Endotoxin-, glutamate- and drug-induced inflammation and cytotoxicity with emphasis on signal transduction mechanisms
Author: Edling, Ylva
Date: 2008-03-14
Location: Farmakologens föreläsningssal, Nanna Svartz väg 2, Karolinska Institutet
Time: 09.00
Department: Institutionen för fysiologi och farmakologi / Department of Physiology and Pharmacology
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Thesis (1.041Mb)
Abstract
Inflammatory cells such as monocytes and glial cells play an important
role in the pathogenesis of ischemic- or drug-induced brain or liver
injury. Excessive concentrations of glutamate is toxic to neural cells,
and triggers a series of transcriptional events beginning with the
expression of the immediate early genes like c-fos and c-jun, which in
turn affect the expression of other genes necessary for the development
of brain injury. Glial cells are important mediators in such response as
they are responsible for the secretion of proinflammatory cytokines and
chemokines which in turn will cause recruitment of immune cells into the
brain. Using primary rat glial cell cultures we found that
lipopolysaccharide (LPS)- induced c-fos expression was p38 MAPK-dependent
and occurred via the activation of the SRE or the CRE elements in the
promoter. In contrast to what has been shown in neurons, we found that
glutamate-induced c-fos expression in glial cells involves a novel
calciumdependent pathway. This mechanism requires the participation of
mGluR5, mobilization of ER-calcium and de-repression of DREAM at the DRE
element in the c-fos promoter.
Similar mechanisms of inflammation as seen in the brain also occur in the liver, where Kupffer cells play a similar role functioning as the hepatic macrophages in their ability to release pro-inflammatory cytokines. Drug-induced hepatotoxicity is a major problem in drug development since preclinical in vitro as well as in vivo animal models usually are of little value for prediction of hepatotoxicity in humans. Pure hepatocyte cultures are generally not a sensitive enough model system to predict drug-induced cytotoxicity. We therefore developed a novel in vitro system containing both monocytes and hepatocytes. Ximelagatran (thrombin inhibitor) as well as the PPAR-gamma agonists troglitazone (hepatotoxic) and rosiglitazone (not hepatotoxic) were used as model compounds.
Studies in single cultures of monocytes (THP-1) showed a ximelagatran dependent release of pro-inflammatory chemokines and decreased cell viability, which was shown by inhibitors to involve the JNK- and ERK-signal transduction pathways.
A novel human in vitro co-culture model system containing THP-1 and hepatocytes (Huh-7) was established where the cells were separated by a permeable membrane. In such cocultures troglitazone-induced cytotoxicity was more apparent and observed earlier than using single cultures of either Huh-7 or THP-1 cells, whereas rosiglitazone showed no cytotoxicity in either system. The troglitazone effect was accompanied by a much greater expression of genes encoding pro-inflammatory cytokines, chemokines and several other stress-related genes using the co-culture system as compared to single cell type cultures. Conditioned medium from troglitazone-treated THP-1 cells decreased the viability of Huh-7 cells indicate the release of monocyte-derived mediators. It is concluded that such co-culture system might constitute a valuable tool for predictions of drug-induced hepatotoxicity.
Similar mechanisms of inflammation as seen in the brain also occur in the liver, where Kupffer cells play a similar role functioning as the hepatic macrophages in their ability to release pro-inflammatory cytokines. Drug-induced hepatotoxicity is a major problem in drug development since preclinical in vitro as well as in vivo animal models usually are of little value for prediction of hepatotoxicity in humans. Pure hepatocyte cultures are generally not a sensitive enough model system to predict drug-induced cytotoxicity. We therefore developed a novel in vitro system containing both monocytes and hepatocytes. Ximelagatran (thrombin inhibitor) as well as the PPAR-gamma agonists troglitazone (hepatotoxic) and rosiglitazone (not hepatotoxic) were used as model compounds.
Studies in single cultures of monocytes (THP-1) showed a ximelagatran dependent release of pro-inflammatory chemokines and decreased cell viability, which was shown by inhibitors to involve the JNK- and ERK-signal transduction pathways.
A novel human in vitro co-culture model system containing THP-1 and hepatocytes (Huh-7) was established where the cells were separated by a permeable membrane. In such cocultures troglitazone-induced cytotoxicity was more apparent and observed earlier than using single cultures of either Huh-7 or THP-1 cells, whereas rosiglitazone showed no cytotoxicity in either system. The troglitazone effect was accompanied by a much greater expression of genes encoding pro-inflammatory cytokines, chemokines and several other stress-related genes using the co-culture system as compared to single cell type cultures. Conditioned medium from troglitazone-treated THP-1 cells decreased the viability of Huh-7 cells indicate the release of monocyte-derived mediators. It is concluded that such co-culture system might constitute a valuable tool for predictions of drug-induced hepatotoxicity.
List of papers:
I. Simi A, Edling Y, Ingelman-Sundberg M, Tindberg N (2005). Activation of c-fos by lipopolysaccharide in glial cells via p38 mitogen-activated protein kinase-dependent activation of serum or cyclic AMP/calcium response element. J Neurochem. 92(4): 915-24.
Pubmed
II. Edling Y, Ingelman-Sundberg M, Simi A (2007). Glutamate activates c-fos in glial cells via a novel mechanism involving the glutamate receptor subtype mGlu5 and the transcriptional repressor DREAM. Glia. 55(3): 328-40.
Pubmed
III. Edling Y, Andersson T. B., Porsmyr-Palmertz M, Ingelman-Sundberg M (2007). Pro-inflammatory response and adverse drug reactions: mechanisms of action of ximelagatran on chemokine and cytokine activation in a monocyte in vitro model. [Submitted]
IV. Edling Y, Sivertsson L, Butura A, Ingelman-Sundberg M, Ek M (2008). Increased sensitivity for drug-induced hepatotoxicity using a novel human in vitro coculture model. [Submitted]
I. Simi A, Edling Y, Ingelman-Sundberg M, Tindberg N (2005). Activation of c-fos by lipopolysaccharide in glial cells via p38 mitogen-activated protein kinase-dependent activation of serum or cyclic AMP/calcium response element. J Neurochem. 92(4): 915-24.
Pubmed
II. Edling Y, Ingelman-Sundberg M, Simi A (2007). Glutamate activates c-fos in glial cells via a novel mechanism involving the glutamate receptor subtype mGlu5 and the transcriptional repressor DREAM. Glia. 55(3): 328-40.
Pubmed
III. Edling Y, Andersson T. B., Porsmyr-Palmertz M, Ingelman-Sundberg M (2007). Pro-inflammatory response and adverse drug reactions: mechanisms of action of ximelagatran on chemokine and cytokine activation in a monocyte in vitro model. [Submitted]
IV. Edling Y, Sivertsson L, Butura A, Ingelman-Sundberg M, Ek M (2008). Increased sensitivity for drug-induced hepatotoxicity using a novel human in vitro coculture model. [Submitted]
Issue date: 2008-02-22
Rights:
Publication year: 2008
ISBN: 978-91-7357-484-6
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