Identification of Rat IL-1β, IL-2, IFN-γ and TNF-α in Activated Splenocytes by Intracellular Immunostaining

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Abstract
We have developed a sensitive three-step indirect immunofluorescence method to identify individual rat cells that produce cytokines including IL-1β, IL-2, IFN-γ and TNF-α. Cultured rat splenocytes were polyclonally activated to cytokine synthesis by mitogens such as lipopolysaccharide or a combination of a protein kinase C activator (phorbol 12-myristate 13-acetate) and a calcium ionophore (ionomycin). Careful selection of either antigen affinity-purified polyclonal or monoclonal cytokine-detecting antibodies combined with gentle fixation and permeabiliza-tion of the cells enabled discrimination of cytokine-producing cells based on distinct morphological staining criteria. Cells making IL-2, IFN-γ and TNF-α could be identified by a characteristic, intracellular, rounded, juxtanuclear immunofluorescence signal. This staining pattern reflected the accumulation of the intracellularly processed cytokines in the Golgi organelle of producer cells. The staining of cells that synthesized IL-1β, which is not transported intracellularly via the endoplasmatic reticulum-Golgi pathway, generated a different, but distinct and reproducible staining pattern, IL-1β producing macrophages expressed intense nuclear immunofluorescence with additional reticular, cytoplasmic signals. Furthermore, the use of biologically neutralizing detecting antibodies against the cytokines under study prevented recognition of surface-stained target cells that had bound secreted cytokines by cytokine-specific receptors. This modified staining technology enabled analysis of the kinetic pattern and the frequency of cytokine-producing cells in cultures of rat splenocytes after various modes of polyclonal activation.