Allergic contact dermatitis to nickel: Modified in vitro test protocols for better detection of allergen-specific response

R. Spiewak^1,2, H. Moed³, B. M. E. von Blomberg⁴, D. P. Bruynzeel³, R. J. Scheper⁴, S. Gibbs³, T. Rustemeyer³

¹Institute of Dermatology, Krakow, Poland, ²Celimun Biomedical Research, Krakow, Poland, ³Department of Dermatology, VU University Medical Centre, Amsterdam, the Netherlands, ⁴Department of Pathology, VU University Medical Centre, Amsterdam, the Netherlands

For more than a century, patch tests have been the method of choice for the diagnosis of allergic contact dermatitis (ACD). Though indispensable, they have, however, certain limitations, such as inter-observer variability (1), site-to-site variability (2), and test-to-test variability (3). Patch test results may be influenced by the time of reading (4, 5), quality of allergens used (6), ultraviolet irradiation (7), topical and oral steroids (8, 9). In some cases, excessive irritation of the skin makes the interpretation of patch tests difficult or impossible - a situation referred to as "angry back" or "excited skin syndrome" (10, 11). Taking the above limitations into account, a reliable in vitro test for contact allergy would be greatly appreciated.

In the past decades, various in vitro tests have been used for the detection of contact allergy, starting with macrophage migration inhibition test (12) and lymphocyte blastic transformation test (13). Lymphocyte proliferation test (LPT) was introduced in the 1970s (14) and has been used until today, however, mainly for experimental purposes. Later, the above-mentioned methods were followed by analyses of cytokine and chemokine secretion, surface cell markers and gene expression. Unfortunately, none of these methods have proven sufficient for diagnostic use, mainly due to poor sensitivity and/or specificity. Combinations of 2 or 3 different in vitro methods or parameters have been proposed to overcome this problem (15, 16), however, also this approach has not found its way into routine clinical applications.

In a previous study (17), we have demonstrated that skewing lymphocytes towards "type 1" (IFN-γ-secreting cells) and "type 2" (IL-5 and IL-13 secreting cells) could improve detection of nickel-specific T cell response in contact allergy. Observations were also published suggesting that enzyme-linked immunospot assay (ELISpot) could offer advantages in detection of allergen-specific responses than other in vitro assays (18-20). In the present study, we combined both above-mentioned approaches to see whether this could offer any progress to the present state of the art. The aim was to determine whether combining modified culture conditions with ELISpot assay could improve the detection of nickel-specific response in vitro, as compared to the previously described methods.

Patients and Methods

Combinations of particular culture conditions and in vitro assays are in this article referred to as "test protocols". The design of the study and data analysis were aimed at answering the 3 following questions: Q1 "In which test protocol the differences between Ni-ACD patients and Controls are most pronounced and significant?" and Q2 "In which test protocol the results correlate best with clinical diagnosis?". As LPT has probably been the most popular in vitro assay in contact allergy for past decades, the question Q3 was "Which non-radioactive test protocol produces results that correlate best with LPT?".

Study group
14 female patients with confirmed nickel-allergic contact dermatitis (Ni-ACD) and 14 female Controls were studied. The inclusion criteria for the Ni-ACD group were: clear history of metal dermatitis and an at least ++ patch test reaction to 5% NiSO4 pet. after 48, 72 and/or 144 h ("golden standard" for this study). The control group consisted of 7 eczema patients and 7 healthy volunteers, with no signs of metal intolerance and negative patch test to nickel. Three persons among Ni-ACD and 4 among Controls had positive history of atopic diseases. The age range was 20-59 (median 36.5) years in the Ni-ACD group and 25-62 (median 36.5) years among Controls. The participants gave informed consent, and the study was accepted by the local ethics committee.

Cell cultures
PBMC were separated from the participants' blood samples in Ficoll-Paque Plus (Amersham, Uppsala, S) and cryopreserved in liquid nitrogen until testing. The cells were cultured in IMDM medium with penicillin, streptomycin and 1,4-dithiothreitol at 37°C, 95% RH, 5% CO₂. The final cell density was 10⁴ cells/well for IFN-γ ELISpot and 2×10⁵ cells/well for all remaining ELISpot, ELISA and LPT assays. The cells were cultured in triplicate both with and without the presence of NiS04 (50 µM), and both with and without the addition of IL-7 and IL-4 or IL-12, referred to as "cytokine cocktails". The selection of nickel concentration was based on study results by Lindemann et al. (19), who compared seven different concentrations of nickel sulphate (from 6 to 200 µM) and found 50 µM most suitable for cell cultures, in terms of specific lymphocyte stimulation versus cytotoxicity. Two "cytokine cocktails" were used in the study: "7/4 cocktail" - a "type 2"-skewing combination of IL-7 and IL-4, and "7/12 cocktail" - a "type 1"-skewing combination of IL-7 and IL-12. All cytokines used were human recombinant proteins (Strathmann Biotec, Hannover, D) with specific activities of 5 × 10⁷ units/mg for IL-7, 2 × 10⁷ units/mg for IL-4, and 1 × 10⁷ units/mg for IL-12. Final concentrations of the cytokines in cell cultures were 240 units ml^-1 IL-4, 0.5 units ml^-1 IL-7, and 10 units ml^-1 IL-12. These concentrations were established in a series of introductory tests and proved effective in preceding studies done in our group (17, 21). A similar approach was also used by Jennes et al. for improving detection of virus-specific lymphocytes through culturing PBMC with IL-7 and IL-15 (22).

Enzyme-Linked Immunospot Assay (ELISpot)
The cells were cultured initially for 20 h in round-bottom 96-well culture clusters in order to enable good antigen presentation. Subsequently, the cells were moved to 96-well PVDF microfilter plates (Millipore, Molsheim, F), each coated with respective antibodies. After additional 40 h of culture for IFN-γ, and 5 days for IL-2, IL-5 and IL-13, the ELISpot assay was done following the manufacturers' guidelines. Antibodies for IFN-γ, IL-5, IL-13 ELISpot and streptavidin-alkaline phosphatase (s-ALP) conjugate were from Mabtech (Näcka, S), antibodies for IL-2 ELISpot were from R&D (Minneapolis, MN), the BCIP/NBT colour AP substrate was from Bio-Rad (Hercules, CA). Spots were counted automatically using the AID ELISpot Reader (AID, Straβberg, D).

Enzyme-Linked Immunosorbent Assay (ELISA) and Lymphocyte Proliferation Assay (LPT)
Cultures for ELISA and LPT were grown in round-bottom 96-well culture clusters for 6 days. IFN-γ in supernatant was measured with antibody pairs from Sanquin (Amsterdam, NL) and IL-5 with antibodies from BD (San Diego, CA) according to the manufacturers' recommendations. Colour reaction was developed with horseradish peroxidase (s-HRP) polymer (Sanquin) with OPD/H2O2 substrate, and measured with Microplate Autoreader EL311 (Bio-Tek, Winooski, VT). 3H-thymidine (Amersham, Little Chalfont, UK) was added to cell cultures for the last 5 h, after which the amount of incorporated radioactivity was measured using TopCount NXT from Packard Biosciences (Downers Grove, IL).

Data analysis

Altogether, results of 15 in vitro test protocols were compared: IL-2 ELISpot in cultures without and with the "7/12 cocktail", IL-5 ELISpot and ELISA without and with the "7/4 cocktail", IL-13 ELISpot without and with the "7/4 cocktail", IFN-γ ELISpot and ELISA without and with the "7/12 cocktail", and finally LPT without cocktail, with the "7/4 cocktail", and with the "7/12 cocktail". To answer question Q1 (influence of in vitro test protocols on measurable differences between Ni-ACD patients and Controls), test protocols which produced significant differences (P≤ 0.05, Mann-Whitney "U" test, 2-tailed), were ranked according to magnitude of differences between median results in Ni-ACD and Controls, expressed as per cent of the median result in Ni-ACD group. To answer question Q2 (concordance of in vitro results with clinical diagnosis), in vitro test protocols were ranked according to the coefficient of correlation (Pearson correlation, significance test 2-tailed) with the golden standard (positive history of nickel intolerance combined with at least a ++ patch test to Ni). To answer the question Q3 (concordance between non-radioactive test protocols and the LPT), non-radioactive test protocols were ranked from the highest to the lowest coefficient of correlation with LPT (Pearson, 2-tailed). Statistical package SPSS+ for Windows (SPSS Inc, Chicago, IL) was used for the above analyses.

RESULTS

Influence of protocol on differences between Ni-ACD patients and Controls (Q1)
In 6 of 15 in vitro protocols tested, differences between median results for Ni-ACD patients and Controls were larger than 90% and significant at P≤0.05 (Table 1). The differences of more than 100% in the case of IL-13 ELISpot with "7/4 cocktail" are due to the fact that numbers of cells secreting IL-13 in response to nickel were decreased in PBMC cultures from nonallergic subjects after addition of "7/4 cocktail" (possibly a cytotoxic effect, compare Fig. 1). Individual results for IL-5, IL-2, and LPT test protocols are shown in Figures 2-4. Regarding secretion of the "type 1" cytokine IFN-γ, no relevant differences were observed between Ni-ACD and Controls in any of the test protocols studied. IFN-γ ELISA without cytokine cocktails was closest to the selected significance level with P=0.062.

Concordance between in vitro results and clinical diagnosis (Q2)
The strongest correlation with "golden standard" was observed in the case of LPT with the "7/4 cocktail", followed by IL-13 ELISpot with "7/4 cocktail" (Table 2). For all other protocols analysed, correlation coefficients r were below 0.5, with the lowest correlation observed for IFN-γ ELISpot.

Concordance between non-radioactive test protocols and the LPT (Q3)
The strongest correlation was found between LPT and IL-2 ELISpot without cytokine cocktails, followed by IL-13 ELISpot with and without "7/4 cocktail" (Table 3). The correlation coefficients between LPT and IFN-γ ELISpot and ELISA assays were low (r ≤ 0.051) and not significant.

DISCUSSION

Nickel is the most important contact allergen with sensitisation rates estimated at 17% among adults (23) and 8-10% among children (24, 25). In the present study, we compared clinical diagnosis of ACD with results of 15 in vitro test protocols, i.e. combinations of 3 in vitro assays (ELISpot, ELISA, LPT) with 3 various culture conditions (standard culture conditions, and 2 modifications with cytokine cocktails). Both cytokine cocktails used contained IL-7, which together with IL-4 or IL-12 enhances priming of naive cells (26, 27). IL-7 also counteracts apoptosis of allergen-specific naive and effector T lymphocytes (28).The development of "type 1" and "type 2" lymphocyte subpopulations was supported by adding IL-12 or IL-4, respectively (17). There is ongoing discussion, which lymphocyte subsets act as effector cells in ACD: T helper cells (CD4+), T cytotoxic cells (CD8+), other lymphocytes like NK or NKT cells, or maybe more than one subpopulation (29). Although this study was not specifically aimed at addressing this question, our results show that IL-5 and IL-13 production was the most prominent response to Ni, thus indicating on the prevailing "type 2" activation (possible effector cell phenotypes Th2, Tc2, NK2, and/or NKT2). The exact phenotype of these effector cells will be subject to further investigations.

Despite allergic contact dermatitis has traditionally been regarded as an IFN-γ-driven disease, we haven't observed any significant differences between Ni-ACD patients and Controls regarding IFN-γ production in response to nickel. Technically, this could be explained through e.g. a high spontaneous secretion of IFN-γ (possibly by NK cells), which could "obscure" the secretion by nickel-specific lymphocytes. However, there were previous hints on preferred "type 2" response to nickel of peripheral blood lymphocytes (30, 31), and also of Ni-specific lymphocytes from isolated eczematous skin (32, 33). The "type 2" cytokine pattern in response to Ni seems independent of atopic status of the sensitised person (34, 35). Moreover, this pattern seems not restricted to nickel only: Masjedi et al. found that allergen-specific IL-13 production discriminated best between cultures of PBMC from people with contact allergy to methylisothiazolinones and from controls (36). This encourages further studies of "type 2" test protocols also with other contact allergens.

The in vitro test protocol that correlated best with clinical diagnosis ("golden standard") was LPT done with "type 2" skewing (addition of "7/4 cocktail"). This observation was disappointing to some extent, as one of the aims of the study was to identify a non-radioactive alternative for the LPT. However, the "classical" LPT, i.e. done without enhancement with cytokine cocktails, was outperformed by IL-13 ELISpot with "7/4 cocktail". Finally, when looking for a non-radioactive alternative for the "traditional" LPT, the highest correlation was found between this method and IL-2 ELISpot (without cocktail). This finding fits well to the knowledge that memory cells secrete IL-2 to stimulate proliferation and differentiation after encounter with specific antigen (37). Relatively high correlations were also observed between LPT and IL-13 ELISpot and IL-5 ELISA, but again not in case of the "type 1" cytokine IFN-γ.

In concluding the above results, combining ELISpot assay with modified culture conditions may constitute a relevant progress in the detection of contact allergy to nickel in vitro. Among the test protocols analysed, IL-13 ELISpot in cultures with "7/4 cocktail" was most effective in detecting Ni-specific response and should be further evaluated regarding its possible use for in vitro diagnosis of contact allergy.

Acknowledgement

This study was funded by a grant from the European Commission (Marie Curie Individual Fellowship QLK4-CT-2002-51504 for Dr Radoslaw Spiewak).

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