The Way Forward: Applying Non-Animal Approaches to the Assessment of Immunotoxicity

By Emanuela Corsini, University of Milan

The immune system has evolved to protect the host against invasive microorganisms such as bacteria and viruses as well as malignant cells. Immune responses are the result of an effective collaboration between innate (natural and relatively nonspecific) and acquired (adaptive, and extremely specific) components of the immune system. Epidemiological evidence in Western countries indicates that the prevalence of diseases associated with alterations of the immune response, such as asthma, certain autoimmune diseases, and cancer are increasing to such an extent that it cannot be attributed to improved diagnostics alone. There is some concern that this trend could be at least partially attributable to new and modified patterns of exposure to chemicals.

Immunotoxicology studies the effects of xenobiotics on the immune system. An immunotoxic compound can be defined as a compound that can alter one or more immune functions resulting in an adverse effect for the host.

In particular, two main immunotoxic effects can be identified:

1. decreased immunocompetence (immunosuppression), which may result in repeated, more severe, or prolonged infections as well as the development of cancer;
2. immunoenhancement, which, as an adverse effect, may lead to immune-mediated diseases, such as hypersensitivity reactions and autoimmune diseases. Hypersensitivity reactions are the result of normally beneficial immune responses acting inappropriately, causing inflammatory reactions and tissue damage. The two most frequent manifestation of chemical-induced allergy are contact hypersensitivity and respiratory sensitization, both of which can have serious impact on quality of life and represent a common occupational health problem. Hypersensitivity reactions are often considered to have increased at such a rate as to become a major health problem in relation to environmental chemical exposure.

At least two specific properties make the immune system vulnerable to chemical or physical insults: 1) the immune system develops rather late in life (thymus development lasts at least until puberty) and immune components are continuously renewed (e.g., granulocytes), and 2) each pathogen attack, as well as immune surveillance, demands delicate control of the balance between activation, silencing, and regulation of immune reactivity.

The immune system can be the target of many chemicals, including environmental contaminants and drugs, with potentially adverse effects on the host's health. Due to the important health consequences of an immunotoxic effect, which are underscored by clinical data that unequivocally demonstrate that immunotoxicity is associated with significant morbidity and even mortality (1), immunotoxicity is of considerable importance to the toxicologist who has the responsibility of identifying and characterizing the immunotoxic potential of chemicals and estimating the risk they pose to human health.

At present, assessment of immunotoxic effects relies on different animal models, and several assays have been proposed to characterize immunosuppression and sensitization. Current available animal models and assays are not valid to assess the potential for systemic hypersensitivity and, at this time, autoimmunity is 'not predictable' at all, with the reporter antigen-popliteal lymph node assay (RA-PLNA) showing strong promise (2). The use of whole animals, however, presents many secondary issues, such as expense, ethical concerns, and eventual relevance to risk assessment for humans. Furthermore, due to the new policy on chemicals (REACH) and the 7th Amendment on the Cosmetic Legislation in the European Union, together with the Toxicity Testing in the 21st Century envisaged by the U.S. National Academy of Sciences, in vitro methods will play a major role in the near future (3).

Hypersensitivity and immunosuppression, for which animal models have been developed and validated, are considered the primary focus for developing in vitro methods in immunotoxicology. Nevertheless, in vitro assays, as well as in vivo models, to detect immunostimulation and autoimmunity are also needed.

For comprehensive reviews of the state-of-the-art in the field of in vitro immunotoxicity, including sensitization, readers should refer to recent reviews (4, 5). Here I provide a brief summary of possibilities to identify sensitizers and immunosuppressive agents.

In Vitro Opportunities to Identify Sensitizers

The evaluation of contact sensitization by a drug, cosmetic material, pesticide or industrial chemical is usually done using the local lymph node assay (LLNA) as described in the OECD guideline 429, and the new test guideline using non-radioactive endpoints (OECD 442A and 442B). The LLNA is a murine model developed to evaluate the skin sensitization potential of chemicals (6). The LLNA is a well-established alternative approach to traditional guinea pig methods (OECD 406) and, in comparison, provides important animal welfare benefits. The assay relies on measures of events during the induction phase of skin sensitization - specifically lymphocyte proliferation in the draining lymph nodes - as a hallmark of a skin sensitization response.

In the field of in vitro toxicology, sensitization still remains a challenge, and no validated tests are currently available. However, in the last decade important progress has resulted in the development of alternative test methods that could make a valuable contribution to the replacement of the existing animal models. In the following table the key events of contact sensitization and in vitro opportunities are described.

Table 1. Key events in chemicals-induced skin sensitization and in vitro opportunities.

At present, three non-animal test methods, namely the Direct Peptide Reactivity Assay (DPRA), the Myeloid U937 Skin Sensitisation Test (MUSST) and the human Cell Line Activation Test (hCLAT) are under formal validation at the European Centre for the Validation of Alternative Methods (ECVAM) for their potential to predict skin sensitization potential. Results are expected at the end of 2011. It has been anticipated, however, that it will take at least another 7-9 years for the full replacement of the in vivo animal models currently used to assess sensitization (23). Nevertheless, the above-mentioned in vitro methods can be used for hazard identification, i.e. to discriminate between sensitizers and non-sensitizers, ahead of these dates. Furthermore, it is important to note that some of these in vitro methods can also be used for potency classification (24, 25, 26).

It is desirable in the future to be able to shift the focus from hazard identification toward risk assessment - enabling better health protection. A first indication of potency may come, for example, from the concentration required to induce a threshold of positive response (CD86 ≥150) in the h-CLAT system. A good correlation (R = 0.839, p < 0.01) was indeed found between the h-CLAT thresholds and LLNA EC3 values (24). The quantitative dose-response data should be then integrated into a testing strategy along with the peptide reactivity data, bioavailability data, and some informed rating of structural alerts in order to establish an acceptable exposure level. Further studies testing a broader range of chemicals are, however, necessary.

In vitro opportunities to indentify immunosuppressive agents

Due to the complexity of the immune system, it is generally assumed that it would be very difficult to reproduce it in vitro. To a larger extend, in vitro systems do not take into account the interactions of the different cellular and soluble components involved in the immune response, or the potential for neuro-immuno-endocrine interactions. Therefore, an assessment of in vitro immunotoxicity will be valuable only in the cases of a direct immunotoxicant (27). To this purpose, several isolated processes can be studied in vitro such as lymphocyte proliferation, cytokine production, phagocytosis, lytic functions, and even primary antibody production.

Before starting with in vitro tests, bioavailability should be considered. If the compound does not have appreciable bioavailability, immunotoxicity is unlikely to occur. As a general strategy, in vitro testing for direct immunotoxicity should be done in a tiered approach (27), the first tier measuring myelotoxicity. Compounds that are capable of damaging or destroying the bone marrow will often have a profound immunotoxic effect, since the effectors of the immune system itself will no longer be available. Therefore, if a compound is myelotoxic, the material will be a de facto immunotoxicant. The methodology for evaluating myelotoxicity in vitro using bone marrow culture systems is well characterized and scientifically validated (28, 29, 30).

Compounds that are not overtly myelotoxic may still selectively damage or destroy lymphocytes, which are the primary effectors and regulators of acquired immunity. Compounds are therefore tested for lymphotoxicity (second tier). This toxicity may result from the destruction of rapidly dividing cells by necrosis or apoptosis; alternatively, chemicals may interfere with cell activation affecting signal transduction pathways. A variety of methods are available for assessing cell viability (e.g., colorimetric, flow cytometric assays). After myelotoxicity and overt cytotoxicity are excluded as endpoints, basic immune cell functionality should then be assessed by performing specific functional assays, i.e., proliferative responses, cytokine production, NK cell activity, etc. (third tier), using non-cytotoxic concentrations of the tested chemicals (viability > 80%).

As a general consideration regarding the choice of the cellular models, it is recommended that human cells be used for all in vitro test systems, to maximize human relevance. Although the use of primary human cells will be of the highest clinical relevance, consideration may eventually be given to the use of sufficiently well-characterized and validated cell lines (human or animal) for certain aspects of the test systems (27).

In Table 2 relevant immune components and opportunities for in vitro assessment of immunotoxicity (immunosuppression) are described.

Table 2. Key targets in chemical-induced immunosuppression and in vitro opportunities

The in vitro system, named "fluorescent cell chip", is based on a number of cell lines derived from T-lymphocytes, mast cells, monocytes, each transfected with various cytokine reporter cell constructs for measuring cytokine expression (35). Although further refinement of this system is required, this assay holds promises for in vitro screening of chemicals for their immunotoxicity.

The human whole blood cell culture, introduced more than 20 years ago, may also be useful in studying the biological effects of potential immunomodulatory chemicals based on immune cell activation and cytokine secretion. Whole blood assays can be very useful tests due to the easy access of samples from healthy donors and the minimal processing of the sample required. Both plant lectins (e.g., PHA, ConA, pokeweed mitogen [PWM], etc.) as well as LPS (a purified protein derivative [PPD] of tuberculin), anti-CD3, and/or anti-CD28 antibodies, etc. can be used to stimulate T- or B-lymphocyte or monocyte functions in whole blood (36, 37).

Cytokine production together with lymphocyte proliferation are currently in a pre-validation phase (33). Based on results of two previous studies, the human T cell activation assay was selected as the most promising of the investigated in vitro immunotoxicity test. This assay is based on CD3/CD28-mediated T cell activation using proliferation and cytokine release (TNF-α and IFN-γ) as read-out parameters. To pre-validate the human T cell activation assay, 20 compounds were selected, of which 10 were immunosuppressive and 10 non-immunosuppressive. Statistical analyses revealed that the human T cell activation test had a 'sensitivity' (correct prediction of immunosuppressive chemicals) of 76% and a 'specificity' (correct prediction of non-immunosuppressive chemicals) of 83% (manuscript in preparation). The human T cell activation assay may be a promising candidate for in vitro evaluation of immunosuppressive activity.

Immunotoxicogenomics represents a novel approach to investigate immunotoxicity. Hochstenbach et al. (38) have recently reported the possibility to use a set of 48 genes to distinguish immunotoxic from non-immunotoxic compounds using human peripheral blood mononuclear cells. These genes might be considered as candidate biomarker genes for immunotoxicity screening. However, even if many of the annotated genes appear to be immunologically relevant, in vivo studies in the human population or in experimental models are necessary to demonstrate their effective relevance.

In animals, production of T-dependent antibodies is considered to be the "gold standard". However, there are currently no good systems for in vitro antibody production using human cells. Recently, Koeper and Vohr (39) reported that using a modification of the Mishell-Dutton assay with female NMRI mice splenocytes, all six immunosuppressive compounds tested (with the exception of cyclophosphamide) and all four non-immunotoxic compounds were correctly identified. Further explorations of this model are, therefore, recommended.

Perspectives

Even though no validated alternative in vitro tests to assess immunotoxicity exists, much progress has been made toward these assays in the last decade. Such models can, at least, be used for the pre-screening and hazard identification of unintended immunosuppression and contact hypersensitivity of direct immunotoxicants.

Methods such the whole blood assay, lymphocyte proliferation, and cytokine production can be used for the hazard identification of immunosuppressive potential of chemicals, whereas several in vitro methods are already available to identify allergens and, possibly, to discriminate contact form respiratory allergens and to classify sensitizers accordingly to potency.

Despite all these efforts, however, there is still a clear need for continued investment in the development of methods and approaches that will allow the correct identification in vitro of potential immunotoxic compounds, including immunogenicity and autoimmunity. Intensive international and inter-laboratory cooperation and coordination will be necessary to reach this goal.

References

Author Biography

©2011 Emanuela Corsini