Laboratoire d'Immunologie, Hôpital Universitaire Dupuytren, Limoges, France.
Inflamm Res. 2010 Mar;59 Suppl 2:S193-5. doi: 10.1007/s00011-009-0126-3.
Human leukocyte activation induced by specific and non-specific stimuli is characterized by the formation of lipid rafts defined as lipid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. These lipid rafts are formed in parallel with profound membrane reorganization.
Analyse the rafting and non-rafting proteins present on the activated and resting basophil membrane and study their interest for the flow cytometric analysis of basophil activation.
Human basophils obtained from samples used for diagnostic cellular tests such as basophil or lymphocyte activation tests were stimulated either by the formyl-methionyl-leucyl-phenylalanine peptide (fMLP), by an anti-IgE or by an allergen. After 40 min at 37 degrees C, they were labelled by different antibodies conjugated to fluorescent dyes as an anti-IgE FITC, an anti-CCR3 PE, an anti-CD63, an anti-CD203c PE, an anti-11b, annexin V FITC or by cholera toxin FITC. Moreover, several experiments were analysed using an Amnis cytometer, allowing one to obtain the picture of the analysed cells.
Anti-IgE or specific allergen elicits a membrane neo expression of CD63 at a high density and is poorly represented on resting basophil membrane. Upon an IgE-dependant activation some of the markers already present on resting basophil membrane, as CD203c, are up regulated and others, such as the IgE/IgE FcepsilonRI receptor and CCR3 are down regulated and submitted to the formation of clusters demonstrated by the pictures taken with the Amnis cytometer. For non-IgE dependant activators, such as fMLP, the picture was different since IgE was not down regulated, whereas CCR3 was down regulated. As demonstrated using annexin V or the cholera toxin used for analysing apoptosis, these phenomenon were paralleled by the formation of lipid rafts, gangliosides domains, such as GM1, which is accessible from the extra cellular medium.
Basophil activation leads to membrane events close to the apoptosis phenomenon. The flow cytometric analysis of these membrane events may lead to protocols for allergen-induced activation and, may significantly increase cellular test sensitivity, particularly for drugs allergy diagnosis for which the usual protocols, such as those using CD63 alone, are insufficiently sensitive.
人类白细胞受特异性和非特异性刺激激活的特征是形成脂筏，脂筏被定义为比双层的周围非筏相更紧密排列的脂质有序域。这些脂筏与深刻的膜重排平行形成。
分析激活和静止嗜碱性粒细胞膜上存在的筏蛋白和非筏蛋白，并研究它们对嗜碱性粒细胞激活的流式细胞分析的意义。
从用于诊断细胞测试的样本中获得的人嗜碱性粒细胞，如嗜碱性粒细胞或淋巴细胞激活测试，通过甲酰基-甲硫氨酸-亮氨酸-苯丙氨酸肽（fMLP）、抗 IgE 或过敏原刺激。在 37°C 孵育 40 分钟后，用不同的荧光染料标记抗体，如抗 IgE-FITC、抗 CCR3-PE、抗 CD63、抗 CD203c-PE、抗 11b、膜联蛋白 V-FITC 或霍乱毒素-FITC。此外，使用 Amnis 细胞仪分析了几个实验，该仪器可以获得被分析细胞的图像。
抗 IgE 或特异性过敏原引发 CD63 的高密度膜新生表达，在静止嗜碱性粒细胞膜上的表达较少。在 IgE 依赖性激活后，一些已经存在于静止嗜碱性粒细胞膜上的标记物，如 CD203c，被上调，而其他标记物，如 IgE/IgE FcepsilonRI 受体和 CCR3，则被下调，并通过 Amnis 细胞仪获得的图像显示形成簇。对于非 IgE 依赖性激活剂，如 fMLP，情况则不同，因为 IgE 没有下调，而 CCR3 则下调。如使用膜联蛋白 V 或用于分析细胞凋亡的霍乱毒素所示，这些现象伴随着脂筏的形成，神经节苷脂域，如 GM1，从细胞外介质中可获得。
嗜碱性粒细胞的激活导致与细胞凋亡现象密切相关的膜事件。这些膜事件的流式细胞分析可能会导致过敏原诱导激活的方案，并且可能显著提高细胞测试的敏感性，特别是对于药物过敏诊断，通常的方案，如单独使用 CD63，不够敏感。
