Elsevier

Toxicology

Volume 158, Issues 1–2, 2 February 2001, Pages 65-69
Toxicology

The popliteal lymph node assay: a tool for predicting drug allergies

https://doi.org/10.1016/S0300-483X(00)00409-1Get rights and content

Abstract

A considerable number of drugs is able to induce systemic hypersensitivity in man. Systemic hypersensitivity can be drug- or autoantigen-specific, but in either case a complex of immunological processes and predisposing factors are involved and it is rarely if ever noticed in standard toxicity testing. The popliteal lymph node assay (PLNA) is regarded a suitable test to screen for the immunostimulating ability of a chemical, which may indicate its immunosensitizing potential. The most simple, primary PLNA measures popliteal lymph node hyperplasia after subcutaneous injection of a chemical into the footpad of the hindpaw of a mouse or rat. In order to assess the involvement of T cells, and hence immunosensitizating potential of a chemical, anamnestic immune reactions to a chemical or its metabolite can be measured in previously exposed (and sensitized) animals or in naive animals that received an adoptive transfer of syngeneic T cells from previously exposed animals. In the recently introduced modified PLNA, defined reporter antigens TNP-OVA (T cell-dependent antigen) and TNP-Ficoll (T cell-independent antigen) are used to distinguish between sensitizing and non-sensitizing (IgG1-response or not to TNP-Ficoll, respectively) and between mere inflammatory and complete innocent (no IgG1-response to TNP-Ficoll and an IgG1-response or not to TNP-OVA, respectively) drugs. Results with about 130 compounds (drugs and environmental pollutants) with the various types of the PLNA show a good correlation with documented immunostimulating (both autoimmunogenic and allergic) potential and no false negative chemicals were detected if metabolism was considered. The PLNA awaits further validation before this test can be recommended as a tool for prediction of drug allergy.

Introduction

Certain pharmaceutical low molecular weight compounds (LMWC, <1000 dalton) are able to sensitize the immune system (Gleichmann et al., 1989, Kammüller et al., 1989a, Griem et al., 1998). Occasionally, this leads to allergic or autoimmune diseases (AIDs). The immunological mechanisms that are involved in these two types of adverse immunostimulatory drug reactions are quite similar but they differ in the type of antigen that is recognized by the immune system. A chemically induced allergic response is drug-specific and therefore regarded as a form of true drug hypersensitivity, whereas in chemically induced autoimmune reactions the epitope specificity of the immune response is eventually spread to include auto-antigens and this type of hypersensitivity is also designated as autoallergy (Coleman and Sim, 1994). These chemically induced adverse immune reactions are under strong influence of multiple inherent (i.e. MHC, gender) and environmental (i.e. infections, food) predisposing factors (Kammüller et al., 1989a). Due to these predisposing factors combined with the complexity of the immunological processes involved and the regulatory capacity of the immune system it is difficult to reproduce drug-induced autoimmunity or AID in animals and only few examples in certain specific animal models have been described (Kosuda and Bigazzi, 1996). Conceivably, generally applicable animal models. Systems to predict drug-induced adverse immune effects do not exist. Assessment of drug-specific systemic hypersensitivity may seem much easier, but validated models are also not available as yet. At present, it seems best to focus on the assessment of the potential of a drug to initiate an immune response rather than on the ability to elicit systemic hypersensitivity or actual AID and the popliteal lymph node assay (PLNA) appears to be very useful for this purpose (Bloksma et al., 1995, Pieters and Albers, 1999).

Although much knowledge already exists concerning drug-induced immune derangements, the mechanism is not understood in all its details, but it is clear that stimulation of T cells is required for immunosensitization to occur. But because of their low molecular weight drugs are not recognizable by T cells as such. In order to be recognized they need to alter the structure of autoantigens. Alteration of an autoantigen may occur through covalent or non-covalent binding, in which the LMWC are considered haptens, or by chemically altering the autoantigen so that previously cryptic epitopes are released (Griem et al., 1998). In either case the immune system encounters new antigens, which are collectively referred to as neo-antigens and specific T cells may receive a so-called signal 1.

Sensitization of T (and also B) cells also requires proper adjuvant activity, which includes the upregulation of certain proinflammatory cytokines and costimulatory molecules (eventually providing a decisive signal 2). Important to note here is that most immunostimulatory drugs are chemically reactive or converted into chemically reactive metabolites, thus displaying an intrinsic capacity to provide both signal 1 and 2.

Thus, a LMWC-specific signal 1 combined with an efficient signal 2 would suffice to initiate a drug-specific hypersensitivity response. An autoantigen-specific immune response may result from release of non-tolerated cryptic epitopes (so still not directed to unaltered autoantigens), or prolonged induction of costimulatory signal 2 (possibly including reaction to unaltered autoantigens). T cells specific to unaltered (epitopes or parts of) autoantigens can be sensitized through a process called epitope or determinant spreading (Lehmann et al., 1993). This process may also start with the activation of drug- or LMWC-specific T cells that (also) recognize the LMWC presented in the context of the MHC molecule on an autoantigen-specific B cell (Fig. 1). Normally, this autoreactive B cell will not receive T cell help but now this B cell will obtain a stimulatory help signal (i.e. signal 2) from the LMWC-specific T cell and start to produce autoantibodies. In addition, the B cell will upregulate its costimulatory molecules and become an effective APC. Once activated, the B cell is able to activate (i.e. deliver signal 2) to autoreactive T cells that recognize unaltered parts of the autoantigen that are presented by the same B cell.

The ensuing evolvement of the immune response from stimulation of LMWC-specific or autoantigen-specific T cell to activation of destructive effector mechanisms is under strict control of many regulatory processes. In most individuals this strict regulation prevents the occurence of adverse immune responses, in particular AID. Conceivably, disturbance of this regulation or interference with the ultimate effector-mechanisms (such as complement activation, reviewed by Coleman and Sim, 1994) should also be included in the understanding of the process of chemically initiated adverse immune responses.

Section snippets

The PLNA

The PLNA is an animal test system that detects local lymph node hyperplasia as a simple straightforward way to measure the potency of a LMWC to stimulate the immune system. In the most simple or primary PLNA, the LMWC is injected without adjuvant subcutaneously into the footpad of the hindpaw of a mouse or a rat. Six to 8 days later the ratio of weight or cell number of the draining lymph node of the LMWC-treated animals over that of the vehicle-treated animals is determined as the PLN-index

Concluding remarks

The main advantage of the primary PLNA is that it is an easy to perform, immunologically straightforward and short-term test which shows a good correlation with documented adverse immune effects in man. Important to note, the assay also fits to detect structure-activity (Kammüller and Seinen, 1988) and dose-reponse relationships (Kammüller et al., 1989b) and to assess the influence of predispositing factors (such as MHC haplotype, metabolic rate, gender) (Hurtenbach et al., 1987,

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