Angela Papale

Role of ROS and HMGB1 in Contact Allergen–Induced IL-18 Production in Human Keratinocytes

Keratinocytes have a key role in all phases of allergic contact dermatitis. We have recently identified the possibility to use IL-18 production for the in vitro identification of contact allergens. The purpose of this study was to characterize the molecular mechanisms underlying allergen-induced IL-18 production, in order to identify the cellular source of reactive oxygen species (ROS) and the danger signals involved. The NCTC2544 cell line was exposed to three contact allergens, namely p-phenylenediamine (PPD), 2,4-dinitrochlorobenzene (DNCB), and citral, in the presence or absence of diphenylene iodonium (DPI), allopurinol, and rotenone to identify the source of ROS, and to anti-Toll-like receptor 4 antibody and glycirrizic acid to characterize the danger-associated molecular pattern molecules. In the case of PPD, the induction of IL-18 can be modulated by rotenone, allopurinol, and DPI. In the case of DNCB, rotenone completely prevents the induction of IL-18, whereas for citral, DPI completely prevents the induction of IL-18. We demonstrated the ability of all allergens tested to induce the release of high-mobility group protein B1 (HMGB1). Its sequester by glycirrizic acid significantly modulates PPD-induced IL-18 production and completely prevents DNCB- and citral-induced IL-18. We found that different intracellular sources of ROS are triggered by contact allergens, and an important role for HMGB1 in chemical allergen-induced IL-18 production was demonstrated.

Role of androgens in dhea-induced rack1 expression and cytokine modulation in monocytes

BackgroundOver the past fifteen years, we have demonstrated that cortisol and dehydroepiandrosterone (DHEA) have opposite effects on the regulation of protein kinase C (PKC) activity in the context of the immune system. The anti-glucocorticoid effect of DHEA is also related to the regulation of splicing of the glucocorticoid receptor (GR), promoting the expression of GRβ isoform, which acts as a negative dominant form on GRα activity. Moreover, it is very well known that DHEA can be metabolized to androgens like testosterone, dihydrotestosterone (DHT), and its metabolites 3α-diol and 3β-diol, which exert their function through the binding of the androgen receptor (AR). Based on this knowledge, and on early observation that castrated animals show results similar to those observed in old animals, the purpose of this study is to investigate the role of androgens and the androgen receptor (AR) in DHEA-induced expression of the PKC signaling molecule RACK1 (Receptor for Activated C Kinase 1) and cytokine production in monocytes.ResultsHere we demonstrated the ability of the anti-androgen molecule, flutamide, to counteract the stimulatory effects of DHEA on RACK1 and GRβ expression, and cytokine production. In both THP-1 cells and human peripheral blood mononuclear cells (PBMC), flutamide blocked the effects of DHEA, suggesting a role of the AR in these effects. As DHEA is not considered a direct AR agonist, we investigated the metabolism of DHEA in THP-1 cells. We evaluated the ability of testosterone, DHT, and androstenedione to induce RACK1 expression and cytokine production. In analogy to DHEA, an increase in RACK1 expression and in LPS-induced IL–8 and TNF–α production was observed after treatment with these selected androgens. Finally, the silencing of AR with siRNA completely prevented DHEA-induced RACK1 mRNA expression, supporting the idea that AR is involved in DHEA effects.ConclusionsWe demonstrated that the conversion of DHEA to active androgens, which act via AR, is a key mechanism in the effect of DHEA on RACK1 expression and monocyte activation. This data supports the existence of a complex hormonal balance in the control of immune modulation, which can be further studied in the context of immunosenescence and endocrinosenescence.

Hyaluronan regulates chemical allergen-induced IL-18 production in human keratinocytes

Interleukin-18 (IL-18) has been shown to play a key proximal role in the induction of allergic contact dermatitis. Low molecular weight hyaluronan (LMWHA), an endogenous molecule and a member of the so-called damage associated molecular patterns (DAMPs), has been suggested to elicit immune-stimulatory effects. The purpose of this study was to examine the role of hyaluronan (HA) degradation in IL-18 production in human keratinocytes following stimulation with the contact sensitizers 2,4-dinitrochlorobenzene (DNCB) and PPD. IL-18 production in the human keratinocyte cell line NCTC2544 was measured by ELISA, whereas changes in HA metabolism were determined by Real-time PCR and immunofluorescence. Both contact allergens were able to enhance hyaluronidase (HYAL) 1 and 2 expression inducing HA degradation. Modulation of HA production, by HYAL or aristolochic acid pre-treatment, resulted in a significant reduction of contact allergen-induced IL-18 production. Oxidative stress appears to be the initial step in KC activation, as all the sequels of events can be blocked using antioxidants. This is the first indication that LMWHA can act as a DAMP in keratinocytes. In conclusion LMWHA fragments are important mediators in the process of contact sensitisation leading to IL-18 dependent responses.

Development of an in vitro method to estimate the sensitization induction level of contact allergens

No standardized in vitro methods to assess potency of skin sensitizers are available. Recently, we standardized a procedure which combines the epidermal equivalent potency assay with assessment of IL-18 to provide a single test for identification and classification of skin sensitizers. This current study aimed to extend tested chemicals, and to provide a simple in vitro method for estimation of the expected sensitization induction level interpolating in vitro EC50 and IL-18 SI2 values to predict LLNA EC3 and/or human NOEL from standards curves generated using reference contact allergens. Reconstituted human epidermis was challenged with 14 chemicals not previously tested benzoquinone, chlorpromazine, chloramine T, benzyl salicylate, diethyl maleate, dihydroeugenol, 2,4-dichloronitrobenzene, benzyl cinnamate, imidazolidinyl urea, and limonene as contact sensitizers while benzyl alcohol, isopropanol, dimethyl isophthalate and 4-aminobenzoic acid as non-sensitizers in the LLNA. Where for benzyl salicylate and benzyl cinnamate no sensitization was observed in human predictive studies, positive responses to benzyl alcohol and dimethyl isophthalate were reported. The proposed method correlates better with human data, correctly predicting substances incorrectly classified by LLNA. With the exception of benzoquinone (interference with both MTT and IL-18 ELISA), and chloramine T (underestimated in the interpolation), a good estimation of LLNA EC3 and in vivo available human NOEL values was obtained.

Insights on wood combustion generated proinflammatory ultrafine particles (UFP).

This study aimed to collect, characterize ultrafine particles (UFP) generated from the combustion of wood pellets and logs (softwood and hardwood) and to evaluate their pro-inflammatory effects in THP-1 and A549 cells. Both cell lines responded to UFP producing interleukin-8 (IL-8), with wood log UFP being more active compared to pellet UFP. With the exception of higher effect observed with beech wood log UFP in THP-1, the ability of soft or hard woods to induce IL-8 release was similar. In addition, on weight mass, IL-8 release was similar or lower compared to diesel exhaust particles (DEP), arguing against higher biological activity of smaller size particles. UFP-induced IL-8 could be reduced by SB203580, indicating a role of p38MAPK activation in IL-8 production. The higher activity of beech wood log UFP in THP-1 was not due to higher uptake or endotoxin contamination. Qualitatively different protein adsorption profiles were observed, with less proteins bound to beech UFP compared to conifer UFP or DEP, which may provide higher intracellular availability of bioactive components, i.e. levoglucosan and galactosan, toward which THP-1 were more responsive compared to A549 cells. These results contribute to our understanding of particles emitted by domestic appliances and their biological effects.

In vitro Models to Evaluate Drug-Induced Hypersensitivity: Potential Test Based on Activation of Dendritic Cells

Hypersensitivity drug reactions (HDRs) are the adverse effect of pharmaceuticals that clinically resemble allergy. HDRs account for approximately 1/6 of drug-induced adverse effects, and include immune-mediated (“allergic”) and non-immune-mediated (“pseudo allergic”) reactions. In recent years, the severe and unpredicted drug adverse events clearly indicate that the immune system can be a critical target of drugs. Enhanced prediction in preclinical safety evaluation is, therefore, crucial. Nowadays, there are no validated in vitro or in vivo methods to screen the sensitizing potential of drugs in the pre-clinical phase. The problem of non-predictability of immunologically-based hypersensitivity reactions is related to the lack of appropriate experimental models rather than to the lack of -understanding of the adverse phenomenon. We recently established experimental conditions and markers to correctly identify drug associated with in vivo hypersensitivity reactions using THP-1 cells and IL-8 production, CD86 and CD54 expression. The proposed in vitro method benefits from a rationalistic approach with the idea that allergenic drugs share with chemical allergens common mechanisms of cell activation. This assay can be easily incorporated into drug development for hazard identification of drugs, which may have the potential to cause in vivo hypersensitivity reactions. The purpose of this review is to assess the state of the art of in vitro models to assess the allergenic potential of drugs based on the activation of dendritic cells.

The scaffold protein RACK1 is a target of endocrine disrupting chemicals (EDCs) with important implication in immunity

ABSTRACT We recently demonstrated the existence of a complex hormonal balance between steroid hormones in the control of RACK1 (Receptor for Activated C Kinase 1) expression and immune activation, suggesting that this scaffold protein may also be targeted by endocrine disrupting chemicals (EDCs). As a proof of concept, we investigated the effect of the doping agent nandrolone, an androgen receptor (AR) agonist, and of p,p′DDT (dichlorodiphenyltrichloroethane) and its main metabolite p,p′DDE (dichlorodiphenyldichloroethylene), a weak and strong AR antagonist, respectively, on RACK1 expression and innate immune response. In analogy to endogenous androgens, nandrolone induced a dose‐related increase in RACK1 transcriptional activity and protein expression, resulting in increased LPS‐induced IL‐8 and TNF‐&agr; production and proliferation in THP‐1 cells. Conversely, p,p′DDT and p,p′DDE significantly decrease RACK1 expression, LPS‐induced cytokine production and CD86 expression; with p,p′DDE exerting a stronger repressor effect than p,p′DDT, consistent with its stronger AR antagonistic effect. These results indicate that RACK1 could be a relevant target of EDCs, responding in opposite ways to agonist or antagonist of AR, representing a bridge between the endocrine system and the innate immune system. HIGHLIGHTSRACK1 expression can be induced by AR agonists with a consequent enhancement of the response to LPS.RACK1 can be negatively modulated by the AR antagonists DDT and its main metabolite p,p′DDE.RACK1 can be a relevant target of EDCs, representing a bridge between the endocrine system and the immune system.

The plasticizer dibutyl phthalate (DBP) potentiates chemical allergen-induced THP-1 activation.

Recent studies suggest that phthalates may have a role in the development of allergic diseases, probably due to an adjuvant effect. The present study aimed to investigate the possible adjuvant effect of dibutyl phthalate (DBP) in two in vitro models of contact-allergen induced cell activation, namely the NCTC 2544 IL-18 assay and the THP-1 activation assay. Results show no adjuvant effect in the human keratinocyte cell line NCTC 2544, indicated by lack of increase in interleukin 18 (IL-18) production after exposure to p-Phenylenediamine (PPD) in association with DBP. On the contrary, increased upregulation of CD86 and interleukin 8 (IL-8) production were observed in THP-1 cells exposed to combinations of citral (Cit) or imidazolidinyl urea (IMZ) with DBP, indicative of an adjuvant effect. Additionally, higher production of reactive oxygen species (ROS) in THP-1 cells treated with DBP associated to Cit supports that oxidative stress could be part of the molecular mechanism of the observed adjuvant effect. In conclusion, we demonstrate that DBP presents in vitro an adjuvant effect for immune stimulation in dendritic cells but not in keratinocytes. Future studies are necessary to elucidate the precise mechanism underlying the adjuvant effect of DBP in vitro and in vivo.