免疫细胞内源性儿茶酚胺的免疫调节作用

机体内儿茶酚胺（catecholamines,CAs）包括去甲肾上腺素（norepinephrine,NE）、肾上腺素（epinephrine,E）和多巴胺（dopamine,DA）。CAs由神经元和内分泌细胞合成和分泌,其主要功能是调节心血管、呼吸和消化等内脏活动。近三十年来的研究说明,CAs也参与调控机体的免疫功能,但CAs的这种免疫调节作用一般视为神经和内分泌系统调节的介导作用。然而,近年来的研究发现,免疫细胞也能合成CAs,这是对传统观念的一种补充和提高。免疫细胞内存在经典的CAs代谢途径,既有合成CAs的酪氨酸羟化酶（tyrosine hydroxylase,TH）又有降解CAs的单胺氧化酶（monoamine oxidase,MAO）和儿茶酚氧位甲基移位酶（catechol-O-methyl transferase,COMT）。免疫细胞合成的内源性CAs可以调控细胞的增殖、分化、凋亡和细胞因子生成等多种免疫功能。CAs的这些作用可能主要通过自分泌或旁分泌途径作用于免疫细胞上相应受体和细胞内环磷酸腺苷（cyclicAMP,cAMP）实现。细胞内氧化应激机制可能也参与免疫细胞内源性CAs的免疫调节作用。此外,一些自身免疫性疾病如多发性硬化、风湿性关节炎可能也与免疫细胞内CAs的代谢异常有关。上述发现不仅为免疫系统有可能成为除神经和内分泌系统以外的第三个CA能系统提供了证据,而且为免疫系统内源性CAs的功能意义拓展了认识。     

儿茶酚胺调节免疫功能的细胞和分子机制

目前认为儿茶酚胺(cateeholamines,CAs)不只是引起普遍的免疫抑制,而是抑制细胞免疫但促进体液免疫。CAs可抑制1型辅助T(T helper 1,Th1)细胞、细胞毒性T(T cytotoxie,Tc)细胞、自然杀伤(natural kiHer,NK)细胞和单核细胞的作用,并增强Th2和B细胞的作用。CAs通过免疫细胞上的β2-肾上腺素受体(β2-adrenoreceptors,β2-ARs)引起细胞内cAMP增加,cAMP激活蛋白激酶A(protein kinase A,PKA),后者调节核转录因子的活性,从而影响细胞因子的基因表达,即抑制白介素-2(interleukin-2,IL-2)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和IL-12的基因转录;增强IL-6、IL-10和IL-4的基因表达。     

小脑顶核损毁对淋巴细胞功能的影响

目的：研究小脑顶核对淋巴细胞功能的调节作用,并初步探讨介导这种调节的中枢途径。方法：用海人酸（KA）毁损大鼠双侧小脑顶核,于术后第8d,取动物肠系膜淋巴结细胞和脾脏自然杀伤（NK）细胞进行体外培养,分别用四甲基偶氮唑（MTT）比色法检测由刀豆蛋白A（Con A）诱导的淋巴细胞的增殖反应,用流式细胞术测定NK细胞杀伤YAC-1肿瘤细胞的活性。同时用高效液相色谱法检测下丘脑中兴奋性神经递质谷氨酸的含量。结果：小脑双侧顶核注入KA后的第8d,小脑切片经Nissl染色,可见顶核内神经元胞体被有效破坏。此时,淋巴细胞对Con A诱导的增殖反应较双侧顶核注入生理盐水的对照组明显增强;而且NK细胞对YAC-1靶细胞的杀伤活性也明显高于对照组;同时下丘脑中谷氨酸含量较对照组明显减少。结论：小脑双侧顶核毁损可导致T和NK淋巴细胞功能明显增强,且下丘脑中谷氨酸含量显著下降,提示小脑顶核对淋巴细胞功能具有调节作用,小脑-下丘脑的谷氨酸能神经投射可能介导小脑顶核的免疫调节作用。     

小脑间位核对淋巴细胞功能的调节作用

目的:研究小脑深部核团之一间位核对淋巴细胞功能的调节作用,以拓宽对小脑功能的认识进而增加神经免疫学的知识.方法:在大鼠双侧小脑间位核内注射海人酸(KA)以损毁间位核内神经元的胞体,并设对照组,于小脑间位核内注入等量生理盐水.在手术后的第8、16、32 d分别用血细胞计数法检测动物外周血中淋巴细胞的数量;用四甲基偶氮唑(MTT)比色法检测动物肠系膜淋巴结细胞对刀豆蛋白A(Con A)刺激的增殖反应;用ELISA法检测动物血清中抗绵羊红细胞(SRBC)特异性IgM抗体的生成能力;用流式细胞术测定脾脏自然杀伤(NK)细胞的活性.结果:小脑间位核损毁后的第8、16、32 d,动物外周血中淋巴细胞数都明显低于损毁手术前的淋巴细胞数,也显著低于生理盐水对照组相应时间段的淋巴细胞数.在小脑间位核注射KA后的第8、16、32 d,动物的肠系膜淋巴结细胞由Con A诱导的增殖反应、血清中特异性抗SRBC IgM抗体的生成能力和脾脏NK细胞杀伤靶细胞YAC-1的活性均明显低于生理盐水对照组,但比较损毁后不同时间段的T、B和NK细胞功能的变化,没有发现显著的差异.结论:小脑双侧间位核损毁可导致总淋巴细胞数以及T、B和NK细胞功能均发生不可逆的降低,充分说明小脑间位核可调节淋巴细胞的功能,并提示在正常体内,小脑间位核对淋巴细胞功能具有增强效应.     

儿茶酚胺神经递质对促性腺激素释放激素分泌的影响

下脑脑儿茶酚胺神经递质是调节促性腺激素释放激素合成和分泌的重要因素。形态学和药理学证据以及离体实验都证明,儿茶酚胺类神经递质通过直接的突触联系或通过间接的途径影响ＧｎＲＨ释放,并参与ＧＧｎＲＨ的基因;儿茶酚胺的调节作用与体内类固醇水平有关。     

阿片受体介导大鼠海马内脑啡肽对细胞免疫功能的调节

以刀豆蛋白Ａ（ＣｏｎＡ）刺激的脾淋巴细胞增殖活性及自然杀伤细胞（ＮＫｃｅｌ）活性为细胞免疫功能检测指标,观察了阿片受体阻断剂纳洛酮（Ｎａｌｏｘｏｎｅ,ＮＬＸ）对大鼠海马内微量注射甲硫脑啡肽所致的免疫功能增强作用的影响。结果发现：（１）海马内微量注射白细胞介素１（ＩＬ１）诱导剂（细菌内毒素）脂多糖（ｌｉｐｏｐｏｌｙｓａｃｈａｒｉｄｅ,ＬＰＳ,５０ｎｇ／１μｌ）可降低机体免疫功能。（２）双侧海马内预先注射甲硫脑啡肽（ＭＥＮＫ,浓度：１０μｇ／μｌ）各１μｌ,可阻止脑内ＬＰＳ降低免疫功能的作用。（３）脑啡肽的这种作用可被阿片受体阻断剂纳洛酮（１０μｇ／１μｌ）阻断。（４）海马内单纯注射纳洛酮对机体免疫功能也起抑制作用。上述结果提示,海马内脑啡肽对免疫功能的增强作用是通过阿片受体介导的。     

胶原诱导性关节炎小鼠CD4^＋T细胞亚群表达酪氨酸羟化酶的作用

目的：本研究应用胶原诱导性关节炎（CIA）的动物模型,通过研究CD4＋T细胞亚群表达酪氨酸羟化酶（TH）的变化与作用,探讨CIM＋T细胞亚群来源的儿茶酚胺与CIA的炎症反应之间的关系。方法：雄性DBA／1小鼠36只随机分为对照组、35天模型组和55天模型组（n=12）。用Ⅱ型胶原（cⅡ）乳剂免疫DBA／1小鼠诱导CIA,在初次免疫后第35天和55天进行关节临床评分并检测血清中抗cⅡ IgG抗体水平的变化。用Western blot法检测肠系膜淋巴结中1h1、Th17、Th2和Treg细胞的特异性转录因子及其细胞因子以及TH表达的变化。用流式细胞术检测肠系膜淋巴结中表达TH的CD4＋T细胞亚群数目的变化。结果：CIA小鼠在发病早期（初次免疫后第35天）和发病晚期（初次免疫后第55天）临床评分和血清中抗cⅡ IgG抗体水平显著升高。CIA小鼠肠系膜淋巴结中1h1和Th17细胞的特异性转录因子和细胞因子表达增加而Th2和Treg细胞的细胞因子表达减少。CIA小鼠肠系膜淋巴结中TH的表达增加,且CD4＋T细胞中TH＋的细胞数目增多,这主要是来自CD4＋T细胞亚群中Thl和rIh17细胞的作用。结论：CIA小鼠肠系膜淋巴结中存在CIM＋T细胞亚群来源的儿茶酚胺的增加,可能在cn的发展过程中具有一定的抗炎作用。     

去甲肾上腺素对T淋巴细胞增殖的影响

本实验研究了不同浓度和不同作用时间的去甲肾上腺素（ＮＡ）对大鼠脾脏Ｔ淋巴细胞增殖的影响,并探讨α和β受体在此影响中的作用。结果表明,１０＾－８－１０＾－５ｍｏｌ／ＬＮＡ可显著抑制由刀豆素Ａ诱导的Ｔ细胞增殖反应,其中１０＾－７ｍｏｌ／ＬＮＡ的抑制作用最强。ＮＡ与已被ＣｏｎＡ刺激１ｈ的Ｔ细胞作用后,或者ＮＡ与脾细胞作用１ｈ后被洗去,Ｔ细胞的增殖仍受到抑制,但是ＮＡ不能抑制已被ＣｏｎＡ刺激６小时的Ｔ细胞     

去甲肾上腺素对骨髓间充质干细胞增殖的影响

目的:观察去甲肾上腺素(norepinephrine,NE)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖的影响及其作用途径.方法:分离培养正常大鼠BMSCs,采用3H-TdR掺入实验检测不同浓度的NE(10-7-10-4 M)作用8h及10-5M的NE作用不同时间(0-24h)BMSCs细胞增殖情况,real time RT-PCR检测肾上腺素能受体α1A-AR,α1B-AR和α1D-AR mRNA表达变化情况.结果:10-7-10-4M的NE作用8h后均促进了BMSCs细胞的增殖.并且在10-5M时NE对BMSCs的促增殖效应最为显著;正常组BMSCs细胞的α1A-AR,α1B-AR,α1D-AR mRNA表达维持在较低水平,加入10-5M的NE作用后α1-AR三个亚型mRNA表达水平均有不同程度的升高(P<0.05).结论:NE能够促进BMSCs的增殖,并且这种促增殖作用是通过AR依赖的信号通路来调节的.     

甲状腺素对小鼠IgM的产生及淋巴细胞上β—肾上腺素能受体的影响

本文利用实验性“甲高”和“甲低”小鼠模型,观察了甲状腺素(L-T4)对绵羊红细胞(SRBC)诱导小鼠脾细胞产生 IgM 的影响。并采用放射配体结合法,测定了不同甲状腺机能状态的小鼠在免疫反应过程中,脾淋巴细胞上 β-肾上腺素能受体(β-Adr.R.)的密度及亲和力的变化。结果发现,用 SRBC 免疫后第3—5天,甲高小鼠脾细胞产生的 IgM 及脾淋巴细胞上β-Adr.R.密度的增加均明显高于对照小鼠(P<0.01),甲低小鼠则相反,其 IgM 的生成及脾淋巴细胞上β-Adr.R.密度的增加均明显低于对照小鼠(P<0.01),受体亲和力不随小鼠甲状腺机能状态及免疫状态的改变而改变(P>0.05)。以上结果提示,L-T_4能促进小鼠对SRBC 的免疫反应,其机制可能与淋巴细胞上 β-Adr.R.的密度变化有关。     

Newly synthesized AIFM1 determines the hypersensitivity of T lymphocytes to STING activation-induced cell apoptosis

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Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.     

Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 microg/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.     

Immortalization of human T lymphocytes by oncogenes

Immortalized human T cell lines were established by cotransfecting c-Ha-ras and c-myc oncogenes to lymph node lymphocytes. The cell lines kept growing for 3 months after establishment without a decrease in growth rate. The cells did not require interleukin-2(IL-2) for their growth, but addition of IL-2 stimulated the growth of these cells. Flow cytometric analysis revealed that these cells were T cells expressing CD4 or CD8 antigens. A CD4 positive (CD4⁺) cell line produced IL-6, indicating that the cell line belongs to helper T cells. The CD8 positive (CD8⁺) cell line possessed cytotoxicity to tumor cells, indicating that the cell line were killer T cells. Both cell lines were able to proliferate in serum-free medium indefinitely.     

Anandamide is an endogenous inhibitor for the migration of tumor cells and T lymphocytes

Cell migration is of paramount importance in physiological processes such as immune surveillance, but also in the pathological processes of tumor cell migration and metastasis development. The factors that regulate this tumor cell migration, most prominently neurotransmitters, have thus been the focus of intense investigation. While the majority of neurotransmitters have a stimulatory effect on cell migration, we herein report the inhibitory effect of the endogenous substance anandamide on both tumor cell and lymphocyte migration. Using a collagen-based three-dimensional migration assay and time-lapse videomicroscopy, we have observed that the anandamide-mediated signals for CD8⁺ T lymphocytes and SW 480 colon carcinoma cells are each mediated by distinct cannabinoid receptors (CB-Rs). Using the specific agonist docosatetraenoylethanolamide (DEA), we have observed that the norepinephrine-induced migration of colon carcinoma cells is inhibited by the CB₁-R. The SDF-1–induced migration of CD8⁺ T lymphocytes was, however, inhibited via the CB₂-R, as shown by using the specific agonist JWH 133. Therefore, specific inhibition of tumor cell migration via CB₁-R engagement might be a selective tool to prevent metastasis formation without depreciatory effects on the immune system of cancer patients.     

Signal transduction through the T cell receptor is dynamically regulated by balancing kinase and phosphatase activities

Within seconds of T cell receptor engagement, a well-characterized series of tyrosine phosphorylation events mediate cellular activation. It is widely accepted that these initial phosphorylations remain stable until protein tyrosine phosphatases return the cell to its basal level. Based on a model of peripheral blood T cell activation, in which the kinetics of phosphorylation can be modulated, we propose an alternate hypothesis that T cell signaling is highly dynamic. Our results demonstrate that tyrosine phosphorylation and dephosphorylation are occurring co-temporally after T cell receptor cross-linking, regulated by a delicate balance of kinases and phosphatases.     

Roles of CD147 on T lymphocytes activation and MMP-9 secretion in systemic lupus erythematosus

The cellular and molecular mechanisms involved in many abnormalities described in Systemic Lupus Erythematosus (SLE) are still unclear. Some of these abnormalities referred to the hyperactivation of T lymphocytes and the enhanced secretion of MMP-9 by peripheral blood mononuclear cells (PBMCs). Therefore, in this paper we investigated the potential role of CD147 molecule in these abnormalities. Our results demonstrated that CD147 molecule is overexpressed on CD3+T lymphocytes from SLE patients when compared with CD3+T lymphocytes from healthy donors. Monoclonal anti-CD147 antibodies, MEM-M6/1 clone, were able to inhibit protein tyrosine phosphorylation only in CD3 x CD28 costimulated T lymphocytes from SLE patients. However, this monoclonal antibody was unable to inhibit the enhanced activity of MMP-9 secreted by SLE PBMCs.