Effects of long-term estrogen treatment on IFN-gamma, IL-2 and IL-4 gene expression and protein synthesis in spleen and thymus of normal C57BL/6 mice.

Estrogens have been shown to markedly modulate the immune system. One mechanism by which estrogens could modulate the immune system is by regulating cytokines, an aspect not well-studied thus far. To address this issue, normal C57BL/6 orchiectomized mice were given estrogen and its effects on selected cytokines, interferon-gamma (IFN-gamma), interleukin 2 (IL-2) and IL-4 in lymphocytes from a developmental organ (thymus) and a mature lymphoid organ (spleen) examined. Estrogen significantly increased IFN-gamma and IL-2 mRNA in concanavalin-A (Con-A) activated thymocytes, splenic lymphocytes, and in enriched splenic T cells. Estrogen had no marked effect on IL-4 mRNA. While estrogen increased IFN-gamma mRNA in Con-A activated unseparated splenic lymphocytes and enriched splenic T cells, a numerical increase in IFN-gamma was noticed only in the supernatants of Con-A activated unseparated splenic lymphocytes, but not in enriched splenic T cells. This suggests that for optimal secretion of IFN-gamma in estrogen-treated mice, co-stimulatory signals from antigen presenting cells are needed. Gender differences in IFN-gamma and IL-2 mRNA were also evident. Con-A activated splenic lymphocytes from gonadal-intact, untreated female had a pattern of numerical increase in IFN-gamma mRNA, and IFN-gamma and IL-2 protein levels compared to their male counterparts. Taken together, our data suggests that estrogens regulate the expression of cytokines, which could account in part, for the gender differences in immune capabilities.

A dye-based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis, and immunophenotyping studies.

Alamar Blue in the microenvironment of activated cells, undergoes color change and also becomes fluorescent. By using the Alamar Blue dye, we have reported a non-radioactive colorimetric assay to indirectly determine proliferation of murine lymphocytes. We further show that the pattern of mitogen-induced proliferation assessed fluorometrically was comparable to the 3H-thymidine incorporation assay (3H-Tdr assay). Of practical importance is that the color/fluorescence changes were stable at 4 degrees C in the dark for 3-4 weeks. In immunological studies, it is important to further analyze lymphocytes that have undergone activation and/or proliferation. This is not possible with the standard 3H-Tdr assay, which requires lysis of cells. In contrast, the Alamar Blue-based non-radioactive assay does not require cell lysis. We therefore tested the hypothesis that further analysis of lymphocytes is possible, after assessing the proliferation using Alamar Blue. Following assessment of proliferation in a 72-h culture, the Alamar Blue dye was washed-off and cells were re-utilized to perform additional immunological analysis. Short-term exposure of lymphocytes to Alamar Blue was not detrimental to lymphocytes, as assessed by trypan blue exclusion and the propidium iodide (PI) assays. Exposure of dexamethasone-treated cells to Alamar Blue did not interfere with the performance of apoptosis assays, such as flow cytometric analysis of PI-stained cells and microscopic examination of ethidium bromide/acridine orange-stained cells. In addition, prior exposure of lymphocytes to Alamar Blue did not affect the analysis of chromosomal aberrations or the visualization of cell surface antigens by flow cytometry. Further, the expression of cytokine mRNA in lymphocytes previously exposed to Alamar Blue was similar to unexposed cells. Together, a notable advantage of this assay is that it now enables the investigator to maximize information by following or correlating proliferation with other immunologic events in the same cells.