Downregulation of the ubiquitin-proteasome system in normal colonic macrophages and reinduction in inflammatory bowel disease.

BACKGROUND: In normal mucosa, intestinal lamina propria macrophages (IMACs) maintain tolerance against food antigens and the commensal bacterial flora. Several mechanisms have been identified that mediate tolerance. The ubiquitin-proteasome system (UPS) is a large multiprotein complex that degrades cellular proteins. As the UPS may modulate immune functions of IMACs, we performed a detailed investigation of UPS expression and function under normal conditions and in cells derived from patients suffering from inflammatory bowel disease (IBD). METHODS: IMACs were isolated from intestinal mucosa. mRNA expression of macrophages differentiated in vitro (i.v. MACs) and IMACs was compared by Affymetrix® oligonucleotide arrays. Quantitative Taqman-PCR was performed on five exemplary proteasomal and five ubiquitinylation genes each. Proteins were analyzed by immunohistochemistry and Western blotting. Proteasome function was assessed by a fluorimetric test. RESULTS: Affymetrix analysis showed downregulation of mRNA expression of almost all represented proteasomal and of 22 ubiquitination-associated genes in IMACs as compared to i.v. MACs and monocytes. By quantitative PCR, up to tenfold higher mRNA expression of 10 exemplary genes of the UPS (UBE2A, UBE2D2, UBE2L6, USP14, UBB and ATPase2, ß2, ß5, ß2i/MECL-1, ß5i/LMP7) was demonstrated in i.v. MACs as compared to IMACs. Immunohistochemistry and Western blots confirmed these findings in intestinal mucosa of controls and patients suffering from diverticulitis. In contrast, a significant increase in protein amounts was found in mucosa of patients with IBD. CONCLUSION: Reduced expression of subunits of the UPS in IMACs of normal mucosa supports the concept of the presence of a nonreactive, anergic macrophage phenotype in the gut under normal conditions. Reinduction in IMACs of IBD mucosa reflects activated IMACs which can present antigenic peptides and thus support inflammation.

Inflamed intestinal mucosa features a specific epithelial expression pattern of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first step in the degradation of tryptophan, an essential amino acid. During inflammation IDO can be induced in different cell types resulting in local tryptophan depletion. This inhibits T cell proliferation and may induce apoptosis. High expression of IDO was previously found in inflammatory bowel disease and is thought to represent a mechanism for downregulation of the local immune response. Our aim is to investigate the expression pattern of IDO in normal and inflamed murine and human intestinal mucosa. Immunohistochemical staining for IDO was performed on paraffin sections of colon of two mouse models for colitis and their controls and on paraffin sections of human ileum and colon in normal and two different inflammatory conditions, namely inflammatory bowel disease and diverticulitis. IDO immunohistochemistry showed similar results in murine and human tissue. In normal, as well as in inflamed mucosa, some mononuclear cells, fibroblasts and endothelial cells were positive for IDO. In inflamed mucosa a specific expression pattern of epithelial IDO was found where epithelial cells flanking ulcers or bordering crypt abscesses showed high IDO expression. Moreover, in human intestinal inflammation, IDO was expressed in ulcer associated cell lineage. Since bacterial invasion is more pronounced in erosions and in crypt abscesses and since IDO activity and the resulting local tryptophan depletion can cause growth arrest of several tryptophan-dependent microorganisms, IDO expression in the vicinity of interruptions of the epithelial barrier may point to a role for IDO as a local anti-infectious agent. Furthermore, expression of IDO at the margin of ulcerations and in the reparative ulcer-associated cell lineage suggests involvement of IDO in repair processes.