Acute alcohol inhibits the induction of nuclear regulatory factor kappa B activation through CD14/toll-like receptor 4, interleukin-1, and tumor necrosis factor receptors: a common mechanism independent of inhibitory kappa B alpha degradation?
ABSTRACT Nuclear translocation and DNA binding of the nuclear factor kappaB (NF-kappaB) is an early event in inflammatory cell activation in response to stimulation with bacterial components or cytokines. Cell activation via different receptors culminates in a common pathway leading to NF-kappaB activation and proinflammatory cytokine induction. We have previously shown that acute alcohol inhibits NF-kappaB activation by lipopolysaccharide (LPS) in human monocytes. Here we investigated whether acute alcohol treatment of human monocytes also inhibits NF-kappaB when induced through activation of the interleukin (IL)-1 or tumor necrosis factor (TNF) receptors.
Human peripheral blood monocytes were treated with LPS, TNFalpha, and IL-1beta in the presence or absence of 25mM alcohol for 1 hr. NF-kappaB activation was determined by electrophoretic mobility shift assays using nuclear extracts. Inhibitory kappaB(alpha) (IkappaB(alpha)) was estimated by Western blotting in cytoplasmic extracts. Chinese hamster ovary cells expressing human CD14 were treated with LPS in the presence or absence of alcohol to study NF-kappaB and IkappaB(alpha) regulation.
Our results indicate that acute alcohol inhibits IL-1beta- and TNFalpha-induced NF-kappaB activation. We further show in CD14/toll-like receptor 4-expressing Chinese hamster ovary cells the specificity of alcohol-mediated inhibition of NF-kappaB via the toll-like receptor 4/CD14 receptors. Inhibition of NF-kappaB by acute alcohol was concomitant with decreased levels of the IkappaB(alpha) molecule in the cytoplasm of LPS, IL-1, and TNFalpha-activated monocytes.
These data suggest a unique, IkappaB(alpha)-independent pathway for the inhibition of NF-kappaB activation by acute alcohol in monocytes. Universal inhibition of NF-kappaB by acute alcohol via these various receptor systems suggests a target for the effects of alcohol in the NF-kappaB activation cascade that is downstream from IkappaB(alpha) degradation. Further, these results demonstrate that acute alcohol is a potent inhibitor of NF-kappaB activation by mediators of early (LPS) or late (IL-1, TNF(alpha)) stages of inflammation in monocytes.
- SourceAvailable from: Stephen Pruett
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- "Acute EtOH interferes with the innate immune response: in vivo EtOH administration suppresses cytokine responses induced through Toll-like receptor (TLR)-4 (Kolls et al., 1995; Pruett et al., 2004) and inhibits TLR4 signaling (Yamashina et al., 2000; Mandrekar et al., 2002; Goral et al.. 2004; Goral and Kovacs, 2005). Whereas most studies show that acute EtOH suppresses cytokine responses, the situation is more controversial and more complex in case of chronic EtOH. "
ABSTRACT: Both binge and chronic heavy drinking can adversely affect the immune system, but the effects seem to be at least partly dependent on the manner of ethanol (EtOH) consumption. Previous study results from several labs have clearly demonstrated that acute administration of EtOH interferes with innate immune responses. Specifically, EtOH has a general inhibitory effect on cytokine and chemokine production induced by various Toll-like receptor (TLR) ligands, and it suppresses signaling on several levels along the TLR signaling pathways. However, it is not clear whether chronic exposure to ethanol has the same effects or not. The purpose of this study was to investigate the difference between the effect of chronic versus acute EtOH exposure on LPS-induced cytokine production and clustering of components of the TLR4 complex, which is an important early signaling event. Some groups of mice received acute EtOH by oral gavage using our binge drinking model and/or chronic administration of EtOH at 20% (w/v) in the drinking water as the sole liquid source for 4 wk. The cellular distribution of CD14 and TLR4 were studied by confocal microscopy following exposure of peritoneal cells to LPS locally in vivo, and cytokine production in peritoneal fluid and serum was measured by ELISA after LPS injection via a tail vein. Chronic EtOH exposure did not consistently cause significant changes in LPS-induced cytokine production. However, mice previously exposed to chronic EtOH treatment became partially resistant to the suppressive effects of acute EtOH administration with regard to cytokine production. As we have reported previously, acute EtOH treatment suppressed the LPS-induced clustering of TLR4 and CD14 in peritoneal macrophages. However, peritoneal cells from mice treated with chronic EtOH exhibited a greater amount of intracellular expression of CD14 instead of CD14/TLR4 clustering on the membrane following LPS exposure. The results demonstrate different effects of chronic versus acute EtOH treatment on LPS-induced cytokine production in mice. Partial tolerance to the effect of acute EtOH administration caused by chronic EtOH treatment suggests a compensatory mechanism is induced by chronic EtOH administration. Acute EtOH exposure acts probably by disrupting the receptor clustering following LPS recognition, whereas adaptations induced by chronic EtOH treatment seem to involve alteration of LPS receptor expression.Journal of Immunotoxicology 01/2007; 3(4):217-25. DOI:10.1080/15476910601080156 · 1.91 Impact Factor
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- "This is consistent with our previous report indicating that EtOH inhibits cellular signaling in a manner that is consistent with action very early in the TLR signaling pathway (Dai et al., 2005; Pruett et al., 2004a). However, it is important to note that EtOH also inhibits cellular signaling mediated through IL-1 and TNF receptors (Mandrekar et al., 2002). This indicates either that similar inhibition of these receptors at the membrane occurs or that EtOH acts by directly inhibiting NF-kB activation (the signaling end point evaluated in the study cited above). "
ABSTRACT: Ethanol (EtOH) suppresses cytokine responses induced through most Toll-like receptors (TLRs), but the mechanism of action is unclear. We recently found that acute EtOH alters lipopolysaccharide (LPS)-induced partitioning of CD14, a critical component of the LPS receptor complex, within lipid raft fractions in the macrophage-like cell line RAW264.7. Here we investigated the role of receptor clustering in alteration of the responses of cells to LPS caused by EtOH both in vitro and in vivo. The cellular distribution of CD14, TLR4, actin cytoskeleton, and tumor necrosis factor-alpha (TNF-alpha) were studied by confocal microscopy following exposure of cells to LPS with or without EtOH. TLR4 and CD14 were clustered into highly colocalized patches on the cell membrane accompanied by the reorganization of the actin cytoskeleton in some of the RAW264.7 cells as well as peritoneal cells following LPS treatment. Addition of EtOH reduced the number of cells that had LPS-induced receptor patches and in which this reorganization occurred. Cells on which CD14 and TLR4 formed clusters or caps had substantially higher levels of membrane-bound TNF-alpha compared with cells without clustering or capping of these molecules. Interference with the actin cytoskeleton by cytochalasin D suppressed the production of TNF-alpha and receptor clustering, as EtOH did. These data confirm our previous observations, suggest a novel mechanism of EtOH action that involves interference with receptor clustering, and indicate a potential role of actin filaments in the formation of receptor patches, subsequent activation of macrophages by LPS, and production of TNF-alpha.Alcoholism Clinical and Experimental Research 09/2006; 30(8):1436-44. DOI:10.1111/j.1530-0277.2006.00172.x · 3.31 Impact Factor
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- "The addition of LPS also stimulated CHO/ TLR2 cells (Fig. 2A) owing to the endogenous expression of TLR4 in CHO cells. These results suggested that CHO cells expressing TLRs and CD14 closely resemble TLR functions seen in monocytes, as previously reported by us, and others (Mandrekar et al., 2002; Triantafilou et al., 2002). Similar to monocytes (Fig. 1), alcohol inhibited TLR4-induced cell activation and did not affect TLR2- induced signaling in CHO cells (Fig. 2A). "
ABSTRACT: Alcohol, a substance that is most frequently abused, suppresses innate immune responses to microbial pathogens. The host senses pathogens via Toll-like receptors (TLRs). Recent studies indicate that alcohol affects TLR signaling. Here, we hypothesized that acute alcohol treatment may interfere with early steps of membrane-associated TLR2 and TLR4 signaling at the level of lipid rafts. Human monocytes and Chinese hamster ovary (CHO) cells, transfected with human TLR2, TLR4, or CD14, were stimulated with peptidoglycan (PGN, TLR2 ligand) or lipopolysaccharide (LPS, TLR4 ligand) with or without alcohol (50 mM) and analyzed for cytokine production (enzyme-linked immunosorbent assay), nuclear factor-kappaB (NF-kappaB) activation (electrophoretic mobility shift assay), membrane fluidity (fluorescent pyrene eximer formation), and partition of cellular membrane into cholesterol-rich, detergent-resistant domains (DRMs; Western blot). We determined that both TLR2 and TLR4 were located outside the rafts; flotillin, a DRM marker, was resident in the rafts, while CD14 was equally distributed in and outside the rafts in a steady-state condition. PGN forced TLR2 to migrate into DRMs. Engagement of TLR4 and CD14 with LPS induced their migration into the rafts. Alcohol prevented TLR4 partitioning; however, it did not affect TLR2 migration into the rafts. Furthermore, alcohol downregulated TLR4-induced, but not TLR2-induced, NF-kappaB activation and cytokine production in monocytes. We found that alcohol increased membrane fluidity and depleted cellular cholesterol in CHO cells without affecting cell viability. These data demonstrate for the first time that alcohol disturbs TLR4 and CD14 association with lipid rafts. We propose that alcohol-induced effects on lipid rafts may contribute to modulation of TLR4-CD14-triggered early cellular responses.Alcoholism Clinical and Experimental Research 02/2006; 30(1):76-85. DOI:10.1111/j.1530-0277.2006.00003.x · 3.31 Impact Factor