Subtractive screening reveals up-regulation of NADPH oxidase expression inCrohn's disease intestinal macrophages

Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 o r CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a p opulation of macrophages displaying these markers, secreting higher cytokin e levels and representing an activated cell population. CD33(+) cells (macr ophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed sub tracting mRNA from normal macrophages from those of Crohn's disease macroph ages. Oxidative burst activity was determined by flow cytometry. Seventy cl ones were obtained by the subtractive mRNA screening. Sequencing showed > 9 9% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed > 99% homology to mRN A of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed b y RT-PCR and 'virtual' Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0.9 +/- 0.16 in control macrophages indicati ng a lack of oxidative burst activity. In Crohn's disease this ratio was si gnificantly increased to 1.80 +/- 0.8 (P = 0.004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macro phages from normal mucosa correlating with a lack of oxidative burst activi ty. In IBD macrophage-oxidative burst activity is increased and NADPH oxida se mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical t arget in IBD and reduce mucosal tissue damage in active IBD.