Sphingosine Chain Research Articles

Identification of novel glycolipid ligands activating a sulfatide‐reactive, CD1d‐restricted, type II natural killer T lymphocyte

Sulfatide-reactive CD1d-restricted natural killer T (NKT) lymphocytes belong to the type II NKT cell subset with diverse TCRs, and have been found to regulate experimental auto-immune encephalomyelitis, tumor immunity, and experimental hepatitis in murine models. NKT cells can be activated by self-lipids presented by CD1d, manifested as autoreactivity. The identity of most of these self-lipids remains unknown. By isolating lipids from a CD1d-expressing, highly stimulatory antigen presenting cell, we identified isoforms of β-glucosylceramide (GlcCer), with sphingosine and fatty acid chain lengths of C24:0 and C16:0, that activated a sulfatide-reactive type II NKT cell hybridoma. A screen of structurally related glycosphingolipids demonstrated β-galactosylceramide (GalCer) as another ligand, and further, that the lysoforms were the most potent isoform of the glycosphingo-lipid ligands, followed by isoforms with a long fatty acid chain of C24. Thus, the same type II NKT cell was activated by several ligands, namely sulfatide, GlcCer, and GalCer. However, CD1d-dependent reactivity to antigen presenting cells lacking all GlcCer-based glycosphingolipids, or all glycosphingolipids, was maintained. This suggests that other endogenous, nonglycosphingolipid, lipid ligands contribute to steady-state autoreactivity by type II NKT cells.

Enhanced Selectivity for Sulfatide by Engineered Human Glycolipid Transfer Protein

Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D‖D48V), each containing bound N-nervonoyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of "portal entrance" residues, A47 and D48, reversibly regulate sphingosine access to the hydrophobic pocket via a mechanism that could involve homodimerization. "Door-opening" conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of "engineered GLTPs" with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.

Ceramide N-Acyl Chain Length: A Determinant of Bidimensional Transitions, Condensed Domain Morphology, and Interfacial Thickness

Several lipids of biological interest are able to form monomolecular surfaces with a rich variety of thickness and lateral topography that can be precisely controlled by defined variations of the film composition. Ceramide is one of the simplest sphingolipids, consisting of a sphingosine base N-linked to a fatty acid, and is a membrane mediator for cell-signaling events. In this work, films of ceramides N-acylated with the saturated fatty acids C10, C12, C14, and C16 were studied at the air-aqueous interface. The dipole moment contribution (from surface potential measurements) and the surface topography and thickness (as revealed by Brewster angle microscopy) were measured simultaneously with the surface pressure at different molecular areas. Several surface features were observed depending on the asymmetry between the sphingosine and the N-linked acyl chains. At 21 °C, the C16:0 and C14:0 ceramides showed condensed isotherms and the film topography revealed solid film patches (17.3-15.7 Å thick) that coalesced into a homogeneous surface by further compression. On the other hand, in the more asymmetric C12:0 and C10:0 ceramides, liquid expanded states and liquid expanded-condensed transitions occurred. In the phase coexistence region, the condensed state of these compounds formed flowerlike domains (11.1-13.3 Å thick). C12:0 ceramide domains were larger and more densely branched than those of C10:0 ceramide. Both the film thickness and the surface dipole moment of the condensed state increased with ceramide N-acyl chain length. Bending of the sphingosine chain over the N-linked acyl chain in the more asymmetric ceramides can account for the variation of the surface electrostatics, topography, and thickness of the films with the acyl chain mismatch.

Synthesis of truncated analogues of the iNKT cell agonist, α-galactosyl ceramide (KRN7000), and their biological evaluation

Stimulation of iNKT cells by α-galactosyl ceramide (α-GalCer), also known as KRN7000, and its truncated analogue OCH induces both Th1- and Th2-cytokines, with OCH inducing a Th2-cytokine bias. Skewing of the iNKT cells' response towards either a Th1- or Th2-cytokine profile offers potential therapeutic benefits. The length of both the acyl and the sphingosine chains in α-galactosyl ceramides is known to influence the cytokine release profile. We have synthesized analogues of α-GalCer with truncated sphingosine chains for biological evaluation, with particular emphasis on the Th1/Th2 distribution. Starting from a common precursor, d-lyxose, the sphingosine derivatives were synthesised via a straightforward Wittig condensation.

Induction of Th1-biased cytokine production by -carba-GalCer, a neoglycolipid ligand for NKT cells

NKT cells are characterized by their production of both T(h)1 and T(h)2 cytokines immediately after stimulation with alpha-galactosylceramide (alpha-GalCer), which is composed of alpha-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of alpha-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized alpha-carba-GalCer, which has an alpha-linked carba-galactosyl moiety; here, the 5a'-oxygen atom of the D-galactopyranose ring of alpha-GalCer is replaced by a methylene group. The alpha-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-gamma compared with alpha-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The alpha-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was alpha-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens.

Synthesis of α-Galactosyl Ceramide (KRN7000) and Analogues Thereof via a Common Precursor and Their Preliminary Biological Assessment

A new practical synthesis of alpha-GalCer and of its analogues is presented, opening the chance to easily modify the sphingosine chain. The common precursor is a disaccharide, obtained by coupling tetra-O-benzyl-D-galactose with allyl 2,3-O-isopropylidene-D-lyxofuranoside. Introduction of alkyl chains via Wittig reaction (for alpha-GalCer and OCH) or via Williamson reaction (for oxa analogues) followed by standard synthetic steps allows one to efficiently obtain such compounds. The analogues are able to activate iNKT cells when presented by CD1d expressing cells.

Synthetic Glycolipid Ligands for Human iNKT Cells as Potential Therapeutic Agents for Immunotherapy

Invariant natural killer T (iNKT) cells are an attractive therapeutic target in autoimmune diseases, since they play a major role in immune regulation. iNKT cells recognize glycolipid antigens presented by CD1d molecules that resemble the non-polymorphic MHC class I protein. alpha-galactosylceramide (alpha-GalCer) isolated from marine sponge has long been used as a prototype iNKT cell ligand in the laboratory. As alpha-GalCer is the most efficacious ligand for iNKT cells, its potential to treat autoimmune disease has been evaluated in animal models. Previous studies showed that alpha-GalCer effectively suppressed disease in some autoimmunity models, but not in others. This inconsistency may be attributed to the ability of alpha-GalCer to induce the production of both proinflammatory Th1 and anti-inflammatory Th2 cytokines by iNKT cells. To overcome this issue, we and other groups have synthesized new, unnatural glycolipids by modifying the structure of alpha-GalCer. These efforts have led to an identification of glycolipid compounds that provoke the production of Th2 (but not Th1) cytokines by iNKT cells. Among these novel ligands, an alpha-GalCer analogue named OCH, which contains a truncated sphingosine chain, induces a Th2 biased response by murine iNKT cells. Here we describe that OCH also polarizes human iNKT cells towards Th2, which opens up a new avenue for the clinical application of glycolipid compounds in treating of autoimmune diseases such as multiple sclerosis. The pursuit of synthetic glycolipid antigens has the great potential to lead to a better understanding of the regulatory effects of human iNKT cells and development of a new therapeutic agent for autoimmune diseases.

Molecular dynamics simulation study on the interaction of KRN 7000 and three analogues with human CD1d

Many analogues of KRN 7000, a synthetic glycolipid (α-galactosylceramide) exhibiting immuno-stimulatory activity and antitumor properties, were previously synthesized and tested in order to understand the reasons for the resulting biological activity and Th1/Th2 cytokine profile. Principles have been established for the interaction of such glycolipids with the human CD1d molecule but the exact mechanism by which different ligands with the same polar head elicit distinct biological responses remains unclear. Based on these experiments and on the available crystal structures, protein–ligand interactions are explored using molecular dynamics simulations. Hydrogen bond interactions are examined with regard to the polar group orientation. The influence of modulations on the dynamic behavior of the CD1d–glycolipid complex is addressed. Overall, our data support the mechanism by which the shortening of the α-GalCer sphingosine chain causes a significant twist of the CD1d α1 helix structure from residue Phe84 that affects the position of CD1d residues involved in the TCR recognition.

Natural Sphingomonas Glycolipids Vary Greatly in Their Ability to Activate Natural Killer T Cells

Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.

Chemical modification of iGb3 increases IFN-γ production by hepatic NKT cells

Isoglobotrihexosylceramide (iGb3) has been identified as an endogenous ligand recognized by NKT cells; however, it is a weak agonist compared to the exogenous α-galactosylceramide. Modification of the structure of iGb3 might improve its stimulatory activity. In this study, we assessed the stimulating activity of chemically-modified iGb3 analogues on murine hepatic NKT cells. We analyzed the percentage of IFN-γ- or IL-4-producing cells in hepatic iNKT cell population and found that two chemically-modified iGb3 analogues, especially 4‴-dh-iGb3, induced significantly greater intracellular IFN-γ+ NKT cells in liver by flow cytometry. In vivo experiments also showed that 4-HO-iGb3 and 4‴-dh-iGb3 are selectively strong inducer for rapid serum IFN-γ production compared with unmodified iGb3. Comparing the structure of iGb3 and its two iGb3 analogues, 4-HO-iGb3 has an extra hydroxy group on C4, suggesting that the additional hydroxy group of phytosphingosine might augment the stability of the CD1d/glycoceramide complex forming and thereby possibly promote IFN-γ producing. By further modifying the polysaccharide of glycolipid as did in 4‴-dh-iGb3, we found that 4‴-dh-iGb3 elicited more Th1-biased responses than iGb3 and 4-HO-iGb3. This modification might more strongly strengthen the affinity of the TCR/glycoceramide complex and ultimately polarize iNKT cells to release more Th1 cytokines. Our data suggests that a combination modification on both polysaccharide and sphingosine chain of iGb3 elicits preferential Th1-biased responses.

MR1‐restricted Vα19i T cells – a second population recognizing lipid antigens?

There is increasing evidence that T cells recognizing lipid antigens contribute to the immunological regulation of different disease conditions including autoimmunity. The best-known subset is CD1d-restricted lipid-reactive T cells characterized by the expression of an invariant TCRalpha chain. Much less is known about the biology of another invariant T cell subset, which is restricted to the MHC class I-like molecule MR1. A beneficial role of MR1-restricted T cells has been suggested in a mouse EAE model. However, the nature of antigens that can be presented by MR1 to this invariant T cell subset remained largely unclear. An article in this issue of the European Journal of Immunology presents strong indications that derivatives of alpha-mannosyl ceramide (alpha-ManCer), i.e. glycolipids, can serve as ligands for MR1-restricted invariant T cells. In addition to that, the structure of the alpha-ManCer sphingosine chain influences the Th1-Th2 polarization of the cytokine response. These important new findings will foster further research on the identity of physiological ligands for MR1-restricted T cells and on their relation with immunoregulation. See accompanying article: (http://dx.doi.org/10.1002/eji.200636689).

Activation of invariant natural killer T cells by synthetic glycolipid ligands suppresses autoantibody‐induced arthritis

Stimulation of invariant natural killer T (iNKT) cells with SGL-S23, a novel synthetic glycolipid analog of alpha-galactosylceramide with an elongated sphingosine chain, has been shown to strongly suppress K/BxN serum transfer arthritis. This study was designed to evaluate the protective effects of SGL-S23 in an effector phase of arthritis. To induce arthritis, C57BL/6 mice were injected with 150 mul of serum from K/BxN mice (KRN TCR-transgenic mice crossed with nonobese diabetic mice). Subsequently, synthetic glycolipid ligands were administered intraperitoneally twice, either 3 times starting on day 0 (the day of K/BxN serum injection) or twice starting on day 3. Neutralizing antibody against interferon-gamma (IFNgamma) interleukin-4 (IL-4), IL-10, or transforming growth factor beta was administered 4 hours before injection of SGL-S23. Recombinant IFNgamma was administered subcutaneously every day. The severity of arthritis was monitored using a macroscopic scoring system. Cytokine production and plasma histamine levels were measured by enzyme-linked immunosorbent assay. SGL-S23 strongly suppressed K/BxN serum transfer arthritis by inhibiting inflammatory cell infiltration and subsequent destruction of cartilage and bone. The inhibitory effect mediated by SGL-S23 was abolished by neutralization of IFNgamma. Systemic administration of IFNgamma prevented the development of inflammatory arthritis. Histamine release was suppressed by administration of SGL-S23 or IFNgamma. Degranulated mast cells in the synovium were significantly reduced in SGL-S23-treated mice, suggesting that suppression of mast cell activation contributed to the inhibition of arthritis. These findings suggest that activation of iNKT cells with glycolipid ligands holds promise with regard to the treatment of autoimmune diseases such as rheumatoid arthritis. SGL-S23 has clinical benefit over alpha-galactosylceramide since it induces a weaker cytokine production response in iNKT cells, therefore reducing potential side effects caused by excessive cytokine release.

Comparison of the interaction of dihydrocholesterol and cholesterol with sphingolipid or phospholipid Langmuir monolayers

We report here a study of the interaction of dihydrocholesterol (DChol) with palmitoyl-oleoyl-phosphatidylcholine (POPC) or sphingomyelin (SM) in Langmuir monolayers. DChol and cholesterol (Chol) have very close chemical structures, and DChol is often used in place of Chol because of its better stability. Surface pressure measurements and experiments of desorption induced by β-cyclodextrin show that POPC–DChol monolayers behave similarly to POPC–Chol ones: condensing effects of DChol and Chol on POPC and desorption percentages are in the same range. Moreover Brewster angle microscopy (BAM) experiments performed on these monolayers show that on the whole they are both homogenous. The analysis of mean molecular areas versus DChol percentage shows that this sterol is also able to induce SM condensation at low surface pressure. The condensation of SM molecules is particularly strong at 30 mol% of DChol. At higher surface pressure, the condensation efficiency of DChol decreases and monolayers behave more ideally, even if an inflection point is always observed at 30 mol% of DChol. However, desorption percentages, clearly lower than those obtained with POPC–DChol monolayers, show that DChol is kept at the interface. At last BAM images show also differences in the behaviour of SM–DChol and SM–Chol monolayers. These differences could be due to the different compressibility and conformation of the A/B rings in the two sterols and the rigidity of the sphingosine chain. They suggest that the use of DChol in place of Chol has to be done carefully in the presence of SM.

IFN-gamma inhibits inflammatory arthritis via suppression of mast cell activation. (129.23)

Abstract KBxN mice spontaneously develop arthritis similar to RA in humans, and transfer of serum from arthritic K/BxN mice into healthy animals provokes arthritis within days independently on T and B cells. K/BxN serum-induced arthritis is mediated by anti-glucose-6-phsophate-isomerase IgGs. Here we demonstrate that synthetic glycolipid ligand, a-galactocylceramide (alpha-GalCer) or its analogue with an elongated sphingosine chain strongly suppressed K/BxN serum transfer arthritis by inhibiting inflammatory cellular infiltration and subsequent destruction of cartilage and bone. The inhibitory effect mediated by these glycolipd ligands was abolished by neutralization of IFN-gamma and systemic administration of IFN-gamma prevented the development of inflammatory arthritis. Furthermore, we demonstrate that histamine release involved in the induction of arthritis was suppressed by administration of glycolipid ligands or IFN-gamma. In addition, degranulation of mast cells in synomium was significantly suppressed in glycolipid ligands-treated mice, suggesting that the suppression of mast cell activation contributes to the inhibition of arthritis.

Too Long, Too Short, or Just Right: Glycosphingolipid–Protein Binding Varies with Acyl Chain Length

Glycosphingolipids (GSLs) reside in the membranes of all mammalian cells, where they play roles in both structure and signaling. They traffic between the plasma membrane—where most are found—and vesicle membranes within the cell. One of the carriers of GSLs is glycolipid transfer proteins. The interactions between these two molecules have only recently begun to be elucidated. In a new study, Lucy Malinina, Margarita Malakhova, Rhoderick Brown, Dinshaw Patel, and colleagues reveal a highly unusual binding characteristic of the protein: the sphingosine chain of the GSL either buries itself inside the protein or is left outside of it, depending on the length of the acyl chain. Every GSL has three parts: a sugar head and two long hydrocarbon chains (an 18-carbon, nitrogen-containing sphingosine chain, and an “acyl” chain whose length can vary from 16 to 26 carbons). Using x-ray crystallography, the authors recently elucidated the structure of human glycolipid transfer protein, both with and without an attached GSL, and showed that it has a novel protein fold adapted to interacting with membranes and binding with lipids. In that study, which used a GSL containing a lactose sugar and an 18-carbon monounsaturated acyl chain, they found that the sugar binds to the exterior, while the sphingosine and acyl chains lay parallel inside a hydrophobic tunnel made from an interior fold of the protein. To explore how the protein accommodated other GSLs, they varied acyl length and sugar groups and determined the structure of these protein–GSL complexes. To their surprise, they found that when the acyl chain was either longer (24 carbons) or shorter (8 or 12 carbons) than the one in their initial experiment, the sphingosine chain was not included in the tunnel, but instead jutted out away from the surface of the protein. While the effect on sphingosine is the same, the cause appears to be slightly different in the two cases. When the shorter acyl chain sits in the tunnel, it is joined by an extraneous free hydrocarbon, which denies sphingosine an entrance. The exact origin and role of this hydrocarbon is unknown, but it also occupies the tunnel in the unbound protein. In contrast, there is no extraneous hydrocarbon when the longer acyl chain is in the tunnel, but the chain curls around within, apparently blocking out sphingosine with its bulk. When the authors reverted to the 18-carbon acyl chain but introduced an additional chain-kinking double bond, once again sphingosine was excluded, suggesting that its ability to fit depends on both the length and shape of the acyl group. The tunnel itself expands and contracts with the changes in size of the chains within. Unlike the highly variable interactions of tunnel and hydrocarbon chains, the binding of sugar to the protein appears to rely mainly on a small set of invariant attractions, whether from the double sugar, lactose, or from the single sugars, galactose or glucose. In addition, in each case there are conserved hydrogen bond contacts involving an amine and carbonyl (amide linkage) in the GSL ceramide and specific amino acids of the protein, helping to position the GSL hydrocarbons for entry into the tunnel. The binding of the amide group also triggers a conformational shift in one loop of the protein at the head of the tunnel. From these observations, the authors propose a stepwise binding sequence for GSLs, in which the sugar binds first, acting as the primary determinant of GSL-protein specificity. The amide group binds next, orienting the GSL tails with the tunnel and shifting the loop to help open the tunnel for the hydrocarbons. The acyl hydrocarbon binds next. The sphingosine follows the acyl group into the tunnel in some cases, while in others, it remains outside. These results should accelerate understanding of GSL trafficking within the cell and provide insight into how these molecules play their parts in cell growth and development.

Caveolar Endocytosis and Microdomain Association of a Glycosphingolipid Analog Is Dependent on Its Sphingosine Stereochemistry

We have previously shown that glycosphingolipid analogs are internalized primarily via caveolae in various cell types. This selective internalization was not dependent on particular carbohydrate headgroups or sphingosine chain length. Here, we examine the role of sphingosine structure in the endocytosis of BODIPYtrade mark-tagged lactosylceramide (LacCer) analogs via caveolae. We found that whereas the LacCer analog with the natural (D-erythro) sphingosine stereochemistry is internalized mainly via caveolae, the non-natural (L-threo) LacCer analog is taken up via clathrin-, RhoA-, and Cdc42-dependent mechanisms and largely excluded from uptake via caveolae. Unlike the D-erythro-LacCer analog, the L-threo analog did not cluster in membrane microdomains when added at higher concentrations (5-20 microm). In vitro studies using small unilamellar vesicles and giant unilamellar vesicles demonstrated that L-threo-LacCer did not undergo a concentration-dependent excimer shift in fluorescence emission such as that seen with BODIPYtrade mark-sphingolipids with natural stereochemistry. Molecular modeling studies suggest that in d-erythro-LacCer, the disaccharide moiety extends above and in the same plane as the sphingosine hydrocarbon chain, while in L-threo-LacCer the carbohydrate group is nearly perpendicular to the hydrocarbon chain. Together, these results suggest that the altered stereochemistry of the sphingosine group in L-threo-LacCer results in a perturbed structure, which is unable to pack closely with natural membrane lipids, leading to a reduced inclusion in plasma membrane microdomains and decreased uptake by caveolar endocytosis. These findings demonstrate the importance of the sphingolipid stereochemistry in the formation of membrane microdomains.

Structural basis for CD1d presentation of a sulfatide derived from myelin and its implications for autoimmunity

Sulfatide derived from the myelin stimulates a distinct population of CD1d-restricted natural killer T (NKT) cells. Cis-tetracosenoyl sulfatide is one of the immunodominant species in myelin as identified by proliferation, cytokine secretion, and CD1d tetramer staining. The crystal structure of mouse CD1d in complex with cis-tetracosenoyl sulfatide at 1.9 Å resolution reveals that the longer cis-tetracosenoyl fatty acid chain fully occupies the A′ pocket of the CD1d binding groove, whereas the sphingosine chain fills up the F′ pocket. A precise hydrogen bond network in the center of the binding groove orients and positions the ceramide backbone for insertion of the lipid tails in their respective pockets. The 3′-sulfated galactose headgroup is highly exposed for presentation to the T cell receptor and projects up and away from the binding pocket due to its β linkage, compared with the more intimate binding of the α-glactosyl ceramide headgroup to CD1d. These structure and binding data on sulfatide presentation by CD1d have important implications for the design of therapeutics that target T cells reactive for myelin glycolipids in autoimmune diseases of the central nervous system.

The crystal structure of human CD1d with and without α-galactosylceramide

The glycolipid alpha-galactosylceramide binds with high affinity to CD1d and stimulates natural killer T cells. Here we report the crystal structure of human CD1d in complex with synthetic alpha-galactosylceramide at a resolution of 3.0 A. The structure shows a tightly fit lipid in the CD1d binding groove, with the sphingosine chain bound in the C' pocket and the longer acyl chain anchored in the A' pocket. We also present the CD1d structure without lipid, which has a more open conformation of the binding groove, suggesting a dual conformation of CD1d in which the 'open' conformation is more able to load lipids. These structures provide clues as to how CD1 molecules load glycolipids as well as data to guide the design of new therapeutic agents.

The clinical implication and molecular mechanism of preferential IL-4 production by modified glycolipid-stimulated NKT cells

OCH, a sphingosine-truncated analog of α-galactosylceramide (αGC), is a potential therapeutic reagent for a variety of Th1-mediated autoimmune diseases through its selective induction of Th2 cytokines from natural killer T (NKT) cells. We demonstrate here that the NKT cell production of IFN-γ is more susceptible to the sphingosine length of glycolipid ligand than that of IL-4 and that the length of the sphingosine chain determines the duration of NKT cell stimulation by CD1d-associated glycolipids. Furthermore, IFN-γ production by NKT cells requires longer T cell receptor stimulation than is required for IL-4 production by NKT cells stimulated either with immobilized mAb to CD3 or with immobilized “αGC-loaded” CD1d molecules. Interestingly, transcription of IFN-γ but not that of IL-4 was sensitive to cycloheximide treatment, indicating the intrinsic involvement of de novo protein synthesis for IFN-γ production by NKT cells. Finally, we determined c-Rel was preferentially transcribed in αGC-stimulated but not in OCH-stimulated NKT cells and was essential for IFN-γ production by activated NKT cells. Given the dominant immune regulation by the remarkable cytokine production of ligand-stimulated NKT cells in vivo, in comparison with that of (antigen-specific) T cells or NK cells, the current study confirms OCH as a likely therapeutic reagent for use against Th1-mediated autoimmune diseases and provides a novel clue for the design of drugs targeting NKT cells.

The clinical implication and molecular mechanism of preferential IL-4 production by modified glycolipid-stimulated NKT cells.

OCH, a sphingosine-truncated analog of alpha-galactosylceramide (alphaGC), is a potential therapeutic reagent for a variety of Th1-mediated autoimmune diseases through its selective induction of Th2 cytokines from natural killer T (NKT) cells. We demonstrate here that the NKT cell production of IFN-gamma is more susceptible to the sphingosine length of glycolipid ligand than that of IL-4 and that the length of the sphingosine chain determines the duration of NKT cell stimulation by CD1d-associated glycolipids. Furthermore, IFN-gamma production by NKT cells requires longer T cell receptor stimulation than is required for IL-4 production by NKT cells stimulated either with immobilized mAb to CD3 or with immobilized "alphaGC-loaded" CD1d molecules. Interestingly, transcription of IFN-gamma but not that of IL-4 was sensitive to cycloheximide treatment, indicating the intrinsic involvement of de novo protein synthesis for IFN-gamma production by NKT cells. Finally, we determined c-Rel was preferentially transcribed in alphaGC-stimulated but not in OCH-stimulated NKT cells and was essential for IFN-gamma production by activated NKT cells. Given the dominant immune regulation by the remarkable cytokine production of ligand-stimulated NKT cells in vivo, in comparison with that of (antigen-specific) T cells or NK cells, the current study confirms OCH as a likely therapeutic reagent for use against Th1-mediated autoimmune diseases and provides a novel clue for the design of drugs targeting NKT cells.