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

Neutrophils are the first cell type to migrate out of the vascular space an d into the inflammatory site during an acute inflammation, However, in chro nic inflammatory diseases, such as chronic obstructive pulmonary disease (C OPD), a lack of clearance of neutrophils, imbalance between inflammatory me diators produced by neutrophils and their natural inhibitors make these cel ls a potential cause of tissue destruction in lung disease. Neutrophilic in flammation is generally characterised by high levels of local expression of activating: cytokines (e.g., GM-CSF), Only a few studies have been publish ed so far that have investigated the expression of genes preferentially exp ressed in activated neutrophils. The isolation of such genes, however, can lead to a better understanding of inflammatory disease and the identificati on of potential novel therapeutic targets or markers of the disease. We per formed 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 abov e nine transcripts three were chosen and shown to be up-regulated in neutro phils cocultured with stimulated primary human bronchial epithelial cells u sing a semiquantitative RT-PCR approach. Among the known genes identified w ere HM-74, CIS1, Cathepsin C, alpha -enolase, CD44, and the gene Translocat ion Three Four (TTF), most of them previously not known to be involved in G M-CSF induced neutrophil activation. Along with its tissue and cellular dis tribution we also derived the complete cDNA sequence and genomic structure of CIS1 using an in silico approach. In addition, we also report the initia l characterisation of a novel gene, P1-89 that is primarily expressed in gr anulocytes and is up-regulated in activated cells. Our results identify sev eral important genes associated with neutrophil activation and can lead to a better understanding of the molecular mechanisms of neutrophilic inflamma tions. (C) 2000 Academic Press.