Takako Toda

Intracellular Protein Phosphorylation in Eosinophils and the Functional Relevance in Cytokine Production

Background: Eosinophils play a pivotal role in the pathogenesis of asthma. Thus, it is of paramount importance to investigate the mechanism of eosinophil activation. Although a number of factors including cytokines/chemokines activate eosinophils, the potency of each stimulus to phosphorylate intracellular molecules and activate eosinophils remains to be elucidated. In the present study, we performed inclusive analyses of protein phosphorylation in eosinophils and studied the functional relevance of such phosphorylation in cytokine production. Methods: Blood eosinophils were purified using Percoll and anti-CD16 antibody-coated magnetic beads. Purified eosinophils were stimulated with various stimuli. The eosinophil lysates were subjected to phosphoprotein analysis using the Luminex system. In some of these experiments, we studied the effect of a few signaling inhibitors on cytokine production from eosinophils. Results: We found that several factors such as IL-5, eotaxin, platelet-activating factor (PAF), and PGD2 phosphorylated Akt, ERK1/2, p38 MAPK, and glycogen synthase kinase-3 (GSK-3) in the eosinophils. Because eotaxin most potently induced the production of various cytokines, we performed the inhibition study using eotaxin-stimulated eosinophils. Eotaxin-induced production of IL-1β, IL-6, and MIP-1β was significantly reduced by the MEK inhibitor PD98059, p38 MAPK inhibitor SB203580, or PI3K inhibitor LY294002. In contrast, the GSK-3 inhibitor SB216763 blocked only IL-1β production from the eosinophils. Conclusions: In terms of the phosphorylation of intracellular signaling molecules, we could quantify the potency of various stimuli that activate eosinophils. We are the first to demonstrate the role of GSK-3 in cytokine production from eosinophils. The Luminex system aids in examining the mechanism of eosinophil activation.

A Case of Anaphylactic Reaction Following Matsutake Mushroom Ingestion: Demonstration of Histamine Release Reaction of Basophils

BACKGROUND Matsutake mushroom is not recognized as a common food allergen. However, several case reports have suggested that this mushroom can induce anaphylaxis on rare occasions. CASE SUMMARY We report a woman with bronchial asthma, who experienced two episodes of Matsutake-induced anaphylaxis. Both the prick-to-prick test and basophil histamine release test showed positive reactions to this mushroom in this patient, but not in control subjects. DISCUSSION Matsutake mushroom can, on rare occasions, cause anaphylaxis in sensitized people, a reaction so far observed only in Japan. Not ony the in vivo prick-to-prick test but also the in vitro basophil activation test utilizing the patients blood represent useful methods for allergen identification and also for identification of sensitized subjects.

The in vitro Effects of Advanced Glycation End Products on Basophil Functions

Background: Basophils are thought to play pivotal roles in the pathogenesis of allergic reactions, but their roles in inflammation associated with systemic abnormalities such as metabolic disorders remain largely unknown. Advanced glycation end products (AGEs) are potentially important substances produced in high-glucose disease conditions. In this in vitro study, we investigated whether the biological functions of human basophils can be influenced by AGEs. Methods: We analyzed the effects of AGEs on various functions and markers of human basophils, including CD11b expression, apoptosis, degranulation, and cytokine production. Results: Flow cytometric analysis indicated that the level of the receptor for AGEs (RAGE) on the surface of freshly isolated basophils was very low but was clearly upregulated by IL-3. Apoptosis of basophils was induced by high concentrations of glycated albumin. Although glycated albumin failed to affect the level of surface CD11b expression or to trigger degranulation or production of IL-4 and IL-13 in basophils, it dose-dependently induced IL-6 and IL-8 secretion. Conclusions: AGEs seem to act on human basophils; they suppress the cells’ longevity but elicit secretion of inflammatory cytokines. Through these biological changes, basophils might play some roles in inflammatory conditions associated with metabolic disorders presenting elevated levels of AGEs.