From the Department of Medicine and Therapeutics and Division of
Biochemistry and Molecular Biology, University of Glasgow, Glasgow
G12 8QQ, Scotland, United Kingdom, Physiological
Laboratory, University of Cambridge, Cambridge CB2 3EG, United Kingdom,
and the § Department of Immunology, University of Glasgow,
Glasgow G11 6NT, Scotland, United Kingdom
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ABSTRACT |
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Aggregation of receptors specific for the
constant region of immunoglobulin G activates a repertoire of monocyte
responses that can lead ultimately to targeted cell killing via
antibody-directed cellular cytotoxicity. The high affinity receptor,
FcRI, contains no recognized signaling motif in its cytoplasmic tail
but rather utilizes the
-chain of Fc
RI as an accessory molecule
to recruit tyrosine kinases for signal transduction. We show here that,
in a human monocytic cell line primed with interferon-
, Fc
RI
mobilizes intracellular calcium stores using a novel pathway that
involves tyrosine kinase coupling to phospholipase D and resultant
downstream activation of sphingosine kinase. Moreover, Fc
RI is not
coupled to phospholipase C; hence, calcium release from intracellular stores occurred in the absence of any measurable rise in inositol triphosphate. Finally, as this novel activation pathway is also shown
to be responsible for mediating the vesicular trafficking of
internalized immune complexes for degradation, it is likely to play a
key role in controlling intracellular events triggered by Fc
RI.
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INTRODUCTION |
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The macrophage-specific receptor
(FcRI)1 for the constant
region (Fc) of IgG plays a central role in the clearance of immune complexes (1, 2). Fc
RI belongs to a family of receptors for IgG that
are distinguished by the affinity for ligand. While Fc
RI is a high
affinity IgG receptor, Fc
RII and Fc
RIII are both low affinity IgG
receptors (reviewed in Refs. 1 and 2). Aggregation of Fc
RI activates
macrophages to undergo a repertoire of responses that can ultimately
lead to cell killing through the process of antibody-directed cellular
cytotoxicity, a critically important feature in the body's defense
against virus-infected cells and in cancer surveillance (3, 4). Immune
complex aggregation of Fc
RI initiates signal transduction events,
which include protein tyrosine phosphorylation (5, 6) and tyrosine kinase-dependent calcium transients (7, 8). However, the cDNA for Fc
RI predicts an integral type I glycoprotein in which, unlike Fc
RIIa, the cytoplasmic tail contains no recognized signaling motifs (9). Fc
RI has been shown to associate noncovalently with the
signal-transducing
-chain (10), which contains an immunoreceptor
tyrosine activation motif (11, 12) in its cytoplasmic tail, and this
association is thought to allow aggregated Fc
RI to recruit and
activate soluble tyrosine kinases (13). The
chain was originally
identified in mast cells as a component of the high affinity IgE
receptor, Fc
RI, but has subsequently been found in macrophages in
the absence of the
-chain of Fc
RI (14). Thus, although expressed
in different cell types, the ligand recognition subunits (
-chains)
of Fc
RI and Fc
RI are able to use the same signal-transducing
molecule. Recently, Fc
RI has been shown to mobilize calcium
transients in a mast cell line through the activation of a novel
pathway involving sphingosine kinase (15). However, the precise details
of the signaling pathway and its relationship to tyrosine kinase
activation are as yet unclear.
In this study, we demonstrate that FcRI mobilizes calcium from
intracellular stores by activating sphingosine kinase in the absence of
phospholipase C activation and resultant generation of inositol
1,4,5-triphosphate (InsP3). We also show that
Fc
RI-stimulated activation of sphingosine kinase is downstream of
phospholipase D activation and that both these enzymes are dependent on
tyrosine kinase activation. Moreover, activation of this pathway is
necessary and sufficient to account for intracellular calcium
mobilization after Fc
RI aggregation in cytokine-primed U937 cells
and for efficient vesicular trafficking of internalized immune
complexes for degradation.
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MATERIALS AND METHODS |
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Receptor Aggregation--
U937 cells, a human monocyte cell line
(16), treated with 200 ng/ml interferon- for 18 h were used for
all experiments (8, 17). For the biochemical assays, approximately
3 × 106 cells were harvested and incubated with 1 µM human monomeric IgG (Serotec) to occupy surface
Fc
RI. Unbound IgG was removed by dilution and centrifugation of the
cells. The cells were resuspended in ice-cold Hepes-buffered saline
(HBS), and cross-linking antibody (goat anti-human IgG, 1:100 dilution)
was added. The cells were then warmed to 37 °C and harvested at
specified times for biochemical assay. Where the low affinity receptor
was specifically aggregated using anti-Fc
RIIa, the cells were loaded
with the monoclonal antibody 2e1 (1 µg) (Serotec) in the presence of
saturating concentrations (3 µM) of human IgG4 (to block
binding of the Fc portion of 2e1 to Fc
RI). After removal of excess
antibody, anti-Fc
RIIa was aggregated by the addition of goat
anti-mouse IgG F(ab) (1:100 dilution).
Measurement of Sphingosine Kinase--
Sphingosine kinase was
assayed as described by Olivera et al. (18). Briefly,
reactions were terminated at the times specified in the figures by the
addition of ice-cold phosphate-buffered saline (PBS). After
centrifugation, the cells were resuspended in ice-cold 0.1 M phosphate buffer (pH 7.4) containing 20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, phosphatase
inhibitors (20 mM ZnCl2, 1 mM
sodium orthovanadate, and 15 mM sodium fluoride), protease
inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF), and 0.5 mM 4-deoxypyridoxine. Cells
were disrupted by freeze thawing and centrifuged at 105,000 × g for 90 min at 4 °C. Supernatants were assayed for
sphingosine kinase activity using sphingosine (Sigma) and
[32P]ATP (2 µCi, 5 mM) as specified by
Olivera et al. (18). After incubation, products were
separated by TLC on silica gel G60 using chloroform/methanol/acetic
acid/water (90:90:15:6) and visualized by autoradiography. The
radioactive spots corresponding to sphingosine phosphate were scraped
and counted in a scintillation counter.
Measurement of Sphingosine 1-Phosphate-- Sphingosine 1-phosphate concentrations were measured as described by Olivera and Spiegel (19). Briefly, cells were preincubated overnight (15 h) in media containing [3H]serine (20 µCi ml) to label cellular sphingolipids and free sphingosine pools. Following labeling, the cells were washed in ice-cold RPMI 1640, 10 mM HEPES, 0.1% bovine serum albumin (RHB medium) and resuspended in ice-cold RHB medium containing 0.1 mM L-canaline and the pyridoxal phosphate analog 4-deoxypyridoxine (0.5 mM) to inhibit the pyridoxal-dependent sphingosine-1-phosphate lyase. Cells were then stimulated by the addition of cross-linking antibody and warming to 37 °C, and the reactions were terminated at specified times. Cells were harvested by centrifugation, and the lipids were extracted and analyzed by TLC on silica gel G60 using chloroform/methanol/acetic acid/water (90:90:15:6). Standard sphingosine 1-phosphate was applied with the samples, and the lipids were visualized using iodine vapors. Bands corresponding to sphingosine 1-phosphate were excised from the plate and counted by liquid scintillation spectrometry. Results were calculated as a percentage of the total radioactivity incorporated in the lipids. Data presented are the mean ± S.D. of triplicate measurements, and the results shown are representative of three different experiments.
Measurement of Inositol Phosphate and DAG Generation-- Inositol phosphates were assayed essentially as described by Harnett and Harnett (20). Briefly, U937 cells were labeled with myo-[3H]inositol (1 µCi/106 cells) for 16 h at 37 °C. The cells were washed three times and resuspended (at 1-3 × 107 cells/ml) in RHB medium, pH 7.4, at 4 °C. Following stimulation, the cells were harvested, resuspended in 100 µl of HBS, transferred to glass trident vials, and extracted by the addition of 0.94 ml of chloroform/methanol (1:2) on ice for 10 min. A Bligh-Dyer phase separation was achieved by the addition of 0.31 ml of chloroform and 0.31 ml of water, vortexing, and centrifugation at 270 × g for 5 min. Levels of [3H]InsP3 or total [3H]inositol phosphates (reaction mixture containing 10 mM LiCl) were determined by liquid scintillation counting of fractions eluted following Dowex (formate form) ion exchange chromatography of aliquots of the aqueous phase. Results were calculated as a percentage of the total radioactivity incorporated in the lipids. Data presented are the mean ± S.D. of triplicate measurements, and the results shown are representative of three different experiments.
DAG Assay--
Mass DAG was measured as described by Briscoe
et al. (21). The lower organic phase of Bligh-Dyer
extractions was dried in vacuo, and the lipids were
solubilized in a Triton X-100/phosphatidylserine mixture. Briefly,
phosphatidylserine (30 µl; supplied as 25 mM stock from
Lipid Products) was dried under nitrogen and then probe-sonicated in
2.5 ml of 10 mM imidazole buffer, pH 6.6, containing 0.6%
(w/v) Triton X-100, until the solution was optically clear. Aliquots (50 µl) were added to the lipid samples, which were then sonicated in
a bath for 30 min. Once sonicated, 20 µl of 250 mM
imidazole buffer, pH 6.6, containing 250 mM NaCl, 62.5 mM MgCl2, 5 mM EGTA, and 10 µl of
freshly prepared 100 mM dithiothreitol was added to the
solubilized lipid. Escherichia coli diacylglycerol kinase (Calbiochem) was added to a final concentration of 50 milliunits/ml, and the reaction was started by the addition of 10 µl of 5 mM ATP containing 1 µCi of [-32P]ATP
made up in 100 mM imidazole, pH 6.6; this results in a
final ATP concentration of 0.5 mM in a final reaction
volume of 100 µl. The tubes were incubated at 30 °C for 30 min.
The reaction was stopped by the addition of 1 ml of
chloroform/methanol/HCl (150:300:2). After 10 min, 300 µl of
chloroform and 400 µl of H2O are added. The tubes are
vortexed and centrifuged at 270 × g for 5 min to
promote phase splitting and washed once with 1 ml of a synthetic upper
phase. The samples were then dried in vacuo and solubilized
in 40 µl of chloroform/methanol (19:1), and 20 µl was spotted onto
silica TLC plate (Whatman catalog no. 4861720, 10 × 20 cm K6F).
The plates were developed in chloroform/methanol/acetic acid
(38:9:4.5), and radiolabeled bands were located by autoradiography or
phosphor imaging. The phosphatidic acid (PtdOH) band (relative to
standards) was scraped into scintillation vials, scintillant was added,
and the associated radioactivity was determined by liquid scintillation
counting.
Measurement of Phospholipase D (PLD) Activity-- PLD activity was measured by the transphosphatidylation assay (21). Briefly, U937 cells were labeled (106 cells/ml) with [3H]palmitic acid (5 µCi/ml) in RPMI 1640 medium containing 10% (v/v) fetal calf serum for 16 h. Following labeling, the cells were washed in ice-cold RHB medium, resuspended at 2 × 106 cells/ml, and incubated at 37 °C for 15 min in RHB medium containing butan-1-ol (0.3% final). Specific Fc receptors were cross-linked as described above, and after the times indicated, cells were extracted by Bligh-Dyer phase separation. An aliquot of the lower organic phase was removed and dried down under vacuum (Jouan, catalog number RC1022), and the samples were redissolved in 25 µl of chloroform/methanol (19:1, v/v), containing 40 µg of unlabeled phosphatidylbutanol (Lipid Products, South Nutfield, Surrey, UK) as standard, and applied to prerun, heat-activated TLC plates (20 × 20 cm, Silica gel 150A grooved plates, Whatman). The plates were developed in the organic phase of the solvent, ethyl acetate/2,2,4-trimethylpentane/acetic acid/water (11:5:2:10) for approximately 90 min, and the position of the phosphatidylbutanol product was detected using iodine vapor. [3H]PtdBut-containing silica indicated by the phosphatidylbutanol standard was then scraped into scintillation fluid and counted. Results were calculated as a percentage of the total radioactivity incorporated in the lipids. Data presented are the mean ± S.D. of triplicate measurements, and the results shown are representative of three different experiments.
Measurement of Tyrosine Phosphorylation by Western Blot-- U937 cells were loaded with human IgG and cross-linked as described earlier. After washing in PBS, the cells were lysed with ice-cold radioimmune precipitation lysis buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml CLAP (1 mg/ml each of chymostatin, leupeptin, antipain, and pepstatin), 1 mM sodium orthophosphate, and 1 mM sodium fluoride for 30 min. Cellular debris was removed by centrifugation at 13,000 rpm for 15 min, and the cell lysates were incubated with an agarose-conjugated anti-phosphotyrosine monoclonal antibody (clone 4G 10; Upstate Biotechnology, Inc.) at 4 °C overnight. Phosphotyrosine proteins were then harvested by centrifugation of the agarose beads and were then dissociated from the beads by boiling in sample buffer (22) containing 50 mM dithiothreitol for 15 min. Samples were run in a 10% SDS-polyacrylamide gel (23). After electrophoresis, the proteins were transferred to a nitrocellulose membrane (0.2-µm pore size) as described by Towbi et al. (24). The presence of tyrosine-phosphorylated proteins was then detected by Western blotting with a monoclonal anti-phosphotyrosine antibody (clone 4G 10; Upstate Biotechnology). Western blots were developed using the ECL system (Amersham Pharmacia Biotech).
Measurement of Cytosolic Calcium-- Cytosolic calcium was measured in cell populations at 37 °C using a Cairn Research Spectrophotometer as described previously (8). Cells were loaded with Fura2 and human monomeric IgG in HBS supplemented with 1 mM Ca2+ to prevent depletion of calcium stores. After dilution and centrifugation to remove excess dye and antibody, the cells were resuspended in a small volume of HBS, 1 mM Ca2+ to give a final density of 106 cells/100 µl. From this, cells were added to stirred cuvettes containing 1.4 ml of nominally Ca2+-free HBS (at 37 °C) in a Cairn Spectrophotometer system (Cairn Research Ltd.). Excitation wavelengths of 340, 360, and 380 nm were provided by a filter wheel rotating at 35 Hz in the light path. Emitted light was filtered by a 485-nm-long pass filter, and samples were averaged to give a data point every 500 ms. The background-corrected 340/380 ratio was calibrated using the method of Grynkiewicz et al. (25). Following each experiment, cells were lysed by the addition of 50 µM digitonin in the presence of external 2 mM Ca2+ to give an Rmax value. Rmin was subsequently determined by the addition of 20 mM EGTA (pH 7.4) in the presence of an equimolar concentration of Tris base.
Measurement of Endocytosis and Rate of Trafficking for
Degradation--
Interferon- (IFN-
)-treated cells were harvested
and washed in phosphate-buffered saline (PBS), 1% bovine serum
albumin. The cells were then loaded with 125I-labeled IgG
as described previously (17). After removal of nonbound radiolabel by
dilution and centrifugation, cross-linking antibody was added, and the
cells were warmed to 37 °C for the given times.
Endocytosis--
The rate of endocytosis was assessed by
measuring the rate of internalization of radiolabeled surface immune
complexes. At time 0, triplicate aliquots of cells were harvested into
ice-cold PBS (pH 7.4), and this was counted in a Packard -counter to
provide a measure of the total counts bound to the cell surface. To
measure the proportion of radiolabeled immune complexes internalized
after incubation at 37 °C, any surface-bound radiolabeled immune
complexes can be stripped from the cell by incubating the cells in
ice-cold acidified PBS (pH 2.0) (17). Radiolabeled immune complexes
that have been internalized remain trapped inside the cell and cannot be released by this acid wash. Thus, to assess the rate of
internalization, aliquots of cells were transferred at given times into
ice-cold acidified PBS (pH 2.0) for 5 min to strip off cell surface
radiolabeled immune complexes (17). The cells were then centrifuged,
and the pellets were counted in a Packard
-counter to yield the
counts that had been internalized, or the cell-associated counts. The cell-associated counts for each time point were then expressed as the
percentage of total counts bound at time 0 to provide a measure of the
rate of internalization of the immune complexes.
Degradation-- After warming the cells to 37 °C for long time intervals, the proportion of cell-associated counts was observed to fall. To determine whether this reduction in cell-associated counts represented degradation of the immune complexes, the supernatant following the cell incubation was examined for the presence of trichloroacetic acid-soluble radiolabel indicating that the radiolabeled IgG had been degraded. Thus, cells were also harvested at the same time points to measure the rate of degradation of the internalized counts. Cells were centrifuged, the supernatants were harvested, and trichloroacetic acid was then added to these supernatants. After incubation on ice for 60 min, the samples were centrifuged at 12,000 × g at 4 °C, and the supernatants were counted to provide a measure of the trichloroacetic acid-soluble counts in the supernatant. The results were expressed as a percentage of the initial cell-associated counts at time 0.
The results shown are the mean ± S.D. of triplicate measurements and are representative of three different experiments. ![]() |
RESULTS |
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Aggregation of FcRI Activates Sphingosine Kinase in a Tyrosine
Kinase-dependent Manner--
In IFN-
-primed U937 cells,
aggregation of Fc
RI with surface-bound immune complexes results in
calcium transients in the form of a single spike (see Ref. 8 and Fig.
1A). The Fc
RI-associated accessory transducing molecule,
-chain, has recently been reported to mobilize calcium via activation of sphingosine kinase when coupled
to the high affinity IgE receptor, Fc
RI (15). Thus, to compare the
nature of this Fc
RI-calcium response to that of Fc
RI, the effect
of DL-threo-dihydrosphingosine (DHS) on the release of calcium from intracellular stores was determined.
Pretreatment of cells with 25 µM DHS completely abolished
the Fc
RI-mediated rise in cytosolic calcium, indicating that
intracellular calcium stores are mobilized in these cells in a similar
fashion to that observed for Fc
RI in mast cells (15). The calcium
stores were intact in cells treated with DHS, since the subsequent
addition of thapsigargin (250 nM) resulted in a prompt
increase in cytosolic calcium, thereby demonstrating that the failure
to observe a rise in calcium following aggregation of Fc
RI in cells
pretreated with DHS was not secondary to depletion of intracellular
calcium stores.
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Aggregation of FcRI Activates Phospholipase D and Not
Phospholipase C in a Tyrosine Kinase-dependent
Manner--
Since immune complex aggregation of Fc
RI has previously
been reported to lead to tyrosine phosphorylation of phospholipase C
1 (5) with presumed generation of InsP3 and DAG, the
role of this phospholipid signaling pathway in mediating the cytosolic calcium response was also investigated. Surprisingly, no increase in
InsP3 could be detected (data not shown). Since
InsP3 generation can be transient in nature, the
accumulation of total inositol phosphates (InsPs) was measured to
ensure that any small transient InsPs signals did not go undetected. No
accumulation of total InsPs over 20 min could be detected in
IFN-
-primed U937 cells after aggregation of Fc
RI (Fig.
2A). Phospholipase C signaling was, however, functional in these cells, since aggregation of a related
immune receptor, the low affinity IgG receptor (Fc
RIIa), using
monoclonal antibodies resulted in an easily measurable accumulation of
InsP3 (data not shown) and total InsPs (Fig.
2A). Unlike Fc
RI, the low affinity receptor possesses an
integral, albeit unconventional, immunoreceptor tyrosine activation
motif in its cytoplasmic tail; the tyrosine residues are separated by
an unusually long intervening sequence (26). Taken together, these data
indicate that the high affinity receptor, Fc
RI, mobilizes calcium
stores through a novel pathway that, unlike the low affinity receptor
(Fc
RIIa), does not involve InsP3.
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Activation of Sphingosine Kinase Is Downstream of Phospholipase D Activation-- To assess the relative relationship of activation of PtdCho-PLD and sphingosine kinase, studies were initially undertaken to explore comparative kinetics of activation and use of selective inhibitors. However, comparison of the relative kinetics was complicated by the difference in the assay characteristics for measuring sphingosine kinase and phospholipase D. Thus, sphingosine kinase is measured as an in vitro kinetic kinase assay, whereas the assay for phospholipase D relies on the accumulation of a nonhydrolyzable product. The difference in assay characteristics, therefore, precluded definitive early comparative time course analysis. The relationship of phospholipase D and sphingosine kinase activation was therefore addressed by examining selective inhibitors of the two enzymes.
To determine whether sphingosine kinase activation was upstream or downstream of phospholipase D, cells were preincubated with butan-1-ol or butan-2-ol for 20 min before aggregation of Fc
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Tyrosine Phosphorylation Is Triggered Promptly by Aggregation of
FcRI and Is Upstream of both Phospholipase D and Sphingosine
Kinase--
Tyrosine phosphorylation events were monitored in these
cytokine-primed U937 cells after aggregation of Fc
RI by
immunoprecipitating tyrosine-phosphorylated proteins with a monoclonal
antibody to phosphotyrosine. Consistent with other reports (5-7, 27,
28), the addition of cross-linking antibody to form surface immune complexes resulted in the prompt appearance of a large number of
tyrosine-phosphorylated proteins. Preincubating cells with either
butan-1-ol (0.3%) or DHS (25 µM) did not influence the pattern of tyrosine phosphorylation (Fig.
4), results consistent with our findings
that both PtdCho-PLD (Fig. 2C) and sphingosine kinase (Fig.
1C) activation are downstream of tyrosine kinase activation.
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Activation of Phospholipase D Is Necessary for both Mobilization of
Intracellular Calcium and for Trafficking of Immune Complexes for
Degradation--
The release of intracellular stores of calcium by
aggregation of FcRI was significantly inhibited by pretreating the
cells with 0.3% butan-1-ol (Fig.
5A), thus providing further
support for the role of this pathway in mobilizing calcium and the
concept that PtdCho-PLD is upstream of sphingosine kinase. The
possibility that butan-1-ol affected calcium mobilization through
nonspecific effects was ruled out, since the subsequent addition of
thapsigargin (250 nM) resulted in a prompt response in
cytosolic calcium. In addition, butan-1-ol had no effect on the
InsP3-dependent mobilization of calcium
following aggregation of the related low affinity receptor, Fc
RIIa
(Fig. 5A). The difference in release of calcium after thapsigargin is not likely to be significant following manual injection
as undertaken here. Although the speed of calcium release by
thapsigargin can be influenced by a number of intracellular factors
such as the amount of calcium in the stores, it is well recognized that
there is considerable variability between runs for
thapsigargin-mediated calcium release, and the largest influence is the
rate of addition of thapsigargin and its mixing in the cuvette (29,
30).
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DISCUSSION |
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Taken together, these data indicate that FcRI in
cytokine-primed U937 cells is coupled through tyrosine kinase
activation to a novel pathway responsible both for mobilizing calcium
transients through an InsP3-independent route and for
trafficking internalized immune complexes for degradation. This novel
pathway involves the activation of PtdCho-PLD, in the absence of
measurable activation of phospholipase C, and this is upstream of
activation of sphingosine kinase, which generates sphingosine
1-phosphate.
Sphingosine 1-phosphate has been proposed previously to play a role in
mobilizing calcium from intracellular stores (32-34). However, this
proposal has proven highly controversial due to the presence of
extracellular G protein-coupled receptors for sphingosine 1-phosphate
(35, 36), which are able to mobilize calcium through conventional
InsP3 receptor-dependent pathways. The recent
cloning of the SCaMPER receptor (37) provides additional evidence that
sphingoid derivatives are able to engage intracellular receptors and
effect calcium release from stores independently of InsP3
generation. The data presented here provide evidence for specific
immune receptor triggering of this pathway in myeloid cells. Thus,
aggregation of FcRI resulted in the rapid activation of sphingosine
kinase and consequent cellular increases in sphingosine 1-phosphate
concentrations. In these same cells, neither product of phospholipase C
activation could be detected; no accumulation of total InsPs could be
measured even in the presence of lithium chloride to prevent breakdown.
Moreover, the observed increase in DAG could be completely blocked by
pretreatment of cells with butanol, indicating PtdCho-PLD rather than
phospholipase C activation as the source of the DAG. In contrast,
aggregation of an alternative immune receptor, Fc
RIIa, on these
cells, resulted in increases in both phospholipase
C-dependent DAG and inositol phosphate generation, indicating that this pathway is intact and functional in these cells
and that the assays used were potentially able to detect any such
receptor-triggered changes.
Taken together, the data presented here suggests that the high affinity
receptor, FcRI, mobilizes intracellular calcium through this
sphingosine kinase-dependent, InsP3-independent
pathway. In this respect, Fc
RI is behaving like the high affinity
IgE receptor, Fc
RI, in mast cells (15). Of interest, both these receptors use the same signal-transducing molecule (
-chain) (10) to
recruit soluble tyrosine kinases to mediate cellular activation. However, the mechanism of coupling of tyrosine kinases to sphingosine kinase activation following Fc
RI aggregation in mast cells was unclear (15). Here, we demonstrate that PtdCho-PLD is activated following aggregation of Fc
RI in myeloid cells and that sphingosine kinase activation is dependent on PtdCho-PLD activation. The immediate product of PtdCho-PLD is phosphatidic acid, and this is subsequently converted to DAG through the action of phosphatidic acid
phosphohydrolase. Previous studies have shown that sphingosine kinase
is activated by phosphatidic acid (38) and not by DAG (38), a product
of both phospholipase D and phospholipase C. Our finding that
sphingosine kinase is downstream of PtdCho-PLD is, therefore,
consistent with this in vitro work. Moreover, both
components of this novel Fc
RI-coupled intracellular signaling
pathway involving the sequential activation of PtdCho-PLD and
sphingosine kinase depend on tyrosine kinase activation. This finding
is consistent with previous in vitro studies demonstrating
that v-Src can activate PLD (39).
Aggregation of FcRI in myeloid cells triggers a number of effector
functions. The novel intracellular signaling pathway demonstrated here
appears to be functionally interactive/associated with these. Thus,
previous studies have implicated phosphatidic acid in modulating neutrophil function, in particular by influencing the respiratory burst/NADPH oxidase cascade (40). In the study reported here, inhibiting this pathway at either the PtdCho-PLD or sphingosine kinase
level reduced or abolished the ability of this receptor to mobilize
calcium from intracellular stores. In addition, the inhibition of
PtdCho-PLD significantly slowed the rate of trafficking of internalized
immune complexes for degradation. Of interest, ADP-ribosylation factor
plays a major role in regulating vesicular trafficking, and this small
molecular weight G protein has also been demonstrated to regulate
phospholipase D activity (41). The finding that Fc
RI is coupled to
the release of intracellular calcium stores and vesicular trafficking
via a novel pathway that does not use InsP3 has profound
implications for the development of strategies for therapeutic
intervention against differential myeloid responses to immune
complexes.
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ACKNOWLEDGEMENTS |
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We thank Sandra Seatter and T. McShane for
technical support and Dr. Stewart Sage for use of the Cairn
spectrophotometer and for helpful discussions. We are grateful to Dr.
W. Cushley and Professor F. Y. Liew for helpful suggestions in
preparing this paper. We thank Bender Wein for the IFN-.
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FOOTNOTES |
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* This work was supported by grants from the MacFeat Bequest at the University of Glasgow and from the Scottish Hospital Endowment Research Trust and Biotechnology and Biological Sciences Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ A Medical Research Council Senior Fellow.
To whom correspondence should be addressed: Davidson Bldg.,
University of Glasgow, Glasgow G12 8QQ, Scotland, UK. Tel.:
0141-330-5189; Fax: 0141-330-4620; E-mail:
janet.allen{at}bio.gla.ac.uk.
1
The abbreviations used are: FcRI, high
affinity immunoglobulin G receptor; Fc
RI, high affinity
immunoglobulin E receptor; Fc, constant region; IFN-
,
interferon-
; InsP3, inositol 1,4,5-triphosphate; InsPs,
total inositol phosphates; DAG, diacyl glycerol; PBS,
phosphate-buffered saline; HBS, HEPES-buffered saline; DHS,
DL-threo-dihydrosphingosine; PtdOH,
phosphatidic acid; PtdBut, phosphatidylbutanol; PLD,
phospholipase D; PtdCho-PLD, phosphatidylcholine-specific
phospholipase D.
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REFERENCES |
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