Identification of rat IL-1beta, 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-1beta, IL-2, IFN-gamma and TNF-alpha. 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 permeabilization of the cells enabled discrimination of cytokine-producing cells based on distinct morphological staining criteria. Cells making IL-2, IFN-gamma and TNF-alpha 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-1beta, which is not transported intracellularly via the endoplasmatic reticulum-Golgi pathway, generated a different, but distinct and reproducible staining pattern, IL-1beta 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.
Palmblad
K
K
Centre of Molecular Medicine, Karolinska Institute, and Department of Pediatric Rheumatology, Astrid Lindgren's Children's Hospital, Karolinska Hospital, Stockholm, Sweden. karin.palmblad@cmm.ki.se
Andersson
U
U
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018929:Cell Culture Techniques; D003593:Cytoplasm / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D007375:Interleukin-1 / Q000032:analysis* / Q000276:immunology; D007376:Interleukin-2 / Q000032:analysis* / Q000276:immunology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000737:chemistry; D051381:Rats; D012680:Sensitivity and Specificity; D013154:Spleen / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D013194:Staining and Labeling; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D007375:Interleukin-1; D007376:Interleukin-2; D007476:Ionophores; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D015759:Ionomycin; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate