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Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of REBIRTH Cluster of Excellence

1.When the immune system encounters incoming infectious agents, this generally leads to immunity. The evoked immune response is usually robust, but can be severely perturbed by potentially harmful environmental agents such as chemicals, pharmaceuticals and allergens. 2.Immunosuppression, hypersensitivity and autoimmunity may occur due to changed immune activity. Evaluation of the immunotoxic potency of agents as part of risk assessment is currently established in vivo with animal models and in vitro with cell lines or primary cells. 3.Although in vivo testing is usually the most relevant situation for many agents, more and more in vitro models are being developed for assessment of immunotoxicity. In this context, hypersensitivity and immunosuppression are considered to be a primary focus for developing in vitro methods. Three-dimensional organotypic tissue models are also part of current research in immunotoxicology. 4.In recent years, there has been a revival of interest in organotypic tissue models. In the context of immunotoxicity testing, precision-cut lung slices in particular have been intensively studied. Therefore, this review is very much focused on pulmonary immunotoxicology. Respiratory hypersensitivity and inflammation are further highlighted aspects of this review. Immunotoxicity assessment currently is of limited use in other tissue models, which are therefore described only briefly within this review.

Assessment of immunotoxicity using precision-cut tissue slices.

Occupational asthma can be induced by a number of chemicals at the workplace. Risk assessment of potential sensitizers is mostly performed in animal experiments. With increasing public demand for alternative methods, human precision-cut lung slices (PCLS) have been developed as an ex vivo model. Human PCLS were exposed to increasing concentrations of 20 industrial chemicals including 4 respiratory allergens, 11 contact allergens, and 5 non-sensitizing irritants. Local respiratory irritation was characterized and expressed as 75% (EC25) and 50% (EC50) cell viability with respect to controls. Dose-response curves of all chemicals except for phenol were generated. Local respiratory inflammation was quantified by measuring the production of cytokines and chemokines. TNF-α and IL-1α were increased significantly in human PCLS after exposure to the respiratory sensitizers trimellitic anhydride (TMA) and ammonium hexachloroplatinate (HClPt) at subtoxic concentrations, while contact sensitizers and non-sensitizing irritants failed to induce the release of these cytokines to the same extent. Interestingly, significant increases in T(H)1/T(H)2 cytokines could be detected only after exposure to HClPt at a subtoxic concentration. In conclusion, allergen-induced cytokines were observed but not considered as biomarkers for the differentiation between respiratory and contact sensitizers. Our preliminary results show an ex vivo model which might be used for prediction of chemical-induced toxicity, but is due to its complex three-dimensional structure not applicable for a simple screening of functional and behavior changes of certain cell populations such as dendritic cells and T-cells in response to allergens.

Assessment of immunotoxicity induced by chemicals in human precision-cut lung slices (PCLS).

One of the most insidious characteristics of cancer is its spread to and ability to compromise distant organs via the complex process of metastasis. Communication between cancer cells and organ-resident cells via cytokines/chemokines and direct cell-cell contacts are key steps for survival, proliferation and invasion of metastasized cancer cells in organs. Precision-cut liver slices (PCLS) are considered to closely reflect the in vivo situation and are potentially useful for studying the interaction of cancer cells with liver-resident cells as well as being a potentially useful tool for screening anti-cancer reagents. Application of the PCLS technique in the field of cancer research however, has not yet been well developed.

Murine precision-cut liver slices (PCLS): a new tool for studying tumor microenvironments and cell signaling ex vivo.

In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (μM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results.

Katherina Sewald

Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM),
German Center for Lung Research (DZL),
Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), German Center for Lung Research (DZL)

Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of REBIRTH Cluster of Excellence

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Katherina Sewald

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM),German Center for Lung Research (DZL),Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), German Center for Lung Research (DZL)

Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of REBIRTH Cluster of Excellence

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM),
German Center for Lung Research (DZL),
Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), German Center for Lung Research (DZL)