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The Journal of Immunology, 2000, 164: 5389-5397.
Copyright © 2000 by The American Association of Immunologists

Glycosphingolipid-Induced Relocation of Lyn and Syk into Detergent-Resistant Membranes Results in Mast Cell Activation

Eva E. Prieschl1,*, Robert Csonga*, Veronica Novotny*, Gary E. Kikuchi{dagger} and Thomas Baumruker*

* Department of Immunology, Novartis Research Institute, Vienna, Austria; and {dagger} Genetic Therapy Inc. (A Novartis Company), Gaithersburg, MD 20878


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Over the last few years, members of the sphingolipid class of compounds, which is composed of >300 naturally occurring substances, have been shown to be involved in signal transduction processes regulating cell growth, differentiation, and apoptosis (1). A paradigm illustrating this potential on immune cells was the discovery of an intracellular second messenger function for ceramide in TNF-{alpha} and IL-1ß signaling as well as in Fas-induced cell death (2, 3). Recent findings, which demonstrate that the balance of sphingosine (S)2 and sphingosine-1-phosphate (S1P) is decisive for allergic triggering of mast cells, have extended this aspect of sphingolipid biology to other leukocytes (4). This has introduced a new facet into their ubiquitous role as structural constituents of all eukaryotic membranes, including the Golgi and lysosomal vesicles.

S, which comprises the central component of this lipid class, has been described as both an activator and an inhibitor of different signaling pathways. It stimulates the 80-kDa isoform of diacylglycerol kinase, p21-activated kinases, and mobilizes Ca2+ (5, 6, 7), whereas it inhibits classical and novel protein kinase C (PKC) (8). Previously, the latter was thought to be the prime cause for its ability to down-regulate cellular processes. However, this is not restricted to S itself, but is also observed for many other lysosphingolipids, such as S1P, which, in contrast to S leads to cellular activation (8, 9, 10, 11, 12). While sphingolipids in general act either intracellularly or via G-protein coupled receptors; sugar derivatives of sphingolipids are additionally implicated as being essential constituents of the outer leaflet of detergent-resistant membranes (DRMs), where they are specifically enriched. In the course of activation, many triggered receptors and their associated protein tyrosine kinases, such as the Fc{epsilon}RI and Lyn in mast cells, translocate into this part of the cellular membrane where they are brought into close vicinity to each other for activation (13). The importance of the specific architecture of the DRMs for the activation process from the Fc{epsilon}RI has recently been demonstrated by cholesterol deprivation experiments (cholesterol is a major constituent of DRMs). As a result, not only the translocation of the Fc{epsilon}RI and associated tyrosine kinases, but also the initial tyrosine phosphorylation steps usually observed after IgE plus Ag (IgE/Ag) stimulation, were abolished (14). A contribution of the outer leaflet (glycosylated sphingolipids) to signaling events has also long been implicated in mast cells. In RBL cells, binding of a mAb directed against the ganglioside GD1b (Ab AA4; GD1b is localized in the outer leaflet of DRMs) is able to induce comparable signaling events as the allergic trigger IgE/Ag (increased ruffling, redistribution of the cytoskeletal elements, a rise in intracellular calcium, phosphatidylinositol breakdown, and PKC activation (15)). To further elucidate the contribution of the outer leaflet component (glycosphingolipids) to a potential signal initiation, we used the mouse mast cell line CPII as well as bone marrow-derived mouse mast cells (BMMCs) and applied galactosylsphingosine (Gal-S), a small naturally occurring sugar derivative of S. Here we show that exogenous Gal-S specifically accumulates in DRMs leading to the relocation of the tyrosine kinases Lyn and Syk into this membrane compartment independent of an Fc{epsilon}RI triggering by IgE/Ag. As a consequence, an enhanced tyrosine-phosphorylation and kinase activity of Lyn and Syk is observed in these DRMs, which subsequently results in a suboptimal, extremely transient activation of the mitogen-activated protein kinase (MAPK) pathway(s). The overall weaker induction at membrane-distal levels after a Gal-S stimulation, in comparison to an IgE/Ag trigger, leads to a predominantly JunD-driven transcriptional response concerning the AP-1 transcription factor family. This suggests that only constitutively expressed transcription factors become activated in the course of such stimulation. This chain of events primes and partially activates the CPII cell line as well as primary BMMCs for all investigated effector functions (degranulation, S1P secretion, and cytokine production).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culturing conditions of CPII cells as well as BMMCs, transient transfections, reporter gene constructs, gel shift analyses, hexosaminidase release assays, reporter gene assays, use of ionomycin (Iono.), apigenin, TLC to determine S1P secretion, and Western blot analyses were conducted as described (4, 16, 17, 18, 19, 20).

Materials

Gal-S was purchased from Sigma (St. Louis, MO), piceatannol was obtained from Roche Diagnostics (Roche, Vienna, Austria), PP2 (a chloride derivative of PP1) was purchased from Calbiochem-Novabiochem International (La Jolla, CA), and pertussis toxin was purchased from Sigma.

Abs

Abs directed against Raf, MAPK kinase (Mek) 1, and phosphotyrosine used in Western blot analyses were purchased from Transduction Laboratories (Lexington, KY). Abs directed against extracellular signal-regulatory kinase (Erk) 1,2, c-Jun NH2-terminal kinase (Jnk) 1,2, p38, and c-Jun (phosphospecific and nonphosphospecific) were provided by New England Biolabs (Beverly, MA). Abs directed against other AP-1 components, as well as Lyn and Syk, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Abs directed against the FcR {gamma}-chain, linker of activated T cells (LAT), and phosphatidylinositol 3-kinase (PI3K) were bought from Upstate Biotechnology (Lake Placid, NY).

Immunoprecipitation/kinase assays

The kinase assay for c-Raf activity was done using the c-Raf 1 immunoprecipitation kinase cascade assay kit (Upstate Biotechnology) according to the manufacturer’s protocol. The Mek1 kinase activity was determined using the MAPKK immunoprecipitation kinase cascade kit (Upstate Biotechnology). For both assays, 2 x 107 stimulated (5 min) or nonstimulated CPII cells were lysed in a buffer consisting of 50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% 2-ME, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyruvate, 10 mM sodium ß-glycerol phosphate, 0.1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µM microcystin. For precipitation, 2 µg anti-Raf Ab (Upstate Biotechnology) or 10 µl anti-Mek antiserum (Upstate Biotechnology) coupled to 50 µl Sepharose G beads (Pharmacia, Uppsala, Sweden) were incubated with the cell lysate for 2 h at 4°C. After washing four times with lysis buffer, the immunoprecipitate was resuspended in 20 mM MOPS, pH 7.2, 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM Na3VO4, and 1 mM DTT containing additionally 0.4 µg nonactive Mek1 (for the Raf assay only; Upstate Biotechnology) and 1 µg nonactive Erk2 (for both assays; Upstate Biotechnology). The reaction was incubated at 30°C for 30 min. Subsequently, an aliquot was incubated with 10 µCi [{gamma}-32P]ATP and 20 µg myelin basic protein (MBP) as a substrate. After 10 min incubation at 30°C, the reaction was separated by PAGE, the gel was fixed in 40% methanol, 10% acetic acid for 1 h, dried, and subjected to autoradiography. For normalization, a Western blot was performed using monoclonal anti-Raf and anti-Mek Abs (Transduction Laboratories). For precipitation of the FcR {gamma}-chain and LAT, 1 x 107 CPII cells (either nonstimulated or stimulated for 1 min) were lysed in a buffer consisting of 150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM sodium vanadate, and 1 mM sodium fluoride for 1 h at 4°C. For each immunoprecipitation, 5 µg rabbit anti-FcR {gamma}-chain Ab or 4 µg rabbit anti-LAT Ab, respectively, were coupled to 50 µl Sepharose G beads (Pharmacia). After washing the beads, they were added to the cell lysates and incubated for 1 h (for FcR {gamma}-chain) or 2 h (for LAT) at 4°C with constant rotation. The beads were subsequently collected by centrifugation and washed three times with lysis buffer and two times with kinase buffer (25 mM HEPES, pH 7.3, 150 mM NaCl, 5 mM MnCl2). Then 10 µl beads were subjected to a kinase reaction (addition of 10 µCi [{gamma}-32P]ATP (Nycomed Amersham, Little Chalfont, U.K.); 10 min at 30°C) with subsequent PAGE. After electrophoresis, the gel was fixed in 40% methanol, 10% acetic acid overnight, dried, and subjected to autoradiography. Then 20 µl beads were used in a Western blot analysis for normalization.

Membrane preparation and fractionation

A total of 4 x 106 CPII cells were stimulated for 1 min before lysing them in a buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM EDTA, 1 mM sodium vanadate, 30 mM sodium pyruvate, 10 mM glycerophosphate, 1 mM PMSF, 0.02 U/ml aprotinin, 0.01% sodium azide, and 0.05% Triton X-100 on ice for 10 min. Subsequently, the lysate was mixed 1:1 with an 80% sucrose solution (sucrose had been dissolved in 25 mM Tris-HCl, pH 7.5, 125 mM NaCl, 2 mM EDTA) before loading onto a sucrose gradient. The gradient was performed stepwise with 2 ml of each 80%, 60%, 40% (containing the cell lysate), 30%, 20%, and 10% sucrose. Centrifugation was done using a SW40 rotor with 37,500 rpm for 18 h at 4°C. After centrifugation, 666-µl fractions were collected from top of the gradient. Protein content of the fractions was determined with a Bradford assay (Bio-Rad Laboratories, Hercules, CA) according to the protocol provided by the manufacturer.

Kinase assay and immunoprecipitation of sucrose fractions

For the in vitro kinase reaction, 20 and 40% sucrose fractions were pooled (10 µl of each fraction) and diluted to 120 µl kinase buffer (25 mM HEPES, pH 7.3, 150 mM NaCl, 5 mM MnCl2). Then 10 µCi of [{gamma}-32P]ATP (Nycomed Amersham) was added and the reaction incubated for 10 min at 30°C. Next, 20 µl of each reaction were used for PAGE. After electrophoresis, the gel was fixed in 40% methanol, 10% acetic acid before drying. The gel was then subjected to autoradiography. For immunoprecipitation, 4 µg rabbit anti-Lyn Ab or 4 µg rabbit anti-Syk Ab, respectively, were coupled to 50 µl Sepharose G beads (Pharmacia). After washing the beads, they were resuspended in 1 ml buffer either consisting of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM sodium vanadate, and 1 mM sodium fluoride (Lyn) or 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM sodium vanadate, 1 mM sodium molybdate, 1 mM PMSF, 1 µg/ml aprotinin, and 1 mM MgCl2 (Syk). Then, 240 µl of a radiolabeled kinase reaction from the 20% sucrose fraction (see above) was added and incubated with the beads for 1 h at 4°C with constant rotation. The beads were subsequently collected by centrifugation and washed four times with the precipitation buffer. The beads were resuspended in SDS sample buffer with subsequent PAGE. After electrophoresis, the gel was fixed in 40% methanol, 10% acetic acid for 1 h, dried, and subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A small glycosphingolipid primes and activates mast cells for effector function

A structure activity relationship study has recently demonstrated that, besides other features, the phosphorus group in position 1 of S1P is essential for its activation potential (21). In the case of glycosylated sphingolipids, one (e.g., Gal-S) or more sugar group(s) (e.g., gangliosides) substitute this particular moiety, while the other characteristics still remain preserved. Therefore, we started to investigate if such molecules, represented by Gal-S, result in a similar activation of the CPII mouse mast cell line, as we had recently observed for S1P (4). Using a TNF-{alpha} reporter gene assay as the activation readout, a bell-shaped dose-response curve is observed with a concentration optimum of 20 µM Gal-S, in case this glycolipid is combined with the calcium ionophore Iono. (Fig. 1GoA). Both stimuli alone fail to significantly stimulate TNF-{alpha} transcription (Fig. 1GoA), as does the combination of PMA together with Gal-S at all concentrations tested (data not shown). Toxicity of Gal-S at 20 µM was excluded due to no changes in the mitochondrial activity as determined in an cell proliferation assay (XTT) test, and no gross membrane or surface molecule alterations as measured by propidium iodide and FACS staining after 24 h Gal-S treatment (data not shown). To further extend these results, additional pathologically relevant mast cell activation parameters were investigated, not only in the CPII cell line but also in primary BMMCs. As readouts, hexosaminidase release (degranulation) and S1P secretion, as well as TNF-{alpha} secretion, in the case of BMMCs, were employed (4, 17). Gal-S was again either applied alone at the 20 µM dose or together with the suboptimal stimuli Iono. An IgE/Ag stimulation was done in parallel for direct comparison. As is clearly visible from Fig. 1GoB (for CPII cells) and Fig. 1GoC (for BMMCs), Gal-S primes/activates CPII mast cells as well as BMMCs with respect to the parameters investigated. Its activities closely resemble those of S1P, which recently has been described to activate this cell type (compare Ref. 4). While significant amounts of hexosaminidase (Fig. 1Go, B and C, left panels) and considerable S1P secretion (Fig. 1Go, B, right panel and C, middle panel) are observed after Gal-S stimulation, TNF-{alpha} transcription and secretion (Fig. 1Go, A and C, right panel) depend on the additional application of the calcium ionophore Iono. Taken together, the data therefore suggest that Gal-S induces some, but not all, of the signaling cascades required for complete mast cell activation (especially Ca2+ influx has to be elicited by the addition of Iono.).



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  		FIGURE 1. Gal-S leads to mast cell priming/activation. A, Titration of Gal-S on CPII mast cells using a TNF-{alpha} reporter gene assay with 4 x 104 CPII cells per value stimulated as indicated at the x-axis for 4 h. Luciferase values corresponding to TNF-{alpha} transcription are indicated at the y-axis. Values represent means of quadruplicate experiment ± SD. nst, Nonstimulated. B, Left, Degranulation (early phase, hexosaminidase assay) with 1 x 105 CPII cells per value stimulated for 1 h. Stimulation conditions are indicated at the x-axis; hexosaminidase released is indicated at the y-axis. Values represent means of quadruplicate experiment ± SD. Right, S1P secretion of 4 x 105 CPII cells stimulated for 4 h as indicated at the top. Std, Standard. The arrows mark the position of S and S1P on the TLC plate. C, Same experimental setting as under B for BMMCs with the late phase measured by a TNF-{alpha} ELISA in pg/ml (equivalent to 5 x 105 cells) given at the y-axis (right).

 
Exogenously applied Gal-S accumulates in the DRM fraction leading to the relocation of Lyn and Syk

A G protein-coupled receptor for Gal-S has been postulated, which mediates a pertussis toxin-sensitive activation of phospholipase C and Ca2+ mobilization in HL60 cells (22). To investigate whether a similar receptor-dependent process applies to the stimulation of CPII mast cells, a preincubation with 100 nM pertussis toxin 24 h before a Gal-S plus Iono. stimulation was performed in direct comparison to an identical reaction without the inhibitor (Fig. 2GoA). Using a TNF-{alpha} reporter gene assay as the readout, it is observed that the drug has no influence on stimulation, strongly suggesting that Gal-S in this cell type does not act via G protein-coupled receptors. In an attempt to define its mode of action, we began to investigate where the exogenously applied Gal-S accumulates in the cells. Its distribution was monitored using [3H]Gal-S as a tracer, before fractionating cells using a sucrose gradient (1 min application of Gal-S; Fig. 2GoB). In contrast to other sphingolipids (such as S), which are easily taken up by CPII cells (4), only 0.6% of the applied Gal-S (20 µM) is found cell bound. A unique and specific enrichment of Gal-S is observed in the 20% sucrose fraction, which in this type of fractionation represents DRMs (solid line). Residual Gal-S present in the cell culture medium is predominantly found in the 40% sucrose fraction (broken line), as determined in a mock gradient omitting cells, underlining the specificity of the observed finding (see Fig. 2GoB) (23, 24). In the 40% fraction, the majority of cellular protein is also localized (bars in Fig. 2GoB). Following the deposition of Gal-S into the DRMs, both protein tyrosine kinases, Lyn (almost completely) and Syk (to ~50%), clearly translocate into the 20% fraction (DRMs) as determined by Western blot analyses (Fig. 1GoC, upper two panels). A direct comparison to IgE/Ag-triggered cells shows a similar movement of Lyn, but, as has already been reported for this type of triggering, no relocation of Syk (23). The specificity of this translocation of the kinases is highlighted by Western blot analyses using Abs directed against two further proteins, which do not translocate in the course of an activation, PI3K and LAT. The latter is also a marker protein for DRMs and therefore served as an internal quality control for the successful separation of this membrane compartment from other cellular fractions (Fig. 2GoC, lower two panels) (25).



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  		FIGURE 2. Gal-S stimulation mediates a shift of Lyn and Syk into the 20% sucrose fraction (DRMs) in a G protein-coupled receptor-independent way. A, A pertussis toxin-insensitive activation of mast cells is mediated by Gal-S. TNF-{alpha} reporter gene assay using 4 x 104 CPII cells, either nontreated or pertussis toxin pretreated (24 h with 100 nM). Drug treatment and stimulation conditions are indicated at the x-axis; luciferase values are indicated at the y-axis. Values represent means of quadruplicate experiment ± SD. nst, Nonstimulated. B, The distribution of exogenously added [3H]-labeled Gal-S in a Gal-S stimulation of CPII cells ({blacksquare}) was determined in comparison to soluble [3H]Gal-S omitting CPII cells (•) in a sucrose gradient. Corresponding percentage of sucrose of each fraction is indicated at the x-axis; cpm is indicated at the y-axis to the left. Protein concentration of the fractions is indicated at the y-axis to the right as OD (595 nm) and shown as gray bars for each fraction. C, Western blot analyses of pooled fractions corresponding to 20% (DRMs) or 40% (cytosolic) sucrose of either nonstimulated (nst) or stimulated cells (1 min) as indicated at the top. Proteins detected by the Abs used in the analysis are indicated to the right.

 
Tyrosine phosphorylation and tyrosine kinase activity follow the relocation of Lyn and Syk after Gal-S stimulation

Lyn and Syk relocation into DRMs is believed to be a prerequisite for signal initiation at the cross-linked Fc{epsilon}RI. However, it does not address the activation status (phosphorylation) and kinase activity of those molecules (23). Therefore, in Western blot analyses and in vitro kinase assays we investigated the induction and distribution of tyrosine phosphorylation and intrinsic kinase activity in the DRM fraction (20%) vs the 40% sucrose fraction of Gal-S-treated CPII cells in comparison to IgE/Ag-triggered and nonstimulated cells (both stimuli were applied for 1 min). As shown in Fig. 3GoA, an identical and induced pattern of tyrosine phosphorylation (p53/56 and p72) is observed in both IgE/Ag-triggered and Gal-S-treated CPII cells. This phosphorylation is accompanied by an induced kinase activity in those fractions, which is specific for the DRMs of induced cells, resulting in a closely analogous picture (p53/56 and p72). Only one additional protein (with a size of ~120 kDa) is detected in IgE/Ag-stimulated cells that is absent in Gal-S-treated cells (Fig. 3GoB). An immunoprecipitation with specific Lyn and Syk Abs directly from the in vitro kinase reaction of the 20% fraction from Gal-S-stimulated cells finally proved that the p53/56 and p72 proteins correspond to Lyn and Syk, respectively (Fig. 3GoB). Taken together, this demonstrates that after 1 min of Gal-S treatment tyrosine-phosphorylated p53/56 Lyn and p72 Syk are located in the DRM fraction exhibiting (intrinsic) kinase activity.



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  		FIGURE 3. Stimulation with Gal-S leads to an enhanced tyrosine phosphorylation and kinase activity of Lyn and Syk in the 20% (DRMs) sucrose fraction. A, Western blot analysis of pooled fractions corresponding to 20% (DRMs) or 40% (cytosolic) sucrose of either nonstimulated (nst) or stimulated cells (1 min) as indicated at the top. An anti-phosphotyrosine Ab was used in this analysis. Corresponding m.w. of the proteins specifically detected in the 20% fraction are indicated to the right. Molecular size standard is given to the left. B, Kinase assay of pooled fractions corresponding to 20% (DRMs) or 40% (cytosolic) sucrose of either nonstimulated (nst) or stimulated cells (1 min) as indicated at the top (lanes 1–6). Immunoprecipitations (lanes 7 and 8) with Abs directed against Lyn (Lyn IP) and Syk (Syk IP) from the 20% sucrose fraction of Gal-S stimulated cells after an in vitro kinase reaction. The positions of phosphorylated kinases Lyn and Syk are indicated to the right. Molecular size standard is given to the left.

 
The tyrosine kinases Lyn and Syk are involved in a Gal-S plus Iono. stimulation

To further investigate whether the increased kinase activity of Lyn is also a prerequisite for the activation of the effector functions, a TNF-{alpha} reporter gene assay was performed using the compound PP2, which has been described to inhibit this kinase (26). A dose-dependent inhibition of the Gal-S plus Iono. (combined stimulus to achieve cytokine transcription)-induced TNF-{alpha} transcription is observed (Fig. 4GoA, left) with an IC50 value even lower than in IgE/Ag-triggered cells (Fig. 4GoA, right). This suggests that Lyn, which is known to phosphorylate the {gamma}-chain of the Fc{epsilon}RI after IgE/Ag stimulation, is also an essential molecule in the initiation of the signaling cascades after the Gal-S plus Iono. stimulus (27). This picture is further strengthened by immunoprecipitations of the {gamma}-chain from whole-cell lysates coupled to in vitro kinase assays. In such a setting, a greatly enhanced activity of associated kinases resulting in the phosphorylation of the {gamma}-chain after a Gal-S plus Iono. stimulation was observed (Fig. 4GoB, left, lanes 1 and 2). The inhibitor PP2 prevented this phosphorylation in the same dose range (10 and 3 µM) as it abolishes the Gal-S plus Iono.-induced TNF-{alpha} transcription in CPII cells (Fig. 4GoB, left, lanes 3 and 4).



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  		FIGURE 4. The tyrosine kinases Lyn and Syk are involved in mast cell activation after Gal-S plus Iono. stimulation. A, TNF-{alpha} reporter gene assay using different concentrations of the Lyn kinase inhibitor PP2 ({diamondsuit}) or corresponding solvent ({blacksquare}) as indicated at the x-axis. The solid lines depict nonstimulated (nst; •) and Gal-S+Iono. (left; •)- or IgE/Ag (right; •)-stimulated cells, respectively. The broken line indicates IC50 value. A total of 4 x 104 CPII cells were used per value, and stimulation was done for 4 h. Luciferase values measuring TNF-{alpha} transcription are indicated at the y-axis. Values represent mean of quadruplicate experiments ± SD. B, Left, Kinase assay with immunoprecipitated FcR {gamma}-chain from either nonstimulated (nst) and Gal-S+Iono.-stimulated CPII cells (1 min). Stimulation conditions as well as input of PP2 into the reaction is indicated at the top. Right, Immunoprecipitates were normalized by Western blot. ctr., Ab control. C, TNF-{alpha} reporter gene assay using different concentrations of the Syk kinase inhibitor piceatannol (pic; {diamondsuit}) or corresponding solvent ({blacksquare}) as indicated at the x-axis. The solid lines depict nst (•) and Gal-S+Iono. (left; •)- or IgE/Ag (right; •)-stimulated cells, respectively. The broken line indicates the IC50 value. A total of 4 x 104 CPII cells were used per value, and stimulation was done for 4 h. Luciferase values measuring TNF-{alpha} transcription are indicated at the y-axis. Values represent mean of quadruplicate experiments ± SD. D, Left, Kinase assay with immunoprecipitated LAT from either nonstimulated (nst) and Gal-S+Iono.-stimulated (1 min) CPII cells. Stimulation conditions as well as input of piceatannol (pic.) into the reaction is indicated at the top. Right, Immunoprecipitates were normalized by Western blot.

 
Lyn activation is sequentially followed by the recruitment of Syk to the phosphorylated {gamma}-chain, which subsequently leads to the activation of the MAPK cascade (via Shc and Grb) and phospholipase C{gamma}. In a similar setting to Lyn, a Syk-specific inhibitor, piceatannol, was employed to investigate the involvement of this kinase (28). A dose-dependent inhibition of both Gal-S plus Iono. (combined stimulus to achieve cytokine transcription)-induced and IgE/Ag-induced activation of TNF-{alpha} transcription is observed in CPII cells treated with this inhibitor (Fig. 4GoC). Again, no difference in the sensitivity to the drug is seen for the two stimuli, strongly suggesting that an analogous mode of action located around the {gamma}-chain, Lyn, and Syk takes place. Coupled immunoprecipitation/kinase assays were performed using LAT, which is a target for Syk, as a readout. As observed for the {gamma}-chain (mediated by Lyn), an increase of LAT phosphorylation (mediated by Syk) is detected in case of Gal-S plus Iono.-stimulated cells compared with nonstimulated ones (Fig. 4GoD, left, lanes 1 and 2). Application of the Syk inhibitor piceatannol abrogated this phosphorylation completely in the same dose range as seen to be effective on cells. (Fig. 4GoD, left, lanes 3 and 4). In summary, these findings show that initial signaling steps provoked after an Fc{epsilon}RI cross-linking are also induced by Gal-S accumulation in the DRMs of mast cells.

The MAPK pathway(s) is (are) suboptimally triggered by Gal-S

Ca2+ influx and the MAPK pathway(s) has (have) recently been shown to be the essential signaling cascades in CPII mouse mast cells for TNF-{alpha} activation after IgE/Ag stimulation (20, 29). The observed synergy between Gal-S and Iono. for up-regulating this cytokine suggested that Gal-S exerted its function by inducing the MAPK pathway(s). Coupled immunoprecipitation/in vitro kinase assays demonstrated that Gal-S application resulted in a suboptimal but significant stimulation of MAPK kinase kinase (Raf) and MAPK kinase (Mek1) compared with the IgE/Ag stimulus in CPII cells (Fig. 5GoA). Consequently, as visualized in Western blot analyses with phospho-specific Abs, an activation of the two MAPKs, Erk1,2 and Jnk1, was detected (Fig. 5GoB). Remarkably, regarding the induction of the MAPKs is a shift in the time kinetics with an earlier peak after Gal-S stimulation (5 min) in comparison to IgE/Ag (15 min). In addition, a more pronounced phosphorylation of Jnk1 and lower levels of phospho-Erk1,2 are observed. Classical and nonclassical PKCs, which recently have been implicated in transient MAPK induction in RBL cells, seem unlikely to be the basis for the detected differences (30). Gal-S not only inhibits all recombinant PKC isozymes in in vitro kinase reactions (8), but PKC-depleted CPII cells still respond equally well to a Gal-S plus Iono. stimulation compared with nondepleted cells (data not shown). The slightly fluctuating phosphorylation pattern of the third MAPK, p38, after both stimulation conditions seems to be irrelevant as demonstrated by the failure of a specific p38 inhibitor (SB203580) to prevent TNF-{alpha} induction (Fig. 5GoB and data not shown) (20). Therefore, Gal-S alone triggers the MAPK pathways similarly, although more rapidly and to a lower degree than usually seen after an IgE/Ag stimulus.



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  		FIGURE 5. The MAPK pathway(s) is (are) activated by Gal-S. A, Top, Kinase assay with immunoprecipitated (IP) Raf kinase from either nonstimulated (nst) or stimulated (5 min) CPII cells as indicated at the top. MBP was used as a substrate, and immunoprecipitates were normalized by Western blots (Raf IP). ctr., Ab control. Bottom, Kinase assay with immunoprecipitated (IP) Mek1 from either nonstimulated (nst) or stimulated (5 min) CPII cells as indicated at the top. MBP was used as a substrate, and immunoprecipitates were normalized by Western blots (Mek1 IP). B, Western blot analyses of the phosphorylation status of Erk 1,2, Jnk 1,2, and p38 over the first 30 min after Gal-S, and IgE/Ag stimulation as indicated.

 
Gal-S treatment leads predominantly to JunD activation

The AP-1 transcription factor family is one of the major targets of activated MAPKs (31). Based on the altered activation of these kinases after Gal-S compared with IgE/Ag stimulation, we addressed the question of whether this "ultratransient" response is sufficient to allow AP-1 activation. Therefore, in a transient transfection experiment, we used a 3x TRE (12-O-tetradecanoylphorbol-13-acetate responsive element)-driven luciferase construct, which solely depends on AP-1 for activation. Consistent with the results of the MAPK phosphorylation pattern after Gal-S stimulation, such a treatment provokes a significant although weaker induction of the reporter gene construct compared with IgE/Ag (Fig. 6GoA). A similar result is obtained in EMSA using an AP-1 consensus binding site as a radiolabeled probe and nuclear extracts of Gal-S- and IgE/Ag-stimulated cells (18, 29). A clear complex formation at this site is detected after both stimuli again; however, Gal-S consistently causes a weaker induction of AP-1 than IgE/Ag triggering (Fig. 6GoB, left). This suggests that the changes in the kinetics of MAPK activation subsequently lead to an altered pattern of AP-1 transcription factors. In a comparative supershift analysis, FosB, c-Fos, c-Jun, JunD, and Fra2 are detected after IgE/Ag stimulation (Fig. 6GoC, left) (32), while, in contrast, JunD binding to the AP-1 consensus site is predominantly found after Gal-S treatment. This picture remains unchanged even after the combined stimulus Gal-S plus Iono. excluding an influence of Ca2+ on the AP-1 component (Fig. 6GoC, middle and right panel and data not shown). After an extended exposure, other members of the AP-1 transcription factor family become detectable in this supershift analysis; however, JunD still remains the dominant binding protein (Fig. 6GoC, right). To strengthen this finding, corresponding Western blot analyses of the different AP-1 components were performed over the first 60 min of a Gal-S stimulation in comparison to the IgE/Ag stimulus (again no difference to a combined stimulus Gal-S plus Iono. is observed, Fig. 6GoD and data not shown). In agreement with the supershift data, most of the inducible AP-1 components are significantly less up-regulated at the protein level after Gal-S stimulation compared with IgE/Ag. As already described for other cell types, JunD, in contrast to other AP-1 proteins, is constitutively expressed and therefore apparently needs only posttranslational modification/activation for interacting with its corresponding site (33, 34).



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  		FIGURE 6. Gal-S stimulation specifically promotes JunD activation. A, Transient transfection experiment using a 3x TRE reporter gene construct. Stimuli (applied for 3 h) are indicated at the x-axis; normalized luciferase values are indicated at the y-axis. Values represent mean of triplicate experiments ± SD. B, EMSA with nuclear extracts of either nonstimulated (nst) or stimulated (1 h) CPII cells as indicated above the panels using radiolabeled probes indicated at the bottom. SP1 served as control for the quality of the nuclear extracts. C, Supershift analysis with nuclear extracts of either nonstimulated (nst) or stimulated (1 h) CPII cells at an AP-1 consensus-binding site as radiolabeled probe. Stimuli and Abs for the supershifts are indicated at the top; f, Free probe. Left and middle panels show a comparable short exposure of an autoradiogram (overnight); right panel shows a long exposure of the middle panel. D, Western blot analysis of a time kinetic of CPII cells after IgE/Ag and Gal-S stimulation for time points as indicated. Abs used are indicated to the right.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Initiation of signaling cascades at the tetrameric Fc{epsilon}RI fully depends on the clustering of this holo-receptor; a step triggered if receptor-bound IgE becomes cross-linked with polyvalent allergen. As one of the first consequences of this event, the src-family member Lyn phosphorylates tyrosine residues in the immunoreceptor tyrosine-based activation motifs of the Fc{epsilon}RI ß- and {gamma}-chains (27). In contrast to other receptors and their associated kinases, there is no increase observed in the specific kinase activity of Lyn for soluble substrates accompanying such an engagement, although phosphorylation of the receptor subunits is only detected after cross-linking (35). Currently, this peculiarity is addressed by two (complementary) models (35). First, the trans-phosphorylation model, in which receptor associated Lyn only phosphorylates receptor units in trans after being brought into close proximity by clustering (36). Second, the coalescence model, which emphasizes lipid:protein (or lipid:lipid interaction), compared with the protein:protein interaction of the first model in the initial activation. Here, receptor cross-linking leads to the coalescence of receptor (Fc{epsilon}RI)-associated lipids with lipid rafts containing Lyn. The subsequently formed DRM microdomains facilitate and stabilize an interaction of the receptor (Fc{epsilon}RI) with the kinase (Lyn) (23, 24). High levels of cholesterol and glycosphingolipids at the outer leaflet characterize these DRMs (37). In allergically activated mast cells, the importance of cholesterol in signaling was recently demonstrated by deprivation experiments leading to a clear decrease in the initial tyrosine phosphorylation steps (14). Our finding that glycosphingolipids added exogenously result in mast cell priming (concerning cytokine production) and activation (concerning degranulation and S1P secretion) not only implies that the outer leaflet component of DRMs has a similar key function, but if exogenously added is capable of initiating certain signaling steps independently. The characteristics of these glycosphingolipid-mediated signaling steps, as measured by induced tyrosine phosphorylation, enhanced kinase activity of Lyn and Syk, and the shift of Lyn into the DRM fraction are qualitatively indistinguishable and quantitatively even more pronounced and rapid than observed in allergically activated mast cells. In addition, the tyrosine kinase Syk, which in the case of IgE/Ag stimulation is mainly localized outside of the DRM fraction, is translocated into these microdomains after a Gal-S stimulation, illustrating that considerable differences already exist in the membrane-proximal events between the two stimuli. This variation at the early entry signal is most likely to be responsible for the more pronounced differences in terms of signaling strength and time kinetics further downstream, such as a relatively weak but extremely rapid activation of the MAPK pathway. The overall recruitment of more Lyn and Syk protein into the DRMs in the case of Gal-S stimulation without a correlated increase in the overall tyrosine phosphorylation could negatively impact on the balance between active (phosphorylated) and inactive kinases. An internal competition for substrate(s) and binding sites would thereby prohibit a prolonged transmission of the entry signal, which in analogy to Fc{epsilon}RI engagement/disengagement experiments might result in an inappropriate (untimely) termination of the initiated signaling cascades (27). As a consequence, this signal would be insufficient to promote de novo protein synthesis as observed for the AP-1 proteins (c-Jun, c-Fos, FosB) after Gal-S stimulation.

A second explanation for the differences of a Gal-S plus Iono. compared with an IgE/Ag stimulation may be that the addition of Gal-S does not mediate the formation of fully functional DRMs due to the lack of other glycosphingolipids (cerebrosides, gangliosides) and cholesterol, known to participate in the complex architecture of these microdomains. However, application of several other outer leaflet components alone (cerebrosides and gangliosides), or in combination with Iono. provided no priming or activation of CPII mast cells. In particular, a combination of Gal-S with different concentrations of cholesterol also showed no synergistic effect (data not shown).

Based on the recent isolation of several G protein-coupled receptors for different sphingolipids, binding of Gal-S to a G protein-coupled receptor might account for the differences between a Gal-S plus Iono. and an IgE/Ag stimulation (9, 38, 39, 40). G protein-coupled receptors are known to translocate into DRMs, and Gq-type receptors have been demonstrated to activate the MAPK pathway via Lyn and Syk, all facts in agreement with our results (41). However, the insensitivity of Gal-S plus Iono.-mediated TNF-{alpha} induction to pertussis toxin and the heavily phosphorylated FcR {gamma}-chain precipitated from Gal-S plus Iono.-stimulated CPII cells argues against such an involvement of a G protein-coupled receptor structure.

Both Gal-S and Gal-S plus Iono. stimulation lead to a predominantly JunD (which is expressed constitutively)-driven AP-1 response. This is most likely the consequence of the altered initial signal that is insufficient to induce de novo protein synthesis of other members of this transcription factor family (see above). From the concept of transient vs sustained signaling, it is known that in any given cell type different sets of transcription factors are selected according to the strength and duration of the initial signal (42, 43). As a result, either effector functions or proliferation and differentiation occur, respectively. In comparison to the transient type of signaling by IgE/Ag (see Erk1,2 and Jnk1 kinetics), which elicits mast cell effector functions, the described effects of Gal-S and Gal-S plus Iono. on characteristic kinases such as MAPKs have to be classified as "ultratransient," describing a third type of excitability of mast cells (transient after IgE/Ag or sustained after S1P treatment being the other two (4, 20)). Beaven and colleagues have recently described a similar time kinetic (ultratransient) for RBL-2H3 cells triggered via the G protein-coupled muscarinic m1 or the adenosine A3 receptors. However, in contrast to Gal-S activation these responses are pertussis toxin sensitive (30).

The "ultratransient" activation of MAPKs results in the elicitation of a subgroup of effector functions and priming for other suboptimal stimuli such as Iono. At the molecular level, it seems to be based on the fact that only those transcription factors already expressed are (posttranscriptionally) activated but new sets of transcription factors are not produced. JunD seems to play a key role in this type of response, in agreement with an already described dual role of this protein dependent on whether it is activated alone or in conjunction with other AP-1 transcription factors. In one case, JunD is regarded as acting antimitogenically and represses transcription in concert with other AP-1 factors (34). Contrary to this, it has been recently described, if solely activated, to be responsible for the transcriptional induction of nur77 in nerve growth factor receptor (TrkA)-triggered neuronal cells, for the stimulation of the proenkephalin gene, and the induction of IL-6 after TGFß stimulation in primary lung fibroblasts. (44, 45, 46). It is noteworthy that this is a clear difference to the situation observed after S1P treatment in CPII cells. In such a setting, it has recently been shown that the AP-1 components (FosB, c-Fos, c-Jun, and JunD) are induced in a pattern very similar to an IgE/Ag triggering, most likely due to a much longer activation of the MAPK pathways compared with Gal-S triggering (4). This difference is strongly underlined by the fact that Gal-S, in contrast to S1P, cannot overcome the inhibition observed after S treatment (E. E. Prieschl, unpublished observations) (4). Therefore, one has to conclude that though both molecules activate mast cells and chemically only differ by a sugar moiety substituting the phosphorus group, their mode of action is fundamentally different.

It is tempting to speculate that high levels of certain glycosphingolipids, as observed in various lipid storage diseases, not only activate/prime mast cells but also comprise triggers for other hemopoietic cell types. This is suggested by the fact that in twitcher mice, resembling an authentic animal model to Krabbe disease (lipid storage disease with high levels of Gal-S), not only degranulating mast cells, but also infiltration of eosinophils and polymorphonuclear cells and elevated levels of the cytokines TNF-{alpha} and IL-6 are detected (47, 48). In Gaucher patients (lipid storage disease with high levels of the Gal-S-related glucosphingosine), the predominant target population seems to be of the monocytic/myeloid lineage as glycosphingolipid-laden macrophages infiltrate the liver, spleen, and bone marrow resulting in hepatosplenomegaly, bone lesions, osteonecrosis, and cytopenia (49). Recent studies link the increased presence of proinflammatory cytokines such as TNF-{alpha}, M-CSF, IL-1ß, IL-6, and IL-8 found in many patients to the severity and progression of the disease (50, 51). In this respect, glycosphingolipids of various kinds seem to have an activating/priming potential on immune cells on a scale beyond the described influence of Gal-S on mast cells.


   Acknowledgments
 
We thank W. Phares, C. Williams, and E. Liehl for critical reading of the manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Eva E. Prieschl, Novartis Research Institute, Brunner Strasse 59, A-1235 Vienna, Austria. Back

2 Abbreviations used in this paper: S, sphingosine; BMMC, bone marrow-derived mouse mast cell; DRM, detergent-resistant membrane; Erk, extracellular signal-regulatory kinase; Gal-S, galactosylsphingosine; Iono., ionomycin; Jnk, c-jun NH2-terminal kinase; LAT, linker of activated T cells; MAPK, MAP kinase; MBP, myelin basic protein; Mek, MAPK kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; S1P, sphingosine-1-phosphate. Back

Received for publication December 20, 1999. Accepted for publication March 3, 2000.


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