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Simultaneous production of interleukin 2, interleukin 4 and interferon-gamma by activated human blood lymphocytes
U Andersson1, J Andersson, A Lindfors
1Department of Immunology, University of Stockholm, Sweden.
Insights
This study quantifies cytokine production in human immune cells, revealing distinct patterns for interleukin 2 (IL 2), IL 4, and interferon-gamma (IFN-gamma). Neonatal cells show impaired IFN-gamma and absent IL 4 production compared to adults.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Human blood mononuclear cells produce key cytokines like IL 2, IL 4, and IFN-gamma.
- Understanding single-cell cytokine production kinetics and phenotypes is crucial for immune response analysis.
Purpose of the Study:
- To investigate the single-cell production kinetics and phenotypes of IL 2, IL 4, and IFN-gamma in human lymphocytes.
- To compare cytokine production between adult and neonatal immune cells.
Main Methods:
- Intracellular immunofluorescent staining was used to identify individual lymphokine-synthesizing cells.
- Two-color immunofluorescence studies were performed to analyze co-production of cytokines.
- Mononuclear cells from adult and umbilical cord blood were cultured and stimulated in vitro.
Main Results:
- Adult lymphocytes showed biphasic IL 2 and IFN-gamma production (peaks at 4-6h and 24-30h), with 20%-40% producing these cytokines.
- IL 4 production was transient (peak at 6h) and involved only 1%-3% of lymphocytes.
- Both CD4+ and CD8+ T cells produced all three cytokines, with specific subsets dominating IL 2, IL 4, or IFN-gamma synthesis. Neonatal cells exhibited significantly reduced IFN-gamma and no IL 4 production.
Conclusions:
- Distinct kinetic and phenotypic profiles characterize IL 2, IL 4, and IFN-gamma production in human lymphocytes.
- Significant differences in cytokine production exist between adult and neonatal immune cells, particularly for IL 4 and IFN-gamma.
- The Golgi system accumulation of cytokines aids in their detection and analysis.
Abstract:
The production of interleukin 2 (IL 2), IL 4 and interferon-gamma (IFN-gamma) by in vitro activated unselected human blood mononuclear cells was studied at a single-cell level. Individual lymphokine-synthesizing cells were identified by intracellular immunofluorescent staining using cytokine-specific monoclonal or polyclonal antibodies. Cultures from adult blood donors revealed a biphasic kinetic production pattern for IL 2 and IFN-gamma with peaks occurring 4-6 and 24-30 h after initiation of the cultures. Approximately 20%-40% of the lymphocytes produced IL 2 and IFN-gamma. In contrast, only 1%-3% of the lymphocytes synthesized IL 4 with maximal frequency after 6 h of culture. CD4+ as well as CD8+ T cells contributed to the synthesis of all three lymphokines studied. CD4+CD45R- T cells were the major producers of IL 2 and IL 4, while CD8+CD45R- T cells were the most common phenotype of IFN-gamma-synthesizing cells. By performing two-color immunofluorescence studies we observed that among IL 4-producing cells every second one made simultaneously IL 2 and every fourth one made IFN-gamma. Mononuclear cells from umbilical cord blood could be stimulated to make IL 2 to the same extent as cells from adult blood donors. No IL 4 production and a strikingly reduced frequency of IFN-gamma producers were noted in cell cultures from neonates. IL 2, IL 4 and IFN-gamma accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.
