Oligonucleotide-microarray analysis of peripheral-blood lymphocytes in severe asthma

Abstract

CD4 + lymphocytes play a key role in asthma pathogenesis, but much remains unknown about the genetic mechanisms that affect disease severity. In this study we sought to investigate global patterns of gene expression in CD4 + lymphocytes isolated from subjects with severe asthma through the use of microarray technology. CD4 + lymphocytes were separated from peripheral blood, total RNA was purified, and biotinylated complementary RNA was prepared and hybridized to Affymetrix HU133 chips (Affymetrix, Santa Clara, Calif). Using the robust multi-chip average procedure, we compared the messenger RNA expression profiles of more than 33,000 genes of CD4 + lymphocytes in subjects with severe ( n = 5) and mild ( n = 5) asthma. Forty genes had 2-fold mean expression differences or greater. Thirty-seven genes were up-regulated, including transforming growth factor-beta and those involved in T-cell activation, proliferation, and cytoskeletal changes. Three genes were down-regulated, including the T-cell-receptor delta locus. This study demonstrates a method by which CD4 + lymphocytes can be extracted from blood for the purpose of microarray analysis. Furthermore, we show that T-lymphocytes from the peripheral blood of subjects with severe and mild asthma differ in their gene-expression profiles, supporting the view that asthma is a systemic disease. These differentially expressed genes identify potential molecular targets for preventive and therapeutic options for severe asthma.