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

Abstract

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.

Gp91phox contributes to the development of experimental inflammatory bowel disease

“…21 These studies indicate that part of the treatment efficacy of drugs used in the treatment of IBD could be mediated by decreasing the production or effects of ROS. 22 Such information could contribute to future developments in anti-inflammatory therapy. However, no significant difference in colonic inflammation between p47 phoxÀ/À and WT mice in response to DSS challenge was reported, 9 indicating that gp47 phox and gp91 phox differ in their roles in intestinal acute inflammation, despite both being components of nicotinamide adenine dinucleotide phosphate oxidases.…”

ERK Activation Following Macrophage FcγR Ligation Leads to Chromatin Modifications at the IL-10 Locus

“…1). Consequently they can contribute to the pathology associated with a number of autoimmune diseases, such as rheumatoid arthritis (2), multiple sclerosis (3), and inflammatory bowel disease (4) to mention a few. We have recently shown that macrophages activated in the presence of immune complexes secreted high levels of IL-10.…”

Physiological Role of Macrophage Inflammatory Protein-3α Induction during Maturation of Intestinal Macrophages

“…A total of 10 7 cells were resuspended in 80 l of PBS and labeled with 20 l of immunomagnetic MicroBeads armed with CD33 Ab (isotype, mouse IgG1; clone, P67.6; Miltenyi Biotec). IMAC were purified twice with the help of type AS separation columns (Miltenyi Biotec) as described recently (14). Briefly, LPMNCs with magnetically labeled macrophages were passed through an AS separation column placed in the permanent magnet Super-MACS.…”

“…In addition, IMAC from normal mucosa lack expression of receptors for bacterial wall products such as TLRs 2 and 4 (13). Colonic macrophages from normal mucosa also have low antibacterial toxicity and lack enzymes for oxidative burst reaction such as NADPH oxidase (14).…”

“…For this study, we purified IMAC from normal mucosa according to a protocol recently established in our laboratory (9,14) and performed a subtractive hybridization to study mRNA expression in normal IMAC vs in vitro differentiated macrophages. In the subtraction of cDNA from in vitro differentiated macrophages from intestinal macrophages from normal mucosa, we obtained 76 gene products.…”