Structure-function analysis of Lyn kinase association with lipid rafts and initiation of early signaling events after Fcepsilon receptor I aggregation.

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcepsilonRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.

Changes in lymphoid organs, peripheral blood and in homing of lymphocytes after administration of local anaesthetics.

The newly synthetized local anaesthetics of the carbanilate type--pentacaine and heptacaine, and the currently used trimecaine have been previously found to exhibit markedly different effects on immune reactions in vitro and in vivo when administered in equitoxic doses. These compounds are now assayed for their influence on the weight and cell count in lymphoid organs, on peripheral blood leucocyte count and homing of 51Cr-labelled syngeneic lymph node cells. We have found that the weight and cell count in the thymus, spleen, lymph nodes, bone marrow and the levels of peripheral leucocytes are reduced after administration of pentacaine, whereas heptacaine and trimecaine have no effect on these parameters. In contrast to heptacaine and trimecaine, pentacaine significantly reduces also the homing of lymph node cells to the recipients' lymph nodes and increases their homing to bone marrow.

Effect of local anaesthetics on immune reactivity.

Two local anaesthetics, pentacaine and trimecaine, were assayed for their effects on immune reactivity of mice in the regional popliteal lymph node after local injection of the antigen, on the regional graft-versus-host reaction and on the ability to reject skin allografts. These local anaesthetics exhibited a dose-dependent, immunosuppressive effect on all the systems used, although the mechanism of their action was different: pentacaine exerted a destructive effect on lymphocytes, whereas trimecaine induced a blockade of the lymphocyte functions.

The effect of carbanilic acid derivatives with local anaesthetic action on concanavalin A-induced blastic transformation of lymphocytes.

We have examined the effect of six carbanilic acid derivatives (differing in the substitution of the aromatic ring, in the coupling chain or the basic moiety of the molecule) and trimecaine, i.e. agents with local anaesthetic action, on concanavalin A-induced blastic transformation of lymphocytes. We have found that these compounds differ in the ability to block blastic transformation and agglutination of lymphocytes when added to cell cultures at different concentrations simultaneously with Con A. At the minimal inhibitory concentrations, the derivatives exhibit a different degree of toxicity which, however, provides no explanation for the inhibitory effect of most of the compounds. The known effect of these compounds on the membranes together with observed inhibition of agglutination (a relatively early sign of activation), and their lower inhibitory activity, when added several hours after Con A, support the idea that they affect the early cell membrane-mediated processes during blastic transformation. In agreement with this finding is also the observed correlation between local anaesthetic activity and the ability of individual carbanilic acid derivatives to block the blastic transformation.

Engagement of phospholipid scramblase 1 in activated cells: implication for phosphatidylserine externalization and exocytosis.

Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca(2+)-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcepsilonRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcepsilonRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation.

Non-apoptotic phosphatidylserine externalization induced by engagement of glycosylphosphatidylinositol-anchored proteins.

The exposure of phosphatidylserine (PS) on the cell surface is a general marker of apoptotic cells. Non-apoptotic PS externalization is induced by several activation stimuli, including engagement of immunoreceptors. Immune cells can also be activated by aggregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs). However, it is unknown whether cell triggering through these proteins, lacking transmembrane and cytoplasmic domains, also leads to PS externalization. Here we show that engagement of GPI-APs in rodent mast cells induces a rapid and reversible externalization of PS by a non-apoptotic mechanism. PS externalization triggered by GPI-AP-specific monoclonal antibodies was dependent on the activity of H(+)-ATP synthase and several other enzymes involved in mast cell signaling but was independent of cell degranulation, free cytoplasmic calcium up-regulation, and a decrease in lipid packing as determined by merocyanine 540 binding. Surprisingly, disruption of actin cytoskeleton by latrunculin B or plasma membrane integrity by methyl-beta-cyclodextrin had opposite effects on PS externalization triggered through GPI-AP or the high affinity IgE receptor. We further show that PS externalization mediated by GPI-APs was also observed in some other cells, and its extent varied with antibodies used. Interestingly, effects of different antibodies on PS externalization were additive, indicating that independent stimuli converge onto a signaling pathways leading to PS externalization. Our findings identify the cell surface PS exposure induced through GPI-AP as a distinct mechanism of cell signaling. Such a mechanism could contribute to "inside-out" signaling in response to pathogens and other external activators and/or to initiation of other functions associated with PS externalization.

Topography of plasma membrane microdomains and its consequences for mast cell signaling.

Thy-1 (CD90) is a glycoprotein bound to the plasma membrane by a GPI anchor. Aggregation of Thy-1 in mast cells and basophils induces activation events independent of the expression of Fcepsilon receptor I (FcepsilonRI). Although we and others have previously suggested that plasma membrane microdomains called lipid rafts are implicated in both Thy-1 and FcepsilonRI signaling, properties of these microdomains are still poorly understood. In this study we used rat basophilic leukemia cells and their transfectants expressing both endogenous Thy-1.1 and exogenous Thy-1.2 genes and analyzed topography of the Thy-1 isoforms and Thy-1-induced signaling events. Light microscopy showed that both Thy-1 isoforms were in the plasma membrane distributed randomly and independently. Electron microscopy on isolated membrane sheets and fluorescence resonance energy transfer analysis indicated cross-talk between Thy-1 isoforms and between Thy-1 and FcepsilonRI. This cross-talk was dependent on actin filaments. Thy-1 aggregates colocalized with two transmembrane adaptor proteins, non-T cell activation linker (NTAL) and linker for activation of T cells (LAT), which had been shown to inhabit different membrane microdomains. Thy-1 aggregation led to tyrosine phosphorylation of these two adaptors. The combined data indicate that aggregated GPI-anchored proteins can attract different membrane proteins in different clusters and thus can trigger different signaling pathways.

Cyclosporin A inhibits rat mast cell activation.

To gain further insight into the mechanism of immunosuppression by cyclosporin A (CyA), the effect of CyA on activation of isolated rat mast cells was studied. CyA alone, up to a concentration of 80 microM, had no effect on histamine release from unstimulated mast cells in both calcium-supplemented and calcium-free media. However, in the presence of extracellular calcium CyA inhibited, in a dose-dependent manner (range from 8 nM to 80 microM), histamine release induced by three unrelated secretagogues, the compound 48/80, calcium ionophore A23187 and concanavalin A plus phosphatidylserine. In the absence of extracellular calcium no, or only a marginal, effect of CyA on histamine release induced by the secretagogues was observed. CyA also inhibited the uptake of radiolabeled calcium by the secretagogues-treated cells. However, CyA did not interfere with the activation-related early increase in the intracellular free calcium. Thus, CyA-mediated inhibition of mast cell activation is related to external calcium uptake. These results indicate that rat mast cells belong to the immune cell types whose activity can be modulated by physiologically relevant concentrations of CyA.

The involvement of Thy-1 antigen in the activation of rat mast cells.

The Thy-1 antigen expressed on the surface of mouse T lymphocytes has been previously found to be involved in T cell activation. In the present study we have employed the anti-Thy-1.1 monoclonal antibody MRC OX7 and analyzed the expression and properties of Thy-1 antigen on the surface of peritoneal and pleural rat mast cells. Direct radioantibody binding assays, indirect immunofluorescence studies and flow cytometry analysis revealed that isolated rat mast cells express on their surface large amounts of the Thy-1 antigen. Scatchard analysis of the binding data indicated that at least one million Thy-1 molecules are expressed per cell. Immunoprecipitation studies carried out on mast cells demonstrated that MRC OX7 antibody recognizes a surface molecule of approximately 25 kDa that appears to correspond to Thy-1 antigen, and an additional molecule of 50 kDa. Incubation of the isolated mast cells with various concentrations of anti-Thy-1.1 antibody induced a rapid and concentration-dependent increase in the intracellular free calcium concentration ([Ca2+]i). The early increase in [Ca2+]i was observed in both Ca2+-supplemented and Ca2+-free media. This indicated that the initial increase in [Ca2+]i is due to a release of Ca2+ from internal stores. The [Ca2+]i increase was followed by an increase in the histamine release which was also dependent on antibody concentration. These data suggest that Thy-1 may act as an activation receptor on mast cells, analogously to its receptor function on T cells.

Thy-1-mediated activation of rat basophilic leukemia cells does not require co-expression of the high-affinity IgE receptor.

The glycosylphosphatidylinositol (GPI)-anchored protein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia cells, RBL-2H3. Antibody-mediated aggregation of Thy-1 induces in these cells release of secretory components; so does aggregation of the receptor with high affinity for IgE (Fc epsilon RI). To examine whether there is any relationship between Thy-1- and Fc epsilon RI-mediated activation, we have isolated from mutagenized RBL-2H3 cells a variant cell line deficient in the expression of surface Fc epsilon RI, and analyzed its ability to be activated by an antibody to Thy-1. Northern and immuno-blot analyses revealed that the variant cells were deficient in the expression of a structural or a regulatory gene for Fc epsilon RI gamma subunit. The cells did not respond by release of secretagogues and protein-tyrosine phosphorylation to IgE and antigen and anti-Fc epsilon RI monoclonal antibody (mAb) but their response to anti-Thy-1.1 mAb and calcium ionophore A23187 was retained. Transfection of the cloned Fc epsilon RI gamma subunit into the variant cells restored the surface expression of Fc epsilon RI and responsiveness to both the antigen and anti-Fc epsilon RI mAb but had no effect on responsiveness to anti-Thy-1 mAb. The combined data indicate that aggregation of surface Thy-1 glycoproteins activates a metabolic pathway which is independent of the presence of Fc epsilon RI gamma subunit and surface expression of Fc epsilon RI.

Functional expression of the endogenous Thy-1 gene and the transfected murine Thy-1.2 gene in rat basophilic leukemia cells.

An interaction of MRC OX7 monoclonal antibody with Thy-1.1 antigen of rat peritoneal and pleural mast cells has been previously shown to induce rat mast cell activation (L. Dráberová, Eur. J. Immunol. 1989, 19: 1715). In the present study we analyzed the expression and function of the Thy-1 antigen in rat basophilic leukemia cells, clone RBL-2H3. Two RBL-2H3-derived cell lines with stable expression of murine Thy-1.2 antigen, after transfection of a genomic clone of murine Thy-1.2 or Thy-1.2 cDNA under the control of simian virus 40 promoter, were also analyzed. Direct radioantibody binding assays, indirect immunofluorescence studies and flow cytometry analyses revealed that both endogenous Thy-1.1 and transfected murine Thy-1.2 gene products were expressed on the surface of the cells under study. Analysis of the distribution of the Thy-1 antigen in situ in cells grown attached to tissue culture vessels located the Thy-1 predominantly in regions of cell-cell contacts. Incubation of RBL-2H3 cells with Thy-1.1-specific antibodies, or of the transfected cells with both Thy-1.1- and Thy-1.2-specific antibodies, induced a rapid early increase in the concentration of intracellular free calcium [( Ca2+]i) released from internal stores. Sustained increase of [Ca2+]i required the presence of Ca2+ in the extracellular medium. The increase in [Ca2+]i was followed by histamine release from the target cells. The combined data indicate that RBL-derived cells can be used as a useful model system for analysis of Thy-1 antigen-mediated activation of rat mast cells.

Cross-linking of Thy-1 glycoproteins or high-affinity IgE receptors induces mast cell activation via different mechanisms.

Rat peritoneal and pleural mast cells and rat basophilic leukemia cells, RBL-2H3, have been previously shown to be activated by Thy-1-specific monoclonal antibodies (mAb). In the present study we investigated the mechanism of Thy-1-mediated activation and compared it with activation induced by cross-linking of the high-affinity IgE receptor. Binding of an IgG Thy-1 x 1-specific mAb, MRCOX7 (OX7), to RBL-2H3 cells and mast cells, and activation of RBL-2H3 by the OX7 were abrogated by pretreatment of the cells with phosphatidyl inositol-specific phospholipase C (PI-PLC). The F(ab')2 fragment of OX7, in contrast to the Fab' fragment, induced cell activation as well as intact OX7 mAb. Cells sensitized with IgE exhibited an increased responsiveness to anti-Thy-1 antibodies suggesting formation of functional complexes of IgE receptor/IgE/Thy-1/anti-Thy-1. Pretreatment of RBL-2H3 cells with cholera toxin potentiated activation induced by IgE+antigen (Ag) and IgE+OX7, but had no effect on activation induced by OX7 antibody alone. Similarly, dexamethasone had no effect on OX7-induced activation but inhibited IgE+Ag- and IgE+OX7-induced activation. Analysis of phosphotyrosine-containing proteins in RBL-2H3 cell lysates revealed that IgE+Ag and IgE+OX7 induced a marked increase in tyrosine phosphorylation of several proteins that were not tyrosine phosphorylated in cells exposed to OX7 mAb alone. Similar results were obtained when RBL-2H3-derived cells, expressing transfected mouse Thy-1.2, were activated with Thy-1.2-specific IgM antibody. The combined data suggest that Thy-1-specific antibodies activate cells by a mechanism that is different from activation induced by cross-linking of high-affinity IgE receptor.

Thy-1 glycoprotein and src-like protein-tyrosine kinase p53/p56lyn are associated in large detergent-resistant complexes in rat basophilic leukemia cells.

Thy-1 is a surface glycoprotein that is attached to the plasma membrane by a glycosyl-phosphatidyl-inositol anchor. Crosslinking of Thy-1 in rat mast cells and basophilic leukemia cells (RBL-2H3) induces cell activation including histamine release and tyrosine phosphorylation of several proteins. Here we show that glycosyl-phosphatidylinositol-linked Thy-1 forms noncovalent complexes with src-related protein-tyrosine kinase p53/p56lyn and other protein-tyrosine kinases and/or their substrates. These complexes are resistant to solubilization by a nonionic detergent, sedimentable at 200,000 x g, and very large ( > 10 MDa) as determined by gel chromatography. Activation of RBL-2H3 cells by crosslinking of the high-affinity IgE receptors resulted in decreased recovery of the complexes. The combined data indicate the existence of large detergent-resistant domains in the surface membrane of mast cells that may play an important role in their activation.

Protein tyrosine kinase Syk is involved in Thy-1 signaling in rat basophilic leukemia cells.

Thy-1, a glycosyl-phosphatidylinositol-anchored surface glycoprotein, has been shown to possess transmembrane signaling capacity. In rat mast cells and rat basophilic leukemia cells (RBL) aggregation of surface Thy-1 with antibodies triggers a series of intracellular events, resembling those induced by aggregation of the high-affinity receptor for IgE (Fc epsilonRI), including tyrosine phosphorylation of multiple proteins and release of secretory components. Unlike the Fc epsilonRI-mediated activation, where both the membrane-associated protein tyrosine kinase (PTK) Lyn and the cytoplasmic PTK Syk are responsible for initiating the signaling cascade, only Lyn has been implicated in Thy-1-mediated activation in RBL cells. Here we report that Syk is also rapidly tyrosine phosphorylated upon Thy-1 cross-linking. Increased Syk tyrosine phosphorylation is observed only in cells in which extensive aggregation of Thy-1 is induced by two layers of cross-linking reagents. RBL-derived mutant cells deficient in the expression of surface Thy-1 and transfectants re-expressing surface Thy-1 were used to exclude the possibility that Syk activation reflects an interaction of the cross-linking reagents with surface molecules other than Thy-1. As Fc epsilonRI gamma subunits are well known to promote activation of Syk and its recruitment to membrane complexes, we also investigated the role of these subunits in Thy-1-mediated Syk activation, using RBL-derived mutant cells deficient in the expression of Fc epsilonRI gamma subunits and their revertants. Consistent with the lack of Fc epsilonRI expression, no IgE-induced response could be elicited, while Thy-1-inducible Syk phosphorylation was preserved. Our results suggest that Syk might be one of the kinases responsible for signal propagation upon Thy-1 cross-linking in a Fc epsilonRI-independent pathway.

Thy-1-mediated activation of rat mast cells: the role of Thy-1 membrane microdomains.

The glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia (RBL) cells. The finding that Thy-1 from detergent-solubilized RBL-2H3 cells forms complexes with src-related protein-tyrosine kinase p56/p53lyn suggested that this kinase may play a key role in Thy-1-mediated mast-cell activation. The molecular mechanism of this activation is, however, unknown. Here we show that in RBL-2H3-derived cells extracted by the standard procedure with several non-ionic detergents, the majority of Thy-1 and p56/p53lyn were not released into postnuclear supernatant but remained associated with the detergent-resistant cytoskeletal/nuclear fraction. Pretreatment of the cells with the cholesterol-complexing agents, saponin or digitonin, resulted in complete solubilization of Thy-1 and p56/p53lyn in non-ionic detergents and dissociation of the complexes; this implies that cholesterol plays a crucial role in stabilization of the complexes. This conclusion was supported by double immunofluorescence colocalization experiments which also allowed us to estimate the size of the insoluble complexes to be about 0.1 micron. Sequential treatment with saponin and Nonidet P-40 was used to fractionate tyrosine-phosphorylated proteins during Thy-1-mediated activation of RBL-2H3 cells. Among the soluble cytoplasmic proteins the most dramatic change in tyrosine phosphorylation was found in pp72, whereas pp40 and pp33 were found mainly in the membrane fraction. Our data suggest that surface aggregation of GPI-anchored Thy-1 molecules leads to aggregation of p56/p53lyn kinase located in the same membrane microdomain, followed by transphosphorylation of both soluble and membrane-bound substrates.