自身免疫甲状腺病患者血清中IL-12和IL-18水平的分析

目的:提供自身免疫甲状腺病(AITD)患者体内Th1/Th2平衡紊乱的依据。方法:应用酶联免疫吸附法(ELISA)测定27例Graves病(GD)、24例甲状腺功能正常的桥本甲状腺炎(HT)、25例甲状腺功能低下的HT患者及20例正常对照者血清中IL12和IL18的浓度,并检测GD患者的甲状腺刺激性抗体。结果:GD患者与甲状腺功能正常的HT患者血中IL12、IL18水平无明显差异,但均高于正常对照者的相应水平。甲状腺功能低下的HT患者血中IL12和IL18的水平与正常对照者无差异。在GD和甲功正常的HT,IL18与IL12呈明显正相关。在GD,IL12和IL18均与其甲状腺刺激性抗体(TSAb)活性呈正相关。在甲状腺功能正常的HT还存在IL12和IL18二者与甲状腺球蛋白抗体(TgAb)的显著性正相关。结论:提示Th1型细胞在GD和HT两种AITD的发病中均起重要作用。通过抑制Th2型免疫反应,促进向Th1型的转变来治疗GD时,有可能导致病情恶化。     

IL-25和IL-33促进Th2细胞分化的研究进展

Th2细胞介导的免疫应答是机体清除寄生虫感染和变态反应性疾病发生的主要机制.T细胞产生的一些细胞因子能够有效地调节和促进Th2细胞介导的免疫应答.最近的研究发现,肠粘膜上皮细胞分泌的IL-25和IL-33可以诱导几种新型的固有免疫细胞来促进Th2细胞的分化和激活,增加IL-4、IL-5和IL-13的分泌,从而清除体内寄生虫.     

Demonstration of the presence of IL-16, IL-17 and IL-18 at the murine fetomaternal interface during murine pregnancy

PROBLEM: To determine if interleukin-16 (IL-16), IL-17, and IL-18 are present at the murine fetomaternal interface during pregnancy as a first step towards investigating their roles in fetomaternal relationship. METHODS: Expression of IL-16, IL-17, and IL-18, was assessed by immunohistochemistry (IHC) in the BALB/c x BALB/k (H2d x H2k), and the CBA/J x BALB/c non-abortion prone, and CBA/J x DBA/2 abortion prone matings. Enzyme-linked immunosorbent assay (ELISA) were performed for the two latter cytokines to compare local production in the abortion prone CBA/J x DBA/2 versus the non-abortion prone CBA/J x BALB/c matings. RESULTS: Expression of IL-17 was borderline. The anti-IL-16 staining specifically localized in the uterine stroma and glandular epithelium and was rather low in the placenta. IL-18 staining started in the peri-implantation uterus in the basal proliferative stroma, and was also traced, although weaker, in the glandular epithelium. In the immediate post-implantation period, a weak stromal staining persisted but there was a strong labeling of the ectoplacental cone. Interestingly, when the ectoplacental cone differentiates into placenta having a major histocompatibility complex (MHC) class I + spongiotrophoblast and a (MHC class I-) labyrinth, a very strong transient labeling of uterine natural killer (u-NK) cells was found. Later in gestation, IL-18 was also produced by giant cell and spongiotrophoblast. Finally, we compared by ELISA the production of IL-17/-18 in CBA/J x DBA/2 and CBA/J x BALB/c matings. We detected significantly more IL-18 in the non-abortion prone combination decidua or placenta. CONCLUSION: The three cytokines IL-16, IL-17, and IL-18 were detected at the fetomaternal interface with a tissue specific, stage-dependent distribution. The predominance of IL-18 secretion in the non-resorption prone matings lead us to question the general validity of the classical T-helper (Th)1/2 paradigm.     

IL-18BP对急性移植物抗宿主病的影响及机制研究

目的：探讨白细胞介素18结合蛋白（IL-18BP）对小鼠异基因骨髓移植后急性移植物抗宿主病（aGVHD）的影响及作用机制.方法：C57BL/6小鼠随机分为A、B 2组,经致死量^60Coγ射线照射后,经尾静脉移植入BALB/c小鼠骨髓细胞和脾脏细胞.移植后,A组注射NS;B组注射IL-18BP.比较A、B 2组小鼠aGVHD的程度,酶联免疫法（ELISA）测定血清中Th1类细胞因子干扰素-γ（IFN-γ）、白细胞介素2（IL-2）及Th2类细胞因子白细胞介素10（IL-10）、白细胞介素4（IL-4）的浓度.结果：移植后,B组小鼠较A组小鼠的aGVHD程度轻,血清IFN-γ、IL-2的浓度低,血清IL-10、IL-4的浓度高.结论：①IL-18BP可以减轻异基因骨髓移植后aGVHD反应;②IL-18BP促进Th1/Th2平衡向Th2漂移,可能是IL-18BP减轻aGVHD反应的机制之一.     

荧光定量-逆转录-聚合酶链反应测定IL-2mRNA和IL-4 mRNA方法的建立及其应用

目的建立荧光定量-逆转录-聚合酶链反应（FQ-RT-PCR）测定TH细胞中IL-2 mRNA和IL-4 mRNA方法，并作临床初步应用。方法制备IL-2 cDNA和IL-4 cDNA，分别构建含有人IL-2基因和叫基因片段的pMD18-T质粒，克隆后作为定量阳性模板；设计和制备用于FQ-RT-PCR的引物和FAM、TAMRA标记的探针，优化实验条件，建立FQ-RT-PCR方法；用固相单抗方法从健康人、妇科良性疾患和恶性肿瘤患者以及慢性肾功能衰竭（cRF）患者PBMC中富集CD4（TH）细胞，经PMA和calcium ionophore诱导，提取总RNA，继用所建FQ-RT-PCR方法，作IL-2 mRNA和IL-4 mRNA定量测定。实验设阻actin为内控基因以监测RNA的质量。结果根据系列模板浓度的对数与CT值作直线回归建立标准曲线，线性范围为10^2～10^7copies/μl；不精密度试验显示，高值样品的批内、批间CV分别为7．8％和12．5％，低值样品的批内、批间CV分别为10．8％和19．5％；妇科恶性肿瘤患者与健康对照组和妇科良性疾患组比较，CRF患者与健康对照组比较，IL-2 mRNA表达均显著降低，IL-4 mRNA表达均显著升高（P〈0．001）。结论用所建立的FQ-RT-PCR法可对TH细胞内IL-2 mRNA和IL-4 mRNA作定量测定，方法简便、敏感、准确；妇科恶性肿瘤患者和CRF患者TH细胞内IL-2mRNA和IL-4 mRNA表达有明显的极化现象，呈TH2反应，提示恶性肿瘤患者和CRF患者TH细胞功能处于失衡状态。可通过改善和调整TH细胞的失衡提高恶性肿瘤患者的免疫监视功能和纠正CRF患者免疫紊乱。     

IL-15-IgG2b fusion protein accelerates and enhances a Th2 but not a Th1 immune response in vivo,while IL-2-IgG2b fusion protein inhibits both

We have explored how IL-15 influences Th1 or Th2 type immune response in vivo. Intraperitoneal application of an IL-15-IgG2b fusion protein (FP) to mice did neither significantly affect the footpad swelling nor the production of hemagglutinizing antibodies in a delayed type hypersensitivity reaction to sheep red blood cells. In contrast, in an established murine Th2 model of sensitization to ovalbumin (OVA), IL-15-IgG2b FP plus OVA sensitization resulted in massively accelerated and enhanced allergen-specific IgE and IgG1 antibody production. In vitro, stimulation of spleen cells from OVA-sensitized mice with OVA+IL-15 or OVA+IL-15-IgG2b resulted in a significantly enhanced IgE production. IL-4 secretion was significantly induced by IL-15 but not by IL-15-IgG2b. An IL-2-IgG2b FP with the same Fc tail as the IL-15-IgG2b FP was used as control in both models. In striking contrast to the IL-15-IgG2b FP, IL-2-IgG2b significantly inhibited the Th2 type antibody production in vivo. The current study suggests that IL-15-IgG2b may be employed as a potent accelerator and enhancer of Th2 type immune responses in vivo, while IL-2-IgG2b can suppress the latter.     

Th2 Cytokines IL-4 and IL-13 Downregulate Paxillin Expression in Bronchial Airway Epithelial Cells

Asthma is characterized by infiltration and shedding of the bronchial epithelium. The Th2 cytokines IL-4 and IL-13 are involved in the cellular recruitment and infiltration seen in asthma. The effects of IL-4 and IL-13 on cell-matrix interactions and epithelial shedding are unknown. We hypothesize that bronchial airway epithelial cells (BAEC) express paxillin, a structural focal adhesion protein, and downregulation of paxillin by Th2 cytokines lead to BAEC hyperpermeability. We showed by confocal microscopy the presence of paxillin in BAEC. We demonstrated by Western blot analysis that IL-4 and IL-13 stimulation results in downregulation of paxillin production. IL-4 and IL-13 stimulation decreased epithelial cell-matrix attachment as measured by electrical cell-substrate impedance sensing system (ECIS). Our results suggest that Th2 cytokines IL-4 and IL-13 downregulate paxillin production by BAEC, thereby disrupting the cell-matrix interactions. This may help explain the epithelial shedding and epithelial membrane hyperpermeability that occurs in asthma.     

慢性乙型肝炎患者血清HBVDNA含量和IL-12对其PBMC产生Th1/Th2类细胞因子影响的相关性研究

目的：研究慢性乙型肝炎患者血清HBVDNA含量对IL－12诱导其PBMC产生Th1/Th2类细胞因子协同效应的影响。方法：分离50例慢性乙型肝炎患者外周血单个核细胞，分别与PHA（100μg/ml）、HBcAg（1μg/ml）、HBeAg（1μg/ml）单独或联合IL－12（10ng/ml）体外培养48h，ELISA法检测培养上清液细胞因子IL－2、IFN－γ、IL－10。荧光定量PCR检测患者血清DNA含量，并分成HBVBDNA小于10^3拷贝／ml、1063-10^5拷贝／ml、1065-10^7拷贝／ml、大于10^7拷贝/ml4组。结果：以HBVDAN小于10^3拷贝／ml组做对照组，比较发现无论抗原（PHA、HBcAg、HBeAg）单独诱导还是联合IL－12共同诱导，随着血清HBVDNA含量的增高，PBMC产生IL－2和IFN-γ水平逐渐降低，产生IL－5和IL－10水平逐渐升高，并且IL－12对PBMC产生IFN－γ的增殖效应逐渐减弱，特别是血清HBVDNA大于10^7拷贝／ml患者几乎无明显增殖效应。结论：高水平血清HBVDNA含量对IL－12诱导慢性乙型肝炎PBMC产生IFN－γ协同效应有抑制作用。     

IL-23/IL-17轴在银屑病免疫发病机制中的作用

银屑病是一种以T淋巴细胞异常活化和浸润为主要特征的慢性炎性反应皮肤病。Th17细胞及IL-23/IL-17轴在银屑病的发病机制中可能处于关键地位,并成为新的治疗靶标。IL-23诱导Th17细胞分化增殖,分化成熟的Th17可以分泌IL-17、IL-21、IL-22等多种细胞因子,Th17类细胞因子在银屑病等多种自身免疫疾病和炎性疾病中起重要作用。     

混合皮肤移植中IL-10在自体角朊细胞诱导局部免疫耐受中的作用

目的：探讨IL-10在混合皮肤移植中角朊细胞诱导的局部免疫耐受中的作用。方法：利用已经建立的MEIC体外模拟系统，检测在引入和未引入自体角朊细胞的实验系统上清中IL-2、IL-4、IL-10和IFN-γ的浓度，并用IL-10基因敲除小鼠实验观察IL-10在诱导抑制中的作用。结果：在未引入自体角朊细胞的MELC体系中，表现的是典型的Th1相关的细胞因子动力学曲线，在加入自体角朊细胞后，则出现了细胞因子由Th1和Th2转换的动力学曲线，且IL-10起着关键的作用，在以IL-10基因knockout小鼠的角朊细胞去替代实验体系中的正常小鼠角朊细胞时，对自体淋巴细胞的同种异体增殖反应的抑制率虽有所下降，但无显著差异，而以IL-10基因knockout小鼠的淋巴细胞替代实验体系中的正常小鼠的淋巴细胞时，对自体淋巴细胞的同种异体增殖反应的抑制作用被显著缓解，表明对抑制起关键调节作用的是自体淋巴细胞来源的IL-10，而非自体角朊细胞分泌的IL-10。结论：在混合皮肤移植中，自体角朊细胞是通过激活Th2细胞，后者分泌大量的IL-10和IL-4等细胞因子来抑制Th1细胞增殖，从而抑制Th1亚群所介导的细胞免疫及移植物排斥来诱导局部免疫耐受的。     

IL-9 exerts biological function on antigen-experienced murine T cells and exacerbates colitis induced by adoptive transfer

IL-9 is involved in various T cell-dependent inflammatory models including colitis, encepahlitis, and asthma. However, the regulation and specificity of IL-9 responsiveness by T cells during immune responses remains poorly understood. Here, we addressed this question using two different models: experimental colitis induced by transfer of naive CD4⁺CD45RB^high T cells into immunodeficient mice, and OVA-specific T cell activation. In the colitis model, constitutive IL-9 expression exacerbated inflammation upon transfer of CD4⁺CD45RB^high T cells from WT but not from Il9r^−/− mice, indicating that IL-9 acts directly on T cells. Suprisingly, such naïve CD4⁺CD45RB^high T cells failed to express the Il9r or respond to IL-9 in vitro, in contrast with CD4⁺CD45RB^low T cells. By using OVA-specific T cells, we observed that T cells acquired the capacity to respond to IL-9 along with CD44 upregulation, after long-lasting (5 to 12 days) in vivo antigenic stimulation. Il9r expression was associated with Th2 and Th17 phenotypes. Interestingly, in contrast to the IL-2 response, antigen restimulation downregulated IL-9 responsiveness. Taken together, our results demonstrate that IL-9 does not act on naïve T cells but that IL-9 responsiveness is acquired by CD4⁺ T cells after in vivo activation and acquisition of memory markers such as CD44.     

Increased levels of interferon-gamma (IFN-gamma), IL-4 and transforming growth factor-beta (TGF-beta) mRNA expressing blood mononuclear cells in human HIV infection.

Evidence has been presented for the involvement of IFN-gamma, IL-4 and TGF-beta in AIDS. Measured plasma levels may, however, poorly reflect in vivo production, since cytokines act auto- and paracrinally and have very short half life in plasma. In situ hybridization with complementary DNA oligonucleotide probes was used to enumerate blood mononuclear cells expressing cytokine messenger RNA (mRNA). HIV-infected patients had elevated blood levels of cells expressing each of the cytokines, with predominance for cells expressing TGF-beta mRNA. All AIDS patients included had elevated numbers of IL-4 mRNA-expressing cells, and levels of cells expressing this cytokine correlated inversely with counts of CD4+ cells in blood, reflecting the involvement of Th2-like cells in later stages of HIV infection. The described approach should be useful in further studies of cytokines in HIV infection and other diseases.     

人IL-2mRNA的特性研究

通过测定人IL-2和IL-2mRNA的诱生动力学,在高峰时相从PHA+TPA刺激的人脾脏单个核细胞内用冷酚法提取了大量富含IL-2mRNA的胞浆RNA。用制备型柱状7M尿素—SDS—PAGE分离胞浆RNA,并结合麦胚无细胞转译体系和IL-2生物活性检测,测定出人IL-2mRNA全长片段约为14S。将转译IL-2(tIL-2)与基因工程IL-2(rIL-2)和部分纯化的天然IL-2(nIL-2)进行分子筛层析,证实三种IL-2洗脱峰基本相同,分子量约为14000道尔顿。tIL-2与IL-2R具良好的结合特性。     

L-17与自身免疫性疾病关系的研究进展

IL-17是CD4 T细胞亚群Th17分泌的细胞因子。Th17的发育、扩增及分泌IL-17主要受TGF-β、IL-6、IL-15、IL-23等细胞因子的调控。IL-17调节前炎性因子、趋化因子等的产生及分泌,在白细胞的迁移、破骨细胞的活化和骨质的吸收等方面发挥重要作用。研究证实,IL-17在自身免疫性疾病的发生、发展中也具有一定的影响和作用。     

Early IL-2 treatment of mice with Pseudomonas aeruginosa pneumonia induced PMN-dominating response and reduced lung pathology

IL-2 is a pro-inflammatory and a Th1 inducing cytokine, which is important for activation of the cell-mediated immunity. IL-2 in serum and sputum has been observed to be reduced in cystic fibrosis (CF) patients. The present IL-2 treatment study of Pseudomonas aeruginosa (PA) lung infected mice was performed in order to evaluate the effect of IL-2 supplement. One hundred-and-twenty female BALB/c mice were divided into three groups: 1) IL-2 treatment/infection (TIG), 2) non-treatment/infection (NTIG), and 3) IL-2 treatment/non-infection (TNIG). The mice were challenged intra-tracheally with PA (PAO579) embedded in seaweed alginate to resemble the biofilm mode of growth. At day 0 and 1, the treatment groups received IL-2 s.c. Mice were killed on day 1 or 2, and cytokine production, lung pathology, and quantitative lung bacteriology were estimated. IL-2 treatment of infected mice reduced the number of mice with signs of macroscopic lung pathology at day 2 (p < 0.05). The reduced macroscopic pathology was paralleled by a reduced IL-1β and TNF-α. In contrast, an increased PMN response at day 2 was observed in the IL-2 treated mice (p < 0.01). This was preceded by a significantly higher degree of microscopic inflammation at day 1 (p < 0.02). The IL-12 levels decreased in both groups of infected mice at day 2 (p < 0.01), however, significantly more in the IL-2 treated mice (p < 0.02). In accordance, but surprisingly, IFN-γ was significantly reduced in the IL-2 treated PA infected group at day 2 (p < 0.05). The present results indicate that early IL-2 treatment prolongs the PMN response but also reduces pro-inflammatory IL-1β and TNF-α and macroscopic signs of pathology.     

IL-12 prevents neonatal induction of transplantation tolerance in mice

We investigated the effect of IL-12 on the induction of transplantation tolerance by neonatal injection of allogenic cells. We first observed that injection of newborn BALB/c mice with IL-12 and (A/J × BALB/c) F1 spleen cells prevented the Th2 alloimmune response induced by neonatal inoculation of F1 cells alone and allowed the differentiation of T cells secreting high amounts of IL-2 and IFN-γ in mixed lymphocyte cultures with donor-type stimulators. Furthermore, IL-12 administration resulted in the emergence of anti-donor cytotoxic T lymphocyte responses although at lower levels than in control uninjected mice. In parallel, we found that mice injected at birth with IL-12 and F1 cells did not develop chimerism and were able to reject a donor-type skin graft as efficiently as control mice. We conclude that IL-12 inhibits the Th2 polarization of the newborn response to alloantigens and prevents thereby the establishment of transplantation tolerance.     

细粒棘球蚴感染对哮喘大鼠血清白介素-17水平及Th1/Th2平衡的影响

目的建立细粒棘球蚴感染并发过敏性哮喘实验动物模型及探讨细粒棘球蚴感染对过敏性哮喘的作用。方法 24只健康Wistar大鼠,随机分为3组,A组为正常对照组,B组为单纯哮喘组,C组为细粒棘球蚴感染并发哮喘组。C组大鼠经腹腔注射感染细粒棘球蚴,70 d后,以卵白蛋白(OVA)分别对B组、C组大鼠进行致敏及激发,建立过敏性哮喘模型。取大鼠肺组织作病理切片,观察大鼠肺组织的炎症变化,采用酶联免疫吸附试验(ELISA)检测大鼠血清白介素-17(IL-17)、白介素-4(IL-4)及干扰素-γ(IFN-γ)的水平。结果与B组比较,C组大鼠血清IL-17和IL-4的水平明显下降(P<0.05),而血清IFN-γ的水平明显上升(P<0.05)。各组大鼠血清IL-4与IFN-γ水平呈密切负相关(r=-0.915),IL-4和IFN-γ水平与IL-17呈负相关关系(r=-0.406,-0.603),P<0.05。结论细粒棘球蚴感染对过敏性哮喘的发生有抑制作用。