contact Dermatitis

As described above, the skin, which is the outermost outpost of the immune system, is an organ essential for the initiation and maintenance of contact dermatitis.Although much research has been focused on allergic contact dermatitis, numerous studies have characterized the cellular infiltrate in irritant contact dermatitis, especially the experimentally induced acute irritant reaction. The histological manifestation of the irritant reaction is often impossible to dis- tinguish from the manifestation observed in the contact allergic reaction [28, 29]. In addition, diversity of the histopathological changes is seen following skin exposure to different irritants [38]. However, the cellular infiltrate is characterized mainly by mononuclear cells in particular T lymphocytes belonging to the CD4⁺subset [39, 40]. These T lymphocytes detected in irritant contact dermatitis seem to belong to a Th1-like subpopulation, as the major T lymphocyte cytokines detected are IFN-γ and IL-2 [41]. This observation parallels findings in allergic contact dermatitis. Furthermore, a study has shown that in both allergic and irritant skin reactions, an increase in number of CLA⁺T lymphocytes is observed in the skin [42]. This study was, however, performed on

atopic individuals. Another study also found an increase in CD3⁺cells in skin biopsy samples from irritant reactions, however in this study they actually observed a decreased percentage of CLA⁺cells as compared to samples from atopic dermatitis skin [43].

Furthermore, the same study found marked expression of integrin α4β7 by T lymphocytes present in the skin [43].α4β7 is a gut homing marker and skin expression of this molecule suggests that a nonspe- cific influx of T lymphocytes has occurred and that CLA is not a prerequisite for cutaneous T lymphocyte infiltration [43, 44]. Thus, the precise role of CLA in irritant contact dermatitis is still not clearly understood.

In addition to CLA-positive T cells, new informa- tion has implicated cells expressing IL-2 receptor (CD25) in the regulation of inflammation in tissues, including the skin. The CD25-positive T cells seem to be downregulators of inflammation and thus involved in the regulation and termination of inflammatory processes. In allergic contact dermatitis, a decreased number of CD25-positive cells has been observed in involved skin (nickel allergic patch tests) compared to normal skin. However, it is imperative to state that a role for CD25-positive T cells in the development and maintenance of the irritant reaction is currently unknown.

Many studies have implicated the keratinocyte as an important player in the induction of immunological changes observed in irritant contact dermatitis (Fig. 1). The effect of irritants on the epidermal keratinocytes varies depending on the exposure. Strong acids or alkalis often result in necrosis of keratinocytes. In contrast, following damage to the skin barrier by tape-stripping or irritant challenge using SLS, an increased mitotic activity in keratinocytes has been observed [36, 45]. At the histopathological level, irritants exhibit different effects on keratinocyte mor- phology. Willis et al. [38] evaluated clinical and histological changes in skin following 48 h of exposure to different irritants [38]. Nonanoic acid induced eosin- ophilic degeneration of keratinocytes with nuclear degeneration and only minimal spongiosis. Croton oil produced considerable spongiosis, and the pres- ence of intracytoplasmic vesicles in the upper dermis was observed. SLS induced minor morphological changes in the keratinocytes and induced parakerat- osis, suggesting increased epidermal turnover. Final- ly, ditranol induced a marked swelling of the keratinocytes in the upper epidermis. Thus, specific changes of keratinocytes can be observed following exposure to structurally different irritants. In addition to inducing morphological changes in the skin, irritants are also capable of upregulating cell surface molecules on epidermal cells. One important observation

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is the capacity to upregulate MHC class II expression on keratinocytes [46]. This upregulation is also observed in the contact allergic reaction. Furthermore, induction of adhesion molecules such as ICAM-1 on keratinocytes has been demonstrated [47] and this molecule, possibly in combination with irritant-induced upregulation of E-selectin on endothelial cells [48], is known to be involved in T lymphocyte accu- mulation within the skin. Finally, irritant challenge results in the release of several keratinocyte-derived cytokines, as discussed later.

The involvement of the epidermal Langerhans cell in irritant contact dermatitis is still unclear. Some studies have indicated that the number of epidermal Langerhans cells remain unaltered in the skin. In contrast, other studies have demonstrated a decrease in epidermal Langerhans cell numbers following irritant challenge [22, 49–51]. The effect of irritants on Langerhans cell number was long lasting, and full recovery was first obtained 4 weeks following irritant challenge [22]. In support of the latter observation, increased numbers of Langerhans cells have been identified in the afferent lymphatic system following irritant challenge of human skin [52, 53]. However, one must consider that chemically different irritants might have different capacities to modulate Lange- rhans cell numbers. Accordingly, different effects on Langerhans cell numbers have been observed when comparing SLS and nonanoic acid (NAA) [54].

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The histological manifestation of the irritant reaction is often impossible to distin- guish from the contact allergic reaction.

Furthermore, diverse histopathological

changes are seen following skin exposure to different irritants. In general, during the acute phase of the irritant reaction, a decrease in epidermal Langerhans cells number is observed, and upregulation of MHC class II and ICAM-1 on keratinocytes is demonstrated.

4.4.3 Epidermal Cytokines Involved in Irritant Contact Dermatitis

As discussed before, both keratinocytes and Lange- rhans cells exhibit the capacity to secrete a variety of immunologically active cytokines. In irritant contact dermatitis many cytokines have been found to be upregulated as compared to normal, uninvolved skin (Table 5).Although demonstration of increased levels

Fig. 1.

Keratinocyte responses to skin irritants

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Table 5.Cytokines upregulated in irritant contact dermatitis In vivo In vitro

Interleukin-1α [41, 55]

Interleukin-1β [56, 57]

Interleukin-2 [41]

Interleukin-6 [57, 58]

Interleukin-8 [59]

Interleukin-10 [56]

Tumor necrosis factor-α [60, 61] [62]

Granulocyte-macrophage colony [60]

stimulating factor

Interferon-γ [41, 60]

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of cytokines in the irritant reaction is well established both in vivo and in vitro, different results are published in the literature as to which cytokines actually are increased. Many studies have investigated one or two irritants, and generalized from these data.

However, today it is known that the application of different irritants to the skin results in the induction of different cytokine profiles. One example is a study by Grängsjö et al. demonstrating that in contrast to SLS, NAA is capable of upregulating IL-6 mRNA in human skin [63]. Similar, several irritants including SLS, but not benzalkonium chloride, have been demonstrated to upregulate TNF-α [58]. The complexity of irritant-induced cytokine profiles in skin is further underscored by the findings that SLS, phenol, and croton oil all upregulate IL-8 whereas only croton oil upregulates GM-CSF [64]. Thus, differences exist in the capability of irritants to induce cytokines. Of the many irritant-inducible cytokines (see Table 5), the pro-inflammatory cytokines IL-1α, IL- 1β, and TNF-α are of particular interest.

4.5 Irritant-induced Interleukin-1

Interleukin-1, which was first isolated from monocy- tes, is now known to be synthesized in several cell types, including keratinocytes. IL-1 exists in two functionally active forms: IL-1α and IL-1β.

In normal skin, IL-1α is constitutively produced by the keratinocytes, and damaging the cell mem- brane can result in the release of pre-formed IL-1α to the intercellular space. IL-1α is the major form of IL-1 produced by keratinocytes and is secreted as an active molecule. In contrast, IL-1β is secreted as a 31-kDa biologically inactive precursor, which has to be cleaved into an active 17.5-kDa molecule by a pro- tease, not present in resting human keratinocytes.

However, in activated keratinocytes, mRNA of IL-1β- converting-enzyme was readily detected following incubation with the hapten urushiol or the irritants phorbol myristate acetate (PMA) or SLS [65]. Thus, even though the keratinocyte is not capable of syn- thesizing immunological active IL-1β in intact skin, this capacity can be induced by external inflammatory signals. The mechanism for this induction remains unclear. IL-1 is a multifunctional cytokine [66], implicated in T lymphocyte activation and IL-2 production. In addition, IL-1 is involved in upregulation of IL-2 receptors on activated T lymphocytes and is chemotactic for T lymphocytes. IL-1β is also produced by the Langerhans cell and involved in antigen presentation and Langerhans cell migration.

Furthermore, IL-1 is capable of inducing other keratinocytes to release or synthesize IL-1 in a paracrine

or even autocrine fashion [67] as well as upregulating other cytokines including epidermal growth factor, IL-6, IL-8, and GM-CSF [68]. Thus, the release of IL-1 can lead to amplification of the ongoing immunological process. In addition to its capacity to regulate other cytokines, IL-1 upregulates cell adhesion molecules on the keratinocyte. In vitro analyses have demonstrated that IL-1 upregulates ICAM-1 expression on keratinocytes, thereby further contributing to the maintenance of the inflammatory cells in the skin.

When analyzing cytokine profiles in the early phases of the allergic as well as irritant reaction in mice, Enk and Katz demonstrated that IL-1β is upregulated as early as 15 min following application of an allergen but not an irritant. Cell depletion studies revealed the Langerhans cell as the cellular source [60].

Furthermore, blocking IL-1β inhibited the elicitation of the allergic reaction, thereby substantiated by the importance of IL-1β. Similar, injection of recombinant IL-1β in vivo led to the development of a clinical reaction, indistinguishable from the contact dermatitis reaction. This observation has supported the hypothesis that expression of IL-1β could differentiate between contact allergic and irritant reactions. How- ever, later studies have indeed found IL-1β in the irritant reaction, though at later time points [56, 57].

Thus, early synthesis of IL-1β seems to be an important initial step in the induction of allergic contact dermatitis, but is not specific for allergic reactions.

쐽

Both IL-1α and IL-1β have been found to be upregulated in the contact irritant reaction.

In murine studies, IL-1β was the first cytokine upregulated and injection of IL-1β in vivo resulted in clinical eczema indistinguishable from the irritant reaction.

4.6 Irritant-induced TNF-α

TNF-α was first described as a molecule exhibiting anti-tumor activity in vivo and in vitro. TNF-α is a highly pleomorphic cytokine [66], produced by a variety of cell types, including T lymphocytes, monocy- tes, Langerhans cells, fibroblasts, and keratinocytes.

TNF-α is synthesized as a 26-kDa pro-peptide. Be- fore secretion the pro-peptide is converted into a 17- kDa protein by metalloproteases [69]. In its active form, TNF-α is composed of three 17-kDa subunits.

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TNF-α exerts its function by binding to specific cell surface receptors. Two distinct TNF-α receptors are described. TNF-R1 (414 amino acids) has a mo- lecular weight of approximately 55–60 kDa and TNF- R2 (461 amino acids) is a 75- to 80-kDa receptor.

These receptors have similar extracellular structures but distinct cytoplasmic domains. The TNF receptors are expressed on a variety of cells, however mainly the TNF-R1, which is involved in metabolic altera- tions, cytokine production, and cell death, is expressed on the keratinocytes [70]. TNF-α stimulates the production of collagenase and prostaglandin E2

by synovial cells and dermal fibroblasts and thus contributes to inflammation and tissue destruction in general. TNF-α increases both MHC class II antigen expression and upregulates the surface expression of ICAM-1 on keratinocytes [71, 72]. Thus, TNF- α is an important cytokine involved in the maintenance of inflammatory processes in the skin. The pro-inflammatory role of TNF-α is stressed by its capacity to induce other inflammatory markers, including IL-1α, IL-6, and the chemoattractant IL-8 [66].

Finally, it has been demonstrated that blocking TNF-α results in inhibition of Langerhans cell migration towards the local lymph nodes following epi- cutaneously applied allergens or irritants [73, 74].

The importance of TNF-α in irritant contact dermatitis has been further emphasized by studies by Pi- guet et al. demonstrating that primary irritant reactions to trinitrochlorobenzene (TNCB) could be inhibited in vivo by injection of antibodies to TNF or recombinant soluble TNF receptors [61]. Thus, TNF- α seems to be a key player in the induction of irritant reactions in the skin.

Several irritants exhibit the capacity to upregulate TNF-α in skin. These irritants include dimethylsulfoxide (DMSO), PMA, formaldehyde, phenol, tribu- tylin, and SLS [56, 62, 75]. The list of skin irritants that upregulate TNF-α is still growing, and studies reveal that this upregulation is also found by application of allergens to the skin and when analyzing the irritant capacity of sensitizers, e.g., TNCB, DNTB, and nickel [61, 62]. Although many irritants upregulate TNF-α in skin, no increase in TNF-α expression has been observed following skin application of benzalkonium chloride [58]. Thus, as previously discussed, different irritants interact or regulate the immune system at different levels.

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Several irritants can induce keratinocyte expression of TNF-α both in vitro and in vivo. The importance of irritant-induced TNF-α is stressed by observations by Piguet et al. [61], who could block elicitation of irritant reactions by administration of anti-TNF antibodies.

4.7 Mechanisms of Irritant-induced TNF-α in Keratinocytes

Most previous studies addressing the upregulation of cytokine expression in skin have focused on protein measurements – often by ELISA. In addition, cytokine mRNA expression has been determined by ei- ther Northern blotting or reverse transcriptase poly- merase chain reaction (RT-PCR). Increased protein and mRNA expression has been interpreted as an increase in synthesis of the investigated cytokine. How- ever, increased mRNA stability or other posttranscriptional modifications have hardly been ad- dressed. The importance of such investigations is stressed by findings that both transcriptional and translational mechanisms were involved the lipopol- ysaccharide-induced upregulation of TNF-α mRNA in macrophages [76]. Recently it was determined whether transcriptional or posttranscriptional mechanisms are involved in the irritant-induced upregulation of TNF-α in keratinocytes [62]. This study was performed on murine keratinocytes that were transfected with a chloramphenicol acetyl transfe- rase (CAT) reporter construct containing the full- length TNF-α 5´-promoter region. Increased TNF-α promoter activity was indeed observed following in vitro exposure to the irritants PMA and DMSO, strongly suggesting that the PMA- and DMSO-induced upregulation of TNF-α mRNA in keratinocytes is due to increased transcription of the TNF-α gene. These findings were further substantiated by the observation that no significant difference in TNF-α mRNA stability was observed between un- stimulated and stimulated keratinocytes [62]. It is generally accepted that the irritant PMA mediates most of its effects via PKCα-dependent signal trans- duction pathways. Accordingly, it was found that PMA, as well as the common irritants DMSO and SLS, induced an increase in TNF-α mRNA in keratinocytes via a PKC-dependent signaling pathway (Fig. 2).

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It is known that nickel, in addition to being a fre- quent contact sensitizer, can act as an irritant in nonsensitized animals. Furthermore, nickel exhibits the capacity to upregulate TNF-α mRNA and protein in purified keratinocytes. Inhibitors of PKC and of the cyclic nucleoside-dependent protein kinase were reported not to block this nickel-induced increase in TNF-α mRNA. In addition, this study demonstrated no increase in TNF-α promoter activity following stimulation with nickel. Of particularly interest was the finding that nickel stimulation of keratinocytes in vitro resulted in a pronounced increase in the stability of TNF-α mRNA as compared to unstimu- lated control cultures [62]. The precise mechanism of the nickel-induced increased stability of TNF-α mRNA remains unclear. One possibility is modifica- tion of peptides binding to an AUUUA-sequence in the 3´-region of the mRNA thereby blocking/inhibiting degradation of the mRNA transcript. Another possibility is that nickel stimulation could result in sequestering TNF-α mRNA in the ribosomal com- partment, thereby stabilizing the mRNA. Indepen- dently of the mechanism, the overall result was an increase in the release of biologically active TNF protein.

Thus, when comparing the irritant effect of nickel in nonsensitized animals with irritants such as DMSO and PMA, different intracellular signaling mechanisms are involved in upregulation of TNF-α peptide expression (Fig. 2).

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Not all skin irritants induce measurable TNF-α. Furthermore, different signaling mechanisms have been described, including direct gene activation (transcription) and stabilization of the TNF-α mRNA (posttranscriptional regulation).

4.7.1 Regulation of the Inflammatory Milieu Locally in Inflamed Skin

As described in this chapter, an upregulation of pro- and inflammatory cytokines is present in the irritant reaction. It is noteworthy that this type of reaction often tends to exhibit a prolonged course, even despite removal of the irritant exposure. Thus, the clinical reaction may continue for several years. Until recently, no explanation for this phenomenon has been forwarded. However, data are now available suggesting that elements in the local inflammatory milieu may actually contribute to the persistence of skin inflammation. Previous, it was shown that autocrine regulation of IL-1, both IL-1α and IL-1β, is present in vitro [77, 78]. Therefore, a study was enforced to de- scribe whether such autocrine regulation of the pro- inflammatory cytokine – TNFα – was present in keratinocytes. Indeed, it was found that stimulating keratinocytes with TNF-α in vitro led to an increase in

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Fig. 2.Mechanisms of irritant-induced TNF-α in keratinocytes. Irritants (e.g., PMA, DMSO, SLS) upregulate TNF-α mRNA in keratinocytes via a PKC-dependent signaling pathway resulting in increased mRNA transcription. In contrast, nickel salts mediate their effects by increasing the stability of TNF-α

mRNA. Both pathways ultimately lead to increased release of TNF protein. (DMSO Dimethylsulfoxide, PKC protein kinase C, PMA phorbol myristate acetate,SLS sodium lauryl sulfate,TNF tumor necrosis factor)

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TNF-α mRNA expression [79]. This potential, inter- esting signaling pathway was critically dependent upon signaling through PKC-dependent pathways and involved increased gene transcription. Thus, it was shown that induction of the pro-inflammatory cytokine TNF-α, e.g., by skin irritants, could lead to induction of an autocrine signaling pathway locally in the skin, thereby substantiating the inflammatory reaction and as such contributing to the persistence of the clinical irritant skin reaction.

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Skin irritants can induce an inflammatory milieu, following which further amplification is possible. Today, data exist demonstrating autocrine regulation of both IL-1 and TNF-α in keratinocytes.

4.8 Hypothesis of the Immunological Events Leading to Irritant Contact Dermatitis

Following application of irritants to the skin, penetration of the stratum corneum is the primary event.

During this, perturbation of the skin barrier occurs.

This further facilitates the penetration of the skin by the irritant and other external agents. Following penetration of the stratum corneum, the irritant most likely induces the release of pre-formed IL-1α from the keratinocytes, and induces the synthesis of several other immunoregulatory keratinocyte-derived cy-

tokines (Fig. 3). TNF-α in particular seems essential, because in a murine system injection of antibodies to TNF in vivo completely blocks the development of irritant reactions [61]. The mechanism of irritant-induced upregulation of TNF-α seems to involve increased transcription of the gene; however, irritant- induced stabilization of cytokine mRNA may also contribute [62]. Next, induction of cell adhesion molecules such as ICAM-1 on the keratinocytes and E-selectin on the endothelial cells facilitates the ex- travasation of inflammatory T lymphocytes to the skin. This process may be enforced by the release of the chemoattractant IL-8 by the keratinocytes [80].

During the first 24–72 h, an epidermal influx on non- Langerhans cell-derived antigen-presenting cells occurs. In addition, the number of epidermal Lange- rhans cells decreases and these cells possibly migrate towards the draining local lymph node. A cellular infiltrate comprised mainly of mononuclear cells, in particular CD4⁺T lymphocytes, is then seen in the involved skin area. These cells are activated and they release inflammatory cytokines. In particular, increased levels of IFN-γ and IL-2 have been observed [41]. Ultimately, these events lead to the histological picture of acute irritant contact dermatitis.

The often-observed chronicity of irritant contact dermatitis is elusive. However, the irritant-induced inflammation may expose the immune system to im- munogenic skin peptides that it does not normally see. The chronicity may therefore involve presentation of such self-peptides to the immune system resulting in the development of an autoimmune skin disease. Alternative, the irritant-induced TNF-α is regulated in an autocrine way and thereby involved in the maintenance of an inflammatory milieu locally in the skin. The resulting irritant contact dermatitis reaction may continue for years.

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Fig. 3.

Epidermal changes following exposure to irritants

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