Is there an in vitro test for type IV allergy discriminating between sensitization and allergic disease?

Contact dermatitis is defined as an inflammatory response of the upper layers of the skin – namely epidermis and dermis – that occurs as a result of contact with exogenous substances. It accounts for approximately 80% of environment/occupation-based dermatoses and 30% of all occupational diseases [1]. Contact dermatitis includes irritant contact dermatitis and allergic contact dermatitis – irritant contact dermatitis being the most common form. Irritant contact dermatitis is induced by either an obligatory irritant inducing an inflammatory response in every individual or is the result of a cumulative toxic process in susceptible individuals. Because of the fact that irritative contact dermatitis is per definitionem a non-immunologic mechanism, no immunologic sensitization can be detected in vitro. Thus, the only tool for diagnosis – in case of irritative contact dermatitis – is a thorough history, perhaps accompanied by tests for individual disturbed barrier function (e.g. alkali resistance method, nitrazine yellow [2]). Allergic contact dermatitis is a delayed hyper-sensitivity (type IV) reaction and involves sensitization before the development of clinical symptoms. Since its introduction into medicine in 1895 by Joseph Jadassohn, the patch test is the method of choice in the diagnosis of contact sensitization [3]. However, considering the more than 100 years bygone since then, it seems to be time to come up with an objective tool for diagnosis of contact sensitization with predictive value for clinical relevance. Until today, the combination of clinical history and patch test results constitutes the two cornerstones in the diagnosis of type IV allergy. Simple, safe and accurate in vitro methods identifying contact allergy would offer both theoretical and practical advantages. At present, the only substantial in vitro test – although primarily in the experimental setting – is the lymphocyte transformation test (LTT). Currently, research focuses in many areas on the identification of biomarkers for disease or progression of diseases – whereas the necessity of an in vitro diagnostic tool for contact dermatitis is largely neglected. This editorial aims to discern – on the basis of the publication in this issue of Lindemann et al. [4] – current and given up methods for in vitro diagnosis of allergic contact dermatitis. Development of contact allergy is the result of an interplay between environmental exposure and individual susceptibility because only a fraction of exposed individuals become sensitized. Epidemiologic studies concluded that contact allergy is influenced by sociodemographic parameters [5], a not clearly defined genetic susceptibility [6–8] and plays an important role in the general population. The most frequent elicitor of contact sensitization is fragrance mix, followed by nickel, thimerosal and balsam of Peru [1, 9], with women being more often sensitized than men [5]. Chemicals that possess the capacity to cause skin sensitization have been recognized to be reactive (electrophilic) or at least the precursor of an electrophile. Contact allergens are almost exclusively haptens – i.e. they need binding to a protein in order to become a full antigen. Hapten-induced immune reactions are classic examples of adaptive immunity. However, for this process to occur, it has been noted that in addition to haptenation of skin proteins, secondary stimuli ‘danger signals’ are required [10]. Such signals might be derived from keratinocytes and/or Langerhans cells perturbed by a chemical sensitizer. Thus, a substance that induces allergic contact dermatitis in susceptible individuals mostly shares both irritant and sensitization potential – this characteristic reflects the set of problems we are confronted with in practice regarding both in vitro and in vivo test techniques. The current paradigm of contact sensitization follows a two-step mechanism: a sensitization and an effector phase [11]. During sensitization, the hapten penetrates the skin, binds to a protein to become a full antigen and is taken up by local dendritic cells (DCs). Antigen-loaded DCs migrate while undergoing a maturation process to regional lymph nodes, where they present the antigen to naïve T cells that consequently expand clonally. By expressing skin-homing factors such as CLA [12] or CCR4 [13], they are enabled to recirculate into the skin. At the second and every subsequent encounter of susceptible individuals with the allergen, the CLA/CCR4+T cells migrate into the skin. Here, they induce a cascade of cellular reactions, leading finally to the eczematous inflammation characterized by the formation of spongiosis and apoptosis of keratinocytes [11, 14, 15]. The patch test today is still the gold standard in the diagnosis of allergic contact dermatitis. On the basis of almost 100 years of practice, this test is well standardized and – although with a subjective readout – a reliable test. It has a predictive value with regard to clinical relevance [16]. However, we have to be aware of the fact that patch testing harbours several caveats and risks. First of all, it bears the risk of iatrogenic sensitization due to improper testing or testing of unknown material. Furthermore, the problem of local reactions to some allergens, e.g. paraphenylenediamine, exists. Patch test reactions to this contact allergen are known to be often strong and sometimes may even lead to scarring processes. As mentioned above, a contact-sensitizing agent exhibits both irritative and sensitizing potential. For example, in the case of chromium – when used at a standard concentration of 0.5% chromium dichromate – one out of two reactions is considered to be irritant. In this case, both sensitization and clinical relevance can be difficult to interpret. Another disadvantage of the epicutantest is the fact that it is often not possible to be performed in individuals at the most prominent need, namely, in the case of generalized skin reactions. Furthermore, false-positive reactions are observed in the context of ‘angry back syndrome’ or ‘excited skin syndrome’. False-negative results can occur at low concentrations of the substance, systemic or local therapy with corticosteroids or after sun exposure [17]. Thus, the need for a reliable in vitro test for sensitization and prediction of clinical relevance becomes obvious. The probably first in vitro assay for a delayed-type hyper-sensitivity reaction was established as early as in 1932 when Rich and Lewis showed that an antigen (tuberculin) inhibited migration of cells from tissue explants taken from sensitized animals [18]. Leucocyte migration inhibition in metal-allergic patients was first described by Mirza et al. (1995) [19]. In the 1970s and early 80s, this test was kind of en vogue [20] in the diagnosis of contact sensitization. Today, however, this test belongs to the not entirely understood and – presumably therefore – forgotten in vitro tests. In the history of in vitro diagnosis of contact allergy the leucocyte pro-coagulant activity also has to be mentioned. This test, described by Aldridge et al. in 1985 [21], was a unique application in the in vitro diagnosis of contact allergy with promising results, however, still awaiting confirmation. The basis for the LTT was given in 1960 when Nowell [22] first described that lymphocytes cultured in the presence of phytohaemaglutinin (PHA) transform into blasts. The finding that uptake of radio-labelled thymidine by lymphocyte's nucleic acids in culture correlates well with lymphocyte stimulation has led to this as a standard method in cellular immunology [23]. In the 60s and 70s of the last century, the LTT was established in order to determine metal allergy in vitro– first described by Aspegren and Rorsman [24]. However, the problem of non-specific proliferation of lymphocytes in the presence of nickel became evident already at that time [25] and still remains a major problem nowadays [26]. Valentine-Thon et al. [27] aimed at evaluating LTT in a cohort of 700 patients with suspicion for metal allergy and found a good reproducibility and sensitivity to detect allergy – however, the negative controls, i.e. non-sensitized individuals in this large study were lacking. The question remains whether the LTT is a predictive test for clinically relevant sensitization to metals. Furthermore, the timing of the LTT in relation to epicutaneous testing or accidental exposure seems to be important, adding to the problem of a predictive test with clinical relevance. A proposed solution for the low specificity of the test is the combination of antigen-specific proliferation and cytokine release. McKimm-Breschkin et al. were the first researchers to describe a hapten (Oxazolone)-specific production of IFN-γ after lymphocyte stimulation. Notably, at that time, IFN-γ was measured by means of its ability to inhibit the growth of viral plaques [28]. Cedebrant et al. aimed at analysing whether the secretion of cytokines, especially IL-10 and IL-17, or the use of T cell receptor (TCR) Vβ families in Ni-stimulated primary peripheral blood mononuclear cell (PBMC) cultures might be more useful for discriminating between allergic and non-allergic subjects [29]. They concluded that the determination of IL-10 production in primary PBMC cultures is a potentially promising in vitro method for discrimination of Ni allergy in females, as compared with cell proliferation. Despite the unexplained gender-specification, these results are in line with the observation of Cavani et al. They describe that nickel-allergic patients exhibited primarily a Th1 outcome while non-allergic patients showed a predominance of so-called T-regulatory 1 (Tr1) cells with high IL-10 production [30–32]. Thus, an IFN-γ/IL-10 quotient combined with classical LTT could be a promising endpoint to distinguish sensitized vs. non-sensitized individuals. Spiewak et al. [33] aimed at strengthening the in vitro test by undertaking it with polarizing in vitro conditions either for Th1 or Th2. They found significant differences between patients with Ni-allergic contact dermatitis and controls for ‘type 2’ cytokines IL-13 and IL-5, with further increase of allergen-specific responses occurring when cultures were supplemented with IL-7 and IL-4. These results are intriguing and expand our knowledge on the nature of T cell responses towards metals; however, it is – to date – not suitable for routine applications. An important further caveat is that the immune systems may disparately react to different antigens [34]; thus, a general ratio of cytokine secretions might be predictive for one but not the other antigen/allergen. Unlike allergic contact dermatitis to Ni, where the in vitro cytokine responses by PBMCs or specific T cell clones have been described extensively [11, 35], few studies have addressed the immunological cytokine profile induced in vitro by other metal sensitizers. Also, in this regard the publication of Lindemann et al. in this issue [4] investigating the in vitro response to chromium adds significantly to our understanding of in vitro response to metals. In contrast to the results seen for nickel [29], Lindemann et al. found that the ELISpot assay was of minor value for the prediction of an allergy against chromium. Volunteers with sensitization with and without allergy did not differ significantly in terms of IFN-γ, IL-2, IL-4, IL-10 and IL-12 production following stimulation with tri- and hexavalent chromium compounds. These findings correspond with the observation of Minang et al., who found a mixed Th1- and Th2-type cytokine profile in response to Cr thus, making it difficult to take cytokine-endpoints as predictive values for sensitization and disease [36]. In contrast, Lindemann et al. concluded that the LTT was able to better discriminate between sensitization without symptoms and sensitization with allergic disease and gave additional information on the patch test. At the end of the day, it turns out that we need specific tests or combinatory endpoints for different allergens. However, introducing laboratory tests such as the LTT into routine clinical practice may be challenging for practicing dermatologists/allergists – especially in view of decreasing resources in the health care system in many countries. Lindemann et al. conclude that the diagnosis of an allergy against chromium appears likely if the following three criteria are fulfilled: (1) a positive patch test (verification of sensitization), (2) a positive reaction in the LTT and (3) actual exposure to chromium. This conclusion reflects where we are standing with regard to in vitro methods in type IV allergy. They are for sure – to date – no ‘stand-alone’ diagnoses. However, they may be suitable in combination with clinical findings and a detailed history. To date, none of the in vitro tests for contact allergy fulfils the requirements of an easy, accurate and safe test with sufficient specificity and sensitivity. However, studies such as that reported by Lindemann et al. [4] are certainly helpful in the development of a routine in vitro diagnostic test of contact allergy.