Differential effects of interleukin 12 and interleukin 10 on superantigen-induced expression of cutaneous lymphocyte-associated antigen (CLA) and alphaEbeta7 integrin (CD103) by CD8+ T cells

At both cutaneous and mucosal sites, interleukin (IL)-10, IL-12 and transforming growth factor (TGF)-beta are important regulators of chronic inflammatory disease, where cutaneous lymphocyte-associated antigen (CLA) and alphaE integrin (CD103) may be expressed. Stimulation with streptococcal pyrogenic exotoxin C (SpeC) increased the expression of CD103 by CD8+ but not CD4+ T cells. While adding IL-12 augmented the expression of CLA, superantigen-induced expression of CD103 was markedly suppressed by IL-12, which could be reversed by TGF-beta. Antibodies against TGF-beta inhibited, and a combination of anti-TGF-beta and IL-12 completely abrogated the induced CD103 expression. IL-10 strongly decreased the frequency of CLA+ and although not increasing the frequency of CD103+CD8+ T cells, the amount of CD103 expressed per cell was markedly increased. Thus, the expression of CLA and CD103 may be antagonistically regulated by IL-10 and IL-12 and the balance between these cytokines could influence the T cell migration of inflammatory cells into epithelial tissues.     

Linomide,a novel immunomodulator that prevents death in four models of septic shock

Intravenous injections of 50 μ.g Staphylococcus aureus enterotoxin B (SEB) or bacterial lipopolysaccharide (LPS) are lethal, provided that mice are simultaneously sensitized with either N-galactosamine (GalN) or the anti-glucocorticoid RU-38486. Similar to the synthetic glucocorticoid (GC) receptor agonist dexamethasone, pharmacological doses of the immunomodulator linomide (quinoline-3-carboxamide) prevent death in all four models of lethal septic shock (LPS + GalN, LPS + RU-38486, SEB + GalN, and SEB + RU-38486) and inhibit the secretion of tumor necrosis factor, one of the major intermediate effector molecules of SEB and LPS toxicity. In this system, cyclosporine A (CsA), although effective in suppressing SEB toxicity, fails to counteract the lethal effect of LPS. This observation, together with the fact that linomide acts in the presence of excess amounts of GC receptor antagonist, indicates that linomide functions in a different way to that of known immunosuppressive agents like CsA and GC.     

超抗原金黄色葡萄球菌肠毒素基因B的原核表达

目的构建重组pET-30a-SEB原核表达载体,转化感受态大肠杆菌BL-21(DE3),诱导表达超抗原金黄色葡萄球菌肠毒素B(staphylococcalenterotoxinB,SEB)。方法利用PCR技术从产SEB的金黄色葡萄球菌标准菌株CMCC-26075基因组DNA中克隆SEB全长序列,将其克隆到pGEM-TEasy载体中并进行测序。构建pET-30a-SEB原核表达质粒,转化感受态大肠杆菌BL21(DE3),异丙基硫代β-D半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达,经镍离子螯合亲和层析纯化后免疫鉴定。结果PCR获得超抗原SEB基因片段,与克隆载体连接后经测序与文献报道的基本一致;成功构建了pET-30a-SEB原核表达质粒且成功诱导表达出相对分子质量(Mr)约31×103的蛋白。结论成功克隆了seb基因序列,并进行了原核表达和鉴定,获得了SEB蛋白,为后续对超抗原SEB的进一步研究奠定了基础。     

Fas Ligand-dependent and -independent mechanisms of toxicity induced by T cell lymphomas in lymphoid organs and in the liver

In the current study, we investigated the effect of growth of FasL⁺ tumors in vivo on the functions of peripheral lymphoid organs and the liver. Injection of FasL⁺ LSA tumor cells into syngeneic C57BL/6 wild-type mice but not C57BL/6 lpr/lpr (Fas-deficient) mice caused apoptosis in splenocytes. Spleen cells expressing CD3, CD4, CD8, CD19, Mac-3, and CD44 were all susceptible to tumor-induced apoptosis. Also, activated T cells were more sensitive to apoptosis induced by LSA tumor cell lysate when compared to naïve T cells. In contrast, anti-Fas Abs (Jo2) induced apoptosis in only activated but not naïve T cells. When the LSA tumor-bearing mice were injected with a superantigen (SEA), these mice showed a significant decrease in the expansion of SEA-reactive Vβ3⁺ and Vβ11⁺ T cells. When injected into syngeneic mice, the FasL⁺ LSA tumor cells caused hepatotoxicity, as indicated by an increase in serum aspartate aminotransferase (AST) levels. Interestingly, Fas-deficient C57BL/6 lpr/lpr mice also showed significant AST levels in the serum following LSA tumor growth. Moreover, hepatocytes isolated from C57BL/6 wild-type and C57BL/6 lpr/lpr mice were equally susceptible to apoptosis induced by LSA tumor cell lysate in vitro. Using cDNA array, LSA tumor cells were found to express several cytokine genes including IL-2, IL-7, IL-11, IL-13, IL-16, lymphotoxin β, and tumor necrosis factor β. Together, these data suggested that, in mice bearing FasL⁺ LSA tumor, the immunotoxicity is FasL-based, whereas the hepatotoxicity, at least in part, may be FasL-independent.     

Synergistic effect between CD40 and class II signals overcome the requirement for class II dimerization in superantigen-induced cytokine gene expression

Although staphylococcal enterotoxin A (SEA), B (SEB), and toxic shock syndrome toxin 1 (TSST-1) bind to major histocompatibility complex (MHC) class II molecules, they differ in their mode of binding. Signaling induced by these toxins via MHC class II molecules seems to be largely mediated by their mode of interaction. In the present study, we have demonstrated that contrary to SEA, stimulation of the human monocytic cell line THP-1 with SEB or TSST-1 failed to induce interleukin-1β or tumor necrosis factor-α gene expression. Treatment of THP-1 cells with interferon-γ increased the level of MHC class II expression but did not enhance the SEB and TSST-1 response. However, cross-linking of SEB or TSST-1 bound to MHC class II molecules with specific antibodies leads to cytokine gene expression, indicating that dimerization of class II molecules is a requirement for this superantigen-induced response. The presence of anti-CD40 antibodies in the course of SEB or TSST-1 stimulation overcomes this requirement, indicating that certain signal(s) induced via CD40 molecules can replace those induced by dimerization of class II molecules. Pretreatment with anti-lymphocyte functional antigen-1 (LFA-1) antibodies completely inhibited SEA-induced response as well as that induced by SEB or TSST-1 in the presence of CD40 antibodies, supporting the involvement of LFA-1 intercellular adhesion molecule system in these responses. The entirety of these results demonstrate clearly that dimerization of class II molecules is a prerequisite for superantigen-induced T cell-independent cytokine gene expression which can be replaced by signaling via CD40 in an LFA-1-dependent system.     

超抗原葡萄球菌肠毒素A对肝癌细胞免疫原性影响的研究

目的给肝癌细胞转入超抗原分子葡萄球菌肠毒素A(staphylococcal enterotoxin A,SEA)的编码基因,使其表达这种毒素分子,以增强肝癌细胞的免疫原性,达到增强肝癌细胞诱导免疫排斥反应的目的.方法构建SEA编码基因的逆转录真核表达载体,转染病毒包装细胞系PA317后,行滴度测定.以高滴度克隆培养上清转导肝癌细胞株HHCC,筛选获得阳性表达株.然后利用外周血混合淋巴细胞进行杀伤实验.结果成功的构建了SEA的逆转录真核表达载体pLXSN-SEA,转导肝癌细胞后,获得表达SEA分子的阳性克隆HHCC-SEA.培养上清中SEA含量在pg的水平.外周血混合淋巴细胞杀伤实验结果显示,T淋巴细胞对转导后肝癌细胞的杀伤率可达45.6%.高于对HHCC 20.7%的杀伤率,T细胞对转导后肝癌细胞杀伤反应的Km值为5.18×104,而HHCC组的Km值为2.92 × 105,与前者的差别有统计学意义.结论肝癌细胞在转入SEA分子的编码基因后,表达出微量的SEA蛋白,却具有较强的免疫激活能力.即表达SEA的肝癌细胞在很低的浓度下,激活的T细胞即可对其达到最大杀伤反应速度,对T细胞的激活能力远远高于HHCC.     

超抗原活化淋巴细胞对喉癌Hep-2细胞系的抑制作用

目的 观察超抗原活化淋巴细胞后对喉癌细胞的抑制作用.方法 金黄色葡萄球菌肠毒素A与人淋巴细胞混合培养24 h,激活淋巴细胞,再加入人喉鳞状细胞癌Hep-2细胞混合培养72 h,通过MTT法观察喉癌细胞的抑制情况.结果 ①实验组金黄色葡萄球菌肠毒素A激活淋巴细胞后对人喉鳞癌Hep-2细胞的生长有明显的抑制作用,抑制率是42.68%,高于对照组4.82%(P＜0.01).②抑制作用呈现时间的依赖性.③金黄色葡萄球菌肠毒素A在不同浓度时对喉癌细胞的生长均有抑制作用,特别在浓度是1 μg/ml、72 h培养时抑制率最高,达82.83%.结论 超抗原活化淋巴细胞后明显抑制喉癌细胞的生长.     

SEC治疗胶质瘤的体外实验

目的观察SEC活化的淋巴细胞对胶质瘤的杀伤作用。方法分离并培养外周血淋巴细胞,培养液中加入不同浓度的SEC,将淋巴细胞与胶质瘤细胞(SHG-44、MGR2、MGR3、SF767、SF295、SKMG-1、T98G及UW28)混合培养,按MTT法测定淋巴细胞对瘤细胞的杀伤作用。结果经SEC活化的淋巴细胞对8株胶质瘤均产生强大的杀伤,其中以10μ/ml剂量组作用最明显。SEC作用的第一天,淋巴细胞就被激活并对胶质瘤细胞产生杀伤,第三天杀伤率达到峰值。结论SEC活化的淋巴细胞对胶质瘤细胞有明显的抗瘤活性。