AC—cAMP—PKA途径对淋巴细胞DNA合成的调节作用

以ＣＤ３ＭｃＡｂ为激动机，以淋巴细胞体外ＤＮＡ合成的研究手段，探讨了ＡＣｃＡＭＰ－ＰＫＡ信号途径在ＣＤ３ＭｃＡｂ诱导的淋巴细胞活化中的意义，研究结果表明ＡＣ，ｃＡＭＰ和ＰＫＡ在决定细胞对外界刺激反应中起着重要作用，在淋巴细胞活化早期细胞内ｃＡＭＰ出现一过性升高，随着细胞活化增殖，ｃＡＭＰ降至正常水平以下，活化ＡＣ，升高细胞内，ｃＡＭＰ水平可显著过性升高，随着细胞活化增殖，ｃＡＭＰ降至正常水平以下，     

淋巴细胞经TCR－CD3活化增殖作用的分析

本文探讨了抗ＣＤ３单抗诱导的淋巴细胞活化增殖及有关影响因素。实验结果表明：①淋巴细胞内钙升高是淋巴细胞活化增殖的重要条件，ＣＤ３ＭｃＡｂ引起的早期胞浆游离钙迅速升高主要由内质网释放钙离子所致，而淋巴细胞增殖不仅需要细胞内钙释放，还需要细胞外钙内流；②ＧＴＰ结合蛋白是淋巴细胞激活过程的一重要环节，经Ｇ蛋白作用物霍乱毒素作用后，淋巴细胞ＤＮＡ合成显著降低；③新霉素和ＰＳＳ可抑制ＰＬＣ和ＰｋＣ的活性，对淋巴细胞ＮＤＡ合成造成剂量依赖性抑制作用。此外，抗ＣＤ３ＭｃＡｂ诱导的淋巴细胞ＤＮＡ合成需要辅佐细胞的存在，高度纯化的Ｔ细胞对ＣＤ３ＭｃＡｂ的刺激不发生增殖反应。     

淋巴细胞经TCR—CD3活化增殖作用的分析

本文探讨了抗ＣＤ３单抗诱导的淋巴细胞活化增殖及有关影响因素。实验结果表明：①淋巴细胞内钙升高是淋巴细胞活化增殖的重要条件，ＣＤ３ＭｃＡｂ引起的早期胞浆游离钙迅速升高主要由内质网释放钙离子所致，而淋巴细胞增殖不仅需要细胞内钙释放，还需要细胞外钙内流；②ＧＴＰ结合蛋白是淋巴细胞激活过程的一重要环节，经Ｇ蛋白作用物霍乱毒素作用后，淋巴细胞ＤＮＡ合成显著降低；③新霉素和ＰＳＳ可抑制ＰＬＣ和ＰＫＣ的活性，对     

霍乱毒素对淋巴细胞活化增殖的影响及其机制

研究探讨霍乱毒素（ＣＴＸ）对淋巴细胞活化、增殖的影响及可能的作用机制。研究结果表明，　　ＣＴＸ可显著升高静息ＰＢＭＣ和／或ＣＤ３单抗激活的琳巴细胞内ｃＡＭＰ水平，该效应与ＣＴＸ抑制淋巴细胞增殖和ＩＬ－２的作用密切相关；　　ＡＣ激活剂Ｆｏｒｓｋｏｌｉｎ通过升高细胞内ｃＡＭｐ水平，对于ＣＤ３单抗激活的淋巴细胞增殖有明显的抑制作用；ＣＴＸ不能干扰ＩＬ－２依赖株ＣＴＬＬ细胞的增殖效应，也不影响ＣＴＬＬ细胞内ｃＡＭＰ水平。实验结果提示，ＣＴＸ敏感的Ｇ蛋白亚类参与了ＣＤ３单抗对淋巴细胞活化的调节，ＣＴＸ的抑制作用与其升高细胞内ｃＡＭｐ水平有关。     

创伤后活化T细胞内cAMP代谢,蛋白激酶A活性的变化及意义

研究了创伤小鼠活化Ｔ细胞内ｃＡＭＰ代谢、蛋白激酶Ａ（ＰＫＡ）活性的变化及同Ｔ细胞功能的关系。结果显示，创伤后活化Ｔ细胞内ｃＡＭＰ含量增加，这一变化同创伤后Ｔ细胞白介素２（ＩＬ－２）生成减少，ＩＬ－２受体（ＩＬ－２Ｒ）表达受抑，Ｔ淋巴细胞转化（ＴＬＴ）降低密切相关。创伤后活化Ｔ细胞内腺苷酸环化酶（ＡＣ）、ＰＡＫ活性增加，ｃＡＭＰ－磷酸二酯酶（ｃＡＭＰ－ＰＤＥ）活性降低。ＰＫＡ抑制剂Ｈ－８在体外可明显     

CD4单克隆抗体在体内诱导小鼠胸腺细胞凋亡

为研究抗ＣＤ４ ＭｃＡｂ引起胸腺细胞减少的机理，本课题探讨了ＣＤ４ ＭｃＡｂ诱导胸腺细胞发生凋亡的可能性。小鼠注射抗ＣＤ４ ＭｃＡｂ后，皮质胸腺细胞体积缩小，染色质凝聚；ＰＩ染色后经流式细胞计测定，显示大量的亚二倍体细胞（凋亡细胞，Ａｐｏｐｔｏｔｉｃ ｃｅｌｌｓ），与正常小鼠相比Ｐ〈０．００１；片断化ＤＮＡ的百分比也明显高于正常小鼠，Ｐ〈０．０１。片断化ＤＮＡ在凝胶电泳上呈现典型的阶梯现象。细胞内     

膜辅蛋白─补体活化调节剂家族新成员

膜辅蛋白（ＭＣＰ，ＣＤ４６）是一种广泛分布于Ｃ３ｂ／Ｃ４ｂ结合细胞表面的糖蛋白。它属于补体活化调节剂（ＲＣＡ）基因簇家族。ＭＣＰ在ＳＤＳ－ＰＡＧＥ上呈两条完全不同的带，其表达量在不同的细胞类型有所不同。已用探针从ｃＤＮＡ文库中获得了其ｃＤＮＡ片段并测序，基因组构成也已确定。ＭＣＰ在补体调控途径中发挥很重要的作用，它可与Ｃ３结合，并具有Ⅰ因子依赖性辅因子活性。     

抗Thy－1单克隆抗体的免疫抑制作用

从体内、体外研究了抗Ｔｈｙ－１单克隆抗体（ＭｃＡｂ）的免疫抑制作用。结果表明：（１）体外抗Ｔｈｙ－１ＭｃＡｂ在补体参与下能够杀伤小鼠胸腺细胞，无补体时能抑制ＣｏｎＡ诱导的Ｔ淋巴细胞增殖反应；（２）在体内，可以抑制小鼠脾细胞对ＣｏｎＡ和ＰＨＡ诱导的Ｔ淋巴细胞增殖、但不影响ＬＰＳ诱导的Ｂ淋巴细胞增殖反应。结果说明：抗Ｔｈｙ－１ＭｃＡｂ的免疫抑制作用可能包括补体依赖性细胞毒作用和通过Ｔｈｙ－１分子对Ｔ淋巴细胞功能的抑制。     

枸杞多糖对小鼠淋巴细胞信号系统的效应

对枸杞多糖发挥免疫调节效应的信号传导系统进行探讨，结果显示，５０￣４００μｇ／ｍＬ ＬＢＰ可剂量依赖性的升高小鼠淋巴细胞内ｃＡＭＰ和ｃＧＭＰ的水平，５０μｇ／ｍｌ可升高ＰＭＡ活化的脾淋巴细胞内ｃＧＭＰ的水平，另外，１００μｇ／ｍｌ ＬＢＰ可增加ＣｏｎＡ活化后的小鼠脾淋巴细胞膜上的ＰＫＣ活性，说明ＬＢＰ的作用途径可能是通过影响ｃＡＭＰ／ｃＧＭＰ系统以及促进的ＰＫＣ活性来发挥免疫调节效应的。     

天花粉蛋白抑制TCR介导的Jurkat T细胞蛋白酪氨酸磷酸化以及PLCγ1磷酸化

目的研究天花粉蛋白（Ｔｋ）抑制Ｔ细胞增殖的作用机理。方法以抗ＣＤ３的ＭｃＡｂ刺激ＪｕｒｋａｔＴ细胞增殖，以３Ｈ－ＴｄＲ同位素掺入法观察Ｔｋ对其抑制作用。用免疫沉淀和免疫印迹法测定胞浆蛋白酪氨酸磷酸化情况。结果Ｔｋ能够抑制ＣＤ３ＭｃＡｂ刺激的ＪｕｒｋａｔＴ细胞的增殖，并且经Ｔｋ脉冲处理后的ＪｕｒｋａｔＴ细胞对ＣＤ３ＭｃＡｂ活化作用的反应降低。深入分析后发现，在Ｔｋ作用下胞浆蛋白酪氨酸磷酸化水平降低，经ＰＬＣγ１免疫沉淀后，发现ＰＬＣγ１的磷酸化也能够被Ｔｋ作用所抑制。结论Ｔｋ对Ｔ细胞活化的抑制涉及并经由ＴＣＲ的激活信号传导，其机理与阻断胞浆蛋白酪氨酸激酶磷酸化有关     

Accessory Cell-Dependent T-Cell Activation via Ti-CD3

The activation of resting T cells to interleukin 2 (IL-2) production and DNA synthesis via Ti-CD3 is dependent on accessory cells (AC). Using positively selected, resting T cells activated with particle-bound anti-CD3, we investigated the ability of various cell lines to function as AC. We found that cell lines able to act as AC all expressed LFA-3, while cell lines not expressing LFA-3 were unable to provide AC signals. This applied to CD3+, CD4+, and CD8+ T cells. Sheep red blood cells (SRBC), which express LFA-3-like molecules, also had a weak, but significant AC function in this test system. Both CD4+ and CD8+ T cells activated with particle-bound anti-CD3 could be induced to enter DNA synthesis in the absence of AC when monoclonal antibodies reacting with CD2 were present instead of AC. IL-2 production could be detected in the latter cultures but not when positively selected CD3+ or CD2+ T cells were cultured alone. Our data suggest that activation of resting T cells via CD3 will lead to IL-2 receptor expression, while the interactions between LFA-3 and its ligand CD2 provide the necessary secondary signals for IL-2 production and induction of DNA synthesis.     

Epitope-specific signaling through CD45 on T lymphocytes leads to cAMP synthesis in monocytes after ICAM-1-dependent cellular interaction

We recently demonstrated that different CD45 monoclonal antibodies (mAb) are able to induce cellular aggregation in human peripheral blood mononuclear cells (PBMC) through LFA-1/ICAM-1 interactions. Such interactions could be down-modulated by protein kinase (PK) A/G inhibitors, but were unaffected by inhibitors of PKC, suggesting the involvement of PKA or PKG in CD45 mAb-induced adhesion. In this study we show that after incubation of PBMC with several (but not all) mAb to CD45, CD45RO and CD45RA, intracellular cAMP, but not cGMP concentrations readily increase, reaching a maximum 30 min after start of activation. As evidenced by several lines of investigation cAMP accumulation was independent of Fc receptor-associated signaling as well as tyrosine phosphatase activity of CD45. In highly pure T lymphocytes, CD45 mAb were unable to induce cAMP synthesis, but readily did so after addition of autologous monocytes. After paraformaldehyde fixation of both quiescent or IFN-γ/TNF-α-preactivated monocytes, cAMP production was no longer detectable, suggesting monocytes as the cell of origin for the increased cAMP synthesis. Further, cAMP accumulation in monocytes occurred after reconstitution to T lymphocytes preincubated with CD45 mAb and extensively washed. Importantly, pretreatment of T lymphocyte/monocyte mixtures with LFA-1 mAb and/or ICAM-1 mAb down-regulated CD45 mAb-induced cAMP synthesis. Finally, we demonstrate that CD45 mAb are not only capable of inducing cAMP production, but also of directly stimulating PKA enzyme activity. Based on the data presented, we propose that CD45 signaling in T lymphocytes subsequently activates cAMP accumulation and PKA activation in monocytes via LFA-1/ICAM-1-dependent cellular interactions.     

Activation of Resting, Pure CD4+, and CD8+ Cells via CD3

We studied the requirements for secondary activation signals in pure CD4+ and CD8+ T cells after stimulation with anti-CD3 antibodies. Stimulation of CD4+ or CD8+ cells with anti-CD3 monoclonal antibodies (MoAb) bound to polystyrene monosized particles never resulted in a proliferative response. However, DNA synthesis was observed when recombinant interleukin 2 (IL-2) or other secondary signals, such as those provided by phorbol myristate acetate (PMA) or autologous accessory cells (AC), were also added. These secondary signals were not in themselves capable of inducing DNA synthesis in the absence of particle-bound anti-CD3. We also found that the signals provided by AC may be dependent on the activation state of these cells. Thus, the effects of accessory cells were enhanced by a factor present in fetal calf serum (FCS), most likely endotoxin or lipopolysaccharide (LPS), which alone, however, were not able to activate T cells, even in the presence of particle-bound anti-CD3. Recombinant IL-1 over a broad dose range was unable to replace PMA or activated AC after stimulation with particle-bound anti-CD3. Purified CD4+ and CD8+ T cells behaved identically in all the experiments, indicating that the basic mechanisms for activation in the two T-cell subsets are identical.     

TCRVβ基因亚家族的优势取用和PTK信号传导途径的激活及体外对肝癌细胞凋亡的诱导

目的：研究特异识别肝癌细胞株肿瘤相关抗原的TCRVβ基因亚家族的优势取用及对肝癌细胞凋亡的诱导。方法：流式细胞术检测淋巴细胞表型,RT-PCR和Southem印迹分析TCRVβ基因亚家族表达水平,Westem blot检测PTK含量,透射电镜观察凋亡细胞超微结构。结果：McAb共刺激T细胞与肝癌细胞混合培养后,T细胞表面CD3和CD8分子表达量明显升高,而CD4玩明显变化,TCRVβ6选择性扩增,表达水平由5%升高至13%-25%,且在第4天达到高峰,同时相应的PTK信号传导途径被激活,其含理由11%升至58%,亦在第4,5天达到高峰,以抗CD3 CD28,抗CD28 CD80,抗CD2 CD58共刺激的淋巴细胞均在体外诱导了肝癌细胞的凋亡。结论：TCRVβ7为特异的肿瘤抗原识别受体,TCR-CD4复合物与抗原结合后激活PTK信号传导途径。并诱导了肝癌细胞的凋亡。     

Cyclic AMP Sensitive Signalling by the CD28 Marker Requires Concomitant Stimulation by the T-Cell Antigen Receptor (TCR/CD3) Complex

We have previously demonstrated that activation of cAMP-dependent protein kinase (cAK) type I (cAKI, RIα₂-Cβ₂) mediates the inhibitory effects of cAMP on T-cell replication induced through the TCR/CD3 complex. In the present study we have investigated the effect of cAMP on T-cell DNA synthesis, tyrosine phosphorylation of a 100 kDa protein (pp100) and IL2 mRNA expression, induced through stimulation of the TCR/CD3- and/or the CD28 molecules. Our results demonstrate that tyrosine phosphorylation of pp100 stimulated by anti-CD3 is inhibited by cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T-cell replication. Combined stimulation with anti-CD3 and anti-CD28, which gives a synergistic response in T-cell replication, gave pp100 phosphorylation and IL2 mRNA expression sensitive to cAMP-dependent inhibition. When PMA was added in addition to anli-CD3 and anti-CD28, the inhibitory effect of cAMP on both T-cell replication and pp100 phosphorylation was completely abolished. The fact that pp100 phosphorylation in response to TCR/CD3-, CD28- and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T-cell proliferation, makes this protein an interesting candidate in downstream signalling from these receptors. In addition, our results are compatible with a model where cAMP, through activation of cAKI, eliminates both the PTK and PKC activating capability of the T-cell receptor at a site(s) proximal to PKC activation. Furthermore, the CD28 molecule which activates PTKs, enters the PTK cascade at a point distal to the target(s) for cAKI action. Therefore, during CD28 signalling PKC activation can be achieved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP)     

Cyclic AMP has the ability to influence multiple events during B cell stimulation

Negative regulators of cellular proliferation are important in maintaining a balanced growth control. In this study we have examined the effects of the diterpene forskolin on various parameters of B cell activation. Forskolin is known to elevate intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels and thereby to influence B cell stimulation. We found that forskolin exerted an inhibitory effect on early as well as late events during stimulation of resting normal human B cells. Cells were activated either by antibodies to surface immunoglobulins (anti-mu), by the monoclonal antibody 1F5 reactive with the CD20 antigen or by 12-O-tetradecanoylphorbol 13-acetate. While anti-mu stimulation induces increased phosphatidylinositol (PI) turnover and [Ca2+]i fluxes, the latter two reagents confer an activation of B cells independent of the PI/Ca2+ pathway. We found a clear inhibitory effect of forskolin on the anti-mu-induced PI turnover and [Ca2+]i fluxes as well as on later parameters of cell activation. There was also a clear inhibition of G1 entry and DNA synthesis when PI/Ca2+-independent activation was employed, indicating that cAMP interferes with B lymphocyte stimulation in several ways. Importantly, forskolin maintained its inhibitory effect when added late after anti-mu stimulation, implying an effect also at multiple stages of activation. When examining the inhibitory effect of forskolin on neoplastic B cells, we found essentially no differences from the responses in normal cells.     

Induction of Interleukin-2 Production in CD4+ and CD8+ T-Cell Subsets after Activation via CD3 and CD2

The activation signals necessary for interleukin-2 (IL-2) receptor induction, IL-2 production, and DNA synthesis in resting T cells were investigated. IL-2 receptors were induced after activation via CD2 or CD3 alone, while IL-2 production in both CD4+ and CD8+ T cells required activation via both CD3 and CD2. The sequence of activation signals via CD3 and CD2 was shown to be important since DNA synthesis was induced when the primary activation signal was delivered via CD3, and the CD2 signal within 8 h. In contrast, no DNA synthesis was demonstrated when the primary activation signal was delivered via CD2 and the CD3 signal later. Ciclosporin A (CyA) inhibited T-cell DNA synthesis after activation via CD2 and CD3. The inhibition seemed to be due to the prevention of IL-2 synthesis.     

Effect of cyclic adenosine monophosphate on the in vitro DNA synthesis in lymphocytes of patients with chronic lymphocytic leukemia

Deoxyribonucleic acid (DNA) synthesis was investigated in vitro in T and B lymphocyte populations isolated from peripheral blood. The synthesis was determined by measurements of 3H-thymidine incorporation. Only T lymphocytes isolated from peripheral blood of healthy subjects as well as patients with chronic lymphocytic leukemia (cll) showed proliferative ability after stimulation with phytohemagglutinin (PHA). DNA synthesis rate in normal T lymphocytes was enhanced by exogenous cyclic adenosine monophosphate (cAMP) added to lymphocyte culture together with PHA in concentrations of 10(-5) and 10(-4)M. Higher concentrations (10(-3) and 10(-2)M) of this nucleotide inhibited DNA synthesis in the lymphocytes of healthy subjects as well as cell patients. Impairment of 3H-thymidine incorporation observed in cll (in non-isolated cell populations) seems to be due to the presence of non-proliferating lymphocyte B population.