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Biomedical subjects

A Trautmann

Publications and source records attributed to A Trautmann.

At least 19 recordsLinked to original sources
PubMed1997

Reconstituted killer cell inhibitory receptors for major histocompatibility complex class I molecules control mast cell activation induced via immunoreceptor tyrosine-based activation motifs.

Natural killer and T cells express at their surface, members of a multigenic family of killer cell inhibitory receptors (KIR) for major histocompatibility complex Class I molecules. KIR engagement leads to the inhibition of natural killer and T cell activation programs. We investigated here the functional reconstitution of KIR in a non-lymphoid cell type. Using stable transfection in the RBL-2H3 mast cell line, we demonstrated that (i) KIR can inhibit signals induced by FcepsilonRIgamma or CD3zeta polypeptides that bear immunoreceptor tyrosine-based activation motifs; (ii) two distinct immunoreceptor tyrosine-based inhibition motifs-bearing receptors, i.e. KIR and FcgammaRIIB, use distinct inhibitory pathways since KIR engagement inhibits the intracellular Ca2+ release from endoplasmic reticulum stores, in contrast to FcgammaRIIB, which only inhibits extracellular Ca2+ entry; (iii) KIR require co-ligation with an immunoreceptor tyrosine-based activation motif-dependent receptor to mediate their inhibitory function. This latter finding is central to the mechanism by which KIR selectively inhibit only the activatory receptors in close vicinity. Taken together our observations also contribute to define and extend the family of immunoreceptor tyrosine-based inhibition motif-bearing receptors involved in the negative control of cell activation.

Animals↗
PubMed1997

Heterotypic cell-cell adhesion of human mast cells to fibroblasts.

Skin mast cells are typically located in the perivascular or perineural connective tissue. We observed that HMC-1 mast cells growing in suspension adhered efficiently to (> 90% of cells) and spread on top of fibroblast monolayers and to a lesser degree on purified extracellular matrix proteins. Since adhesive interactions determine cell migration and tissue localization we studied the mechanism. It was found that HMC-1 cells attach to collagen I and fibronectin, laminin, collagen IV and vitronectin, but not to collagens III and VI or hyaluronic acid. Adhesion to fibronectin, collagen I and laminin was completely inhibited by mAbs blocking beta 1-integrins, whereas adhesion of HMC-1 cells to vitronectin was inhibited by anti-alpha v-chain mAbs. However, attachment of HMC-1 cells to fibroblasts was not influenced by mAbs blocking beta 1- or alpha v-chain function, by RGD peptides or by mAbs interfering with other receptors, most notably c-kit. Identical results were obtained with normal mast cells isolated from human foreskin. These results indicate that human mast cells attach to fibroblasts independently of beta 1- or alpha v-integrins as well as of c-kit receptor-mediated mechanisms. The functional characteristics observed (i.e. only partial sensitivity to trypsin and EDTA, no increase in trypsin sensitivity by pretreatment with EDTA) suggest that cadherin receptors were not involved, and it is likely that the adhesion process observed involved not-yet-defined heterotypic cell-cell adhesion receptors.

Antibodies, Monoclonal↗
PubMed1996

The CD8 beta polypeptide is required for the recognition of an altered peptide ligand as an agonist.

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 alpha and beta chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8beta in the CD8 co-receptor dimers expressed at the T cell surface.

Animals↗
PubMed1995

Rapid refilling of Ca2+ stores in macrophages stimulated by ATP involves the sequential activation of phospholipase D and protein kinase C.

Ca2+ movements between intracellular stores, the cytoplasm and external solution were analysed in murine peritoneal macrophages stimulated by various agonists. The Ca2+ content of intracellular stores was estimated from the amplitude of Ca(2+)-transients elicited by ionomycin applied in Ca(2+)-free solution. Both uridine 5'-triphosphate (UTP) and platelet-activating factor (PAF) triggered the release of Ca2+ followed by a sustained influx, during which intracellular stores remained totally empty. In contrast, in the continuous presence of adenosine 5'-triphosphate (ATP), Ca2+ was initially released and then rapidly sequestered again by the stores. ATP-induced store refilling was not related to cell depolarization or to an increase in the intracellular Na+ concentration (two specific consequences of ATP stimulation which are not induced by PAF and UTP). Store refilling was not caused by a signal that ATP would fail to induce (e.g. as a result of receptor desensitization), but was positively controlled by ATP, even in the simultaneous presence of a concentration of PAF which, on its own, would have caused a persistent store depletion. The hypothesis that the signal delivered by ATP involves the sequential activation of phospholipase D and protein kinase C is consistent with the present pharmacological evidence. However, although we found conditions in which Ca2+ stores did not refill in the presence of ATP, this maintained store depletion was not accompanied by a sustained Ca2+ response similar to that elicited by PAF or UTP, suggesting that store depletion is a condition which is necessary, but not sufficient, for inducing Ca2+ influx.

Adenosine Triphosphate↗
PubMed1995

The intracellular Ca2+ concentration optimal for T cell activation is quite different after ionomycin or CD3 stimulation.

The relationship between the initial increase of intracellular Ca2+ concentration ([Ca2+]i) (measured at the single-cell level with an imaging system) and the ensuing proliferation was examined in a human T cell clone stimulated by a phorbol ester in combination with ionomycin, thapsigargin or an anti-CD3 mAb (monoclonal antibody against the CD3 molecule, UCHT1). From the responses to various ionomycin concentrations, one can define a range of [Ca2+]i values (400-900 nM) which appears optimal for T cell proliferation; lower [Ca2+]i values are suboptimal, higher values are cytotoxic. It was then examined if the [Ca2+]i requirements were similar following anti-CD3 stimulation. [Ca2+]i oscillations elicited by a concentration of UCHT1 (1/1,000) optimal for mitogenicity fall precisely within the 400-900 nM range. However, very low concentrations of UCHT1 (1/100,000) which evoke barely detectable [Ca2+]i responses still cause the cells to proliferate. The possibility that the lower [Ca2+]i requirements observed following anti-CD3 stimulation was due to [Ca2+]i oscillations was tested under conditions which prevented the appearance of these oscillations. It turns out that an oscillatory Ca2+ signal is not more mitogenic than a sustained augmentation of [Ca2+]i. Finally, it was examined if overstimulation via CD3 could have toxic consequences similar to those elicited after ionomycin overstimulation. Large transient [Ca2+]i responses can be observed following anti-CD3 stimulation in appropriate conditions, and namely in T cells pretreated with interleukin-2. These [Ca2+]i augmentations are not cytotoxic. A role for the plasmalemmal Ca2+ pump in the prevention of cytotoxicity can be demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1994

Reconstitution of CD3 zeta coupling to calcium mobilization via genetic complementation.

The integrity of the T cell receptor complex (CD3-TCR) transduction machinery is central to T cell development and to T cell effector function. Molecular dissection of the multimeric CD3-TCR complex revealed that at least two associated polypeptides, CD3 zeta and CD3 epsilon, autonomously couple antigenic recognition event to early and late events of the intracytoplasmic activation cascade. A 18-amino acid motif based on a tandem YXXL stretch, the activation receptor homology sequence 1 (ARH-1) motif, is necessary and sufficient to the transducing properties of both CD3 zeta and CD3 epsilon. Stimulation of chimeric molecules made of ecto- and transmembrane domains of various cell surface proteins and intracytoplasmic domains of CD3 epsilon or CD3 zeta leads to an increase in the intracellular Ca2+ concentration ([Ca2+]i) in Jurkat cells. We describe here that a similar CD25/zeta chimeric molecule was unable to induce a detectable [Ca2+]i rise upon CD25 cross-linking once expressed in the murine thymoma BW-. A Ca2+ influx could, however, be triggered in BW- cells by thapsigargin, i.e. following depletion of Ca2+ stores. Somatic cell hybrids made from BW- and either thymocytes or mature lymph node T cells reconstituted the coupling of CD3 zeta to the Ca2+ signal via an ARH-1 motif-dependent pathway. However, pervanadate-induced Ca2+ mobilization, a phenomenon attributed to tyrosine phosphorylation, was impaired in BW-cells and reconstituted in hybridomas. In contrast to the Ca2+ response, IL-2 production was induced in both BW- and hybrids cells, which questions the functional relevance of [Ca2+]i augmentation in T cell activation. In conclusion, the properties of the BW- thymoma, which define a novel group of CD3 zeta transduction cell mutants, as well as its complementation by somatic cell fusion demonstrate that this cell line represents a useful model to dissect the signaling pathway that couples CD3 zeta to Ca2+ mobilization by genetic reconstitution.

Animals↗
PubMed1994

Antigen recognition by helper T cells elicits a sequence of distinct changes of their shape and intracellular calcium.

BACKGROUND: Helper T-cell activation is initiated in vivo when the T-cell receptor complex recognizes an antigen fragment associated with MHC class II molecules on the surface of an antigen-presenting cell. In most previous studies of this phenomenon, T cells were stimulated not with antigen-presenting cells, but with CD3-specific antibodies. This approach provided considerable understanding of the cascade of molecular events triggered by T-cell receptor stimulation. However, the specific consequences of cell-cell interactions are still poorly understood. We therefore used a dual imaging system that provides simultaneous transmission and fluorescence images to study the morphological changes and variations of intracellular calcium concentration ([Ca2+]i) triggered in a human CD4+ antigen-specific T-cell clone in response to antigen presented by a class II-transfected murine fibroblast. RESULTS: T cells loaded with the Ca(2+)-sensitive fluorescent dye Fura-2 were individually monitored for half an hour following their contact with a monolayer of antigen-pulsed antigen-presenting cells. The response was found to have three distinct phases. During the first few minutes after contact, the T cell moves over the antigen-presenting cells, as if 'scanning' them. After several minutes, an oscillating [Ca2+]i response begins, accompanied by the immobilization of the cell and the retraction of pseudopodia. This rounding-up was probably Ca(2+)-dependent, as it could also be triggered by ionomycin or thapsigargin. Later during the [Ca2+]i response, the T cell becomes flattened and further elongated, suggesting increased adhesion to antigen-presenting cells. CONCLUSIONS: The physiological signal for T-cell activation, antigen recognition, is a three-step process reminiscent of the three steps previously observed in the interaction between neutrophils and endothelial cells. During these successive steps, a mobile, weakly interacting T cell is transformed into an immobile cell fully engaged in the activation pathway. Thus, antigenic recognition is not instantaneous, but evolves slowly by progressive amplification of the signal given by a few antigen molecules, eventually resulting in T-cell activation.

Animals↗
PubMed1993

Calcium responses elicited by nucleotides in macrophages. Interaction between two receptor subtypes.

The responses elicited by ATP and UTP in macrophages (measured by microfluorescence and in patch-clamp) present marked differences. The release of Ca2+ from intracellular stores induced by ATP is due to the activation of P2U receptors. These receptors can be activated by ATP4- and by MgATP2-, with apparent K0.5 values of 0.65 and 6.5 microM, respectively. The release of Ca2+ due to activation of P2U receptors by either ATP or UTP is followed by the opening of ionic channels leading to an influx of Ca2+. A second pathway for Ca2+ influx results from the opening of P2Z receptor channels triggered by adenosine-5'-O(1-thiotriphosphate) or ATP but not by UTP. The form of ATP that activates P2Z receptors is ATP4- (with a K0.5 of 0.5 microM). In voltage-clamped cells, the inward current activated by ATP4- is transient, partly because it inactivates and partly because it is rapidly masked by the development of a quinine-sensitive Ca(2+)-dependent K+ current. In current-clamp, macrophages stimulated by UTP remain normally polarized, whereas ATP depolarizes them. This P2Z-mediated depolarization results in an inhibition of the influx of Ca2+, which explains part of the difference between the time courses of the Ca2+ responses elicited by ATP and UTP.

Adenosine Triphosphate↗
PubMed1993

Is there a Na+/Ca2+ exchanger in macrophages and in lymphocytes?

In two blood cell types, peritoneal murine macrophages and Jurkat cells (a human T cell line), we have examined whether a Na+/Ca2+ exchange was present and what could be its functional importance. In non-stimulated macrophages, the intracellular Ca2+ concentration, [Ca2+]i, was unchanged when Li+ was substituted for external Na+. However, after stimulation by platelet-activating factor (PAF), the Ca2+ response was larger when the extracellular solution contained Li+ rather than Na+ ions. In stimulated macrophages, the rate of Ca2+ extrusion was smaller in a Li(+)- than in a Na(+)-containing medium. The net electrochemical gradient for ionic movements through the Na+/Ca2+ exchanger, during the course of the response of macrophages to PAF, was determined by combining the measurements of membrane potential (in patch-clamp), of [Ca2+]i (with fura-2), and of the intracellular Na+ concentration (with sodium-binding benzofuran isophthalate). These results show that macrophages possess a Na+/Ca2+ exchange that only functions as a Ca2+ extruder, and this only when [Ca2+]i has been increased, for instance following PAF stimulation. In T lymphocytes, before or after stimulation by an anti-CD3 antibody, no Na+/Ca2+ activity could be detected by measuring either [Ca2+]i, or the rate of Ca2+ extrusion. Even if a Na+/Ca2+ exchanger was present in these cells, its equilibrium potential would be such that it would not allow Ca2+ influx but only Ca2+ extrusion.

Animals↗
PubMed1993

Calcium imaging.

We review herein the following questions related to Ca2+ imaging: what are the key elements to consider in a Ca2+ imaging system? What are the limits of such systems? What are the main artifacts and to what specific applications are a Ca2+ imaging systems best adapted?

Artifacts↗
PubMed1992

Calcium fluxes in T lymphocytes.

Mechanisms controlling Ca2+ fluxes through the plasma membrane of lymphocytes have been characterized in a human T-cell clone and in the Jurkat T-cell line. Due to endogenous buffers, about 1/125 of the Ca2+ ions that enter the cell are free. Ca2+ fluxes were estimated from the variations in intracellular Ca2+ concentration ([Ca2+]i) elicited by concentration jumps in extracellular Ca2+ ([Ca2+]o). Thapsigargin was used to inhibit Ca2+ uptake into intracellular stores and to stimulate Ca2+ entry. Ca2+ extrusion was strictly due to the activity of plasma membrane Ca(2+)-ATPases since there was no detectable Na+/Ca2+ exchange activity in these cells. The rate of Ca2+ extrusion was mainly influenced by [Ca2+]i and less by [Ca2+]o but was insensitive to cell depolarization. In depolarized cells, thapsigargin-induced Ca2+ influx was reduced to 10% of the value measured in normally polarized cells, suggesting that depolarization not only reduces the electrochemical gradient for Ca2+ ions, but also inhibits Ca2+ permeation. When Ca2+ ions enter the cell, they bind to a site inside the channel, with a Kd of 3.3 mM. Stimulation of clonal T-cells with low concentrations of either anti-CD3 antibodies or thapsigargin elicited Ca2+ oscillations. Both the amplitude and the frequency of CD3-induced Ca2+ oscillations were sensitive to [Ca2+]o. These oscillations were immediately interrupted when extracellular Ca2+ was removed. The properties of Ca2+ oscillations in T lymphocytes suggest that they are mainly due to variations of Ca2+ influx, modulated by variations in [Ca2+]i.

Antibodies, Monoclonal↗
PubMed1992

High density of Ca(2+)-dependent K+ and Cl- channels on the luminal membrane of lacrimal acinar cells.

Tight-seal whole-cell recording and Ca2+ imaging were simultaneously performed on cell clusters or individual acinar cells of rat lacrimal glands during application of the secretagogue acetylcholine. Activation of Ca(2+)-dependent K+ and Cl- currents was selectively followed as a function of time by placing the cell potential near the equilibrium potential for Cl- or for K+ ion, respectively. Upon acetylcholine application to cell clusters, K(+)- and Cl(-)-selective currents displayed a distinctive initial rise ("hump"). At this time, there was only a small elevation of Ca2+ concentration, [Ca2+]i, that was restricted to the luminal end of acinar cells. A quantitative analysis of Ca2+ and current signals during the hump suggested that the luminal membrane contained high densities of K(+)- and Cl(-)-selective channels, roughly 10 times higher than those found in the basolateral domain. Distinct luminal and basolateral membrane domains were preserved in isolated cells, but with less contrasted densities than in cell clusters. The results suggest that Ca(2+)-dependent K+ channels are implicated not only in the transfer of salt from the blood compartment to the interior of acinar cells, as commonly accepted, but also in the electrolyte secretion from the cell interior to the acinar lumen.

Acetylcholine↗
PubMed1992

Imaging early steps of human T cell activation by antigen-presenting cells.

In this work the Ca2+ response and the morphological changes elicited by Ag in human CD4+ T cells are described at the single cell level. The APC used to present the diphtheria toxoid Ag to a human diphtheria toxoid-specific T cell clone were murine L cell fibroblast transfectants expressing MHC class II molecules. The increase of the intracellular Ca2+ concentration, [Ca2+]i, which is one of the earliest steps of the response to TCR stimulation, was followed by fluorimetry with fura-2 on an imaging system. This response was a specific consequence of successful Ag presentation, because it only took place when fibroblasts expressed both class II MHC molecules and Ag. CD4 molecules were also involved in this intercellular interaction, because the Ca2+ response could be inhibited by preincubating the T cells with an anti-CD4 antibody. The response induced by APC started after a delay of at least 6 min, after which large Ca2+ oscillations took place, with a pseudo period of 100 s at 35 degrees C. The frequency of these oscillations decreased with temperature. The oscillations became progressively more damped during the first 30 to 40 min of cell-to-cell interaction, after which they completely stopped; however, [Ca2+]i remained well above its resting level for more than 1 h after the contact. The Ca2+ oscillations were entirely dependent on Ca2+ influx because they immediately disappeared when external calcium was removed. Similar oscillations were observed when the cells were stimulated with an anti-CD3 antibody. After stimulation with APC, many T cells abandoned their spherical shape and tended to flatten and elongate. This aspect of the T cell response was not observed after stimulation with an anti-CD3 antibody. In the presence of cytochalasin B, the morphologic changes elicited by the APC were blocked, whereas the Ca2+ response was slightly enhanced. However, when T cells were loaded with the Ca2+ chelator BAPTA, both Ca2+ and morphologic changes were inhibited, suggesting that the Ca2+ response plays a permissive role for the morphologic changes.

Animals↗
PubMed1991

Cyclic-GMP-dependent refilling of calcium stores in macrophages.

The effects of cyclic GMP on the release of calcium from intracellular stores, induced in murine peritoneal macrophages by either ATP or platelet-activating factor, were studied by microfluorimetry with fura-2. When macrophages were incubated for 10-20 min with 10 microM LY83583, an inhibitor of guanylate cyclase, the rise in intracellular calcium induced by agonist application was strongly depressed. This inhibition of the response to platelet-activating factor could be reversed by the addition of 0.1 mM cyclic 8-bromo-GMP. In the presence of cyclic 8-bromo-GMP, the decay of the calcium transient was speeded. Furthermore, when two calcium transients were evoked within 1 min by stimulating the cells with 10 microM ATP, the second calcium transient was more depressed than the first one in the presence of LY83583. These findings are compatible with the hypothesis that cyclic GMP is necessary for the activation of the calcium pump of the intracellular stores.

Adenosine Triphosphate↗
PubMed1991

Nitrendipine-induced inhibition of calcium influx in a human T-cell clone: role of cell depolarization.

An increase in intracellular calcium concentration stimulated by anti-CD2 or CD3 antibodies has been measured with Fura-2 in P28 cells, a human CD4+ T cell clone. This intracellular calcium increase was sensitive to membrane potential changes, being increased when the cells were hyperpolarized and decreased when they were depolarized. The intracellular calcium increase was inhibited by nitrendipine (1-50 microM). Nitrendipine also induced a depolarization of the cells, due to the blockade of a potassium conductance. The inhibition of the calcium increase caused by nitrendipine could be partially reversed by hyperpolarizing the cells with valinomycin. It is concluded that the effects of nitrendipine on potassium channels may account for a large part of the inhibition that nitrendipine exerts on the calcium increase elicited by CD2 or CD3 stimulation.

Antibodies, Monoclonal↗
PubMed1991

Ca(2+)-induced Ca2+ release amplifies the Ca2+ response elicited by inositol trisphosphate in macrophages.

We have studied the rise in intracellular calcium concentration ([Ca2+]i) elicited in macrophages stimulated by platelet-activating factor (PAF) by using fura-2 measurements in individual cells. The [Ca2+]i increase begins with a massive and rapid release of Ca2+ from intracellular stores. We have examined the mechanism of this Ca2+ release, which has been generally assumed to be triggered by inositol trisphosphate (IP3). First, we confirmed that IP3 plays an important role in the initiation of the PAF-induced [Ca2+]i rise. The arguments are 1) an increase in IP3 concentration is observed after PAF stimulation; 2) injection of IP3 mimics the response to PAF; and 3) after introduction of heparin in the cell with a patch-clamp electrode, the PAF response is abolished. Second, we investigated the possibility of an involvement of Ca(2+)-induced Ca2+ release (CICR) in the development of the Ca2+ response. Ionomycin was found to elicit a massive Ca2+ response that was inhibited by ruthenium red or octanol and potentiated by caffeine. The PAF response was also inhibited by ruthenium red or octanol and potentiated by caffeine, suggesting that CICR plays a physiological role in these cells. Because our results indicate that in this preparation IP3 production is not sensitive to [Ca2+]i, CICR appears as a primary mechanism of positive feedback in the Ca2+ response. Taken together, the results suggest that the response to PAF involves an IP3-induced [Ca2+]i rise followed by CICR.

Animals↗