Paraformaldehyde fixation of LPS-stimulated human monocytes: technical parameters permitting the study of membrane...

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.