Sens-it-iv: A Pathway Towards In Vitro Assessment of the Sensitising Potency of Proteins and Chemicals

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Skin Sensitization

Sens-it-iv: A Pathway Towards In Vitro Assessment of the Sensitising Potency of Proteins and Chemicals

Erwin L. Roggen, Novozymes A/S

Published: January 24, 2008

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

Introduction and aims

As yet, risk assessment for potential skin- or lung-sensitizers completely depends on animal testing.  As a direct consequence, the additional testing for allergenicity required by the EU-legislation on chemicals (REACH) would require a large number of animals even though Art. 1 states that “The purpose of this Regulation is to ensure a high level of protection of human health and the environment, including the promotion of alternative methods for assessment of hazards of substances,…”. Clearly this is inconsistent in the light of the European Directive 86/609/EEC aimed at a reduction of animal testing. In addition, the 7th Amendment to the Cosmetics Directive (Directive 76/768/EEC2) will ban all animal testing for cosmetic ingredients by 2009, including testing for skin sensitisation. A ban for finished cosmetic products is already in place. Against this background there is a clear need for researchers to focus on developing alternative methods that will eliminate the need for animals in safety 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. The Sens-it-iv Consortium comprises 28 research groups overall, of which 7 are from industry, 16 groups are universities or research institutes, and 4 represent organizations. The Consortium is coordinated by Novozymes (DK) [Dr. Erwin L Roggen, Coordinator,] and Max-Planck-Institut für Immunbiologie (D) [Prof. Hans-Ulrich Weltzien, Vice-coordinator,].

The scientific approach of the Sens-it-iv project is to build on and exploit our increasing understanding of the mechanisms involved in sensitisation and allergy development. This involves an improved appreciation of the biological processes that occur when tissue is exposed to sensitising materials and to compare this with molecular indicators on the cells involved in these reactions. This work is carried out in Workpackages, which are grouped in a Science Module. The knowledge generated in the Science Module will be used in a Technology Module to develop (mostly cell-based) assay systems that model sensitisation, rather than irritation and toxicity of chemicals and proteins.

Science module – achievements

Already during the first year (01.10.2005 – 30.09.2006) Sens-it-iv made significant contributions to our knowledge about human cells and cell-lines, and our understanding of the responses of these cells to selected compounds. This knowledge was further expanded during the 2nd reporting period.

Identification of the proper test compounds.

Above all, in vitro assay development requires a set of well characterized compounds. The first selected set of compounds comprises 14 chemicals including positive compounds either as skin or respiratory sensitisers, negative controls, 5 pre-pro-haptens1 and 4 proteins. Moreover Sens-it-iv, ECVAM and Colipa agreed upon a common larger list of reference chemicals for the assessment of newly developed in vitro methods for skin sensitization only.

A database was also established in the Sens-it-iv internal website with all chemicals of the mouse local lymphnode assay (LLNA) database (N=211) supplemented with a selection of respiratory sensitizers (chemicals and proteins) (N=10).

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

Previously, the technical challenges related to the implementation of the precision cut lung slices (PCLS) technology in the field of sensitization was assessed on lung slices obtained from mice. The resulting standard operating procedure (SOP) was refined for implementation on human lung. The availability of human PCLS is essential for assessing the relevance of in vitro identified markers, pathways and networks for sensitization of humans.

Ongoing studies of in situ cytokine profiles of lung EC, DC and T-cells in mouse PCLS before and after contact with the learning set of compounds revealed promising differences in cytokine regulation between sensitizers and irritants. As compared to cytokine release, the induction of surface expression of activation markers or co-stimulatory molecules such as ICAM-1, MHC class II or CD86 was less effective and in some cases required the presence of allergens specific T cells.

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

The catalogue of available primary lung EC (N=5) and lung EC lines (N=14) was completed in collaboration with the EU project Carcinogenomics. Similarly, expansion of the catalogue with factors affecting in vitro cell phenotype and functionality was finalized.

Based upon the available information it was decided to continue studies on lung sensitizers with the human bronchial Calu-3 cell line. This decision was based upon its availability and its capability to form tight and polarized monolayers. These characteristics are considered very relevant for development of immuno-competent 3D test systems.

The catalogue of markers responding to exposure of EC to sensitizers, irritants and control substances was expanded. Very promising new leads were identified in the MAPK p38 and ERK1/2 mediated signaling pathways. Among the cytokines, IL-8 secretion appeared to be the most indicative marker in Calu-3 cells. For primary keratinocytes and the NCTC 2544 cell line, IL-1α and IL-18 appeared as potential candidates, but as yet responses are not robust enough to identify allergens. The Sens-it-iv epidermal equivalent was employed to study effects of the training compounds on cytokine secretion, but so far the tests did not discriminate between sensitizers and irritants.

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

As for the EC, gene expression profiles of blood, tonsillar and skin DC populations (N= 9), precursor-derived DC (N=6) and DC lines (N=3) were catalogued. As yet, the DC line showing the most in vivo-like phenotype and functionality is the MUTZ-3 cell line. The various differentiation states of MUTZ-3 cells appear very promising in resembling different DC/LC states in vivo.

Expression of CD-markers on MUTZ-3 cells upon treatment with chemicals was tested with CD-arrays containing 140 CD markers. Several markers of potential relevance for sensitization were identified and are to be further analysed by FACS. Genomic, FACS and ELISA determinations of MUTZ-3 responses to chemicals so far indicate a combination of IL-8 and CD86 as the most promising indicators for skin sensitizers.

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

Principally three different systems were used to address EC-DC cross-talk. A co-culture in a 3D reconstituted epidermis model so far gave no indication for sensitizer-specific signals on MUTZ-3/MUTZ-3-LC. A co-culture in the Sens-it-iv epidermal equivalent model revealed specific upregulation of CD80/CD86 on MUTZ-imLC by the contact sensitizer NiSO4. Co-cultures of 2 cell types in an airlifted two-layer system were established employing EC (cell lines A549, Calu-3 or NCTC 2544, or primary keratinocytes) and DC (cell lines THP-1 or MUTZ-3, or blood-derived DC).

While most of these studies still are in a very experimental phase, their was evidence that THP-1 cells in the presence, not in the absence, of keratinocytes respond to pro-haptens by upregulation of IL-8 and CD86.

Establishing and implementing tools for addressing DC – T-cell interactions.

A serious obstacle in studying the role of T-cells in the DC – T-cell interplay is the low frequency of allergen-specific T-cells. An effective system was established to expand the absolute number of antigen-specific T-cells from the naïve T-cell pool of peripheral blood samples. The system was successfully applied to determine T-cell frequencies for several allergens in human blood samples. In different attempts magnetic isolation of peptide-allergen specific T-cells by use of tetrameric MHC/peptide complexes showed promising results.

Neither sensitizers nor stromal cells alone induce homing receptors on T-cells. However, hapten stimulation in the presence of stromal cells, depending on their origin, induces either skin or gut homing receptors in murine systems.

The observation that the activation of Toll like receptors (TLR) on DC via specific ligands alter their reactivity towards the contact sensitizers indicates a role of innate immune responses in allergy development. In addition, TLR7 specific ligands were found to functionally redirect lung homing Der p1 specific T-cells from Th2 towards Th1. These findings may lead to the detection of new marker molecules to discriminate sensitizers from non-sensitizers.

Addressing the impact of sensitizers on cellular protein profiles.

Preliminary data on effects of exposure of cells or proteins to sensitizers were obtained. These results were compared to data obtained after exposure to irritant, solvent or culture medium. Proteome analyses in primary keratinocytes and monocyte-derived DC resulted in the identification of numerous, partially cell-specific hapten-binders.

Understanding the role of metabolism in chemical sensitization.

A combined bio-analytical and cellular immunological platform was established to determine how chemicals interact with immune cells (both antigen presenting cells and T-cells). Initial studies focused on the ability of the pro-hapten p-phenylenediamine (PPD) to stimulate T-cells. Lymphocytes from allergic patients were found to proliferate with PPD and the oxidation product Bandrowski’s base, while lymphocytes from non-allergic individuals proliferated only with Bandrowski’s base. Cord blood lymphocytes failed to respond.

Utilizing the THP-1 cell line and DNCB, DNFB, DNBrB and DNIB as model test compounds, a matrix of assays was developed to relate metabolism, compound distribution and covalent binding to transcription factor regulation and immune cell activation.

To explore the association between protein-adduct formation and stimulation of polarized immune responses, synthetic strategies to introduce 13C labels on the reactive positions of respiratory allergens were devised. Investigations exploring the reactivity of these compounds toward amino acids and peptides suggest differences between skin and respiratory sensitizers.
1Pre- and pro-haptens are chemicals that require activation (typically by spontaneous or enzyme mediated oxido-reduction reactions) resulting in a hapten.
©2008 Erwin Roggen