Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication

Intracellular replication of Brucella requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that Brucella internalizes into macrophages by swimming on the cell surface with generalized membrane ruffling for several minutes, after which the bacteria are enclosed by macropinosomes. Lipid raft-associated molecules such as glycosylphosphatidylinositol (GPI)-anchored proteins, GM1 gangliosides and cholesterol were selectively incorporated into macropinosomes containing Brucella. In contrast, lysosomal glycoprotein LAMP-1 and host cell transmembrane protein CD44 were excluded from the macropinosomes. Removing GPI-anchored proteins from the macrophage surface and cholesterol sequestration markedly inhibited the VirB-dependent macropinocytosis and intracellular replication. Our results suggest that the entry route of Brucella into the macrophage determines the intracellular fate of the bacteria that is modulated by lipid raft microdomains.     

Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.     

Stabilized beta-catenin potentiates Fas-mediated T cell apoptosis

In response to Ag stimulation, Ag-specific T cells proliferate and accumulate in the peripheral lymphoid tissues. To avoid excessive T cell accumulation, the immune system has developed mechanisms to delete clonally expanded T cells. Fas/FasL-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) T cells. Using transgenic mice expressing a stabilized beta-catenin (beta-cat(Tg)), we show here that beta-catenin was able to enhance apoptosis of activated T cells by up-regulating Fas. In response to staphylococcal enterotoxin B stimulation, beta-cat(Tg) mice exhibited accelerated deletion of CD4(+)Vbeta8(+) T cells compared with wild type mice. Surface Fas levels were significantly higher on activated T cells obtained from beta-cat(Tg) mice than that from wild type mice. Additionally, T cells from beta-cat(Tg) mice were more sensitive to apoptosis induced by crosslinking Fas, activation-induced cell death, and to apoptosis induced by cytokine withdrawal. Lastly, beta-catenin bound to and stimulated the Fas promoter. Therefore, our data demonstrated that the beta-catenin pathway was able to promote the apoptosis of activated T cells in part via up-regulation of Fas.     

Membrane domains in lymphocytes - from lipid rafts to protein scaffolds

Lateral compartmentalization of the plasma membrane into domains is a key feature of immune cell activation and subsequent immune effector functions. Here, we will review the high diversity of membrane domains, ranging from elementary lipid rafts, envisioned as dynamic and small domains (in the tens of nm), to relatively stable μm-scale membrane domains, which form the immunologic synapse of T lymphocytes. We will discuss the relationship between these different types of plasma membrane domains and how raft lipid- and protein-controlled interactions and cell biological processes cooperate to generate functional domains that mediate lymphocyte activity.     

Modulation of Abeta-induced neurotoxicity by estrogen receptor alpha and other associated proteins in lipid rafts

Some evidences have demonstrated the participation of estrogen receptors (ERs) in rapid, non-genomic actions of estrogen to promote neuroprotection against different toxic agents. However, there is still very little information about the structural nature of these receptors and the manner these proteins may be integrated into the plasma membrane. One of the plausible possibilities is that they may be localized in lipid rafts microstructures where they would be associated with other, still unknown, molecules which may modulate their physiological activities related to cell survival. In this work, we have identified in caveolar fractions of murine septal and hippocampal neurons a membrane-related ER shown to physically interact with, both, a voltage-dependent anion channel and scaffold protein caveolin-1.     

CD28 and lipid rafts coordinate recruitment of Lck to the immunological synapse of human T lymphocytes

In T lymphocytes, the Src family kinase Lck associates lipid rafts and accumulates at the immunological synapse (IS) during T cell stimulation by APCs. Using CD4- or CD28-deficient murine T cells, it was suggested that recruitment of Lck to the IS depends on CD4, whereas CD28 sustains Lck activation. However, in human resting T cells, CD28 is responsible for promoting recruitment of lipid rafts to the IS by an unknown mechanism. Thus, we performed a series of experiments to determine 1) whether Lck is recruited to the IS through lipid rafts; and 2) whether Lck recruitment to the IS of human resting T cells depends on CD4 or on CD28 engagement. We found that CD28, but not CD4, stimulation induced recruitment of Lck into detergent-resistant domains as well as its accumulation at the IS. We also found that Lck recruitment to the IS depends on the CD28 COOH-terminal PxxPP motif. Thus, the CD28-3A mutant, generated by substituting the prolines in positions 208, 211, and 212 with alanines, failed to induce Lck and lipid raft accumulation at the synapse. These results indicate that CD28 signaling orchestrates both Lck and lipid raft recruitment to the IS to amplify T cell activation.     

Sequestration of NF-kappaB signaling complexes in lipid rafts contributes to repression of NF-kappaB in T lymphocytes under hyperthermia stress

Sepsis causes extensive apoptosis of lymphocytes, a pathological condition that is frequently associated with hyperthermia. Heat stress has been implicated to repress the activation of an inflammatory mediator, nuclear factor of kappaB (NF-kappaB), which sensitizes cells to apoptosis mediated by inflammatory cytokine, tumor necrosis factor alpha. However, the molecular mechanism of hyperthermia-associated loss of T cells remains unclear. We show that hyperthermia causes rapid translocation of IkappaB kinase (IKK) and NF-kappaB complexes into the plasma membrane-associated lipid rafts in T cells. Heat stress induces aggregation of Carma1 in lipid rafts, which in turn recruits protein kinase C theta (PKC theta) and Bcl10 to the microdomains, causing subsequent membrane translocation of the IKK and NF-kappaB signalosomes. Depletion of Carma1 and inhibition of PKC theta impair accumulation of NF-kappaB complexes in lipid rafts. Heat stress prohibits IkappaB kinase activity by sequestrating the IKK and NF-kappaB complexes in lipid rafts and by segregating the chaperone protein Hsp90, an essential cofactor for IKK, from the IKK complex. This process ultimately results in functional deficiency of NF-kappaB and renders T cells resistant to tumor necrosis factor alpha-induced activation of IKK, thereby contributing to the apoptotic loss of T lymphocytes in sepsis-associated hyperthermia.     

Cetuximab enhances TRAIL-induced gastric cancer cell apoptosis by promoting DISC formation in lipid rafts

TRAIL is a member of the tumor necrosis factor family that selectively induces cancer cell apoptosis. However, gastric cancer cells are insensitive to TRAIL. Our and others studies showed that the inhibition of EGFR pathway activation could increase the sensitivity of TRAIL in cancer cells. But the detailed mechanism is not fully understood. In the present study, compared with TRAIL or cetuximab (an anti-EGFR monoclonal antibody) alone, treatment with the TRAIL/cetuximab combination significantly promoted death receptor 4 (DR4) clustering as well as the translocation of both DR4 and Fas-associated death domain-containing protein (FADD) into lipid rafts. This in turn resulted in caspase-8 cleavage and the formation of the death-inducing signaling complex (DISC) in these lipid rafts. Cholesterol-depletion with methyl-β-cyclodextrin partially prevented DR4 clustering and DISC formation, and thus partially reversed apoptosis induced by the TRAIL/cetuximab dual treatment. These results indicate that cetuximab increases TRAIL-induced gastric cancer cell apoptosis at least partially through the promotion of DISC formation in lipid rafts.     

Proteases in Fas-mediated apoptosis

Involvement of a unique family of cysteine proteases in the multistep apoptotic process has been documented. Cloning of several mammalian genes identifies some components of this cellular response. However, it is currently unclear which protease plays a role as a signal and/or effector of apoptosis. We summarize contributions to the data concerning proteases in Fas-mediated apoptosis. J. Cell. Biochem. 64:43–49. © 1997 Wiley-Liss, Inc.     

Integrin alpha2beta1 inhibits Fas-mediated apoptosis in T lymphocytes by protein phosphatase 2A-dependent activation of the MAPK/ERK pathway

The mechanisms by which T lymphocytes escape apoptosis during their activation are still poorly defined. In this study, we elucidated the intracellular signaling pathways through which beta1 integrins modulate Fas-mediated apoptosis in T lymphocytes. In experiments done in Jurkat T cells and activated peripheral blood T lymphocytes, engagement of alpha2beta1 integrin with collagen type I (Coll I) was found to significantly reduce Fas-induced apoptosis and caspase-8 activation; Annexin V binding and DNA fragmentation were reduced by approximately 42 and 38%, respectively. We demonstrated that the protective action of Coll I does not require new protein synthesis but was dependent on the activation of the MAPK/Erk pathway. Furthermore, we found that activation of protein phosphatase 2A (PP2A) by Coll I was required for both Coll I-mediated activation of Erk, and inhibition of Fas-induced caspase-8 activation and apoptosis. Other ligands of beta1 integrins, fibronectin (Fbn), and laminin (Lam), did not sustain significant Erk activation and had no effect on Fas-induced apoptosis. Taken together, these results provide the first evidence of a PP2A-dependent activation of the MAPK/Erk pathway downstream of alpha2beta1 integrin, which has a functional role in regulating Fas-mediated apoptosis in T lymphocytes. As such, this study emphasizes the potential importance that Coll I interactions may have on the control of T lymphocyte homeostasis and their persistence in chronic inflammatory diseases.     

牙龈卟啉单胞菌诱导T淋巴细胞活化、凋亡的体外研究

目的检测牙龈卟啉单胞菌对人外周血中T淋巴细胞活化及凋亡的作用,并检测Fas/FasL在牙龈卟啉单胞菌(Porphyrom onas gingivalis,Pg)诱导的T淋巴细胞凋亡中的表达。方法选取10例全身及牙周组织健康受试者,分离外周血中T淋巴细胞,在有/无Pg情况下培养0~96 h,用荧光探针(Annexin V-FITC、PI、CD69)及特殊的单克隆抗体(Fas、FasL)进行标记,并进行流式细胞仪检测。结果 CD69+淋巴细胞+Pg组Annexin V+/PI-细胞百分数在各个时间点上都明显高于T淋巴细胞+Pg组(P0.01)。Fas和FasL的表达量明显上调。用抗Fas单克隆抗体阻滞Fas-FasL相互作用导致T细胞凋亡的明显减少,百分比为(20.56±2.43)%,未加抗体的为(50.41±2.68)%。但残余的细胞凋亡活动与阴性对照相比仍高。结论 Pg能够诱导人外周血中T淋巴细胞活化,并且能够通过活化促进其凋亡,Pg诱导T淋巴细胞凋亡主要通过Fas-FasL途径,并具有时间依赖性。     

Signal-transducing adaptor protein-2 modulates Fas-mediated T cell apoptosis by interacting with caspase-8

We found that an adaptor protein, signal-transducing adaptor protein (STAP)-2, is a new member of the Fas-death-inducing signaling complex and participates in activation-induced cell death in T cells. STAP-2 enhanced Fas-mediated apoptosis and caspase-8 aggregation and activation in Jurkat T cells. Importantly, STAP-2 directly interacted with caspase-8 and Fas, resulting in enhanced interactions between caspase-8 and FADD in the Fas-death-inducing signaling complex. Moreover, STAP-2 protein has a consensus caspase-8 cleavage sequence, VEAD, in its C-terminal domain, and processing of STAP-2 by caspase-8 was crucial for Fas-induced apoptosis. Physiologic roles of STAP-2 were confirmed by observations that STAP-2-deficient mice displayed impaired activation-induced cell death and superantigen-induced T cell depletion. Therefore, STAP-2 is a novel participant in the regulation of T cell apoptosis after stimulation.     

The death-inducing signalling complex is recruited to lipid rafts in Fas-induced apoptosis

Membrane microdomains known as lipid rafts have been shown recently to be involved in Fas signalling and apoptosis in T and B cell lines. Here, we have investigated further the role of lipid rafts in Fas-induced apoptosis in non-transformed human CD4 T cells. We show that Fas-induced apoptosis in CD4 T cells was inhibited by the lipid raft disrupter methyl-beta-cyclodextrin. When lipid rafts were isolated from control and Fas ligand treated cells, we found that a small proportion of Fas was present in the raft fraction in untreated cells and that this was greatly increased upon Fas ligation. The other components of the Death Inducing Signalling Complex (DISC), FADD, and procaspase 8, were also present at higher levels in the raft fraction isolated from Fas ligand treated cells. We conclude that formation of the DISC occurs in lipid rafts and that these membrane microdomains are required for efficient Fas signalling and apoptosis.     

Segregation of Bad from lipid rafts is implicated in the induction of apoptosis

Many molecules relocate subcellularly in cells undergoing apoptosis. Using coimmunoprecipitation experiments we demonstrate that Bad is not associated to 14-3-3 protein, suggesting a new mechanism for the control of the proapoptotic role of Bad. Here we show, by confocal microscopy and cellular fractionation, that Bad is attached to lipid rafts in IL-4-stimulated cells and thymocytes while associated with mitochondria in IL-4-deprived cells. Disruption of lipid rafts by methyl-beta-cyclodextrin treatment induces segregation of Bad from rafts, which correlates with apoptosis. Our results suggest that the interaction of Bad with rafts is a dynamic process regulated by IL-4 and involved in the control of apoptosis.     

Differential role of lipid rafts in the functions of CD4+ and CD8+ human T lymphocytes with aging

Lipid rafts are critical to the assembly of the T-cell receptor (TCR) signaling machinery. It is not known whether lipid raft properties differ in CD4+ and CD8+ T cells and whether there are age-related differences that may account in part for immune senescence. Data presented here showed that time-dependent interleukin-2 (IL-2) production was different between CD4+ and CD8+ T cells. The defect in IL-2 production by CD4+ T cells was not due to lower levels of expression of the TCR or CD28. There was a direct correlation between the activation of p56(Lck) and LAT and their association/recruitment with the lipid raft fractions of CD4+ and CD8+ T cells. p56Lck, LAT and Akt/PKB were weakly phosphorylated in lipid rafts of stimulated CD4+ T cells of elderly as compared to young donors. Lipid rafts undergo changes in their lipid composition (ganglioside M1, cholesterol) in CD4+ and CD8+ T cells of elderly individuals. This study emphasizes the differential role of lipid rafts in CD4+ and CD8+ T-cell activation in aging and suggests that the differential localization of CD28 may explain disparities in response to stimulation in human aging.     

Oxaliplatin enhances TRAIL-induced apoptosis in gastric cancer cells by CBL-regulated death receptor redistribution in lipid rafts

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family that selectively induces apoptosis in cancer cells. However, gastric cancer cells are insensitive to TRAIL. In the present study, we show that oxaliplatin enhanced TRAIL-induced apoptosis of MGC803, BGC823, and SGC7901 cells. Oxaliplatin promoted death receptor 4 (DR4) and death receptor 5 (DR5) clustering into aggregated lipid rafts, while the cholesterol-sequestering agent nystatin partially prevented lipid raft aggregation, DR4 and DR5 clustering, and reduced apoptosis. Furthermore, the expression of the casitas B-lineage lymphoma (Cbl) family was downregulated by oxaliplatin. Transfection of c-Cbl or Cbl-b partially reversed oxaliplatin-induced lipid raft aggregation. These results indicated that oxaliplatin enhanced TRAIL-induced gastric cancer cell apoptosis at least partially through Cbl-regulated death receptor redistribution in lipid rafts.     

Inhibition of Fas-mediated apoptosis by Trypanosoma cruzi infection

Trypanosoma cruzi-infected and normal control mammalian cells were subjected to analysis of Fas-mediated apoptosis stimulated by an agonistic anti-Fas monoclonal antibody. The infected cells showed markedly hampered apoptotic changes in nuclear morphology, phosphatidylethanolamine translocation from the inside to the outside of the plasma membrane, and DNA fragmentation into multiples of 180 bp, relative to normal control cells. Upstream of these morphological and biochemical consequences, the caspase-3 activity was elevated by the Fas stimulation in a significantly greater proportion of intact control cells, but at a highly reduced rate of infected cells. The rapid elevation of caspase-8 activity in control, apoptotic cells was completely inhibited in infected cells. In an examination of the specificity of other stimulants, X-ray radiation or chemicals such as hydrogen peroxide, colchicine or etoposide did not cause significant differences in apoptotic rates between control and infected cells; tumor necrosis factor-alpha, however, induced a high rate of apoptosis in control cells, with an extremely lowered rate in infected cells. This study demonstrates, for the first time, that T. cruzi infection inhibits one of the earliest steps of death receptor-mediated apoptosis, an effect that most probably involves the inhibition of caspase-8. Differential apoptotic responses in cells infected with T. cruzi and other intracellular parasites are discussed.     

How T lymphocytes recognize lipid antigens

Recognition of lipid antigens by T lymphocytes is well established. Lipids are recognized by T cells when presented in association with CD1 antigen-presenting molecules. Both microbial and self lipids stimulate specific T lymphocytes, thus participating in immune reactions during infections and autoimmune diseases. The immune system uses a variety of strategies to solubilise lipid antigens, to facilitate their internalization, processing, and loading on CD1 molecules. Recent studies in the field of lipid antigen presentation have revealed new mechanisms which allow the immune system to sense lipids as stimulatory antigens.