Homocysteine Stimulation of T Cell Function & Apoptosis

同型半胱氨酸（Hcy）是氨基酸蛋氨酸的直接前体。在人类中，由于叶酸、维生素B6或维生素B12的缺乏，血液中Hcy的浓度可能会升高，最近已被确定为许多与年龄相关的疾病状态的假定危险因素，包括动脉硬化、心肌梗死、动脉闭塞性疾病、阿尔茨海默病和神经管缺陷。Hcy或其任何代谢物，如s -腺苷型Hcy （SAH）或Hcy硫代内酯，在这些情况下的具体作用尚未确定。尤其缺乏对Hcy水平升高对免疫功能影响的描述。一些研究已经检查了Hcy对单核细胞、中性粒细胞和B细胞功能、炎症和趋化因子产生的影响；然而，我们对Hcy对T淋巴细胞的作用知之甚少。我们的初步研究表明，用Hcy（100-1000微摩尔）治疗静息的人T细胞导致凋亡细胞死亡的剂量依赖性增加。在这方面，Hcy比Hcy硫内酯更有效，而SAH在许多情况下活性明显较低或无活性。我们还发现，在细胞培养中加入血清、泛半胱天冬酶抑制剂和聚adp核糖聚合酶（PARP）抑制剂后，Hcy的促凋亡作用被消除。这些结果表明，D，L-Hcy与其他凋亡应激因子一样，导致caspase级联的激活，最终导致关键细胞蛋白（如PARP）的裂解，最终导致凋亡细胞中典型的形态学变化。我们还发现hcy介导的细胞凋亡受到磷酸酶抑制剂原钒酸钠、细胞内钙螯合剂BAPTA-AM和蛋白质合成抑制剂环己亚胺的抑制。此外，我们还发现Hcy似乎可以增强由许多其他已确定的凋亡信号诱导的细胞死亡，包括激活诱导细胞死亡（AICD）、热休克、Fas配体和hiv介导的T细胞死亡。除了Hcy的促凋亡作用外，在Hcy或硫内酯（而非SAH）存在的情况下，用固定的抗cd3单抗刺激单个核细胞或分离的T细胞，可导致几种1型细胞因子（包括IL-2、ifn - γ、tnf - α和IL-10）的显著增加，但2型细胞因子（IL-4或IL-5）的显著增加。对Hcy对T细胞活化的影响的更详细的研究表明，这种1型细胞因子的产生部分是通过产生IL-18和可能的IL-12介导的。这些细胞因子产生的确切机制目前还在研究中，但我们认为Hcy效应部分是由Hcy治疗后的特定应激相关信号介导的。总的来说，Hcy似乎对免疫细胞产生了许多不同的影响，这可能会随着年龄和疾病病理改变循环和组织微环境中的免疫功能。更深入地了解Hcy及其代谢物对免疫功能的潜在调节作用，可能会导致开发潜在的治疗策略，以控制和优化年龄、艾滋病和各种年龄相关疾病状态下的免疫反应。

Homocysteine (Hcy) is the immediate precursor of the amino acid, methionine. In humans, blood concentrations of Hcy may become elevated as a result of deficiency in folate, vitamin B6 or vitamin B12 and has recently been identified as a putative risk factor for a number of age-associated disease states including arteriosclerosis, myocardial infarction, arterial occlusive disease, Alzheimer's disease and neural tube defects. A specific role for Hcy or any of its metabolites, such as S-adenosyl Hcy (SAH) or Hcy thiolactone, in these conditions has not yet been firmly established. Particularly absent is a description of the effects of elevated Hcy levels on immune function. Several studies have examined the effects of Hcy on monocyte, neutrophil and B cell function, inflammation and chemokine production; however, little is known about the Hcy effects on T lymphocytes. Our initial studies revealed that treatment of resting human T cells with Hcy (100-1000 micromolar) resulted in a dose-dependent increase in apoptotic cell death. D, L Hcy was more potent than Hcy thiolactone in this respect while SAH was found to be significantly less active or inactive in many cases. We also found that the pro-apoptotic effects of Hcy were abrogated with the addition of serum, pan-caspase inhibitors, and poly-ADP-ribose polymerase (PARP) inhibitors to the cell cultures. These results suggest that D,L-Hcy, like other apoptotic stressors, leads to the activation of the caspase cascade and eventually to the cleavage of the key cellular proteins, like PARP, eventually leading to the typical morphological changes observed in cells undergoing apoptosis. We have also found that Hcy-mediated apoptosis is inhibited by the phosphatase inhibitor, sodium orthovanadate, the intracellular calcium chelator, BAPTA-AM, and the protein synthesis inhibitor, cycloheximide. Moreover, we have also found that Hcy appears to potentiate cellular death induced by a number of other established apoptotic signals including activation-induced cell death (AICD), heat shock, and Fas ligand- and HIV-mediated T cell death. In addition to the pro-apoptotic effects of Hcy, stimulation of mononuclear cells or isolated T cells with immobilized anti-CD3 mAb in the presence of Hcy or thiolactone but not SAH resulted in a significant increase in several type 1 cytokines including IL-2, IFN-gamma, TNF-alpha and IL-10 but not the type 2 cytokines, IL-4 or IL-5. More detailed examination of the Hcy effects on T cell activation revealed that this type 1 cytokine production profile is mediated, in part, through the production of IL-18 and possibly IL-12. The precise mechanism involved in the generation of these cytokines is currently under investigation but we believe the Hcy effect is being mediated, in part, by specific stress-associated signals post Hcy treatment. Overall, Hcy appears to exert a number of differential effects on immune cells, which may alter immune function in the circulation and tissue microenvironment with age and disease pathology. A greater understanding of the potential modulatory effects of Hcy and its metabolites on immune function may result in the development of potential therapeutic strategies to control and optimize immune responses with age, AIDS and in various age-associated disease states.