Expression of Actin and Tubulins in Purified Human Basophil Leukocytes upon Stimulation with IL-3

Abstract

Background: Pharmacological and morphological results indicate that the cytoskeleton proteins tubulins and actin play a role in the histamine release process in basophil leukocytes and mast cells. In this report, we investigate the expression of these cytoskeleton proteins in purified human basophils upon stimulation with anti-IgE and IL-3. Methods: Human basophils were purified using a negative selection procedure. They were incubated for 6 h with anti-IgE and/or IL-3 in radiolabeling media. Proteins in the cells were analyzed by two-dimensional gel electrophoresis and autoradiography. Actin and α- and β-tubulin were identified by amino acid sequence analysis or matrix assisted laser desorption/ionisation time-of-flight mass spectrometry. The synthesis of these proteins under different experimental conditions was evaluated by densitometry. Results: Actin is regulated dose dependently by IL-3 with an optimum concentration of 100 ng/ml, corresponding to the optimal IL-3 concentration for enhancement of histamine release. The synthesis of tubulins was not significantly upregulated by IL-3 or anti-IgE alone, but there was a slight and significant upregulation of tubulins upon stimulation with both IL-3 and anti-IgE in doses optimal for maximal histamine release. Conclusions: The de novo synthesis of actin is regulated dose dependently by IL-3 in purified human basophils in short-term culture. The optimum IL-3 concentration is the same as for enhancement of histamine release, suggesting that the same processes that regulate IL-3-induced enhancement of histamine release also regulate the expression of actin. The finding that the expression of the tubulins is not upregulated by IL-3 alone suggests that regulation of tubulin synthesis is not a pathway by which IL-3 in itself primes basophil histamine release.