Hervé Lebrec

Immunotoxicological investigation using pharmaceutical drugs. In vitro evaluation of immune effects using rodent or human immune cells.

In order to evaluate the relevance of in vitro methods for immunotoxicity assessment, the effects of pharmaceutical drugs on lymphoproliferative and cytotoxic functions of mouse splenocytes and human peripheral blood mononuclear cells (hPBMC) were studied. A comparison of sensitivity of immune cells from different origins to an in vitro exposure to different xenobiotics was performed using non-immunosuppressive (cimetidine and furosemide) and immunosuppressive (azathioprine (AZA), cyclosporine A (CSA), and dexamethasone (DEX)) drugs. For CSA, sensitivity of both rat and mouse splenocytes following in vitro exposure was compared to the one of hPBMC. Immune function tests included lymphoproliferative response to mitogenic lectins (concanavalin A (Con A) and phytohemagglutinin (PHA-P)) or to allogeneic cells (mixed leukocyte response (MLR)) and cytotoxicity assays (cytotoxic-T lymphocyte (CTL) and natural killer (NK) cell-mediated cytolysis). Additionally, to evaluate how well in vitro assays represent the in vivo situation, a comparison of the effect of cyclosporine A on the same immune function tests following in vivo or in vitro exposure was performed. The data obtained show numerous similarities in the effects observed following in vitro exposure of rodent or human cells to the drugs and a very similar sensitivity of rat and mouse cells to CSA in vitro. Discrepancies between human and rodent cells such as lymphoproliferative response to PHA-P following exposure to DEX or sensitivity of CTL-mediated cytolysis to CSA do exist. In vitro assays were very representative of the in vivo situation, both in the rat and in the mouse, following CSA exposure, except for NK cell activity in the rat. These data show the usefulness of in vitro systems for immunotoxicity assessment. They allow direct comparison of rodent and human systems, and could be representative, for drugs altering specifically the immune system like CSA does, of the in vivo situation.

Th1/Th2 responses to drugs

T lymphocytes can be characterized by their pattern of cytokine secretion and he divided into type I (Th 1 /Tc 1 ) and type 2 (Th 2 /Tc 2 ) subsets. The involvement of type-1 or type 2-like responses in sensitization has been studied in the mouse, with reference contact and respiratory contact sensitizers. One interesting feature with certain drugs, such as β-lactam antibiotics, is the diversity of clinical manifestations associated with immune-mediated hypersensitivity reactions in humans: immediate reactions such as urticaria, Quincke oedema and anaphylactic shock, and delayed hypersensitivity reactions, such as maculopapular rashes, allergic contact dermatitis and skin reactions of other types. In the mouse, Th, and Th 2 cytokines have been shown to regulate primary and secondary benzylpenicilloyl- (BPO-) specific antibody responses. Peripheral blood lymphocytes isolated from patients with a clear history of β-lactam allergy were assessed for type-I and type-2 phenotypes. Immediate reactions involved mixed Th 1 , Tc 1 . and Tc 2 responses, whereas allergic contact dermatitis involved Tc 1 and Th 1 cells. Other delayed hypersensitivity reactions to β-lactams were restricted to Th 1 responses. It has been demonstrated that both CD4 + and CD8 + -lidocaine-specific T cell clones isolated from patients with allergic contact dermatitis produced IFN-γ, even though CD8 + clones only produce IFN-γ, while IFN-y producing CD4 + cells concomitantly produced IL-5 and IL-4. Together these data illustrate the heterogeneity of drug-specific T-cell responses.

Interleukin-4 plays a dominant role in Th1- or Th2-like responses during the primary immune response to the hapten penicillin

Despite a large number of studies on the Thl/Th2 balance during immune response to pathogens or protein antigens, little is known concerning the early events which regulate Thl/Th2 differentiation following a single injection of haptenic compounds. In this work, we studied how two mouse strains with different MHC haplotypes, SJL (H-2s) and Balb/c (H-2d), could develop different primary immune responses to subcutaneously injected benzylpenicillin coupled to tetanus toxoid (BPO-TT). The SJL mice showed a high BPO-specific IgG1 response that was maximum on day 10 and no BPO-specific IgG2a response. In contrast, Balb/c mice showed a high BPO-specific IgG2a response on days 15 and 22 and a weak IgG1 production. In SJL mice, the response to BPO-TT was characterized by a very early and high IL-4 mRNA expression. In Balb/c, a delayed and weaker expression of IL-4 mRNA was observed. Kinetics of IL-2 and IFN-gamma mRNA expression were comparable in both strains, but IFN-gamma mRNA expression was higher in SJL than in Balb/c. In vivo neutralization of IL-4 induced a significant BPO-specific IgG2a production and a two-fold reduction of IgG1 production in SJL mice while it accelerated production of BPO-specific IgG2a in Balb/c mice. In addition, studies of IL-12 p4O and IL-10 mRNA expression following immunization with BPO-TT showed a greater IL-12 p4O mRNA expression in Balb/c mice and a slightly higher IL-10 mRNA expression in SJL. Taken together, our data suggest that Th1 or Th2 differentiation in primary immune responses to haptenic compounds such as penicillin may be driven by the kinetics and the level of IL-4 production rather than by the level of IFN-gamma. Additional cytokines such as IL-10 and IL-12 are likely to contribute to the regulation of this response.

Testing strategies in immunotoxicology

Developing a battery of immune function assays to screen potential immunotoxic compounds has been a major issue these past years. Improving this approach is possible using new probes and parameters that are now available from recent knowledge on how the immune system is working (apoptosis, RT-PCR for cytokine mRNA expression). Immunotoxic outcome generally results in serious adverse effects, thus it seems appropriate to evaluate this risk early in drug development. This is especially true in the context of the emerging combinatorial chemistry techniques and high throughput screening in pharmacology resulting in probably numerous molecules to test in toxicology. In this case, screening for adverse effects (genotoxicity, hepatotoxicity, immunotoxicity) that may compromise definitely the development of a molecule should be a help in the decision process since more than one molecule with equivalent pharmacological properties may be available.

Mechanisms of drug-induced allergic contact dermatitis

Allergic contact dermatitis is induced by a wide variety of drugs that trigger specific immune responses following topical exposure. Identified chemical stuctures involved in such reactions include the mercuric and thiosalicylic acid groups of thimerosal, the diphenylketone group of the anti-inflammatory drug ketoprofen, the amide or ester structure of local anesthetics, and the side-chain and thiazolidine ring of β-lactams. The T cell responses to such compounds involve CD4+ and CD8+ αβ+ T lymphocytes and also CD4–/CD8– γδ+ T cells. Although T helper 2 cytokine production by drug-specific human T cells from patients with allergic contact dermatitis has been described, T helper 1-like and T cytotoxic 1-like responses clearly play key roles in this cutaneous reaction.

Immune parameters are affected differently after cyclosporine A exposure in Fischer 344 rats and B6C3F1 mice: implications for immunotoxicology

Knowledge of interspecies differences, commonly evaluated in other disciplines such as carcinogenesis, is a prerequisite for an appropriate assessment of immunotoxicological risks. The purpose of this study was to assess interspecies differences following exposure of Fischer 344 rats and B6C3F1 mice to cyclosporine A. These animals were exposed daily to cyclosporine A by oral gavage at 0, 5, 10, 25 mg/kg/day for 14 consecutive days. The results showed that splenocytes lymphoproliferation in response to concanavalin A or phytohemagglutinin, and IgM antibody-forming cells to sheep red blood cells, were affected in both species. Cytotoxic T-lymphocyte activity and mixed lymphocyte response were significantly inhibited in the rat following cyclosporine A exposure while they remained unaffected in the mouse. In contrast, natural killer cell activity was significantly depressed in the B6C3F1 mouse but not in the Fischer 344 rat. The discrepancies between the two species in cytotoxic T-lymphocyte activity and mixed lymphocyte response assays could partially be explained by the constantly higher blood level of cyclosporine A in the rat than in the mouse. When these tests were performed using rat and mouse splenocytes exposed to cyclosporin A in vitro (10(-9) to 10(-5) M) it was possible to correlate in vivo and in vitro data for concanavalin A- and phytohemagglutinin-induced lymphoproliferation and for cytotoxic T-lymphocyte activity but not for mixed lymphocyte response. Natural killer activity was 10-fold more sensitive in mice than in rats in vitro but these results did not clarify the in vivo difference. In conclusion, these results emphasize that the utilization of more than one species should be considered when assessing immunotoxicity.

Evaluation of the Immunotoxic Potential of Pharmaceuticals: Functional Aspects

Developing a battery of immune function assays to screen potential immunotoxic compounds has been a major issue these past years. Combining histopathology of lymphoid organs with several functional tests fosters the detection of an immunosuppressive drug and in addition provides information concerning the nonobservable adverse effect dose, thus fostering the performance of risk assessment. More work is urgently needed to carefully evaluate differences between results obtained in rodents and humans due to the origin of the cells used to perform the immune function assays, to develop more physiological tests, and to quantify the occupational risk linked to immunosuppressive molecules. The sensitization potential of a drug depends on the activation of specific lymphocyte but the clinical outcome is related to Th1/Th2 differentiation and the “cytokine” background of the individual. Measuring cytokine production in “lymph node assays” will help to assess the potential of pharmaceuticals to produce contact and respiratory allergy.