Novel Testing Strategies for In Vitro Assessment of Allergens

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Skin Sensitization

Novel Testing Strategies for In Vitro Assessment of Allergens

Erwin L. Roggen, (Novozymes A/S) for the Sens-it-iv Consortium

Published: June 25, 2010

About the Author(s)
Erwin Roggen took a degree in Biochemistry at the University of Antwerp (1984). Over time, he expanded his scientific expertise into the areas of protein chemistry, molecular biology, immunology and cell biology, and he acquired management skills as Project Manager of an international research project funded by the Swedish Agency for Research aiming at understanding better the interaction between HIV and other sexually transmitted diseases.

As science manager for In Vitro (Immuno)Toxicology, coordinator of the FP6 funded project Sens-it-iv, and board member of the Carcinogenomics project, Dr. Roggen acquired extensive expertise in the fields of in vitro/in silico immunology, (immuno-)toxicology, and risk assessment and management.

Throughout the years, Dr. Roggen has actively pursued the development, implementation and acceptance by regulatory bodies of the 3Rs (Refinement, Reduction and Replacement of Animal Experimentation) as chairman of the task force on respiratory sensitisation and  member of the task force on skin sensitisation (ECVAM), as member of the ECVAM Scientific Advisory Board (ESAC), co-chair of the EPAA Platform on 3Rs in Regulation, active member of the EPAA Platforms on Science, and Communication and Dissemination, president of IVTIP, and as board member of the Dutch Center for Toxicogenomics and the Dutch ‘Assuring Safety without Animal Testingi initiative. He is also member of the editorial board of Toxicology In Vitro and Frontiers in Pharmacology (In Vitro Toxicology), and invited reviewer for Toxicological Sciences, among others.

Dr. Erwin L. Roggen
Novozymes A/S
Krogshoejvej 36
DK-2880 Bagsvaerd
Denmark
Email: elro@novozymes.com

Summary description of the objectives of the Sens-it-iv project

As yet, risk assessment for potential skin- or lung-sensitizers completely depends on animal testing. The overall objective of Sens-it-iv is to produce in vitro alternatives for these assays, and to develop them up to the level of pre-validation. Besides reducing animal experimentation, an increase in the accuracy of predicting sensitising potencies is expected.

In vitro mechanisms relevant for in vivo sensitisation will be identified at the level of human lung and skin epithelial cells (EC), dendritic cells (DC) and T-cells.

These efforts pursue specific scientific (S) and technologic (T) objectives:

  1. Existing data on sensitising, irritating and toxic compounds are collected and stored in a compound database (S).
  2. In vivochanges induced by selected compounds in specified cell types are described using functional genomics (S).
  3. The impact of compounds on individual cells, and the interaction between these cells, is assessed in vitro(S).
  4. The physico-chemical properties of chemicals responsible for metabolic activation and hapten-formation are determined (S).
  5. The data are collected in an Inductive Database allowing queries for data patterns and predictive models (T).
  6. Mechanisms specifically involved in skin and respiratory sensitization are identified using bio-informatics (S).
  7. The information is used to adapt/improve existing techniques, and to develop organotypic models derived from human cells assays (T).
  8. A proof of principle is established on a set of skin and respiratory sensitizers, irritants and toxic compounds (T).

Consistency with existing and ongoing projects and optimal exploitation of these achievements are ensured by the involvement of European Centre for the Validation of Alternative Methods, COLIPA (the European cosmetics association), the European Consensus-Platform for Alternatives, In Vitro Testing Industrial Platform and the Organisation for Economic Cooperation and Development, in various levels of the project.

Overall status

During it’s third year (01.10.2007 – 30.09.2008) Sens-it-iv expanded the knowledge about human cells and cell lines, and their responses to selected compounds to the point that a first set of test systems could be transferred to the technology module (Work Package [WP] 8) for further development. During the 4th year (01.10.2008 – 30.09.2009) Basic Research was limited to a few specific issues, while the major focus was on the evaluation and refinement of the selected test systems in the technology module (Applied Research). In addition, a concentrated effort was initiated to identify novel markers for sensitization. The progress of each of these efforts is summarized below.

Science module: Achievements of the remaining activities

 

Identification of the proper test compounds.

The tutorial list of skin sensitizing chemicals was expanded in view of the needs of WP8. As yet, 50 chemicals and 20 proteins are available for test evaluation and refinement. General guidelines for dissolving chemicals were developed for the new compounds. Moreover, specific guidelines for dissolving chemicals were produced.

Describing the in vivo phenotype of epithelial cells (EC), dendritic cells (DC) and T-cells.

The Precision Cut Lung Slice (PCLS) technology has been shown to be a useful tool for studying events occurring in vivo during sensitization in an ex vivo context. Studies of in situ cytokine profiles of lung EC, DC and T-cells before and after contact with the learning set of compounds revealed promising differences in cytokine regulation for e.g. TNF and IL-1 between sensitizers and irritants. Our understanding of the key events in respiratory sensitization is growing and new markers are emerging. The availability of human PCLS is essential for assessing the relevance of in vitro identified markers, pathways and networks relevant for sensitization of humans.

Finding the most in vivo-like EC and EC markers.

A potential break-through was realized in the area of in vitro respiratory sensitization. A promising human primary bronchial cell and a human alveolar cell line-based test format were identified. Both tests are currently under evaluation using the learning set of chemicals.

Finding biomarkers for sensitization applicable in DC-based assays.

The understanding of mechanisms related to sensitization and occurring in DC (MUTZ-3 and moDC) upon stimulation with sensitisers is rapidly growing. Implementation of genomics, proteomics and CD array technology has resulted in the identification of potential novel markers for discriminating sensitisers and non-sensitisers.

Finding the in vitro conditions supporting the most in vivo-like EC-DC interactions.

A significant break-through was made for the keratinocytes (KCs)-DC co-culture. A reconstructed human epidermis model containing CD34-derived langerhans cells (LCs) (RHE-LC) responded to sensitizers by induction of LC-specific genes. Sensitizer-specific response patterns were observed. It is therefore an interesting and valuable functional research model for studying case by case the effect of chemicals on a LC/KC 3D co-culture model.

Assessing the sensitization potential of chemicals using T-cells.

Using previously developed methods, WP3 has made tremendous progress in the understanding of T-cell mediated processes involved in sensitization. Furthermore, 3 T-cell based assays were developed: a) priming of naive chemical/drug-specific T-cells, b) amplification of precursor T-cells specific for sensitizers and c) using the amplification of memory T-cells from sensitized patients.

Finding novel DC markers involved in chemical sensitization.

Extensive genomic analysis of the DC-line MUTZ-3 before and after exposure to chemicals has identified a genomic profile predicting skin sensitizing chemicals with 99.9% accuracy.

Finding proteomic profiles for chemical sensitization.

Extensive proteomic analysis of human KCs, MUTZ-3 cells and lung cells is currently ongoing. In addition, a novel proteomic approach was established to search for chemical-specific (allergen versus non-allergen) proteomic profiles further supporting biomarker development. Preliminary data for KCs suggested the existence of sensitizer-specific protein profiles.

Understanding the role of metabolism in chemical sensitization.

Our understanding of the fate of chemicals when brought in contact with a cell culture was considerably increased. Binding of haptens to extra- and intracellular proteins was analysed. It was shown that the nature or type of target protein rather than the number of haptenated proteins is the key determinant in the triggering of DC maturation, with a possible implication of cysteine binding. Preliminary experiments showed the involvement of the NRF2 pathway in DCs. These findings are related to depletion of glutathione and cellular viability. Furthermore, the expression of metabolizing enzymes and importance of efflux transporter expression and activity was demonstrated. It was observed that the DC-line THP-1 nor the KC-line NCTC2544 express detectable levels of enzymes involved in xenobiotic metabolism. A transwell system based on neutrophils and THP-1 cells for simultaneous investigation of the chemistry and immunological readouts was developed.

Technology module: Achievements
Managing the data

The tools allowing for data storage and retrieval were provided, thereby facilitating analysis of the huge amount of data produced by this project. A special emphasis has been put in on implementing data submission, data analysis and report generation tools.

In vitro assay development and refinement

A: Comparison of 3DC maturation based assay – a prevalidation exercise
An inter-laboratory collaboration was initiated with ECVAM in an advisory function. Addressing documentation, Standard Operating Procedures, and standardisation has been a useful learning process for the partners. Inter-laboratory agreements resulted in an excellent intra-and inter- laboratory reproducibility. This success is promising for the future activities related to the preparation of selected tests for pre-validation by ECVAM.

Cells, mRNA and supernatants derived from the MUTZ-3, U937, and THP1 cultures before and after stimulation with compounds were stored frozen. Thus, novel biomarkers identified can and will be assessed on the stored samples to investigate their potential as markers in the DC based assay.

B: Identification of skin sensitizers using the KC-line NCTC2544 with IL-18 expression as read-out
It has been established that human keratinocytes responded to sensitizers by increased production of IL-18. Determination of intracellular IL-18 levels in the human keratinocyte cell line NCTC 2544 identified correctly all skin sensitizers of the Sens-it-iv tutorial chemicals (including the pro-haptens eugenol, isoeugenol, cinnamic alcohol and paraphenylenediamine). Irritants and to respiratory sensitizers were not capable of inducing changes in IL-18 levels.

An inter-laboratory study assessing the ability of this test to distinguish sensitizers from non-sensitizers and respiratory sensitizers was initiated.

C: The EE potency assay
This assay mimicks the penetration of the stratum corneum and general irritation of epidermal cells. The great advantage of this model is that chemicals, independent of their solubility in water, may be topically applied on the very same type of filter discs used in human patch testing. The readout is the concentration leading to 50% reduction of metabolic activity. This assay cannot differentiate irritants from allergens. However, when selectively applied to sensitizers identified by any of the other assays below, the obtained EC50 values correlate well with EC3 data derived in vivo by LLNA testing allowing a classification into strong, intermediate and weak sensitizers. Interestingly, none of the respiratory sensitizers tested so far was active in this assay.

D: The DC migration assay
To simulate hapten-induced migration of LCs from the epidermis to the dermis, a dual chamber experiment was designed. This migration depends on 2 fibroblast-derived chemokins, i.e. CXCL12 and CCL5. Pre-treatment of fluorescently labelled (CSFE) MUTZ-LC (upper compartment) with sensitizers, but not with irritants, induces the expression of a CXCL12 receptor and, hence, enhances migration towards CXCL12 (lower compartment).

For every chemical the index of migration directed towards CXCL12 vs. that directed towards CCL5 can be determined. An index of CXCL12:CCL5 >1, therefore, indicates sensitizers, values

E: The T cell priming assay
The in vitro T cell priming assay employs human DCs and sorted naïve T cells depleted of the CD25+ regulatory subpopulation of the same donor. When in co-culture, chemically activated DCs drive hapten-specific T cells into proliferation and differentiation to produce interleukins such as interferon gamma (IFN-gamma). Combination with a polyclonal T cell amplification step before the addition of specific allergen significantly increase the sensitivity of the system and allows for frequency determination of T cells of extremely rare specificities as well as for isolation and fuctional/genetical analysis of allergen-specific T cell clones.

©2010 Erwin Roggen