Applying Non-Animal Approaches to the Assessment of Skin Sensitization

By Emanuela Corsini, University of Milan

Skin sensitization potential is an endpoint that needs to be assessed within the framework of existing and forthcoming legislation. A range of in vivo methods exist that has been proven to be very accurate in terms of the predictive identification of chemicals that possess skin sensitizing properties, e.g. the local lymph node assay (LLNA). However, the challenge now to be faced is how to obtain the same quality of information on the allergenic potential of chemicals and potency using in silico and in vitro methods. Progress in understanding the mechanisms of skin sensitization, including effects on the production of cytokines by the different cell types within the skin, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitization testing. With the forthcoming elimination of in vivo tests for cosmetic testing in the European Union, several opportunities that have been exploited for in vitro test development focus on key elements of the sensitization process (1). In Table 1 the key passages of contact sensitization and in vitro opportunities are described.

Table 1. Key passages in chemicals-induced skin sensitization and in vitro opportunities

One unifying characteristic of chemical allergens is the requirement that they react with proteins for the effective induction of skin sensitization. The majority of chemical allergens are electrophilic and react with nucleophilic amino acids. One potential alternative approach to skin sensitisation hazard identification is the use of (Quantitative) structure activity relationships (QSARs) coupled with appropriate documentation and performance characteristics. This represents a major challenge. Current thinking is that QSARs might best be employed as part of a battery of approaches that collectively provide information on skin sensitisation hazard. A number of QSARs and expert systems have been developed and are described in the literature, i.e. TOPKAT, Derek for Windows and TOPS-MODE; none appears to perform sufficiently well to act as a stand-alone tool for hazard identification (4-6).

More recently, based on the requirement of chemical reactivity in the induction of skin sensitization, 82 chemicals comprising allergens of different potencies and nonallergenic chemicals were evaluated for their ability to react with reduced glutathione (GSH) or with two synthetic peptides containing either a single cysteine or lysine (peptide binding test). Following a 15-min reaction time with GSH, or a 24-h reaction time with the two synthetic peptides, the samples were analyzed by high-performance liquid chromatography. Generally, nonallergens and weak allergens demonstrated minimal to low peptide reactivity, whereas moderate to extremely potent allergens displayed moderate to high peptide reactivity. Classifying minimal reactivity as nonsensitizers and low, moderate, and high reactivity as sensitizers, it was determined that a model based on cysteine and lysine gave a prediction accuracy of 89%. The results of these investigations reveal that measurement of peptide reactivity has considerable potential utility as a screening approach for skin sensitization testing, and thereby for reducing reliance on animal-based test methods (7, 8).

Besides its barrier function, the skin has been recognised as an immunologically active tissue. Keratinocytes (KC) may convert nonspecific exogenous stimuli into the production of cytokines, adhesion molecules and chemotactic factors (18). After keratinocytes, Langerhans cells (LC) comprise the second most prominent cell type in the skin (2-5 % of the epidermal population). These are the principal antigen presenting cells (APC) in the skin (19). Due to their anatomical location and their significant role in the development of allergic contact dermatitis (ACD), the use of both these cell types to evaluate sensitizing potency in vitro is amply justifiable.

In principle, a test system comprised of KC alone may not be useful in establishing allergenic potency as these cells lack antigen-presenting capacity. However, in addition to chemical processing, LC activation requires the binding of cytokines produced by KC as a result of initial chemical exposure. The irritant capacity of allergens might present an additional risk factor so that irritant allergens may be stronger allergens than non-irritant ones (20). In that case, the potency of chemicals to induce cutaneous sensitization may be assessed as a function of KC cytokine expression. Starting from the in vivo observation that in mouse IL-1α expression by KC was selectively increased after in vivo application of contact sensitizers but not tolerogen or irritant (21), similar results were reproduced in vitro using the murine KC cell line HEL30 (10). Similar results were also obtained by van Och et al., (11) and, furthermore, the authors observed that the ranking of potency was similar to the ranking established using the LLNA. Similarly, using human KC it has been demonstrated that allergens but not irritants or tolerogens induced IL-12 (12, 22). Trinitrobenzene sulphonic acid induced the expression of CD40 on KC, whereas sodium lauryl sulphate did not (23).

More recently, our group has evaluated the possibility to use cytokine production, namely interleukin-18 (IL-18), by human keratinocytes (human keratinocyte cell line NCTC 2455) to assess in vitro the contact sensitization potential of low molecular weight chemicals. IL-18 has been demonstrated to favor Th-1 type immune response by enhancing the secretion of pro-inflammatory mediators such as TNF-α, IL-8 and IFN-γ, and to play a key proximal role in the induction of ACD. Cells were exposed to contact allergens (dinitrochlorobenzene, cinnamaldehyde, tetramethylthiuram disulfide, eugenol, isoeugenol, paraphenylediamine, resorcinol), to respiratory allergens (diphenylmethane diisocyanate, trimellitic anhydride, ammonium hexachloroplatinate) and to irritants (sodium lauryl sulphate, salicylic acid, phenol). Cell associated IL-18 were evaluated 24 later by a commercially available ELISA kit. At not cytotoxic concentrations (cell viability higher of 80% as assessed by MTT reduction assay), all contact sensitizers induced a dose-related increase in IL-18, whereas both irritants and respiratory failed to do so. Results obtained indicated that cell-associated IL-18 may provide an in vitro tool for identification and discrimination of contact versus respiratory allergens and/or irritants (manuscript in preparation). Altogether these studies indicate the possibility to identify contact sensitizers using murine or human keratinocytes.

On the other hand, Dendritic cells (DC) form a sentinel network able to detect, capture, and process antigens such as invading bacteria, viruses, products of tissue damage and haptens (24-26). Upon antigen capture, the DC undergo a maturation process leading to the upregulation of co-stimulatory molecules (CD86, CD80, CD40), MHC class II molecules and the CD83 protein (27). Thereafter, DC migrate to the T-cell areas of lymphoid organs where they lose antigen-processing activity and become potent immunostimulatory cells. Knowledge of DC physiology has progressed considerably because of the discovery of culture techniques, in the early 1990s, which support the in vitro generation of large numbers of DC from hematopoietic progenitors (28). Two main protocols to generate DC, from either monocytes or CD34⁺ hematopoietic cell precursors (HPC), have been described. Generating DC from murine bone marrow CD34⁺ HPC has been used as an alternative, but this procedure is time-consuming. The establishment of human in vitro models of DC had offered the possibility to demonstrate that haptens were able to directly activate cultured DC derived from peripheral blood monocytes or from CD34⁺ HPC (24, 29, 30-32). Several studies confirmed these observations showing the upregulation of maturation markers (CD83, CD80, CD86, CD40) on human DC (23, 33-35). Cytokine production such as IL-12p40, TNF-α, IL-1ß and IL-8 has also been reported upon hapten stimulation (24, 31, 36, 37). However, significant differences exist between experimental systems and authors concerning cytokine production and costimulatory molecules’ upregulation. Furthermore, other major limitations of such tests are the donor-to-donor variability, the low levels in the source, and a possible shortage of human sources.

A recent ECVAM workshop has reviewed the state of the art of the use of dendritic cells and human myeloid cell lines for the predictive identification of skin sensitization hazard (9). At present, only a limited number of cell lines such as THP-1, U937, KG-1 or MUTZ-3 have shown promising results. Among these, THP-1, a human monocytic leukemia cell line commercially available, has been proposed by Ashikaga et al. (38) to identify sensitizers. Recently, Yoshida et al. (39) reported that naïve THP-1 could respond to sensitizers specifically through augmented expression of co-stimulatory molecules, CD54 and CD86, and considered this as a possible tool to be used as an in vitro sensitization test. Always using THP-1 cells, we have recently investigated whether interleukin-8 (IL-8) production could provide a methodology for the detection of both respiratory and contact allergens. Following 48 h of incubation, the release of IL-8 was evaluated by sandwich ELISA. IL-8 production was significantly increased after stimulation with all contact allergens tested. In contrast to IL-8 release, CD54 and CD86 expression did not provide a sensitive method, failing to correctly identify approximately 30% of the tested compounds. Although CD86 appears to be a more sensitive marker than CD54 when discriminating allergens from irritants, neither of these markers provides robust methodology.

We also investigated if a common activation pathway in allergen-induced IL-8 production, involving p38 mitogen-activated protein kinase, could be identified. By Western blot analysis we could indeed demonstrate p38 activation by all chemical allergens tested and, using the selective p38 MAPK inhibitor SB203580, a significant modulation of allergen-induced IL-8 release could be achieved in all cases. Taken together, our data suggest that IL-8 production by naïve THP-1 cells may represent a promising in vitro model for the screening of potential chemical allergens and, that p38 MAPK activation represents a common pathway triggered by allergens (41).

Among the several endpoints investigated in different experimental models, CD86, IL-8 and p38 MAP kinase appear to be the most promising and robust biomarkers described to date in DC based assays. Therefore, it is possible that the combined analysis of these 3 biomarkers rather than analysis of a single biomarker will give even more satisfactory results.

Only limited studies address the potency of the sensitizer, i.e. whether it is an extreme, strong, moderate or weak sensitizer (39-41). In these studies, not all chemicals tested gave results which were in accordance with LLNA data, indicating that DC based assays might not be a sufficient stand-alone assay to determine sensitizer potency. In our work (41), the calculation of concentration of allergen that induced a release of IL-8 of 100 pg/ml by linear regression analysis of data gave the following ranking order: DNCB 0.9 µg/ml, PPD 2.4 µg/ml, TMTD 3.4 µg/ml, HClPt 6.8 µg/ml, Cinnamaldehyde 6.9 µg/ml, Penicillin G 13.4 µg/ml, NiSO₄ 22.6 µg/ml. If compared with the available in vivo LLNA EC3 values (DNCB 0.05 %, TMTD 5.2 %, Cinnamaldehyde 3.0 %, Penicillin G 13.4 % and PPD 0.16%), a significant correlation (R= 0.924, p=0.0248) was obtained. However, investigations need to better address the question of potency.

At present, neither single cell-based assay nor single marker is yet able to distinguish all sensitizers from non-sensitizers in a test panel of chemicals, nor is it possible to rank the sensitizing potential of the test chemicals. However, many promising methods are in various stages of development and use. It is expected that a predictive method to totally replace animal testing will be a test battery composed of molecular, cell-based, and/or computational methods. In this regard, it is important to mention the five years project named SENS-IT-IV, coordinated by Dr. E.L. Roggen (Novozyme, Denmark), recently sponsored by the EU (2005-2009). The Sens-it-iv Consortium comprises 28 research groups overall, of which 7 are from industry, 16 are from universities or research institutes, and 4 represent organizations. The project is aimed to develop "in vitro" alternatives to animal tests currently used for the risk assessment of potential skin or lung sensitizers. Two years are left, and several tests now have entered the work package devoted to in vitro assay development, including CD86 expression and IL-8 release in MUTZ-3, THP-1 and U936 cell lines, and induction of intracellular IL-18 in the human keratinocyte cell line NCTC 2544.

References

Biography

©2008 Emanuela Corsini