ABSTRACT
In inflammatory bowel disease, cells that infiltrate the mucosa regulate intestinal epithelial cell function partly through release of pro- and anti-inflammatory cytokines. The aim of this study is to evaluate the role of the anti-inflammatory cytokine, IL-10, on normal mouse intestinal epithelial cells (Mode-K) in the absence or presence of IL-1. Western blotting assays and immunocytochemistry were used to identify the presence of IL-1 and IL-10 receptors on Mode-K cells; and electrophoretic mobility shift assays were used to study the activation of NF-kappaB transcription factor. Stimulation of Mode-K cells with IL-1 or IL-10 did not modify IL-1 and IL-10 receptor expression levels. IL-1 induced the synthesis of the enzyme cyclooxygenase-2 (COX-2) through the activation and translocation of p65 subunit of NF-kappaB. Inhibition of translocated p65 binding to DNA, inhibited COX-2 production and induced apoptosis. IL-10 inhibited IL-1-induced effects on IKB-alpha and IKB-beta proteins through stabilizing these proteins; subsequently causing inhibition of NF-kappaB translocation to the nucleus and any subsequent induction of COX-2. These data support a role for IL-10 in the regulation of IEC function under inflammatory conditions and the involvement of COX-2 in inhibiting apoptosis in mouse intestinal epithelial cells.
Subject(s)
Epithelial Cells/drug effects , Epithelial Cells/metabolism , Interleukin-10/pharmacology , Intestinal Mucosa/metabolism , Intestines/drug effects , NF-kappa B/metabolism , Animals , Cells, Cultured , Cyclooxygenase 2/metabolism , Dinoprostone/metabolism , I-kappa B Kinase/metabolism , Inflammation/metabolism , Interleukin-1/pharmacology , Mice , NF-kappa B/antagonists & inhibitors , Receptors, Interleukin-1/metabolism , Receptors, Interleukin-10/metabolismABSTRACT
BACKGROUND: Interleukin-1 (IL-1), an inflammatory cytokine whose levels are elevated in inflamed mucosa, causes part of its effect on intestinal epithelial cells (IEC) through inducing ceramide production. AIM: To study the role of nuclear factor-kappaB (NF-kappaB), a pro-inflammatory and anti-apoptotic factor, in IL-1-treated IEC. METHODS: NF-kappaB activity and levels of apoptotic proteins were assessed by electrophoretic mobility shift assay and RNA-protection assay, respectively. RESULTS: IL-1 and ceramide, which have been shown to partially mediate IL-1 effects on IEC, activated NF-kappaB levels significantly. This activation was due to a decrease in IkappaB-alpha and IkappaB-beta protein levels. Moreover, the ratio of mRNA levels of anti-apoptotic to pro-apoptotic proteins was significantly increased in IL-1-treated IEC. CONCLUSION: NF-kappaB may play a key role in the regulation of the expression of pro-inflammatory and/or apoptotic genes in inflammatory bowel disease, making this protein an attractive target for therapeutic intervention.
Subject(s)
Enteritis/metabolism , Epithelial Cells/metabolism , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , NF-kappa B/metabolism , Animals , Apoptosis/immunology , Apoptosis Regulatory Proteins , Cell Line , Ceramides/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Enteritis/pathology , Epithelial Cells/drug effects , Epithelial Cells/immunology , I-kappa B Proteins/metabolism , Interleukin-1/pharmacology , Leupeptins/pharmacology , NF-KappaB Inhibitor alpha , Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/metabolism , bcl-X ProteinABSTRACT
A variety of factors contribute to the complex course of inflammation. Microbiological, immunological and toxic agents can initiate the inflammatory response by activating a variety of humoral and cellular mediators. In the early phase of inflammation, excessive amounts of cytokines and inflammatory mediators are released. These factors activate, in addition to other signaling pathways, the lipid synthesis pathways, which play a crucial role in the pathogenesis of organ dysfunction. Arachidonic acid (AA), the precursor of pro-inflammatory eicosanoids, is released from membrane phospholipids by the action of phospholipase A(2) (PLA(2)), and is metabolized to prostaglandins (PGs) and leukotrienes (LTs) by the action of cyclooxygenase (COX) and lipoxygenase (LO) enzymes, respectively. Disordered activation of PLA(2), LO and COX enzymes have been implicated in many inflammatory diseases. PLA(2) is activated by phospholipase-A(2)-activating protein (PLAP) and LO by 5-lipoxygenase-activating protein (FLAP). The inducible form of COX-2 enzyme, which is usually not present under basal conditions, is induced in inflammation. In this article the function of these enzymes in eicosanoid synthesis, their regulation, and their implication in inflammatory disorders will be reviewed. The properties, function and regulation of the protein activators PLAP and FLAP will also be discussed.
Subject(s)
Eicosanoids/biosynthesis , Inflammation Mediators/metabolism , Inflammation/enzymology , Inflammation/metabolism , Animals , Arthritis, Rheumatoid/enzymology , Arthritis, Rheumatoid/metabolism , Humans , Inflammatory Bowel Diseases/enzymology , Inflammatory Bowel Diseases/metabolismABSTRACT
BACKGROUND: In inflammatory bowel disease (IBD), cytokine levels (such as interleukin-1 (IL-1)) are elevated. We have shown previously that IL-1 activates phospholipid signaling pathways in intestinal epithelial cells (EEC), leading to increased ceramide levels. AIM: To determine whether ceramide induces apoptosis in IEC. METHODS: Apoptosis was evaluated by annexin-V binding or Hoechst nuclear staining. Levels of bcl-2, bcl-x, bax, p53 and p21 were determined by Western blotting, and celi cycle analysis was determined by flow cytometry. RESULTS: IL-1 increased ceramide accumulation in a time-dependent and concentration-dependent manner with a peak response at 4 h, with [IL-1] = 30 ng/ml. Neither IL-1 nor ceramide induced apoptosis in EEC, but they increased bcl-2 levels and decreased bax and p21 levels without affecting bcl-x and p53 levels. They also caused a slight but significant increase in the G2/M phase. These data suggest a role for ceramide in IBD and suggest a possible mechanism for the enhanced tumorigenic activity in IBD patients.
