Ceramide-rich membrane rafts mediate CD40 clustering.

Many receptor systems use receptor clustering for transmembrane signaling. In this study, we show that acid sphingomyelinase (ASM) is essential for the clustering of CD40. Stimulation of lymphocytes via CD40 ligation results in ASM translocation from intracellular stores, most likely vesicles, into distinct membrane domains on the extracellular surface of the plasma membrane. Surface ASM initiates a release of extracellularly oriented ceramide, which in turn mediates CD40 clustering in sphingolipid-rich membrane domains. ASM, ceramide, and CD40 colocalize in the cap-like structure of stimulated cells. Deficiency of ASM, destruction of sphingolipid-rich rafts, or neutralization of surface ceramide prevents CD40 clustering and CD40-initiated cell signaling. These findings indicate that the ASM-mediated release of ceramide and/or metabolites of ceramide regulate clustering of CD40, which seems to be a prerequisite for cellular activation via CD40.     

The transmembranous domain of CD40 determines CD40 partitioning into lipid rafts

Stimulation of CD40 has been previously shown to result in a release of ceramide in small sphingolipid-enriched rafts in the cell membrane [Grassmé et al., J. Immunol. 168 (2002) 298-307]. Those rafts fused to larger signaling platforms that served to cluster CD40. Here, we defined molecular mechanisms of CD40 clustering in membrane platforms. To this end, we replaced the transmembranous domain of CD40 with that of CD45, a molecule known to be excluded from lipid rafts. Murine T cells were stably transfected with wild-type CD40 or chimeric CD40/CD45. Flow cytometry confirmed normal binding properties of the mutant to CD40 ligand. Stimulation with CD40 ligand resulted in clustering of wild-type CD40, while the chimeric CD40/45 receptor failed to cluster. This correlated with a deficiency of the chimeric receptor to activate JNK, p38 MAP kinase and SAPK, known signaling molecules of the intracellular pathway initiated by CD40. Forced crosslinking of the CD40/45 chimeric receptor restored, at least partially, these signaling events. The results suggest that the transmembranous domain of CD40 is central for the recruitment to and clustering of CD40 in membrane platforms.     

Lipid rafts regulate cellular CD40 receptor localization in vascular endothelial cells

Cholesterol enriched lipid rafts are considered to function as platforms involved in the regulation of membrane receptor signaling complex through the clustering of signaling molecules. In this study, we tested whether these specialized membrane microdomains affect CD40 localization in vitro and in vivo. Here, we provide evidence that upon CD40 ligand stimulation, endogenous and exogenous CD40 receptor is rapidly mobilized into lipid rafts compared with unstimulated HAECs. Efficient binding between CD40L and CD40 receptor also increases amounts of CD40 protein levels in lipid rafts. Deficiency of intracellular conserved C terminus of the CD40 cytoplasmic tail impairs CD40 partitioning in raft. Raft disorganization after methyl-beta-cyclodextrin treatment diminishes CD40 localization into rafts. In vivo studies show that elevation of circulating cholesterol in high-cholesterol fed rabbits increases the cholesterol content and CD40 receptor localization in lipid rafts. These findings identify a physiological role for membrane lipid rafts as a critical regulator of CD40-mediated signal transduction and raise the possibility that certain pathologic conditions may be treated by altering CD40 signaling with drugs affecting its raft localization.     

A mathematical model for dynamics of CD40 clustering

Ligand bound-receptors in a signalosome complex trigger signals to determine cellular functions. Upon ligand binding, the ligand–receptor complexes form clusters on cell membrane. Guided by the previous experimental reports on the cluster formation of CD40, a trans membrane receptor for CD40-ligand, we built a minimal model of the receptor cluster formation. In this model, we studied co-operative and non-co-operative clustering of a maximum of four CD40 molecules assuming a positive mediator of clustering such as cholesterol to be present in both cases. We observed that co-operative interactions between CD40 molecules resulted in more of the largest CD40 clusters than that observed with the non-co-operatively interacting CD40 molecules. We performed global sensitivity analysis on the model parameters and the analyses suggested that cholesterol influenced only the initial stage of the co-operatively clustering CD40 molecules but it affects both the initial and the final stages in case of the non-co-operatively clustering CD40 molecules. Robustness analyses revealed that in both co-operative and non-co-operative interactions, the higher order clusters beyond a critical size are more robust with respect to alterations in the environmental parameters including the cholesterol. Thus, the role of co-operative and non-co-operative interactions in environment-influenced receptor clustering is reported for the first time.     

CD95 signaling via ceramide-rich membrane rafts

Clustering seems to be employed by many receptors for transmembrane signaling. Here, we show that acid sphingomyelinase (ASM)-released ceramide is essential for clustering of CD95. In vitro and in vivo, extracellularly orientated ceramide, released upon CD95-triggered translocation of ASM to the plasma membrane outer surface, enabled clustering of CD95 in sphingolipid-rich membrane rafts and apoptosis induction. Whereas ASM deficiency, destruction of rafts, or neutralization of surface ceramide prevented CD95 clustering and apoptosis, natural ceramide only rescued ASM-deficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.     

Formation and function of ceramide-enriched membrane platforms with CD38 during M1-receptor stimulation in bovine coronary arterial myocytes

CD38 contains an ADP ribosylcyclase domain that mediates intracellular Ca(2+) signaling by the production of cyclic ADP-ribose (cADPR), but the mechanisms by which the agonists activate this enzyme remain unclear. The present study tested a hypothesis that a special lipid-raft (LR) form, ceramide-enriched lipid platform, contributes to CD38 activation to produce cADPR in response to muscarinic type 1 (M(1)) receptor stimulation in bovine coronary arterial myocytes (CAMs). By confocal microscopic analysis, oxotremorine (Oxo), an M(1) receptor agonist, was found to increase LR clustering on the membrane with the formation of a complex of CD38 and LR components such as GM(1), acid sphingomyelinase (ASMase), and ceramide, a typical ceramide-enriched macrodomain. At 80 microM, Oxo increased LR clustering by 78.8%, which was abolished by LR disruptors, methyl-beta-cyclodextrin (MCD), or filipin. With the use of a fluorescence resonance energy transfer (FRET) technique, 15.5+/-1.9% energy transfer rate (vs. 5.3+/-0.9% of control) between CD38 and LR component, ganglioside M(1) was detected, further confirming the proximity of both molecules. In the presence of MCD or filipin, there were no FRET signals detected. In floated detergent-resistant membrane fractions, CD38 significantly increased in LR fractions of CAMs treated by Oxo. Moreover, MCD or filipin attenuated Oxo-induced production of cADPR via CD38. Functionally, Oxo-induced intracellular Ca(2+) release and coronary artery constriction via cADPR were also blocked by LR disruption or ASMase inhibition. These results provide the first evidence that the formation of ceramide-enriched lipid macrodomains is crucial for Oxo-induced activation of CD38 to produce cADPR in CAMs, and these lipid macrodomains mediate transmembrane signaling of M(1) receptor activation to produce second messenger cADPR.     

Ceramide-enriched membrane domains

Cellular activation involves the re-organization of receptor molecules and the intracellular signalosom in the cell membrane. Recent studies indicate that specialized domains of the cell membrane, termed rafts, are central for the spatial organization of receptors and signaling molecules. Rafts are converted into larger membrane platforms by activity of the acid sphingomyelinase, which hydrolyses raft-sphingomyelin to ceramide. Ceramide molecules spontaneously associate to form ceramide-enriched microdomains, which fuse to large ceramide-enriched membrane platforms. The acid sphingomyelinase is activated by multiple stimuli including CD95, CD40, DR5/TRAIL, CD20, FcgammaRII, CD5, LFA-1, CD28, TNF, the Interleukin-1 receptor, the PAF-receptor, CD14, infection with P. aeruginosa, S. aureus, N. gonorrhoeae, Sindbis-Virus, Rhinovirus, treatment with gamma-irradiation, UV-light, doxorubicin, cisplatin, disruption of integrin-signaling and under some conditions of developmental death. Ceramide-enriched membrane platforms serve the clustering of receptors, the recruitment of intracellular signaling molecules and the exclusion of inhibitory signaling factors and, thus, facilitate signal transduction initiated by the specific stimulus.     

Doxorubicin enhances TRAIL-induced cell death via ceramide-enriched membrane platforms

Previous studies indicated that signalling via CD95 and DR5 is greatly enhanced by the formation of ceramide-enriched membrane platforms. Here, we employed this concept to convert doses of subtherapeutic TRAIL that were unable to release ceramide and kill leukemic B-cells or ex vivo T lymphocytes, into a very effective apoptotic stimulus. Ceramide production was induced by application of sub-toxic doses of doxorubicin that resulted in an activation of the acid sphingomyelinase (ASM), release of ceramide and formation of ceramide-enriched membrane platforms. The latter served DR5 to cluster after application of very low doses of TRAIL in combination with doxorubicin. Genetic deficiency of the ASM abrogated doxorubicin-induced ceramide release, as well as clustering of DR5 and apoptosis induced by the combined treatment of doxorubicin and TRAIL. These data show that local release of ceramide potentiates very low, otherwise inactive doses of TRAIL that may represent a novel therapeutic concept to treat tumors.     

CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily

CD40, a tumor necrosis factor (TNF) receptor (TNFR) family member, conveys signals regulating diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death. The ability of CD40 to mediate apoptosis in carcinoma cells is intriguing given the fact that the CD40 cytoplasmic C terminus lacks a death domain homology with the cytotoxic members of the TNFR superfamily, such as Fas, TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) receptors. In this study, we have probed the mechanism by which CD40 transduces death signals. Using a trimeric recombinant soluble CD40 ligand to activate CD40, we have found that this phenomenon critically depends on the membrane proximal domain (amino acids 216 to 239) but not the TNFR-associated factor-interacting PXQXT motif in the CD40 cytoplasmic tail. CD40-mediated cytotoxicity is blocked by caspase inhibitors, such as zVAD-fmk and crmA, and involves activation of caspase 8 and caspase 3. Interestingly, CD40 ligation was found to induce functional Fas ligand, TRAIL (Apo-2L) and TNF in apoptosis-susceptible carcinoma cells and to up-regulate expression of Fas. These findings identify a novel proapoptotic mechanism which is induced by CD40 in carcinoma cells and depends on the endogenous production of cytotoxic cytokines and autocrine or paracrine induction of cell death.     

DC-SIGN mediated sphingomyelinase-activation and ceramide generation is essential for enhancement of viral uptake in dendritic cells

As pattern recognition receptor on dendritic cells (DCs), DC-SIGN binds carbohydrate structures on its pathogen ligands and essentially determines host pathogen interactions because it both skews T cell responses and enhances pathogen uptake for cis infection and/or T cell trans-infection. How these processes are initiated at the plasma membrane level is poorly understood. We now show that DC-SIGN ligation on DCs by antibodies, mannan or measles virus (MV) causes rapid activation of neutral and acid sphingomyelinases followed by accumulation of ceramides in the outer membrane leaflet. SMase activation is important in promoting DC-SIGN signaling, but also for enhancement of MV uptake into DCs. DC-SIGN-dependent SMase activation induces efficient, transient recruitment of CD150, which functions both as MV uptake receptor and microbial sensor, from an intracellular Lamp-1+ storage compartment shared with acid sphingomyelinase (ASM) within a few minutes. Subsequently, CD150 is displayed at the cell surface and co-clusters with DC-SIGN. Thus, DC-SIGN ligation initiates SMase-dependent formation of ceramide-enriched membrane microdomains which promote vertical segregation of CD150 from intracellular storage compartments along with ASM. Given the ability to promote receptor and signalosome co-segration into (or exclusion from) ceramide enriched microdomains which provide a favorable environment for membrane fusion, DC-SIGN-dependent SMase activation may be of general importance for modes and efficiency of pathogen uptake into DCs, and their routing to specific compartments, but also for modulating T cell responses.     

Molecular characterization of CD40 signaling intermediates

Signal transduction through the CD40 receptor is initiated by binding of its trimeric ligand and propagated by interactions of tumor necrosis factor receptor-associated factor (TRAF) proteins with the multimerized CD40 cytoplasmic domain. Using defined multimeric constructs of the CD40 cytoplasmic domain expressed as either soluble or myristoylated proteins, we have addressed the extent of receptor multimerization needed to initiate signal transduction and identified components of CD40 signaling complexes. Signal transduction in human embryonic kidney 293 cells, measured by nuclear factor kappaB activation, was observed in cells expressing soluble trimeric CD40 cytoplasmic domain and to a lesser extent in cells expressing dimeric CD40 cytoplasmic domain. Nuclear factor kappaB activation was strongest in cells expressing myristoylated trimeric CD40 cytoplasmic domain. Signal transduction through trimeric CD40 cytoplasmic domains with various point mutations in the TRAF binding sites was similar to signal transduction through analogous full-length receptors. Transiently expressed soluble trimeric CD40 cytoplasmic domain was isolated as complexes that contained TRAF2, TRAF3, TRAF5, TRAF6, and the inhibitor of apoptosis protein (c-IAP1). Association of c-IAP1 with the CD40 cytoplasmic domain complex was indirect and dependent on the presence of an intact TRAF1/2/3 binding site. These results suggest that extracellular ligation of CD40 can be bypassed and that soluble trimerized CD40 complexes can be isolated and used to identify components that link CD40 with signaling pathways.     

CD40-mediated apoptosis in murine B-lymphoma lines containing mutated p53

Crosslinking CD40 induces normal B-cells to proliferate and differentiate but causes many tumor cell lines to undergo apoptosis. As p53 is required for many apoptotic pathways, we analyzed the effects of CD40 ligation and their correlation with p53 function in four murine B-lymphoma lines. A20 and M12 cells showed complete growth inhibition and an increase in the proportion of apoptotic cells upon CD40 crosslinking, but WEHI 231 and WEHI 279 cells were unaffected. IkappaBalpha protein levels were reduced in all four lines in response to CD40 crosslinking, indicating that CD40 signaling was normal. NFkappaB activity was quantitated by reporter and gel shift assays and found to be equivalent in all four cell lines. P53 reporter constructs were activated in WEHI231 but not A20 or M12 cells, suggesting that p53 in the latter two cell lines may be mutated. These data are supported by the lack of detectable p21 mRNA in A20 and M12 cells. P21 mRNA was increased to detectable levels in M12 cells upon CD40 ligation; however, blocking this effect with the p53 inhibitor pifithrin had no effect on CD40-mediated apoptosis. Sequencing showed that p53 in A20 and M12 cells contained point mutations leading to amino acid substitutions in DNA binding regions, but was unmutated in WEHI231 and WEHI 279. These results suggest that CD40-mediated apoptosis can occur in the absence of functional p53.     

Characterization of a CD40-dominant inhibitory receptor mutant.

CD40 is an important mediator of immune and inflammatory responses. It is a costimulatory molecule for B cell proliferation and survival. Blockade of CD40 has been shown to induce tolerance and its role in other pathogenic conditions has led to the proposal that CD40 inhibition could be valuable therapeutically. As a first step to this end, we have characterized a CD40-dominant negative receptor. This inhibitory mutant lacks the identified CD40 signaling domains. It inhibits both cotransfected and endogenous CD40 activation of NF-kappaB. This mutant is specific, as it does not affect TNF or latent membrane protein 1 signaling. Its potential usefulness is illustrated by its ability to inhibit the CD40 ligand-stimulated increases of HLA and CD54 expression, molecules involved in Ag recognition and lymphocyte recruitment leading to organ rejection. The inhibitory mutant has no TNFR-associated factor 2-binding capabilities and inhibits the recruitment of TNFR-associated factor 2 to the CD40 signaling complex after stimulation. These studies show that the CD40 inhibitory receptor molecule is effective, specific, and useful both for research and potentially as a clinical tool. And furthermore, it is likely that similar dominant inhibitory receptors can be generated for all of the members of the TNFR superfamily.     

Requirement of oxidation-dependent CD40 homodimers for CD154/CD40 bidirectional signaling

It is well established that the CD154/CD40 interaction is required for T cell-dependent B cell differentiation and maturation. However, the early molecular and structural mechanisms that orchestrate CD154 and CD40 signaling at the T cell/APC contact site are not well understood. We demonstrated that CD40 engagement induces the formation of disulfide-linked (dl) CD40 homodimers that predominantly associate with detergent-resistant membrane microdomains. Mutagenesis and biochemical analyses revealed that (a) the integrity of the detergent-resistant membranes is necessary for dl-CD40 homodimer formation, (b) the cytoplasmic Cys(238) of CD40 is the target for the de novo disulfide oxidation induced by receptor oligomerization, and (c) dl-CD40 homodimer formation is required for CD40-induced interleukin-8 secretion. Stimulation of CD154-positive T cells with staphylococcal enterotoxin E superantigen that mimics nominal antigen in initiating cognate T cell/APC interaction revealed that dl-CD40 homodimer formation is required for interleukin-2 production by T cells. These findings indicate that dl-CD40 homodimer formation has a physiological role in regulating bidirectional signaling.     

Recruitment of CD40 and tumor necrosis factor receptor-associated factors 2 and 3 to membrane microdomains during CD40 signaling

Signals delivered to antigen-presenting cells through CD40 are critical for the activation of immune responses. Intracellular tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are key elements of the signal transduction pathways of many TNF receptor family members, including CD40. We show for the first time that engagement of CD40 in intact B cells induces the rapid translocation of TRAF2 from the cytoplasm to the plasma membrane. We found that CD40 engagement also results in its recruitment, together with TRAF2 and TRAF3, to membrane microdomains, regions of the plasma membrane enriched in signaling molecules such as the Src family kinases. Using a membrane-permeable chelator of zinc or a mutant TRAF2 molecule, we show that the putative zinc-binding domains of TRAFs contribute to their recruitment to microdomains and to the downstream activation of c-Jun N-terminal kinase. We suggest that the zinc RING and zinc finger domains of TRAFs are required for communication between CD40 and microdomain-associated signaling molecules and may serve a similar role in the signal transduction pathways of other TNF receptor family members.     

Crystallographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation

CD40 is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in B cell development. CD154/CD40L is the physiological ligand of CD40. We have determined the crystal structure of the CD40-CD154 complex at 3.5 Å resolution. The binding site of CD40 is located in a crevice formed between two CD154 subunits. Charge complementarity plays a critical role in the CD40-CD154 interaction. Some of the missense mutations found in hereditary hyper-IgM syndrome can be mapped to the CD40-CD154 interface. The CD40 interaction area of one of the CD154 subunits is twice as large as that of the other subunit forming the binding crevice. This is because cysteine-rich domain 3 (CRD3) of CD40 has a disulfide bridge in an unusual position that alters the direction of the ladder-like structure of CD40. The Ser¹³² loop of CD154 is not involved in CD40 binding but its substitution significantly reduces p38- and ERK-dependent signaling by CD40, whereas JNK-dependent signaling is not affected. These findings suggest that ligand-induced di- or trimerization is necessary but not sufficient for complete activation of CD40.     

Signaling through CD40 ligand decreases CD80 expression on murine Langerhans cells and enhances IL-12 p40 production

We previously showed that murine Langerhans cells (LC) express CD40 ligand (CD40L). In this study, we further investigated the function of CD40L on LC using agonistic antibodies and CD40L knockout (KO) mice. Signaling through CD40L decreased CD80 expression on LC 48 h after stimulation and the decrease was more remarkable in the presence of interferon-gamma (IFN-gamma). Signaling through CD40 enhanced the production of IL-12 p40 from LC, and simultaneous signaling through CD40L slightly augmented this effect. Addition of IFN-gamma further enhanced IL-12 p40 production. LC from CD40L KO mice expressed similar levels of surface molecules such as CD40, CD80, CD86, and MHC class II, compared with those from wild-type mice. However, they produced less amount of IL-12 p40 during 48 h after purification. These results suggest that signaling through CD40L on LC is important in regulating IL-12 production, which is critical for Th1 type immune responses.     

CD27 and CD40 inhibit p53-independent mitochondrial pathways in apoptosis of B cells induced by B cell receptor ligation

B cells in the germinal center are known to undergo apoptosis after B cell receptor (BCR) ligation, a process relevant to immunological tolerance. Human CD27 is a B cell co-stimulatory molecule. The aim of this study was to compare the effects of CD27 and CD40 signals on BCR-mediated apoptosis of B cells. BCR ligation activated mitochondrial apoptotic pathways including down-regulation of Bcl-X(L), dissipation of mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-9. Each of these effects was significantly inhibited by CD27 and CD40. Bik expression was weakly but significantly down-regulated by CD27 but up-regulated by CD40. BCR ligation resulted in p53 activation including its phosphorylation at Ser(15), nuclear translocation, and target gene p53AIP1 induction. CD27 and CD40 clearly suppressed these processes. Analyses that used dominant-negative p53 variants revealed a low but still substantial level of BCR-mediated apoptosis and intact mitochondria-mediated apoptotic pathway. These pathways were further inhibited by CD27 and CD40, although the cells showed no p53 phosphorylation or p53AIP1 expression. Our results suggested that, at the mitochondrial level, CD27 and CD40 co-stimulatory signals regulated the p53-amplified apoptotic pathway in B cells through the inhibition of p53-independent apoptotic pathway primarily induced by BCR ligation.     

CD40-40L signaling in vascular inflammation

Ligation of CD40 in circulating cells or in the vessel wall may promote mononuclear cell recruitment, participate in the weakening of the plaque, and contribute to thrombosis. This process appears to be redox-sensitive, but the precise signaling mechanism by which the interaction between CD40L and its receptor CD40 mediates inflammatory secretion is unclear. Our previous studies have shown that the CD40-CD40L interaction modulates release of reactive oxygen species (ROS) and the current findings demonstrate that in endothelial cells CD40L dose dependently induces intracellular CD40L and MCP1 release in a redox sensitive manner. Pharmacological inhibition of phosphatidylinositol 3-kinase and p38 MAPK as well as adenovirus-mediated inactivation of Akt and p38 MAPK inhibited CD40L effects on endothelial cells. Akt, in particular, appeared to mediate CD40L-induced CD40L synthesis and MCP1 release by endothelial cells in a redox sensitive manner via NFkappaB activation. In addition, using confocal microscopy, exogenous addition of recombinant CD40L or adenoviral mediated CD40L overexpression was found to stimulate nuclear translocation of NFkappaB, which was further augmented by Akt overexpression and inhibited by Akt inactivation. These data support a mechanism whereby redox-sensitive CD40-CD40L interactions induce activation of Akt and p38 MAPK, leading to stimulation of NFkappaB and enhanced synthesis of CD40L and MCP1. Increased CD40L and MCP1 may contribute to the adherence of CD40-positive cells, such as platelets and monocytes, to the vessel wall modulating atherothrombosis.