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Published on: March 2, 2011
Immunolocalization of intracellular interleukin-4 in normal human peripheral blood basophils
R Mueller1, C H Heusser, S Rihs
1Ciba Geigy, Research Department, Basel, Switzerland.
Insights
Human basophils can produce interleukin-4 (IL-4), a key cytokine. This study provides direct evidence of IL-4 presence and release from activated human basophils, confirming their role beyond T lymphocytes.
Area of Science:
- Immunology
- Cell Biology
- Cytokine Research
Background:
- Interleukin-4 (IL-4) production is primarily associated with helper T lymphocytes.
- The potential for other cell types, such as basophils, to produce IL-4 has been a subject of recent investigation.
- Direct evidence for IL-4 production by human basophils was previously lacking.
Purpose of the Study:
- To demonstrate the presence and production of IL-4 in activated human peripheral blood basophils.
- To provide direct proof of IL-4 synthesis and release by basophils at the single-cell level.
- To characterize the IL-4 staining pattern in basophils and compare it to activated T cells.
Main Methods:
- Intracellular immunofluorescence staining using IL-4-specific monoclonal antibodies.
- Activation of human peripheral blood basophils in vitro using IL-3 priming and anti-IgE or C5a stimulation.
- Confocal scanning laser microscopy for intracellular IL-4 visualization and cell identification.
- Double labeling studies with antibodies against IL-4 and the high-affinity IgE receptor (Fc epsilon R1).
Main Results:
- IL-4 was detected in the cytoplasm of activated human basophils, exhibiting a characteristic granular staining pattern.
- Stimulation significantly increased the percentage of IL-4-positive basophils (approx. 19%) compared to unstimulated controls (6%).
- Supernatant IL-4 levels showed a five- to sevenfold increase upon stimulation, confirming cytokine release.
- Confocal microscopy confirmed intracellular IL-4 and identified the cells as basophils by nuclear morphology.
Conclusions:
- This study provides direct evidence that activated human basophils produce and release IL-4.
- Basophils contribute to IL-4 production, expanding the known cellular sources of this important cytokine.
- The findings highlight the significant role of basophils in immune responses involving IL-4.
Abstract:
The question as to whether other cell types apart from helper T lymphocytes are capable of producing interleukin-4 (IL-4) has gained much interest over the last years. Recent studies indicate that human basophils also produce IL-4, although direct proof is missing so far. In this study we demonstrate the presence of IL-4 in the cytoplasm of in vitro activated human peripheral blood basophils derived from normal donors. Cytokine-producing cells were revealed at the single-cell level by intracellular immunofluorescence staining using IL-4-specific monoclonal antibodies. Basophils showed a characteristic, apparently granular staining pattern easily discerned from the eccentric dot-shaped staining pattern in activated T cells used in control experiments. Cell counts following priming with IL-3 and stimulation with polyclonal sheep anti-IgE antibody or the anaphylatoxin C5a revealed a significant increase in IL-4-positive basophils to about 19% as compared with unprimed, unstimulated control cells (6%). The amount of IL-4 in the supernatant of these cell preparations paralleled these observations with an at least five- to sevenfold increase following stimulation as compared with control cells (< 5 ng/ml). Using confocal scanning laser microscopy, the intracellular presence of IL-4 was confirmed, and the cells were identified as being basophils on terms of their characteristic multilobed nucleus. This observation was supported by double labeling studies using antibodies to IL-4 and to the high-affinity IgE receptor (Fc epsilon R1). Interestingly, stimulation of cells led to a decrease in the number of Fc epsilon R1-positive cells. The above results show direct evidence that IL-4 is produced by activated human basophils.
