Gangliosides in T cell development and function of mice.

The molecular diversity of glycosphingolipids (GSLs) that arouse during the course of evolution clearly plays an essential role in maintenance of biological homeostasis. Why is such a wide variety of GSLs necessary, and what gave rise to the expression mechanisms that are selective and specific to individual cells, tissues, or organs? What is the biological significance of these mechanisms? The same questions apply to GSLs involved in T cell development and activation. Primary CD4+ T cells and CD8+ T cells preferentially express differing ganglioside series: a-series and o-series, respectively. Conversely, a-series and o-series ganglioside deficiency results respectively in CD4+ and CD8+ T cell dysfunction. Dynamic changes in ganglioside expression occur during T cell development in thymus. Ganglioside GM3 synthase deficiency, which results in lack of a-series gangliosides, ameliorated CD4+ T cell-mediated airway hypersensitivity in a mouse model of allergic asthma. In this review, we summarize findings from these and many studies to illustrate the key roles of gangliosides in T cell differentiation and function.

Homeostatic and pathogenic roles of the GM3 ganglioside.

Two decades ago, we achieved molecular cloning of ganglioside GM3 synthase (GM3S; ST3GAL5), the enzyme responsible for initiating biosynthesis of complex gangliosides. The efforts of our research group since then have been focused on clarifying the physiological and pathological roles of gangliosides, particularly GM3. This review summarizes our long-term studies on the roles of GM3 in insulin resistance and adipogenesis in adipose tissues, cholesterol uptake in intestine, and leptin resistance in hypothalamus. We hypothesized that GM3 plays a role in innate immune function of macrophages and demonstrated that molecular species of GM3 with differing acyl-chain structures and modifications functioned as pro- and anti-inflammatory endogenous Toll-like receptor 4 (TLR4) modulators in macrophages. Very-long-chain and α-hydroxy GM3 species enhanced TLR4 activation, whereas long-chain and unsaturated GM3 species counteracted this effect. Lipidomic analyses of serum and adipose tissues revealed that imbalances between such pro- and anti-inflammatory GM3 species promoted progression of metabolic disorders. GM3 thus functions as a physiological regulatory factor controlling the balance between homeostatic and pathological states. Ongoing studies based on these findings will clarify the mechanisms underlying ganglioside-dependent control of energy homeostasis and innate immune responses.

Plasma membrane sphingomyelin modulates thymocyte development by inhibiting TCR-induced apoptosis.

Sphingomyelin (SM) in combination with cholesterol forms specialized membrane lipid microdomains in which specific receptors and signaling molecules are localized or recruited to mediate intracellular signaling. SM-microdomain levels in mouse thymus were low in the early CD4+CD8+ double-positive (DP) stage prior to thymic selection and increased >10-fold during late selection. T-cell receptor (TCR) signal strength is a key factor determining whether DP thymocytes undergo positive or negative selection. We examined the role of SM-microdomains in thymocyte development and related TCR signaling, using SM synthase 1 (SMS1)-deficient (SMS1-/-) mice which display low SM expression in all thymocyte populations. SMS1 deficiency caused reduced cell numbers after late DP stages in TCR transgenic models. TCR-dependent apoptosis induced by anti-CD3 treatment was enhanced in SMS1-/- DP thymocytes both in vivo and in vitro. SMS1-/- DP thymocytes, relative to controls, showed increased phosphorylation of TCR-proximal kinase ZAP-70 and increased expression of Bim and Nur77 proteins involved in negative selection following TCR stimulation. Addition of SM to cultured normal DP thymocytes led to greatly increased surface expression of SM-microdomains, with associated reduction of TCR signaling and TCR-induced apoptosis. Our findings indicate that SM-microdomains are increased in late DP stages, function as negative regulators of TCR signaling and modulate the efficiency of TCR-proximal signaling to promote thymic selection events leading to subsequent developmental stages.

The regulatory roles of glycosphingolipid-enriched lipid rafts in immune systems.

Lipid rafts formed by glycosphingolipids (GSLs) on cellular membranes play important roles in innate and adaptive immunity. Lactosylceramide (LacCer) forms lipid rafts on plasma and granular membranes of human neutrophils. These LacCer-enriched lipid rafts bind directly to pathogenic components, such as pathogenic fungi-derived ß-glucan and Mycobacteria-derived lipoarabinomannan via carbohydrate-carbohydrate interactions, and mediate innate immune responses to these pathogens. In contrast, a-series and o-series gangliosides form distinct rafts on CD4+ and CD8+ T cell subsets, respectively, contributing to the respective functions of these cells and stimulating adaptive immune responses through T cell receptors. These findings suggest that gangliosides play indispensable roles in T cell selection and activation. This Review introduces the involvement of GSL-enriched lipid rafts in innate and adaptive immunity.

NPC1L1-dependent intestinal cholesterol absorption requires ganglioside GM3 in membrane microdomains.

Intestinal cholesterol absorption is a key regulator of systemic cholesterol homeostasis. Excessive dietary cholesterol and its intestinal uptake lead to hypercholesterolemia, a major risk factor for cardiovascular disease. Intestinal cholesterol uptake is mediated by Niemann-Pick C1-like 1 (NPC1L1), a transmembrane protein localized in membrane microdomains (lipid rafts) enriched in gangliosides and cholesterol. The roles of gangliosides, such as monosialodihexosylganglioside (GM3) and its synthesizing enzyme GM3 synthase (GM3S), in NPC1L1-dependent cholesterol uptake have not been examined previously. Here, we examined NPC1L1-dependent cholesterol uptake in a cell model as well as in wild-type and apoE-deficient mice fed normal or high-cholesterol diets. We showed that NPC1L1-dependent cholesterol uptake was impaired in GM3S-deficient cells and that GM3S deficiency promoted resistance to hypercholesterolemia in both wild-type and apoE-deficient mice fed the high-cholesterol but not the normal diet. Our findings suggest that GM3 and related gangliosides are essential for NPC1L1-mediated intestinal cholesterol absorption and are potential targets for hypercholesterolemia therapy.

Biology of GM3 Ganglioside.

Since the successful molecular cloning in 1998 of GM3 synthase (GM3S, ST3GAL5), the enzyme responsible for initiating biosynthesis of all complex gangliosides, the efforts of our research group have been focused on clarifying the physiological and pathological implications of gangliosides, particularly GM3. We have identified isoforms of GM3S proteins having distinctive lengths of N-terminal cytoplasmic tails, and found that these cytoplasmic tails define subcellular localization, stability, and in vivo activity of GM3S isoforms. Our studies of the molecular pathogenesis of type 2 diabetes, focused on interaction between insulin receptor and GM3 in membrane microdomains, led to a novel concept: type 2 diabetes and certain other lifestyle-related diseases are membrane microdomain disorders resulting from aberrant expression of gangliosides. This concept has enhanced our understanding of the pathophysiological roles of GM3 and related gangliosides in various diseases involving chronic inflammation, such as insulin resistance, leptin resistance, and T-cell function and immune disorders (e.g., allergic asthma). We also demonstrated an essential role of GM3 in murine and human auditory systems; a common pathological feature of GM3S deficiency is deafness. This is the first direct link reported between gangliosides and auditory functions.

Distinct selectivity of gangliosides required for CD4⁺ T and CD8⁺ T cell activation.

T cells compose a crucial part of the immune system and require activation. The first step of T cell activation is triggered by the movement of one of their surface molecules, known as T cell receptor, into localized regions of cell membrane known as lipid rafts. Molecules called gangliosides are known to be major components of lipid rafts, but their role in T-cell activation remains to be elucidated. This review summarizes recent findings that different types of T cells require distinct ganglioside types for the activation. Control of ganglioside expression would offer a strategy targeting for specific T-cell subpopulations to treat immune diseases. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.

Control of homeostatic and pathogenic balance in adipose tissue by ganglioside GM3.

Ganglioside GM3 (Siaα2-3Galß1-4Glcß1-1Cer) has been known to participate in insulin signaling by regulating the association of the insulin receptor in caveolae microdomains (lipid rafts), which is essential for the execution of the complete insulin metabolic signaling in adipocytes. Macrophage-secreted factors including proinflammatory cytokines, tumor necrosis factor-α and interleukin-1ß, in adipose tissues have been known to limit the local adipogenesis and induce insulin resistance; however, the interplay between adipocytes and macrophages upon regulation of GM3 expression is not clear. GM3 was virtually absent in primary adipocytes differentiated from macrophage-depleted mesenteric stromal vesicular cells, which accompanies enhancement of insulin signaling and adipogenesis. We found that the expression of GM3 is governed by soluble factors including steady-state levels of proinflammatory cytokines secreted from resident macrophages. The direct involvement of GM3 in insulin signaling is demonstrated by the fact that embryonic fibroblasts obtained from GM3 synthase (GM3S)-deficient mice have increased insulin signaling, when compared with wild-type embryonic fibroblasts, which in turn leads to enhanced adipogeneis. In addition, GM3 expression in primary adipocytes is increased under proinflammatory conditions as well as in adipose tissue of diet-induced obese mice. Moreover, GM3S-deficient mice fed high-fat diets become obese but are resistant to the development of insulin resistance and chronic low-grade inflammatory states. Thus, GM3 functions as a physiological regulatory factor of the balance between homeostatic and pathological states in adipocytes by modulating insulin signaling in lipid rafts.

Heterogeneity of gangliosides among T cell subsets.

Gangliosides are major components of highly organized membrane microdomains or rafts, yet little is known about the role of gangliosides in raft organization. This is also the case of gangliosides in TCR-mediated activation. Comprehensive structural analysis of gangliosides in the primary thymocytes and CD4(+) T and CD8(+) T cells was not achieved due to technical difficulties. We have found that CD8(+) T cells express very high levels of o-series gangliosides, but on the other hand, CD4(+) T cells preferably express a-series gangliosides. In the TCR-dependent activation, CD4(+) T cells selectively require a-series gangliosides, but CD8(+) T cells do require only o-series gangliosides but not a-series gangliosides. Ganglioside GM3 synthase-deficient mice lacking a-series gangliosides neither exhibited the TCR-dependent activation of CD4(+) T nor developed ovalbumin-induced allergic airway inflammation. These findings imply that the distinct expression pattern of ganglioside species in CD4(+) and CD8(+) T cells define the immune function of each T cell subset.

CD4 and CD8 T cells require different membrane gangliosides for activation.

Initial events of T-cell activation involve movement of the T-cell receptor into lipid rafts. Gangliosides are major components of lipid rafts. While investigating T-cell activation in ganglioside-deficient mice, we observed that CD4(+) and CD8(+) T cells required different ganglioside subsets for activation. Activation of CD4(+) T cells from GM3 synthase-null mice, deficient in GM3-derived gangliosides, is severely compromised, whereas CD8(+) T-cell activation is normal. Conversely, in cells from GM2/GD2 synthase-null mice, expressing only GM3 and GD3, CD4(+) T-cell activation is normal, whereas CD8(+) T-cell activation is deficient. Supplementing the cells with the corresponding missing gangliosides restores normal activation. GM3 synthase-null mice do not develop experimental asthma. Distinct expression patterns of ganglioside species in CD4(+) T and CD8(+) T cells, perhaps in uniquely functional lipid rafts, define immune functions in each T-cell subset. Control of ganglioside expression would offer a strategy targeting for specific T-cell subpopulations to treat immune diseases.

Mice lacking ganglioside GM3 synthase exhibit complete hearing loss due to selective degeneration of the organ of Corti.

The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. In SAT-I null mice, hearing ability, assessed by brainstem auditory-evoked potentials (BAEP), was impaired at the onset of hearing and had been completely lost by 17 days after birth (P17), showing a deformity in hair cells in the organ of Corti. By 2 months of age, the organ of Corti had selectively and completely disappeared without effect on balance or motor function or in the histology of vestibule. Interestingly, spatiotemporal changes in localization of individual gangliosides, including GM3 and GT1b, were observed during the postnatal development and maturation of the normal inner ear. GM3 expressed in almost all regions of cochlea at P3, but at the onset of hearing it distinctly localized in stria vascularis, spiral ganglion, and the organ of Corti. In addition, SAT-I null mice maintain the function of stria vascularis, because normal potassium concentration and endocochlear potential of endolymph were observed even when they lost the BAEP completely. Thus, the defect of hearing ability of SAT-I null mice could be attributed to the functional disorganization of the organ of Corti, and the expression of gangliosides, especially GM3, during the early part of the functional maturation of the cochlea could be essential for the acquisition and maintenance of hearing function.

Physiological levels of insulin and IGF-1 synergistically enhance the differentiation of mesenteric adipocytes.

Visceral adipose tissue, particularly mesenteric adipose tissue, is important in the pathogenesis of metabolic syndrome. Here, we present a physiologically relevant differentiation system of rat mesenteric-stromal vascular cells (mSVC) to mesenteric-visceral adipocytes (mVAC). We optimized the insulin concentration at levels comparable to those in vivo ( approximately 0.85 ng/ml) by including physiological concentrations of IGF-1. We found that the insulin-like growth factor (IGF-1) and insulin worked synergistically, since IGF-1 alone could induce CCAAT/enhancer binding protein alpha (C/EBPalpha) and adipocyte lipid binding protein (aP2) mRNA expression but not lipid droplet accumulation associated with maturation. Using real-time PCR analyses on 180 adipocyte-related genes, we identified a dramatic effect by IGF-1 plus insulin. We also demonstrated the state of insulin resistance at pathologically high insulin concentrations. This culture system will contribute to understanding the physiological differentiation process and the patho/physiology of mVAC.

Circulating levels of ganglioside GM3 in metabolic syndrome: A pilot study.

BACKGROUND: Insulin resistance is a characteristic feature of metabolic syndrome. Ganglioside GM3 [α-Neu5Ac-(2-3)-ß-Gal-(1-4)-ß-Glc-(1-1)-ceramide] may impair insulin sensitivity in adipose tissue. We investigated the relationship between serum GM3 levels and adiposity indices, as well as between serum GM3 levels and metabolic risk variables. METHODS: Study 1: we assessed serum GM3 levels in normal subjects and in patients with hyperglycemia and/or hyperlipidemia (HL). Study 2: we investigated the relationship between serum GM3 levels and metabolic risk variables in patients with type 2 diabetes. RESULTS: Study 1: serum GM3 levels were higher in hyperglycemic patients (1.4-fold), hyperlipidemic patients (1.4-fold) and hyperglycemic patients with hyperlipidemia (1.6-fold), than in normal subjects. Study 2: serum GM3 levels were significantly increased in type 2 diabetic patients with severe obesity (visceral fat area (VFA) >200 cm(2), BMI > 30). The GM3 level was positively correlated with LDL-c (0.403, p = 0.012) in type 2 diabetes mellitus, but not affected by blood pressure. In addition, the high levels of small dense LDL (>10 mg/dL) were associated with the elevation of GM3. CONCLUSIONS: Serum GM3 levels was affected by glucose and lipid metabolism abnormalities and by visceral obesity. GM3 may be a useful marker for severity of metabolic syndrome.

Structure and function of lipid rafts in human activated T cells.

Lipid rafts, specialized membrane microdomains enriched in sphingolipids and cholesterol, have been shown to function as signaling platforms in T cells. Surface raft expression is known to be increased in human T cells upon activation, and this increased raft expression may account for efficient signaling capability and decreased dependency for co-stimulation in effector and/or activated T cells. However, raft-mediated signaling ability in activated T cells remains to be clarified. In this study, we analyzed the structure and function of lipid rafts in human activated T cells. We demonstrated that raft protein constituents are dramatically changed after activation along with an increase in lipid contents. T cells stimulated with anti-CD3 plus anti-CD28 antibodies showed an increase not only in surface monosialoganglioside GM1 expression but also in total amounts of raft-associated lipids such as sphingomyelin, cholesterol and glycosphingolipids. Raft proteins increased after activation include Csk, Csk-binding protein and Fyn, the molecules known to be involved in negative regulation of T cell activation. Consistent with the increase in expression of these proteins, TCR-mediated Ca(2+) response, a response dependent on raft integrity, was clearly inhibited in activated T cells. Thus, the structure and function of lipid rafts in human activated T cells seem to be quite distinct from those in naive T cells. Further, human activated T cells are relatively resistant to signaling, at least transiently, by TCR re-stimulation even though their raft expression is increased.

Anti-HER-2/neu immune responses are induced before the development of clinical tumors but declined following tumorigenesis in HER-2/neu transgenic mice.

HER-2/neu oncogene products have been implicated as a potential target of T cell-mediated immune responses to HER-2/neu-induced tumors. Using HER-2/neu transgenic mice (oncomice), we investigated whether, and if so how, anti-HER-2/neu immune responses are induced and modulated in these oncomice from birth to tumor initiation. Female oncomice carrying the activated HER-2/neu oncogene displayed apparent hyperplasia in mammary glands at 10 weeks of age and developed mammary carcinomas around an average age of 26 weeks. Unfractionated spleen cells from 10- to 15-week-old oncomice that were cultured without any exogenous stimuli exhibited cytotoxicity against the F31 tumor cell line established from an HER-2/neu-induced mammary carcinoma mass. The final antitumor effectors were a macrophage lineage of cells. However, this effector population was activated, depending on the stimulation of oncomouse CD4(+) T cells with oncomouse-derived antigen-presenting cell (APC) alone or with wild-type mouse APC in the presence of F31 membrane fractions, suggesting the presence of HER-2/neu-primed CD4(+) T cells and HER-2/neu-presenting APC in 10- to 15-week-old oncomice. These antitumor cytotoxic responses were detected at approximately 5 weeks of age and peaked at age 10 to 15 weeks. However, the responses then declined at tumor-bearing stages in which the expression of target proteins could progressively increase. This resulted from the dysfunction of CD4(+) T cells but not of APC or effector macrophages. These results indicate that an anti-HER-2/neu CD4(+) T cell-mediated immune response was generated at the pretumorigenic stage but did not prevent tumorigenesis and declined after the development of clinical tumors.

Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor.

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.

Analysis of the mobility of signaling molecules in lymphocytes using fluorescence photobleaching techniques.

The earliest biochemical events at the plasma membrane that lead to gene activation appear to depend not only on the local concentration of signaling molecules, but also on the mobility of these molecules at the site of signaling. To elucidate the process of signal transduction after receptor engagement in the immune system, it is important to analyze the mobility of signaling molecules in living lymphocytes. Current knowledge of the changes in intracellular localization and dynamic movements of signaling molecules during lymphocyte activation is limited. Here, we describe a method for known as fluorescence recovery after photobleaching, used to measure the diffusion mobility of a signaling molecule in a T cell line after T cell receptor stimulation. This method is a useful tool in studies of spatiotemporal regulation in immunoreceptor signaling.

Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation.

Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.