Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals

The local lymph node assay (LLNA) is a murine model developed to evaluate the skin sensitization potential of chemicals. The LLNA is an alternative approach to traditional guinea pig methods and in comparison provides important animal welfare benefits. The assay relies on measurement of events induced during the induction phase of skin sensitization, specifically lymphocyte proliferation in the draining lymph nodes which is a hallmark of a skin sensitization response. Since its introduction the LLNA has been the subject of extensive evaluation on a national and international scale, and has been successfully validated and incorporated worldwide into regulatory guidelines. Experience gained in recent years has demonstrated that adherence to published procedures and guidelines for the LLNA (e.g., with respect to dose and vehicle selection) is critical for the successful conduct and eventual interpretation of the data. In addition to providing a robust method for skin sensitization hazard identification, the LLNA has proven very useful in assessing the skin sensitizing potency of test chemicals, and this has provided invaluable information to risk assessors. The primary method to make comparisons of the relative potency of chemical sensitizers is to use linear interpolation to estimate the concentration of chemical required to induce a stimulation index of three relative to concurrent vehicle-treated controls (EC3). In certain situations where there are available less than optimal dose response data a log-linear extrapolation method can be used to estimate an EC3 value which can reduce significantly the need for repeat testing of chemicals. The LLNA, when conducted according to published guidelines, provides a robust method for skin sensitization testing that not only provides reliable hazard identification information but also data necessary for effective risk assessment and risk management.     

Cytokine fingerprinting: characterization of chemical allergens.

Chemical allergy is a common and important occupational health issue. Allergic sensitization induced by chemicals may take a variety of forms, including allergic contact dermatitis (skin sensitization) and allergic asthma and rhinitis (sensitization of the respiratory tract). There is a need to identify and characterize chemicals that have the potential to cause such sensitization reactions. Although a number of methods are available for the prospective analysis of skin sensitizing activity, there are currently no widely accepted tests for the identification of chemical respiratory allergens. We here describe a novel approach, cytokine fingerprinting, that has the potential to distinguish between chemical contact and respiratory allergens. The pattern of cytokine production by draining lymph node cells (LNCs) is evaluated following repeated topical exposure of mice to test chemicals. Experience to date reveals that contact allergens stimulate the selective development of type 1 immune responses associated with the secretion by draining LNCs of interferon gamma (IFN-gamma), but little interleukin-4 (IL-4) or interleukin-10 (IL-10). In contrast, chemical respiratory allergens are found to induce the appearance of preferential type 2 immune responses characterized by IL-4 and IL-10 production, but comparatively low levels of IFN-gamma. It is proposed that cytokine fingerprinting may permit the simultaneous identification and characterization of those chemicals that have the potential to cause allergic sensitization.     

Reduction,refinement and replacement: putting the immune system to work

Many chemicals are known to be, or have been implicated as, contact allergens, and allergic contact dermatitis is an important occupational and environmental health issue. It is the responsibility of toxicologists to identify those chemicals that have the potential to induce skin sensitisation, and to assess the conditions under which there will exist a risk to human health. This article describes progress that has been made in the development of new approaches to the toxicological evaluation of skin sensitisation, and the benefits to animal welfare that such developments have already produced, and are likely to produce in the future. In this context, the local lymph node assay is described with regard to hazard identification and risk assessment, and possible strategies for the development of in vitro approaches to safety assessment are discussed.     

Detection of autoimmunity by pharmaceuticals

Despite the important health and economic impact of autoimmunogenicity or allergenicity by pharmaceuticals models to detect such adverse effects are not available yet. The most important reason for this is the related complex interplay of multiple factors, for which reason these adverse effects are also referred to as idiosyncratic in nature. Moreover, clinical effects are quite diverse, and involve both organ-specific and systemic effects, including a diversity of skin diseases. Because of its complexity on the one hand and the fundamental knowledge on certain particular mechanistic effects it may be more relevant to design a rationalistic toolbox of test models from which a predictive strategy can be composed. Since one mechanistic aspect centers around T cell sensitization a straightforward lymph node assay such as the reporter antigen-popliteal lymph node assay (RA-PLNA) would fit in such a toolbox. This RA-PLNA holds a strong promise to distinguish sensitizing and/or neoantigen-forming capacity of low molecular weight pharmaceuticals. In addition, from the pharmacokinetic point of view a rationalistic toolbox should also contain oral exposure models with immunological read out parameters in normal or in genetically predisposed animal strains. This review focuses on these two categories of candidate test methods, PLNA and oral exposure models, and proposes to use these in tandem in order to predict the hazard of induction of allergy or autoimmune phenomena by new pharmaceutical candidates.     

The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test – human cell line activation test (h-CLAT)

Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20–95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65–90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of “CD86 ≥ 150 or CD54 ≥ 200” resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.     

Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization

These studies address the hypothesis that Ag-bearing epidermal Langerhans cells migrate to the regional lymph node during contact sensitization and function as APC. Skin from C3H mice was grafted onto BALB/c nude mice, and 7 or 14 days later, the recipients were sensitized with FITC through the grafts. APC from lymph nodes draining the site of sensitization were capable of sensitizing C3H recipients to FITC. Because sensitization is MHC restricted, only cells reaching the lymph node from the grafted skin could have induced contact hypersensitivity in C3H mice. Examination of the FITC+ draining lymph node cells by immunofluorescence and immunoelectron microscopy demonstrated that all were Ia+, most were F4/80+, and some contained Birbeck granules. These studies demonstrate that Ia+, FITC+ cells from the skin, at least some of which are Langerhans cells, leave the skin after epicutaneous sensitization with FITC and participate in the initiation of the contact hypersensitivity response within the regional lymph node.     

Antigen presented in the local lymph node by cells from dimethylbenzanthracene-treated murine epidermis activates suppressor cells

Application to skin depleted of LC by treatment with the chemical carcinogen DMBA of a dose of contact sensitizer optimal for inducing contact sensitivity activates transferrable suppressor cells. Excision of solvent- or DMBA-treated skin at various times following application of the contact sensitizer DNFB indicated that the fraction of antigen which leaves the skin within the first few hours induces tolerance. An initial signal inducing unresponsiveness, observed within 1/2 hr, was overturned 3-6 hr later. A more permanent tolerogenic signal in the DMBA- but not solvent-treated lymph node resulted from an epidermal cell from DMBA-treated skin presenting antigen to suppressor cells. Therefore it is likely that suppressor cells are activated in DMBA-treated mice by an epidermal cell which migrates to the local lymph node. Local lymph node cells from DMBA-treated mice also have a diminished ability to present antigen in vivo but they do not activate suppressor cells.     

Lymphatic mapping for Merkel cell carcinoma

Merkel cell trabecular carcinoma of the skin has a prognosis poorer than expected for a small skin lesion. An early diagnosis and prompt treatment can contribute to improve survival in cases of this aggressive skin tumor. A wide local excision is indicated for localized disease. Elective lymph node dissection is controversial. The authors report a rare case of Merkel cell tumor treated with wide local excision and sentinel lymph node biopsy.     

Production of immunity and unresponsiveness in the mouse by feeding contact sensitizing agents and the role of suppressor cells in the peyer's patches, mesenteric lymph nodes and other lymphoid tissues.

Mice were fed the contact sensitizing agents “oxazolone” or picryl chloride by tube. A single feed gave rise to contact sensitivity. However, the contact sensitivity and antibody production which occurred in mice painted with oxazolone were almost abolished when the mice were fed oxazolone 14 days before the skin painting. Feeding also reduced the DNA synthesis response in the regional lymph nodes. Two types of suppressor cells were found in mice after feeding. After a single feed of picryl chloride the Peyer's patches and mesenteric lymph nodes contained suppressor cells which suppressed the passive transfer of contact sensitivity. After three feeds of either agent spleen cells also caused inhibition. These suppressor cells were presumptive B cells as shown by their ability to form rosettes with red cells coated with antibody and complement and their resistance to anti-θ serum and complement. However, separated T cells from the same spleen transferred contact sensitivity. In addition to these B suppressor cells the spleens and peripheral lymph node cells of mice fed with contact sensitizing agent and then painted on the skin contained T cells which limited DNA synthesis in lymph nodes. This was shown by injecting their cells into normal recipients which were then painted with contact sensitizing agent and measuring DNA synthesis 4 days later in the regional lymph nodes. It was concluded that suppressor B and T cells were an important part of the mechanism of unresponsiveness caused by feeding contact sensitizing agents.     

Prediction of Chemical Respiratory Sensitizers Using GARD,a Novel In Vitro Assay Based on a Genomic Biomarker Signature

Background

Repeated exposure to certain low molecular weight (LMW) chemical compounds may result in development of allergic reactions in the skin or in the respiratory tract. In most cases, a certain LMW compound selectively sensitize the skin, giving rise to allergic contact dermatitis (ACD), or the respiratory tract, giving rise to occupational asthma (OA). To limit occurrence of allergic diseases, efforts are currently being made to develop predictive assays that accurately identify chemicals capable of inducing such reactions. However, while a few promising methods for prediction of skin sensitization have been described, to date no validated method, in vitro or in vivo, exists that is able to accurately classify chemicals as respiratory sensitizers.

Results

Recently, we presented the in vitro based Genomic Allergen Rapid Detection (GARD) assay as a novel testing strategy for classification of skin sensitizing chemicals based on measurement of a genomic biomarker signature. We have expanded the applicability domain of the GARD assay to classify also respiratory sensitizers by identifying a separate biomarker signature containing 389 differentially regulated genes for respiratory sensitizers in comparison to non-respiratory sensitizers. By using an independent data set in combination with supervised machine learning, we validated the assay, showing that the identified genomic biomarker is able to accurately classify respiratory sensitizers.

Conclusions

We have identified a genomic biomarker signature for classification of respiratory sensitizers. Combining this newly identified biomarker signature with our previously identified biomarker signature for classification of skin sensitizers, we have developed a novel in vitro testing strategy with a potent ability to predict both skin and respiratory sensitization in the same sample.     

Graft-versus-host response,measured by splenomegaly assay,by populations of allosensitized mouse lymphocytes

The graft-vs-host (G-v-H) reactivity of sensitized or nonsensitized mouse lymphoid cell populations was measured using a splenomegaly assay. Sensitized populations were obtained either from the local lymph nodes of alloimmunized animals or from the spleens of heavily irradiated mice previously infused iv with allogeneic lymphocytes (educated cells). Immunization of animals resulted in increased G-v-H responses of the cells in their local lymph nodes. This effect was more pronounced when the immunizing cells differed only at non-H-2 transplantation antigens than when H-2-disparate strain combinations were tested. There was no evidence of a changed doseresponse profile of lymphocytes obtained from immunized mice. The G-v-H reactivity of educated cell populations was complex. The slopes of the dose-response lines obtained for lymph node cells or thymic cells educated in an H-2-disparate strain were generally lower than those obtained for nonsensitized cells. This difference was particularly evident when testing educated thymocytes. By studying the G-v-H indices obtained in A/Sn × C57B1 hybrids after inoculation of nonsensitized C57B1 lymph node cells or specifically educated C57BL lymph node cells, it was observed that the latter cells were approximately 30 times more reactive when small cell inocula were compared. On the contrary, education of lymphocytes in H-2-compatible allogeneic hosts did not result in any increment of their G-v-H reactivity. The results indicate that different methods of sensitizing lymphocyte populations against alloantigens may lead to activation of different subclasses of T-cells which differ in their mode of antigen reactivity.     

Local lymph node assay: differentiating allergic and irritant responses using flow cytometry.

The murine local lymph node assay (LLNA) is a method for assessing the contact sensitization potential of chemicals. Based on events that occur during the induction phase of a contact sensitization response, the LLNA measures the in vivo proliferation of cells in the draining lymph nodes (DLNs) of mice following topical exposure to chemicals. In terms of predictive identification of important skin sensitizers, the LLNA has been shown to be at least as sensitive as, and much more reliable than, current guinea pig tests. However, proliferation has also been observed following treatment with some irritants. In an attempt to distinguish allergic from irritant-induced proliferation, flow cytometric techniques have been used to examine the phenotype of lymphocyte subsets in the DLNs as well as markers of T-lymphocyte activation and memory. Mice were treated on the ears for 3 consecutive days with allergens or irritants. The DLNs were harvested 72 h after the final treatment. Single-cell suspensions were prepared, counted, and stained for analysis of the percentages of T cells and B cells and T-cell expression of two adhesion molecules that have been associated with differentiating na?ve and activated/memory T cells, CD62L (L-selectin) and CD44 (H-cam). Increases in lymph node cellularity were observed in both allergen- and irritant-treated mice relative to na?ve and vehicle-treated animals. Mice treated with allergens showed a preferential increase in the percentage of B220(+) B cells compared with irritant-treated mice. Treatment with allergens, but not irritants, resulted in a selective increase in the percentages of CD4(+) and CD8(+) cells expressing the T-cell activation/memory phenotype CD62L(lo)CD44(hi). Taken together, flow cytometric analysis of cell phenotype and expression of T-cell activation/memory markers may provide important information for differentiating allergen- and irritant-induced proliferative responses in the DLNs of chemically treated mice.     

LLNA：BrdU-ELISA 改良法的建立及其在化妆品安全性评价中的应用

目的：建立可快速检测化学物致敏性和刺激性的局部淋巴结试验（ LLNA：BrdU-ELISA）改良法,并对化妆品产品进行评价。方法3种化学物（2,4二硝基氯苯（ DNCB）、丁子香酚、己基肉桂醛）和3种化妆品产品作为受试物,雌性BALB／c小鼠连续染毒3天,测量小鼠耳缘厚度,第5天腹腔注射BrdU,第6天称耳廓重,分离颌下淋巴结称重并制取单细胞悬液,用ELISA试剂盒检测淋巴细胞增殖。结果 DNCB（1.0％）、己基肉桂醛（25％、50％）和3号粉底霜引起耳肿胀和耳廓重显著增加（P ＜0.05或P ＜0.01）,可能为刺激物,其他均无刺激性;3种化学物和3号粉底霜致敏检测阳性,其他均为阴性。结论结合小鼠耳肿胀和耳廓重的LLNA：BrdU-ELISA改良法可较好地评价化学物和化妆品产品的致敏性和刺激性,有望在化妆品安全性评价中发挥重要作用。     

Differences in responsiveness of mouse strain against p-benzoquinone as assessed by non-radioisotopic murine local lymph node assay.

The non-radioisotopic modification of murine local lymph node assay (LLNA) by using 5-bromo-2'-deoxyuridine (BrdU) was conducted to investigate the strain-related difference of the responsiveness of mice to p-benzoquinone (PBQ) with BALB/cAnN, CBA/JN and CD-1 mouse strains. Strain and dose related differences were analyzed by two-way analysis of variance (two-way ANOVA). CBA/JN was considered to be the highest responsive strain to PBQ, and interaction was detected between CD-1 and each of the other inbred strains. These results support the recommendation in the OECD test guideline 429 and the skin sensitization test guideline of US-EPA with regard to the selection of mouse strain for LLNA.     

Primary defect in UVB-induced systemic immunomodulation does not relate to immature or functionally impaired APCs in regional lymph nodes

UVB irradiation of the shaved dorsal skin of mice can cause both local and systemic suppression of contact hypersensitivity responses; the former demonstrated by administration of the sensitizing Ag/hapten to the irradiated site and the latter by its administration at least 72 h later to distal unirradiated sites. The immunological basis of systemic immunomodulation is not clear. When haptens (trinitrochlorobenzene, FITC) were administered to the shaved ventral skin 4 days after irradiation (8 kJ/m(2)) to the shaved dorsum of BALB/c mice, CD11c(+)/FITC(+) cells in the skin-draining lymph nodes from control and irradiated mice produced on a per cell basis similar levels of IL-12 and PGE(2) were phenotypically mature and efficient at presenting FITC to lymphocytes from FITC-sensitized mice. Ag presentation by FACS-sorted CD11c(+) lymph node cells isolated 4 days after UVB irradiation was as efficient as were cells from unirradiated mice at presentation in vitro of an OVA peptide (OVA(323-339)) to CD4(+) cells from OVA-TCR-transgenic DO11.10 mice. Further, IFN-gamma levels were increased in the cultures containing CD11c(+) cells from UVB-irradiated mice, suggesting that inflammation may precede downstream immunosuppression. These results suggest that the primary cause of reduced contact hypersensitivity responses in mice in which UV irradiation and the sensitizing Ag are applied to different sites several days apart must originate from cells other than CD11c(+) APCs that directly or by production of soluble mediators (IL-12, PGE(2)) affect cellular responses in the nodes of UVB-irradiated mice.     

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.     

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz-(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.     

Development of ecotoxicity assay based on inhibition of respiring activity in microbial community using XTT reduction

The aim of this study is to develop ecotoxicity assay for evaluating the influence of chemicals on a microbial ecosystem based on XTT reduction inhibition (XTT assay). XTT reduction method is used for quantification of the microbial respiratory activity. Since the XTT assay indicates the inhibition of microbial respiratory activity, it could evaluate the toxicity of chemicals. Suitable conditions for the XTT assay were determined to be 200 mg/L of particulate organic carbon as test microbe concentration and 15 min of assay time using activated sludge. Toxicities of several chemicals evaluated by activated sludge as test microbes were examined under these conditions. Sensitivity for the toxicity evaluated by the XTT assay using activated sludge microbes was almost the same value was that for the OECD activated sludge respiration inhibition test (ASRI test). XTT assay was also applied for evaluating the influence of chemicals on the soil microbial community and the XTT assay was used to evaluate a median effective concentration (EC(50)) value of 3,5-dichlorophenol (3,5-DCP). The EC(50) value of 3,5-DCP was almost the same as the value using activated sludge as test microbes. These results suggest that the XTT assay using both mixed cultures of non-contaminated environments and chemical extracts from various contaminated environments could evaluate the influence on microbial ecosystems affected by toxic chemicals.     

1-Chloromethylpyrene: a reference skin sensitizer and genotoxin

1-Chloromethylpyrene (1-CMP) has been evaluated as a model mutagen and toxin related to the ultimate electrophiles derived from benzo[a]pyrene and 1-nitropyrene. It was mutagenic to Salmonella (greater than 100 pg/plate) and exceptionally reactive to DNA when assessed by the 32P-postlabelling technique. 1-CMP was inactive in a mouse bone micronucleus assay when administered by gavage, probably due to hydrolysis, whose kinetics have been studied (t1/2 approximately 23 min at 37 degrees C). However, as expected, it was a potent skin toxin as determined by its activity as a mitogen to mouse skin and its contact allergenicity, as determined using the local lymph node proliferative assay. It is concluded that 1-CMP will probably be a potent human skin carcinogen and contact allergen.