cytokines

/Tag:cytokines

Selective Induction of Interleukin-12 in Reconstructed Human Epidermis by Chemical Allergens

Emanuela Corsini, Elena Limiroli, Marina Marinovich, Catherine
Cohen, Roland Roguet and Corrado L. Galli

Keratinocytes play an important role in skin inflammatory and immunological reactions through the release of cytokines and response to them. These cells have been shown to direct T-cell priming by producing cytokines such as interleukin (IL)-10 and IL-12. The purpose of this work was to explore the potential use of IL-12 production to discriminate between skin irritants and contact allergens in vitro. Initially, a reconstituted human epidermis was treated with a known human skin irritant, sodium lauryl sulphate (SLS), and a known human contact allergen, 1-chloro-2,4-dinitrobenzene (DNCB). The expression of IL-12p40 was assessed at specific time intervals by the semi-quantitative reverse transcriptase-polymerase chain reaction (rt-PCR). The data obtained indicated that only DNCB induced an up-regulation of IL-12p40. This up-regulation occurred after exposure to DNCB for 3 hours. Importantly, the application of SLS or vehicles did not induce IL-12 mRNA up-regulation. An increase in total IL-12 protein content was detected in supernatants of allergen-stimulated, but not vehicle-stimulated, reconstituted epidermis. To confirm these results, the effects of benzalkonium chloride, oxazolone and eugenol were assessed. At concentrations that resulted in equivalent IL-1α release, only contact allergens increased IL-12 expression, which confirmed the previous results. These data suggest that IL-12, which is crucial for T-helper type 1 cell responses, could be a useful marker for discriminating between contact allergens and irritants.
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Evaluation and Prevalidation of an Immunotoxicity Test Based on Human Whole-blood Cytokine Release

Ingrid Langezaal, Sebastian Hoffmann, Thomas Hartung, & Sandra Coecke

Immunotoxicology is a relatively new field in toxicology, and is one of emerging importance, because immunotoxicity appears to contribute to the development of cancer, autoimmune disorders, allergies and other diseases. At present, there is a lack of human cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical and reliable way. Existing immunotoxicity tests are mainly performed in animals, although species differences favour humanbased testing. Whole-blood cytokine release models have attracted increasing interest, and are broadly used for pharmacological in vitro and ex vivo studies, as well as for pyrogenicity testing. We have adapted those methods for immunotoxicity testing, to permit the potency testing of immunostimulants and immunosuppressants. Following stimulation with a lipopolysaccharide or staphylococcal enterotoxin B, monocytes and lymphocytes release interleukin-1β and interleukin-4, respectively. Thirty-one pharmaceutical compounds, with known effects on the immune system, were used to optimise and standardise the method, by analysing their effects on cytokine release. The in vitro results were expressed as IC50 values for immunosuppression, and SC4 (fourfold increase) values for immunostimulation, and compared with therapeutic serum concentrations of the compounds in patients, and in vivo LD50 values from animal studies. The in vitro results correlated well with the in vivo data, so the test appears to reflect immunomodulation. Results were reproducible (CV = 20 ± 5%), and the method could be transferred to another laboratory (r2 = 0.99). We therefore propose this method for further validation and for use in immunotoxicity testing strategies.
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The In Vitro Acute Skin Irritation of Chemicals: Optimisation of the EPISKIN Prediction Model within the Framework of the ECVAM Validation Process

José Cotovio, Marie-Hélène Grandidier, Pascal Portes, Roland Roguet and Gilles Rubinstenn

In view of the increasing need to identify non-animal tests able to predict acute skin irritation of chemicals, the European Centre for the Validation of Alternative Methods (ECVAM) focused on the evaluation of appropriate in vitro models. In vitro tests should be capable of discriminating between irritant (I) chemicals (EU risk: R38) and non-irritant (NI) chemicals (EU risk: “no classification”). Since major in vivo skin irritation assays rely on visual scoring, it is still a challenge to correlate in vivo clinical signs with in vitro biochemical measurements. Being particularly suited to test raw materials or chemicals with a wide variety of physical properties, in vitro skin models resembling in vivo human skin were involved in prevalidation processes. Among many other factors, cytotoxicity is known to trigger irritation processes, and can therefore be a first common event for irritants. A refined protocol (protocol15min–18hours) for the EPISKIN model had been proposed for inclusion in the ECVAM formal validation study. A further improvement on this protocol, mainly based on a post-treatment incubation period of 42 hours (protocol15min–42hours), the optimised protocol, was applied to a set of 48 chemicals. The sensitivity, specificity and accuracy with the MTT assay-based prediction model (PM) were 85%, 78.6% and 81.3% respectively, with a low rate of false negatives (12%). The improved performance of this optimised protocol was confirmed by a higher robustness (homogeneity of individual responses) and a better discrimination between the I and NI classes. To improve the MTT viability-based PM, the release of a membrane damage marker, adenylate kinase (AK), and of cytokines IL-1α and IL-8 were also investigated. Combining these endpoints, a simple two-tiered strategy (TTS) was developed, with the MTT assay as the first, sort-out, stage. This resulted in a clear increase in sensitivity to 95%, and a fall in the false-positive rate (to 4.3%), thus demonstrating its usefulness as a “decision- making” tool. The optimised protocol proved, both by its higher performances and by its robustness, to be a good candidate for the validation process, as well as a potential alternative method for assessing acute skin irritation.
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