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- Materials and methods
We have recently reported that ethanol-induced inflammatory processes in the brain and glial cells are mediated via the activation of interleukin-1 beta receptor type I (IL-1RI)/toll-like receptor type 4 (TLR4) signalling. The mechanism(s) by which ethanol activates these receptors in astroglial cells remains unknown. Recently, plasma membrane microdomains, lipid rafts, have been identified as platforms for receptor signalling and, in astrocytes, rafts/caveolae constitute an important integrators of signal events and trafficking. Here we show that stimulation of astrocytes with IL-1β, lipopolysaccharide or ethanol (10 and 50 mM), triggers the translocation of IL-1RI and/or TLR4 into lipid rafts caveolae-enriched fractions, promoting the recruitment of signalling molecules (phospho-IL-1R-associated kinase and phospho-extracellular regulated-kinase) into these microdomains. With confocal microscopy, we further demonstrate that IL-1RI is internalized by caveolar endocytosis via enlarged caveosomes organelles upon IL-1β or ethanol treatment, which sorted their IL-1RI cargo into the endoplasmic reticulum–Golgi compartment and into the nucleus of astrocytes. In short, our findings demonstrate that rafts/caveolae are critical for IL-1RI and TLR4 signalling in astrocytes, and reveal a novel mechanism by which ethanol, by interacting with lipid rafts caveolae, promotes IL-1RI and TLR4 receptors recruitment, triggering their endocytosis via caveosomes and downstream signalling stimulation. These results suggest that TLRs receptors are important targets of ethanol-induced inflammatory damage in the brain.
Ethanol is known to affect the innate immune response in several organ systems (MacGregor 1986; Nagy 2003). Nevertheless, little is known about the potential action of ethanol on the CNS immune system, or how inflammation participates in alcohol-induced brain damage. Our recent studies demonstrate that astrocytes respond to ethanol by secreting cytokines and other inflammatory mediators (Blanco et al. 2004, 2005; Valles et al. 2004), and by contributing to an inflammatory environment in the brain of alcohol-fed animals (Valles et al. 2004). These effects seem to be mediated by the ethanol-induced activation of interleukin-1 beta receptor type I (IL-1RI), and by toll-like receptor type 4 (TLR4) signal-transduction pathways, as blocking these receptors with neutralizing antibodies abolishes most inflammatory signal events and prevents cell death (Blanco et al. 2005).
Interleukin-1 beta receptor type I and TLR4, the specific receptors of IL-1β and bacterial lipopolysaccharide (LPS), are members of a defined group of receptors which shares a cytoplasmic toll/IL-1 receptor domain, that participates in host responses to injury and infection (O’Neill 2000; Akira and Sato 2003). Activation of TLRs/IL-1Rs leads to the recruitment of specific adaptor molecules into the receptor complex, including IL-1R-associated kinase (IRAK), the adaptor molecule myeloid differentiation primary-response protein 88 (MyD88), and tumour-necrosis factor-receptor-associated factor 6 (Martin and Wesche 2002; Akira and Takeda 2004), which trigger the downstream stimulation of protein kinases (MAPK and stress-activated protein kinases/c-Jun N-terminal kinase), that ultimately lead to the activation of transcription factors, such as nuclear factor-κB and activator protein-1 (Martin and Wesche 2002; Akira and Takeda 2004).
Increasing evidence indicates the role of lipid rafts, cholesterol/sphingomyelin-enriched membrane microdomains, in immune system activation (Manes et al. 2003). The recruitment of TLR4 and other adaptor proteins, such as CD14, into lipid rafts has been observed upon LPS stimulation (Triantafilou et al. 2002). We have recently proposed that the effects of ethanol on the immune system and on TLRs receptors are, in part, mediated by the interaction of ethanol with lipid rafts (Blanco et al. 2005; Blanco and Guerri 2007). Indeed, high ethanol concentration seems to disrupt lipid rafts clustering, leading to the suppression of TLR4 signalling (Dai et al. 2005; Dolganiuc et al. 2006). However, low ethanol concentrations (10–50 mM) might facilitate protein–protein and protein–lipid interaction within the membrane microdomains to promote receptor recruitment into the lipid rafts, and to allow for IL-1RI/TLR4 activation and signalling.
Membrane rafts are specialized signalling platforms which are implicated in protein sorting, membrane trafficking and signal-transduction events (Pelkmans and Helenius 2002; Parton and Richards 2003; Pike 2003; Pelkmans et al. 2004). Different subtypes of lipid rafts have been described according to their protein and lipid composition. Caveolae are a major subclass of rafts enriched in the caveolin-1 (cav-1) protein, which are a specialized form of flask-shaped invaginations involved in endocytosis and transcytosis in many eukaryotic cell types (Nichols and Lippincott-Schwartz 2001; Pelkmans and Helenius 2002; Pelkmans et al. 2004). The endocytosed caveolar vesicles accumulate in a pre-existing population of cav-1-containing endosomes, termed caveosomes (Pelkmans et al. 2001). These structures serve as an intermediate station during the internalization of endocytosed ligands in the caveolar/raft endocytic pathway, and have some properties of a sorting compartment, delivering their cargo to the endoplasmic reticulum (ER)–Golgi apparatus, and also in the cellular nucleus (Pelkmans et al. 2001; Rajendran and Simons 2005). When compared with the clathrin-mediated pathway however, the non-clathrin endocytic pathways are poorly understood, and the emergence of caveosomes as a new organelle raises the question of the functional significance of uptake by a caveolar mechanism as opposed to clathrin-coated tips.
The present study was undertaken to test the hypothesis that ethanol-induced activation of IL-1RI/TLR4 in astroglial cells, is mediated by promoting receptors recruitment into lipid rafts caveolae, leading to receptor internalization and signalling. Here we show that astrocyte stimulation with ethanol (10 mM), IL-1β or LPS, induces a rapid translocation of IL-1RI and TLR4 into lipid rafts cav-rich fractions, as well as the recruitment and activation of signalling molecules [P-IRAK and phospho-extracellular regulated-kinase (P-ERK)] into these microdomains. Disruption of lipid rafts with nystatin, saponin or methyl-β-cyclodextrin (MβCD) abolishes the expression and activation of both receptors, suggesting the role of lipid rafts caveolae in IL-1RI and TLR4 signalling. Immunofluorescence studies and confocal microscopy further revealed that, in either ethanol or IL-1β-stimulated astrocytes, it is observed a rapid internalization of IL-1RI into enlarged cytoplasmic ring structures. These structures are positive for cholera toxin subunit B (Ct-B) (raft/caveolae marker) and cav-1 (caveosomes marker), and negative for the early/late endosomes and lysosomes, suggesting that ethanol- or IL-1β-induced internalization of IL-1RI in astrocytes occurs via the caveolar endocytic pathway.
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- Materials and methods
Clinical and experimental studies revealed that ethanol intake affects both the adaptive and innate immune system, resulting in specific alterations in the TLRs response (Medzhitov 2001; Nelson and Kolls 2002; Campbell 2004). We have recently demonstrated that ethanol is capable of activating astrocytes to release cytokines and inflammatory mediators, and that these effects are mediated by the activation of IL-1RI/TLR4 since blocking these receptors abolishes the production of inflammatory mediators and cell death induced by ethanol (Blanco et al. 2004, 2005; Valles et al. 2004). Although the mechanisms by which ethanol activates the IL-1RI/TLR4 response are presently unknown, we have suggested that low concentrations of ethanol could promote the aggregation and clustering of lipid rafts microdomains, leading to the recruitment and activation of IL-1RI and TLR4 into these rafts (Blanco and Guerri 2007). We herein demonstrate this hypothesis by showing that ethanol (10 mM), as well as IL-1β or LPS treatment of astrocytes, induce a rapid translocation of IL-1RI and/or TLR4 into lipid rafts caveolae to trigger a recruitment of activated signalling molecules (P-IRAK and P-ERK) into these microdomains. We further demonstrate that the recruitment and activation of IL-1RI upon IL-1β- or ethanol-stimulation is followed by its internalization and intracellular trafficking via caveolar-dependent endocytosis.
Increasing evidence indicates the role of lipid rafts as a coordinated signalling platform that regulates the dynamics and agonist-induced translocation of specific signalling proteins (Dykstra et al. 2001; Lai 2003; Pike 2003; Laude and Prior 2004). The existence of different classes of lipid rafts has significant implications for the function of these membrane domains in cell signalling (Pike 2003). In primary astrocytes, the lipid rafts caveolae constitute an important membrane compartment of signalization, necessary for the coordinated activation of the intracellular pathways (Cameron et al. 1997; Teixeira et al. 1999). Indeed, we demonstrate, for the first time, the importance of lipid rafts caveolae in the recruitment and signalling of IL-1RI and TLR4 upon IL-1β and LPS stimulation in primary astrocytes. Thus, after ligand binding we observe two peaks of induction in which IL-1RI, TLR4 and activated signalling molecules translocate into caveolae-rich fractions, one at 5 min and the other at 30 min. In the latter peak, we observe the maximal recruitment of receptors and signalling molecules into rafts/caveolae fractions. We also note a disassembly of receptors to rafts/caveolae, as a resting condition, at the intervals between the two peaks of activation (10, 15 and 60 min). It is known that the interactions that drive raft assembly are dynamic and reversible, and indeed, raft clusters can disassemble by the negative modulators implicated in the signalling response and/or by the removal of raft components (Simons and Toomre 2000).
Notably, the present results also demonstrate that ethanol treatment triggers a similar translocation and recruitment of IL-1RI/TLR4 and activated signalling molecules into rafts/caveolae as their specific ligands. These results suggest that low concentrations of ethanol, through its interaction with membrane lipids, might facilitate lipid–protein and protein–protein interactions, allowing receptor aggregation and signalling. Indeed, slight alterations in the structure of these membrane microdomains can facilitate the initiation of signal transduction pathways (Simons and Toomre 2000). Nevertheless, the interaction of ethanol with rafts seems to depend on ethanol concentration since high ethanol concentration (> 50 mM) can disrupt membrane lipid microdomains, interfering with lipid raft clustering and leading to the suppression of the IL-1RI/TLR4 response (Blanco et al. 2005; Dai et al. 2005; Dolganiuc et al. 2006; Fernandez-Lizarbe et al. 2008). Indeed, our results demonstrate that moderate levels of ethanol (50 mM) result in a partial disruption of lipid rafts caveolae, affecting the recruitment of IL-1RI and TLR4 into these rafts compared with 10 mM ethanol. Finally, the role of lipid rafts in the ethanol- or ligand-induced IL-1RI and TLR4 recruitment and signalling in astrocytes is further supported by the findings demonstrating that the disruption of rafts/caveolae with nystatin, saponin or MβCD abolishes the expression and activation of both receptors. These results indicate that lipid rafts caveolae are an essential platform in the IL-1RI and TLR4 signalling response in astrocytes, and that they also play a key role in the induction of inflammatory mediators induced by ethanol in these cells (Blanco et al. 2005).
Another important finding of this study is that ethanol- or ligand-mediated IL-1RI recruitment leads to the internalization and intracellular trafficking of this receptor via caveolar endocytosis. Internalization of membrane receptors occurs through both clathrin- and caveolar/rafts-mediated pathways (Nichols and Lippincott-Schwartz 2001; Parton and Richards 2003; Neel et al. 2005; Parton and Simons 2007). However, in comparison with the clathrin-mediated pathway, alternative non-clathrin endocytic pathways, such as caveolar endocytosis, are poorly understood (Lajoie and Nabi 2007; Parton and Simons 2007). In the latter pathway, it has been shown that endocytic caveolar vesicles accumulate in a discrete population of pre-existing cytoplasmic structures that are enriched in cav-1, called caveosomes (Pelkmans et al. 2001). These structures, which are devoid of markers of other endocytic and biosynthetic organelles, serve as an intermediate station during the internalization of endocytosed ligands in the caveolar/raft endocytic pathway, delivering their cargo into the ER–Golgi compartment, and in the nucleus (Pelkmans et al. 2001; Rajendran and Simons 2005). According to this pathway, after 5 min of ethanol or IL-1β stimulation, we observe that IL-1RI is located in cytoplasm cav-1-positive ring structures or caveosomes. These enlarged structures were negative to the early/late endosomal and lysosomal markers, as demonstrated by the lack of staining with early endosome associated protein-1, LAMP-1 and Lysotracker™. At longer times (30 min), IL-1RI labelling was located in the ER–Golgi compartment and in the nucleus of astrocytes. Therefore, these findings provide strong evidence that IL-1RI is exclusively internalized via caveolar endocytosis upon stimulation with either IL-1β or ethanol. In addition, we also observe a correlation between the translocation and signalling of IL-1RI in the caveolae-enriched sucrose fractions and the appearance of caveosomes containing IL-1RI at 5 and 30 min of stimulation.
An interesting aspect of our results is the enlarged size of caveosomes. Large caveosomes and invaginations-involved caveolins have also been observed in the caveolar endocytosis used by viruses to enter cells, such as SV-40 and polyoma virus (Anderson et al. 1996; Stang et al. 1997; Pelkmans et al. 2001). By passing through this pathway, viruses avoid their degradation in lysosomes to accumulate in enlarged caveosomes (Parton and Simons 2007). Interestingly, the cytoplasmic region of IL-1RI and both viruses contains similar consensus sequences which are known to mediate their nuclear transport (Heguy et al. 1991), and which might explain the similar characteristics of caveolae-dependent internalization. In fact, it is known that IL-1RI is transported to the nucleus and regulates the IL-1-induced gene transcription (Curtis et al. 1990). Alternatively, the enlarged caveosomes could be the result of the fusion of other caveosomes. This idea is supported by a recent study showing that both LPS and TLR4 are trafficked to early/sorting endosomes upon LPS stimulation, and that LPS-induced signalling increases the size of these endosomes, by endosome–endosome fusions (Husebye et al. 2006).
Finally, although evidence from our laboratory suggests that ethanol can interact with non-caveolae lipid rafts activating TLR4 signalling in RAW 264.7 macrophages (Fernandez-Lizarbe et al. 2008), the present study demonstrates for the first time that IL-1RI and TLR4 activation and signalling occurs exclusively via lipid rafts caveolae in astrocytes, and that ethanol is able to act as a ligand-mediated activation of these receptors. In addition, we provide new insights into the mechanisms of ethanol action, demonstrating that activation of IL-1RI by either IL-1β or ethanol triggers the internalization and intracellular trafficking of this receptor via caveolar-dependent endocytosis in astrocytes.
In conclusion, the present data provide a novel mechanism by which ethanol, through its interaction with rafts/caveolae in astrocytes, promotes IL-1RI and TLR4 recruitment into these microdomains, and triggers their internalization and downstream signalling pathways associated with inflammation. This mechanism could participate in ethanol-induced neuroinflammation and brain damage (Valles et al. 2004). The results might also contribute to understanding the mechanisms of the actions of ethanol.