The Regulatory Functions And Mechanisms Of Interleukin-12 Treatment On NK Cells In Mice

The innate immune system is the first line against invading microorganisms. Natural killer cells(NK cells) are a key component of the innate immune system, play critical roles in immune regulation as well as in the immune response against cancer and infections. NK cells are particularly important in resistance to infection, in recognizing and eliminating cancer cells, and in immune surveillance plus hematopoietic regulation. Therefore, NK cells have attracted much attention in immunotherapy, and become a research hotspot. The regulatory function of NK cells in immune responses are dependent on cytokines and chemokines that are produced by antigen presenting cells. IL-12, a multifunctional cytokine acting as a key regulator in the activities of natural killer cells, is produced by macrophages, dendritic cells and B cells. IL-12 promotes NK cell secreting IFN-γ as well as the cell-mediated cytotoxicity and their proliferation. IL-12 plays an important role in regulation the innate and adaptive immune system. The activity of NK cells can show the strength of cellular immune response, NK cells has been widely applied to the treatment of various diseases by immune cells, which has good application prospects. However, IL-12 consecutive injections can cause the reducion in the reactivity of IL-12 and treatment effect during antineoplastic therapy, but the relative molecular mechanism remains to be further research.In previous studies, we found that the IL-12 continuous stimulation of human NK cells in vitro induced NK cell apoptosis through induction of ROS accumulation, or induced microRNA to repress IL-12 transcription, resulting in IL-12 hyporesponsiveness in NK cells, and resulting gradually reduce of IFN-γ secretion. In this study, we test the effects of rmIL-12 continuous stimulation on the activity of normal mice NK cells in vitro and vivo. The results of the studies are listed as follows:1. rmIL-12 with different concentrations stimulated splenic lymphocytes for indicated times in vitro. The results showed that 1 ng/ml of rmIL-12 significantly induced the secretion of IFN-γ in spleen lymphocytes in the presence of either IL-2 or IL-15, suggueting that 1 ng/ml of rmIL-12 is an effective concentration for subsequent experiments. In addition, the secretion of IFN-γ induced by rmIL-12 reached peak at 16 h, then reduced different degree in subsequent every 8 h.2. rmIL-12 with different concentrations stimulated splenic lymphocytes for 24 h and 48 h, the secretion of IFN-γ increased as the stimulus concentration increases(P<0.05), and then the second stimulus was carried out with 1ng/ml of rmIL-12 for 12 h, the secretion of IFN-γ have no significant difference when compared to cells without the first stimulus of rmIL-12.3. The splenic lymphocytes of mice were respectively incubated with rmIL-12 and two different Toll-like receptor agonists, and further suffered from the second stimulus. The results showed that the continuous stimulation of rmIL-12 on mice splenic lymphocytes in vitro have no significant effects on the production of IFN-γ induced by Toll-like receptor agonists joint stimulation.4. rmIL-12 stimulated mice splenic lymphocytes for 24 h and 48 h, and then suffered the second stimulus from 1 ng/ml of rmIL-12. The result showed that the continuous stimulation of rmIL-12 on mice splenic lymphocytes in vitro caused a significant reduction of the proportion of NK cells in spleen lymphocytes(P<0.05) and reduced the ability of IFN-γ production.5. C57BL/6 female mice were devided into control groups injected with saline and experimental groups injected with rmIL-12. Then we measured the changes of NK cells number and percentage, subtype distributions of NK cells, apoptosis rate of NK cells, the changes of active moleculars of NK cells(such as IFN-γ, CD69, GZMB), and cytotoxicity of NK cells in mice injected with rmIL-12 continuously. The result showed that continuous IL-12 treatment increased the proportions of CD69 positive cells in NK cells(P<0.01), but NK cells numbers and proportion(P<0.05), growth activities and cytotoxicities significantly decreased(P<0.05), reduced the expressing of IFN-γ and GZMB(P<0.05).The research results demonstrate that continuous stimulation of rmIL-12 on mice splenic lymphocytes in vitro have no significant effects on IFN-γ production in spleen lymphocytes but caused a significant reduction of the proportion of NK cells in spleen lymphocytes and modest reducion of IFN-γ production in NK cells, whereas continuous stimulation of rmIL-12 in vivo can induce the decrease in the number of NK cells, the declining secretion of cytokines, weakened immune reactivity and cytotoxicity in NK cells.