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, β2, β5, β2i/MECL-1, β5i/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.


CONCLUSIONS
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.
Department of Internal Medicine I, University of Regensburg, Regensburg, Germany.
A M
AM
Hetzenecker
M C
MC
Seidl
K
K
Kosovac
H
H
Herfarth
S
S
Kellermeier
F
F
Obermeier
W
W
Falk
J
J
Schoelmerich
M
M
Hausmann
G
G
Rogler
D000251:Adenosine Triphosphatases / Q000235:genetics; D002454:Cell Differentiation; D002478:Cells, Cultured; D003093:Colitis, Ulcerative / Q000201:enzymology / Q000235:genetics*; D003106:Colon / Q000201:enzymology; D003424:Crohn Disease / Q000201:enzymology / Q000235:genetics*; D004238:Diverticulitis / Q000201:enzymology / Q000235:genetics; D015536:Down-Regulation; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology*; D008264:Macrophages / Q000201:enzymology*; D046228:Microarray Analysis; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000097:blood / Q000235:genetics* / Q000378:metabolism*
C506526:ATP5F1B protein, human; D012333:RNA, Messenger; C479419:UBB protein, human; C509299:USP14 protein, human; D025801:Ubiquitin; C471545:UBE2A protein, human; C097309:UBE2D2 protein, human; C471622:UBE2L6 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D043222:Ubiquitin Thiolesterase; C077436:LMP7 protein; C098948:PSMB10 protein, human; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human