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of July 31, 2017.
Impaired FcεRI-Dependent Gene Expression
and Defective Eicosanoid and Cytokine
Production as a Consequence of Fyn
Deficiency in Mast Cells
Gregorio Gomez, Claudia Gonzalez-Espinosa, Sandra
Odom, Gabriela Baez, M. Eugenia Cid, John J. Ryan and
Juan Rivera
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 175:7602-7610; ;
doi: 10.4049/jimmunol.175.11.7602
http://www.jimmunol.org/content/175/11/7602
The Journal of Immunology
Impaired Fc⑀RI-Dependent Gene Expression and Defective
Eicosanoid and Cytokine Production as a Consequence of Fyn
Deficiency in Mast Cells1
Gregorio Gomez,* Claudia Gonzalez-Espinosa,† Sandra Odom,* Gabriela Baez,†
M. Eugenia Cid,† John J. Ryan,‡ and Juan Rivera2*
A
ggregation of Fc⑀RI on mast cells results in release of
preformed inflammatory mediators (e.g., histamine, vasoactive amines, proteases, serotonin) from cytoplasmic
granules and de novo production of inflammatory lymphokines
and leukotrienes (LT)3 that serve to mobilize and activate additional circulating leukocytes, thus promoting inflammation (1).
The Src family protein tyrosine kinase (SrcPTK) Lyn, which phosphorylates the ITAM motifs of the Fc⑀RI ␤- and ␥-chains (2) initiates the propagation of signals through the subsequent activation
of Syk kinase and the formation of a macromolecular “signalsome” anchored by the adapter linker for activation of T cells
(LAT) (3). We found that, in addition to Lyn kinase, the SrcPTK
Fyn was also activated upon Fc⑀RI aggregation and that it was
*Molecular Inflammation Section, Molecular Immunology and Inflammation Branch,
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892; †Pharmacobiology Department, Centro de
Investigacion y Estudios Avanzados Zona Sur, Mexico Distrito Federal, Mexico; and
‡
Department of Biology, Virginia Commonwealth University, Richmond, VA 23284
Received for publication July 25, 2005. Accepted for publication September 12, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Department of Health and Human Services and the
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health and the United States-Israel Binational Science Foundation Grant
Number 2000016 (to J.R.). C.G.-E. was supported by Grant Number 39726-Q from
Consejo Nacı́onal de Ciencia y Tecnologia. J.J.R. was supported by National Institutes of Health Grants 1RO1AI43433 and 1R01CA91839.
2
Address correspondence and reprint requests to Dr. Juan Rivera, National Institute
of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health,
Building 10, Room 9N228, 10 Center Drive, MSC 1820, Bethesda, MD 20892-1820.
E-mail address: [email protected]
3
Abbreviations used in this paper: LT, leukotriene; SAP, signaling lymphocyte activation molecule (SLAM)-associated protein; LAT, linker for activation of T cells;
BMMC, bone marrow-derived mast cell; WCL, whole cell lysate; cPLA2, cytosolic
phospholipase A2; wt, wild type; IKK, I␬B kinase; RPA, RNase protection assay;
SrcPTK, Src family protein tyrosine kinase; PLD, phospholipase D; PDK1, PI3Kdependent kinase 1; PKC, protein kinase C; SCF, stem cell factor; ATF, activated
transcription factor.
Copyright © 2005 by The American Association of Immunologists, Inc.
required for optimum mast cell degranulation (4). Others have
demonstrated that Fyn kinase can contribute to the activation of
phospholipase D (PLD) by phosphorylating this enzyme (5, 6) and
we recently found that PLD activity is defective in Fyn-deficient
mast cells (A. Olivera and J. Rivera, submitted for publication).
This is important because PLD has been demonstrated to contribute to mast cell degranulation (7, 8). Nonetheless, the extent of our
knowledge about the Fyn-dependent pathway is limited. In previous work, we demonstrated that Fyn kinase was important in the
phosphorylation of the adapter protein, Grb2-associated binder
protein 2, the phosphorylation of which is critical for activation of
PI3K and for membrane targeting of the SH2 domain-containing
protein tyrosine phosphatase 2 (4, 9). The findings also demonstrated a defect in protein kinase B (Akt) phosphorylation as well
as a loss in PI3K-dependent kinase 1 (PDK1) phosphorylation of
protein kinase C ␦ (PKC␦).
Despite the similarities in structure and function among
SrcPTKs, Lyn and Fyn appear to have opposing roles in regulating
the degranulation of mast cells (10). Lyn-deficient mast cells
showed a hyperdegranulation phenotype that is mediated, at least
in part, as a consequence of increased Fyn kinase activity (10). Lyn
was found to be important for the phosphorylation of the adapter
C-terminal Src kinase-binding protein, which functions as a scaffold for recruitment of C-terminal Src kinase to the membrane
where it exerts negative regulatory control on SrcPTKs, like Fyn,
by phosphorylation of a negative regulatory tyrosine at the C terminus (10). These findings provide evidence for a dominant-negative regulatory role of Lyn kinase in mast cell responses. Given
this conclusion and our prior demonstration that fyn⫺/⫺ mice were
resistant to anaphylactic challenge, and that bone marrow-derived
mast cells (BMMCs) derived from these mice were markedly defective in degranulation, we sought to learn whether Fyn kinase
serves as a positive regulator of mast cell responses. We also
wished to explore the role of Fyn kinase in delayed responses,
which govern the chronic state of allergic inflammation.
0022-1767/05/$02.00
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Fyn kinase is a key contributor in coupling Fc⑀RI to mast cell degranulation. A limited macroarray analysis of Fc⑀RI-induced gene
expression suggested potential defects in lipid metabolism, eicosanoid and glutathione metabolism, and cytokine production.
Biochemical analysis of these responses revealed that Fyn-deficient mast cells failed to secrete the inflammatory eicosanoid products leukotrienes B4 and C4, the cytokines IL-6 and TNF, and chemokines CCL2 (MCP-1) and CCL4 (MIP-1␤). Fc⑀RI-induced
generation of arachidonic acid and normal induction of cytokine mRNA were defective. Defects in JNK and p38 MAPK activation
were observed, whereas ERK1/2 and cytosolic phospholipase A2 (S505) phosphorylation was normal. Pharmacological studies
revealed that JNK activity was associated with generation of arachidonic acid. Fc⑀RI-mediated activation of I␬B kinase ␤ and
I␬B␣ phosphorylation and degradation was defective resulting in a marked decrease of the nuclear NF-␬B DNA binding activity
that drives IL-6 and TNF production in mast cells. However, not all cytokine were affected, as IL-13 production and secretion was
enhanced. These studies reveal a major positive role for Fyn kinase in multiple mast cell inflammatory responses and demonstrate
a selective negative regulatory role for certain cytokines. The Journal of Immunology, 2005, 175: 7602–7610.
The Journal of Immunology
The studies in this report analyzed the role of Fyn kinase in
signaling events controlling gene expression, arachidonic acid production leading to de novo synthesis and release of LTs, and cytokine production and secretion. These responses are essential for
IgE-mediated host defense through effective recruitment of an inflammatory immune response (11). Thus, dysregulation of the production and secretion of these products is a major contributory
factor to the persistent inflammation that characterizes chronic allergic inflammation. We now describe that, beyond its role in degranulation, Fyn kinase is required for various signaling pathways
leading to gene expression. In the absence of Fyn, defective activation of selected MAP kinase signaling pathways was observed.
In addition, the gene expression of various Fc⑀RI-stimulated inflammatory transcription factors and secreted products was defective. These findings demonstrate a key role for Fyn as a positive
regulator of multiple mast cell effector responses. They also reveal
a negative regulatory role for Fyn in selected cytokine responses.
This provides a basis to further explore the therapeutic potential of
this kinase in allergy and asthma.
Abs and reagents
DNP-specific mouse IgE was produced essentially as previously described
(12). Abs to I␬B␣, p44/42, p38, JNK, cytosolic phospholipase A2 (cPLA2),
phospho-Akt (Thr308 and Ser473), phospho-I␬B␣ (Ser32/36), phosphoIKK␣␤ (Ser180/Ser181), phospho-p44/42 (Thr202/Tyr182), phospho-p38
(Thr180/Tyr182), phospho-JNK (Thr183/185), phospho-cPLA2 (Ser505), and
ubiquitin were purchased from Cell Signaling Technology. Abs to Akt and
Vav1 were purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine Abs and Abs to LAT and phospho-LAT (Tyr191) were from Upstate
Biotechnologies. Secondary Abs used were Alexa Fluor 680-conjugated
goat anti-rabbit IgG (Molecular Probes) and IRDye800-conjugated goat
anti-mouse IgG (Rockland Immunochemicals). The proteosome inhibitor
MG-132, p38 inhibitor SB20358, and JNK inhibitor SP600125 were purchased from Calbiochem. Cell culture medium (RPMI 1640) was from
Mediatech. FCS was purchased from Invitrogen Life Technologies. Murine
IL-3 and stem cell factor (SCF) were from PeproTech. DNP36-HSA (Ag),
Triton X-100, n-octyl-␤-D-glucopyranoside, indomethacin, and all other
chemicals were purchased from Sigma-Aldrich. TNF, IL-6, LTB4, LTC4,
CCL4, and IL-13 ELISAs were from R&D Systems and CCL2 ELISA was
from Biosource. [14C]Arachidonic acid was purchased from PerkinElmer
Life Sciences.
Mice, BMMC cultures, and IgE sensitization
Bone marrow was isolated from femurs and tibias of sex and age-matched
(8- to 12-wk-old) fyn⫹/⫹, fyn⫺/⫺, lyn⫺/⫺, and fyn⫺/⫺/lyn⫺/⫺ mice
(SV129 ⫻ C57/BL6, (N5)) that were maintained and used in accordance
with National Institutes of Health guidelines and a National Institute of
Arthritis and Musculoskeletal and Skin Diseases-approved animal study
proposal. Bone marrow cells were cultured in complete RPMI 1640 supplemented with 20% FBS and 20 ng/ml each of SCF and IL-3. Cultures
were monitored by flow cytometry for surface expression of Fc⑀RI and
used for experiments when ⬎95% of the cells were Fc⑀RI⫹. BMMCs were
sensitized with IgE before all experiments by incubating in Tyrode’s-BSA
buffer (20 mM HEPES, 135 mM NaCl, 1 mM MgCl2, 5 mM KCl, 1.8 mM
CaCl2, 5.6 mM glucose, 0.05% BSA (pH 7.4)) containing anti-DNP IgE
(0.1 ␮g/106 cells) for 3 h at 37°C.
Cell lysates, immunoprecipitation, and immunoblotting
Cells (i.e., 3 ⫻ 107 per sample) were first deprived of SCF overnight in
complete RPMI 1640 culture medium and sensitized with IgE. After washing to remove unbound IgE, BMMCs were stimulated with 25 ng/ml Ag in
Tyrodes-BSA for the indicated times at 37°C. For the experiments in which
p38, JNK, and proteosomal activities were inhibited, the cells were preincubated for 20 min at 37°C and activated in Tyrodes-BSA ⫾ pharmacological inhibitor at the indicated concentration. Immediately after stimulation, cells were placed in an ice-water bath and ice-cold PBS was added to
stop the reaction. Pelleted cells were lysed in 1.0 ml of borate-buffered
saline that contained 1% Triton X-100, 60 mM n-octyl-␤-D-glucopyranoside, 2 ␮g/ml leupeptin and pepstatin, 10 ␮g/ml aprotinin, 2 mM PMSF, 5
mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 50 mM
sodium fluoride for 20 min on ice. Lysates were centrifuged for 10 min
(4°C) at 12,000 ⫻ g, and the supernatant was collected. Total protein
concentration was determined using the DC Protein Assay (Bio-Rad). For
immunoprecipitation, 0.5 mg of total protein in 1.0 ml of lysis buffer was
incubated for 3 h with Abs prebound to protein G-Sepharose (mAbs) or
protein A-Sepharose (polyclonal Abs). Proteins were recovered with equal
volume (50 ␮l) of 2⫻ SDS sample buffer containing 1% 2-ME and 1 mM
sodium orthovanadate and resolved by SDS-PAGE. In the case of immunoprecipitation of tyrosine phosphorylated proteins for analysis of Fc⑀RI␥
phosphorylation, a mixture of the Abs (20 ␮g of total) to phosphotyrosine
(4G10 and PY20) was used. The lysates were normalized to equal receptor
numbers before immunoprecipitation based on 125I-␥IgE labeling of receptors. For whole cell lysates (WCL), 50 ␮g of total protein in an equal
volume of 2⫻ SDS sample buffer was resolved by SDS-PAGE. Proteins
were electrophoretically transferred onto nitrocellulose membranes (Invitrogen Life Technologies). Membranes were blocked with Odyssey
Blocking Buffer (Li-Core Biosciences), probed with the desired primary
Ab and appropriate infrared-labeled secondary Ab and visualized using the
Odyssey Infrared Imaging System (Li-Cor Biosciences). Quantitation was
done using Odyssey software.
Gene expression array
The mouse 1.2k BD Atlas nylon macroarray, carrying a diverse array of
signaling genes, was performed by BD Biosciences/Clontech Custom Atlas
Array Hybridization and Analysis services. RNA purification and cDNA
probe synthesis used the Atlas Pure Total RNA Labeling System and hybridization was done using the ExpressHyb solution. Gene expression profiles were obtained from RNA extracted from IgE-sensitized wild-type (wt)
and fyn⫺/⫺ BMMCs. Activation was with 10 ng/ml Ag (DNP-HSA) for 1 h
based on our previous observation of maximal cytokine gene responses
(13). After normalization to housekeeping genes present on the array, the
gene expression profiles of Fc⑀RI-activated wt and fyn⫺/⫺ BMMCs were
compared using AtlasImage software. Two individual analyses of gene
expression were conducted. The cumulative data are reported as the ratio
of the spot intensities using a cutoff ratio of 2.00 with a r2 coefficient of at
least 0.93. Genes listed in Table I had a variance of no ⬎36% and a
minimum cutoff ratio of 3.0.
Cytokine, LT, and arachidonic acid release assays
Fc⑀RI-mediated release of cytokine and LTs was measured by specific
ELISA. For lymphokine measurements, IgE-sensitized cells (2.0 ⫻ 106 per
sample) were stimulated with the indicated concentration of Ag in Tyrodes-BSA for 3 h at 37°C. For LT measurements, IgE-sensitized cells (106
per sample) were stimulated with the indicated concentrations of Ag in
Tyrode’s-BSA containing 10 ␮g/ml indomethacin (Sigma-Aldrich) for 30
min at 37°C. After incubation, the cells were centrifuged (2000 ⫻ g, 2 min,
4°C) and TNF, IL-6, IL-13, CCL2, CCL4, LTB4, and LTC4 were measured
in the collected cell-free medium according to the manufacturer’s instructions. Arachidonic acid release was quantitated using 14C-labeled arachidonic acid. BMMCs (106 per sample) were loaded with 1 ␮Ci 14C-labeled
arachidonic acid overnight at 37°C. After incubation, the cells were washed
twice with Tyrode’s-BSA, sensitized with IgE, and activated with 25 ng/ml
Ag for the indicated time. After activation, cells were pelleted and the
supernatant was removed. The cell pellet was lysed with 1% Triton X-100
detergent on ice for 20 min. Samples (20 ␮l) from supernatant and lysate
were counted and the percentage of arachidonic acid released into the medium from total cellular content was calculated.
RNase protection assay (RPA)
RPA was performed as previously described (13). Briefly, custom-made
templates were used (BD Biosciences), and probe synthesis was with RiboQuant In vitro Transcription Kit (BD Biosciences) using 100 ␮Ci
[␣-33P]UTP (ICN Biomedicals) following the manufacturer’s suggested
protocol. Hybridization was conducted with 10 ␮g of total RNA and 106
cpm of probe in 13 ␮l of hybridization buffer at 56°C overnight. RNaseI
digestion and subsequent steps were conducted according to the manufacturer’s instruction. Protected fragments were precipitated in the presence of
1.5 ␮g of the glycoblue carrier (Ambion) and resolved on a denaturing 6%
polyacrylamide gel, which was autoradiographed and developed in Kodak
Biomax Transcreen-LE using Kodak BioMax MS film (Eastman Kodak).
Quantitation of the autoradiograph was by densitometry using ImageQuant
from Molecular Dynamics. Data was normalized to the control genes L32
or GAPDH. To detect genes whose mRNA expression was not detected by
RPA, we used RT-PCR. BMMC were incubated with IgE as above, washed
once with Tyrode’s-BSA and stimulated with the indicated concentrations
of Ag. After 1 h, cells were harvested and total RNA was extracted using
Tri-Reagent. Total RNA was used for the cDNA synthesis. PCR and gel
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Materials and Methods
7603
7604
Fyn IS REQUIRED FOR LATE-PHASE MAST CELL RESPONSES
Table I. Expression profile of inhibited genes in Fc␧RI-stimulated
fyn⫺/⫺ BMMC
Genea
GenBank
No.
4.6
3.3
3.5
3.3
3.0
5.8
3.3
4.9
D31788
X52264
M29697
X62700
U52826
X03151
M59378
X57349
5.6
U19118
3.0
3.0
J04115
M64292
3.2
3.4
5.5
5.3
U36277
J03752
U36203
U44088
3.4
3.4
3.7
U10551
L21027
U88328
4.7
11.4
3.2
3.8
5.7
3.0
3.2
7.8
5.5
8.5
3.0
X06086
X82402
Z29532
X72795
M15131
M25892
X06203
M35590
M11434
J04806
X52308
a
The RNA purification and cDNA probe synthesis used the Atlas Pure Total RNA
Labeling System and hybridization on the 1.2k BD Atlas nylon macroarray was done
using the ExpressHyb Solution. Gene expression profiles were obtained from RNA
extracted from IgE/Ag-stimulated (10 ng/ml-Ag, 1 h) wt and fyn⫺/⫺ BMMCs
normalized to nonstimulated BMMCs and to housekeeping genes present on the array.
The gene expression profiles of two individual analyses were compared using
AtlasImage software. The cumulative data interpreted to be significant was determined from the ratio of the spot intensities using a cutoff ratio of 2.00 with an r2
coefficient of at least 0.93. Genes reported in this table had no ⬎36% variance among
membranes and a minimum cutoff ratio of 3.0.
analysis was performed using the conditions and primers previously described (14).
Nuclear NF-␬B DNA binding assay
Nuclear NF-␬B DNA binding activity was determined using the TransAM
NF-␬B p65 Transcription Factor ELISA kit (Active Motif) according to the
manufacturer’s instruction. Nuclear lysates were prepared from IgE-sensitized BMMCs that had been activated with 25 ng/ml Ag for the indicated
times. NF-␬B p65 binding activity was determined by incubating nuclear
lysates in wells containing the NF-␬B consensus binding site oligonucleotide. The plates were washed to remove unbound p65 and DNA-bound
p65 was detected with Ab to p65. After washing, a secondary HRP-conjugated Ab was added to the wells and a colorimetric reaction was performed. Absorbance was read at 450 nm.
Results
Altered gene expression upon Fc⑀RI stimulation of Fyn-deficient
mast cells
To further understand the role of Fyn kinase in Fc⑀RI-dependent
mast cell function, we initiated a limited screening of 1176 murine
genes by membrane array analysis. The RNA purification, hybrid-
Evidence of a role for Fyn kinase in mast cell signaling
pathways leading to gene expression
Signaling competence of the Fc⑀RI is primarily provided by the
␥ subunit, whereas the ␤ subunit functions as an amplifier (22,
23). We previously demonstrated that the Fc⑀RI␤ appeared to
be phosphorylated normally in Fyn-deficient mast cells (4);
thus, we directly assessed the consequence of Fyn deficiency on
Fc⑀RI␥ phosphorylation. This was important because deletion
or mutations of the Fc⑀RI␥, which prevent its phosphorylation,
resulted in inhibition of mast cell degranulation and cytokine
production (24, 25). To assess this possibility, phosphorylated
Fc⑀RI from activated BMMCs was immunoprecipitated using antiphosphotyrosine Abs and equal numbers (as determined by 125IlabeledIgE binding) of receptors were resolved by SDS-PAGE. As
shown in Fig. 1A, the amount of Fc⑀RI␥ phosphorylated in the
absence of Fyn was comparable to that of wt cells. Although the
representative example shown in Fig. 1A suggests a modest increase in phosphorylated receptors in the absence of Fyn, this trend
(104 ⫾ 9% of wt levels, n ⫽ 4) was not statistically significant. In
contrast, loss of Lyn had a pronounced inhibitory effect on the
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Cell surface Ag
BST-1, (CD157)
ICAM1
IL-7R ␣
uPAR1 (CD87)
Syndecan 3
Thymus cell Ag 1, ␪ (Thy 1)
TNFR2
Transferrin receptor (CD71)
Transcription factor/regulator
cAMP-dependent transcription factor 3
(ATF3)
AP-1
Anti-proliferative B-cell translocation
gene 2
I␬B␣
Microsomal GST1
Ski-related oncogene, snoN
T cell death-associated protein
(TDAG51)
Cell signaling/metabolic
Gem (Ras family member)
D-3-phosphoglycerate dehydrogenase
Socs 3
Secreted product
Cathepsin L precursor
Fibronectin 1 precursor
Follistatin precursor
Gelatinase B
IL-1␤
IL-4
IL-6 precursor
MIP-1␤
Nerve growth factor ␣ subunit
Osteopontin precursor
Prothrombin precursor
Fold
Decrease
ization, and data analysis on the mouse 1.2k BD Atlas nylon macroarray was performed by BD Biosciences Clontech Custom Atlas
Array Hybridization and Analysis services.
Table I reports Fc⑀RI-induced genes the expression of which
was reduced in Fyn-deficient mast cells, expressed as fold reduction relative to activated wt cells. Although a 2-fold difference in
mRNA expression is considered significant in this screening based
on AtlasImage quantitative analysis, we used a cutoff of at least
3-fold and corroborated the observed results for a number of these
genes by independent methods. The Fc⑀RI-inducible genes affected by Fyn deficiency could be classified into four classes, cell
surface receptors/Ags, transcription factors/regulators, cell signaling/metabolic genes, and secreted factors. Of the cell surface Ag
genes, Thy-1 (5.8-fold), transferrin receptor (4.9-fold), and the
ADP-ribosyl cyclase-bone stromal cell Ag (BST)-1 (4.6-fold) were
most significantly reduced in expression. Of the transcription factors, activated transcription factor (ATF)-3 (5.6-fold), a member of
the CREB/ATF family of transcription factors induced by JNK
(15), and SnoN (5.5-fold), a negative regulator of TGF-␤ signaling
(16), were most affected by Fyn deficiency. The induction of I␬B␣,
which is required for sequestering NF-␬B in the cytosol (17), was
also found to be defective (3.2-fold). Of the cell signaling/metabolic genes, the T cell death-associated protein (TDAG51), which
regulates Fas expression and is Akt regulated (18, 19), and Socs 3
were reduced 5.3- and 3.7-fold, respectively. For genes of secreted
products, fibronectin precursor (11.4-fold), osteopontin precursor
(8.5-fold), and CCL4 (7.8-fold) were most dramatically reduced.
Moreover, defects in induction of IL-1␤, IL-4, IL-6, and CCL4
mRNAs suggested the requirement for Fyn in cytokine gene expression. Interestingly, a reduction in the expression of microsomal glutathione S-transferase (a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (Ref. 20)
and of D-3-phosphoglycerate dehydrogenase (a key enzyme in Lserine biosynthesis and thus in lipid and sphingolipid biosynthesis
(21)), suggested a possible defect in the production of sphingolipids, phospholipids, and possibly eicosanoids by Fyn-deficient mast
cells. In general, these preliminary findings pointed to a key role
for Fyn kinase in Fc⑀RI-dependent induction of a variety of genes
that contribute to the overall inflammatory ability of mast cells. In
this study, we focused on a biochemical analysis of Fc⑀RI-activated signaling pathways that lead to cytokine gene expression and
in determining the consequence of Fyn deficiency on the latephase responses of mast cells.
The Journal of Immunology
recovery of phosphorylated Fc⑀RI␥, with the inhibition averaging
⬃96 ⫾ 3% (n ⫽ 5). To determine whether the small amount of
Fc⑀RI␥ phosphorylated in the absence of Lyn was due to the previously described increase in Fyn kinase activity (10), we analyzed
mast cells derived from Fyn/Lyn double-deficient mice. Fig. 1A
demonstrates that Fyn was not responsible for the small amount of
phosphorylated Fc⑀RI␥ recovered from lyn⫺/⫺ mast cells. The loss
of both Lyn and Fyn kinases caused a slight but consistent enhancement in the amount of phosphorylated receptor, relative to
Lyn-deficient mast cells, suggesting that removal of both kinases
makes Fc⑀RI available as a substrate for a yet unidentified kinase.
The immediate consequence downstream of Fc⑀RI phosphorylation is the activation of Syk kinase and the formation of a macromolecular signaling complex scaffolded by the adapter LAT (3),
both of which contribute to cytokine responses. We previously
demonstrated that Syk and LAT phosphorylation appeared intact
in the absence of Fyn. However, it was possible that membrane
targeting of Syk was altered or that phosphorylation of a specific
tyrosine residues on LAT (such as Y191), which is one of four
known to contribute to mast cell degranulation and lymphokine
production (26), might be selectively impaired. We investigated
the phosphorylation of LAT (Y191) and Vav1 because this adapter
and this guanine nucleotide exchange factor, respectively, interact
with, and are substrates of, Syk and form a complex with LAT
(26). As shown in Fig. 1B, Fyn deficiency did not alter the ability
of these proteins to become phosphorylated in response to Fc⑀RI
stimulation. Tyrosine 191 of LAT is a key contributor to the stability of the macromolecular complex assembled by LAT (26);
thus, its phosphorylation suggests that this complex is intact. This
was further supported by normal ERK activation (Fig. 2B, lower
panels), which we previously demonstrated to be dependent on
FIGURE 2. Activation of Akt and MAPK in IgE/Ag-activated fyn⫺/⫺
BMMCs. IgE-sensitized wt and fyn⫺/⫺ BMMCs were activated with 25
ng/ml Ag for the indicated times, and the phosphorylation of Akt (A),
p38MAPK (B, top panels), ERK1/2 (B, bottom panels), and JNK1/2 (C)
was assessed by SDS-PAGE and Western blotting with Abs directed
against total and phospho-specific Akt (Ser473 and Thr308), p38 (Thr180/181),
ERK1/2 (Thr202/Tyr204), and JNK1/2 (Thr183/185). Fold induction of phosphorylation of ERK1/2 and JNK1/2 is the mean of all experiments. Fold induction
was determined as in Fig. 1. Blots are representative of three or more individual experiments with different BMMC cultures.
LAT (27) and to be part of a complex that included Vav1 (28).
These findings extend our previous observations by demonstrating
that Fyn does not participate in Fc⑀RI phosphorylation and that
phosphorylation of proteins like LAT, Vav1, and ERKs are not
affected by Fyn deficiency. Thus, the apparent defects in Fc⑀RImediated gene induction noted in Table I appear to be independent
of the Lyn-Syk-LAT axis.
Our prior studies (4) also demonstrated that Akt (S473) phosphorylation was defective in Fyn-deficient mast cells. Serine 473
on Akt was recently shown to be a target of PKC␤II in mast cells
(29). However, the key site for Akt activation is T308, which is a
target of PDK1. Fig. 2A demonstrates that Fc⑀RI-dependent Akt
phosphorylation at both T308 and S473 is defective (with 47– 68%
inhibition at 1 min postactivation, respectively) in the absence of
Fyn. The inhibition at the T308 site is consistent with our prior
report of defective PDK1 activation in fyn⫺/⫺ mast cells, which is
required for full activation of multiple PKC isozymes (4). Given
that PKC␤II phosphorylates Ser473 of Akt in mast cells (29), these
results provide evidence of a broader defect in PKC activation in
the absence of Fyn that is likely due to diminished PDK1 activity.
Thus, two signaling pathways (PKCs and Akt) that are known to
be involved in gene expression in many cell types were found to be
defective in the absence of Fyn.
We subsequently analyzed the phosphorylation (activation) of
MAP kinases (p38MAPK, JNK, ERK), the function of which is
key for activation of various transcription factors, like ATF3 and
AP-1, and whose activity has been linked to NF-␬B (30 –32).
Phosphorylation of p38 MAPK and ERK in wt cells was detectable
as early as 1 min postactivation, whereas JNK phosphorylation
was apparent at 5 min postactivation (Fig. 2, B and C). It was
qualitatively and quantitatively apparent that phosphorylation of
both p38 MAPK and JNK1/2 was defective in fyn⫺/⫺ BMMCs
relative to wt cells with the most severe defect (77 ⫾ 9% inhibition, n ⫽ 4) in JNK phosphorylation (Fig. 2, B and C). In contrast
to p38 MAPK and JNK, and consistent with normal LAT phosphorylation and function (27), ERK1/2 phosphorylation was intact
in fyn⫺/⫺ BMMCs (Fig. 2B). Collectively, the findings demonstrate that the activities of PKC, Akt, JNK, and p38MAPK were
defective in the absence of Fyn.
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FIGURE 1. Phosphorylation of Fc⑀RI␥, Vav1, and LAT in IgE/Ag-activated fyn⫺/⫺ BMMCs. IgE-sensitized BMMCs were activated with Ag
for the indicated times and the phosphorylation of Fc⑀RI␥ (A), LAT (B,
upper panel), and Vav1 (C, lower panel) was assessed by SDS-PAGE and
Western blot analysis. Fc⑀RI␥ phosphorylation of equal numbers of receptors (see Materials and Methods) was detected by immunoprecipitating
tyrosine phosphorylated proteins from 3.0 ⫻ 107 wt, lyn⫺/⫺, lyn/fyn⫺/⫺,
and fyn⫺/⫺ BMMCs using a combination of Abs to phosphotyrosine (clone
4G-10 and PY20) and probing with Ab to FcR␥ (A). WCL (50 ␮g of total
protein) was probed with Ab to phospho-LAT (Tyr191) and LAT (B, upper
panels). Phosphorylated Vav1 was detected by immunoprecipitating Vav1
from 0.5 mg of total WCL protein and probing blots with Ab to phosphotyrosine (clone 4G-10) and Vav1 Abs (B, lower panels). Blots are representative of at least three separate experiments with three individual
BMMC cultures. Phosphorylated and total proteins were detected using the
fluorescence-based Odyssey. Fold induction of phosphorylated protein normalized to the amount of each individual protein in a given lane was determined by the relative ratio of fluorescence intensity and compared with
0 min (arbitrarily set to 1).
7605
7606
Arachidonate and LT production and secretion is defective in
the absence of Fyn
FIGURE 3. Assessment of cPLA2 phosphorylation, arachidonic acid
production, and LT secretion in IgE/Ag-activated fyn⫺/⫺ BMMCs. A,
Western blot analysis of cPLA2 phosphorylation at S505 from IgE-sensitized BMMCs activated with 25 ng/ml Ag. Shown is a representative blot.
Fold induction was calculated as in Fig. 1. B, Percent of arachidonic acid
released after IgE/Ag activation. IgE-sensitized BMMCs were loaded with
14
C-labeled arachidonic acid and activated with 25 ng/ml Ag. Arachidonic
acid released into the extracellular medium was calculated at 30-min intervals up to 4 h and is expressed as the percent of total 14C-labeled arachidonic acid incorporated in the cell. IgE-mediated release of LTB4 (C)
and LTC4 (D) was determined by ELISA from BMMCs activated for 30
min with varying concentrations of Ag in Tyrodes buffer containing 10
␮g/ml indomethacin. Mean ⫾ SEM from at least three individual experiments is shown.
agents that selectively inhibit these kinases. As shown in Fig. 4A,
the use of the p38MAPK-selective inhibitor SB203580 in wt mast
cells failed to significantly suppress arachidonic acid release. In
contrast, the JNK-selective inhibitor, SP600125, showed effective
inhibition (66 ⫾ 7%, n ⫽ 3) of arachidonic acid release (Fig. 4B).
Fig. 4, C and D, shows that these inhibitors (at the concentrations
used) selectively inhibited the intended target but had no significant effect on the S505 phosphorylation of cPLA2. These findings
demonstrate an important role for Fyn-dependent JNK activity in
regulating arachidonic acid release in BMMC that is independent
of cPLA2 (S505) phosphorylation.
Fyn is required for initiating NF-␬B nuclear translocation in
IgE/Ag-activated BMMCs
p38 MAPK and JNK have also been implicated in regulating
NF-␬B activity in several cellular systems (30 –32). Moreover, the
down-regulation of I␬B␣ gene expression (Table I) upon Fc⑀RI
stimulation suggested a possible defect in NF-␬B function, given
the regulatory control of I␬B␣ on the activation of this transcription factor (17). The common pathway leading to NF-␬B activation requires the phosphorylation and degradation of the inhibitor
of NF-␬B, I␬B␣, by phosphorylated I␬B kinase (IKK␣␤). This is
a key step in release of NF-␬B/Rel subunits (p50, p65), which then
translocate from the cytosol to the nucleus and initiate cytokine
gene transcription. Degradation of I␬B␣ requires its ubiquitination, which marks it for a proteosomal pathway. Recovery of I␬B␣
protein is dependent on nuclear NF-␬B activity, which initiates
transcription of this gene (17). Because our gene array data showed
a defect in Fc⑀RI-induced I␬B␣ gene expression, this suggested a
possible decrease in nuclear NF-␬B activity. We found that Fc⑀RIinduced phosphorylation of IKK␣ and ␤ subunits was defective in
the absence of Fyn (Fig. 5A). IKK␤ phosphorylation was most
severely inhibited (90 ⫾ 6%) by Fyn deficiency. Although I␬B␣
was moderately phosphorylated in fyn⫺/⫺ BMMCs, it was not degraded as observed in wt cells (Fig. 5A).
FIGURE 4. Arachidonic acid release from wt BMMCs in the presence
of pharmacological inhibitors of p38 and JNK1/2. 14C-Labeled arachidonic
acid release was measured from IgE-sensitized wt and fyn⫺/⫺ BMMCs
activated for 30 min with 25 ng/ml Ag and preincubated for 20 min in the
presence or absence of 5 ␮M p38 inhibitor (SB203580) (A) or 10 ␮M JNK
inhibitor (SP600125) (B). Western blot analysis was done to assess specificity of SB203580 for p38 (C) and SP600125 for JNK (D) with Abs
directed against total and phospho-specific p38 and JNK1/2. p38MAPK
and cPLA2 phosphorylation was determined at 1 min and JNK phosphorylation was determined at 12 min. No differences were observed in cPLA2
phosphorylation with SP or SB. Data in A and B is mean ⫾ SEM of at least
three individual experiments with different BMMC cultures. Significance
was determined by unpaired t test; ⴱ, p ⬍ 0.05.
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Depending on the cell type, ERK, JNK, and p38MAPK have all
been implicated in the activation of cPLA2 (33, 34), the major
enzyme responsible for the production of arachidonate (precursor
of eicosanoids) in mast cells (35, 36). JNK and p38MAPK have
also been implicated in the expression of eicosanoid-related enzymes (37, 38). Moreover, because the genetic profiling data in
Table I suggested a defect in the induction of genes in the membrane-associated proteins in eicosanoid and glutathione metabolism family as well as in L-serine biosynthesis, it seemed reasonable to determine whether cPLA2 and/or arachidonic acid
production was defective. Phosphorylation of S505 of cPLA2 induces increased enzymatic activity and membrane targeting affinity (39). Fig. 3A shows that S505 phosphorylation appears to be
intact in the absence of Fyn. Although a slight loss of cPLA2
protein was noted, this was not significant. This demonstrates that
cPLA2 phosphorylation of S505 in fyn⫺/⫺ BMMC is normal and
thus independent of Fyn kinase, JNK, or p38MAPK. This is consistent with the previous demonstration of a major role for ERK
kinase in the activation of cPLA2 in mast cells (40), an enzyme
whose activity is intact in the absence of Fyn. However, the release
of arachidonic acid was not induced in fyn⫺/⫺ BMMCs even up to
4 h post-Fc⑀RI stimulation (Fig. 3B). In contrast, arachidonic acid
release from wt BMMC peaked at 30 min and was considerably
sustained for up to 4 h. As might be expected from the loss of
arachidonic acid production, the release of LTs, LTB4 and cysLTs
(LTC4), from fyn⫺/⫺ BMMCs was defective over a wide range of
Ag concentrations when compared with wt cells (Fig. 3, C and D).
Of note, LTB4 release was optimal at lower Ag concentrations than
cysLTs from wt cells.
Whether the defective p38MAPK and JNK activation seen in
Fyn-deficient BMMC had any role in the decreased arachidonic
acid production was further explored by use of pharmacological
Fyn IS REQUIRED FOR LATE-PHASE MAST CELL RESPONSES
The Journal of Immunology
7607
FIGURE 6. Fc⑀RI-stimulated cytokine mRNA induction in fyn⫺/⫺ BMMCs.
A, RPA analysis of cytokine (left panel) and chemokine (right panel) mRNA
responses from IgE-sensitized wt and fyn⫺/⫺ BMMCs activated with varying concentrations of Ag. B, RT-PCR analysis was used to detect IL-2, IL-3, and IL-4
mRNA levels in fyn⫺/⫺ BMMCs. Panels show a representative experiment of
three individual experiments.
Because I␬B␣ was rapidly degraded in wt BMMC, its level of
phosphorylation is likely under-represented relative to Fyn-deficient BMMC, in which this protein is stable. To address this possibility, we assessed I␬B␣ phosphorylation and degradation in
BMMCs activated in the presence of the proteosomal inhibitor
MG-132. We predicted that inhibiting I␬B␣ degradation in wt cells
should reveal the extent of the defect in its phosphorylation in
fyn⫺/⫺ BMMCs. As the results demonstrate, 5 ␮M MG-132 was
sufficient to effectively inhibit I␬B␣ degradation (Fig. 5B). I␬B␣
phosphorylation in fyn⫺/⫺ BMMCs was defective (69 ⫾ 7% inhibition, n ⫽ 3) as indicated by the more transient and hypophosphorylated state of this protein. Additional support for defective
phosphorylation and degradation of I␬B␣ came from the observation that Fc⑀RI-inducible ubiquitination of I␬B␣ in fyn⫺/⫺ BMMCs was reduced compared with wt cells (Fig. 5C). Moreover, the
DNA binding activity of nuclear NF-␬B(p65) was remarkably reduced in fyn⫺/⫺ BMMCs (Fig. 5D). This was consistent with the
finding that I␬B␣ phosphorylation and degradation was defective,
thus sequestering NF-␬B (p65) in the cytosol and inhibiting further
I␬B␣ gene expression in response to Fc⑀RI stimulation.
Defective cytokine production and secretion as a consequence of
Fyn deficiency
Given the above results, and those in Table I, we set out to investigate the impact of Fyn deficiency on cytokine gene expression
FIGURE 7. Fc⑀RI-stimulated cytokine secretion in fyn⫺/⫺ BMMCs. Secretion of TNF (A), IL-6 (B), CCL4 (C), CCL2 (D), and IL-13 (E) was
determined by ELISA. IgE-sensitized BMMCs were activated with the
indicated concentrations of Ag for 3 h, and the amount of lymphokine
released into the cell-free culture medium was quantitated. The data are
representative of at least three or more individual experiments conducted
with different BMMC cultures. Data are the mean ⫾ SEM.
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FIGURE 5. Assessment of NF-␬B pathway activation in fyn⫺/⫺ BMMCs.
To analyze the phosphorylation of IKK␣␤ and I␬B␣, BMMCs from wt and
fyn⫺/⫺ were activated with 25 ng/ml Ag for the indicated times, and WCL (50
␮g of total protein) was probed in a Western blot analysis with phosphospecific Abs (A). Western blot analysis was performed on WCLs from wt and
fyn⫺/⫺ BMMCs activated with 25 ng/ml Ag in the presence of 5 ␮M MG-132
proteosomal inhibitor to determine whether the observed defect in I␬B␣ degradation in fyn⫺/⫺ BMMCs was due to inefficient phosphorylation of the protein or defective degradation process (B, upper panel). The ratio of phosphorylated to nonphosphorylated I␬B␣ in the presence of MG-132 was calculated
(B, lower panel). C, Ubiquitination of I␬B␣ was determined by immunoprecipitation of I␬B␣ from 0.5 mg of total WCL protein and Western blot analysis
with Ab to ubiquitin. D, Nuclear NF-␬B p65 DNA binding activity was measured by ELISA. Nuclear lysates were incubated in wells containing the
NF-␬B consensus binding site oligonucleotide followed by binding of secondary HRP-conjugated Ab and colorimetric reaction (see Materials and Methods). Activity is reported as the mean absorbance at 450 nm from four individual experiments. Blots are representative of at least three individual
experiments. Mean ⫾ SEM is shown.
induced by Fc⑀RI stimulation. As shown in Fig. 6, A and B, fyn⫺/⫺
BMMC were defective in production of mRNA for several cytokines including IL-4 and IL-6, confirming the data in Table I.
Quantitation of the IL-6 response by densitometry found a decrease of 59 ⫾ 7% at suboptimal and optimal concentrations of
Ag, which was consistent with the decrease of IL-6 precursor seen
in the gene array analysis (Table I). TNF mRNA was also greatly
reduced in fyn⫺/⫺ BMMCs with as much as 70% inhibition; however, this gene was not on the macroarray. Both of these genes are
known targets of NF-␬B activity in mast cells (41, 42). Interestingly, IL-13 mRNA was normally induced in fyn⫺/⫺ BMMCs and
showed a trend for increased mRNA levels relative to wt cells.
Chemokine mRNAs were also reduced in the absence of Fyn, especially at suboptimal concentrations of Ag, suggesting decreased
sensitivity of these genes in the absence of Fyn kinase. This is
consistent with our previous finding that chemokine production
can occur under conditions of suboptimal occupancy with IgE or
Ag and that these conditions favor the activation of Fyn, Grb2associated binder protein 2, and PI3K (13). Thus, for genes like
CCL2, a significant reduction of mRNA expression was observed
at 1 and 3 ng/ml Ag (⬃55 ⫾ 9%, n ⫽ 3), whereas these differences
were narrowed at higher concentrations (10 ng/ml) of Ag (⬃27 ⫾
6%, n ⫽ 3) (Fig. 6A). In contrast, quantitation of CCL4 revealed
7608
Fyn IS REQUIRED FOR LATE-PHASE MAST CELL RESPONSES
Discussion
The late phase of the mast cell response is largely based on the de
novo synthesis and secretion of inflammatory mediators (1). The
link between gene expression and Src family kinases has long been
recognized (43); however, this varies with cell lineage. Fyn has
been demonstrated to contribute to TCR and CD43-mediated production of regulatory cytokines, like IL-2 (44, 45). Fyn also appears to contribute in the generation of survival signals for naive T
cells, which is likely to be dependent on cytokine production (46).
The role of Fyn in BCR-mediated gene expression seems to be
minor relative to its role in regulating IL-mediated gene expression
(like IL-4 and IL-5) (47, 48). BCR stimulation of Fyn-deficient B
cells showed a modest impairment of downstream signals, whereas
IL-5 signaling was completely blocked. B cell stimulation with
IL-4 selectively activated Fyn kinase but not Lyn kinase, and its
increased activity was associated with the induction of Ig C⑀ gene
expression (48). There is also increasing evidence that differential
compartmentalization of Fyn, in a given cell type, is important for
its particular function in that cell (49). These observed differences
in the cellular role of Fyn, as well as its differential use by particular receptors, emphasizes the importance of exploring the role of
Fyn in mast cell Fc⑀RI-induced gene expression.
It was important to assess the role of Fc⑀RI␥ because of its
essential nature in transduction of signals that initiate the latephase events (24, 50). We observed no significant alterations in the
amount of Fc⑀RI␥ phosphorylated in the absence of Fyn compared
with wt cells. In contrast, only a small fraction of Fc⑀RI␥ is phosphorylated in Lyn-deficient mast cells, consistent with the previous
report of residual Fc⑀RI␤ phosphorylation in these cells (51). Previously (52), we failed to detect phosphorylated Fc⑀RI in Lyndeficient mast cells. Two technical differences may account for the
apparent discrepancy: 1) in this study, only phosphorylated receptor was immunoprecipitated. This contrasts to the previous study
where immunoprecipitation of total (both nonphosphorylated and
phosphorylated) receptors was performed. 2) We now used a mixture of two Abs to phosphotyrosine for immunoprecipitation
(4G10 and PY20), whereas, in the past experiments, only one Ab
was used for detection (PY20). Together, these modifications
likely increased the sensitivity of phosphotyrosine detection. However, importantly, no correlation was established, in either this or
the previous study (52), between the extent of Fc⑀RI phosphorylation and the ability of the mast cells to degranulate or induce
cytokine gene expression and secretion. An apparent incongruity
arises from the loss of JNK activation in Fyn-deficient BMMC
under circumstances where LAT and Vav1 phosphorylation is normal. Our previous findings (14, 27, 28) demonstrated that LAT- or
Vav1-deficiencies caused a loss in JNK activation and that Vav1
contributed to IL-6 production through Rac/JNK-mediated signals.
This suggests that, in mast cells, there is cross-talk between LAT
or Vav1 and Fyn kinase. The view of Fyn-Vav1 cross-talk is supported by multiple studies in lymphocytes demonstrating the importance of Fyn activity in Vav1 phosphorylation (53, 54), the
presence of both Vav1 and Fyn in macromolecular signaling complexes (55), and the role of both Fyn and Vav1 in JNK activation
(56). This defines a possible point where Fyn kinase and Lyndependent LAT and Vav1 signals may intersect to influence gene
expression, as LAT and Vav1-deficiencies also caused loss of cytokine gene expression (14, 27).
Gene array analysis revealed that Fyn deficiency in mast cells
resulted in selectively impaired Fc⑀RI-induced gene expression.
We verified only a small number of the identified genes, focusing
primarily on cytokines. Among the nonverified down-regulated
genes, the T cell death-associated protein (TDAG51), which was
demonstrated to link TCR signaling to Fas (CD95) expression and
whose expression is regulated by Akt (18, 19), was viewed as an
internal control because Fc⑀RI-dependent Akt activation is defective in Fyn-deficient mast cells (Ref. 4 and Fig. 2A) and thus the
expression of Akt-regulated genes should be impaired. In the absence of Fyn, its Fc⑀RI-dependent expression was reduced by 5.3fold. The early response genes ATF3 and AP-1 are transcription
factors whose expression is dependent on JNK activity (15, 57).
Thus, the loss of their Fc⑀RI-mediated expression is consistent
with the observed defect in JNK activation in Fyn-deficient
BMMC. AP-1 expression is also regulated by PKC, with the ␤ and
⑀ isoforms contributing to induction of c-fos and c-jun in mast cells
(58). The finding that the PKC␤II-dependent phosphorylation of
Akt (Ser473) was defective is consistent with the view that activation of this isoform PKC is impaired and likely affects AP-1 expression. This is also consistent with the reduced PDK1 activity in
these cells and the requirement for this enzyme in PKC
activation (59).
Multiple signaling pathways converge in the metabolic/catabolic regulation of lipid metabolism. Among them, a role for MAP
kinases has been demonstrated in generation of arachidonate (34,
39, 60, 61). Our findings demonstrate that Fc⑀RI-dependent JNK
activation is significantly impaired and that p38MAPK activation
is partly impaired in the absence of Fyn. Depending on the cell
type, one or both of these pathways have been implicated in the
activation of cPLA2, which is the essential provider of arachidonic
acid to the 5-lipoxygenase and cyclooxgenase pathways and is
required for activation-dependent induction of prostaglandin endoperoxidase synthetase 2 in mast cells (35, 62). Given the defect
in JNK and p38MAPK activation in the absence of Fyn, our finding that cPLA2 S505 phosphorylation was normal argues against
this site being a target of JNK or p38MAPK activity in these cells.
Nonetheless, the JNK inhibitor SP600125 inhibited arachidonic
acid release in wt BMMCs, whereas the p38MAPK inhibitor
SB203580 did not, demonstrating a dominant role for JNK in arachidonic acid metabolism. JNK activity has been linked both upstream and downstream of arachidonic acid production, suggesting
a key relationship between the activity of this kinase and generation of this lipid metabolite. The failure of fyn⫺/⫺ BMMC to release arachidonic acid and to generate both LTB4 and LTC4, along
with the normal phosphorylation of cPLA2 in these cells, points to
a defect that is likely to be upstream of cPLA2 activation. Based on
requirement of calcium mobilization and S505 phosphorylation
(39) for cPLA2 activation and the ability of Fyn-deficient BMMC
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significant inhibition at suboptimal (⬃85 ⫾ 3%, n ⫽ 3) and optimal doses (⬃64 ⫾ 7%, n ⫽ 3) of Ag. To corroborate these data
with protein secretion, we measured Fc⑀RI-mediated release of
IL-6, IL-13, TNF, CCL2, and CCL4 in the extracellular medium
(Fig. 7). Release of both TNF and IL-6 from fyn⫺/⫺ BMMCs was
severely impaired at all Ag concentrations, measuring ⬍25 ⫾ 4%
(n ⫽ 4) of that released by wt cells when stimulated at optimal Ag
concentrations (Fig. 7, A and B). CCL4 secretion from fyn⫺/⫺
BMMCs was essentially undetectable even though mRNA production was still observed (Fig. 7C vs 6A). Release of CCL2 from
fyn⫺/⫺ BMMCs was also defective, with the largest differences
observed at low Ag concentrations (Fig. 7D), consistent with the
more modest differences in mRNA expression with increasing Ag
dose. Mirroring the mRNA studies, IL-13 secretion was significantly enhanced (Fig. 7E). At optimal doses of Ag, a 3-fold increase in secreted IL-13 was observed, suggesting a negative regulatory role for Fyn in IL-13 expression. Collectively, the findings
demonstrate an important role for Fyn in generating the signals
required for normal mast cell cytokine production and reveal that
Fyn kinase exerts a negative regulatory role for selected cytokines.
The Journal of Immunology
Disclosures
The authors have no financial conflict of interest.
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to initiate these responses, our findings suggest that either a substrate for cPLA2 is limiting or that cPLA2 is mislocalized. Interestingly, the observed phenotype of mast cell cPLA2 deficiency
mirrors that of Fyn deficiency with respect to arachidonic acid
release and eicosanoid secretion (35, 36).
Fyn kinase is also important for de novo production of various
cytokines. IL-2, IL-3, IL-4, IL-6, TNF, CCL2, and CCL4 mRNAs
were considerably reduced and the secretion of several of these
cytokines was dramatically affected confirming the gene array
analysis. A key regulatory signal for cytokines, downstream of
Fyn, is the transcription factor NF-␬B, a well-characterized regulator of inflammation in health and disease (63). NF-␬B is an essential regulator of IL-6 and TNF production in mast cells (41, 42).
Fyn controls the activation of NF-␬〉 through signals leading to the
phosphorylation of I␬B and its degradation. The link between Fyn
kinase and these events is not yet clear. In SrcPTK triple-deficient
B cells (Blk, Fyn, and Lyn), IKK␣␤ activation and NF␬B activity
were also defective (64). The impaired NF-␬B induction could be
overcome by expression of PKC␭, suggesting that a defect in its
activation linked the SrcPTKS to IKK␣␤ activation. The role for
PKCs in NF-␬B activation is also addressed in a study analyzing
the function of a Fyn binding protein called signaling lymphocyte
activation molecule-associated protein (SAP), whose mutation
causes X-linked lymphoproliferative disease (65). SAP-deficient T
cells have impaired NF-␬B activation as well as impaired recruitment of PKC␪ to the immunological synapse (65). Indeed, this
phenotype was reproduced in Fyn-deficient T cells demonstrating
that Fyn-SAP interactions may play an important role in NF-␬B
activation in these cells. These findings may extrapolate to mast
cells, as we previously demonstrated (4), and presently expand the
evidence for decreased PKC activity in Fyn-deficient mast cells.
Given that both PKC␭ and PKC␪ have been implicated in different
cell types, it is of considerable interest to determine which isoform(s) is/are responsible in mast cells. Regardless, the defective
NF-␬B activation in Fyn-deficient mast cells makes a strong case
for the reduced inflammatory competence of these cells.
The key findings of this study argue that Fyn kinase exerts a
major influence on the delayed inflammatory responses of mast
cells. To a large extent, this is mediated through its role in arachidonic acid production and NF-␬B-mediated induction of proinflammatory cytokines. This is seemingly independent of the LynSyk-LAT signaling axis and thus underscores the importance of
Fyn-mediated signals in mast cell-mediated inflammation. We do
not exclude the contribution of Fyn-independent signals to mast
cell inflammatory responses as we could still detect considerable
production (mRNA) and some release of both proinflammatory
and immunoregulatory cytokines. The enhanced levels of IL-13
mRNA and protein are consistent with a negative regulatory role
of Fyn on expression of this gene. These results also serve to
dismiss the possible generalized inactivity of Fyn-deficient
BMMC to Fc⑀RI stimulation. The dominant role of transcription
factors, like NFAT1 and GATA, in regulating the expression of
IL-13 in mast cells has been demonstrated (66) suggesting possible
candidates for the negative regulatory effects of Fyn. Interestingly,
the gene expression analysis also revealed up-regulation of mast
cell protease 4, granzyme B, and cathepsin D (data not shown),
suggesting other possible candidate genes in which Fyn may play
a negative role. While still early in our understanding, the findings
promote the general conclusion that Fyn kinase is important for
mast cells inflammatory responses. The revelation that Fyn kinase
negatively regulates selected mast cell responses suggests an intriguing complexity that warrants further exploration.
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Fyn IS REQUIRED FOR LATE-PHASE MAST CELL RESPONSES