cDNA representational difference analysis of human neutrophils stimulated by GM-CSF

Neutrophils are the first cell type to migrate out of the vascular space and into the inflammatory site during an acute inflammation. However, in chronic inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), a lack of clearance of neutrophils, imbalance between inflammatory mediators produced by neutrophils and their natural inhibitors make these cells a potential cause of tissue destruction in lung disease. Neutrophilic inflammation is generally characterised by high levels of local expression of activating cytokines (e.g., GM-CSF). Only a few studies have been published so far that have investigated the expression of genes preferentially expressed in activated neutrophils. The isolation of such genes, however, can lead to a better understanding of inflammatory disease and the identification of potential novel therapeutic targets or markers of the disease. We performed representational difference analysis of cDNA, a sensitive PCR-based subtractive enrichment procedure, and isolated 12 genes, 1 EST clone, and 3 sequences not represented in the public databases. Differential expression for 9 of these clones was confirmed by Northern hybridisation. Of the above nine transcripts three were chosen and shown to be up-regulated in neutrophils cocultured with stimulated primary human bronchial epithelial cells using a semiquantitative RT-PCR approach. Among the known genes identified were HM-74, CIS1, Cathepsin C, alpha-enolase, CD44, and the gene Translocation Three Four (TTF), most of them previously not known to be involved in GM-CSF induced neutrophil activation. Along with its tissue and cellular distribution we also derived the complete cDNA sequence and genomic structure of CIS1 using an in silico approach. In addition, we also report the initial characterisation of a novel gene, P1-89 that is primarily expressed in granulocytes and is up-regulated in activated cells. Our results identify several important genes associated with neutrophil activation and can lead to a better understanding of the molecular mechanisms of neutrophilic inflammations.