Immunolocalization of intracellular interleukin‐4 in normal human peripheral blood basophils

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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 (FcεR1). Interestingly, stimulation of cells led to a decrease in the number of FcεR1‐positive cells. The above results show direct evidence that IL‐4 is produced by activated human basophils.

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