Nickel allergy is associated with a broad spectrum cytokine response

Nickel‐induced proliferation or cytokine release by peripheral blood mononuclear cells may be used for in vitro diagnosis of nickel allergy.


| INTRODUCTION
Nickel is the best known and most frequent of the metals causing allergic contact dermatitis (ACD). 1 Nickel can be found in jewellery, medical implants, dental devised, cooking pans and items that are generally used. Nickel allergy is a type-IV hypersensitivity reaction, upon interaction with antigen presenting cells nickel-specific CD4 + T-cells are primed and activated. 2 Nickel is also capable of directly activating dendritic cells via Toll-like receptor-4, thereby inducing inflammatory signalling via NF-κB. 3 The gold standard in diagnosing nickel contact allergy is the patch test. 4 However, this test is particularly suited for ACD, not for complaints due to internal exposure from medical implants and oral exposure from dental devices. Irritant reactions my cause false positive results, while insufficient allergen skin penetration may lead to a false negative result. Furthermore, there is a small risk of patient sensitization. 5 As we described previously, an optimized lymphocyte proliferation test (LPT) using CFSE can also be considered as a good diagnostic tool for nickel allergy. 6 In vitro cytokine production in response to nickel has also been shown to differentiate between nickel allergic and non-allergic patients. Pro-inflammatory cytokines such as IFN-γ, IL-2, Il-12, IL-4, IL-5, IL-8 and regulatory cytokines such as IL-10 and TGF-β1 have been investigated. [7][8][9][10][11] Production of 'type 2' cytokines IL-2, IL-13 and IL-5, showed the best correlation with contact allergy to nickel. 10 However, studies investigating the role of inflammatory mediators in nickel allergy have been limited to a few selected cytokines and chemokines, and thus only provide information on restricted profiles. An evaluation of the broader inflammatory profile of nickel specific T-cells may provide more insight into the pathogenesis and immunological pathways of metal allergy and may help in the development of improved diagnostic tests for metals such as titanium, for which patch tests proved to be unreliable.
Using multiplex assays, a broad range of inflammatory mediators can be determined in a small volume sample. In this study, peripheral blood mononuclear cells (PBMC) of in total 52 nickel allergic patients and controls were stimulated in vitro with nickel sulfate (NiSO 4 ). To study whether sensitivity of the cyto-/chemokine release assay can be enhanced, cytokine cocktails skewing lymphocytes towards type-1, -2 or -17 function were added. A wide range of 33 different chemokines, cytokines and growth factors was analysed, most of which, to our knowledge, have not previously been examined in an in vitro assay in response to nickel. The aim of this study was to identify novel cytokines in the inflammatory mediator profile induced by nickel and to investigate whether existing proliferation and type2 cytokine production diagnostic tests can be improved.

| Patients
The study followed the Declaration of Helsinki Principles. All participants gave written informed consent, and the study was approved by the medical ethical committee of VU University Medical Centre (Central Committee on Research Involving Human Subjects protocol number: NL52668.029.15). Patient inclusion was as described previously. 6 In short, 52 participants who presented at our out patients clinic for evaluation of suspected cutaneous hypersensitivity were patch tested with NiSO 4 petrolatum and included in the study; 27 with a positive patch test and 25 with a negative patch test to nickel. Information on history of AVD to nickel containing metal alloys, based on patients answers to questions regarding the development of skin rash after contact with (metal) jewellery, coins, metal tools, scissors or metals in clothing such as belts and buttons, was recorded, enabling us to divide the participants into four groups: true positives (positive patch test and history), true negatives (negative patch test and history) and two groups in which the patch test did not confirm the history. The subjects were without systemic immunosuppression or UV radiation therapy. Patient characteristics have been previously described, (6) and are listed in Table S1. All participants donated peripheral blood samples (heparinized), which was used for nickel sensitization testing using the lymphocyte cytokine production test. Readings were performed at 48, 72 and 168 h (7 days). Positive reactions were rated as +, ++ or +++.

| Lymphocyte cytokine production test
The lymphocyte cytokine production test (LCPT) was performed in the laboratory of the Oral Cell Biology department of the Academic Centre for Dentistry Amsterdam (ACTA), cultures were set up as previously described. 6 Supernatants of these cultures were collected for multiplex analysis. Briefly, peripheral venous blood (40 ml) samples (heparinized) were collected and then peripheral blood mononuclear cells (PBMCs) were isolated on Ficoll Histopaque (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). After isolation, the cells were cultured in medium (IMDM, 100 IE/ml Na-penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine [Biochrom Seromed, Berlin, Germany] and 50 μM β-mercapto-ethanol) containing 10% autologous serum. The final cell density was 7.5 Â 10 5 cells/ml/well. The PBMC were cultivated with or without 50 μM NiSO 4 (Merck), the same salt as used in the patch test, for 7 days in duplicate. We have previously shown 50 μM NiSO 4 to be the optimal non-toxic concentration. 9,10 In order to increase allergen-induced T-cell cytokine production, growth factor supplements consisting of IL-7 (0.1 ng/ml; Strathmann Biotec AG, Hamburg, Germany), IL-12 (1 ng/ml; PeproTech, NJ, USA), IL-4 (3 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml), all from R&D systems (Minneapolis, MN, USA), were used during culture as described previously. 8 The combination of IL-7, and either IL-12, IL-4 or IL-1β/IL-23 were added to respectively, to skew towards the release of type 1, type 2 or type 17 cytokines. As a positive control, the T-cell mitogen phytohaemagglutinin (PHA; Merck) (3 μg/ml) was used. Negative controls were defined as unstimulated cells. On day 7, culture supernatants were harvested and stored at À30 C until multiplex analysis.  Table S2. The results of the cytokine production test are presented as the stimulation index (SI), which is calculated by the cyto-/chemokine production (pg/ml) in the nickel stimulated cultures divided by the production in unstimulated cultures. To avoid disproportionately high stimulation indexes, a lower cut-off of 1 pg/ml was used.

| RESULTS
A summary of the patient characteristics are listed in Table S1. Of the 52 included patients 15 were defined as 'true negatives' because they had no clinical history of nickel allergy and were patch test negative, this is the control group. Twenty patients were defined as "true positives" because they had a clinical history of nickel allergy and were nickel patch test positive (nickel allergic). The remaining patients had either a clinical history of nickel allergy with a negative patch test (PT À H+, n = 10) or a positive patch test without a clinical history of nickel allergy (PT + HÀ, n = 7).
Five different optimized V-plex kits were used to analyse 44 analytes. Eight of these were excluded from analysis because baseline production (medium) exceeded the upper limit of the standard curve and thus the upper limit of detection of the assay, these were predominantly chemokines (see Table 2). IL-7 was excluded because it was added during culture. IL-17D was excluded because it is not pro- IL-1α, VEGF-A, IL-16, IL-1β, IL-12p70, Eotaxin-1 and Eotaxin-2, were produced but the mean SI was <1.5 in all culture conditions.
All remaining analytes were included in the supervised cluster analysis shown in Figure 1, with the exception of IL-4 production in the type2 skewing culture condition as IL-4 was added to the culture medium, and the IFN-γ production in the type 1 skewing condition due to high background production. IL-17A was analysed with two different assays; IL-17A and IL-17A genB which had different detection ranges (0.74-3633 and 0.413-1950 pg/ml, respectively), both assays were included in the analysis (see also Table S2 for details on included and excluded analytes).
The supervised cluster analysis in Figure 1 shows that a broad spectrum of cytokines is associated with nickel allergy, these cytokines include type 1, type 2, type 9, type 22 and type 17 derived cytokines, demonstrating that the response to nickel is not restricted to one T-cell subset. Unsupervised cluster analysis also shows clustering of controls (true negatives) and patients with both a positive patch test and a history of nickel allergy (true positives). The two intermediate groups having either a history nickel allergy but a negative patch test or no history of nickel allergy with a positive patch test do not cluster preferentially with either the controls of true positives ( Figure S1). Figure 2 summarizes the variety in cytokines associated with nickel allergy, a type 2 response seems to dominate, however.  (Table S3).  Table S4 shows the actual production in pg/ml for medium control and nickel stimulated cultures.
We have previously shown in this patient group that specific proliferation to nickel is associated with nickel allergy. We therefore ana-  Table S5).
In addition, we calculated the percentage of patients and controls that are positive or negative in both the LPT and the four best performing cytokines under non skewing conditions (Table 2). Subsequently, we calculated the sensitivity and specificity for the whole patient group, using nickel allergy in the medical history as reference (Table 3). Sensitivity and specificity of the patch test are then 67% and 68%, respectively. These are increased when IL-5 production is analysed under no skewing conditions (73% and 77%) or type 2 skewing conditions (80% and 73%). We previously demonstrated that the LPT under nonskewing conditions showed an even higher sensitivity (83%) and specificity (86%), demonstrating that the LPT is a good in vitro test to diagnose nickel allergy. A combination of the proliferation test and IL-5 production test increases the specificity (91%) with some loss in sensitivity (70%).

| DISCUSSION
Specific IL-2 production in response to nickel in allergic patients has been shown more than 30 years ago. 12  PBMC has been demonstrated. 7,8,10,11,[13][14][15][16][17][18] Some of these studies could not confirm specific production of one or more of these cytokines (TNF-α, IFN-γ, IL-4, IL-10 or IL-17), possibly due to small group sizes or low sensitivity assays. [11][12][13][14] In this study, using a sensitive analysis platform, we confirm specific production of aforementioned cytokines and 13 additional factors (21 of the 33 cyto-/chemokines analysed were produced specifically in response to nickel in nickel allergic donors), demonstrating that the T-cell response to nickel shows a broad spectrum of type were not associated with nickel allergy. IL-16 is produced by a variety of haematopoietic (a.o., CD8 + T-cells, CD4 + T-cells) cells and nonhaematopoietic (a.o., epithelial cells) cells and is a chemoattractant for CD4 + cells. 19 It was shown to be specifically increased in response to nickel, in tape stripped skin samples of positive patch tests. 20 Since we could not show specific production in our PBMC cultures, it is likely that IL-16 is produced by keratinocytes of allergic individuals upon allergen contact and may be involved in the chemoattraction of (nickel responsive) CD4 + T-cells.
IL-31 belongs to the IL-6 family and is predominantly produced by (particularly under type 2 skewing condition; AUC 0.87) were also associated with nickel allergy (Table S3). IL-12/23p40 and IL-27 are induced in healthy donor dendritic cells upon nickel stimulation while IFN-γ can further boost production of these cytokines by antigen presenting cells. 23,24 The production of IP-10 by antigen presenting cells is also predominantly induced by IFN-γ. 25 As nickel allergy associated IFN-γ production was observed under all culture conditions (except type 1 skewing) it is likely that these associations are related to indi- by qPCR in nickel inflamed skin as compared to paired healthy skin. 27 In skin biopsies of patients with chronic exposure to nickel, TNF-α, IFN-γ, IL-4, IL-13, IL-17A and IL-10 were detected by immunohistochemistry, in challenged skin IL-2, IL-23 and IL-10 were shown to be expressed, however, no controls were included in this study. 28 Overall, these data on local cytokine production upon nickel exposure confirm our data showing a role for a broad spectrum of type 1, type 2, type 9, TH17 and type 22 responses in nickel ACD.
The second aim of this study was to identify a cytokine (profile) after in vitro PBMC stimulation, which can most accurately predict nickel allergy and could be used as a diagnostic test. In the ROC analyses, there were 7 cytokines with an AUC of more than 0.90 with a p < 0.0001 (Table 1) would lead to an increase in processing time. However, if a random access analyser is available to measure IL-5 concentration in culture supernatants, this would be more cost effective than the LPT with a similar processing time, as less hands-on time is needed. For a high specificity, the LPT and IL-5 LCPT (without skewing) can be combined, but this leads to lower sensitivity and higher costs.
In conclusion, nickel allergic contact allergy is characterized by a broad cytokine response with cytokines related to type 1, type 2, type 9, type 17 and type 22 cells; type 2 cytokines showed the strongest correlation. The type 2 cytokine IL-5 is the best biomarker for nickel allergy.

ACKNOWLEDGEMENT
We thank the NWO Domain TTO (formally known as STW) for funding this work.

CONFLICT OF INTEREST
None of the authors have a conflict of interest.

DATA AVAILABILITY STATEMENT
Raw data are provided in the suplemental material.
T A B L E 3 Characteristics of LPT and IL-5 LCPT in the whole patient group, history of nickel allergy is reference Abbreviations: CI, confidence interval; LCPT, lymphocyte cytokine production test; LPT, lymphocyte proliferation test; PT, patch test; Sens, sensitivity; Spec, specificity.