title: Effects of pro-inflammatory cytokines, lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability.
abstract: Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE2, PGF2α) or their corresponding ethanolamides (PGE2-EA or PGF2α-EA) over 48h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10(-5)M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10(-4)M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE2 transiently increased Caco-2 monolayer permeability at 24h, while LPS (10μg/ml) increased permeability over 24-48h. These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE2 transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.
authors.affiliation: Discipline of Pharmacology, School of Medicine, Faculty of Health Sciences, The University of Adelaide, South Australia, Australia.
authors.forename: B K
authors.initials: BK
authors.lastname: Safdari
authors.affiliation: Department of Surgery, Flinders Medical Centre, Bedford Park, South Australia, Australia.
authors.forename: T C
authors.initials: TC
authors.lastname: Sia
authors.affiliation: Department of Surgery, Flinders Medical Centre, Bedford Park, South Australia, Australia.
authors.forename: D A
authors.initials: DA
authors.lastname: Wattchow
authors.affiliation: Discipline of Pharmacology, School of Medicine, Faculty of Health Sciences, The University of Adelaide, South Australia, Australia. Electronic address: scott.smid@adelaide.edu.au.
authors.forename: S D
authors.initials: SD
authors.lastname: Smid
mesh_terms: D018938:Caco-2 Cells; D003092:Colitis / Q000378:metabolism* / Q000473:pathology; D003106:Colon / Q000378:metabolism* / Q000473:pathology; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D008070:Lipopolysaccharides / Q000633:toxicity*; D009469:Neuromuscular Junction / Q000378:metabolism* / Q000502:physiology; D010539:Permeability
chemical_list: D016207:Cytokines; D008070:Lipopolysaccharides; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human
keywords: COX-2; Colitis; Epithelial permeability; Interleukin 17; Prostaglandin ethanolamides; Prostaglandins