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Immunotoxicology studies two different effects of xenobiotics: immunosuppression and dysregulation of immune responses leading to hypersensitivity or autoimmunity. The skin is a major target organ of immunotoxicity which is provoked by small-molecular-weight compounds. Methods may be helpful for immunotoxicological investigations and screenings for adverse effects of xenobiotics which are used for diagnosis or studies on the pathophysiology of skin disorders such as allergic contact dermatitis, cutaneous drug-allergic reactions or autoimmune diseases of the skin. Examples include well-designed patch tests, assays involving antigen-presenting cells such as dendritic cells, but also T lymphocytes, basophiles or keratinocytes., (Copyright 2001 S. Karger AG, Basel)

Allergic contact dermatitis (ACD) is one of the most common skin diseases with a great socioeconomic impact. Although extensively studied, its pathophysiology and the interaction of different cells and factors which lead to sensitization and elicitation reaction are still not completely understood. The advent of transgenic mouse technology has considerably changed the study of ACD. This technology has been used extensively to investigate biomedically important issues in such diverse areas as mammalian development, neurophysiology and immunology. This new approach has already led to fascinating results which are confirmatory but also contradictory to previous thinking on the role of certain cytokines, adhesion and cell surface molecules in the complex pathophysiologic process of ACD. This review will describe how recent experiments employing mice carrying cytokine and accessory molecule transgenes change our current understanding of contact dermatitis which may lead to improved therapeutic strategies.

We tested the hypothesis that topical adenosine monophosphate phosphodiesterase (cAMP PDE) inhibitors are anti-inflammatory. These can be shown by a correlation between PDE inhibitory and anti-inflammatory function of a series of known PDE inhibitors. The effect of various cAMP PDE inhibitors on PDEs isolated from HaCaT cells was first investigated. These compounds were then tested as anti-irritants against topical 8% Balsam of Peru. A direct correlation was observed between the in vitro EC(50) values for PDE inhibition and the in vivo anti-inflammatory potential with a correlation coefficient of r = 0.79. These results stress the value of PDE inhibitors as anti-inflammatory agents in topical use, and also demonstrate that the in vitro PDE assay can be used to predict in vivo anti-inflammatory potential., (Copyright 2000 S. Karger AG, Basel.)

The immunosuppressive effect of topical ethacrynic acid (ECA) was tested on both the induction and elicitation phases of contact sensitization in a mouse model. ECA (0.5% in vehicle) reduced the sensitization response by >50% when the sensitizer was either dinitrochlorobenzene (DNCB), oxazalone (OX) or para-phenylenediamine (PPD), and was applied 1 day later to the ECA-pretreated skin site. The immunosuppressive effect of combining ECA with either hydrocortisone or with cis-urocanic acid was also tested. An additive suppressive effect was observed with ECA in both combinations. The effect of ECA (1% in vehicle) on blocking the elicitation phase was also examined in a mouse ear edema assay. ECA was highly effective in preventing the challenge response in mice previously sensitized to either DNCB, OX or PPD. ECA (1% in vehicle) was also tested for its ability to inhibit contact irritation. ECA (1% in vehicle) was highly effective in preventing ear edema due to topically applied skin irritants including arachidonic acid, capsaicin, lactic acid, phorbol myristate acetate, trans-retinoic acid, and sodium lauryl sulfate. ECA may be useful for both prophylaxis and therapeutic treatment of diverse skin conditions including contact dermatitis, eczema, and other related allergic skin disorders.

Background: Perforin (Pf), a pore-forming protein, is a cytolytic protein of killer cells. Its deposition in lesioned skin has not been studied., Objective: The purpose of this study is to show Pf deposition in the lesioned skin and Pf expression in the dermal infiltrates of various inflammatory skin diseases., Methods: Frozen specimens obtained from 29 patients with 5 different diseases were immunohistochemically stained., Results: Granular deposition of Pf was found in the lesioned skin in 2 out of 5 cases of viral vesicles and in 3 out of 7 cases of erythema multiforme. In the cases with Pf deposition, the percentages of Pf+ cells in the dermis were higher than in those without deposition., Conclusion: Pf released from natural killer cells or cytotoxic T lymphocytes may play a role in tissue damage.

Background: In Spring 1992, an epidemic outbreak of papular and follicular rashes caused by a new line of cosmetics occurred throughout Switzerland., Objective: Epidemiological and clinical data were collected in order to identify the offending agent and to specify the pathophysiological mechanisms., Methods: The data concerning 263 patients seen by dermatologists plus 642 additional cases directly reported by consumers to the manufacturer were analyzed. Seventy-seven patients were patch-tested, 26 extensively, and 15 performed a repeated open application test for a duration of 4 weeks. Control patch and use tests were performed in 73 and 25 patients, respectively. The results were analyzed statistically. In addition, 12 skin biopsies were performed for histological examination. Biochemical studies on the cosmetics (final products and offending ingredient) supplemented the clinical studies., Results: The lesions were mainly papular and follicular, widely distributed, with pronounced pruritus, which was aggravated by sweating or heat exposure, and were long lasting. In a few cases, the papules were located on intensely erythematous, well-defined plaques, suggesting irritation rather than allergy. Both immediate and delayed onsets of the lesions were observed. Skin biopsies showed signs of folliculitis and perifolliculitis with little alteration of the interfollicular epidermis. Patch and use testing disclosed vitamin E linoleate (a mixture of tocopheryl esters, mainly tocopheryl linoleate) as the offending agent. An in vitro time-dependent formation of oxidative products under storage or oxidation-stimulating conditions was observed., Conclusion: Though vitamin E esters have been widely and safely used for decades in dermatological preparations and in cosmetics, vitamin E linoleate was the cause of about 1,000 cases of unusual papular mainly follicular contact dermatitis. Oxidized vitamin E derivatives could act in vivo as haptens and/or irritants, possibly with synergistic effects.

We report a case of primary irritant contact dermatitis caused by an organostannic insecticide, tributyl tin phthalate, which has been thought to be stable and safe. A factory worker has been in contact with raw tributyl tin phthalate on one leg. Soon after taking a hot-water bath, he developed severely painful erythema on this leg. Patch tests and chemical analysis revealed that hot water hydrolyzed tributyl tin phthalate and produced concentrated phthalic acid. Since phthalic acid, a weak acid, was then concentrated, an acute irritant reaction appeared in this patient.

In order to study the relevance of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and endothelial leukocyte adhesion molecule 1 (ELAM-1) for lymphocyte extravasation in the early phase of allergic contact dermatitis, the time courses of lymphocyte infiltration and adhesion molecule expression during initiation of this disorder were determined. Sequential biopsies of positive allergic patch test reactions were obtained 4, 8, 16 and 24 h after antigen application, cryostat sectioned and stained with monoclonal antibodies for ICAM-1, VCAM-1, ELAM-1 and lymphocytes by use of an immunoperoxidase technique. The slides were evaluated semiquantitatively according to appropriate gradation scales that had been defined separately for the staining with each antibody. Our results show that there is a significant upregulation of VCAM-1 and ELAM-1 expression within the first 8 h after antigen application, when lymphocyte extravasation is just beginning. In contrast, ICAM-1 is already expressed in higher levels in normal skin and is hardly enhanced during the first 8 h of patch test reactions. The main influx of lymphocytes occurs 16-24 h after antigen application and is accompanied by a further increase in all three adhesion molecules. We conclude that VCAM-1 and ELAM-1 rather than ICAM-1 may be of particular importance for the start phase of allergic contact dermatitis and that all three of them contribute to an amplification of this inflammation.

We have evaluated a subchronic model of contact hypersensitivity in the guinea pig to mimic human chronic/recurrent eczema. Repeated challenges of the ears of previously sensitized guinea pigs with 0.1% dinitrochlorobenzene (once a week for 4 weeks) induced a typical oedema response, which increased during the first 48 h after each challenge. Crusts were detectable (48 h after challenge) and histological observations (72 h after challenge) revealed hyperplasia, papillomatosis, hyperkeratosis and some mononuclear cell infiltrates in the dermis. In agreement with clinical observations in humans, topical treatment of challenged animals with corticosteroid (1% hydrocortisone) reduced the oedema, hyperplasia, papillomatosis, and leucocyte infiltrates, while application of 5% bufexamac (a non-steroidal drug) was associated with a slight enhancement of the inflammatory response. Thus, this model presents clinical and histological similarities with human eczema. Its pharmacological relevance is also suggested, although further investigations are required to better define its selectivity.

A 57-year-old man suffering from persistent light reaction with photocontact allergy to musk ambrette and contact allergy to fragrance mix was evaluated. A lowered minimal erythema dose to UV-B (MED-UV-B) was seen. Reactions to long-wave UV-A and visible radiation were normal. A skin biopsy from one MED-UV-B, taken 24 h after irradiation, showed acute spongiotic dermatitis.

Availability of reproducible mouse models for allergic contact dermatitis (ACD) to the metal allergens nickel, mercury and chromium, would be of great value for pathogenetic and preventive studies. We explored epicutaneous sensitization to nickel, mercury and chromium in mice in which oral grooming of the sensitization site was prevented by a plaster cast around the abdomen and lower thorax. This procedure was based on earlier findings that oral ingestion of allergen could prevent contact sensitization. The present results show that BALB/c mice can be readily sensitized to mercury and chromium using this epicutaneous casting method, without the further use of adjuvants. With nickel, however, neither this method, nor conventional methods involving the use of Freund's complete adjuvant (FCA) were effective.

It is well known that applications of a single dose of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse ears induces an acute inflammatory reaction consisting of erythema, edema and polymorphonuclear leukocyte (PMN) infiltration. We report here that multiple topical applications of TPA to mouse ears produce a prolonged inflammatory reaction characterized by increases in ear weight, inflammatory cell infiltration and epidermal hyperplasia. TPA was applied 5 times over 10 days to mouse ears. Epidermal thickness and PMN infiltration (myeloperoxidase content) increased 3- and 160-fold, respectively, by day 3 and remained elevated over control values throughout the test period. Ear weight was elevated from day 1 and remained high. Hydrocortisone 17-valerate and betamethasone dipropionate significantly reduced all three parameters of inflammation. Indomethacin and two other cyclo-oxygenase inhibitors, and an antihistamine had little or no effect on any of the parameters. This chronic skin inflammation model may be more relevant for evaluating anti-inflammatory compounds than the acute TPA model because the test compounds are applied after the inflammatory lesion is established, which mirrors the use of clinical anti-inflammatory drugs. Also this model may be more selective than the acute TPA model for compounds which affect leukotriene production since other pharmacological agents which are active in the acute model are not active in the multiple-application model.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the interaction of immunocompetent cells in inflammation. It contributes to lymphocyte recruitment, antigen presentation and T-cell activation. Recently, the murine homologue of ICAM-1, originally designated as MALA-2, has been identified. The monoclonal antibody YN1/1.7 for murine ICAM-1 enables the analysis of ICAM-1 expression in murine allergic contact dermatitis, a prototype of cutaneous T-cell-mediated inflammatory response. At 0, 1, 8, 24, 48, 72 h, and 7 and 14 days after challenge with 2,4-dinitrofluoro-1-benzene (DNFB) cryostat sections of ear skin were immunostained for ICAM-1, I-A and mononuclear cells (L3T4, Lyt-2, BM8). In normal skin ICAM-1 labeling was restricted to endothelial cells and dermal dendritic cells/macrophages; keratinocytes (KCs) did not express ICAM-1. The dermal cellular infiltrate increased progressively from 8 to 72 h after DNFB challenge. The majority of infiltrating cells were BM8+ macrophages (75%) and L3T4+ (10%) or Lyt-2+ T cells (10%); maximally 30% of those stained positive for ICAM-1. At 24 h, focal ICAM-1 expression on KCs developed, reached a maximum at 72 h and faded thereafter. Migration of T cells into the epidermal layer started at 48 h at sites which had already expressed ICAM-1. Our data provide evidence that expression of ICAM-1 by epidermal cells precedes infiltration of the epidermis by T lymphocytes as shown before in human cutaneous disorders. Thus, a mouse model may be useful to investigate the role of ICAM-1 in inflammation further.

Many coelenterates can injure human skin by means of their nematocytes. Our study was based upon clinico-pathological confrontations and was also conducted in vitro with sea anemones. Most acute reactions were toxic in nature and induced sublethal and lethal alterations of the keratinocytes with swelling of their cytoplasms and pyknosis. Another reaction found in 1 patient revealed an acantholytic disruption of the epidermis associated with intercellular deposits of IgG. These lesions were different from the late or recurrent reactions to coelenterates.

Mice of the randomly bred strain ICR and of the inbred strain BALB/c could be successfully sensitized to chromate by repeated topical applications of a solution of potassium bichromate in dimethyl sulfoxide. The specificity of chromium sensitization was ascertained by ear tests, specific desensitization by intravenous injection of potassium bichromate and by histological ear sections showing marked infiltrates of leukocytes following ear application of chromium-dimethyl sulfoxide in already sensitized animals. It is concluded that mice may provide a useful experimental model for study of contact sensitivity to chromium.

Four patients had a recurrence of cutaneous lesions 1 week after being stung by jellyfish. Three patients had flare-up lesions after only one exposure to jellyfish. All of the recurring lesions were vesicular erythema, and the histological findings of case 3 corresponded to that of allergic contact dermatitis.

Mast cells are critical for the expression of certain IgE-mediated responses, but the precise contributions of mast cells to biological processes not involving IgE are obscure. We have employed genetically mast cell-deficient WBB6F1-W/Wv and WCB6F1-S1/S1d mice to investigate the roles of mast cells in several different biological responses. This work strongly suggests that mast cells are not required for the elicitation of contact sensitivity (CS) responses, suppressor T cell-dependent tolerance to CS, reserpine-induced inhibition of CS responses, or bleomycin-induced pulmonary fibrosis. By contrast, mast cells appear to contribute to the acute gastric injury induced by ethanol and the acute inflammation of the skin induced by croton oil.

A comparison has been made of the 'flare-up' of 2,4-dinitrofluorobenzene (DNFB) contact reactions with that of 'flare-up' of potassium dichromate (K2Cr2O7) contact sensitivity reactions following the intravenous injection of dinitrobenzenesulphonic acid (DNBSO3) or K2Cr2O7. Differences in time course were noted and these were confirmed by increase in vascular permeability and histological appearance. Failure to influence the reactions with indomethacin would appear to exclude the participation of an Arthus-type phenomenon, despite evidence of a drop in the circulating antibody levels. Increased numbers of basophils were found particularly in the DNFB/DNBSO3 reaction during its resolution. These reactions could therefore be broadly included among the cutaneous basophil hypersensitivities.

We have studied the specificity of macroscopic responses and the corresponding cellular infiltrates in Giemsa-stained, glycol methacrylate embedded tissue sections of delayed skin reactions elicited by contact sensitizers, homologous and heterologous hapten-protein conjugates, and tuberculin (PPD) in guinea pigs. The animals were sensitized with contact sensitizers emulsified in Freund's complete adjuvant and tested at 19 days and, after repeated contact testing with homologous sensitizer, at 48 days after sensitization. At both times, all delayed reactions contained numerous mononuclear cells. The reactions elicited by contact sensitizer or hapten-protein conjugate also contained significant numbers of basophils which were lacking in PPD reactions. Contact reactions were highly specific macroscopically and microscopically while reactions elicited by conjugates displayed considerable macroscopic and microscopic cross-reactivity. The greater cross-reactivity of conjugate-elicited reactions compared to contact reactions may reflect differences in mechanism of initiation or mediation of these two reactions.

Draining lymph node cells isolated from mice 24 h following topical exposure to a variety of contact-sensitizing chemicals, including the dinitrobenzene derivatives, 2,4-dinitrochlorobenzene and 2,4-dinitrothiocyanobenzene, contained increased numbers of dendritic cells (DCs). The increase in frequency of DCs was time-dependent and preceded significant changes in either lymph node cellularity or lymph node cell proliferative activity. The degree of DC accumulation was also influenced by the chemical used and the concentration employed for sensitization. In the context of contact allergy, the biological relevance of this phenomenon to the induction of hapten-specific responses is indicated by the fact that relatively small numbers of DC--enriched fractions of lymph node cells (comprising approximately 70% DCs), but not unfractionated or DC--depleted populations, transferred sensitization to naive animals. Moreover, using the skin-sensitizing fluorochrome, fluorescein isothiocyanate, it was observed that 24 h following exposure the majority of lymph node cells bearing high concentrations of antigen were within the DC-rich fraction.

The evaluation of contact reactions in previously sensitized mice is assessed conventionally by measurement of increases in ear thickness following challenge. In an attempt to develop a serological method for the investigation of contact sensitization in mice, we have examined whether analysis of changes in the concentration of acute-phase proteins in response to challenge provides a reliable alternative means of evaluating elicitation reactions. Measurement of either the relative serum haptoglobin concentration, using radial immunodiffusion, or the absolute concentration of serum amyloid A, by an enzyme-linked immunosorbent assay, has been found to correlate well with induced increases in ear thickness following challenge. Changes in the concentration of acute-phase proteins proved to be of sufficient sensitivity to reflect the specificity of contact sensitization and its inhibition by antigenic competition.

A 4 year-old boy presented an acute pruriginous dermatitis of the hands, forearms and abdomen. A skin surface biopsy revealed numerous trichomes, small prickles present on plants. These trichomes were those of prickly pears and stuck deeply in the skin, inducing an inflammatory reaction known as Sabra dermatitis. A number of plant dermatitides are due to the presence of trichomes such as those due to linden, primula, wild rose or nettle.

A 5-lipoxygenase inhibitor (5-LPI) and a platelet-activating factor antagonist (PAF-A) were studied in dinitrofluorobenzene (DNFB)-induced allergic and croton-oil-induced irritant murine contact dermatitis. Both inhibitors, at 1 and 5% concentrations, significantly reduced the ear swelling in allergic dermatitis while irritant dermatitis was far less affected. This suggests that 5-lipoxygenase-dependent mediators and PAF are involved in allergic contact dermatitis and that these mediators play only a minor role in irritant dermatitis.

Six hours after challenge with dinitrochlorobenzene (DNCB) in sensitized volunteers, infrequent cells reacting with both OKT6 (CD 1a) and with an antibody to DNCB were demonstrated in the upper dermis in 3 out of 4 subjects, using a double fluorescence technique. Single marker studies using polyclonal and monoclonal antibodies to DNCB showed cytoplasmic reactivity with peripheral accentuation in keratinocytes throughout the epidermis, most intense in the granular and basal layers, and occasional positive cells in the papillary dermis. Immunoperoxidase electron microscopy using polyclonal and monoclonal antibodies to DNCB demonstrated reactivity on keratinocyte and Langerhans cell membranes, with intracellular deposition of reaction product on mitochondria. Reaction product was present on the external leaflets of Birbeck granules, suggesting that the penetration of DNCB was passive rather than due to an active endocytic process. The immunofluorescent studies suggest that the double-fluorescing upper dermal cells were Langerhans cells but this could not be confirmed by electron microscopy.

2 patients with textile contact dermatitis due to formaldehyde developed follicular, papular lesions. Histologically these papules showed eczematous changes in the follicular epidermis and mononuclear cell infiltrates in the dermis around the involved follicle. The skin between the papules was intact. It is suggested that the concept of "dermal" contact sensitivity does not fit for the explanation of papular dermatitis due to formaldehyde.

Hairless albino mice have been used to test the hypothesis of circulating mediators originating from extensive dermatitis, which might be responsible for 'false'-positive patch tests and non specific hypersensitivity. Two open patch tests with 2 or 3% phenol solution were placed on one flank at an interval of 10 days. 48 h before the second application, part of the mice received an intermediate 10% phenol solution on the opposite flank. The remaining animals served as controls. In general, intermediately treated animals showed a more pronounced reaction to the second patch test as compared to the first test, i.e., a larger granulocytic infiltrate, more extensive epithelial lesions and a elevated erythematous wall. In 6 of the 22 animals, the difference was very striking. In controls, only modest differences were observed. These findings favor the hypothesis that mediators of inflammation present in the circulation strengthen the effect of weak skin irritants.

We observed, in addition to the lymphocytes of the classical descriptions, large numbers of basophils in the infiltrate of the specific sensitization reaction with dinitrochlorobenzene and citraconic acid, a few eosinophils in the reaction with dinitrochlorobenzene and rather more eosinophils in the reaction with citraconic acid.