Cutting Edge: Nqo1 Regulates Irritant Contact Hypersensitivity against Croton Oil through Maintenance of Dendritic Epidermal T Cells.

Irritant contact dermatitis (ICD) is associated with local release of inflammatory mediators such as reactive oxygen species and regulated by various antioxidative enzymes and antioxidants. Although Nqo1 is involved in antioxidative reactions and detoxification, its role in ICD remains unknown. Nqo1-deficient mice exhibited augmented ear swelling accompanied by neutrophil infiltration in the croton oil-induced mouse ICD model. In the skin of Nqo1-deficient mice, Vγ5Vδ1+ dendritic epidermal T cells (DETCs), which are known to suppress ICD, were severely reduced. As the transfer of DETCs into Nqo1-deficient mice reversed an increased ICD response, loss of DETCs could account for the increased ICD. DETCs from Nqo1-deficient mice were sensitive to oxidative stress-induced cell death in vitro, and antioxidant NAC treatment in the ears of these mice rescued the number of DETCs and produced a normal ICD response. Taken together, the current results demonstrate that antioxidative enzyme Nqo1 regulates ICD through DETC maintenance.

"Slime" dermatitis, a fad-associated chronic hand dermatitis.

"Slime" is the colloquial name for a non-Newtonian viscoelastic putty-like substance that is currently a popular plaything among pre-teens and adolescents. Several ingredients in homemade slime recipes may cause irritant or allergic contact dermatitis. We report two children who developed slime-associated chronic hand dermatitis, more prominently on their dominant hand. We review the potential for irritant and allergic contact dermatoses as the causes of dermatitis associated with homemade slime.

CysLT1 Receptor Is Protective against Oxidative Stress in a Model of Irritant-Induced Asthma.

The bronchoconstrictive and proinflammatory properties of cysteinyl leukotrienes (cysLTs) in allergic asthma mediate their effects predominantly through the cysLT1 receptor (cysLT1R). However, the role of cysLTs and cysLT1R in innate immune-triggered asthma is largely unexplored. We explored the synthesis of cysLTs and cysLT1R as determinants of airway responses in an oxidative stress-induced model of irritant asthma. Wild-type (WT) mice exposed to 100 ppm Cl2 for 5 min had airway neutrophilia, increased cysLT production, and pulmonary expression of cysLT-related biosynthetic genes. CysLT1R-deficient (CysLTr1(-/-)) mice that were exposed to Cl2 demonstrated airway hyperresponsiveness to inhaled methacholine significantly greater than in WT BALB/c mice. Compared to WT mice, airway neutrophilia and keratinocyte chemoattractant production levels were higher in CysLTr1(-/-) mice and airway hyperresponsiveness was ameliorated using a granulocyte depletion Ab. CysLTr1(-/-) mice also demonstrated prolonged bronchial epithelial cell apoptosis following Cl2 WT mice showed increased antioxidant and NF erythroid 2-related factor 2 (Nrf2) gene expression, Nrf2 nuclear translocation in bronchial epithelial cells, and increased reduced glutathione/oxidized glutathione following Cl2 exposure whereas CysLTr1(-/-) mice did not. Furthermore, CysLTr1(-/-) mice demonstrated increased pulmonary E-cadherin expression and soluble E-cadherin shedding compared with WT mice. Loss of a functional cysLT1R results in aberrant antioxidant response and increased susceptibility to oxidative injury, apparently via a cysLT1R-dependent impairment of Nrf2 function.

Mucosal immune tolerance at the ocular surface in health and disease.

The ocular surface is constantly exposed to environmental irritants, allergens and pathogens, against which it can mount a prompt immune response to preserve its integrity. But to avoid unnecessary inflammation, the ocular surface's mucosal immune system must also discriminate between harmless and potentially dangerous antigens, a seemingly complicated task. Despite its unique features, the ocular surface is a mucosal lining, and as such, it shares some homeostatic and pathophysiological mechanisms with other mucosal surfaces. The purpose of this review is to explore the mucosal homeostatic immune function of the ocular surface in both the healthy and diseased states, with a special focus on mucosal immunology concepts. The information discussed in this review has been retrieved by PubMed searches for literature published from January 1981 to October 2016.

CXCR3 deficiency prolongs Th1-type contact hypersensitivity.

Sensitization and challenge using dinitrofluorobenzene (DNFB) induce contact hypersensitivity (CHS) with Th1 cell infiltration, whereas those using FITC generate CHS with Th2 cell infiltration. In this study, we attempted to determine the role of CXCR3, a chemokine receptor, in Th1- and Th2-type CHS induced by DNFB or FITC using CXCR3-deficient (CXCR3(-/-)) mice. Ear swelling was prolonged after DNFB challenge in CXCR3(-/-) mice, which was accompanied by increased Th1 cytokines and decreased TGF-ß and IL-10 expression at a late time point of CHS, whereas there was no significant difference between wild-type and CXCR3(-/-) mice in FITC-induced CHS. In Th1-type CHS, the number of regulatory T cells (Tregs) was decreased in the challenged ear of CXCR3(-/-) mice compared with that of wild-type mice, suggesting that CXCR3 would be important in migration of Tregs into the site of inflammation. Moreover, we examined the characteristics of CXCR3(+) Tregs both in vitro and in vivo, revealing that CXCR3(+) Tregs expressed high levels of TGF-ß and IL-10 as well as IFN-γ compared with CXCR3(-) Tregs. When CXCR3(-/-) mice were injected with CXCR3(+) Tregs, the prolonged ear swelling induced by DNFB was normalized. Taken together, our results suggest that CXCR3(+) Tregs play a key role for quenching Th1-type CHS.

Contact sensitizer 2,4-dinitrochlorobenzene is a highly potent human TRPA1 agonist.

2,4-Dinitrochlorobenzene (DNCB) is widely used in human clinical studies and in experimental animal studies to evoke allergic contact dermatitis. 2,4-Dinitrochlorobenzene is a potent immunogen capable of inducing contact sensitization in all humans exposed. However, the mechanism by which DNCB evokes such symptoms is presently unknown. TRPA1 is a nonselective cation channel that is expressed in peptidergic sensory neurons and fibroblasts. TRPA1 activation was recently implicated in the pathophysiology of atopic dermatitis especially in transducing cutaneous itch signals. Here, we test the hypothesis that DNCB acts as a TRPA1 agonist and thereby evokes allergic symptoms. We found that DNCB activates human TRPA1 dose dependently in FLIPR experiments with an EC50 of 167 nM, an effect that was fully blocked by selective TRPA1 antagonists Chembridge-5861528 and A-967079. Similarly, DNCB activated nonselective TRPA1 current in patch clamp studies. Neutralization of 3 critical cysteines in TRPA1 resulted in a loss of DNCB agonism.

Investigation of allergenicity of some cosmetic mixtures by using ex vivo local lymph node assay-BrdU endpoints.

BACKGROUND: Balsam of Peru and fragrance mix are commonly used in cosmetic products. Allergy to fragrance is the most common cause of cosmetic contact dermatitis. METHODS: In the present study, ex vivo local lymph node assay-5-bromo-2'-deoxyuridine (LLNA-BrdU) was used to evaluate the dermal sensitization potential of these cosmetic mixtures. The stimulation index values and estimated concentration (EC3) values were calculated and the potency classification was found for each mixture. At the same time, in order to measure the irritant effect without having to use additional animals, a combination of ex vivo LLNA-BrdU and the irritancy assay was conducted. RESULTS: Th1 [interleukin (IL)-2, interferon-γ] and Th2 cytokine (IL-4, IL-5) releases from lymph node cell culture were investigated as non-radioactive endpoints. According to the results of ex vivo LLNA-BrdU assays, EC3 values were found to be 3.09% (moderate) for balsam of Peru and 4.44% (moderate) for fragrance mix. Cytokine analysis results indicate that both Th1 and Th2 cytokines are involved in the regulation of murine contact allergy and can be considered as useful endpoints. CONCLUSION: In conclusion, according to our results, fragrance mix and balsam of Peru can be considered as moderate sensitizers; however, in high concentrations, both of them have irritation properties. The cytokines investigated can be considered as the endpoints of the ex vivo LLNA-BrdU assay.

The hapten-atopy hypothesis III: the potential role of airborne chemicals.

One explanation for the large increase in the prevalence of atopic disease in developed countries during the last 50 years is the 'hygiene hypothesis'. This proposes that a reduced exposure to pathogenic microorganisms at a key period(s) during development results in the maintenance or acquisition of an atopic phenotype. Alternatively, or additionally, we have postulated that increased exposure to chemicals generally, and to irritant/haptenic chemicals in particular, during critical windows of maternal pregnancy/early life have also contributed to changes in the prevalence of atopic disease. Having previously reviewed the potential roles of oral and cutaneous exposure to chemicals on the subsequent diagnosis of atopic disease, we here consider possible evidence of a role for exposure to airborne chemicals as a contributory factor in acquired susceptibility to atopic allergy. After controlling for known confounders, five specific maternal occupations during pregnancy have been implicated as being associated with subsequent atopic disease in the offspring. Each of these occupations is characterized by high and persistent exposure to airborne chemicals. High-level exposure to volatile organic compounds in the domestic environment, either during pregnancy or in early life, is also associated with development of childhood atopic disease. Similarly, sustained exposure to airborne chlorinated chemicals from swimming pools during childhood has been associated with the development of atopic allergy. A possible immunological basis for these associations is that exposure to certain airborne chemicals, even at low levels, can result in the delivery of 'danger' signals that, in turn, bias the immune response towards the selective induction or maintenance of preferential T helper 2-type immune responses consistent with the acquisition of allergic sensitization.

Comparison of irritatant reactions between using lyophilized and commercial food allergen extracts in atopy patch tests in a normal population.

BACKGROUND: Several authors have investigated the use of the atopy patch tests (APT) for the diagnosis of non-IgE mediated food allergy, primarily in patients with atopic dermatitis and digestive disorders. However, one of the difficulties in atopy patch testing is the lack of standardization. Several commercial APTs containing freeze-dried food extracts are now available, but their diagnostic accuracy is still largely undefined. The objective of this study is to evaluate the irritant reactions and safety of atopy patch tests in healthy subjects by using lyophilized and commercial food allergen extracts. METHODS: A cross-sectional study was carried out in healthy volunteers. Atopy patches using lyophilized and commercial allergens, including cow's milk, egg, wheat, soy and shrimp, were assessed. Additionally, commercial extracts of house dust mite (D. pteronyssinus 10,000 AU/ml, D. farinae 10,000 AU/ml) and American cockroach were also evaluated. RESULTS: Eighteen healthy volunteers (13 women, median age 26 years) were enrolled. All APT results, both from using lyophilized and commercial allergen extracts, showed no reactions. There were no systemic allergic reactions or irritant reactions observed. CONCLUSION: APTs using lyophilized and commercial food allergen extracts and commercial extracts of house dust mite and American cockroach showed no irritant reactions in Thai non-atopic subjects.

The cutaneous biochemical redox barrier: a component of the innate immune defenses against sensitization by highly reactive environmental xenobiotics.

Contact allergy to environmental xenobiotics is a common and important problem, but it is unclear why some chemicals are potent sensitizers and others weak/nonsensitizers. We explored this by investigating why similar chemicals, 2,4-dinitrochlorobenzene (DNCB) and 2,4-dinitrothiocyanobenzene (DNTB), differ in their ability to induce contact hypersensitivity (CHS). DNCB induced CHS in humans, whereas at similar doses DNTB did not. However, following DNCB sensitization, DNTB elicited CHS in vivo and stimulated DNCB-responsive T cells in vitro, suggesting that differences in response to these compounds lie in the sensitization phase. In contrast to DNCB, DNTB failed to induce emigration of epidermal Langerhans cells in naive individuals. Examination for protein dinitrophenylation in skin revealed that DNCB penetrated into the epidermis, whereas DNTB remained bound to a thiol-rich band within the stratum corneum. DNTB reacted rapidly with reduced glutathione in vitro and was associated with a decrease in the free thiol layer in the stratum corneum, but not in the nucleated epidermis. By contrast, DNCB required GST facilitation to react with gluthathione and, following penetration through the stratum corneum, depleted thiols in the viable epidermis. Chemical depletion of the thiol-rich band or removing it by tape stripping allowed increased penetration of DNTB into the epidermis. Our results suggest that the dissimilar sensitizing potencies of DNCB and DNTB in humans are determined by a previously undescribed outer epidermal biochemical redox barrier, a chemical component of the innate immune defense mechanisms that defend against sensitization by highly reactive environmental chemicals.

The regulatory use of the Local Lymph Node Assay for the notification of new chemicals in Europe.

The regulatory use of the Local Lymph Node Assay (LLNA) for new chemicals registration was monitored by screening the New Chemicals Database (NCD), which was managed by the former European Chemicals Bureau (ECB) at the European Commission Joint Research Centre (JRC). The NCD centralised information for chemicals notified after 1981, where toxicological information has been generated predominantly according to approved test methods. The database was searched to extract notifications for which the information for skin sensitisation labelling was based on results derived with the LLNA. The details of these records were extracted and pooled, and evaluated with regard to the extent of use of the LLNA over time, as well as for analysing the information retrieved on critical aspects of the procedure e.g. strain and amount of animals used, lymph node processing, solvent and doses selected, stimulation indices, and for assessing their level of compliance to the OECD Test Guideline 429. In addition the accuracy of the reduced LLNA when applied to new chemicals was investigated.

Hazard identification of strong dermal sensitizers.

Dermal reactions are the most frequently reported chemical health-related occupational hazard. Identifying dermal sensitizers is important for improving workplace safety. This paper takes a close look at the physico-chemical properties and results from the Local Lymph Node Assay (LLNA) to better understand and predict potent dermal sensitizers. The LLNA was used to identify 28 pharmaceutical agents or chemical intermediates as potent dermal sensitizers, EC3 < 1%. Certain parameters were examined to determine if there was any predictability to identify potent dermal sensitizers. These included a computer structure activity analysis using Derek for Windows, molecular weight (Mw), calculated log P, and the log-linear extrapolation approach for estimating the potency. With Derek for Windows, 13 compounds were identified as negative and 15 as positive for structural alerts, the most common being haloalkanes, and hydrazines. Additional mechanisms of reactivity were postulated for the remaining compounds. The examination of the Mw showed that all molecules had Mw < 550 Da. For 21 compounds, the interpolated vs extrapolated methods for determining the EC3 value were compared. For eight of the 21 compounds, the extrapolated EC3 was in the correct order of magnitude, eight were incorrect (five were too high and three were too low) and five could not be calculated. The use of a tiered approach including examination of the structural and physico-chemical properties and the LLNA to identify potent dermal sensitizers is integral in the selection of effective safe handling guidance to protect from sensitization hazards.

Skin Sensitization Tests: The LLNA and the RhE IL-18 Potency Assay.

Contact allergy is of considerable importance to the toxicologist, and regulatory authorities worldwide require testing for skin sensitization potential and appropriate hazard labeling to enable management of the risk to human health. Although traditionally the identification of skin-sensitizing chemicals has been carried out using animal models, in Europe legislative changes have promoted, and now require, the use of non-animal methods (i.e., Cosmetic Directive, REACH). Several in vitro alternatives for hazard identification have now been validated, but do not provide information on the potency of a skin sensitizer. Here, we describe an animal model, the local lymph node assay (LLNA), and an in vitro model, the RhE IL-18 potency assay, in the context of the identification and potency classification of skin sensitizers. These two assays have been chosen among the different available tests as representative of an alternative in vivo model (the LLNA) and a promising in vitro method with the potential of both hazard identification and potency classification.

Signal transducer and activator of transcription 6 is essential in the induction of contact hypersensitivity.

Contact hypersensitivity (CHS) is thought to be mainly associated with the activation of T helper type 1 (Th1) cells. However, there is also evidence that Th2 cells or Th2 cytokines play a role in the development of CHS. To analyze the functional contribution of Th2 cytokines interleukin (IL)-4 and IL-13, signal transducer and activator of transcription 6 (STAT6)-deficient (STAT6(-/)-) and wild-type (wt) control C57BL/6 mice were contact sensitized with 5% 2,4,6-trinitrochlorobenzene (TNCB), 0.5% 2,4-dinitrofluorobenzene, or 5% 4-ethoxyl methylene-2-phenyl-2-oxazolin-5-one, and any skin reactions were examined. Ear swelling was significantly reduced with a delayed peak response in STAT6(-/)- mice compared with wt mice.A histological analysis revealed that the infiltration of both eosinophils and neutrophils in the skin challenged after 24 h in STAT6(-/)- mice decreased substantially compared with that in wt mice. The expression of Th2 cytokines (IL-4, IL-5) in TNCB-challenged skin tissues and the supernatants from T cells stimulated by 2,4,6-trinitrobenzene sulfonate-modified spleen cells, as well as the immunoglobulin (Ig)E and IgG1 response after challenge, were also profoundly reduced in STAT6(-/)- mice, whereas the expression of interferon gamma was the same in STAT6(-/)- and wt mice after challenge. Furthermore, adoptive transfer experiments revealed that STAT6(-/)- mice induced CHS after injection of lymph node cells obtained from sensitized wt mice. Our data suggest that the STAT6 signal plays a critical role in the induction phase of CHS.

Classification of sensitizing and irritative potential in a combined in-vitro assay.

We have developed a coculture system which in parallel indicates the sensitizing and irritative potential of xenobiotics. The assay is named loose-fit coculture-based sensitization assay (LCSA) and may be performed within 5 days. The system is composed of human monocytes that differentiate to a kind of dendritic cells by 2-day culturing in the presence of allogenic keratinocytes. The culture medium is enriched by a cocktail of recombinant cytokines. On day 3, concentration series of probes are added. On day 5, cells are harvested and analyzed for expression range of CD86 as a marker of sensitizing potential and for uptake of the viability stain 7-AAD as a marker of irritative potential. Estimation of the concentration required to cause a half-maximal increase in CD86 expression allowed quantification of sensitizing potential, and estimation of the concentration required to reduce viability to 50% allowed quantification of irritative potential. Examination of substances with known potential resulted in categorization of test scores. To evaluate our data, we have compared results with those of the validated animal-based sensitization test, the murine local lymph node assay (LLNA, OECD TG 429). To a large extent, results from LCSA and from LLNA achieved analogous grouping of allergens into categories like weak-moderate-strong. However, the new assay showed an improved capacity to distinguish sensitizers from non-sensitizers and irritants. In conclusion, the LCSA contains potential to fulfil the requirements of the EU's programme for the safety of chemicals "Registration, Evaluation, Authorization and Restriction of chemical substances" (REACH, 2006) to replace animal models.

The possible relevance of sex hormones on irritant and allergic responses: their importance for skin testing.

Dermatological responses are affected by the menstrual cycle phase in female patients, an unsurprising observation as oestrogen and progesterone affect the skin and immunological function, with oestrogen suppression of cellular immunity in particular. Exacerbation of dermatological symptoms is typically observed in either the latter phase of the menstrual cycle or during menstruation. The allergic response is diminished in the ovulatory phase and heightened in the progestinic phase. Definitive conclusions with regard to the effect of reproductive hormones on skin disorders have been somewhat hampered by a body of research that has employed diverse research parameters, such as dosage, testing sites, concentration, vehicle of irritant delivery, and method of assessment, however, individual patient sensitivity varies widely. Standardization of measurement techniques is necessary to provide reproducible results as much as individual patient variation and technique will allow.

Mechanisms of occupational asthma.

Inhalation of agents in the workplace can induce asthma in a relatively small proportion of exposed workers. Like nonoccupational asthma, occupational asthma is probably the result of multiple genetic, environmental, and behavioral influences. It is important that occupational asthma be recognized clinically because it has serious medical and socioeconomic consequences. Environmental factors that can affect the initiation of occupational asthma include the intrinsic characteristics of causative agents as well as the influence of the level and route of exposure at the workplace. The identification of host factors, polymorphisms, and candidate genes associated with occupational asthma may improve our understanding of mechanisms involved in asthma. High-molecular-weight compounds from biological sources and low-molecular-weight chemicals cause occupational asthma after a latent period of exposure. Although the clinical, functional, and pathologic features of occupational asthma caused by low-molecular-weight agents resemble those of allergic asthma, the failure to detect specific IgE antibodies against most low-molecular-weight agents has resulted in a search for alternative or complementary physiopathologic mechanisms leading to airway sensitization. Recent advances have been made in the characterization of the immune response to low-molecular-weight agents. In contrast, the mechanism of the type of occupational asthma that occurs without latency after high-level exposure to irritants remains undetermined.

Management of work-related asthma.

The physician managing work-related asthma (WRA) assumes many roles. The first is to confirm an accurate diagnosis, recognizing that WRA has multiple phenotypes, including sensitizer-induced occupational asthma (OA) caused by high-molecular-weight (HMW) proteins or low-molecular-weight (LMW) chemicals; irritant-induced asthma; and work-exacerbated asthma. Pharmacotherapy for WRA is identical to nonwork-related asthma and should be guided by current asthma guidelines emphasizing control of both asthma impairment and risk domains. It is well established that the majority of workers diagnosed with OA caused by sensitizers experience persistent asthma after leaving the workplace. However, the long-term risk of persistent unremitting asthma can be prevented in a minority of cases, particularly with OA caused by LMW sensitizers, by establishing an early diagnosis of OA and reducing or eliminating exposure. The physician consultant may advise employers on workplace interventions needed to minimize effectively an affected employee's exposure to a causative agent or condition, and what measures are required to prevent new cases of WRA (ie, primary prevention). Although allergen immunotherapy has a putative role in treating and preventing WRA caused by HMW sensitizers, further study is needed.

Filaggrin deficiency confers a paracellular barrier abnormality that reduces inflammatory thresholds to irritants and haptens.

BACKGROUND: Mutations in the human filaggrin gene (FLG) are associated with atopic dermatitis (AD) and are presumed to provoke a barrier abnormality. Yet additional acquired stressors might be necessary because the same mutations can result in a noninflammatory disorder, ichthyosis vulgaris. OBJECTIVE: We examined here whether FLG deficiency alone suffices to produce a barrier abnormality, the basis for the putative abnormality, and its proinflammatory consequences. METHODS: By using the flaky-tail mouse, which lacks processed murine filaggrin because of a frameshift mutation in the gene encoding profilaggrin that mimics some mutations in human AD, we assessed whether FLG deficiency provokes a barrier abnormality, further localized the defect, identified its subcellular basis, and assessed thresholds to irritant- and hapten-induced dermatitis. RESULTS: Flaky-tail mice exhibit low-grade inflammation with increased bidirectional, paracellular permeability of water-soluble xenobiotes caused by impaired lamellar body secretion and altered stratum corneum extracellular membranes. This barrier abnormality correlates with reduced inflammatory thresholds to both topical irritants and haptens. Moreover, when exposed repeatedly to topical haptens at doses that produce no inflammation in wild-type mice, flaky-tail mice experience a severe AD-like dermatosis with a further deterioration in barrier function and features of a T(H)2 immunophenotype (increased CRTH levels plus inflammation, increased serum IgE levels, and reduced antimicrobial peptide [mBD3] expression). CONCLUSIONS: FLG deficiency alone provokes a paracellular barrier abnormality in mice that reduces inflammatory thresholds to topical irritants/haptens, likely accounting for enhanced antigen penetration in FLG-associated AD.