Suppression of pro-inflammatory cytokine release by selective inhibition of inducible nitric oxide synthase in mucosal explants from patients with ulcerative colitis.
BACKGROUND
In ulcerative colitis (UC), inflammatory damage is associated with increased production of pro-inflammatory cytokines and nitric oxide through the inducible nitric oxide synthase (iNOS) pathway. In an animal model of acute experimental colitis we have previously shown amelioration of inflammation with the highly selective iNOS inhibitor 1400W. The aim of the present study was to investigate the effects of selective iNOS inhibition on the production of pro-inflammatory cytokines by the colon mucosa in UC.


METHODS
Inflamed and uninflamed mucosa from patients with severe UC were incubated with a highly selective iNOS inhibitor N-[3-(aminomethyl)benzyl]acetamidine (1400W), with a relatively selective cNOS inhibitor N(G)-nitro-L-arginine-methyl-esther (L-NAME), or with an NO-donor, S-nitroso-acetylpenicillamine (SNAP). Cytokine concentrations in the incubation medium were quantitated with ELISA.


RESULTS
Compared to uninflamed mucosa there was an increase in iNOS protein and nitrotyrosine levels in inflamed mucosal samples. Immunolocalization of iNOS and nitrotyrosine showed their expression in inflammatory cells in the lamina propria. Expression of iNOS was also found in the epithelial brush border. Selective inhibition of iNOS suppressed the release of tumour necrosis factor alpha (TNF-alpha, by 66%) and interleukin-6 (IL-6, by 27%). The NO-donor, SNAP, augmented the secretion of TNF-alpha, IL-6 and IL-1-beta (by 62%, 52% and 175%, respectively) and decreased the release of IL-1 receptor antagonist (IL-1Ra, by 34%) by the inflamed mucosa. Moreover, in uninflamed samples, 1400W suppressed the production of TNF-alpha (by 69%) and incubation with SNAP decreased IL-6 concentrations by 48%. The cNOS over iNOS selective inhibitor L-NAME had no significant effects on the accumulation of cytokines.


CONCLUSIONS
Selective inhibition of iNOS suppresses mucosal TNF-alpha and IL-6 release in active UC, whereas NO seems to exacerbate the inflammatory response. These results suggest that selective iNOS inhibition may have therapeutic promise in the treatment of UC.
Kankuri
E
E
Institute of Biomedicine, Pharmacology, University of Helsinki, BIOMEDICUM Helsinki, Finland. esko.kankuri@helsinki.fi
Hämäläinen
M
M
Hukkanen
M
M
Salmenperä
P
P
Kivilaakso
E
E
Vapaatalo
H
H
Moilanen
E
E
D000328:Adult; D000578:Amidines / Q000494:pharmacology; D001596:Benzylamines / Q000494:pharmacology; D003093:Colitis, Ulcerative / Q000378:metabolism* / Q000473:pathology; D003106:Colon / Q000378:metabolism* / Q000473:pathology; D016207:Cytokines / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D018836:Inflammation Mediators / Q000378:metabolism*; D007375:Interleukin-1 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000578:Amidines; D001596:Benzylamines; D016207:Cytokines; D004791:Enzyme Inhibitors; D018836:Inflammation Mediators; D007375:Interleukin-1; D015850:Interleukin-6; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D020030:Nitric Oxide Donors; D014409:Tumor Necrosis Factor-alpha; C002744:3-nitrotyrosine; D014443:Tyrosine; D026423:S-Nitroso-N-Acetylpenicillamine; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D019331:NG-Nitroarginine Methyl Ester