Nickel, palladium and rhodium induced IFN-gamma and IL-10 production as assessed by in vitro ELISpot-analysis in contact dermatitis patients

Allergic contact dermatitis (ACD) affects a large fraction of the general population being most frequently elicited by nickel (Ni), which is responsible of a delayed-type hypersensitivity clinically revealed by the demonstration of eczematous reactions upon patch testing and by laboratory testing of antigen-specific T-cell proliferation [1–3]. These alterations are the result of the interaction between the metal ions, which act as haptens, and the skin surface, thus favouring the contact and binding with cellular and extracellular matrix proteins [4–6], and the induction of a cellular immune response against the hapten-carrier protein complex [7, 8]. Recent attempts to diminish Ni use in most industrial products have led to an increase in the use of different metal compounds for specific destinations such as the alloys used in orthopaedics, jewellery and dentistry [9]. For instance, the use of palladium (Pd) increased in the past 10 years [10] also due to the fact that dentistry alloys containing Pd appear more resistant to mechanical wearing effects [11, 12]. Similarly, rhodium (Rh), which is primarily used as an alloying agent to harden platinum and Pd, it is now employed in electrical material because of its low electrical and contact resistance, in jewellery (platings of silver or white gold because of its silvery white colour), in optical instruments and dental prostheses.

Nevertheless, it has been recently recognized that also exposure to metal ions such as Pd, chromium, cobalt and gold, can induce a cellular immune response and clinical manifestations of ACD, even if less frequently than Ni [13–15]. In fact, positive patch testing to PdCl₂ and clinical manifestation of ACD to Pd have been described with increasing frequency [16, 17]. Despite its low sensitization potential, also Rh has been reported as sensitizer in the form of salt [18].

In Ni sensitized subjects a simultaneous patch test reactivity to Pd has been frequently reported [19, 20] and a double sensitization can be hypothesised especially in individuals wearing dental prosthesis. Nevertheless, a cutaneous cross-reactivity cannot be excluded for the chemical and structural similarities of Ni and Pd [21]. Metal ions are incomplete antigens which have to bind to endogenous peptides to be "visible" to the immune system [7] and Ni and Pd encountering the same cellular environment might form in vivo complexes with similar proteins thus eliciting cross-recognizing immune responses [17]. Moreover, two independent in vitro studies demonstrated an immunologic cross reactivity between these metals at the T cell clonal level [22, 23].

Identifying both a double sensitization and a lymphocyte cross-reactivity to different metal ions in vivo is an important issue for clinicians in directing the choice of implant materials in order to avoid incompatible reactions. Consequently, the diagnosis of ACD to both Ni and Pd needs in vitro assays complementary to patch testing [24, 25]. Indeed, the variability of the readouts obtained with patch testing have stimulated a number of studies aimed at developing assays characterizing the immune response to metal salts. Recently, Minang et al. [26] analyzed the different cytokine profiles induced by nickel in allergic or tolerant subjects by enzyme-linked immunospot (ELISpot) assay. In fact, the sensitivity and specificity of the ELISpot assay allow the measurement of very low frequencies of cytokine-producing cell, therefore offering a powerful tool for the detection and characterization of Ni sensitization with important implications for diagnostic strategies [27, 28].

The present study was undertaken with the aim to evaluate the potential of the ELISpot assay in determining sensitivities to Ni or Pd and to Ni and Pd on the basis of specific induction of inflammatory/regulatory cytokines. Since all subjects were skin challenged to Ni, Pd and Rh salts, and none of them had positive skin reactivity to Rh, this metal was employed to assess the cytokine profile in non allergic subjects and to explore the possibility to identify a cellular immunity against materials with traces of heavy metals.

In the present study, we have investigated the profiles of cytokine production by PBMC of ACD patients and healthy controls in response to the well known contact allergens Ni and Pd, or to Rh, considered to have a low sensitization potential, through the ELISpot assay.

The results showed that PBMC from well recognized allergic individuals affected by ACD have a higher frequency of IFN-γ releasing cells upon in vitro stimulation with Ni or Pd, as compared to control groups. Most interestingly, we found that PBMC from all the individuals exhibiting a negative skin response to a metal salt (group 4) revealed a clear IL-10 response when challenged with the same metal, in the absence of any detectable IFN-γ response. Accordingly, all subjects included in the study had a negative skin response to Rh, which was invariably associated to Rh-induced IL-10 production in vitro.

The immunologic mechanisms responsible for the clinical hypersensitivity towards metals have been recently reviewed [7]. It is known that Ni-specific, IFN-γ-producing CD4⁺ and CD8⁺ T cells are major effectors of sensitization and directly responsible for skin manifestations [6, 29], whereas IL-10 and TGF-β-secreting regulatory T cells are thought to down-modulate the response [2, 30, 31]. The deleterious expansion of Ni-specific, effector CD8+ T cells might be thus prevented by regulatory mechanisms, including those induced by hapten-specific suppressor T cells [32]. In humans IL-10 has been identified as a potent anti-inflammatory cytokine produced by both Th1 and Th2 CD4+ T cells [33], which inhibits T cell proliferation of both subsets, and regulates the development of Th1 cells by blocking IL-12 driven Th1 responses [34]. IL-10 is thought to exert its effects by down-regulating the expression of co-stimulatory molecules required for appropriate antigen presentation [35]. Ferguson TA et al and Schwarz A et al [36, 37] demonstrated a key regulatory role for IL-10 in both the induction and the effector phases of murine contact hypersensitivity. Our results provide evidence supporting the hypothesis that a regulatory mechanism mediated by IL-10 contributes to the control of hypersensitivity in response to Ni, Pd and Rh therefore contrasting the development of ACD. Even if the protective role of IL-10 is well established, studies addressing the cytokine profile in subjects with or without ACD have been partly contradictory [26, 31, 38]. It should be considered that different protocols used for the in vitro stimulation or the different sensitivities of the immunoassays for IL-10 measurements could be responsible for the different results obtained.

The finding that not only Ni but also other metals could elicit a response in PBMC from sensitized subjects [15], was not surprising and prompted us to explore the profile of cytokine production in patients which scored negative at patch testing toward Pd and Rh, which are present in several synthetic materials including new metallic alloys [39, 40]. Remarkably, we found very clear-cut, divergent, IL-10 "protective" or IFN-γ "reactive" profiles in patients showing a negative or positive skin reactivity to metal salts (group 1, 2, 4), respectively. Moreover, all the subjects with positive patch test to both Ni and Pd (group 3) showed an in vitro response similar to monosensitized patients to Ni, characterized by the release of IFN-γ after Ni stimulation and production of IL-10 in response to Pd or Rh. Interestingly, comparing the cytokine response to Pd, subjects with the same patch test reactivity (n = 3: ++ in the group 2; n = 6: ++ in the group 3) displayed a reversed profile (Table 2). On the basis of these data, it is conceivable that a positive (IFN-γ) response in vitro may predict the development of adverse reaction to prosthesis alloys or to chemotherapeutic agents containing toxic metals. In fact, previous studies on multiple sensitivities to transition metals reported that the majority of Ni/Pd reactions occurred without clinical evidence of Pd sensitization [10, 19, 41].

We analyzed the IFN-γ response in patients with an unambiguous Ni patch test reaction (group 1 and 3 patients with +++ reactions) to provide positive controls for the in vitro assay, in order to define optimal conditions for antigen-specific cytokine production and to offer a highly sensitive and specific test suitable for clinical determinations. On the basis of this setting, stimulation values greater than 3 to 7 were commonly obtained with metal salts or with lectin (PHA) polyclonal stimulation, respectively, suggesting that cryopreservation did not affect the cytokine response of lymphocytes, as sustained by the study from Kreher et al [42].

The data gathered by the ELISpot analysis did not show any cross-reactivity of T-cells neither the co-existence of T cell clones with differential responsiveness. Since any Ni contamination was demonstrated in the Pd salt used for patch testing (data not shown), it is presumable that an immunologic cross-reactivity based on molecular mimicry among different hapten-carrier complexes might take place in vivo, at the skin level. Due to their small size, metal ions are incomplete antigens which have to bind to carrier molecules, which, in turn, are capable to capture metal ions, and shuttling this complex to the epidermal basement membrane [4, 7]. On the other hand, the concomitant reactivity to Ni and Pd, observed in a number of patients by skin testing could concern also the possibility of a delayed or impaired response of skin regulatory cells. A dysfunction in T-regulatory cell can lead to an immune activation often associated with the clinical expression of skin diseases such as atopic dermatitis and psoriasis [43]. Experimental data suggested also the possibility that, in analogy to superantigens, Ni or Pd may also directly link TCR and MHC in a peptide independent manner and can effectively amplify the hapten-specific immune response [44, 45]. It is also known that a strong binding of different superantigens to the TCR could render T cells insensitive to suppression by CD4⁺CD25⁺ Treg cells [46] and IL-10 [47].

Knowledge about the immunologic mechanism responsible for ACD is still fragmentary. It is accepted that the sensitising chemical penetrates into the epidermis, and, depending on its properties, it "perturbates" the environment. However, neither the molecular nature of Ni-induced antigenic determinants nor the very early molecular events of metal transport through the human epidermis to Langerhans cells have yet been satisfactorily resolved indicating that still many aspects of metal allergy remain unclear.

Taken together, these results demonstrate that different cytokine profiles elicited in vitro can reflect different immune responses in vivo in ACD. The development of sensitive and specific in vitro assays based on cytokine response could be extremely useful in complementing patch testing for the clinical diagnosis and characterization of metal sensitization.