Identification of rat IL-1 beta, IL-2, IFN-gamma and TNF-alpha in activated splenocytes by intracellular immunostaining

We have developed a sensitive three-step indirect immunofluorescence method to identify individual rat cells that produce cytokines including IL-1 bet a, IL-2, IFN-gamma and TNF-alpha. Cultured rat splenocytes were polyclonall y activated to cytokine synthesis by mitogens such as lipopolysaccharide or a combination of a protein kinase C activator (phorbol 12-myristate 13-ace tate) and a calcium ionophore (ionomycin), Careful selection of either anti gen affinity-purified polyclonal or monoclonal cytokine-detecting antibodie s combined with gentle fixation and permeabilization of the cells enabled d iscrimination of cytokine-producing cells based on distinct morphological s taining criteria, Cells making IL-2, IFN-gamma and TNF-alpha could be ident ified by a characteristic, intracellular, rounded, juxtanuclear immunofluor escence signal. This staining pattern reflected the accumulation of the int racellularly processed cytokines in the Golgi organelle of producer cells. The staining of cells that synthesized IL-1 beta, which is not transported Intracellularly via the endoplasmatic reticulum-Golgi pathway, generated a different, but distinct and reproducible staining pattern, IL-1 beta produc ing macrophages expressed intense nuclear immunofluorescence with additiona l reticular, cytoplasmic signals. Furthermore, the use of biologically neut ralizing detecting antibodies against the cytokines under study prevented r ecognition of surface-stained target cells that had bound secreted cytokine s by cytokine-specific receptors. This modified staining technology enabled analysis of the kinetic pattern and the frequency of cytokine-producing ce lls in cultures of rat splenocytes after various modes of polyclonal activa tion.