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Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI on rat basophilic leukemia (RBL) cells
initiates a signaling cascade leading to degranulation of the cells and
the release of inflammatory mediators. Inhibitors that disrupt
microfilaments, such as latrunculin and cytochalasin D, do not cause
any degranulation on their own, but they do enhance FcRI-mediated
degranulation. Dose-response studies show a good correlation between
inhibition of actin polymerization and increased degranulation. In RBL
cells, latrunculin causes a decrease in basal levels of filamentous
actin (F-actin), while cytochalasin D does not. This is particularly
evident in the Triton-insoluble pool of F-actin which is highly
cross-linked and associated with the plasma membrane. A concentration
of 500 nM latrunculin decreases the basal level of Triton-insoluble
F-actin by 60–70% and total F-actin levels by 25%. Latrunculin
increases both the rate and extent of Ag-induced degranulation while
having no effect on pervanadate-induced degranulation. Pervanadate
activates the signaling pathways directly and bypasses the
cross-linking of the receptor. RBL cells, activated through FcRI in
the presence of latrunculin, show increased phospholipase activity as
well as increased tyrosine phosphorylation of Syk and increased
tyrosine phosphorylation of the receptor itself by the tyrosine kinase
Lyn. This indicates that the very earliest signaling events after
receptor cross-linking are enhanced. These results suggest that actin
microfilaments may interact, either directly or indirectly, with the
receptor itself and that they may regulate the signaling process at the
level of receptor phosphorylation. Microfilaments may possibly act by
uncoupling Lyn from the cross-linked receptor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) that binds IgE in a 1:1 ratio. The
receptor consists of an 
-chain, which binds the IgE, a ß-chain,
and two 
-chains. The ß- and -chains are involved in signal
transduction. Cross-linking of these receptors by multivalent Ag, which
binds to the IgE, is the critical event in the activation of these
cells. It is currently thought that there is some Lyn, a Src family
tyrosine kinase, that is constitutively associated with the ß-chain
5, 6, 7 . Upon cross-linking of the receptors, there is
transphosphorylation of the immunoreceptor tyrosine-based activation
motifs (ITAM) found on the ß- and -chains 8 . This leads to the
recruitment of more Lyn as well as Syk, which binds to the -chain
ITAM region through an SH2 domain. Syk is activated by binding to the
-chain as well as its tyrosine phosphorylation by Lyn. Activated Syk
is then involved in the action of phospholipases C, A2, and
D. Phospholipase C hydrolysis of the polyphosphoinositides leads to an
increase in intracellular Ca2+ and activation of protein
kinase C (PKC). Phospholipase D hydrolyzes phosphatidylcholine, which
is responsible for most of the diacylglycerol production and the
sustained activation of PKC. Phospholipase A2 is
responsible for the generation of arachidonic acid, which is converted
to the leukotrienes and PGs. These mediators are important elements of
the inflammatory response. These signals lead to degranulation and the
release of preformed mediators, such as histamine, serotonin, and
various proteases 9 . Activation of mast cells also leads to the
synthesis and release of various cytokines.
Some of the signals that are generated lead to degranulation, some lead
to increased gene transcription, and some lead to the polymerization of
actin and various morphological changes in the cell. In general,
approximately 50% of the actin in a cell is monomeric (G-actin) and
the rest is polymerized into filaments (F-actin) 10, 11, 12 .
There are well over 60 actin binding proteins in the cell that help
regulate its state inside the cell 13, 14, 15 . These proteins are known
to cap, bundle, cross-link, as well as sequester actin monomers. Upon
activation through FcRI, this large pool of monomeric actin is
polymerized into microfilaments. It has been shown that activation of
PKC 16, 17 is important in FcRI-mediated actin polymerization and
that this signal is upstream of phosphatidylinositol kinase and
phosphatidylinositol monophosphate kinase 18 . These kinases are
involved in the production of phosphatidylinositol monophosphate and
phosphatidylinositol bisphosphate, which have been shown to dissociate
many actin binding proteins from actin, thus allowing polymerization to
occur. Morphologically, the cells tend to flatten after activation and
form actin-rich plaques on their ventral surface 19 . These plaques
contain F-actin as well as focal adhesion proteins such as vinculin and
talin 20 . In addition, while control cells show numerous microvilli,
in FcRI-activated cells these microvilli are rapidly transformed
into large membrane ruffles 16 .
The exact role for these morphological changes and for the receptor-mediated actin polymerization is not known. It has been hypothesized that actin microfilaments might be involved in the down-regulation of the degranulation response. The purpose of this study was to investigate at what stage microfilaments are involved in the degranulation process. It was determined that there is a good correlation between inhibition of actin polymerization and increases in phospholipase activity, tyrosine kinase activity, and degranulation. Microfilaments appear to down-regulate the response by affecting the level of receptor tyrosine phosphorylation, thus affecting all the signaling pathways involved.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RBL-2H3 cells were grown in Eagle’s MEM supplemented with 20% FCS, 4 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, MEM nonessential amino acids, and penicillin-streptomycin. The cells were harvested by incubating them in PBS containing 5 mM EDTA for 10 min at 37°C. RBL cells were sensitized by resuspending them in complete Eagle’s MEM at a concentration of 3 x 106 cells/ml. IgE that is specific for DNP 21 was added to a concentration of 1 µg/ml, and the cells were rotated at 37°C for 2 h. The cells were then washed three times in PBS before resuspension in HBSS containing 0.2% BSA.
Reagents
Latrunculin was purchased from Biomol (Plymoth Meeting, PA). 7-Nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) was obtained from Molecular Probes (Eugene, OR), while DNP-BSA, containing at least 40 mol of DNP/mol of protein, was purchased from Calbiochem (La Jolla, CA). Protease inhibitor mixture tablets were purchased from Boehringer Mannheim (Indianapolis, IN). [3H]myoinositol was purchased from Amersham (Arlington Heights, IL), while [3H]arachidonic acid, [3H]myristic acid, and the Renaissance chemiluminescence kit were obtained from New England Nuclear-DuPont (Boston, MA). Rabbit anti-Syk was obtained from both Santa Cruz Biotechnology (Santa Cruz, CA) and Dr. Reuben Siraganian (National Institutes of Health, Bethesda, MD), PY20 (anti-phosphotyrosine) was from Transduction Laboratories (Lexington, KY), and 4G10 (anti-phosphotyrosine) was obtained from Upstate Biotechnology (Lake Placid, NY). The mouse mAb, JRK, which reacts with the first 23 amino acids of the N-terminal cytoplasmic domain of the ß-chain, was provided by Dr. Juan Rivera (National Institutes of Health). Protein A was purchased from Pierce (Rockford, IL). All secondary Abs and conjugates were obtained from Zymed (South San Francisco, CA). All other reagents were purchased from Sigma (St. Louis, MO).
Pervanadate was prepared according to previously published methods 22 . Equal volumes of 10 mM sodium orthovanadate in HBSS containing 50 mM HEPES (pH 7.2) and 10 mM H2O2 in the same buffer were mixed together at room temperature for 15 min. Any remaining H2O2 was removed by adding catalase (1000 U/ml, final concentration) for 5 min. The pervanadate solution was then used immediately.
F-actin assay
A slightly modified version of the assay developed by Howard and his colleagues 23 has been used. IgE-sensitized RBL cells were added to BSA-coated 12- x 75-mm polystyrene tubes in 200 µl (5 x 105 cells/tube). The cells were preincubated with latrunculin for 15 min at 37°C before the addition of DNP-BSA (50 ng/ml). To measure total F-actin, the reaction was stopped by the addition of formaldehyde to a final concentration of 3.7% for 15 min at room temperature. The cells were permeabilized by the addition of ice-cold solubilizing buffer (10 mM imidazole (pH 7.2), 40 mM KCl, 10 mM EGTA, 1% Triton X-100, 1 mM PMSF, and protease inhibitor mixture tablet). After a single wash with PBS at 2200 rpm for 10 min, the supernatant was removed, and 100 µl of NBD-phallacidin (3.3 x 10-7 M) was added to the pellet for 2 h at room temperature. The material was then washed twice with PBS, and the bound NBD-phallacidin was extracted by incubating the pellet with 1.5 ml of methanol overnight in the dark. Triton-insoluble F-actin was measured by activating the cells and then stopping the reaction with ice-cold solubilizing buffer for 15 min at 4°C. The cells were fixed with 3.7% formaldehyde for 15 min and washed, and NBD-phallacidin was added as outlined above. The extracts from both the prefix and postfix procedures were centrifuged to remove any insoluble material, and the relative fluorescence was measured using either a Perkin-Elmer (Norwalk, CT) or an AMINCO-Bowman series 2 spectrofluorometer (Rochester, NY) with an excitation wavelength of 465 nm and an emission wavelength of 535 nm.
Degranulation assay
IgE-sensitized RBL cells were added to BSA-coated tubes and preincubated with latrunculin for 15 min. The cells were activated with DNP-BSA for 45 min and pelleted by centrifugation, and the supernatant was tested for ß-hexosaminidase activity. Total activity was determined by lysing the cells in 300 µl of 0.1% Triton. Briefly, 10 µl of supernatant or cell lysate and 30 µl of 3 mM p-nitrophenyl-N-acetyl-ß-D-glucosaminide (in 0.1 M citrate (pH 4.5)) were added to each well of a 96-well plate for 1 h at 37°C. Color was developed by adding 150 µl of 0.1 M Na2CO3 and 0.1 M NaHCO3 (pH 10.0), and the assay was read at 405 nm using a Titertek Multiskan Plus plate reader (Huntsville, AL). Neither latrunculin nor cytochalasin D had any effect on the ß-hexosaminidase assay.
Intracellular Ca2+ measurements
Intracellular Ca2+ was measured in Ag-activated RBL cells using indo-1. IgE-sensitized cells (3 x 106 cells/ml) were incubated in HBSS containing 0.25 mM sulfinpyrazone and 1 mM indo-1/AM for 30 min at 37°C. The cells were washed twice, resuspended in buffer in a cuvette, and placed in a stirred, cuvette holder at 37°C. Intracellular Ca2+ was monitored using an AMINCO-Bowman AB2 spectrofluorometer using an excitation wavelength of 350 nm and emission wavelengths of 410 and 485 nm.
Assays for phospholipase activity
Phospholipase C activity was measured by monitoring the production of inositol phosphates as previously described 24 . Briefly, cells were cultured overnight in medium containing [3H]myoinositol. Cells were washed, suspended in buffer containing LiCl, activated with DNP-BSA, and extracted with chloroform/methanol. Radiolabeled inositol phosphates were isolated using Dowex chromatography 25, 26 . Phospholipase A2 activity was assayed by following the release of [3H]arachidonic acid and its metabolites. Cellular phospholipids were labeled with [3H]arachidonic acid. The cells were washed, suspended in buffer in tubes, and activated with DNP-BSA, and [3H]arachidonic acid released into the supernatant was determined by liquid scintillation counting. Phospholipase D activity was monitored by the production of phosphatidylethanol 24, 27 . Briefly, cellular phospholipids were labeled by growing the cells overnight in medium containing [3H]myristic acid. The cells were sensitized with IgE, washed, resuspended in buffer containing 0.5% ethanol, and activated with DNP-BSA. Phospholipids were extracted using chloroform/methanol, and [3H]phosphatidylethanol was isolated by TLC using a double one-dimensional system developed by Kennerly and colleagues 28 .
Immunoprecipitations and immunoblotting
IgE-sensitized RBL cells in suspension were activated with 50
ng/ml DNP-BSA at 37°C. The reaction was stopped by the addition of
ice-cold solubilizing buffer (1% Triton-X-100, 100 mM NaCl, 120 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 2 mM sodium orthovanadate, 100 mM sodium
fluoride, 50 mM sodium pyrophosphate, PMSF, and protease inhibitor
tablets) for 30 min at 4°C. The samples were spun in a microfuge at
20,000 x g for 15 min, and the supernatants were
placed into new tubes. RBL cell lysates from 2 x 106
cells were immunoprecipitated with either 5 µg/ml of a mouse
monoclonal anti-FcRIß for 1 h at 4°C or overnight with
2.5 µg/ml of a rabbit anti-Syk. This was followed by a 1-h
incubation with either goat anti-mouse Ig or goat anti-rabbit
Ig bound to Sepharose beads. The beads were washed three times, and
bound proteins were eluted by adding SDS sample buffer and placing the
tubes in a boiling water bath for 3 min. The proteins were resolved on
a 12.5% SDS-PAGE gel and then transferred to an Immobilon P membrane
using a semidry transfer apparatus. The samples were immunoblotted with
4G10, an anti-phosphotyrosine Ab, followed by goat anti-mouse
horseradish peroxidase. The New England Nuclear Renaissance
chemiluminescence kit was used to visualize the results.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Latrunculin reduces total F-actin
levels only moderately in unstimulated cells even at concentrations as
high as 1 µM. At 500 nM, total F-actin levels are reduced by
approximately 20%. Howard and colleagues 23, 31, 32 have determined
that there are at least two different types of F-actin, which can be
distinguished by their solubility in Triton-containing buffers.
Triton-soluble F-actin consists of short oligomers that are
associated with gelsolin and are found throughout the cytoplasm of
neutrophils. Triton-insoluble F-actin forms a cross-linked web of actin
that is found in association with tropomyosin, filamin, and
-actinin. Triton-insoluble F-actin also tends to be
associated with the plasma membrane. In RBL cells, approximately
60–70% of the basal level of F-actin is Triton soluble. In
unstimulated RBL cells, incubated with 500 nM latrunculin, there is no
change in Triton-soluble F-actin levels, but there is a 60%
decrease in the Triton-insoluble pool. Thus, latrunculin does not
affect all forms of F-actin equally. It appears to be exerting its
effects primarily on the Triton-insoluble fraction of F-actin.
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RI causes a rapid increase in the
level of total F-actin. Most of this change occurs in the
Triton-insoluble pool of F-actin (data not shown). As shown in Fig. 2, after activation of the cells with Ag,
the level of Triton-insoluble F-actin rises to between 150 and 200% of
the basal level in 3–5 min. The F-actin level peaks and then slowly
declines to basal levels over 30–45 min (data not shown). In the
presence of 500 nM latrunculin, the basal level is much lower than the
control value, as also shown in Fig. 1
. After the addition of DNP-BSA,
there is almost complete inhibition of actin polymerization in treated
cells. Fig. 2A shows the total level of Triton-insoluble
F-actin. In Fig. 2B, the basal level of
Triton-insoluble F-actin has been subtracted from both sets of samples
so that only Ag-induced actin polymerization is seen. Latrunculin
completely inhibits the response at early time points, although some
actin polymerization is seen at later time points.
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A). However, preincubation of
the cells with 500 nM cytochalasin D does effectively inhibit
Ag-induced actin polymerization. In Fig. 3B, the basal level
of F-actin has been subtracted so that only the Ag-induced
F-actin response is seen. As in the case with latrunculin, cytochalasin
D is more effective in inhibiting the early rise of F-actin.
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and 3. To determine their effects on cell activation,
IgE-sensitized RBL cells in suspension were preincubated with either
latrunculin (Fig. 4A) or
cytochalasin D (Fig. 4B) before activation with DNP-BSA.
Neither latrunculin nor cytochalasin D caused any degranulation in the
absence of Ag. However, upon addition of Ag, there was increased
degranulation, in a dose-dependent manner, in cells that had been
preincubated with either cytoskeletal inhibitor. In both cases, the
peak response occurred at approximately 500 nM, and degranulation was
increased by two- to threefold. In addition, the amount of Ag-induced
F-actin was monitored in cells preincubated with the different
concentrations of latrunculin and cytochalasin D. As can be seen,
inhibition of actin polymerization occurred in the same dose range as
did the increase in degranulation for both inhibitors.
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shows the degranulation responses
at different concentrations of Ag. As can be seen, latrunculin and
cytochalsin D were able to increase degranulation at all concentrations
of DNP-BSA. All these experiments were performed using RBL cells in
suspension. This was done to avoid any effects that these microfilament
inhibitors may have on adhesion and spreading, which is also known to
affect degranulation. If latrunculin was added to RBL cells that were
attached and spread, there was relatively little change seen in the
number of adherent cells. Changes were seen in the shape of the cells,
although most remained spread. Adherent and nonadherent cells were
preincubated with latrunculin before activation with DNP-BSA (Fig. 6). Latrunculin caused increased
degranulation from both adherent and nonadherent cells, although the
level of release in nonadherent cells was considerably less than in
adherent cells.
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RI or by
directly activating the signaling pathways, thus bypassing the
receptor. As shown in Table I,
latrunculin caused increased degranulation in nonadherent RBL cells
stimulated through FcRI but not with pervanadate. Pervanadate
inhibited certain phosphatases, thus causing increased tyrosine
phosphorylation, which activated the signaling pathways leading to
degranulation. This mechanism bypassed the cross-linking of the
receptor. However, in contrast to latrunculin, cytochalasin D also
increased degranulation when the cells were stimulated with
pervanadate. Degranulation could also be induced, in a nonreceptor
manner, with a combination of A23187 and PMA. In nonadherent cells,
latrunculin and cytochalasin D had their optimal effects on Ag-induced
degranulation at a concentration of 500 nM. At this concentration,
increased degranulation was seen when the cells were preincubated with
cytochalasin D and activated with A23187 and PMA. Augmented
degranulation was also seen using latrunculin, although the increase
was considerably less than with cytochalasin D. In adherent cells, 150
nM was the optimal concentration for increased degranulation when the
cells were activated with DNP-BSA. Using adherent cells and 150 nM
cytochalasin D, increased degranulation was seen when the cells were
activated with A23187 and PMA. However, at this concentration,
latrunculin had no effect. Thus, cytochalasin may act at more than one
point in the degranulation process, while latrunculin, depending on the
concentration, appears to be acting mainly at the level of the receptor
itself.
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RI. In
Fig. 7A, RBL cells were activated with Ag
for various lengths of time, and the kinetics of degranulation were
followed. Latrunculin increased both the rate and the extent of
degranulation in Ag-activated RBL cells. The maximal and half-maximal
times of degranulation were approximately the same in treated and
untreated cells. Fig. 7B shows the results of a similar
kinetic experiment in which intracellular Ca2+ was
measured. Latrunculin caused an increase in the rate as well as the
level of intracellular Ca2+ in treated cells (Fig. 7B).
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RI is known
to activate phospholipase C, A2, and D. Phospholipase C
activity can be measured by assaying for inositol phosphate production,
phospholipase A2 activity can be measured by the release of
arachidonic acid and its metabolites into the supernatant, and
phospholipase D activity can be monitored by the production of
phosphatidylethanol when the cells are activated in the presence of
ethanol. The results in Table II show
that activation of RBL cells caused an increase in the activity of
these phospholipases. If the cells were preincubated with latrunculin,
there was no change in the basal activity of these phospholipases.
However, the activities of all three phospholipases were increased in
cells activated through FcRI. Thus, inhibition of actin
polymerization by latrunculin increases not only degranulation but also
the signals leading to degranulation.
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).
In the presence of latrunculin, increased tyrosine phosphorylation of
Syk was observed.
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-chains of the receptor upon
activation. This is the earliest known event to occur after receptor
cross-linking. Therefore, to determine whether microfilaments regulate
degranulation by decreasing Lyn-mediated phosphorylation of the
ß-chain, cells were preincubated with 500 nM latrunculin and
activated with DNP-BSA. Fig. 9 shows an
increase in ß-chain tyrosine phosphorylation in untreated cells after
activation. Pretreatment of the cells with latrunculin, followed by
activation through FcRI, caused increased tyrosine phosphorylation
of the ß-chain. These results suggest that latrunculin is exerting
its effect at the level of receptor phosphorylation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the problems in using cytochalasin D is that it also enhances
degranulation triggered by substances that bypass the receptor, such as
pervanadate or calcium ionophore. This indicates that cytochalasin may
be acting at more than one point in the signaling pathway. Cytochalasin
D also increases receptor tyrosine phosphorylation, thus indicating
that it is acting at a very early stage (data not shown). However, the
degranulation results with pervanadate indicate that it is also acting
at a downstream stage. Latrunculin only causes increased degranulation
when the cells are activated through FcRI and not when the receptor
is bypassed with pervanadate. The effect with latrunculin and A23187
plus PMA is concentration dependent. Latrunculin is, therefore,
generally more specific than cytochalsin D. These results indicate that
the effect of latrunculin is mainly at the level of the receptor itself
and that microfilaments may interact, either directly or indirectly,
with cross-linked receptors. In addition to degranulation, the
activities of all the phospholipases as well as tyrosine
phosphorylation of Syk were also increased in latrunculin-treated cells
that had been activated with Ag. Increased phosphorylation of the
ß-chain of FcRI, which is the earliest known event after receptor
cross-linking, was also seen in activated cells treated with
latrunculin. In some experiments, although not all, preincubation with
latrunculin also caused a small increase in the basal level of
FcRIß tyrosine phosphorylation. Although much more pronounced in
activated cells, it is possible that microfilaments are also regulating
the level of receptor tyrosine phosphorylation in resting cells.
However, increases in none of the other signaling pathways, including
tyrosine phosphorylation of Syk, was ever seen in unactivated cells
treated with latrunculin. Since the tyrosine kinases associate with the
cross-linked receptors, this further supports the idea that
microfilaments are acting on the receptors themselves and not on a
downstream event. Our current hypothesis is that microfilaments are
uncoupling Lyn from the cross-linked receptors, thus shutting down the
response. If actin polymerization is inhibited with either cytochalasin
D or latrunculin, then no uncoupling takes place, and the receptors
remain active, thus leading to increased signaling and degranulation.
In RBL cells, the addition of monovalent DNP-lysine to cells activated with multivalent Ag, such as DNP-BSA or DNP-phycoerythrin, causes immediate cessation of all signaling and degranulation. Oliver and co-workers 35 coupled DNP to the fluorescent protein phycoerythrin so that they could monitor the displacement of this multivalent Ag. What was found was that the addition of DNP-lysine stopped all further degranulation, but a considerable amount of DNP-phycoerythrin remained on the cell surface. This indicated that some of the receptors on the cell surface remained cross-linked, but they were no longer actively signaling. With increasing time of exposure to DNP-phycoerythrin, an increasing number of receptors became DNP-lysine resistant, as the multivalent Ag could not be displaced by the monovalent Ag. Furthermore, this rate of conversion to DNP-lysine resistance was affected if the cells were preincubated with cytochalasin D. Thus, some cross-linked receptors are actively signaling while others have been turned off, and this process appears to be dependent on microfilaments. In neutrophils, it has also been suggested that actin microfilaments might be involved in down-regulation 36, 37, 38 . Neutrophils can be activated through the f-Met-Leu-Phe receptor, which can exist in a high affinity or a low affinity state. The different receptor states can be isolated from different plasma membrane fractions using sucrose density centrifugation. Desensitized receptors are found in a fraction that is rich in actin but contains no G proteins, while the active receptors are found in a fraction that contains G proteins but no actin. The conclusion from these studies was that actin microfilaments physically separated receptors from G proteins, thus turning off the response.
In the present study we found that in cells treated with latrunculin,
increased tyrosine phosphorylation of the ß-chain of FcRI was
observed. FcRI has no intrinsic kinase activity, and therefore,
initiation of the signaling cascade is dependent on Lyn 5, 6, 7 .
Studies in RBL cells have shown that some receptors have Lyn
constitutively associated with the ß-chain 39, 40 and that after
receptor cross-linking, there is transphosphorylation of the ITAM
regions in the ß- and -chains 8 . Studies have shown that the
ß-chain is not absolutely necessary for signaling to occur but that
it acts as an amplifier that increases the response five- to sevenfold
41 . Tyrosine phosphorylation of the ITAM regions leads to the
recruitment of more Lyn as well as Syk. Exactly how Lyn becomes
associated with the cross-linked receptors is not known, but recent
work indicates that detergent-resistant membrane domains may be
important. The fact that plasma membrane domains exist in RBL cells has
been shown both biochemically 42, 43 as well as by confocal
fluorescent microscopy 44 . FcRI normally has a uniform
distribution on the cell surface, indicating that it is not
prelocalized to these domains. Cross-linking of the receptors leads to
a rapid, although transient, relocalization into these domains that are
enriched in Lyn. This leads to tyrosine phosphorylation of the
receptors and the recruitment and activation of Syk and phospholipase
C1. The conclusion from these studies was that signal transduction
mediated through FcRI is compartmentalized and spatially restricted.
It is not known how actin microfilaments help to regulate the responses
triggered by FcRI cross-linking, although the experiments reported
here indicate that it occurs at a very early stage. However, it should
be pointed out that although the experiments presented here show an
excellent correlation between the inhibition of the F-actin response
and increased degranulation, they do not definitively prove that there
is a connection between these two events. Microfilaments could regulate
tyrosine phosphorylation of the receptors by physically restricting the
entry of cross-linked receptors into the detergent-resistant membrane
domains. Inhibition of actin polymerization by latrunculin would
increase the likelihood of the cross-linked receptors entering the
domains and becoming tyrosine phosphorylated by Lyn. Another
possibility is that actin microfilaments do not control the entry into
these lipid domains but that they are involved in separating the
receptors from the lipid domains. If this is the case, then all the
cross-linked receptors potentially become activated, and the
microfilaments are involved in deactivating them. Latrunculin would
presumably function by inhibiting actin polymerization, thus keeping
the receptors in an activated state for a longer period of time. In any
case, signaling in these cells is compartmentalized, and the role of
actin microfilaments may be to regulate whether cross-linked receptors
are in an active compartment.
Activation of mast cells through FcRI leads to the release of a
variety of inflammatory mediators such as histamine, proteases,
leukotrienes, PGs, and cytokines. Control of the release of these
mediators is important, and mast cells have developed several potential
ways of regulating the signaling pathways leading to their release. The
work reported here details one mechanism in which microfilaments are
involved in controlling the extent of the reaction, possibly by
uncoupling the cross-linked receptors from the tyrosine kinases. There
are, however, several other potential mechanisms. Activation of
phosphatases, which dephosphorylate the cross-linked receptors, has
been reported, and it is known that even stably cross-linked receptors
rapidly undergo rounds of phosphorylation and dephosphorylation 45 .
It has also been found that several receptors containing immunoreceptor
tyrosine-based inhibition motifs leads to down-regulation if they are
co-cross-linked with FcRI 46, 47, 48 . Phosphorylation of
immunoreceptor tyrosine-based inhibition motifs recruits and serves as
a docking site for phosphatases that can turn off the response by
dephosphorylating the FcRI. Finally, it has been reported that the
amount of Lyn that associates with the cross-linked receptors is a
limiting factor and that cross-linked receptors compete for this kinase
49 . Thus, it is believed that Lyn shuttles from one cross-linked
receptor complex to another. It is not known what controls the
available pool of Lyn, but that is another potential source of
regulation.
Finally, the experiments reported here focus on the potential role of actin microfilaments in the down-regulation of the signaling pathways leading to the degranulation response. However, previous studies have shown that adhesion and spreading, which are dependent on microfilament assembly and rearrangement, are involved in up-regulation of these same signaling pathways 23, 50 . Spreading of the cells on extracellular matrix proteins seems to prime the cells for increased degranulation after activation with Ag. It is apparent that actin filaments have a very complex interaction with the signaling machinery leading to degranulation and that some microfilaments are involved in up-regulating the response, while others may be involved in down-regulation.
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Acknowledgments |
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RIß
antiserum (JRK), and Dr. Reuben Siraganian for kindly providing rabbit
anti-Syk antisera. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. John R. Apgar, Scripps Research Institute, Department of Molecular and Experimental Medicine, SBR-4, 10550 North Torrey Pines Rd., La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: RBL, rat basophilic leukemia; FcRI, high affinity immunoglobulin E receptor; ITAM, immunoreceptor tyrosine-based activation motif; PKC, protein kinase C; F-actin, filamentous actin; NBD-phallacidin, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin.
Received for publication May 4, 1998. Accepted for publication November 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI. Semin. Immunol. 7:29.[Medline]
RI and other receptors. Curr. Opin. Immunol. 8:766.[Medline]
R1 cross-linking to increased cell substrate adhesion in RBL-2H3 tumor mast cells. J. Immunol. 152:270.[Abstract]
R1 is due to spreading and not simply adhesion. J. Cell Sci. 110:771.[Abstract]
-actinin, and actin binding protein 280 in stabilizing Triton insoluble F-actin in basal and chemotactic factor activated neutrophils. Cell Motil. Cytoskel. 28:155.[Medline]
RIß subunit functions as an amplifier of FcRI-mediated cell activation signals. Cell 85:985.[Medline]
RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling. Proc. Natl. Acad. Sci. USA 92:9201.This article has been cited by other articles:
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