HSP60 and CpG-DNA-oligonucleotides differentially regulate LPS-tolerance of hepatic Kupffer cells
Introduction
Hepatic Kupffer cells (KC), the monocytic cell population of the liver, are major regulators of the immune response to bacterial compounds such as LPS and DNA that are drained from the gut by the portal vein to the liver [1]. The KCs need to discriminate between physiological levels of bacterial products that should be tolerated and gut-derived bacterial infection that should be attacked. Thus, tight control of KC activation is critical for up-regulation of inflammatory immunity to bacterial infection, but also for down-regulation of the inflammatory response, in order to prevent lethality by septic shock. Alcoholic hepatitis is associated with leakage of the gut mucosal barrier, which results in uncontrolled activation of hepatic Kupffer cells by bacterial products and subsequent hepatitis with a potentially lethal course [2], [3]. Therefore, elucidation of the regulatory mechanisms of KC activation seems important for understanding two major diseases, alcoholic hepatitis and septic shock.
Although in the focus of research for many years, mechanisms and cross-talk within the innate immune system signaling network are still enigmatic. Recently, the Toll-like receptor (TLR) family was identified as the pivotal link between extracellular microbial ligands and the activation of monocytic cells. In mammals, 11 different Toll-like receptors have been described to date and microbial ligands for many of them have been identified. Among them, lipopolysaccharides (LPS) and unmethylated bacterial DNA with CpG motifs are of specific importance. TLR4 and under certain conditions TLR2 [4] were found to be crucial for recognition of LPS [5] whereas TLR9 was identified as the receptor for unmethylated bacterial DNA sequences [6]. At present, it is of great interest to identify differences of intracellular signaling between individual TLRs to set the ground for new specific therapeutic options for inflammatory diseases.
Uncontrolled activation of the innate immune system is naturally prohibited by a previously described phenomenon: exposure of macrophages to LPS induces a state of hyporesponsiveness to a subsequent re-exposure termed LPS-tolerance [7], [8]. The molecular mechanism leading to LPS-tolerance is not completely elucidated, although recently published reports describe several signaling molecules such as IRAK-M [9] and SOCS/JAB [10] to be involved in the induction of tolerance.
Surprisingly TLR4 is not only activated by LPS but also by heat shock protein 60 (HSP60), a highly conserved intracellular stress protein, which is preserved from bacteria to mammals [11], [12]. In addition to its intracellular role during cellular stress HSP60 was therefore ascribed a function as an immunological danger signal [13].
In the present study, we investigated the impact of bacterial LPS, bacterial and autologous HSP60 and bacterial DNA motifs (CpG-DNA-oligonucleotides) on the modulation of Kupffer cell activation. Bacterial DNA which contains CpG motifs was originally shown to trigger B-cell activation [14] and later identified as possible factor in septic shock [15]. We demonstrate that autologous HSP60 induced LPS-tolerance, while in contrast CpG-DNA-oligonucleotides seemed to have an amplifying effect on a subsequent LPS-challenge. The latter in vitro observations were confirmed in an in vivo model of LPS-dependent liver failure.
Section snippets
Animals
Mice were bred and maintained in the animal facility of the University of Mainz. Mice were kept in a 12 h light–dark cycle under standard conditions with access to water and food. Proceedings involving the animals were done in agreement with national and international laws and policies.
Cells
Murine macrophage RAW 264.7 cell line was kindly provided by Charles Dinarello. Cells were cultured in RPMI 1640 medium containing 10% FCS, 1% Glutamin, 1% Penicillin/Streptomycin at a density of 5×105 in 24-well
Expression of TLR on hepatic Kupffer cells
Toll-like receptors form the structural link between extra cellular bacterial particles and intracellular activation of monocytes. Initially it was tested whether TLR4, which was shown to be critical for LPS- and HSP60-signaling, and TLR9, which transmits activation by unmethylated DNA with CpG-DNA motifs, are expressed on KC. In RT-PCR analysis transcripts for TLR4 and TLR9 were detected in isolated KC (Fig. 1).
LPS, HSP60 and CpG-DNA activate RAW264.7 macrophages and primary hepatic Kupffer cells
In initial experiments it was examined whether activation of Kupffer cells can be
Discussion
In higher organisms, control and subsequent down-regulation and containment of an immune response is critical to successfully survive attacks of pathogens. KC are the key regulatory cells in alcoholic hepatitis that is mainly driven by drainage of bacterial toxins from the gut to liver via the portal vein. The modulation of the KC response towards LPS is of particular interest in this context since KC activation is the cellular origin of the lethal cascade of septic shock. It has been known for
Acknowledgements
Some of this work is part of the M.D. thesis of F.H. The authors thank Sonja Bamberger for excellent technical assistance and Regine Weisbrod M.A. for critical reading of the manuscript. These studies were supported by an intramural fund of the University of Mainz (MAIFOR) to M.S., by The European Commission through the Concerted Action “Heat Shock Proteins in Inflammatory Diseases” (project BMH4-CT98-3935) and by the Deutsche Forschungsgemeinschaft (DFG), SFB 490 and 532.
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Cited by (25)
Toll-Like Receptors in Liver Disease
2013, Advances in Clinical ChemistryDiscrepant roles of CpG ODN on acute alcohol-induced liver injury in mice
2012, International ImmunopharmacologyCitation Excerpt :Increasing evidence suggests that the development of ALD is associated with activation of Toll-like receptors (TLRs) and the consequent over-production of inflammatory cytokines such as TNF-α [8–10]. Kupffer cells (KC) expressing TLR2, TLR3, TLR4 and TLR9 [11–13] in liver are thought to be the main hepatic source of TNF-α [14]. In the setting of alcohol consumption, TLR4 activation drives KC to produce TNF-α which plays a crucial role in the development of ALD [15–17] by inducing apoptosis of hepatocytes [18–20].
Taking off the brakes: T cell immunity in the liver
2010, Trends in ImmunologyCitation Excerpt :The exact mechanisms determining the low immune-stimulatory potential and the switch to immunogenicity of liver DCs remain to be discovered. KC are sensitive to stimulation with TLR-ligands [64–66]: initially they up-regulate the expression of proinflammatory mediators (IL-1, IL-6 and TNF [34,67]), and subsequently release immunoregulatory cytokines (IL-10, TGFβ and prostanoids [68]) that – after repeated MAMP-stimulation – may suppress immune functions of other APCs in the liver in a paracrine fashion [51]. Interaction of KCs with other hepatic cell populations, e.g. NKT cells, also results in increased T cell immunity [69].
Role of TLR9 in hepatic stellate cells and experimental liver fibrosis
2008, Biochemical and Biophysical Research CommunicationsCitation Excerpt :TLR9 was mainly detected on cells of the immune system, such as dendritic cells, monocytes and B-lymphocytes. However, TLR9 expression was also found on hepatocytes, hepatic endothelial cells and Kupffer cells [20–22,36]. Therefore, we first tested whether human HSC express TLR9 and found TLR9 transcripts in human HSC (Fig. 1A).
Toll-Like Receptor Signaling in the Liver
2006, Gastroenterology