In Vitro Testing for Direct Immunotoxicity: State of the Art

Abstract

Immunotoxicity is defined as the toxicological effects of xenobiotics including pharmaceuticals on the functioning of the immune system and can be induced in either direct or indirect ways. Direct immunotoxicity is caused by the effects of chemicals on the immune system, leading to immunosuppression and subsequently to reduced resistance to infectious diseases or certain forms of nongenotoxic carcinogenicity.In vitro testing has several advantages over in vivo testing, such as detailed mechanistic understanding, species extrapolation (parallelogram approach), and reduction, refinement, and replacement of animal experiments. In vitro testing for direct immunotoxicity can be done in a two-tiered approach, the first tier measuring myelotoxicity. If this type of toxicity is apparent, the compound can be designated immunotoxic. If not, the compound is tested for lymphotoxicity (second tier). Several in vitro assays for lymphotoxicity exist, each comprising specific functions of the immune system (cytokine production, cell proliferation, cytotoxic T-cell activity, natural killer cell activity, antibody production, and dendritic cell maturation). A brief description of each assay is provided. Only one assay, the human whole blood cytokine release assay, has undergone formal prevalidation, while another one, the lymphocyte proliferation assay, is progressing towards that phase.Progress in in vitro testing for direct immunotoxicity includes prevalidation of existing assays and selection of the assay (or combination of assays) that performs best. To avoid inter-species extrapolation, assays should preferably use human cells. Furthermore, the use of whole blood has the advantage of comprising multiple cell types in their natural proportion and environment. The so-called "omics" techniques provide additional mechanistic understanding and hold promise for the characterization of classes of compounds and prediction of specific toxic effects. Technical innovations such as high-content screening and high-throughput analysis will greatly expand the opportunities for in vitro testing.

A roadmap for the development of alternative (non-animal) methods for systemic toxicity testing

“…The same holds true especially for immunosuppression (Carfi et al, 2007;Galbiati et al, 2010;Gennari et al, 2005;Langezaal et al, 2001;Lankveld et al, 2010) but also for chronic cell injury, increased secretion of trophic hormones, oxidative stress, and reactive oxygen and nitrogen species (ROS and RNS). An assay for CYP450 induction in cryopreserved human hepatocytes also is currently under prevalidation (Abadie-Viollon et al, 2010;Richert et al, 2010).…”

Immunotoxicity, genotoxicity and epigenetic toxicity of nanomaterials: New strategies for toxicity testing?

“…Moreover, the respiratory burst of phagocytes can be measured (using hydroxyethidine) in the same tube. Natural killer cell activity is a reliable method for monitoring tumor defence and has been recommended for assessment of immunotoxic potential (Lankveld et al, 2010). Cytolytic function of NK-cells can be measured by either radiolabel release or flow cytometry.…”

Antimicrobial Models in Nanotechnology

“…It is necessary to control the factors that can affect the in vitro assays used for analyzing the effects of NPs on immune cells and immune responses because variable levels of endotoxin are common contaminants of glassware, culture media, and additives and can activate monocytes/macrophages, altering the results (Peer, 2012). The choice of the biological assay is also of central importance; representative in vitro cellular assays should be based on human blood leukocytes (such as monocytes), which are the first cells to come in contact with the injected NMs and have biological endpoints representing the early innate/inflammatory-type responses (Lankveld et al, 2010). Primary cells should be the first choice in this kind of assay, because the primary response to stimulation is more sensitive than continuous cell lines and is qualitatively different.…”