Rob J. Vandebriel

The Use of In Vitro Systems for Evaluating Immunotoxicity: The Report and Recommendations of an ECVAM Workshop

This is the report of a workshop organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAMs main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods that are of importance to the biosciences and which replace, reduce or refine the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures that would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organization of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (). The workshop on “The use of in vitro systems for evaluating Immunotoxicity” was held at ECVAM (Ispra), Italy, on 24th–26th November 2003. The participants represented academia, national organizations, international regulatory bodies and industry. The aim of the workshop was to review the state-of-the-art in the field of in vitro immunotoxicology, and to develop strategies towards the replacement of in vivo testing. At the end of this report are listed the recommendations that should be considered for prevalidation and validation of relevant and reliable procedures, that could replace the use of animals in chemical and cosmetics toxicity testing.

State of the art in non-animal approaches for skin sensitization testing: from individual test methods towards testing strategies.

The hazard assessment of skin sensitizers relies mainly on animal testing, but much progress is made in the development, validation and regulatory acceptance and implementation of non-animal predictive approaches. In this review, we provide an update on the available computational tools and animal-free test methods for the prediction of skin sensitization hazard. These individual test methods address mostly one mechanistic step of the process of skin sensitization induction. The adverse outcome pathway (AOP) for skin sensitization describes the key events (KEs) that lead to skin sensitization. In our review, we have clustered the available test methods according to the KE they inform: the molecular initiating event (MIE/KE1)—protein binding, KE2—keratinocyte activation, KE3—dendritic cell activation and KE4—T cell activation and proliferation. In recent years, most progress has been made in the development and validation of in vitro assays that address KE2 and KE3. No standardized in vitro assays for T cell activation are available; thus, KE4 cannot be measured in vitro. Three non-animal test methods, addressing either the MIE, KE2 or KE3, are accepted as OECD test guidelines, and this has accelerated the development of integrated or defined approaches for testing and assessment (e.g. testing strategies). The majority of these approaches are mechanism-based, since they combine results from multiple test methods and/or computational tools that address different KEs of the AOP to estimate skin sensitization potential and sometimes potency. Other approaches are based on statistical tools. Until now, eleven different testing strategies have been published, the majority using the same individual information sources. Our review shows that some of the defined approaches to testing and assessment are able to accurately predict skin sensitization hazard, sometimes even more accurate than the currently used animal test. A few defined approaches are developed to provide an estimate of the potency sub-category of a skin sensitizer as well, but these approaches need further independent evaluation with a new dataset of chemicals. To conclude, this update shows that the field of non-animal approaches for skin sensitization has evolved greatly in recent years and that it is possible to predict skin sensitization hazard without animal testing.

Interleukin‐10 is an Unequivocal Th2 Parameter in the Rat, whereas Interleukin‐4 is Not *

Exposure of Wistar rats to the immunotoxic compounds hexachlorobenzene (HCB), bis(tri‐n‐butyltin)oxide, and benzo(a)pyrene was previously found to affect mRNA expression of interleukin (IL)‐2, IL‐2R α‐chain, and interferon (IFN)‐γ, the prototypic Th1 cytokine. In contrast, the mRNA expression of IL‐4, the prototypic Th2 cytokine, was unaffected. This latter finding suggested that the IL‐4 mRNA expression may not be an unequivocal parameter for Th2 responses in the rat. In order to obtain such a parameter the present study was performed, consisting of two types of experiments. Expression and production of IL‐4 as well as IL‐10, a second Th2 cytokine, were measured. First, Lewis (Th1 prone) and Brown Norway (BN; Th2 prone) rats were exposed to HCB. Exposure was previously found to increase the serum immunoglobulin (Ig)E levels, an IL‐4‐dependent response, in BN but not Lewis rats, and in Lewis rats to aggravate experimental allergic encephalomyelitis (EAE), severity being inversely related to IL‐10 levels. Secondly, BN rats were infected with Trichinella spiralis, an infection previously found to induce IL‐4 production. HCB exposure did not affect IL‐4 mRNA expression in either strain, while IL‐4 production was decreased in Lewis and unaffected in BN rats. In Lewis rats both the mRNA expression and the production of IL‐10 were decreased. The T. spiralis infection induced IL‐4 and IL‐10 mRNA expression, as well as IL‐10 production. In contrast, the IL‐4 production was strongly reduced. Thus, both the IL‐10 mRNA expression and production correlated with the EAE development and T. spiralis infection. In HCB exposed Lewis rats and T. spiralis infected BN rats the IL‐4 mRNA expression correlated with IgE levels and T. spiralis infection, respectively, whereas the IL‐4 production lacked correlation in all cases. Collectively, these results suggest that IL‐10 is an unequivocal Th2 parameter in the rat, whereas IL‐4 is not.

Production of specific macrophage-arming factor precedes cytotoxic T lymphocyte activity in vivo during tumor rejection

SummaryRecently we published a hypothesis on the immunological events occurring during tumor rejection. One of the implications of this hypothesis is that specific macrophage-arming factor (SMAF) is produced early during the initiation of the immune response, whereas the “classical” cell-mediated immune response components, such as cytotoxic T lymphocytes (CTL), are produced later, that is, during the amplifier-effector phase. In this paper we establish the kinetics of the induction of (a) lymphocytes producing SMAF and (b) CTL. Groups of DBA/2 mice were injected i.p. once, twice or three times with irradiated and/or non-irradiated syngeneic SL2 tumor cells, the injections being given at intervals of 10 days. After each of these injections the production of SMAF and the expression of CTL activity were established. The results showed that in the peritoneal cavity SMAF-producing lymphocytes appeared earlier than cytotoxic lymphocytes (CTL). In addition, it was shown (a) that SMAF does not interfere with the in vitro cytotoxicity expressed by CTL and (b) that in addition to CTL memory cells, SMAF-producing memory cells were also induced after injection of syngeneic tumor cells. These data support the hypothesis that SMAF is involved in the early phase of the cellular immune response against tumors, whereas CTL are induced later.

Immunotoxicology: Extrapolation from Animal to Man — Estimation of the Immunotoxicologic Risk Associated with TBTO Exposure

Bis(tri-n-butyltin)oxide (TBTO) has been shown to be immunotoxic in rodents, resulting in decreased resistance to infections. The no-effect level assessed by estimating effects on host resistance in rats has been found to lie between 0.5 and 5.0 mg TBTO/kg food (0.025 and 0.25 mg/kg body weight). For risk assessment such animal data need to be extrapolated to the human situation. In risk assessment procedures uncertainty factors are used to account for interspecies variation (extrapolation from animal to man) and for variation within the human species. For both factors a value of 10 is often used, based on international guidelines. Hence, exposures below 0.00025 mg/kg body weight should not pose a risk for the human population. In the present study we have taken an alternative approach. We have produced dose-response curves for the effect of TBTO exposure on resistance to Trichinella spiralis. To extrapolate this curve to the human situation, we produced additional dose response data concerning in vitro effects of TBTO exposure on the mitogen responsiveness of both rat lymphoid cells and human blood cells. Using regression analyses of these dose-response data, we calculated a factor that accounts for interspecies variation (IEV) and a factor that accounts for intraspecies variation (IAV) within the human samples. Using these factors, we estimated the dose that decreases resistance in man to an infection. We choose 10% increase of the infectious load as a reference point which in our view is of biological significance. Based on these considerations, we estimated the dose that may affect resistance in adult humans at 0.04 mg/kg body weight. Pre- and postnatal exposure will probably result in effects at lower concentrations, due to the vulnerability of the developing immune system.

Differences in the Induction of Macrophage Cytotoxicity by the Specific T Lymphocyte Factor, Specific Macrophage Arming Factor (SMAF), and the Lymphokine, Macrophage Arming Factor (SMAF), and the Lymphokine, Macrophage Activating Factor (MAF)

Abstract Specific T cell factors, such as specific macrophage arming factor (SMAF), are involved inthe initiation of the immune response. Induction of SMAF-producing T lymphocytes in vivo and of SMAF production by T lymphocytes in vitro is dependent on the presence of intact tumor cells, and is independent of antigen presentation by macrophages. SMAF renders peritoneal macrophages cytotoxic for tumor cells. The armed peritoneal macrophages expressed a specific cytotoxicity. However, antigen-presenting cells can trigger lymphokine-producing T lymphocytes. TheseT lymphocytes produce lymphokines (e.g. macrophage activating factor (MAF)) that activate macrophages. The MAF-activated macrophages express a non-specific tumoricidal activity. In the present study, we investigated the difference in the induction of macrophagecytotoxicity by SMAF and MAF. The following differences were found: 1) SMAF renders peritoneal resident macrophages cytotoxic, whereas MAF could only render peritoneal exudate macrophages cytotoxic. 2) SMAF requires only a 4-h incubation with macrophages, whereas MAF activates macrophages optimally after 12 h. 3) SMAF-armed macrophages recognize only the specific target cell(s), and thus, the cytotoxicity is specific in its expression. MAF activated macrophages were non-specifically cytotoxic. 4) Lipopolysaccharide (LPS) in the culture medium did not enhance the cytotoxicity of SMAF-armed macrophages. In contrast, MAF induced tumoricidal activity was enhanced by adding LPS to the culture medium. 5) After adsorption chromatography with anti-murine interferon-gamma (IFN-γ), the arming capacity of SMAF supernatant was not reduced, whereas the activating capacity of the MAF supernatant was significantly reduced or abrogated. After immunization of mice with allogeneic tumor cells, SMAF-producing lymphocyteswere detected in the draining lymph nodes already 4 days after immunization and up to 12 days. Lymphocytes with the capacity to produce MAF were present in the draining lymph nodes 14-24 days after immunization. Our data indicate that the T cell factors SMAF and MAF can both render macrophages cytotoxic, but act in a different way and during different stages of the cellular immune response against allogeneic tumor cells.

Keratinocytes, Innate Immunity and Allergic Contact Dermatitis - Opportunities for the Development of In Vitro Assays to Predict the Sensitizing Potential of Chemicals

Allergic contact dermatitis (ACD) is the most prevalent form of immunotoxicity in humans characterized by clinical manifestations such as red rashes, itchy skin and blisters. The disease is caused by skin sensitizers which are allergenic low-molecular weight chemicals. ACD is an important occupational disease that gives problems at different workplaces, including hair dressers, metal workers, construction workers, and cleaners. In addition, ACD can develop in the general population as well, since several consumer products contain skin sensitizers. Important skin sensitizers are metals (nickel, chromium), fragrances, hair dye ingredients and preservatives (Kimber et al., 2002a; Vandebriel & van Loveren, 2010).

Initial immunochemical characterization of specific macrophage-arming factor

SummaryThis paper describes the initial immunochemical characterization of specific macrophage-arming factor (SMAF). SMAF is an antigen-specific factor that is released by (sensitized) T lymphocytes after contact with the specific antigen. It renders macrophages specifically cytotoxic. The specificity is dependent on the tumor-mouse combination. In allogeneic systems the specificity is H-2-directed, whereas in the syngeneic systems the specificity is tumor-specific. SMAF has a molecular mass of 65–85 kDa (established by gel filtration). By affinity chromatography SMAF could not be adsorbed with anti-(κ + λ light chain) immunoglobulins or anti-IgG from SMAF-containing supernatants. SMAF could be adsorbed with the monoclonal antibody 14–30 (directed against specific T-cell factors), and could be eluted from columns containing the latter. Furthermore, SMAF could also be adsorbed with and eluted from affinity chromatography columns to which specific tumor cell membranes or KCl extracts of these tumor cell membranes were coupled. Other tumor cell membranes could not adsorb SMAF. Together these data show that SMAF is not an antibody but a T-cell factor with an antigen-specific recognition site.

Specific T-cell factor production and lymphocytes in the direct surroundings of a subcutaneous allogeneic tumor

Antigen-specific T-cell factors (TCF) play a role in the initiation of cellular immune responses. In allogeneic mouse-tumor models lymphocytes from the direct tumor surroundings of both euthymic and nude mice produce TCF. These lymphocytes produce TCF when collected already 1 day after subcutaneous (sc) injection of tumor cells. In contrast to euthymic mice, draining lymph nodes and spleen of nude mice did not contain TCF-producing lymphocytes at any stage after sc tumor cell injection. In sensitized euthymic mice TCF production by lymphocytes is significantly higher in the direct tumor surroundings than in draining lymph nodes or spleen. At 2 and 5 days after tumor cell injection, the mononuclear cell infiltrate of the tissue surrounding the tumor in euthymic mice showed low expression of Thy 1, CD3, TCR alpha beta, TCR gamma delta, CD4, CD8, and asialo GM1, whereas several lymphocytes and mast cells were positive for monoclonal antibody (mAb) 14-30 (directed against TCF). In both euthymic and nude mice, sc injected tumor cells showed apoptosis. In conclusion, the direct tumor surroundings are the first (and, for nude mice, the only) site of TCF production, sc injection of tumor cells attracts mAb 14-30-positive lymphocytes and renders mast cells positive for mAb 14-30.

In vitro responses to immunotoxicants

This paper describes in general terms the function of the immune system, and to what consequences adverse effects of chemical exposure on the immune system can lead. It introduces approaches to test for potential immunotoxicity, and describes in more detail what in vitro test methods are available. The benefits and limitations of in vitro immunotoxicity testing are discussed.