Study of in vitro methods to evaluate immunosuppression

Abstract

The immune system is one important target of xenobiotic toxicity. In fact, immunotoxicity evaluation is becoming a growing concern for international regulatory authorities. At present, immunotoxicity of chemicals is evaluated through standard toxicity studies (STS) on laboratory animals. European Union legislation aims at reducing the number of animals used for experiments, encouraging the development of alternative methods. Since validated alternative methods to evaluate immunotoxicity are not available, except for the CFU-GM (granulocyte macrophage-colony forming unit) test, this study has been developed on the basis of these objectives: 1. Determination of the predictivity of a set of in vitro assays, to evaluate suppressive potential on lymphocyte activation of compounds known for their effects on the immune system; 2. Developing a long-term culture method (LTC) using human mononuclear bone marrow cells to assess effects of chemicals on lymphopoiesis in vitro; 3. Performing a preliminary evaluation of the LTC developed, using the organotin compound tri-butyltin chloride (TBTC), which is known for its immunosuppressive activity in rats. We evaluated the capability of some in vitro tests to classify a panel of compounds known for their immunosuppressive effects. The tests were performed on human, rat and murine cells. Different endpoints were assessed: cytotoxicity, cytokine release, myelotoxicity and mitogen responsiveness. IC50s values were calculated and results compared by species. In vitro tests performed well in classifying the selected compounds, confirming that it is necessary to investigate different end-points to study immunosuppression. Most methods available use mature immune system cells. Since there are no methods to study toxicity on lymphoid progenitors, we developed a long term culture (LTC) from human bone marrow mononuclear cells to maintain lymphopoiesis. The LTC consisted of a two stages: a myeloid stage to allow the formation of a stromal layer and a lymphoid stage to allow expansion of lymphocytes. Results show that the use of IL-7 in LTC inhibits precursor and mature B cells, while it supports the proliferation of CD3+CD8+ and CD3+CD4+ T-cells. TBTC was tested on LTC method to verify if this culture system could be useful in the chemical toxicity characterization. TBTC induced adipocyte differentiation causing an alteration of the stromal cellular components, and it reduced the expression of some cytokines and of leptin, which is important for lymphopoiesis. To verify if TBTC can really reduce lymphocyte population, some B and T cell subsets were analyzed. Some cytokines important for lymphopoiesis, whose production was inhibited by TBTC, were tested to verify if they can prevent TBTC toxicity on lymphocytes. FACS analysis showed a reduction of CD19/CD22-positive B cells by TBTC, both in the presence or absence of cytokines. The treatment did not cause a toxic effect on mature CD3+CD4+ and CD3+CD8+ T cells, suggesting selective TBTC toxicity on B lymphocytes. TBTC caused an alteration of the stromal microenvironment inducing adipocyte differentiation and reducing the production of different cytokines, withdrawing the culture of essential components for B lymphopoiesis. A direct TBTC toxic activity on lymphocytes is also possible, but the mechanism of action should be still explained.