Lichenoid red tattoo reaction: histological and immunological perspectives

ARTICLE

ejd.2011.1558

Auteur(s) : Simone Garcovich¹, Teresa Carbone², Simona Avitabile², Francesca Nasorri², Nadia Fucci³, Andrea Cavani² cavani@idi.it

¹ Institute of Dermatology, Policlinico A. Gemelli University Hospital, Rome

² Laboratory of Experimental Immunology, IDI-IRCCS, Via dei Monti di Creta 104, 00167 Rome

³ Forensic Laboratory, Institute of Legal Medicine - Catholic University of Rome, Italy

Reprints: A. Cavani

Adverse delayed-type inflammatory reactions to tattoo pigments, especially red tattoo dyes, have been previously reported and described as eczematous, lichenoid, granulomatous and pseudolymphomatous reactions, on the basis of clinical and histological findings [1]. Cutaneous lesions are typically limited to the specific coloured area of the tattoo, occurring after a variable time period (weeks-months) after the tattooing. Despite the popularity of tattooing and its high prevalence rate in adults (8.5%) [1], adverse inflammatory reactions to tattoo pigments are relatively infrequent [2]. Identification of causal antigens is difficult because of the variable chemical nature of tattoo dyes, which is often unknown to the tattoo recipient, of the intradermal injection route of the tattoo pigments and of possible eliciting factors such as trauma and sun-exposure. We report a case of a lichenoid tissue reaction to an organic red tattoo pigment, which was further analyzed by means of different in vivo (patch-tests) and in vitro assays (immunohistochemistry and immunophenotyping), documenting a typical Th1-mediated lichenoid reaction to chemically reactive substances of the tattoo dye.

Materials and methods

For immunostaining, paraffin-embedded 3-m formalin fixed samples were de-waxed in xylene and ethanol and rehydrated. The slides were incubated with hydrogen peroxidase (Ultra Tek HPR, Scytek Laboratories) to quench endogenous peroxidase activity and then with Super block (Ultra Tek HPR, Scytek Laboratories) at room temperature to inhibit non-specific binding. The sections were additionally boiled in Tris-EDTA-citrate buffer, PH 7.8 (UCS Diagnostic) at 96̊C to optimize antigen retrieval. Every step was followed by extensive washing in PBS. Sections were immunostained using as primary antibodies mouse pure mAb anti-human CD56 (CD564, IgG2b), (Novocastra, Newcastle, UK) and CD8 (Biocare medical) and polyclonal rabbit anti-human CD3 (DAKO, Glostrun, Denmark) with highly sensitive biotin-avidin immunoperoxidase tecnique (ScyTek Laboratoires). Nuclei were counterstained with hematoxylin. To better define the inflammatory infiltrate, a biopsy was cultured in RPMI 1640 supplemented with 2mM glutamine, 1mM sodium pyruvate, 1% non-essential amino acids, 0.05 mM 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Lonza, Basel, Swisserland) (complete RPMI) plus 5% human serum (Sigma-Aldrich, St. Louis, MO) supplemented with 100U IL-2/mL. Migrated cells were collected after 7-9 days and then subjected to cytofluorimetric analysis. Antibodies used were: mouse FITC- PE- APC- PB- or APC-H7 monoclonal anti-human CD3 (SK7, IgG1), CD16 (3G8, IgG1), CD56 (B159, IgG1), CD8 (RPA-T8, IgG1), CD4 (SK3, IgG1), IFN-g (B27, IgG1), TNF-a (Mab11 IgG1), IL-4 (8D4-08, IgG1) from BD Bioscience (San Diego, CA). Mouse PE-conjugated mAb anti-human IL-22 (142928, IgG1) was from R&D systems. APC- anti-human IL17A (eBio64DEC17, IgG1) was from e-Bioscience (San Diego, CA). Mouse or rat IgG/IgM isotype controls were purchased from BD Bioscience. For surface marker staining, cell populations were washed with PBS 1% human serum/0.01% NaN₃ and then stained directly with FITC-, PE-, APC-, PB- or APC-H7 labeled mAb for 20 minutes on ice in the dark. Staining with matched isotype control Ig was included. Intracellular cytokine staining was performed with a kit (BD Biosciences), according to the manufacturer's instructions. Briefly, cells were stimulated with PMA (10 mg/mL) and ionomycin (1 mg/mL) for 6 hours in the presence of monensin. After 2 hours, brefeldin A was added for the last 4 hours. After staining with antibodies towards surface markers, cells were collected, fixed and permeabilized with Cytofix/Cytoperm (BD bioscience) and stained for 20 min with the indicated anti-cytokine mAb in the presence of Perm/Wash solution (BD bioscience). Acquisition and analysis was done with a FACSAria (BD Biosciences).

Case report

A 32-year old man presented with sharply demarcated papular-infiltrative lesions restricted to the areas of red colour of two distinct polychromatic tattoos of the right arm (figure 1). Infiltration of the red tattoo areas with itching sensations began two months after tattoo-application, triggered by leisure sun exposure. The red portions of both tattoo figures had been created using an organic azo-dye Pigment Red 5 (PR5) (commercial name “poppy red”, CI 12490, CAS No. 6410-41-9; N-(5-Chloro-2,4-dimethoxyphenyl)-4-[[5-[(diethylamino)sulfonyl]-2-methoxyphenyl]azo]-3-hydroxy-2-naphthalenecarboxamide, in two different application sessions by the same tattoo artist. The patient presented other multicoloured tattoos without signs of vesiculation, infiltration or crusting. There was no history of contact or immediate allergy, nor atopic diathesis. The patient took no medication. A skin biopsy was taken from two different lesional red tattoo areas, one processed for routine histology and immunohistochemistry, the other used for isolation and immunophenotyping of skin-infiltrating lymphocytes. Conventional histology revealed a dense, band-like lymphocytic infiltrate in the upper dermis, amidst exogenous red pigment deposition, with a thickened basement membrane zone and necrosis of basal keratinocytes (figure 2A). Immunohistochemical analysis showed abundant CD3⁺ T lymphocytes (figure 2B), some of which were CD8⁺ T cells in close contact with keratinocytes at the dermo-epidermal junction (figure 2C). CD56⁺ natural-killer cells were mostly seen in the upper dermis, with isolated elements in proximity to basal keratinocytes (figure 2D). Flow-cytometric analysis of surface markers and cytokine production of isolated lesional lymphocytes revealed CD4⁺ (34.9%) and CD8⁺ (42.1%) T cell subpopulations, as well as a minor fraction of CD56⁺ innate lymphocytes (11.5%). The latter component of the cellular infiltrate belonged mostly to a CD56⁺CD16^- NK subset (86.4% of the total NK cells) (figure 3). Although this subset represents only a minority of circulating NK cells (10% in peripheral blood), in this lichenoid skin reaction, as in many other cutaneous immune diseases, these cells were preferentially recruited into the inflamed skin. Cutaneous lymphocytes were able to release type 1 cytokines, in particular IFN-γ and TNF-α (figure 4 A). IL-17 release was highly correlated with TNF-α production but not with that of IL-22. Among gated IL-17⁺TNF-α⁺ cells, 72% were pure Th17 cells while the remaining 28% were Th1/Th17 lymphocytes. All IL-17 producing cells were negative for IL-4 (figure 4B). In contrast, only a minority of gated IL-22⁺TNF-α⁺ cells (36.4%) were pure Th22 lymphocytes, whereas the majority co-produced IFN-γ but not IL-4 (figure 4C).

Determination of the chemical nature of the culprit tattoo pigment by means of gas-liquid-chromatography (GLC) and mass spectrometry (MS) revealed the presence of intermediate reactive compounds such as naphthalene, 2-naphtol, chlorobenzene and benzene, as well as traces of saturated fatty acids and fatty acid amides.

Allergological work-up included patch tests with standard series and specific panels for organic and inorganic dyes, emulsifiers and solvents, which resulted negative after 72h. The organic azo-dye PR5 (2% in petrolatum) used in the tattoos was tested by means of patch-tests and photo-patch tests, with the aid of the tape stripping technique to improve allergen penetration. Both tests yielded negative results after delayed reading (after 2, 3 and 6 days). Finally the patient was treated with 0.1% tacrolimus ointment under occlusion for 3 months with marked decrease of infiltration and itching in the affected area.

Discussion

Lichenoid/interface dermatitis type reactions to intradermal tattoo pigments have been reported previously and associated with the use of red pigments [3]. In the past red tattoo colorants consisted predominantly of inorganic compounds such as red mercuric sulphide (cinnabar), recognized as a causative agent of lichenoid tissue reactions. Nowadays organic pigments, azo-dyes belonging to the group of Naphthol-AS pigments, are commonly used in multicoloured tattoos due to their low-solubility and colour-retaining properties [4]. In our case the red organic pigment was Pigment Red 5 (CAS No. 6410-41-9), a mono-azo pigment, commonly used in the manufacturing, textile, printing and cosmetic industries. Since tattoo pigments, in comparison with cosmetics, are not yet officially controlled, the origins and chemical structures of these colouring agents are hardly known [5]. Consequently, neither the tattoo artist nor the tattooed patient usually has information about the chemical compounds injected into the skin. Lack of regulations and standardization for pigments used in tattoos poses a major hurdle for diagnosis and allergological work-up in the case of inflammatory reactions developing in tattoo areas. Histology is usually required to assess the pattern of tissue reaction as the differential diagnosis of idiopathic lichen planus, sarcoidosis and pseudo-lymphoma may be difficult on a clinical basis. In our case, the cell-rich infiltrate intermingled with intradermal pigment was compatible with a lichenoid tissue reaction-pattern, with abundant dermal CD3⁺ and in particular CD8⁺ T lymphocytes, as well as infiltrating cutaneous CD56⁺ NK-cells, in close contact with keratinocytes. The contribution of these effector cells to the full expression of the disease has been demonstrated, thanks to their cytotoxic capacity towards basal keratinocytes and ability to secrete proinflammatory cytokines [6, 7]. Type-1 cytokine patterns define the hallmark of the microenvironment of lichen planus. In other reports, lichenoid reactions to tattoo pigments are commonly associated with negative patch test results, arguing against allergic contact hypersensitivity mechanisms towards organic tattoo pigments [8]. Allergic contact dermatitis is frequently reported in association with temporary henna tattoos containing PPD or with inorganic tattoo dyes containing metal compounds (nickel, cobalt, chromium, mercury and cadmium) [9].

On the other hand, conventional patch tests, even when aided by preliminary tape stripping or scarification and with known culprit causal allergens, as in our case, are limited by the poor solubility of organic tattoo pigments and consequently insufficient penetration through the skin barrier. Intradermal testing and exposition to UVB and UVA light sources could represent a more accurate diagnostic method by replicating intradermal deposition of causal antigens, as occurs in tattoos. Our patient refused to undergo such invasive diagnostic testing. Since modern tattoo pigments consist of photoreactive chemical substances, such as monoazo-dyes, the role of UV radiation as an eliciting factor has to be evaluated by means of photo-patch tests. In our case, photopatch tests with PR5 yielded no delayed cutaneous responses as would be expected from photoallergy. Phototoxicity could not be excluded as the in vivo test conditions did not replicate the intradermal deposition of tattoo pigments. In vitro and in vivo experimental studies have proven that monoazo-dyes such as PR5, PR12 and PR22 are subject to photo-decomposition induced by natural sunlight and laser light sources, with the production of reactive, toxic and carcinogenetic compounds, such as 2-methyl-5-nitroaniline, 1-4-dichloro- benzene, 2-5-dichloraniline , 4-nitro-toluene and napthol-AS [10]. These could possibly have triggered the lichenoid tissue reaction in our patient.

Lichenoid tissue reactions are currently interpreted as an autoimmune T-cell mediated cytotoxic attack on the epidermis [11], triggered by unknown antigens (drugs, viruses) or cross-reactive autoantigens [12, 13]. In our case the antigen triggering the inflammatory reaction is represented by the organic tattoo pigment PR5 and possibly the related intermediate-reactive compounds. Furthermore, we have demonstrated, on the basis of experimental immunohistochemistry and flow-cytometry data, evidence of a Th1 inflammatory process. Most of the skin-infiltrating cells are able to release IFN-γ and TNF-α type-1 cytokines, which are relevant for the maintenance and amplification of the type-1-dominated microenvironment, which in turn induces the expression of adhesion molecules on keratinocytes and the release of chemotactic factors by autologous cells. In contrast, IL-17- and IL-22-releasing cells represented only a minor fraction of the infiltrating T lymphocytes.

Clinical management of lichenoid tissue reactions to tattoo pigments is often difficult and several treatment modalities (surgical excision, topical steroids and immunomodulators, selective photothermolysis, CO2 laser ablation) have been used in previous reports [14]. In our case, topical tacrolimus provided a safe and effective treatment option with good clinical outcome, as confirmed by the report of Campbell et al. [15]. Selective photothermolysis represents the gold-standard for tattoo removal and has also been used for the treatment of lichenoid tattoo reactions [16], but it carries the risk of releasing additional reactive or toxic photo-decomposition products locally [17]. The long-term effects of potentially hazardous compounds, such as naphthol-AS derivatives in modern tattoo pigments, with regards to the risk of toxic/phototoxic reactions as well cross-sensitization with other allergens, are still unknown and further evidence-based assessment as well attention from the general public is needed.

Disclosure

Financial support: This work was partially supported by the Italian Minister of Health (Ricerca Corrente). Conflitcs of interest: none.

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