Detection of intracellular interleukin 2: evidence for novel immunologically related forms of the lymphokine.
At present, few data are available on intracellular interleukin 2 (IL-2) and its posttranscriptional regulation. Unlike other lymphokines, IL-2 does not accumulate within the cell, but is rapidly secreted following its production. The process of detection and biochemical characterization of intracellular IL-2 involved using a high producer subclone of the Jurkat T-lymphoma line as a source for IL-2, in combination with a two-step separation protocol and a sensitive detection method. Following phytohemagglutinin (PHA)/4 beta-phorbol 12-myristate 13 acetate (TPA) stimulation, a 14 kDa molecule could be visualized in Western blots by means of two monoclonal anti-IL-2 antibodies possessing different epitope specificities. This molecule exhibited biological activity of IL-2 as determined by a murine cytotoxic T-cell proliferation assay. In addition to this biologically active form of the lymphokine, a strongly immunoreactive protein with a molecular weight of 54 kDa (P54) was found in Jurkat cell lysates. Further biochemical characterization of this intracellular variant revealed an isoelectric point similar to that of secreted forms of IL-2. All attempts to split the 54 kDa molecule into smaller subunits failed, and no biological IL-2 activity could be measured in response to P54. However, the appearance of this high molecular weight variant followed clear-cut time kinetics. The highest concentration of P54 was found to occur after 2 h of stimulation. Thereafter its concentration decreased continuously, while the amount of the biologically active 14 kDa variant increased under ongoing stimulation. One possible explanation for these results is that P54 may represent an immature form of IL-2 that is tightly linked to a carrier molecule.
Kloth
S
S
Department of Immunology and Cell Biology, Forschungsinstitut Borstel, Germany.
Flad
H D
HD
Brandt
E
E
D005110:Extracellular Space / Q000737:chemistry; D006801:Humans; D007120:Immunochemistry; D007376:Interleukin-2 / Q000032:analysis*; D007700:Kinetics; D010835:Phytohemagglutinins / Q000494:pharmacology; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007376:Interleukin-2; D010835:Phytohemagglutinins; D013755:Tetradecanoylphorbol Acetate