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Studies of differential gene expression in clinically derived eosinophil populations
1Discovery Biology, Pfizer Central Research, Sandwich, UK.
Background:
Influx of eosinophils into the post-capillary bronchial epithelium and the subsequent release of inflammatory mediators is characteristic of the late phase of asthmatic attacks. The genes that serve to predispose the peripheral blood eosinophils of asthmatics to undergo this process are poorly defined. The aim of this report is to describe the differential gene expression of both the known pro-inflammatory genes 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) and novel cDNA sequences in eosinophils derived from clinical samples.
Methods:
Novel cDNA sequences representing genes upregulated in peripheral blood eosinophils of asthmatic as compared with nonasthmatic patients were identified by differential display polymerase chain reaction (DDPCR). The differential expression of these sequences, in addition to known pro-inflammatory genes, were then studied by reverse dot blotting of amplified RNA generated from the eosinophils of nonasthmatic donors, asthmatic donors, asthmatic donors taking steroids, interleukin (IL) -3, IL-5, granulocyte-macrophage colony stimulating factor- (GM-CSF) treated eosinophils from asthmatic donors and the eosinophilic cell line AML14.
Results:
Four unique DDPCR-generated 3'UTR DNA fragments were identified that showed differing patterns of expression between the eosinophil populations of interest. Expression of each of the novel clones was increased in the peripheral blood eosinophils of asthmatics and downregulated in those donors taking steroids. Expression of 5-lipoxygenase was not found to vary between the different eosinophil populations, whereas FLAP was induced by treatment with the cytokine cocktail in both primary eosinophils and the eosinophilic cell line AML14.
Conclusion:
The differential regulation of the novel cDNA sequences and FLAP in the range of eosinophil populations studied suggest that they may provide clinically relevant therapeutic targets. Moreover, the procedures used in these studies may provide a general approach to the study of differential gene expression in small numbers of cells such as those obtained from clinical samples.