Characterization of CDw92 as a member of the choline transporter-like protein family regulated specifically on dendritic cells.

CDw92 is a 70-kDa surface protein broadly expressed on leukocytes and endothelial cells. In this manuscript, we present the molecular cloning of the CDw92 molecule by using a highly efficient retroviral expression cloning system. Sequence analysis of the CDw92 cDNA revealed a length of 2679 bp. The 1959-bp open reading frame encodes a protein of 652 amino acids. Computational analysis of the CDw92 protein sequence indicates 10 transmembrane domains, three potential N-linked glycosylation sites, and an amino acid stretch in the C-terminal region that is related to the immunoreceptor tyrosine-based inhibitory motif. Comparison of the sequence of the CDw92 clone presented in this study with various database entries show that it is a C-terminal variant of human choline transporter-like protein 1, a member of a recently identified family of multitransmembrane surface proteins. Furthermore, we found that CDw92 is stably expressed on monocytes, PBLs, and endothelial cells, as we did not yet find modulation of expression by various stimuli on these cells. In contrast to this factor-independent expression of CDw92, we detected a specific regulation of CDw92 on monocyte-derived dendritic cells (Mo-DCs). Maturation of Mo-DCs by ionomycin or calcium ionophore resulted in down-regulation of CDw92 and incubation of these cells with IL-10 in a specific re-expression. Moreover, targeting of CDw92 on LPS-treated Mo-DCs by CDw92 mAb VIM15b augmented the LPS-induced IL-10 production 2.8-fold. Together, these data suggest a crucial role of the CDw92 protein in the biology and regulation of the function of leukocytes in particular DCs.

Sphingolipids: second messengers, mediators and raft constituents in signaling.

Recently, evidence has accumulated to show that sphingolipids exert an important function in signaling. These lipids serve as intracellular second messengers and as extracellular mediators. Furthermore, glycosylated sphingolipids are essential components of membrane rafts, which serve as platforms for the initiation of signaling cascades. Here, Eva Prieschl and Thomas Baumruker summarize current findings in leukocytes illustrating these different facets.

Major histocompatibility complex class II- fetal skin dendritic cells are potent accessory cells of polyclonal T-cell responses.

Whereas dendritic cells (DC) and Langerhans cells (LC) isolated from organs of adult individuals express surface major histocompatibility complex (MHC) class II antigens, DC lines generated from fetal murine skin, while capable of activating naive, allogeneic CD8+ T cells in a MHC class I-restricted fashion, do not exhibit anti-MHC class II surface reactivity and fail to stimulate the proliferation of naive, allogeneic CD4+ T cells. To test whether the CD45+ MHC class I+ CD80+ DC line 80/1 expresses incompetent, or fails to transcribe, MHC class II molecules, we performed biochemical and molecular studies using Western blot and polymerase chain reaction analysis. We found that 80/1 DC express MHC class II molecules neither at the protein nor at the transcriptional level. Ultrastructural examination of these cells revealed the presence of a LC-like morphology with indented nuclei, active cytoplasm, intermediate filaments and dendritic processes. In contrast to adult LC, no LC-specific cytoplasmic organelles (Birbeck granules) were present. Functionally, 80/1 DC in the presence, but not in the absence, of concanavalin A and anti-T-cell receptor monoclonal antibodies stimulated a vigorous proliferative response of naive CD4+ and CD8+ T cells. Furthermore, we found that the anti-CD3-induced stimulation of naive CD4+ and CD8+ T cells was critically dependent on the expression of FcgammaR on 80/1 DC and that the requirement for co-stimulation depends on the intensity of T-cell receptor signalling.

The role of sphingosine kinase in the signaling initiated at the high-affinity receptor for IgE (FcepsilonRI) in mast cells.

Over the last few years, sphingolipids have emerged as an additional class of lipids participating in signaling events in various cell types. The best-investigated examples so far are ceramide and sphingosine-1-phosphate. Ceramide-activated protein kinase and sphingosine kinase are two enzymes which respond to and generate these mediators. In particular, sphingosine kinase, its substrate sphingosine and the product sphingosine-1-phosphate have recently been implicated in the signaling cascades initiated at the FcepsilonRI of mast cells. High intracellular levels of sphingosine seem to serve as an 'intracellular' inhibitor which is 'deactivated' by the action of sphingosine kinase, due to the conversion to sphingosine-1-phosphate. One mode of action of the inhibitory process in this cell type is prevention of the activation of the mitogen-activated protein (MAP) kinase pathway. Sphingosine-1-phosphate itself, the product of this enzymatic reaction, is believed to lead to Ca(2+) mobilization and to stimulate the MAP kinase pathway. The existence and function of this second messenger explains the 'IP3 gap' described in mast cells after FcepsilonRI activation. Therefore, a picture emerges whereby the balance of these two lipid molecules seems to be decisive for the activation of mast cells by IgE plus antigen, with sphingosine kinase acting as a permissive switch for stimulation.

Beyond a structural component: sphingolipids in immunology.

Two major classes of lipids participating in signaling cascades in immune cells are known today. One comprises glycerol-based lipids with diacylglycerol as its most prominent member that mediates the activation of classical and novel protein kinase C molecules. The second group contains the sphingolipids, with the best-investigated representatives being sphingosine, sphingosine-1-phosphate, and ceramide. In the last years the latter two molecules have especially received considerable attention for their modulatory capacity in the course of an apoptotic response. Today it is clear that sphingolipids are ubiquitously distributed in all eukaryotic cells, especially in cellular membranes, where they were previously thought to fulfil an exclusively structural role. Recent findings, however, have demonstrated functions beyond this. Sphingolipid specific G-protein coupled receptors were identified and their role as intracellular second messengers has been further elucidated. In addition, glycosphingolipids, in particular, are enriched in certain membrane compartments, known as detergent resistant membranes. These serve as entry sites for several receptor-mediated signaling events by stabilizing receptor/kinase interactions, suggesting an involvement in the initiation of signaling cascades. Altogether, these findings have led to new insights into both the role of these lipids in signaling as well as the underlying pathology of several diseases with imbalances in the sphingolipid metabolism. The development of these disorders has mainly been attributed to the toxic potential of lysosphingolipids up to now. In addition, attempts have been made to develop compounds and drugs containing the sphingolipid backbone for influencing diseases associated with unwanted cell activation (e.g, cancer, inflammatory processes). These novel findings and developments are reviewed in the following.

Glycosphingolipid-induced relocation of Lyn and Syk into detergent-resistant membranes results in mast cell activation.

Sphingosine, sphingosine-1-phosphate, and the more complex sphingolipid ceramide exert strong immunomodulatory effects on a variety of leukocytes. However, little is known regarding such a potential of glycosphingolipids, a class of sugar derivatives of sphingosine. Here we demonstrate that galactosylsphingosine, one of the smallest representatives of this group, accumulates in the detergent-resistant membranes resulting in the relocation of the tyrosine kinases Lyn and Syk into this compartment. The result of this is an enhanced tyrosine phosphorylation and kinase activity leading to priming and activation of mast cells by conveying a weak yet significant activation of the mitogen-activated protein kinase pathway(s). In comparison to IgE/Ag triggering, galactosylsphingosine stimulates the mitogen-activated protein kinase pathway more rapidly and favors c-Jun NH2-terminal kinase 1 activation over extracellular signal-regulatory kinase 1 and 2. At the transcription factor level, this "ultratransient signaling event" results in an activation of JunD as the predominant AP-1 component. In this respect, the effects of galactosylsphingosine are clearly distinct from the signaling elicited by other sphingolipids without the sugar moiety, such as sphingosine-1-phosphate.

The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon receptor I triggering.

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc epsilon receptor I by modulating the balance of the counterregulatory lipids.

TNF-alpha and IL-5 gene induction in IgE plus antigen-stimulated mast cells require common and distinct signaling pathways.

BACKGROUND: Mast cells produce a variety of cytokines and chemokines in a timely and tightly controlled fashion if stimulated via the FcepsilonRI. Evidence is accumulating that the transcriptional induction of the corresponding genes and the release of these mediators are dependent on common and mediator-specific components of the signal transduction and transcription factor machinery. METHODS: We addressed this issue by comparing the effects of mitogen activated protein (MAP) kinase pathway inhibitors and protein kinase C (PKC) inhibitors on the induction of TNF-alpha and IL-5 after IgE plus antigen (Ag) stimulation in CPII mouse mast cells using Western blot analyses and transient transfections of reporter gene plasmids. RESULTS: TNF-alpha shows a strict dependence on the MAP kinase pathway, while IL-5 is either activated by PMA-dependent PKCs or along the MAP kinase pathway. In addition, both mediators are sensitive to PKCmu inhibition, suggesting involvement of this atypical, non-PMA dependent PKC in the overall induction process. CONCLUSION: While the two cytokines were recently shown to be regulated by a member of the nuclear factor of activated T-cells (NF-AT) transcription factor family, activator protein 1 (AP1) was identified as a cofactor at the TNF-alpha promoter while a GATA family member comprised the cofactor at the IL-5 promoter. This suggests that the differences in requirement for signal transduction cascades are the result of a different usage of NF-AT cofactors for transcription of each cytokine in mast cells.

Nrf1 in a complex with fosB, c-jun, junD and ATF2 forms the AP1 component at the TNF alpha promoter in stimulated mast cells.

Cap 'n' collar-basic leucine zipper (CNC-bZIP) proteins are widely implicated in developmental processes throughout different species. Evidence is accumulating that some of them are also participating in induced gene expression in the adult. Here we show that the three CNC-bZIP members NF-E2, Nrf1 and Nrf2 are constitutively expressed in the murine mast cell line CPII and that they form transcription factor complexes with several AP1 binding proteins. Upon induction, complexes are observed at the 2 x NF-E2 consensus binding site and the extended kappa3/AP1(+) site of the TNFalpha promoter. The interaction of Nrf1 with c -jun, junD, fosB and ATF2 in mast cells is in contrast to the recently reported binding of Nrf1 alone at the kappa3/AP1(-) site in dendritic cells. We speculated that this may be the result of the expression of isoforms of Nrf1 in mast cells. Using a PCR cloning strategy, we have isolated six novel splice variants of this transcription factor. Some of them have deleted the translational stop codon, resulting in an Nrf1 protein with an altered leucine zipper region. Expression of this altered binding/interaction domain interferes with TNFalpha induction, indicating an interaction of this splice variant with the active AP1/NF-AT complex at this promoter.

A novel splice variant of the transcription factor Nrf1 interacts with the TNFalpha promoter and stimulates transcription.

Common signaling chains of various receptor families, despite some similarities, are able to provoke quite different cellular responses. This suggests that they are linked to different cascades and transcription factors, dependent on the context of the ligand binding moiety and the cell type. The ITAM (immunoreceptor tyrosine-based activation motif) containing gamma chain of the FcepsilonRI, FcgammaRI, FcgammaRIII and the T-cell receptor is one of these shared signaling molecules. Here, we show that in the context of the FcgammaRIII, the gamma chain activates the transcription factor Nrf1 or a closely related protein that specifically interacts with the extended kappa3 site in the TNFalpha promoter. A novel splice variant of Nrf1 with a 411 bp deletion of the serine-rich region, resulting in an overall structure reminiscent of the BTB and CNC homology (Bach) proteins, was isolated from the corresponding DC18 cells. In a gel shift analysis, this bacterially expressed splice variant binds to the TNFalpha promoter site after in vitro phosphorylation by casein kinase II (CKII). In addition, cotransfection studies demonstrate that this splice variant mediates induced transcription at the TNFalpha promoter after stimulation/activation in a heterologous system.

Common and distinct signaling pathways mediate the induction of TNF-alpha and IL-5 in IgE plus antigen-stimulated mast cells.

A small number of signaling cascades represented by mitogen-activated protein kinases, phosphoinositol-3-kinase, protein kinase C, signal transducers and activators of transcription, Ca2+/calcineurin, and a few other molecules are linked to an incomparably large number of surface receptors. Parallel activation of several of these pathways and the existence of isozymes for a number of signal transmitting molecules generate the required complexity and specificity matching the receptor variety. Here we show that the proinflammatory mediator TNF-alpha and the growth factor IL-5 are activated along common and distinct signaling cascades in allergically stimulated murine mast cells. Both of them are dependent on Ca2+ influx, activation of calcineurin and nuclear factor of activated T cells as well as a member of the atypical PKC family, most likely PKCmu. Additionally, mitogen-activated protein kinases for TNF-alpha and members of the classical or nonclassical PKCs for IL-5, respectively, were identified as additional required pathways. Inhibition of the classical and nonclassical PKCs, however, does not abrogate IL-5 induction but instead leads to a switch to mitogen-activated protein kinases, which then become essential. The activated branches of this "salvage" signaling cascade are represented by extracellular signal-regulated kinase 1/2 and c-jun NH2 terminal kinase 1 in allergically stimulated mast cells.

Inhibition of Fc epsilon RI-mediated activation of mast cells by 2,3,4-trihydropyrimidino[2,1-a]isoquinolines.

Assays based on reporter gene technology represent today an important tool in the pharmaceutical industry for discovering novel compound classes interfering with the activation and signaling of target cells after stimulation. Here we describe a reporter gene assay targeting mast cell activation of IgE plus antigen, established in an attempt to identify substances preventing type I allergy (allergic rhinitis, allergic conjunctivitis, allergic asthma, and acute and chronic urticaria). The assay is based on a murine mast cell line designated CPII, stimulation by IgE plus antigen, and a reporter gene construct with the TNF alpha promoter linked to luciferase as a read-out system. Via screening about 50,000 substances, compound 2 was found to inhibit the reporter gene induction in the submicromolar range in this assay. Analogues of compound 2 of the 2,3,4-trihydropyrimidino[2,1-a]isoquinoline type were synthesized starting from 2-alkyl-substituted benzonitriles via aminolysis with 1,3-diaminopropane, dimetalation of 2-substituted 2-phenyl-1,4,5,6-tetrahydropyrimidines with n- and sec-butylithium, reaction with carboxylic acid methyl esters, and finally acidic dehydration. From about 50 derivatives, compound 41 was selected as a lead structure with an IC50 of 0.2 microM and a TC50 of 2.7 microM. In a first profiling in secondary assays, it effectively interfered with the production of mediators such as TNF alpha, IL-4, IL-6, IL-13, and leukotriene synthesis as measured by the corresponding ELISAs. In addition, a passive cutaneous anaphylaxis in mice (a typical type I reaction) is inhibited to more than 90% by compound 41, when administered intradermally 90 min before challenge.

Gene regulation after Fc epsilon RI stimulation in the murine mast cell line CPII.

BACKGROUND: Mast cells produce a number of lymphokines and chemokines upon Fc epsilonRI stimulation. However, signal cascades and transcription factors involved in the induction of the corresponding genes are still poorly understood. METHODS: We addressed this issue using transient transfections of a TNF alpha promoter-driven reporter gene and corresponding 5' successive deletions, the two phosphoinositol-3 kinase inhibitors demethoxyviridin and wortmannin, ELISAs and Western and Southwestern blots. RESULTS: Nuclear factor of activated T cells (NF-AT) and AP1 transcription factors together mediate the activation of TNF alpha transcription in mast cells upon IgE plus antigen stimulation which, in contrast to the degranulation reaction and leukotriene synthesis, is independent of phosphoinositol-3-kinase. CONCLUSIONS: TNF alpha regulation in mast cells provides an experimental system for direct comparison of the regulation of this cytokine in T cells. In the context of our recent findings on IL-5 gene regulation in mast cells, a picture emerges in which NF-AT, dependent on the cytokine and not on cell type, interacts with a specific cofactor (AP1 for TNF alpha, GATA for IL-5). Multiple NF-AT family members found to be expressed in mast cells provide the structural basis for these different interactions with cofactors. The insensitivity of TNF alpha gene activation and release to inhibitors of phosphoinositol-3 kinase demonstrates that the activation of NF-AT and/or AP1 transcription factors in mast cells is not triggered along this signaling cascade.

Effects of phosphatidylinositol-3-kinase inhibitors on degranulation and gene induction in allergically triggered mouse mast cells.

BACKGROUND: Phosphatidylinositol-3-kinase (PI3-kinase) comprises an essential component in a number of signaling cascades, primarily of the growth factor type. Two specific inhibitors, wortmannin and demethoxyviridin (DMV), are widely used to block signaling via this molecule and link certain receptors to the PI3-kinase pathway. METHODS: We have studied the extent of involvement of PI3-kinase in signaling events by Fc epsilonRI in mast cells using a mouse mast cell line as a model system. This was done using beta-hexosaminidase release assays, a leukotriene ELISA, transient transfections with reporter gene constructs of TNF alpha and MARC, and in addition a TNF alpha ELISA. RESULTS: Consistent with previously published data in the rat basophilic cell line RBL-2H3, we find that wortmannin as well as DMV prevent the degranulation reaction in the mouse mast cell line CPII. DMV also inhibits the release of leukotrienes, leading to the conclusion that Fc epsilonRI activates PI3-kinase which then mediates these reactions. On the contrary, however, lymphokine and chemokine induction at the gene and protein level is not inhibited, suggesting that the activation of this gene set in mast cells is independent of PI3-kinase. CONCLUSION: PI3-kinase is activated in our mast cell model system via cross-linking of the Fc epsilonRI. This reaction is clearly necessary for the degranulation process and the release of leukotrienes. Activation of lymphokine and chemokine genes as well as secretion of their gene products are not triggered along the PI3-kinase signaling pathway. This is in agreement with our previous findings, showing that the MAP kinase pathway and Ca2+ influx are both involved in gene activation in this cell type.

The murine homolog of TB2/DP1, a gene of the familial adenomatous polyposis (FAP) locus.

The cDNA of the murine counterpart of the human TB2/DP1 (deleted in polyposis) gene, one of the six genes deleted in severe cases of familial adenomatous polyposis (FAP) disease, was isolated and analyzed. The murine transcript is 734-bp long and thereby considerably shorter than the 3100-bp human counterpart. This is due to a completely different 3' untranslated region in mouse which starts immediately after the translational stop codon, thereby deleting a RFLP (restriction-fragment length polymorphism) marker for this disease. The amino acid sequence, however, is 92% conserved between mouse and man. Triggering of murine mast cells by IgE plus antigen results in a decrease of TB2/DP1 mRNA up to 60% after 2 h implying a possible role of this gene in regulation of the allergic effector cell. Reverse transcription-polymerase chain reaction (RT-PCR) analysis shows an ubiquitous expression pattern in a number of mouse cell lines and tissues.

p21ras links Fc epsilon RI to NF-AT family member in mast cells. The AP3-like factor in this cell type is an NF-AT family member.

Very recently, an AP3-like transcription factor regulating the chemokine gene MARC and an NF-AT family member regulating IL-5 were the first components of the transcription factor repertoire to be described as activated in mast cells after an allergic triggering. In this study, we show that with respect to cross-competition in a gel shift analysis using an NF-AT consensus oligonucleotide binding site, the antigenicity to a recently generated serum against T cell NF-AT, and the sensitivity to macrolide immunosuppressants, the AP3-like activity on the MARC promoter is indistinguishable from that described for NF-AT in T cells. Additionally, we show that this factor functions on the MARC chemokine promoter without the AP1 cofactor, a situation reminiscent of the function of NF-AT in Th2-type T cells. In all of these aspects, and strengthened further by a gel shift competition analysis, the AP3-like transcription factor is identical to the NF-AT family member recently described by an analogous set of experiments as regulating IL-5 in mast cells. Our finding that p21ras, but probably not protein kinase C, is necessary to activate this factor after Fc epsilon RI triggering indicates a situation in which a common transcription factor denominator in mast cells induces chemokine (MARC) and lymphokine (IL-5) gene expression in a manner closely similar to Th2-type T cells, which are induced along the ras/raf signal pathway.

The nomenclature of chemokines.

Migration of leukocytes to injured tissues is a hallmark of inflammation. The recruitment phase of cells can be subdivided into three steps: the rolling phase, the firm adhesion phase, and the transendothelial migration phase. Each step is mediated by a complex interplay of endothelial/leukocyte surface molecule interactions (mostly of selectin and integrin families) as well as a group of small, secreted peptides, called chemokines. Chemokines activate on the one hand, the leukocytes to express the appropriate adhesion molecules and on the other hand, they lead to transendothelial migration via chemotaxis (migration along a gradient in solution) and haptotaxis (migration along a gradient bound to extracellular martrices or cell membranes). The structure, biology and pathobiology of the more than 22 known members of this group of soluble mediators, with a particular emphasis on their past and present nomenclature, is the topic of this minireview.

A nuclear factor of activated T cell-like transcription factor in mast cells is involved in IL-5 gene regulation after IgE plus antigen stimulation.

IL-5, which is produced mainly by activated T cells and allergically triggered mast cells, is a major survival and differentiation factor for eosinophils, and therefore, is of relevance to diseases associated with this type of cell infiltration, most importantly asthma. In this study, we have examined the transcriptional regulation of human IL-5 in a mouse mast cell line, CPII, stimulated with IgE and Ag. We report that an inducible activity in the region between -177 and -80, and a constitutive activity between -80 and -70, in the promoter of the human gene, are both necessary for the allergically triggered activation. A computer-assisted search for transcription factor binding motifs revealed a nuclear factor of activated T cell (NF-AT) and a GATA consensus site in the two regions. Corresponding binding activities were detected to be present in nuclear extracts from the mouse mast cell line by defined NF-AT and GATA binding sites as probes for a gel shift analysis. Competition analysis, in combination with probes from the human IL-5 promoter, confirmed that these factors indeed bind to the consensus sequences identified by computer analysis. An oligonucleotide spanning the IL-5 NF-AT consensus site is shown to confer allergic stimulation to a basal IL-5 promoter only in conjunction with the GATA site downstream, indicating that an inducible NF-AT-like factor cooperates with a constitutive member of the GATA transcription factor family in mediating the allergic stimulation of the human IL-5 gene.

A novel transiently expressed, integral membrane protein linked to cell activation. Molecular cloning via the rapid degradation signal AUUUA.

A novel cDNA clone termed E16 which codes for an integral membrane protein of 241 amino acids with six transmembrane domains was isolated from peripheral blood lymphocytes. The cDNA clone is 4000 base pairs in length and exhibits an unusually long 3'-untranslated region of about 3000 nucleotides. Its expression at the mRNA level is closely linked to cellular activation and division. In all myeloid and lymphoid cells, as well as in primary lymphocytes from peripheral blood, E16 transcripts are rapidly induced and rapidly degraded after stimulation. This pattern of expression is unusual for an integral membrane protein and resembles more closely the kinetic seen for protooncogenes and lymphokines in the T cell system. Its isolation was made possible by a novel approach especially designed to selectively clone cDNAs which exhibit such an expression kinetic. It is based on a combination of the differential screening of a subtracted cDNA library and the subsequent hybridization of the resulting phages to a short oligonucleotide (5'-TAAATAAA-TAAATA-3'). This oligonucleotide is complementary to a trimer of the rapid degradation signal (AUUUA) which is present as a single or reiterated motif in the 3'-untranslated region of many short-lived transcripts.