Bailly S, Ferrua B, Fay M, Gougerot-Pocidalo M A
INSERM U.294, CHU Xavier Bichat, Paris, France.
Eur Cytokine Netw. 1990 Mar-Apr;1(1):47-51.
The existence of IL-1 activity on the cell surface of stimulated mononuclear phagocytes is a matter of controversy. In particular, fixation of IL-1-expressing cells for 15 min in 1% paraformaldehyde (PFA) is commonly used to evidence such "membrane-associated" IL-1 activity but other authors have attributed this to passive leakage of IL-1 alpha from the cells and report no activity with longer fixation times. Using specific IL-1 alpha and IL-1 beta assays, we found that after the mild standard PFA fixation procedure, not only IL-1 alpha but also IL-1 beta were released into the supernatants for up to 96 h following fixation; membrane IL-1 activity cannot thus be measured in these conditions. However, using conditions in which neither immunoreactive IL-1 molecules nor IL-1 activity are found in the supernatants (i.e. assay at 144 h, increased fixation time), we were still able to detect IL-1 activity on LPS-stimulated, PFA-fixed monocytes. This activity was independent of the duration of PFA fixation and was inhibited by anti-IL-1 alpha but not anti-IL-1 beta antibodies. Our data thus underline the importance of technical conditions in the study of membrane-associated IL-1 activity.
刺激的单核吞噬细胞细胞表面存在白细胞介素-1（IL-1）活性这一情况存在争议。具体而言，将表达IL-1的细胞在1%多聚甲醛（PFA）中固定15分钟常用于证明这种“膜相关”IL-1活性，但其他作者将此归因于IL-1α从细胞中的被动泄漏，并报告在固定时间更长时无活性。使用特异性IL-1α和IL-1β检测方法，我们发现，经过温和的标准PFA固定程序后，固定后长达96小时内，不仅IL-1α而且IL-1β都释放到了上清液中；因此在这些条件下无法测量膜IL-1活性。然而，使用上清液中未发现免疫反应性IL-1分子和IL-1活性的条件（即144小时检测，增加固定时间），我们仍然能够在LPS刺激、PFA固定的单核细胞上检测到IL-1活性。这种活性与PFA固定的持续时间无关，并且被抗IL-1α抗体而非抗IL-1β抗体抑制。因此，我们的数据强调了技术条件在膜相关IL-1活性研究中的重要性。
