重组蓖麻毒素B链调节免疫细胞活化及其对信号传导通路的影响
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摘要
蓖麻毒素（RT）由RTA、RTB两个肽链以二硫键共价相连接。RTB是能与半乳糖/N-乙酰基半乳糖胺特定结构结合的外源凝集素蛋白，具有凝集素活性。RTB本身无毒性，但可以帮助RTA转运到细胞溶质中，抑制蛋白质的合成。文献报道，RTB可以用作异源性疫苗抗原的载体，可融合不同的轮状病毒抗原VP7、P24、NSP4等，发挥粘膜佐剂作用。课题组前期研究亦发现RTB作为佐剂能诱导产生狂犬病毒中和抗体，抗体水平显著高于普通氢氧化铝佐剂疫苗，并能加速狂犬病毒疫苗诱导的体液免疫应答。然而对于RTB更多的生物学活性及机制研究国内外报道甚少。RTB对免疫细胞活化及细胞内的信号传导通路的影响国内外未见报道。本研究采用分子生物学与免疫学技术探讨了RTB对免疫细胞活化及细胞内的信号传导通路的影响。首先探讨了RTB对小鼠免疫细胞活化功能的影响。研究发现，RTB可明显促进小鼠脾脏淋巴细胞增殖；促进淋巴细胞培养上清中Th1型细胞因子的分泌；并发现RTB可促进CD4+T细胞的增殖和活化。RTB对小鼠RAW264.7巨噬细胞的活化功能与机制研究表明：RTB可促进RAW264.7细胞NO的分泌；促进iNOS基因与蛋白的表达；增强巨噬细胞的吞噬能力；促进IL-6和TNF-α基因与蛋白的表达。采用小鼠蛋白芯片抗体阵列对巨噬细胞与炎症相关的40种蛋白的变化进行检测，结果发现MCP-1、TNF-α、MIP-1α，MIP-1β，MIP-2，RANTES，TIMP-1，CD54的表达增高，G-CSF、KC的表达下调。RTB对RAW264.7巨噬细胞活化过程中的信号转导通路的影响研究表明：酪氨酸蛋白激酶介导的信号通路抑制剂、蛋白激酶C信号通路抑制剂和核因子κB信号通路抑制剂，可以抑制RTB诱导RAW264.7细胞NO分泌和iNOSmRNA表达以及TNF-α和IL-6mRNA和蛋白水平表达，提示酪氨酸蛋白激酶、蛋白激酶C和核因子κB信号通路可能参与RTB诱导巨噬细胞活化。采用RTB对酪氨酸蛋白激酶信号通路相关磷酸化位点分子进行蛋白组学分析，结果提示RTB通过影响RAW264.7细胞酪氨酸蛋白激酶信号途径SRC、JAK-STAT和核因子κB信号途径IκB-α蛋白磷酸化，激活酪氨酸蛋白激酶和NF-κB信号转导途径。
Western blot和免疫荧光实验结果进一步证实RTB通过诱导巨噬细胞IκB-α磷酸化进而促进NF-κB核转位作用。
上述结果提示RTB可以调节免疫细胞活化，对巨噬细胞的活化可通过细胞内的多条信号转导途径参与。提示RTB诱导巨噬细胞活化可能通过多条信号转导途径互相调节、共同作用。本研究为RTB免疫生物学活性研究及其免疫佐剂等方面的应用提供了新的科研资料，并奠定了实验依据与理论基础。
Ricin toxicity (RT) is covalently linked by the two peptide chains RTA and RTB indisulfide bonds.RTB consists of260amino acids is a galactose/N-acetylgalactosaminelectin protein and has a molecular weight about34000. The individualism of RTB has notoxicity and is believed to have two main functions at present: One is that it can helpRTA transfer to cytosol and inhibit the synthesis of protein. Another is that RTBligand has the capacity of specific binding with the receptor of different carbohydratestructure, to mediate various kinds of bioprocess, including interactions between cell,cell host and pathogen, and the innate immunity response. Also it can be used as thecarrier of heterologous antigens to the immune system. For instance, RTB can fusedifferent Rotavims antigens such as VP7, P24, NSP4, etc, and perform as mucosaladjuvants to enhance the immune reaction of organism. Our study also found that theRTB as adjuvant can induce the production of rabies neutralizing antibody, theantibody levels were significantly higher than ordinary aluminum hydroxide adjuvantvaccine and can accelerate the immune response of the body fluid induced by rabiesvaccine.With RTB as the object, this project investigates RTB's influence on mice's Tcell and macrophage activating function as well as on the signal transductionpathways inside the macrophage, in the hope of furnishing the experimental evidenceand theoretic foundation for the research on RTB immunobiological activation.
The aspects of this research as follow:
1. Preparation of RTB
PET28a-RTB was transformed into BL21(DE3) cells to stimulate recombinantprotein synthesis and soluble protein was renaturated.The protein was purified usingnickel-NTA column chromatography. LPS was removed by Detoxi-Gel.
2. RTB protein induced mouse T cell activation
RTB was found was found to enhance the splenocytes proliferation comparedwith control. In the presence of ConA or LPS, the treatment with the RTBsignificantly enhanced the spleen index.The effect of RTB on the splenocytes proliferation was in a dose-dependent manner. The secretion of the IL-2, IL-4, IFN-γ,IL-5, and TNF-α cytokines was measured using a cytometric bead array immunoassay(CBA). After culturing spleen cells in vitro, the highest concentrations of TNF-α, IL-2,and IFN-γ that were induced were significant compared to the concentrations induced bythe control group. No significant differences were observed in the IL-4and IL-5cytokinelevels produced by experimental and control groups.CD4+T cell immunomagnetic negativeseparation and purification beads were used to study the CD4+T cell proliferation andcytokines secretion induced by recombinant RTB protein. The results showed that the RTBprotein can promote CD4+T cell proliferation and Th1-type cytokine secretion. Flowcytometry is adopted to test the number of cell subsets of CD4+and CD8+T, which showsthat RTB can promote the number of CD4+T, but have no significant effect on CD8+T.Flow cytometry is adopted to test the expression of CD25molecule, the mid-term activationmarker of mice's CD3+T cell, as a result of which RTB acting alone or cooperating withConA can both stimulate the expression of CD25molecule (P<0.01). Alarm blue approachis used to test and purify the proliferation activity of CD4+T cell, wherein the resultindicates that RTB can improve the purification of the proliferation activity CD4+T cellsubsets no matter it acts alone or cooperates with CD3monoclonal antibodies or CD28monoclonal antibodies (P<0.05).
3. RTB protein induced mouse macrophages activation
We researched RTB-induced effect on production of NO in mice macrophagic cell lineRAW264.7cells. It turns out that RTB can induce the release of NO in RAW264.7cells. Andit was dose-dependent and time-dependent. The expression of iNOS mRNA was rised with theincreasing density of RTB. Western blot confirm the expression of iNOS protein was rised asthe same reason. It suggested that RTB can induce the expression of iNOS, which can inducethe increasing immunoloregulation. RTB can also induce TNF-α and IL-6production, and thereis a time and dose-dependent relationship.We used flow cytometry to detect fluorescentmicrospheres engulfed by RAW264.7cells after stimulated with RTB, founding thatcomparing with the control, the percentage of fluorescent microspheres engulfed by RAW264.7cells had improved significantly.We used the RD mouse protein chip40proteins associated with inflammation to observed cytokine and chemokines changes. The resultsshowed that the expression of cytokine MCP-1、TNF-α、MIP-1α、 MIP-1β、 MIP-2、RANTES、 TIMP-1and CD54were increased and the expression of chemokines G-CSF andKC were decreased.
4. Effect of signal transduction pathway on mouse macrophages activationinduced by RTB protein
RAW264.7cells treated with in the presence of genistein, SB203580, LY294002,or staurosporine exhibited reduced NO production.The MAP kinase inhibitorsPD98059and SP600125did not affect the observed RTB-induced NO production.RAW264.7cells treated with RTB in the presence of the JAK2inhibitor tyrphostin(AG490) and NF-κB inhibitor BAY showed inhibited NO production. Similar resultswere obtained for iNOS mRNA expression by RT-PCR. The production of TNF-α andIL-6induced by RTB was inhibited by the co-treatment with genistein, SB203580,LY294002, BAY, staurosporine, and AG490. Similar effects were found with regard toTNF-α and IL-6gene transcription. These data suggested the probable role of tyrosinekinases, PI3K, PKC, and JAK2in RTB-mediated macrophage activation.weperformed a phospho-proteomics-based study using a phospho-antibody microarray.Without the inhibitor,20of the228sites were positive for phosphorylation, and thehighest value was observed for JAK2Tyr221. Two tyrosines (Tyr418and529) inSRC were phosphorylated. The JAK1Tyr1022, JAK2Tyr221, STAT1Tyr701, STAT3Tyr705, STAT4Tyr693, STAT5A Tyr694, and IkB-α Tyr42phosphorylation levelswere increased. Genistein reduced the phosphorylation of16/288tyrosine sites.Phosphoproteins expression spectrum indicates that by affecting RAW264.7celltyrosine protein kinase signaling pathway SRC, JAK-STAT and NF-κB signalingpathway IκB-α protein phosphorylation, RTB activates tyrosine protein kinase andNF-κB signal transduction pathway.
5. Recombinant RTB protein induces NF-κB activation in RAW264.7cells
We further confirmed that RTB was able to promote the IκB-α phosphorylationand NF-κB nuclear translocation by using Western blot and immunofluorescence.We considered that NF-κB activation was involved in the RAW264.7cells activationinduced by recombinant RTB.
The above results demonstrate that RTB has regulating and activating functionon mice's immune cells and RAW264.7cell. The RTB's activating mechanism on RAW264.7cell may be closely related to RTB affecting tyrosine protein kinase and related proteinphosphorylation in NF-κB signal transduction pathways, so as to activate SRC, JAK-STATand NF-κB signal transduction pathways. RTB may induce macrophage activation by mutualadjustment and interaction of signal transduction pathways. Whether there are other signalpathways and related proteins participating in remains to be further studied.
Western blot和免疫荧光实验结果进一步证实RTB通过诱导巨噬细胞IκB-α磷酸化进而促进NF-κB核转位作用。
上述结果提示RTB可以调节免疫细胞活化，对巨噬细胞的活化可通过细胞内的多条信号转导途径参与。提示RTB诱导巨噬细胞活化可能通过多条信号转导途径互相调节、共同作用。本研究为RTB免疫生物学活性研究及其免疫佐剂等方面的应用提供了新的科研资料，并奠定了实验依据与理论基础。
Ricin toxicity (RT) is covalently linked by the two peptide chains RTA and RTB indisulfide bonds.RTB consists of260amino acids is a galactose/N-acetylgalactosaminelectin protein and has a molecular weight about34000. The individualism of RTB has notoxicity and is believed to have two main functions at present: One is that it can helpRTA transfer to cytosol and inhibit the synthesis of protein. Another is that RTBligand has the capacity of specific binding with the receptor of different carbohydratestructure, to mediate various kinds of bioprocess, including interactions between cell,cell host and pathogen, and the innate immunity response. Also it can be used as thecarrier of heterologous antigens to the immune system. For instance, RTB can fusedifferent Rotavims antigens such as VP7, P24, NSP4, etc, and perform as mucosaladjuvants to enhance the immune reaction of organism. Our study also found that theRTB as adjuvant can induce the production of rabies neutralizing antibody, theantibody levels were significantly higher than ordinary aluminum hydroxide adjuvantvaccine and can accelerate the immune response of the body fluid induced by rabiesvaccine.With RTB as the object, this project investigates RTB's influence on mice's Tcell and macrophage activating function as well as on the signal transductionpathways inside the macrophage, in the hope of furnishing the experimental evidenceand theoretic foundation for the research on RTB immunobiological activation.
The aspects of this research as follow:
1. Preparation of RTB
PET28a-RTB was transformed into BL21(DE3) cells to stimulate recombinantprotein synthesis and soluble protein was renaturated.The protein was purified usingnickel-NTA column chromatography. LPS was removed by Detoxi-Gel.
2. RTB protein induced mouse T cell activation
RTB was found was found to enhance the splenocytes proliferation comparedwith control. In the presence of ConA or LPS, the treatment with the RTBsignificantly enhanced the spleen index.The effect of RTB on the splenocytes proliferation was in a dose-dependent manner. The secretion of the IL-2, IL-4, IFN-γ,IL-5, and TNF-α cytokines was measured using a cytometric bead array immunoassay(CBA). After culturing spleen cells in vitro, the highest concentrations of TNF-α, IL-2,and IFN-γ that were induced were significant compared to the concentrations induced bythe control group. No significant differences were observed in the IL-4and IL-5cytokinelevels produced by experimental and control groups.CD4+T cell immunomagnetic negativeseparation and purification beads were used to study the CD4+T cell proliferation andcytokines secretion induced by recombinant RTB protein. The results showed that the RTBprotein can promote CD4+T cell proliferation and Th1-type cytokine secretion. Flowcytometry is adopted to test the number of cell subsets of CD4+and CD8+T, which showsthat RTB can promote the number of CD4+T, but have no significant effect on CD8+T.Flow cytometry is adopted to test the expression of CD25molecule, the mid-term activationmarker of mice's CD3+T cell, as a result of which RTB acting alone or cooperating withConA can both stimulate the expression of CD25molecule (P<0.01). Alarm blue approachis used to test and purify the proliferation activity of CD4+T cell, wherein the resultindicates that RTB can improve the purification of the proliferation activity CD4+T cellsubsets no matter it acts alone or cooperates with CD3monoclonal antibodies or CD28monoclonal antibodies (P<0.05).
3. RTB protein induced mouse macrophages activation
We researched RTB-induced effect on production of NO in mice macrophagic cell lineRAW264.7cells. It turns out that RTB can induce the release of NO in RAW264.7cells. Andit was dose-dependent and time-dependent. The expression of iNOS mRNA was rised with theincreasing density of RTB. Western blot confirm the expression of iNOS protein was rised asthe same reason. It suggested that RTB can induce the expression of iNOS, which can inducethe increasing immunoloregulation. RTB can also induce TNF-α and IL-6production, and thereis a time and dose-dependent relationship.We used flow cytometry to detect fluorescentmicrospheres engulfed by RAW264.7cells after stimulated with RTB, founding thatcomparing with the control, the percentage of fluorescent microspheres engulfed by RAW264.7cells had improved significantly.We used the RD mouse protein chip40proteins associated with inflammation to observed cytokine and chemokines changes. The resultsshowed that the expression of cytokine MCP-1、TNF-α、MIP-1α、 MIP-1β、 MIP-2、RANTES、 TIMP-1and CD54were increased and the expression of chemokines G-CSF andKC were decreased.
4. Effect of signal transduction pathway on mouse macrophages activationinduced by RTB protein
RAW264.7cells treated with in the presence of genistein, SB203580, LY294002,or staurosporine exhibited reduced NO production.The MAP kinase inhibitorsPD98059and SP600125did not affect the observed RTB-induced NO production.RAW264.7cells treated with RTB in the presence of the JAK2inhibitor tyrphostin(AG490) and NF-κB inhibitor BAY showed inhibited NO production. Similar resultswere obtained for iNOS mRNA expression by RT-PCR. The production of TNF-α andIL-6induced by RTB was inhibited by the co-treatment with genistein, SB203580,LY294002, BAY, staurosporine, and AG490. Similar effects were found with regard toTNF-α and IL-6gene transcription. These data suggested the probable role of tyrosinekinases, PI3K, PKC, and JAK2in RTB-mediated macrophage activation.weperformed a phospho-proteomics-based study using a phospho-antibody microarray.Without the inhibitor,20of the228sites were positive for phosphorylation, and thehighest value was observed for JAK2Tyr221. Two tyrosines (Tyr418and529) inSRC were phosphorylated. The JAK1Tyr1022, JAK2Tyr221, STAT1Tyr701, STAT3Tyr705, STAT4Tyr693, STAT5A Tyr694, and IkB-α Tyr42phosphorylation levelswere increased. Genistein reduced the phosphorylation of16/288tyrosine sites.Phosphoproteins expression spectrum indicates that by affecting RAW264.7celltyrosine protein kinase signaling pathway SRC, JAK-STAT and NF-κB signalingpathway IκB-α protein phosphorylation, RTB activates tyrosine protein kinase andNF-κB signal transduction pathway.
5. Recombinant RTB protein induces NF-κB activation in RAW264.7cells
We further confirmed that RTB was able to promote the IκB-α phosphorylationand NF-κB nuclear translocation by using Western blot and immunofluorescence.We considered that NF-κB activation was involved in the RAW264.7cells activationinduced by recombinant RTB.
The above results demonstrate that RTB has regulating and activating functionon mice's immune cells and RAW264.7cell. The RTB's activating mechanism on RAW264.7cell may be closely related to RTB affecting tyrosine protein kinase and related proteinphosphorylation in NF-κB signal transduction pathways, so as to activate SRC, JAK-STATand NF-κB signal transduction pathways. RTB may induce macrophage activation by mutualadjustment and interaction of signal transduction pathways. Whether there are other signalpathways and related proteins participating in remains to be further studied.
引文
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