PC Reduces TNF-alpha-Induced Upregulation of Proinflammatory Cytokines
Background: Phosphatidylcholine (PC) is a major lipid of the gastrointestinal mucus layer. We recently showed that mucus from patients suffering from ulcerative colitis has low levels of PC. Clinical studies reveal that the therapeutic addition of PC to the colonic mucus using slow release preparations is beneficial. The positive role of PC in this disease is still unclear; however, we have recently shown that PC has an intrinsic anti-inflammatory property. It could be demonstrated that the exogenous application of PC inhibits membrane-dependent actin assembly and TNF-α-induced nuclear NF-κB activation. We investigate here in more detail the hypothesis that the exogenous application of PC has anti-inflammatory properties.
Methods: PC species with different fatty acid side chains were applied to differentiated and non-differentiated Caco-2 cells treated with TNF-α to induce a pro-inflammatory response. We analysed TNF-α-induced NF-κB-activation via the transient expression of a NF-κB-luciferase reporter system. Pro-inflammatory gene transcription was detected with the help of a quantitative real time (RT)-PCR analysis. We assessed the binding of TNF-α to its receptor by FACS and analysed lipid rafts by isolating detergent resistant membranes (DRMs).
Results: The exogenous addition of all PC species tested significantly inhibited TNF-α-induced pro-inflammatory signalling. The expression levels of IL-8, ICAM-1, IP-10, MCP-1, TNF-α and MMP-1 were significantly reduced after PC pre-treatment for at least two hours. The effect was comparable to the inhibition of NF-kB by the NF-kB inhibitor SN 50 and was not due to a reduced binding of TNF-α to its receptor or a decreased surface expression of TNF-α receptors. PC was also effective when applied to the apical side of polarised Caco-2 cultures if cells were stimulated from the basolateral side. PC treatment changed the compartmentation of the TNF-α-receptors 1 and 2 to DRMs.
Conclusion: PC induces a prolonged inhibition of TNF-α-induced pro-inflammatory signalling. This inhibition may be caused by a shift of the TNF-α receptors at the surface to lipid rafts. Our results may offer a potential molecular explanation for the positive role of PC seen in clinical studies for the treatment of ulcerative colitis.
Inflammatory bowel disease (IBD) is the result of a chronic intestinal inflammatory response. While the exact pathogenesis of IBD remains incompletely understood, it is likely that the initiation of the immune response is triggered by luminal factors. The nature of these initiating agents is unclear, but both orally ingested nutrients and microbial agents have been implicated. It is widely believed that an impaired barrier function, and in particular a defect of the mucus layer, leads to an increased exposure of the mucosal immune system to luminal antigens. In genetically susceptible individuals, this results in an inappropriate and unrestrained inflammatory response.
We have recently shown that both PC and lysophosphatidylcholine (LPC) but neither phosphatidylethanolamine (PE) nor sphingomyelin (SM) are decreased in the mucus of patients with ulcerative colitis (UC) and that the oral substitution of PC using slow release preparations is beneficial. How luminal PC influences the clinical course of UC is not yet known, but two scenarios are possible. First, because of its hydrophobic property, PC coats the mucus layer, thereby preventing the contact of luminal bacteria to the mucosa. With respect to this, luminal PC has been shown to synergize with conjugated primary bile salts in the binding of luminal endotoxin, which in turn leads to a suppressed inflammation beyond the mucosal surface. On the other hand, we could recently demonstrate that PC per se has anti-inflammatory properties. PC has been shown to inhibit membrane-dependent actin assembly and TNF-α-induced MAPkinase and NF-κB activation. In light of these results, we hypothesized that luminal PC might be integrated into the plasma membranes of enterocytes and in turn modulate the signalling state of the mucosa in the human intestine. This assumption is further substantiated by studies using an in vitro phagosome system which show that exogenous PC is indeed involved in the networks which inhibit pro-inflammatory signalling in membranes.
IBD encompasses two chronic intestinal diseases, Crohn's disease (CD) and UC, which differ in their microscopic and macroscopic features although their symptoms are similar. Ulcerative lesions in IBD are accompanied by a prominent infiltrate of inflammatory cells including T lymphocytes, macrophages, neutrophils, and mast and plasma cells. Mechanisms involved in the recruiting and activating of these inflammatory cells are thought to encompass a complex interplay of inflammatory mediators. This is reflected by the elevation of various chemokines in the serum and mucosa of IBD patients (for review see). Over 40 human chemokines are now acknowledged, each with its own specific pattern of cellular chemotaxis. The chemokine family is categorised into four groups depending on the spacing of their first two cysteine residues. Cumulative studies demonstrate that all four types of chemokines are involved in the development of IBD. The expression level of pro-inflammatory chemokines differed significantly between IBD patients and controls. Up-regulated chemokine expression in human biopsies correlated with increasing activity of the disease. Pro-inflammatory cytokines such as TNF-α and interleukin 1β (IL-1β) up-regulate the transcription of chemokine genes and hence the synthesis of chemokines themselves through the activation of NF-κB. As TNF-α has been shown to be an important player in the inflammatory process of IBD, we previously established a model cell system with human intestinal epithelial cells (Caco-2) which we stimulated with TNF-α to induce a pro-inflammatory response. Using this system, we now analysed the effect of various PC species on the transcriptional levels of selected marker genes. After PC treatment, the TNF-α induced up-regulation was significantly reduced in a time- and dose-dependent manner depending on the fatty acid composition. PC was effective when applied to the apical side of polarized Caco-2 cells if they were stimulated from the basal side. We could show that the TNF-α effect was dependent on NF-κB activity and not due to inhibition of the binding of TNF-α to its receptor. PC treatment changed the compartmentation of TNF-α-R1 and TNF-α-R2 to lipid rafts, which is a possible mechanism of action.
Abstract and Background
Abstract
Background: Phosphatidylcholine (PC) is a major lipid of the gastrointestinal mucus layer. We recently showed that mucus from patients suffering from ulcerative colitis has low levels of PC. Clinical studies reveal that the therapeutic addition of PC to the colonic mucus using slow release preparations is beneficial. The positive role of PC in this disease is still unclear; however, we have recently shown that PC has an intrinsic anti-inflammatory property. It could be demonstrated that the exogenous application of PC inhibits membrane-dependent actin assembly and TNF-α-induced nuclear NF-κB activation. We investigate here in more detail the hypothesis that the exogenous application of PC has anti-inflammatory properties.
Methods: PC species with different fatty acid side chains were applied to differentiated and non-differentiated Caco-2 cells treated with TNF-α to induce a pro-inflammatory response. We analysed TNF-α-induced NF-κB-activation via the transient expression of a NF-κB-luciferase reporter system. Pro-inflammatory gene transcription was detected with the help of a quantitative real time (RT)-PCR analysis. We assessed the binding of TNF-α to its receptor by FACS and analysed lipid rafts by isolating detergent resistant membranes (DRMs).
Results: The exogenous addition of all PC species tested significantly inhibited TNF-α-induced pro-inflammatory signalling. The expression levels of IL-8, ICAM-1, IP-10, MCP-1, TNF-α and MMP-1 were significantly reduced after PC pre-treatment for at least two hours. The effect was comparable to the inhibition of NF-kB by the NF-kB inhibitor SN 50 and was not due to a reduced binding of TNF-α to its receptor or a decreased surface expression of TNF-α receptors. PC was also effective when applied to the apical side of polarised Caco-2 cultures if cells were stimulated from the basolateral side. PC treatment changed the compartmentation of the TNF-α-receptors 1 and 2 to DRMs.
Conclusion: PC induces a prolonged inhibition of TNF-α-induced pro-inflammatory signalling. This inhibition may be caused by a shift of the TNF-α receptors at the surface to lipid rafts. Our results may offer a potential molecular explanation for the positive role of PC seen in clinical studies for the treatment of ulcerative colitis.
Background
Inflammatory bowel disease (IBD) is the result of a chronic intestinal inflammatory response. While the exact pathogenesis of IBD remains incompletely understood, it is likely that the initiation of the immune response is triggered by luminal factors. The nature of these initiating agents is unclear, but both orally ingested nutrients and microbial agents have been implicated. It is widely believed that an impaired barrier function, and in particular a defect of the mucus layer, leads to an increased exposure of the mucosal immune system to luminal antigens. In genetically susceptible individuals, this results in an inappropriate and unrestrained inflammatory response.
We have recently shown that both PC and lysophosphatidylcholine (LPC) but neither phosphatidylethanolamine (PE) nor sphingomyelin (SM) are decreased in the mucus of patients with ulcerative colitis (UC) and that the oral substitution of PC using slow release preparations is beneficial. How luminal PC influences the clinical course of UC is not yet known, but two scenarios are possible. First, because of its hydrophobic property, PC coats the mucus layer, thereby preventing the contact of luminal bacteria to the mucosa. With respect to this, luminal PC has been shown to synergize with conjugated primary bile salts in the binding of luminal endotoxin, which in turn leads to a suppressed inflammation beyond the mucosal surface. On the other hand, we could recently demonstrate that PC per se has anti-inflammatory properties. PC has been shown to inhibit membrane-dependent actin assembly and TNF-α-induced MAPkinase and NF-κB activation. In light of these results, we hypothesized that luminal PC might be integrated into the plasma membranes of enterocytes and in turn modulate the signalling state of the mucosa in the human intestine. This assumption is further substantiated by studies using an in vitro phagosome system which show that exogenous PC is indeed involved in the networks which inhibit pro-inflammatory signalling in membranes.
IBD encompasses two chronic intestinal diseases, Crohn's disease (CD) and UC, which differ in their microscopic and macroscopic features although their symptoms are similar. Ulcerative lesions in IBD are accompanied by a prominent infiltrate of inflammatory cells including T lymphocytes, macrophages, neutrophils, and mast and plasma cells. Mechanisms involved in the recruiting and activating of these inflammatory cells are thought to encompass a complex interplay of inflammatory mediators. This is reflected by the elevation of various chemokines in the serum and mucosa of IBD patients (for review see). Over 40 human chemokines are now acknowledged, each with its own specific pattern of cellular chemotaxis. The chemokine family is categorised into four groups depending on the spacing of their first two cysteine residues. Cumulative studies demonstrate that all four types of chemokines are involved in the development of IBD. The expression level of pro-inflammatory chemokines differed significantly between IBD patients and controls. Up-regulated chemokine expression in human biopsies correlated with increasing activity of the disease. Pro-inflammatory cytokines such as TNF-α and interleukin 1β (IL-1β) up-regulate the transcription of chemokine genes and hence the synthesis of chemokines themselves through the activation of NF-κB. As TNF-α has been shown to be an important player in the inflammatory process of IBD, we previously established a model cell system with human intestinal epithelial cells (Caco-2) which we stimulated with TNF-α to induce a pro-inflammatory response. Using this system, we now analysed the effect of various PC species on the transcriptional levels of selected marker genes. After PC treatment, the TNF-α induced up-regulation was significantly reduced in a time- and dose-dependent manner depending on the fatty acid composition. PC was effective when applied to the apical side of polarized Caco-2 cells if they were stimulated from the basal side. We could show that the TNF-α effect was dependent on NF-κB activity and not due to inhibition of the binding of TNF-α to its receptor. PC treatment changed the compartmentation of TNF-α-R1 and TNF-α-R2 to lipid rafts, which is a possible mechanism of action.
SHARE
