| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,||
*
Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121;
Department of Microbiology and Molecular Genetics, University of California, Los Angeles, CA 90095;
Department of Pediatrics, University of California, Los Angeles, CA 90095;
§
National Research Institute of Vegetables, Ornamental Plants and Tea, Kanaya, Shizuoka, Japan;
¶
Department of Laboratory Medicine, University of California, San Francisco, CA 94143; and
||
Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI). In this study, we
have made the following observations on growth properties and
FcRI-mediated signal transduction of primary cultured mast cells
from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency
partially reversed the survival effect of Btk deficiency. Second,
FcRI-induced degranulation and leukotriene release were almost
abrogated in Btk/Lyn doubly deficient mast cells while singly deficient
cells exhibited normal responses. Tyrosine phosphorylation of cellular
proteins including phospholipases C-
1 and C-2 was reduced in
Btk/Lyn-deficient mast cells. Accordingly, FcRI-induced elevation of
intracellular Ca2+ concentrations and activation of protein
kinase Cs were blunted in the doubly deficient cells. Third, in
contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion
and mitogen-activated protein kinase activation. Lyn-deficient cells
exhibited enhanced secretion of TNF-
and IL-2 apparently through the
prolonged activation of extracellular signal-related kinases and c-Jun
N-terminal kinase. Potentially accounting for this phenomenon and
robust degranulation in Lyn-deficient cells, the activities of protein
kinase C and protein kinase CßII, low at basal levels, were
enhanced in these cells. Fourth, cytokine secretion was severely
reduced and c-Jun N-terminal kinase activation was completely abrogated
in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk
and Lyn are involved in mast cell signaling pathways in distinctly
different ways, emphasizing that multiple signal outcomes must be
evaluated to fully understand the functional interactions of individual
signaling components. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI)3 on mast
cells by Ag leads to the activation of mast cells culminating in the
release of a panel of proinflammatory mediators. Mast cell activation
triggers reactions of immediate hypersensitivity (1).
FcRI is composed of an IgE-binding subunit, a
four-transmembrane, signal-amplifying ß subunit, and a
disulfide-bonded pair of subunits (reviewed in Ref.
2). The Src family protein tyrosine kinase (PTK) Lyn is
associated with the ß subunit in resting cells through interaction of
the N-terminal unique region of Lyn with the C-terminal cytoplasmic
domain of the ß subunit (3, 4). Lyn is activated by
transphosphorylation upon FcRI cross-linking (5).
Activated Lyn phosphorylates the tyrosine residues in the
immunoreceptor tyrosine-based activation motifs (ITAM) of the
cytoplasmic regions of the ß and subunits (6).
Phosphorylated ITAMs of the ß and subunits recruit Lyn and Syk,
respectively, through Src homology (SH) 2-phosphotyrosine interactions
(7, 8, 9). Newly recruited PTKs are activated by
transphosphorylation of tyrosine residues in their activation loops and
by conformational changes in the case of Syk (10, 11).
Active Lyn and Syk phosphorylate themselves and other protein
substrates such as phospholipase C (PLC)-1 and -2
(12, 13, 14). Activation of PLC leads to the generation of two
second messengers, inositol 1,4,5-trisphosphate
(IP3) and diacylglycerol.
IP3 mobilizes Ca2+ from
intracellular storage sites, and diacylglycerol together with
Ca2+ activates protein kinase C (PKC) (reviewed
in Ref. 15). Both Ca2+ and PKC
appear to be required for optimal mast cell degranulation
(16).
Bruton’s tyrosine kinase (Btk) is a member of the Tec family of
kinases characterized by N-terminal pleckstrin homology (PH) and Tec
homology domains (17, 18, 19). btk mutations affect
B cell development and result in X-linked agammaglobulinemia in humans
(17, 18) and X-linked immunodeficiency (xid) in
mice (20, 21). Btk is also highly expressed in mast cells
and is required for cytokine production in response to FcRI
cross-linking (22). Unlike B cells, mast cell development
does not require Btk. This suggests redundant roles for Btk and other
Tec family kinases, such as Itk/Emt, in some mast cell signaling
pathways. Mechanistically, btk mutations result in the loss
of extracellular Ca2+ influx and the sustained
phase of Ca2+ response in B cell receptor
(BCR)-stimulated B cells (23, 24). This defective response
may be due in part to reduced PLC- activation. Downstream of these
early activation events in mast cells and B cells, Btk mediates the
activation of JNK1, JNK2, and, to a lesser extent, p38
(25). JNK regulates c-Jun and other transcription factors
that induce the transcription of TNF-, IL-2, and other cytokine
genes (26), accounting for the reduced cytokine production
in Btk-deficient mast cells.
Activation of Btk in BCR and FcRI signaling systems requires both
phosphatidylinositol 3-kinase (PI3-K) and Src family kinases (27, 28). The product of PI3-K, phosphatidylinositol
3,4,5-trisphosphate, is believed to recruit Btk to the plasma membrane
(29), where it is phosphorylated on tyrosine 551 in its
activation loop by activated Lyn molecules (27). Btk
phosphorylated on tyrosine 551 is enzymatically active and
autophosphorylates tyrosine 223 in its SH3 domain (30).
Although these observations suggest a direct enzyme/substrate
relationship for Lyn and Btk, studies in both B and mast cells imply a
more complex interaction between these two kinases. While
btk mutations profoundly impaired cytokine production in
FcRI-stimulated mast cells (22), this process was not
affected by Lyn deficiency as measured by RT-PCR (12).
Similarly, B cell development and activation are differentially
affected by Btk and Lyn deficiencies (31, 32, 33, 34, 35).
Btk-deficient mice lack B-1 cells, while Lyn-deficient mice have normal
or increased numbers of this cell type. Serum IgM levels are low in the
absence of Btk and high in the absence of Lyn. Aged
lyn-/- mice develop
autoantibodies, a process that is impaired by btk mutations
in several models of autoimmunity (36, 37). Lyn clearly
plays a role in the initiation of BCR signals, but its predominant
unique role in this pathway is inhibitory as shown by the
hypersensitivity of Lyn-deficient B cells to anti-IgM stimulation.
The inhibitory function of Lyn seems to involve the tyrosine
phosphorylation of CD22 and FcRIIb and the recruitment of the
tyrosine phosphatase SHP-1 and 5'-inositol phosphatase SHIP,
respectively, to these inhibitory receptors (38, 39, 40). In
contrast, Btk-deficient cells fail to respond to BCR cross-linking.
Differences in the in vivo phenotypes of
btk-/- (or
xid) and
lyn-/- mice and the
in vitro properties of
btk-/- and
lyn-/- mast cells
suggest that these PTKs may have independent or opposing functions.
Indeed, Btk and Lyn are each required for B cell survival and Ag
response but exert opposing functions in generation of autoantibodies
and the tuning of BCR-dependent proliferative responses (36, 37, 41). To determine whether this complexity is observed in
alternative cell types and receptor systems, growth and activation
properties of mast cells derived from Btk/Lyn-deficient mice were
compared with wild-type and singly deficient mast cells. Btk and Lyn
played both redundant and opposing roles in FcRI signaling depending
on the signal output measured, emphasizing that multiple signal
outcomes must be evaluated to fully understand the functional
interactions of individual signaling components. Strikingly, all
aspects of mast cell activation measured were significantly reduced in
Btk/Lyn-deficient cells. This suggests that simultaneous blockade of
Btk and Lyn may be an attractive therapeutic strategy for allergic
diseases.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Culture media and FCS were purchased from Life Technologies
(Grand Island, NY). Anti-FcRIß subunit mAb (42) was
kindly donated by Juan Rivera (National Institutes of Health, Bethesda,
MD). Sources of commercial Abs are as follows: anti-Btk (M-138),
anti-Lyn (44), anti-Syk (C-20), anti-PLC-2
(Q-20), anti-PKC (C20), anti-PKCßII (C-18), anti-c-Jun
N-terminal kinase (JNK) 1 (C-17), anti-extracellular signal-related
kinase (ERK)1 (C-16), and anti-p38 (C-20) from Santa Cruz
Biotechnology (Santa Cruz, CA); anti-phosphotyrosine mAb 4G10 and
anti-PLC-1 mAbs from Upstate Biotechnology (Lake Success, NY);
anti-phospho-mitogen-activated protein (MAP) kinase and
anti-phospho-p38 from New England Biolabs (Boston, MA). Pansorbin
was purchased from Calbiochem (La Jolla, CA). Other chemicals of
highest grade were obtained from Sigma (St. Louis, MO), unless
otherwise mentioned.
Cells
btk-/- and
lyn-/- mice, each
on a mixed C57BL/6 x 129/Sv genetic background, were mated to
generate
btk+/-lyn+/- F1 progeny. These F1 mice
were mated to obtain wild-type,
btk-/-,
lyn-/->, and
btk-/-lyn-/-
mice (36). Genotyping was done by Southern blotting or PCR
analysis of mouse tail-derived DNAs. Mast cells were cultured as
described previously (44). Briefly, bone marrow cells
derived from the femur of the 6- to 10-wk-old mice were cultured in
RPMI 1640 medium supplemented with 10% FCS, 100 µM nonessential
amino acids, 50 µM 2-ME, and 8% conditioned medium of IL-3
gene-transfected cells (bone marrow-derived mast cell medium). More
than 95% of the trypan blue-excluding viable cells were mast cells
after 4 wk of culture. No discernible differences in morphology and
expression of early signaling proteins, including FcRIß,
FcRI, Syk, Grb2, PLC-2, c-Cbl, and Shc, were detected between
these four types of mast cells (see
Figs. 3–5
and data not shown).
Surface expression of FcRI was measured by flow cytometry using a
FACSCalibur apparatus and CellQuest software (Becton Dickinson,
Mountain View, CA). In acute (<60 min) stimulation experiments, mast
cells were sensitized by an overnight incubation with 1 µg/ml
anti-dinitrophenyl (DNP) IgE mAb, washed once in Tyrode buffer (112
mM NaCl, 2.7 mM KCl, 0.4 mM
NaH2PO4, 1.6 mM
CaCl2, 1 mM MgCl2, 10 mM
HEPES, pH 7.5, 0.05% gelatin, 0.1% glucose), resuspended in Tyrode
buffer to 2 x 107 cells/ml, and stimulated
by polyvalent Ag, 100 ng/ml DNP conjugates of human serum albumin
(DNP-HSA), for the indicated time intervals.
|
|
|
Histamine released into medium during a 45-min stimulation was
measured by an automated fluorometric assay (45).
Leukotrienes secreted into medium for 30 min were analyzed by an enzyme
immunoassay kit for leukotrienes
C4/D4/E4
(Amersham Pharmacia Biotech, Piscataway, NJ). TNF- and IL-2 secreted
into the culture medum for 20 h were measured by ELISA kits
(Endogen, Woburn, MA). In this case, mast cells were stimulated in bone
marrow-derived mast cell medium instead of Tyrode buffer.
Immunoblotting and immunoprecipitation
Cells were lysed in ice-cold 1% Nonidet P-40-containing lysis
buffer (20 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 1 mM sodium
orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin,
25 µM p-nitrophenyl p'-guanidinobenzoate, 1
µM pepstatin, and 0.1% sodium azide) immediately after stimulation.
Lysates were centrifuged in an Eppendorf microcentrifuge at 4°C for
10 min. Protein concentrations were measured using
DC protein assay reagents (Bio-Rad, Richmond,
CA). Cleared lysates were either directly analyzed by SDS-PAGE or
immunoprecipitated before SDS-PAGE analysis. For immunoprecipitation,
lysates were incubated on ice with an appropriate Ab for 2–4 h, and
immune complexes were recovered by brief centrifugation following
another 30 min incubation with Pansorbin (Calbiochem) for rabbit
polyclonal Abs or anti-mouse Ig-conjugated agarose (Sigma) for
mouse mAbs. Immune complexes were washed in lysis buffer four times
before SDS-PAGE analysis. Proteins separated by SDS-PAGE were
electrophoretically transferred to polyvinylidene difluoride membranes
(NEN Life Science Products, Boston, MA). Membranes were blocked,
incubated consecutively with primary Ab and HRP-conjugated secondary
Ab, and immunoreactive proteins were visualized by enhanced
chemiluminescence reagents (NEN Life Science Products). To estimate
concentrations of PKC isoforms in mast cells, various amounts of human
recombinant PKC proteins (, ßI, and ßII isoforms) expressed in
insect cells (Panvera, Madison, WI) were run in parallel with mast cell
lysates and followed by immunoblotting with respective Abs.
Immune complex kinase assays
For Syk kinase assays, immune complexes precipitated from 1%
Nonidet P-40 cell lysates were washed five times in lysis buffer and
once with kinase buffer without ATP. Washed immune complexes were
incubated with kinase buffer (50 mM Tris, pH 7.4, 0.1% Nonidet P-40,
10 mM MnCl2, 10 mM MgCl2)
with 2 µg of GST-HS1 (46) containing the sequence from
position 352 to position 486 of the human HS1 protein in the presence
of [-32P]ATP (DuPont NEN, Boston, MA). For
PKC assays, immunoprecipitates after similarly washed were incubated
with kinase buffer (20 mM Tris, pH 7.4, 10 mM
MgCl2, 10 µM ATP) in the presence of
[-32P]ATP. For JNK kinase assays, cells were
lysed in ice-cold whole-cell extraction buffer (25 mM HEPES, pH 7.5,
0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1%
Triton X-100, 2 µM DTT, 0.5 mM PMSF, 20 mM ß-glycerophosphate, and
0.5 mM sodium orthovanadate). Cleared lysates in this buffer were
diluted with three volumes of dilution buffer (20 mM HEPES, [pH 7.5,
0.1 mM EDTA, 2.5 mM MgCl2, 0.05% Triton X-100,
0.5 mM sodium orthovanadate, 0.5 mM PMSF, and 20 mM
ß-glycerophosphate). Cleared lysates were immunoprecipitated with
anti-JNK1 (C-17, Santa Cruz Biotechnology). Immune complexes were
washed five times with lysis buffer and once with kinase buffer without
ATP and substrate. Then, immunoprecipitates were incubated with 3 µg
GST-c-Jun 1–79(1–79) in 15-min reactions at 30°C in 20 mM HEPES, pH 7.4,
10 mM MgCl2, 22 mM DTT, 20 mM
ß-glycerophosphate, 50 µM
Na3VO4, 20 µM ATP, and 10
µCi [-32P]ATP. Reaction products were
analyzed by SDS-PAGE followed by electroblotting onto polyvinylidene
difluoride membranes and autoradiography.
IP3 measurement
A commercial kit (Amersham Pharmacia Biotech) was used. Cells were extracted with chloroform/methanol (1/2 v/v) on ice for 10 min. Methanol fractions containing phosphorylated inositols were lyophilized and mixed with bovine adrenal IP3-binding proteins in the presence of a limiting amount of tracer D-myo-[3H]IP3. The mixtures were centrifuged at 2000 x g for 10 min and radioactivity bound to IP3-binding protein was measured in a ß-scintillation counter.
Intracellular calcium analysis
Mast cells were sensitized with 0.5 µg/ml anti-DNP IgE in
bone marrow-derived mast cell medium for 2 h at 37°C and loaded
with 10 µM INDO-1 acetoxymethyl (Molecular Probes, Eugene, OR) in
RPMI 1640 and 2% FCS for 40 min at 30°C with constant agitation.
Cells were washed once, resuspended in HBSS with 1.7 mM
Ca2+ (Sigma), and maintained on ice at a
concentration of 1 x 107 cells/ml until
analysis. Cells were resuspended at a concentration of 5 x
105 cells/ml in the same buffer and warmed for 2
min at 37°C with rapid stirring before analysis. Bulk intracellular
calcium levels were monitored by excitation at 350 nm with detection of
the 405 nm bound and 440 nm unbound emissions of INDO-1 using a
DeltaRam spectrofluorometer (Photon Technology Instruments, Princeton,
NJ) at a rate of five measurements per second. Analysis at baseline was
acquired for 20 s before FcRI cross-linking with 100 ng/ml
DNP-HSA. Calcium analysis was continued for 280 s, followed by
addition of 10 µM ionomycin to determine the peak population response
using an additional 60-s data acquisition.
Transcriptional activity assay with luciferase reporter constructs
Luciferase reporter constructs, the mouse IL-2 (-321)-Luc and
the human TNF- (-200)-Luc, were described previously
(22). A total of 1–1.5 x 107
mast cells were transfected with 5–10 µg reporter plasmid by
electroporation at 400V, 950 µF using a Gene Pulser II apparatus
(Bio-Rad). Transfected cells were sensitized overnight with
anti-DNP IgE and left unstimulated or stimulated with 30 ng/ml
DNP-HSA for 8 h before cell harvest. Cells were lysed in 0.2%
Triton-X-100 in 100 mM potassium phosphate buffer (pH 7.8)/1 mM DTT.
Luminescence of cleared lysates was measured after addition of
luciferin solution using a model Monolight 2010 luminometer (Analytical
Luminescence Laboratory, San Diego, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Culturing murine bone marrow cells in IL-3-containing medium for 4
wk leads to the generation of a >95% pure population of immature mast
cells. This process is independent of both Btk and Lyn, as bone marrow
cells derived from wild-type,
btk-/-,
lyn-/-, and
btk-/-lyn-/-
littermates gave rise to similarly pure populations of mast cells. The
genotypes of these cells determined by Southern blot or PCR analysis
were confirmed by immunoblotting cell lysates with anti-Lyn or
anti-Btk Abs (data not shown). These mast cells express similar
levels of FcRI on their surfaces (Fig. 1). Although the
btk-/-lyn-/-
cells exhibited a broader distribution of FcRI expression in this
cell preparation, the distribution in another preparation was similar
to that of wild-type cells. As previously shown (47, 48),
FcRI expression on the surface of wild-type mast cells increased
2–4 days after incubation with IgE (0.05–5 µg/ml) in a
concentration-dependent manner. Comparable IgE-mediated enhancement in
FcRI expression was observed in mast cells derived from the other
genotypes as well (data not shown). As described previously
(25),
btk-/- mice yielded
more mast cells than wild-type counterparts under these culture
conditions, while
lyn-/- bone marrow
generated normal numbers of mast cells (Fig. 2A).
btk-/-lyn-/-
bone marrow also generated mast cell numbers that were intermediate
between those present in wild-type and
btk-/- mice. The
large number of mast cells produced in the absence of Btk could have
resulted from increased number of precursors in the bone marrow, faster
cell cycle time, or reduced cell death. The latter hypothesis is
supported by the observation that both
btk-/- and
btk-/-lyn-/-
cells were more resistant to growth factor (IL-3) deprivation-induced
apoptosis (Fig. 2B). Btk has been suggested to play a role
in both proapoptotic (Fig. 2B, Refs. 25 and
49) and anti-apoptotic pathways (50, 51),
implying cell type- or receptor-specific outcomes of Btk signaling.
|
|
FcRI-mediated activation of mast cells results in degranulation
(measured by histamine release) and secretion of leukotriene. We
examined the interaction of Btk and Lyn in regulating these processes.
Histamine release was normal or near normal and leukotriene release was
normal in cells lacking either Btk or Lyn alone (Fig. 3
). Surprisingly, the secretion of both
histamine and leukotriene was nearly completely abrogated in
btk-/-lyn-/-
mast cells (Fig. 3), indicating that Btk and Lyn together are essential
for FcRI-induced mast cell degranulation and leukotriene release.
Normal degranulation in
lyn-/- mast cells
may be due to a function fulfilled by another Src family PTK(s)
expressed in mast cells. Fyn/Lyn doubly deficient mast cells
degranulated significantly less efficiently than wild-type or singly
deficient mast cells (data not shown), indicating the redundant
function exhibited by two Src family PTKs in mast cell degranulation.
Full description of our study on
fyn-/- and
fyn-/-lyn-/-
mast cells will be provided elsewhere.
Btk/Lyn-deficient mast cells exhibit impaired substrate tyrosine phosphorylation and defective IP3/Ca2+ signaling
We examined FcRI-induced tyrosine phosphorylation of several
key substrate molecules to begin to understand the role(s) for Btk vs
Lyn in mast cell degranulation. The overall pattern of tyrosine
phosphorylation was very similar between
btk-/- and
wild-type cells (Fig. 4A).
This response was partially blunted in
lyn-/- cells and
severely affected in Btk/Lyn-deficient cells. Tyrosine phosphorylation
of the FcRI ß subunit (Fig. 4B) and Syk (Fig. 4, C and D) was reduced in both
lyn-/- and
btk-/-lyn-/-
cells but not in
btk-/- cells. This
is consistent with the current model (7, 52) whereby Lyn
phosphorylates the ITAMs of both the ß and subunits of FcRI
and that ITAM phosphorylation leads to recruitment and activation of
Syk. As previously described for
lyn-/- mouse B
cells (31), the kinetics of FcRI-dependent tyrosine
phosphorylation of several proteins including FcRIß (Fig. 4B), Syk (Fig. 4C), and PLC-1 (Fig. 5A) were delayed in
lyn-/- mast cells
and the degree of phosphorylation was lower than in wild-type or
btk-/- cells.
(Tyrosine phosphorylation of PLC-1 in
btk-/- cells was
variable among several experiments, although it was higher than in
wild-type cells in the experiment shown in Fig. 5A.)
Tyrosine phosphorylation of PLC-1 was significantly reduced in
btk-/-lyn-/-
cells relative to cells lacking either Btk or Lyn alone (Fig. 5A). Tyrosine phosphorylation of PLC-2 was lower in
btk-/-,
lyn-/-, and
btk-/-lyn-/-
cells than in wild-type cells (Fig. 5B).
Consistent with the reduced phosphorylation of PLC-2,
IP3 synthesis in
btk-/- cells was
lower than that in wild-type cells, although its kinetics were
similar to those in wild-type cells (Fig. 5C). Consistent
with this data and previous studies demonstrating an important role for
Btk in the generation of BCR-dependent calcium signaling (23, 24), btk-/-
mast cells exhibited a significantly reduced total calcium flux
relative to wild-type mast cells in response to receptor cross-linking
(Fig. 5D). Following BCR cross-linking, phosphatidylinositol
3,4,5-trisphosphate generated by PI3-K initiates Btk activation in
concert with Src kinases by targeting the Btk PH domain to the plasma
membrane (Refs. 24 and 29 , and reviewed in
Ref. 53). This model is consistent with data obtained from
wild-type and Btk-deficient mast cells. Wortmannin pretreatment
resulted in a marked reduction in calcium signaling in wild-type mast
cells. In contrast, only a minimal reduction in sustained calcium
signaling was observed in
btk-/- mast cells
under identical conditions (data not shown).
IP3 synthesis in
lyn-/- and
btk-/-lyn-/-
cells was much delayed but strikingly augmented at later time points
(Fig. 5C). The difference in this response between these
cell types indicates that the augmented IP3
response in lyn-/-
cells are at least partly dependent on Btk. The acute phase of
Ca2+ increase (within 50 s after FcRI
stimulation) was completely blocked in both
lyn-/- and
btk-/-lyn-/-
mast cells (Fig. 5D) despite normal peak responses to
ionomycin (data not shown), indicating an essential role for Lyn in
this event. In most experiments, these cells exhibited a slow, but
sustained, increase in intracellular Ca2+
concentration, which correlates with the delayed response of
IP3 synthesis. These data are similar to those
previously observed in
lyn-/- chicken B
cells (54) and
lyn-/- mast cells
(12). Effects of Btk and Lyn deficiencies on
IP3 production and Ca2+
response suggest that tyrosine phosphorylation (therefore enzymatic
activation) of PLC-2 is more relevant to IP3
production and Ca2+ mobilization than that of
PLC-1 in mast cells.
Regulation of PKC activity by Lyn and Btk
Robust degranulation in spite of delayed (or blunted in some
lyn-/- cell
preparations as shown in one of the tracings in Fig. 5D)
Ca2+ response in
lyn-/- mast cells
and blunted degranulation with similar Ca2+
response in
btk-/-lyn-/-
cells prompted us to evaluate the involvement of
Ca2+ and PKC in degranulation from mast cells.
Chelation of extracellular calcium by EGTA inhibited histamine release
from FcRI-stimulated
lyn-/- mast cells
as well as from wild-type and
btk-/- cells,
indicating that a Ca2+ flux was important for
this response and that the delayed-phase calcium response in
lyn-/- cells was
sufficient (Fig. 6A).
Inhibition of PKC by Ro 31-8425 also blocked histamine release from
FcRI-stimulated wild-type,
btk-/- or
lyn-/- mast cells
(Fig. 6A). PMA treatment of wild-type cells in the presence
of EGTA induced a modest but significant degranulation (data not
shown). These results indicate that PKC activation is required and
sufficient for degranulation in mouse bone marrow-derived mast cells
and that a minimal threshold of Ca2+ flux is also
required for efficient degranulation. In the same line of study, human
basophils are known to degranulate upon FcRI cross-linking without
the requirement for a pharmacologic Ca2+ release
signal (55) and that PKC is essential for degranulation in
these cells.
|
and
50 ng of PKCßII per 106 cells, and a less
abundant (1.25 ng per 106 cells) PKCßI isoform,
as measured by immunoblotting cell lysates with respective Abs
using recombinant PKC isoforms as reference (data not shown). PKCß is
the major isoform of PKC, which together with
Ca2+ is sufficient to elicit degranulation in
permeabilized RBL-2H3 mast cells (16). Therefore, we
compared the activities of PKC and PKCßII in mast cells of the
four genotypes. Autophosphorylating activities of PKC and PKCßII
were slightly (<3-fold) increased at their peak (15 min) after FcRI
cross-linking in wild-type and
btk-/- mast cells
(Fig. 6B). Interestingly, basal activities of PKC and
PKCßII in unstimulated
lyn-/- cells were
significantly lower than wild-type and
btk-/- cells, but
became higher at their peaks upon FcRI stimulation. In contrast,
btk-/-lyn-/-
cells exhibited little or no activation of PKC or PKCßII (a longer
exposure of the autoradiograms shown in Fig. 6B and in three
other experiments). Therefore, the robust degranulation correlates with
the vigorous activity of PKC, more specifically that of PKCßII, in
lyn-/- cells while
the drastically reduced degranulation correlates with the poor PKC
activation in
btk-/-lyn-/-
cells. Immunoblotting of total cell lysates showed that the amounts of
PKC and PKCßII in unstimulated and FcRI-stimulated
btk-/-lyn-/-
cells were reduced apparently by degradation, because the amount of
degradation products reactive with the C-terminal peptide-specific Abs
was dramatically increased (data not shown).
Kinetics of histamine release were determined to examine the
relationship between degranulation, Ca2+
response, and PKCßII activity in
lyn-/- cells vs
other cell types. As shown in Fig. 6C, histamine release
from FcRI-stimulated
lyn-/- cells
exhibited a lag time (>3 min) before a significant amount of histamine
was detected in the medium, whereas it was detected within 1 min in
wild-type cells. This delayed histamine release in
lyn-/- cells
correlates with the delayed Ca2+ response and the
low basal and induced activity of PKCßII, suggesting the importance
of these signals for degranulation.
Opposing effects of btk and lyn mutations on cytokine secretion
Late-phase reactions of immediate hypersensitivity appear to be at
least partly dependent on TNF- secreted from FcRI-stimulated mast
cells (56). We determined the effects of btk
and lyn mutations on cytokine production by activated mast
cells (Fig. 7). As shown previously
(22),
btk-/- mast cells
produced and secreted less TNF- and IL-2 than wild-type
counterparts. Intriguingly,
lyn-/- mast cells
secreted 3-fold more of these cytokines than wild-type cells. This
enhanced response was abrogated in the absence of Btk, indicating that
Btk and Lyn play opposing roles in the production of specific cytokines
in response to FcRI activation.
|
To begin to understand the mechanism(s) by which Btk and Lyn
exerted opposing effects on cytokine secretion, we evaluated the
signaling pathways potentially regulating IL-2 and TNF- expression
in activated mast cells. Because transcriptional regulation of cytokine
genes is a critical step in cytokine production in activated mast
cells, we analyzed transcriptional activity of the IL-2 gene promoter
by transfecting the IL-2/Luc reporter plasmid into mast cells of the
four genotypes. As described previously (22, 26), FcRI
cross-linking induced a robust transactivation of IL-2 promoter in
wild-type mast cells while
btk-/- cells
exhibited much lower activity (Fig. 8).
Consistent with the data on cytokine secretion, transcriptional
activation in
lyn-/- cells was
about twice more than that in wild-type cells. In contrast,
btk-/-lyn-/-
cells gave little activation of the IL-2 promoter. Similar to TNF-
secretion, TNF-/Luc transcriptional activity was less remarkably
affected by Lyn and Btk deficiencies (data not shown). Therefore, these
data confirm that transcriptional regulation of cytokine genes is an
important regulatory step in mast cell activation.
|
Because ERK and JNK pathways can induce transcription of IL-2 and
TNF- genes (57), we evaluated the activity of these
kinases in FcRI-stimulated mast cells of all four genotypes (Fig. 9).
lyn-/- mast cells
exhibited prolonged activation of both ERK (Fig. 9A) and
JNK1 (Fig. 9B), potentially accounting for the enhanced
cytokine secretion from these cells. As shown previously
(25), JNK1 activity was significantly reduced in
Btk-deficient cells (Fig. 9A). Strikingly, JNK1 activity was
completely abrogated in
btk-/-lyn-/-
cells. In contrast, ERK1 and ERK2 phosphorylation was unaltered in
either btk-/- or
btk-/-lyn-/-
mast cells. Notably, FcRI-induced activation of the p38 MAP kinase
pathway was unchanged in singly deficient cells but was significantly
impaired in doubly deficient mast cells (Fig. 9C). p38 has
been shown to regulate c-jun expression via the
phosphorylation of another transcription factor MEF2C
(58). This combined impairment of JNK1 and p38 activation
may be responsible for the significantly reduced production of TNF-
and IL-2 in Btk/Lyn-deficient cells. BCR-dependent p38 activation in
chicken B cells required both Lyn and Syk activity but was unaltered in
Btk, Syk, or Lyn singly deficient cells (59). Our data
suggest that doubly deficient
(btk-/-lyn-/-)
B cells are also likely to exhibit suboptimal BCR-dependent p38
activation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Signaling pathways used by the two major mast cell growth factors, IL-3 and stem cell factor (c-Kit ligand), activate both Src and Tec family PTKs as proximal events (reviewed in Ref. 60). The data presented here demonstrate that neither Btk nor Lyn are essential for IL-3-dependent expansion of bone marrow-derived mast cells. In fact, loss of Btk function leads to the production of a significantly greater number of mast cells (25). Btk-deficient mast cells exhibit similar proliferative response to IL-3 as wild-type cells but survive better than their wild-type counterparts upon growth factor withdrawal (25), supporting a negative role for Btk in regulating cell survival in this system. In contrast to these results, Btk has also been shown to play an anti-apoptotic role in B cells (50). Btk-deficient B cells survive poorly in vitro and in vivo and fail to up-regulate Bcl-xL in response to Ag receptor cross-linking. JNK, which is regulated by Btk, has been implicated in both positive and negative regulation of apoptosis. Stress-induced JNK activation is crucial for induction of apoptosis (61, 62, 63), while SEK-1 (a direct activator of JNK)-deficient T cells exhibit enhanced activation-induced cell death (64, 65). These observations suggest that cell type, signal context, and/or additional factors may be essential for determining the outcome of Btk-dependent signals.
btk-/-lyn-/- mast cells exhibited an intermediate phenotype (in comparison with wild-type cells and btk-/- cells) in production and the rate of growth factor deprivation-induced cell death. This suggests that Btk and Lyn may have opposing functions in mast cell survival. This suggestion of a weak positive role for Lyn in mast cell survival was observed only in the context of Btk deficiency, as lyn-/- cells were indistinguishable from wild-type cells with respect to both overall production and cell death. The mechanism for this effect is currently unclear, but may be related to the differential regulation of specific MAP kinases and/or PKC isoforms by Btk and Lyn. In contrast to these observations, Btk and Lyn appear to have redundant rather than opposing roles in mediating B cell survival (36), again stressing the importance of cellular context in signal outcome.
Redundant roles for Btk and Lyn in mast cell degranulation and leukotriene release
Activated mast cells contribute to allergic responses primarily through the secretion of proinflammatory mediators. We examined the requirement for Btk and Lyn in the secretion of histamine (as a marker for degranulation) and leukotrienes. Btk and Lyn exhibited redundant or independent roles for maximal degranulation and leukotriene release. Singly deficient mast cells produced normal or near normal levels of each of these chemical mediators. In contrast, Btk/Lyn doubly deficient cells had severely blunted activity in all of these assays.
The signaling mechanisms controlling degranulation and leukotriene
release remain poorly understood. Optimal degranulation requires both
modestly increased intracellular Ca2+
concentrations and activation of PKC isoforms (16). The
impaired activation of both PLC-1 and -2, the consequent defects
in IP3/calcium signaling, and little or no
activation of Ca2+-dependent PKC isoforms,
especially PKCßII, may explain the severe reduction in degranulation
in Btk/Lyn-deficient mast cells. Release of arachidonic acid, the
precursor of leukotrienes, involves Syk, the ERK pathway, and
Ca2+ (66). However, it is unlikely
that the abrogation of leukotriene release in Btk/Lyn-deficient cells
resulted simply from decreased Syk activation because
lyn-/- mast cells
also exhibited reduced Syk activity yet released normal levels of
leukotrienes. The reduction in ERK phosphorylation in
btk-/-lyn-/-
cells is likely to be related to the decrease in leukotriene
production.
Most lyn-/- cell
preparations exhibited delayed, but augmented,
IP3 production and delayed
Ca2+ responses, similar to
lyn-/- chicken B
cells (54). This result is distinct from the enhanced peak
and sustained Ca2+ following BCR stimulation
observed using
lyn-/- B cells from
the same mouse strain (38), further emphasizing the
importance of cellular context in determining functions of Lyn. Despite
the loss of the initial calcium response,
lyn-/- mast cells
degranulate almost normally, suggesting that a low level or delayed
Ca2+ signal remains sufficient for
FcRI-induced degranulation. This situation may be similar to human
basophils, in which PMA, a potent PKC activator, can induce histamine
release without pharmacologic Ca2+ release
(55). The reduced initial peak calcium flux in
lyn-/- and
btk-/-lyn-/-
mast cells likely results from the combined effects of reduced
Lyn-dependent transphosphorylation of both Syk and Btk and a reduced
activation of PI3-K isoforms. In contrast, the dramatic late-phase
increase in IP3 production and gradual increase
in intracellular Ca2+ concentration may represent
a loss of Lyn-dependent inhibitory response. This may be mediated at
least in part via reduced activation of the 5' inositol phosphatase
SHIP leading to Btk membrane targeting and activation. This
mechanism would be consistent with the relative reduction in peak
IP3 response in
btk-/-lyn-/- vs
lyn-/- mast cells.
Interestingly, our present study showed that the activity of PKC and
PKCßII, low before stimulation, is strongly increased upon FcRI
stimulation in
lyn-/- mast cells,
consistent with data on
lyn-/- B cells
(67). This data and the following observations suggest
that PKCßII is involved in mast cell degranulation. First, Ozawa et
al. (16) showed that PKCß (ßI or ßII is not clear
from the paper) plus Ca2+ can reconstitute
degranulation in permeabilized RBL-2H3 cells. Second, although the
autophosphorylating activity of PKCßII is not strongly increased by
FcRI stimulation, this PKC isoform is translocated from the cytosol
to the membrane compartment as vigorously as PKC and PKCßI (data
not shown). Third, the translocation of PKCßII is not severely
affected but that of PKCßI is greatly reduced in
btk-/- cells (data
not shown). These differences correlate with the mild effect of Btk
deficiency on degranulation (Ref. 22 and this study).
Finally,
PKCß-/- mast
cells exhibited drastically reduced degranulation (68).
However, relative contribution of PKCßII vs other PKC isoforms to
FcRI-induced degranulation remains to be studied.
Opposing functions of Btk vs Lyn in mast cell cytokine secretion
Cytokines and chemokines comprise an important subset of
proinflammatory mediators that participate in the induction of the
late-phase allergic responses following mast cell activation (1, 56). Our results demonstrate that Btk and Lyn have opposing
roles in cytokine production/secretion in mast cells. Strikingly,
Lyn-deficient cells secreted 3-fold more cytokines than wild-type
cells. This enhanced response was dependent on Btk, as Btk/Lyn doubly
deficient cells exhibited markedly reduced (TNF-) or absent (IL-2)
secretion upon FcRI cross-linking. A similar Btk-dependent,
inhibitory role for Lyn is observed in proliferative response to BCR
cross-linking in B lymphocytes (36, 37). In B cells, Lyn
mediates down-regulation of BCR signals by phosphorylating CD22
(38, 39, 40, 41) and paired Ig-like receptor B (69)
and is also involved in FcRIIb-mediated inhibition (31, 40). Analogous Lyn-dependent signals may be involved
in the negative regulation of FcRI-dependent responses in mast
cells, as FcRIIb was shown to be tyrosine-phosphorylated by
FcRI-associated Lyn upon coligation (70).
MAP kinase activation in response to either FcRI cross-linking in
mast cells (Fig. 9) or BCR cross-linking in B cells is enhanced in the
absence of Lyn (31). This may explain the increased
production of cytokines by activated
lyn-/- mast cells,
because the duration of MAP kinase activation determines the outcome of
receptor stimulation (e.g., proliferation vs differentiation (reviewed
in Ref. 71)), which probably involves the differential
expression of transcription factors such as Fos and Jun families
(72). Transcription of TNF- and IL-2 involves several
transcription factors. For example, TNF- gene activation induced by
the TCR/CD3 complex requires NF-AT, c-Jun, and ATF-2
(73, 74, 75). c-Jun and ATF-2 are phosphorylated and regulated
by JNK (reviewed in Ref. 57). We have evidence that
activators of ERK and JNK can also stimulate transcription from both
the IL-2 and TNF- promoters in mast cells (data not shown). As
previously described (25), Btk-deficient cells exhibited
reduced JNK activation, accounting for the lower cytokine production in
these cells (26). Both JNK activation and IL-2 secretion
were completely abrogated in Btk/Lyn-deficient cells. IL-2 production
in mast cells is more strictly dependent on Btk than TNF- both in
the presence and absence of Lyn. This phenomenon is consistent with the
recent finding that JNK not only regulates the transcriptional
activation of the IL-2 gene but also controls the stability of IL-2
mRNA in T cells (76).
Implications
We demonstrate that Btk and Lyn have both redundant and opposing
functions in mast cell growth and FcRI-dependent signaling. Similar
observations have been made in B and T lymphocytes (36, 37, 41, 43). The present study extends these previous observations in
two important ways. First, the loss of function of Btk and/or Lyn in B
cells significantly alters the generation of B cell developmental
subpopulations, making it difficult to directly compare BCR-dependent
signals in cells derived from these animals. In the current study, the
functional interaction between Btk and Lyn was evaluated in closely
matched bone marrow-derived mast cell populations using identical
receptor activation permitting a detailed analysis of downstream
FcRI-dependent signals. Second, the current work identifies a
striking impairment in all aspects of mast cell activation in
Btk/Lyn-deficient cells. The limited residual function present in these
cells is likely to be dependent on expression of alternative Src and
Tec family PTKs. Together, these finding strongly suggest that Btk and
Lyn represent a critical combined target for pharmacological
intervention in allergic diseases and related disorders involving mast
cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Toshiaki Kawakami, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121.
3 The abbreviations used are: FcRI, high-affinity IgE receptor; Btk, Bruton’s tyrosine kinase; DNP, dinitrophenyl; HSA, human serum albumin; IP3, inositol 1,4,5-trisphosphate; MAP, mitogen-activated protein; PKC, protein kinase C; PLC, phospholipase C; PTK, protein-tyrosine kinase; SH, Src homology; ITAM, immunoreceptor tyrosine-based activation motif; PH, pleckstrin homology; BCR, B cell receptor; PI3-K, phosphatidylinositol 3-kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-related kinase.
Received for publication November 11, 1999. Accepted for publication May 9, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/Igß immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270:11590.RI-dependent mast cell degranulation and cytokine production. J. Exp. Med. 187:1235.2. J. Exp. Med. 184:31. activates Bruton’s tyrosine kinase in concert with Src family kinases. Proc. Natl. Acad. Sci. USA 94:13820.RI cross-linking. J. Immunol. 148:3513.[Abstract]
RI cross-linking. J. Immunol. 152:642.[Abstract]
RI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions. J. Exp. Med. 185:663.RI case. Immunol. Today 14:222.[Medline]
. J. Clin. Invest. 87:446.
radiation-induced apoptosis. J. Biol. Chem. 271:631.RI is not shared by a G protein-coupled receptor. J. Biol. Chem. 270:10960. receptor I-associated lyn-dependent phosphorylation of Fc receptor IIB during negative regulation of mast cell activation. J. Immunol. 160:1647. gene promoter. J. Exp. Med. 178:1365. gene regulation in activated T cells involves ATF-2/Jun and NFATp. Mol. Cell. Biol. 16:459.[Abstract]
gene in B cells and T cells by NFATp and ATF-2/JUN. Mol. Cell. Biol. 16:5232.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  D. L. Barber Truth or dare: role of Liar in EPO-dependent signaling Blood, April 16, 2009; 113(16): 3650 - 3651. [Full Text] [PDF]
|
|
|
|
 |
|
 H. Oda, M. Fujimoto, M. S. Patrick, D. Chida, Y. Sato, Y. Azuma, H. Aoki, T. Abe, H. Suzuki, and M. Shirai RhoH Plays Critical Roles in Fc{epsilon}RI-Dependent Signal Transduction in Mast Cells J. Immunol., January 15, 2009; 182(2): 957 - 962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. S. Kuehn, E. J. Swindle, M.-S. Kim, M. A. Beaven, D. D. Metcalfe, and A. M. Gilfillan The Phosphoinositide 3-Kinase-Dependent Activation of Btk Is Required for Optimal Eicosanoid Production and Generation of Reactive Oxygen Species in Antigen-Stimulated Mast Cells J. Immunol., December 1, 2008; 181(11): 7706 - 7712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Iyer and A. August The Tec Family Kinase, IL-2-Inducible T Cell Kinase, Differentially Controls Mast Cell Responses J. Immunol., June 15, 2008; 180(12): 7869 - 7877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhang, K. Suzuki, T. Hitomi, and R. P. Siraganian TOM1L1 Is a Lyn Substrate Involved in Fc{epsilon}RI Signaling in Mast Cells J. Biol. Chem., December 28, 2007; 282(52): 37669 - 37677. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Hong, J. Kitaura, W. Xiao, V. Horejsi, C. Ra, C. A. Lowell, Y. Kawakami, and T. Kawakami The Src family kinase Hck regulates mast cell activation by suppressing an inhibitory Src family kinase Lyn Blood, October 1, 2007; 110(7): 2511 - 2519. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Hantschel, U. Rix, U. Schmidt, T. Burckstummer, M. Kneidinger, G. Schutze, J. Colinge, K. L. Bennett, W. Ellmeier, P. Valent, et al. The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib PNAS, August 14, 2007; 104(33): 13283 - 13288. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yamashita, N. Charles, Y. Furumoto, S. Odom, T. Yamashita, A. M. Gilfillan, S. Constant, M. A. Bower, J. J. Ryan, and J. Rivera Cutting Edge: Genetic Variation Influences Fc{epsilon}RI-Induced Mast Cell Activation and Allergic Responses J. Immunol., July 15, 2007; 179(2): 740 - 743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Izawa, J. Kitaura, Y. Yamanishi, T. Matsuoka, T. Oki, F. Shibata, H. Kumagai, H. Nakajima, M. Maeda-Yamamoto, J. P. Hauchins, et al. Functional Analysis of Activating Receptor LMIR4 as a Counterpart of Inhibitory Receptor LMIR3 J. Biol. Chem., June 22, 2007; 282(25): 17997 - 18008. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kitaura, Y. Kawakami, M. Maeda-Yamamoto, V. Horejsi, and T. Kawakami Dysregulation of Src Family Kinases in Mast Cells from Epilepsy-Resistant ASK versus Epilepsy-Prone EL Mice J. Immunol., January 1, 2007; 178(1): 455 - 462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kovarova, C. A. Wassif, S. Odom, K. Liao, F. D. Porter, and J. Rivera Cholesterol deficiency in a mouse model of Smith-Lemli-Opitz syndrome reveals increased mast cell responsiveness J. Exp. Med., May 15, 2006; 203(5): 1161 - 1171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Olivera, N. Urtz, K. Mizugishi, Y. Yamashita, A. M. Gilfillan, Y. Furumoto, H. Gu, R. L. Proia, T. Baumruker, and J. Rivera IgE-dependent Activation of Sphingosine Kinases 1 and 2 and Secretion of Sphingosine 1-Phosphate Requires Fyn Kinase and Contributes to Mast Cell Responses J. Biol. Chem., February 3, 2006; 281(5): 2515 - 2525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Hernandez-Hansen, J. D. J. Bard, C. A. Tarleton, J. A. Wilder, C. A. Lowell, B. S. Wilson, and J. M. Oliver Increased Expression of Genes Linked to Fc{epsilon}RI Signaling and to Cytokine and Chemokine Production in Lyn-Deficient Mast Cells J. Immunol., December 15, 2005; 175(12): 7880 - 7888. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Nishimoto, S.-W. Lee, H. Hong, K. G. Potter, M. Maeda-Yamamoto, T. Kinoshita, Y. Kawakami, R. S. Mittler, B. S. Kwon, C. F. Ware, et al. Costimulation of mast cells by 4-1BB, a member of the tumor necrosis factor receptor superfamily, with the high-affinity IgE receptor Blood, December 15, 2005; 106(13): 4241 - 4248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Iwaki, C. Tkaczyk, A. B. Satterthwaite, K. Halcomb, M. A. Beaven, D. D. Metcalfe, and A. M. Gilfillan Btk Plays a Crucial Role in the Amplification of Fc{epsilon}RI-mediated Mast Cell Activation by Kit J. Biol. Chem., December 2, 2005; 280(48): 40261 - 40270. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Xiao, H. Nishimoto, H. Hong, J. Kitaura, S. Nunomura, M. Maeda-Yamamoto, Y. Kawakami, C. A. Lowell, C. Ra, and T. Kawakami Positive and Negative Regulation of Mast Cell Activation by Lyn via the Fc{epsilon}RI J. Immunol., November 15, 2005; 175(10): 6885 - 6892. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. Vonakis, S. P. Gibbons Jr, M. J. Rotte, E. A. Brothers, S. C. Kim, K. Chichester, and S. M. MacDonald Regulation of Rat Basophilic Leukemia-2H3 Mast Cell Secretion by a Constitutive Lyn Kinase Interaction with the High Affinity IgE Receptor (Fc{epsilon}RI) J. Immunol., October 1, 2005; 175(7): 4543 - 4554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Forssell, P. Sideras, C. Eriksson, M. Malm-Erjefalt, K. Rydell-Tormanen, P.-O. Ericsson, and J. S. Erjefalt Interleukin-2-Inducible T Cell Kinase Regulates Mast Cell Degranulation and Acute Allergic Responses Am. J. Respir. Cell Mol. Biol., June 1, 2005; 32(6): 511 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kitaura, K. Eto, T. Kinoshita, Y. Kawakami, M. Leitges, C. A. Lowell, and T. Kawakami Regulation of Highly Cytokinergic IgE-Induced Mast Cell Adhesion by Src, Syk, Tec, and Protein Kinase C Family Kinases J. Immunol., April 15, 2005; 174(8): 4495 - 4504. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kohno, S. Yamasaki, V. L. J. Tybulewicz, and T. Saito Rapid and large amount of autocrine IL-3 production is responsible for mast cell survival by IgE in the absence of antigen Blood, March 1, 2005; 105(5): 2059 - 2065. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Gessner, K. Mohrs, and M. Mohrs Mast Cells, Basophils, and Eosinophils Acquire Constitutive IL-4 and IL-13 Transcripts during Lineage Differentiation That Are Sufficient for Rapid Cytokine Production J. Immunol., January 15, 2005; 174(2): 1063 - 1072. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Gimborn, E. Lessmann, S. Kuppig, G. Krystal, and M. Huber SHIP Down-Regulates Fc{epsilon}R1-Induced Degranulation at Supraoptimal IgE or Antigen Levels J. Immunol., January 1, 2005; 174(1): 507 - 516. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. W. Harder, C. Quilici, E. Naik, M. Inglese, N. Kountouri, A. Turner, K. Zlatic, D. M. Tarlinton, and M. L. Hibbs Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the inhibitory phosphatases SHP-1 and SHIP-1 Blood, December 15, 2004; 104(13): 3901 - 3910. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Furumoto, S. Nunomura, T. Terada, J. Rivera, and C. Ra The Fc{epsilon}RI{beta} Immunoreceptor Tyrosine-based Activation Motif Exerts Inhibitory Control on MAPK and I{kappa}B Kinase Phosphorylation and Mast Cell Cytokine Production J. Biol. Chem., November 19, 2004; 279(47): 49177 - 49187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Urtz, A. Olivera, E. Bofill-Cardona, R. Csonga, A. Billich, D. Mechtcheriakova, F. Bornancin, M. Woisetschlager, J. Rivera, and T. Baumruker Early Activation of Sphingosine Kinase in Mast Cells and Recruitment to Fc{varepsilon}RI Are Mediated by Its Interaction with Lyn Kinase Mol. Cell. Biol., October 1, 2004; 24(19): 8765 - 8777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mangla, A. Khare, V. Vineeth, N. N. Panday, A. Mukhopadhyay, B. Ravindran, V. Bal, A. George, and S. Rath Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses Blood, August 15, 2004; 104(4): 1191 - 1197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Hernandez-Hansen, A. J. Smith, Z. Surviladze, A. Chigaev, T. Mazel, J. Kalesnikoff, C. A. Lowell, G. Krystal, L. A. Sklar, B. S. Wilson, et al. Dysregulated Fc{epsilon}RI Signaling and Altered Fyn and SHIP Activities in Lyn-Deficient Mast Cells J. Immunol., July 1, 2004; 173(1): 100 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tkaczyk, V. Horejsi, S. Iwaki, P. Draber, L. E. Samelson, A. B. Satterthwaite, D.-H. Nahm, D. D. Metcalfe, and A. M. Gilfillan NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc{epsilon}RI aggregation Blood, July 1, 2004; 104(1): 207 - 214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Odom, G. Gomez, M. Kovarova, Y. Furumoto, J. J. Ryan, H. V. Wright, C. Gonzalez-Espinosa, M. L. Hibbs, K. W. Harder, and J. Rivera Negative Regulation of Immunoglobulin E-dependent Allergic Responses by Lyn Kinase J. Exp. Med., June 7, 2004; 199(11): 1491 - 1502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Wu, M. S. Jordan, M. A. Silverman, E. J. Peterson, and G. A. Koretzky Differential Requirement for Adapter Proteins Src Homology 2 Domain-Containing Leukocyte Phosphoprotein of 76 kDa and Adhesion- and Degranulation-Promoting Adapter Protein in Fc{epsilon}RI Signaling and Mast Cell Function J. Immunol., June 1, 2004; 172(11): 6768 - 6774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yamasaki, E. Ishikawa, M. Kohno, and T. Saito The quantity and duration of FcR{gamma} signals determine mast cell degranulation and survival Blood, April 15, 2004; 103(8): 3093 - 3101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sakurai, S. Yamasaki, K. Arase, S. Y. Park, H. Arase, A. Konno, and T. Saito Fc{epsilon}RI{gamma}-ITAM Is Differentially Required for Mast Cell Function In Vivo J. Immunol., February 15, 2004; 172(4): 2374 - 2381. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Hernandez-Hansen, G. A. Mackay, C. A. Lowell, B. S. Wilson, and J. M. Oliver The Src kinase Lyn is a negative regulator of mast cell proliferation J. Leukoc. Biol., January 1, 2004; 75(1): 143 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tkaczyk, M. A. Beaven, S. M. Brachman, D. D. Metcalfe, and A. M. Gilfillan The Phospholipase C{gamma}1-dependent Pathway of Fc{epsilon}RI-mediated Mast Cell Activation Is Regulated Independently of Phosphatidylinositol 3-Kinase J. Biol. Chem., November 28, 2003; 278(48): 48474 - 48484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Suzuki, T. Yoshimaru, T. Matsui, and C. Ra Silver Activates Calcium Signals in Rat Basophilic Leukemia-2H3 Mast Cells by a Mechanism That Differs from the Fc{epsilon}RI-Activated Response J. Immunol., October 1, 2002; 169(7): 3954 - 3962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sada, S. M. S. Miah, K. Maeno, S. Kyo, X. Qu, and H. Yamamura Regulation of Fcepsilon RI-mediated degranulation by an adaptor protein 3BP2 in rat basophilic leukemia RBL-2H3 cells Blood, August 28, 2002; 100(6): 2138 - 2144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kitaura, K. Asai, M. Maeda-Yamamoto, Y. Kawakami, U. Kikkawa, and T. Kawakami Akt-Dependent Cytokine Production in Mast Cells J. Exp. Med., September 5, 2000; 192(5): 729 - 740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-T. Ching, A.-L. Hsu, A. J. Johnson, and C.-S. Chen Phosphoinositide 3-Kinase Facilitates Antigen-stimulated Ca2+ Influx in RBL-2H3 Mast Cells via a Phosphatidylinositol 3,4,5-Trisphosphate-sensitive Ca2+ Entry Mechanism J. Biol. Chem., April 27, 2001; 276(18): 14814 - 14820. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
