Influence Of Autogenic Apoptotic Or Necrotic Cells On Regulatory T Cells

BackgroundApoptosis and necrosis are the two ways of cell death that participate in a series of physiopathological processes and differently influence the immune system. Apoptosis, also known as programmed cell death is an active, programmed and energy consuming cellular process and characterized by condense of nucleus, membrane shrink, flip-out of phosphatidylserine and formation of apoptotic bodies. Importantly, apoptotic cells or bodies should be quickly eliminated. Meanwhile, necrosis is a passive, non-programmed and destructive cellular process that is usually caused by a sudden physical, chemical or biotical damage. Duo to abrupt shut up of energy supply, necrotic cells are characterized by membrane disruption, cell swelling, nucleus lysis and release of cellular ingredients into the periphery tissue that usually causes inflammation.During the past 10 years, it has been found that apoptotic cells themselves release or help their phagocyte release many anti-inflammatory factors that initiate a series of signaling pathways to down regulate immune response or induce immune tolerance. Apoptotic cells have been reported to play important roles in autoimmunity, tumor immunity, infection immunity and process of transplantation immunity. Disruption of phagocytosis machinery for apoptotic cells causes auto-immune diseases. Tumor genesis and metastasis may be related to the abnormally high rate of apoptosis as well as phagocytosis. In addition, apoptotic donor cells can induce transplant tolerance in rodent models, although the detained mechanisms remain largely unknown.Differing from apoptotic cells, necrotic cells initiate aggressive immune responses. DCs can be activated by engulfing necrotic cells. Co-culture of necrotic cells with macrophages increases the anti-tumor capability of macrophages. Macrophages stimulated by necrotic cells that can secret large amount of pro-inflammatory factors. Furthermore, "danger signals" released by necrotic cells can activate innate immune system and initiate immune responses.Therefore, necrosis and apoptosis are two ways of cell death that plays different roles in immune system. The relationship between the effects of necrotic cells and that of apoptotic cells is not clear. Until now, Treg cells have been found to play an important role in apoptotic cell-induced immune tolerance. Experiments have shown that injection of apoptotic cells increases Treg cells. And apoptotic cells can't induce immune tolerance with the deletion of Treg cells. Furthermore, TGF-βessential for generation and growth of Treg cells is secreted by phagocytes engulfing apoptotic cells. However, although apoptotic cells might promote the generation and development of Treg cells, the effects of necrotic cells on Treg cells remain obscure.Many immune researchers wish to utilize apoptotic cells to induce allograft tolerance. However, their only achieve prolonged graft survival in rodent transplant models, but not in big animal transplant models, especially in nonhuman primates. In mice, Treg cells are discovered to participate in apoptotic cell induced immune tolerance, which critically challenges the classical immune recognition theory, the clonal selection model. Further studies on the effect of apoptotic cells on Treg cells in nonhuman primates are necessary to evaluate the potential clinical significance of apoptotic cell mediated immune tolerance. And the investigation of necrotic cells on Treg cells will help us to understand the effects of different ways of cell death on immune regulation.Objectives1.To establish a method to induce apoptosis of peripheral blood mononuclear cell from rhesus monkey; 2.To investigate the effect of the apoptotic cells in combination with rapamycin on the Treg in rhesus monkeys; 3.To study the role of the necrotic cells on Tregs in mice.Methods1. Venous blood was collected from rhesus monkey and peripheral blood mononuclear cell (PBMC) was separated by gradient density centrifuge. PBMC apoptosis was induced with ultraviolet irradiation. The UV exposure time, incubation time after irradiation, and the necessity of mixing the cells by continuous vibration during UV irradiation were determined. Apoptotic cells was double stained with Annexin-V FITC and PI and analyzed by flow cytometry.2. Four rhesus monkeys were divided into two groups, the rapamycin control group and experimental rapamycin in combination with apoptotic cell group. In the control group, the monkey was injected with PBS along with 0.4mg rapamycin was orally administrated daily. In experimental group, 3.5×10⁷ apoptotic cells are intravenously infusion injected along with 0.4mg rapamycin was orally administrated daily. The percentage of CD4⁺Foxp3⁺ Treg in rhesus monkey's peripheral blood was detected periodically after operation and the percentage of Treg in CD4⁺T cells accordingly calculated. The changes of Treg in two groups of rhesus monkeys were compared.3. Thirty six SPF Balb/c mice aged 6 weeks were divided into four groups: blank control group (n=6), PBS+H₂O₂ control group (n=10), necrotic cells group (n=10) and H₂O₂ treated necrotic cells group (n=10). For the blank control group, mice were not injected anything. For the PBS+H₂O₂ control group, mice were intravenously injected with 200μl 50mM H₂O₂ in PBS. In necrotic cells group or H₂O₂ treated necrotic cells group, 2×10⁷ (200μl) necrotic cells or H₂O₂ treated necrotic cells were intravenously injected. The mice were executed two weeks later and blood,spleen and thymus harvested for the detection of CD4⁺Foxp3⁺ Treg. Data were illustrated asmean±standard deviation ((?)±SD) and analyzed with one-way ANOVA (SPSSV16.0).(1) Preparation of necrotic cellsThree SPF Balb/C mice aged five weeks were euthanized and the thymuses harvested and smashed in medium to prepare thymocyte single cell suspension Incubate the cells in 56℃water bath for an hour and analyzed the cell with Annexin-V / PI double staining. Necrotic cell rate was calculated as the percentage of double positive cell in the total cells.(2) Detection of TregAccording to the manufacture's protocol (eBioscience, USA), sample cells by were first labeled with different fluorescence conjugated CD4 monoclonal antibody and then perforated and fixed by specialized fixation fluid. Then, the samples were stained with anti-Foxp3 antibodies as well as an IgG2b antibody as isotope control. The cells were incubated at 4℃in the dark for 1 hr and proceeded to flow cytomerry analysis.Results1. Ultraviolet light irradiation induced apoptosis of PBMC from rhesus monkey. The ideal conditions are irradiation at 20cm distance with constant shaking incubating with RPMI 1640 medium (10% calf serum)for 15 hours after irradiation. The general apoptosis rate is above 85%.2. Apoptotic cells combined with rapamycin might be able to raise the percentage of Treg in CD4⁺T cells in rhesus monkey. In the two monkeys administrated with rapamycin, the percentages of Treg/CD4⁺T cells increased only in one monkey but not in another monkey. Thirty eight days after the first administration of rapamycin, Treg/CD4⁺T cells in the two monkeys were 13%, and 6.92%, respectively. In the two monkeys adminitrated with apoptotic cells and rapamycin, the Treg percentages increased to 21.56% in one rhesus and to 16.67% in another two or three weeks after injecting apoptotic cells.3. The percentage of CD4⁺Foxp3⁺ Treg in total lymphocytes in mice. In the spleen, the percentages are (3.80±0.24) % and (3.41±0.64) % in blank and PBS+H₂O₂ control groups. But it significantly reduced to (2.75±0.38)% in necrotic cell treated group (F=6.895, P_{vs Blank}=0.000, P_{vs PBS+H2O2=0.004} . In the H₂O₂ treated necrotic cell group, Treg percentage (3.42±0.49)% is not different from either blank control or PBS+ H₂O₂ control (F=6.895, P_{vs Blank}=0.139, P_{vs PBS+H2O2}=0.948), but significantly higher than that in necrotic cells group (F=6.895, P=0.004). In blood and thymus, however, there are no statistical differences among different groups (F_blood=0.337, P_blood=0.799; F_thymus=0.507, P_thymus=0.680).There are no statistical differences of the percentages of CD4⁺ T cells in total lymphocytes were in different groups in blood, spleen or thymus.The percentage of CD4⁺Foxp3⁺ Treg/CD4⁺T cells. In peripheral blood, the percentage of Treg/CD4⁺T cells in PBS+H₂O₂ control group was (8.46±2.48)%. In the necrotic cells group, it significantly decreased to (6.19±1.62)% as compared with PBS+H₂O₂ control group (P=0.013). In the spleen, the percentages of Treg/CD4⁺T cells were (16.74±1.54) % in blank control group and (14.16±3.27)% in PBS+H₂O₂ control group. Similarly, it significantly decreased to (10.15±2.64) % in the necrotic cells group (F=8.482, P_{vs Blank}=0.000, P_{vs PBS+H2O2}=0.002). In H₂O₂ treated necrotic cells group, the percentage (12.81±2.44) % is significantly higher than that in necrotic cells group (F=8.482, P=0.043). However, it was still lower than that in the blank control group (F=8.482, P=0.007). In thymus, there are no statistical differences among different groups (F=0.487, P=0.220).Conclusion1. The ultraviolet light (UVC) irradiation can effectively induce apoptosis in peripheral blood mononuclear cell of rhesus monkeys.2. Treatment with apoptotic cells in combination with rapamycin might be able to increase the percentage of Treg in rhesus monkey.3. Treatment with necrotic cells decreased the percentage of Treg in peripheral blood and spleen, but not in the thymus. H₂O₂ treatment can reverse the effect of necrotic cells on Treg cells in spleen.