Interleukin 10 inhibits interferon gamma- and tumor necrosis factor alpha-stimulated activation of NADPH oxidase 1 in human colonic epithelial cells and the mouse colon.
BACKGROUND
NADPH oxidase 1 (Nox1) is preferentially expressed in the colon, but its functional role is not fully understood. This study was designed to elucidate a potential role of Nox1 in inflammation of the colon.


METHODS
Superoxide production by T84 cells was measured by the cytochrome c method. Protein and mRNA levels of Nox1 and Nox organizer 1 (NOXO1) in the cells were measured by real-time reverse transcriptase PCR and Western blotting, respectively. Expression of Nox1, Nox2, dual oxidase 2 (Duox2), NOXO1, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha mRNAs was measured in proximal, middle, and distal portions of colonic mucosas from male wild-type C57BL/6J and interleukin (IL)-10 knockout mice at 6, 10, and 16 weeks of age. Grading of inflammation was done by scoring histological changes.


RESULTS
IL-10 significantly inhibited IFN-gamma- or TNF-alpha-induced up-regulation of superoxide-producing activity in T84 cells by suppressing expression of Nox1 mRNA and protein. IL-10 also inhibited TNF-alpha-stimulated induction of NOXO1 and p38 MAPK phosphorylation. Levels of Nox1, but not Nox2 or Duox2 mRNA, was age-dependently increased following a gradient with low levels in the proximal colon and high levels in the distal colon of the wild-type mice. The absence of IL-10 significantly facilitated Nox1 expression in association with increased IFN-gamma mRNA expression before the development of spontaneous colitis and age-dependently accelerated their mRNA expression.


CONCLUSIONS
IL-10 may be a possible down-regulator of the Nox1-based oxidase in the colon, suggesting a potential role of reactive oxygen species (ROS) derived from Nox1-based oxidase in inflammation of the colon.
Kamizato
Mai
M
Department of Stress Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho, Tokushima, Japan.
Nishida
Kensei
K
Masuda
Kiyoshi
K
Takeo
Keiko
K
Yamamoto
Yuta
Y
Kawai
Tomoko
T
Teshima-Kondo
Shigetada
S
Tanahashi
Toshihito
T
Rokutan
Kazuhito
K
D000818:Animals; D002478:Cells, Cultured; D003092:Colitis / Q000235:genetics; D003106:Colon / Q000201:enzymology / Q000378:metabolism; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D053709:Interleukin-10 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism; D053710:Interleukin-10 Receptor beta Subunit / Q000235:genetics / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D053709:Interleukin-10 Receptor alpha Subunit; D053710:Interleukin-10 Receptor beta Subunit; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D016753:Interleukin-10; D007371:Interferon-gamma; D009247:NADH, NADPH Oxidoreductases; C488842:Cybb protein, mouse; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C000614711:NOX1 protein, mouse; D048051:p38 Mitogen-Activated Protein Kinases