Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn'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 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33(+) cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed > 99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed > 99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and 'virtual' Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0.9 +/- 0.16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1.80 +/- 0.8 (P = 0.004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD.
Hausmann
M
M
Department of Internal Medicine I, University of Regensburg, Germany.
Spöttl
T
T
Andus
T
T
Rothe
G
G
Falk
W
W
Schölmerich
J
J
Herfarth
H
H
Rogler
G
G
D018932:Chemokine CCL2 / Q000235:genetics; D003424:Crohn Disease / Q000201:enzymology* / Q000235:genetics; D020224:Expressed Sequence Tags; D005434:Flow Cytometry / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000201:enzymology*; D008264:Macrophages / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000235:genetics; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics*; D016897:Respiratory Burst; D015854:Up-Regulation
D018932:Chemokine CCL2; D008562:Membrane Glycoproteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases