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H Stockinger

Publications and source records attributed to H Stockinger.

At least 19 recordsLinked to original sources
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

Induction of hyporesponsiveness and impaired T lymphocyte activation by the CD31 receptor:ligand pathway in T cells.

CD31 is a member of the Ig superfamily expressed on various cell types of the vasculature, including a certain subpopulation of T lymphocytes. Previous reports suggest that interaction of CD31 with its heterophilic ligand on T cells (T cell CD31 ligand) plays a regulatory role in T lymphocyte activation. Here we demonstrate that a soluble rCD31-receptorglobulin (CD31Rg) specifically down-regulated the proliferation of human peripheral blood CD31(-) T lymphocytes stimulated via CD3 and CD28 mAbs. Notably, engagement of the T cell CD31 ligand by CD31Rg during primary stimulation also induced a prolonged unresponsive state in T cells. Retroviral transduction of CD31 into CD31(-) Th clones resulted in a significant inhibition of their proliferative capacity. When cocultured with purified CD31(-) T lymphocytes, irradiated CD31-transduced Th clones counterregulated the CD3/CD28-mediated activation of these cells. Furthermore, primary stimulation in the presence of CD31-transduced Th clones induced a comparable state of hyporesponsiveness in the T cell responders as the soluble CD31Rg. Thus, by counterregulating the activation of cognate T lymphocytes, CD31-expressing T cells might contribute to the establishment and maintenance of peripheral tolerance.

Abatacept↗
PubMed2001

Platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin cooperatively regulate fibroblast growth factor-induced modulations of adherens junction functions.

Cellular adherens junctions are formed by cadherins linked to proteins of the catenin family. In endothelial cells, not only vascular endothelial cadherin but also platelet endothelial cell adhesion molecule-1 localizes into junctions and associates with beta-catenin. To explore a putative cooperation of platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin, we analyzed transfectants expressing either platelet endothelial cell adhesion (CD31 cells) or vascular endothelial cadherin (CD144 cells) or both molecules (CD31/CD144 cells), and, for comparison, human umbilical vein endothelial cells. Basic fibroblast growth factor completely dissociated vascular endothelial cadherin/beta-catenin complexes and robustly moved beta-catenin into the nucleus in CD144 cells, whereas in CD31/CD144 cells as well as in human umbilical vein endothelial cells, fibroblast growth factor only partially dissociated the junctional complex followed by a significantly reduced nuclear translocation of beta-catenin. In contrast, in CD31 cells, the subcellular distribution of beta-catenin remained unaffected by fibroblast growth factor. As a functional consequence, fibroblast growth factor induced a complete collapse of the F-actin network in CD144 cells, a limited rearrangement of F-actin fibers in CD31/CD144 cells and no F-actin rearrangement in CD31 cells. We also analyzed the effect of fibroblast growth factor-induced rearrangement of junctions on junction permeability for leukocytes: in line with our observation that vascular endothelial cadherin was required for cells to respond to fibroblast growth factor, only in CD31/CD144 cells, but not in CD31 cells, leukocyte transmigration was significantly enhanced by fibroblast growth factor. In conclusion platelet endothelial cell adhesion molecule-1 cooperates with vascular endothelial cadherin in a mutual fashion; platelet endothelial cell adhesion molecule-1 reduces and temporarily limits fibroblast growth factor-induced dissociation of vascular endothelial cadherin/beta-catenin complexes, but requires vascular endothelial cadherin to control leukocyte transmigration in dependence of fibroblast growth factor.

Adherens Junctions↗
PubMed2000

Suppression of primary T-cell responses and induction of alloantigen-specific hyporesponsiveness in vitro by the Janus kinase inhibitor tyrphostin AG490.

BACKGROUND: Tyrphostin AG490 has recently been shown to block interleukin (IL)-2 receptor gamma-chain-associated Janus kinase 3. Here, we analyzed the effect of AG490 on T-cell alloresponses in vitro. METHODS: For the evaluation of T-cell activation, DNA synthesis, surface marker expression, cytokine secretion, intracellular calcium mobilization, early protein tyrosine phosphorylation, and apoptosis were measured. RESULTS: AG490 effectively inhibited T-cell proliferation in human mixed lymphocyte culture (MLC) even when added 4 days after culture initiation. Inhibition of IL-2-dependent proliferation in T-cell blasts and the incapability of IL-2 or IL-15 to restore proliferation in AG490-treated MLC suggests interference with cytokine receptor signaling. T-cell receptor-triggered early protein tyrosine phosphorylation, calcium mobilization, up-regulation of CD69, and initial CD25 expression were not affected. Interestingly, AG490 substantially inhibited production of IL-2 and interferon-gamma in T cells stimulated with alloantigen or via CD3 and CD28. In CD28-independent activation models (e.g., stimulation with phorbol myristate acetate plus ionomycin), however, cytokine secretion was not inhibited. Pretreatment of primary MLC with AG490 resulted in substantial down-regulation of secondary responses to cells from the original donor as opposed to third-party cells or phytohemagglutinin. Unresponsiveness was induced also in T cells stimulated with CD3 monoclonal antibody. Induction of apoptosis in polyclonally activated T cells and the incapability of IL-2 to reverse specific hyporesponsiveness, suggest programmed cell death as an important mechanism underlying antigen-specific down-regulation of alloresponses. CONCLUSIONS: We demonstrate that AG490 blocks different manifestations of T-cell activation. This and its ability to induce alloantigen-specific hyporesponsiveness point to a potential use for interfering with alloreactivities in vivo.

CD3 Complex↗
PubMed2000

A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain.

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

Allergens↗
PubMed2000

CD99 monoclonal antibody induce homotypic adhesion of Jurkat cells through protein tyrosine kinase and protein kinase C-dependent pathway.

CD99 is a 32 kDa cell surface glycoprotein which is involved in cell adhesion. Engagement of the CD99 molecule by CD99 monoclonal antibodies has been shown to induce homotypic aggregation of various cell types. By using a newly established CD99 monoclonal antibody, MT99/3, we show here that LFA-1/ICAM-1 independent cell adhesion pathways are activated via CD99. Engagement of the CD99 molecule by MT99/3 induced homotypic cell aggregation of Jurkat T-cells within 30 min reaching its maximal level within 4 h. The Jurkat cell aggregation was not blocked by addition of CD11a (LFA-1) and CD54 (ICAM-1) mAbs. Furthermore, MT99/3 treatment did not alter the expression of LFA-1 and ICAM-1 molecules. Induction of Jurkat homotypic aggregation by MT99/3 was, however blocked by the protein kinase C inhibitor, sphingosine, the protein tyrosine kinase inhibitor, genistein, and by actin filament polymerization blocking agent, cytochalasin B. Thus, these observations suggest that CD99 can mediate beta2-integrin independent cell adhesion that depends on activation of protein kinases and reorganization of the cytoskeleton.

12E7 Antigen↗
PubMed1999

Activation of nuclear factor-kappaB by lipopolysaccharide in mononuclear leukocytes is prevented by inhibitors of cytosolic phospholipase A2.

In monocytes, lipopolysaccharide induces synthesis and activity of the 85-kDa cytosolic phospholipase A2. This enzyme releases arachidonic acid and lyso-phospholipids from membranes which are metabolized to eicosanoids and platelet-activating-factor. These lipid mediators increase activity of transcription factors and expression of cytokine genes indicating a function for cytosolic phospholipase A2 in signal transduction and inflammation. We have shown previously that trifluoromethylketone inhibitors of cytosolic phospholipase A2 suppressed interleukin-1beta protein and steady-state mRNA levels in human lipopolysaccharide-stimulated peripheral blood mononuclear leukocytes. In this study, the subcellular mechanisms were analyzed by which trifluoromethylketones interfere with gene expression. We found that they reduced the initial interleukin-1beta mRNA transcription rate through prevention of degradation of inhibitor-kappaB alpha. Consequently, cytosolic activation, nuclear translocation and DNA-binding of nuclear factor-kappaB were decreased. Trifluoromethylketones ameliorate chronic inflammation in vivo. Thus, this therapeutic potency may reside in retention of inactive nuclear factor-kappaB in the cytosol thereby abrogating interleukin-1beta gene transcription.

Animals↗
PubMed1999

M6P/IGFII-receptor complexes urokinase receptor and plasminogen for activation of transforming growth factor-beta1.

Transforming growth factor-beta1 (TGF-beta1) is a critical cytokine for cell proliferation and differentiation. It is secreted by many cells in a latent pro-form (LTGF-beta1) from which biologically active TGF-beta1 is released by an in vivo mechanism that is not known. Here we show that the mannose-6-phosphate/insulin-like growth factor II-receptor (M6P/IGFII-R), which binds LTGF-beta1, complexes with urokinase (plasminogen activator)-receptor (uPA-R) on the surface of human monocytes and directly binds plasminogen (Plg). Plasmin generated from Plg in the complex mediates release of TGF-beta1 when M6P/IGFII-R is associated with uPA-R. Thus, this interaction of M6P/IGFII-R and uPA-R suggests a potential mechanism for the generation of TGF-beta1 by cells.

Amino Acid Sequence↗
PubMed1999

GPI-microdomains: a role in signalling via immunoreceptors.

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids are assembled on the leukocyte surface within membrane microdomains, which also accommodate a set of cytoplasmic signalling molecules (Src family kinases, G-proteins, linker proteins). Recent results suggest that these membrane specializations mediate not only signal transduction via GPI-proteins and glycolipids but also play important roles in initiation of signalling via immunoreceptors.

Animals↗
PubMed1999

Analysis of the early biogenesis of CD1b: involvement of the chaperones calnexin and calreticulin, the proteasome and beta(2)-microglobulin.

beta(2)-Microglobulin (beta(2)m)-associated human CD1b proteins present lipid and glycolipid antigens, which are loaded on CD1b in endosomal compartments. In contrast, the related MHC class I molecules acquire antigenic peptides in the endoplasmic reticulum. Here, we investigated the biogenesis of CD1b before beta(2)m binding in comparison to MHC class I. In beta(2)m-deficient FO-1 cells, we found CD1b heavy chains (HC) complexed with the chaperones calnexin and calreticulin, while MHC class I HC associated only with calnexin. Despite this difference, both CD1b HC and MHC class I HC were degraded when the chaperone interactions were prevented by the glucosidase inhibitor castanospermine. The degradation of both molecules included the proteasome and mannosidases. Chaperone-unassociated CD1b could be rescued from degradation by supplementing FO-1 cells with beta(2)m. Finally, prevention of chaperone interaction significantly reduced neoexpression of CD1b upon differentiation of monocytes to dendritic cells, underlining the importance of chaperones for proper expression of CD1b under physiological conditions.

Antigens, CD↗
PubMed1999

T cell activation-associated epitopes of CD147 in regulation of the T cell response, and their definition by antibody affinity and antigen density.

CD147 is a broadly expressed cell surface glycoprotein of the Ig superfamily whose expression is up-regulated upon T cell activation. In order to elucidate a possible role of CD147 in T cell biology, we established 15 specific mAb. Seven distinct epitopes were defined by the mAb panel. Most of the mAb bound only to phytohemagglutinin (PHA)-activated but not resting T cells. We demonstrate that this was not because of true expression of activation-dependent neoepitopes but rather due to bivalent binding of the relatively low-affinity mAb (affinity constant KA values between 2.25 x 10(8) and 7 x 10(9) M-1) to the more densely expressed and/or more clustered CD147 molecules on the activated T cells. In contrast, the mAb with higher affinity (KA > 7 x 10(9) M-1) could stably bind in a monovalent fashion even to the relatively low dense CD147 molecules on resting T cells. This model might more generally explain the nature of 'activation epitopes' described previously in other leukocyte surface molecules. Finally, we provide evidence that induction of ordered dimerization of CD147 by a mAb directed to a unique epitope results in strong inhibition of CD3-mediated T cell activation.

Animals↗
PubMed1999

Vascular-endothelial cadherin (CD144)- but not PECAM-1 (CD31)-based cell-to-cell contacts convey the maintenance of a quiescent endothelial monolayer.

BACKGROUND: In vivo, all blood vessels are lined by a single layer of flattened noncycling endothelial cells. We tested the hypothesis that the maintenance of such a quiescent endothelial monolayer depends on homotypic contacts between not yet defined growth-inhibitory molecules located at interendothelial junctions. METHODS: ECV304 cells, which lack endogenous vascular endothelial cadherin (VE cadherin) or CD31 expression, were transfected with cDNA encoding for the respective proteins or with the empty vector. RESULTS: In VE cadherin transfectants, beta-catenin was targeted to junctional regions and the F-actin-based cytoskeleton formed parallel bundles reaching from one cell border to the other. In contrast, in CD31 transfectants and in empty vector cells, beta-catenin was dispersed throughout the cytoplasm, and F-actin formed short, plump and criss-cross bundles. On a two-dimensional plastic matrix, both, VE cadherin and CD31 transfectants formed clusters of polygonal cells, whereas in three-dimensional gels, only VE cadherin cells were able to form tubes. Empty vector cells grew in a fibroblast-like pattern and neither formed clusters nor tubes. Most importantly, whereas CD31 and empty vector cells grew on top of each other, formed polylayers and maintained cycling even after reaching confluence, VE cadherin cells strictly maintained a single layer of flattened cells and the numbers of cycling cells dramatically dropped after reaching a continuous monolayer. CONCLUSION: The insertion of VE cadherin into ECV304 cells produces a cell type which mimics endothelial growth characteristics seen in vivo.

Antigens, CD↗
PubMed1998

Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains.

Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects, but the molecular alterations leading to T cell inhibition are not yet elucidated. Signal transduction seems to involve detergent-resistant membrane domains (DRMs) acting as functional rafts within the plasma membrane bilayer with Src family protein tyrosine kinases being attached to their cytoplasmic leaflet. Since DRMs include predominantly saturated fatty acyl moieties, we investigated whether PUFAs could affect T cell signaling by remodeling of DRMs. Jurkat T cells cultured in PUFA-supplemented medium showed a markedly diminished calcium response when stimulated via the transmembrane CD3 complex or glycosyl phosphatidylinositol (GPI)- anchored CD59. Immunofluorescence studies indicated that CD59 but not Src family protein tyrosine kinase Lck remained in a punctate pattern after PUFA enrichment. Analysis of DRMs revealed a marked displacement of Src family kinases (Lck, Fyn) from DRMs derived from PUFA-enriched T cells compared with controls, and the presence of Lck in DRMs strictly correlated with calcium signaling. In contrast, GPI-anchored proteins (CD59, CD48) and ganglioside GM1, both residing in the outer membrane leaflet, remained in the DRM fraction. In conclusion, PUFA enrichment selectively modifies the cytoplasmic layer of DRMs and this alteration could underlie the inhibition of T cell signal transduction by PUFAs.

Binding Sites↗
PubMed1998

CD38 binding to human myeloid cells is mediated by mouse and human CD31.

Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukocyte trafficking. In the present study, we found that the soluble form of the CD38 antigen (sCD38) bears a binding domain of low affinity for a cellular receptor on U937 cells. Cross-linking and peptide-mapping studies confirmed the physical association and the identification of the U937 receptor as a 130 kDa protein. The binding of sCD38 to the receptor was differentially inhibited by several monoclonal antibodies against the CD31 cell-adhesion molecule. Thus the interaction was analysed through direct association of soluble and membrane CD38 with soluble recombinant murine CD31 with three N-terminal and with all six extracellular Ig domains. Cross-linking experiments on U937 intact cells, and ligand blot assays of the immunoprecipitated CD38 molecule, indicated that (i) the recognized epitope is determined by the tertiary structure of the molecule, and that (ii) the binding domain involved resides in the ectocellular portion of the CD31 molecule, more precisely in the first three N-terminal domains. A comparative functional activity between murine and human CD31 was also explored. The data presented suggest that (i) human CD31 bears a highly functional similarity with its murine counterpart, as it is a receptor in myeloid cells with more than one ligand (the alphavbeta3 integrin and the CD38 molecule), and that (ii) the activity of sCD38 as decoy molecule for CD31 may play an important role in cell-cell interactions in physiological and pathological conditions.

ADP-ribosyl Cyclase↗
PubMed1998

Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member.

Human CD38 is a cell surface molecule involved in the regulation of lymphocyte adhesion to endothelial cells. This suggests that HUVEC bear a ligand(s) for CD38 on the cell surface. By means of the mAb Moon-1, which specifically inhibits CD38-mediated cell adhesion, we have identified a trans-membrane 130-kDa molecule acting as a ligand for CD38. Here, we report that the molecule recognized by the Moon-1 mAb is CD31, a member of the Ig superfamily. This conclusion is based on 1) cross-inhibition assays between Moon-1 and reference anti-CD31 mAbs; 2) sequential immunoprecipitation experiments using Moon-1 and known anti-CD31 mAbs, and 3) reactivity of the Moon-1 mAb with CD31 transfectants. Further, CD31 and CD38 cognate interactions were found to modulate heterotypic adhesion as well as to implement cytoplasmic calcium fluxes identical to those obtained by means of agonistic anti-CD38 mAbs. Other effects tested included the synthesis of messages for a panel of cytokines, markedly increased upon receptor-ligand interactions. These results suggest that the interplay between CD38 and its ligand CD31 is an important step in the regulation of cell life and of the migration of leukocytes (and CD38+ cancer cells) through the endothelial cell wall.

ADP-ribosyl Cyclase↗
PubMed1998

New mechanisms by which secretory phospholipase A2 stimulates neutrophils to provoke the release of cytotoxic agents.

BACKGROUND: Secretory phospholipase A2 (sPLA2) is a potent proinflammatory enzyme that stimulates inflammation through the production of reactive lipids. However, enzymatic inhibitors have been disappointing in their effectiveness in halting hyperinflammation. OBJECTIVE: To determine whether sPLA2 acts directly on neutrophil plasma membrane lipids or via a nonenzymatic mechanism. DESIGN: Isolated neutrophils (PMNs) were incubated with 3 types of sPLA2, and elastase and superoxide release from PMNs was measured. Ethyleneglycotetraacetic acid was used as a selective enzymatic inhibitor. The PMNs were exposed to sPLA2 in the presence and absence of ethyleneglycotetraacetic acid and the release of elastase was measured. SETTING: Urban trauma research laboratory. PATIENTS: Normal healthy donors of PMNs. MAIN OUTCOME MEASURES: Stimulated release of superoxide and elastase. RESULTS: The sPLA2 acted directly on plasma membrane lipids to stimulate the PMN to produce superoxide and release elastase. This mechanism is blocked with enzymatic inhibition of sPLA2. The sPLA2 also provokes elastase release from PMNs independently of its enzymatic function. This mechanism is not blocked with traditional enzymatic inhibitors. CONCLUSIONS: These data indicate that the sPLA2 can act directly on PMNs to stimulate the release of inflammatory mediators via enzymatic degradation of plasma membrane lipids. In addition, sPLA2 can act as a ligand and stimulate the PMN independently of its enzymatic activity.

Animals↗