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首页> 外文会议>Optical Investigations of Cells In Vitro and In Vivo >Multiparameter fluorescence imaging for quantification of TH-1 and TH-2 cytokines at the single-cell level
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Multiparameter fluorescence imaging for quantification of TH-1 and TH-2 cytokines at the single-cell level

机译：多参数荧光成像可在单细胞水平上定量TH-1和TH-2细胞因子

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Abstract: Immune responses are strongly influenced by the cytokines following antigenic stimulation. Distinct cytokine-producing T cell subsets are well known to play a major role in immune responses and to be differentially regulated during immunological disorders, although the characterization and quantification of the TH-1/TH-2 cytokine pattern in T cells remained not clearly defined. Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signaling pathways. The aim of this study was (1) to quantify the cytokine expression in T cells at the single cell level using optical imaging, (2) and to analyze the influence of cyclic AMP- dependent signal transduction pathway in the balance between the TH-1 and TH-2 cytokine profile. We attempted to study several cytokines (IL-2, IFN-$gamma@, IL-4, IL-10 and IL-13) in peripheral blood mononuclear cells. Cells were prestimulated in vitro using phytohemagglutinin and phorbol ester for 36h, and then further cultured for 8h in the presence of monensin. Cells were permeabilized and then simple-, double- or triple-labeled with the corresponding specific fluorescent monoclonal antibodies. The cell phenotype was also determined by analyzing the expression of each of CD4, CD8, CD45RO and CD45RA with the cytokine expression. Conventional images of cells were recorded with a Peltier- cooled CCD camera (B/W C5985, Hamamatsu photonics) through an inverted microscope equipped with epi-fluorescence (Diaphot 300, Nikon). Images were digitalized using an acquisition video interface (Oculus TCX Coreco) in 762 by 570 pixels coded in 8 bits (256 gray levels), and analyzed thereafter in an IBM PC computer based on an intel pentium processor with an adequate software (Visilog 4, Noesis). The first image processing step is the extraction of cell areas using an edge detection and a binary thresholding method. In order to reduce the background noise of fluorescence, we performed an opening procedure of the original image using a structuring element. The opened image was therefore subtracted from the original one, and the gray intensities were subsequently measured. Fluorescence intensities are mapped in MD representation using Matlab software. Consequently, quantitative comparative expression of intracellular cytokines and cell membrane markers was achieved. Using this technique, we showed that CD4$PLU and CD8$PLU@T lymphocytes expressed a large panel of cytokines, and that protein kinase A (PKA) activation pathway induced a polarization of activated human T cells to the TH-2 type profile. Data also showed different sensitivities of CD45 RO/CD45RA lymphocytes to the activation of PKA, thus suggesting the implication of memory CD4$PLU@- and CD8$PLU@-T cells in the T cell specific immune and inflammatory processes and their control by PKA activation pathway. Finally, this method represents a powerful tool for the detection and quantification of intracellular cytokine expression and the analysis of the functional properties of T lymphocytes during immune responses. !7
机译：摘要：免疫刺激受抗原刺激后细胞因子的强烈影响。众所周知，产生不同细胞因子的T细胞亚群在免疫反应中起主要作用，并在免疫系统疾病中受到差异调节，尽管尚不清楚T细胞中TH-1 / TH-2细胞因子模式的表征和定量。 。 T淋巴细胞表达细胞因子是一个高度平衡的过程，涉及刺激性和抑制性细胞内信号通路。这项研究的目的是（1）使用光学成像在单个细胞水平上定量T细胞中的细胞因子表达，（2）并分析环AMP依赖信号转导途径对TH-1之间平衡的影响和TH-2细胞因子谱。我们试图研究外周血单核细胞中的几种细胞因子（IL-2，IFN-γ，IL-4，IL-10和IL-13）。使用植物血凝素和佛波醇酯体外刺激细胞36h，然后在莫能菌素存在下进一步培养8h。细胞被透化，然后用相应的特异性荧光单克隆抗体进行简单，双重或三重标记。还通过分析具有细胞因子表达的CD4，CD8，CD45RO和CD45RA各自的表达来确定细胞表型。通过配备有落射荧光的倒置显微镜（Diaphot 300，尼康），用珀尔帖冷却的CCD相机（B / W C5985，滨松光子学）记录细胞的常规图像。使用762像素的采集视频接口（Oculus TCX Coreco）以570像素（以8位（256灰度级）编码）将图像数字化，然后在基于Intel pentium处理器的IBM PC计算机中使用适当的软件进行分析（Visilog 4， Noesis）。第一步图像处理是使用边缘检测和二进制阈值方法提取单元区域。为了减少荧光的背景噪声，我们使用结构元素执行了原始图像的打开过程。因此，从原始图像中减去了打开的图像，然后测量了灰度强度。使用Matlab软件以MD表示法映射荧光强度。因此，实现了细胞内细胞因子和细胞膜标志物的定量比较表达。使用该技术，我们显示CD4 $ PLU和CD8 $ PLU @ T淋巴细胞表达了大量的细胞因子，并且蛋白激酶A（PKA）激活途径诱导了激活的人类T细胞极化至TH-2型谱。数据还显示CD45 RO / CD45RA淋巴细胞对PKA活化的敏感性不同，因此表明记忆CD4 $ PLU @-和CD8 $ PLU @ -T细胞与T细胞特异性免疫和炎症过程有关，并受PKA控制激活途径。最后，该方法代表了检测和定量细胞内细胞因子表达以及免疫应答过程中T淋巴细胞功能特性的强大工具。 ！7

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