Development of a microarray-based method to detect exposure of human basophils to IL-3

Abstract

Human basophils are an accessible participant of the human allergic reaction. There is natural variation in various functional endpoints and in signaling molecule expression but there has been only a limited effort to place this information in the context of mRNA expression profiles. The goal of these studies was to develop gene transcript profiles associated with IL-3 exposure for use in probing basophil phenotypes. Highly purified human basophils were cultured under several conditions and the cells subsequently analyzed by microarray analysis of gene transcript expression. Basophils were cultured with or without IL-3 for 24h and the expression profile, relative to pre-culture, analyzed for transcripts that act in a reciprocal fashion depending on the condition.A 51-gene transcript set was identified that was composed of 34 transcripts that increased with IL-3 and decreased without IL-3 and a further 17 transcripts that did the reverse. This set of transcripts was validated on two microarray chips. The changes induced by IL-3 were stable in time, with 3day cultures generating a signature concordant with 1day cultures. The inverted nature of the response to ±IL-3 suggested that the normal circulating basophil is balanced between a state of high and low IL-3 exposure and thus is very sensitive to changes in IL-3. For example, the basophil could detect the early generation of IL-3 that follows IgE-mediated stimulation of basophils. When this signature tool was applied to freshly isolated basophils, it was observed that the signature was similar for the same subjects’ basophil sampled weeks apart. It was also shown that the 51-gene transcript was insensitive to the method of preparing purified basophils. Finally, these studies provided an estimate for the normal in vivo exposure of circulating basophils to IL-3 or IL-3-like functionality. These studies identified an IL-3 signature to probe changes in basophils occurring in vivo.