RESUMO
Interest in mixed chimerism has evolved from its role in the induction of alloantigen tolerance. However, its precise impact on the host organism remains to be elucidated. In the present work, we analyzed cytokine secretion from chimeric mice cells to assess the influence of different mixed chimerism induction protocols on immune system function in recipient mice. To our knowledge, there have been no reports on using this parameter for the optimization of the mixed chimerism induction method. B6.SJL-PtprcaPep3b or C57BL/6J mice were used as recipients and Balb/c as donors. We utilized four protocols which consisted of: 3Gy total body irradiation (day -1), the injection of 20-30×10(6) bone marrow cells (day 0), and a combination of CD40L (days 0 and 4), CD8 (day -2), and NK1.1 (day -3) blocking antibodies and cyclophosphamide (175mg/kg - day 2). The concentrations of cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF) were evaluated in the supernatants of unstimulated or phytohemagglutinin-stimulated chimeric spleen, bone marrow and peripheral blood cells in the 8th week of experiment. The induction of tolerance to Balb/c mouse antigens was initially tested in chimeric mice by assessing the presence of Vß5 and Vß11 TCR-expressing lymphocytes. The cytokine production was considerably increased, especially in chimeric mice treated by cyclophosphamide. Also the mixed chimerism itself seems to affect IFN-γ, IL-2, IL-4, IL-6, IL-17A, and TNF secretion. Using the optimized induction protocol, we established that chimeric mice cells secreted lower IFN-γ, IL-2, IL-4 and higher IL-6, IL-17A, and TNF levels as compared to control animals. We found that both donor and recipient cells markedly participated in the cytokine production. In conclusion, our optimization study based on cytokine assessment contributes to establishing an effective protocol of mixed chimerism induction with no cyclophosphamide use and better understanding of the influence of this phenomenon on the recipient organism.
