From the Department of Medicine, Harvard Medical School and the Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, Massachusetts 02115
Received for publication, April 20, 2001
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ABSTRACT |
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Leukotriene C4 synthase
(LTC4S), the terminal 5-lipoxygenase pathway enzyme that is
responsible for the biosynthesis of cysteinyl leukotrienes, has been
deleted by targeted gene disruption to define its tissue distribution
and integrated pathway function in vitro and in
vivo. The LTC4S The cysteinyl leukotrienes
(cysLTs),1 leukotriene (LT)
C4, and its metabolites, LTD4 and
LTE4, are potent lipid mediators of tissue inflammation,
particularly implicated in allergic and asthmatic diseases (1, 2). In
humans, inhalation of cysLTs constricts bronchial smooth muscle and
attracts eosinophils, and intradermal injection elicits an increase in
vascular permeability (3-6). The contribution of the cysLTs to the
pathophysiology of bronchial asthma is established by the therapeutic
efficacy of inhibitors of their biosynthesis (7) and antagonists of
their receptor-mediated action (8). The cellular generation of
LTC4 requires activation with
Ca2+-dependent translocation of cytosolic
phospholipase A2 and 5-lipoxygenase (5-LO) to the
perinuclear and endoplasmic reticular membranes. There, in the presence
of 5-LO-activating protein (FLAP), the arachidonic acid released by
cytosolic phospholipase A2 is converted to
5-hydroperoxyeicosatetraenoic acid and then to LTA4
(9-11). LTA4 is processed either to the dihydroxy
leukotriene, LTB4, by LTA4 hydrolase (12) or to
LTC4 through conjugation with reduced glutathione by
LTC4 synthase (LTC4S) (13-15). After
carrier-mediated export of LTC4 (16), glutamic acid and
glycine are sequentially cleaved by LTC4S is an 18-kDa integral membrane protein that shows
glutathione S-transferase (GST) activity that is strictly
specific for LTA4 as a substrate (13-15). The human
LTC4S cDNA encodes a protein of 150 amino acids and
belongs to a recently recognized superfamily of membrane-associated
proteins in eicosanoid and glutathione metabolism that includes FLAP
and microsomal GSTs (MGSTs) (27). LTC4S shows 44% amino
acid identity with MGST2 (28) and 31% identity with FLAP. MGST2 and
MGST3 (29) conjugate glutathione not only to xenobiotics but also to
LTA4 to form LTC4, and they are ubiquitously
expressed even in cells lacking the capacity to provide
LTA4. LTC4 can be formed through the
transcellular metabolism of LTA4 by cells that express
LTC4S, such as platelets (30), or MGST2, such as
endothelial cells (31). With the exception of platelets,
LTC4S has been identified only in hematopoietic cells that
also express 5-LO.
We sought to establish that LTC4S was the dominant
constitutive enzymatic source of LTC4 in situ in
the mouse by targeted gene disruption of the LTC4S gene.
The loss of function in the gene-disrupted mice relative to their
controls was then used to quantitate the roles of LTC4S in
models of innate and adaptive immune inflammation. Zymosan, a yeast
cell wall polysaccharide, was used to elicit an intraperitoneal
extravasation of plasma proteins, and hapten-specific, IgE-mediated
passive cutaneous anaphylaxis provided a permeability increment at the
skin. We also assessed for the catalytic function of LTC4S
in various tissues relative to the bifunctional GSTs by the decrement
in LTC4 biosynthesis in the LTC4S
Generation of LTC4S Culture of BMMC and LTC4S mRNA
Analysis--
Bone marrow cells were collected from femurs and tibiae
of mice and cultured for 4-6 weeks in RPMI 1640 medium containing 10%
fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 units/ml penicillin, 100 µg/ml streptomycin, and 1% culture supernatant from Chinese hamster
ovary cells expressing mouse interleukin-3 (33, 34). The culture medium
for the BMMC was changed every week, and the cell density was adjusted
to 3 × 105/ml at every passage. After 4 weeks, more
than 97% of the cells were BMMC as assessed by staining with
Wright-Giemsa and toluidine blue.
Total RNA was isolated from BMMC with Tri-Reagent (Sigma). A 20-µg
sample of the total RNA was resolved by electrophoresis on a
formaldehyde-denatured gel and transferred to a nylon membrane (Gelman
Sciences) with 20× SSC for 24 h. The membrane was baked at
80 °C for 2 h, prehybridized at 42 °C for 2 h in 5×
SSC, 5 × Denhardt's solution, 50% formamide, 0.2% SDS, 100 µg/ml denatured salmon sperm DNA, and then hybridized at 42 °C for
16 h with a 32P-labeled mouse LTC4S
cDNA fragment prepared with a Megaprime DNA labeling kit (Amersham
Pharmacia Biotech). The blot was washed once in 0.5× SSC, 0.1% SDS at
60 °C for 30 min and twice in 0.2× SSC, 0.1% SDS at 60 °C for
30 min and was then exposed to an Eastman Kodak Co. AR film for 24 h at Enzyme Assay--
Mice were euthanized with CO2, and
various tissues were isolated and homogenated with a Tissue-Tearor
homogenizer (Biospec Products) in five volumes of a buffer containing
50 mM HEPES, 0.1 mM EDTA, 1 mM
2-mercaptoethanol, pH 7.9. BMMC were washed, suspended in
phosphate-buffered saline (PBS), and sonicated for 2 min at 4 °C.
The tissue homogenates and the BMMC lysates were centrifuged at
1,000 × g for 5 min at 4 °C, and the supernatants were collected for measurements of LTC4S activity and
protein concentration. To assay LTC4S activity, the samples
were incubated in 200 µl of HEPES buffer, pH 7.6, containing 10 mM MgCl2, 10 mM glutathione at room
temperature. The reaction was started by the addition of 2 µl of
LTA4-methyl ester (ME) (Dr. J. Rokach, Florida Institute
of Technology, Melbourne, FL), which had been dried in nitrogen and
dissolved in methanol containing 3% triethylamine, to give a final
concentration in the reaction of 20 µM. After incubation
for 10 min at room temperature, the reactions were terminated by the
addition of three volumes of methanol containing 200 ng of
PGB2. Samples were analyzed for LTC4-ME by
reverse phase-high performance liquid chromatography (RP-HPLC) (14).
Protein concentration was determined by the Bradford method (35) with
bovine IgE-dependent Activation of BMMC--
BMMC were
washed, suspended at a concentration of 1 × 107
cells/ml in Hanks' balanced salt solution containing 1 mM
CaCl2, 1 mM MgCl2, and 0.1% bovine
serum albumin (HBSA2+), and sensitized with 2 µg/ml monoclonal anti-dinitrophenyl (DNP) IgE (Sigma) for 1 h at
4 °C. After being washed with HBSA2+, the cells were
resuspended at a concentration of 1 × 107 cells/ml in
HBSA2+ and stimulated with 10 µg/ml goat anti-mouse
immunoglobulin (Jackson ImmunoResearch). After 15 min, the reaction was
stopped by centrifugation at 120 × g for 5 min at
4 °C, and the supernatants were retained for assays of
Zymosan A-induced Peritoneal Inflammation--
Each mouse
received an intravenous injection of 0.5% Evans blue dye (10 ml of dye
solution/kg of body weight) in PBS immediately before the
intraperitoneal injection of 1 ml of zymosan A suspension (1 mg/ml in
PBS; Sigma). Mice were euthanized by CO2 at time points of
10, 30, 60, and 120 min and underwent peritoneal lavage with 4 ml of
cold PBS. Cells were sedimented from the lavage fluid by centrifugation
at 500 × g for 5 min, and Evans blue dye extravasation was assessed by light spectrophotometry of the supernatants at 610 nm.
In separate experiments to determine the levels of LTB4 and
cysLTs in the lavage fluid, mice were injected with zymosan A
suspension without intravenous injection of Evans blue dye. After
2 h, the peritoneal lavage fluid was collected and centrifuged at
500 × g for 5 min. After the addition of ~10,000 dpm
of [3H]LTB4 and 100 ng of PGB2 in
ethanol (4 times the volume of the supernatant), the lavage fluid
supernatant was incubated on ice for 30 min and centrifuged at
10,000 × g for 10 min at 4 °C. The ethanolic
supernatant was evaporated by vacuum centrifugation, dissolved in 200 µl of 50 mM HEPES buffer, pH 7.1, containing 50%
methanol, and analyzed by RP-HPLC as described above. The UV absorbance
at 280 nm and the UV spectra were recorded simultaneously. The fraction
containing [3H]LTB4 was collected, evaporated
by vacuum centrifugation, and dissolved in 200 µl of enzyme
immunoassay buffer for detection of LTB4 (Cayman Chemical).
LTE4, the only cysLT detected, was quantitated against the
internal standard as described above.
Passive Cutaneous Anaphylaxis--
LTC4S
Statistical Analysis--
Results of the experiments were
expressed as means ± S.E. Student's t test was used
for the statistical analysis of the results. P values < 0.05 were considered to be significant.
Generation of LTC4S
We used BMMC, which are known to generate LTC4 (39), to
confirm that the mRNA for LTC4S is not expressed
because of the homologous recombination of the targeting construct.
Northern blot analysis with total RNAs from BMMC of
LTC4S Functional Disruption of LTC4S in Mouse Tissues and
BMMC Assayed in Vitro--
We sought to establish that
LTC4S function was absent in BMMC from the
LTC4S gene-disrupted mice. No LTC4
biosynthesis was detected in extracts of BMMC from
LTC4S
GST activity for LTA4-ME in the testis of
LTC4S
The activation of BMMC through their high affinity receptor for IgE
(Fc Functional Assessment of LTC4S Disruption in
Vivo--
To elucidate the possible role of cysLTs in acute
inflammation, we examined the in vivo responses of
LTC4S
Zymosan is a yeast cell wall polysaccharide that can directly stimulate
monocytes to generate leukotrienes (45) or can act indirectly via
activation of the alternative complement pathway to provide peptides
(46) capable of eliciting leukotrienes (47). Together with the ability
of peritoneal macrophages to generate LTC4 in response to
zymosan ex vivo (45), the findings that the zymosan-induced
elaborations of LTC4 in the peritoneal cavity are similar
in time course and concentration in C5a-deficient and sufficient mice
and also in mast cell-deficient and control mice (48) suggest that the
monocyte/macrophages in the peritoneal cavity are directly activated
with zymosan to produce leukotrienes. In the zymosan-elicited
peritoneal inflammation model, LTC4 was generated with a
peak at 30 min and was converted to LTE4 with a peak at 60 min, whereas vascular permeability, assessed by protein concentration,
increased rapidly after injection and reached a peak at 120 min (45).
In other studies, the level of LTB4 peaked at 120-180 min
(45), followed by the recruitment of neutrophils to a plateau 360-420
min after zymosan injection (48, 49). We focused on the vascular
permeability component of the zymosan effect, which was significantly
impaired from 10 to 60 min in the LTC4S
To examine the contribution of cysLTs to a mast cell-mediated acute
allergic reaction in the skin, we performed passive cutaneous anaphylaxis in the ears of the LTC4S
Topical administration of mast cell-derived mediators such as
LTC4, LTD4, histamine, and serotonin has been
demonstrated to induce ear edema in the mouse as assessed after 30 min
by Evans blue dye extravasation. Serotonin and LTC4 were
about 100 and 10 times, respectively, more potent than histamine on a
weight basis in eliciting increased vascular permeability in the mouse ear (51). The contribution of the cysLTs to the ear edema in a model of
passive IgE-induced cutaneous anaphylaxis had been considered to be
minimal, because CysLT1 receptor antagonists did not block
increased vascular permeability (52, 53). The 5-LO
/
mice developed normally and were fertile. LTC4S activity,
assessed by conjugation of leukotriene (LT) A4 methyl ester
with glutathione, was absent from tongue, spleen, and brain and
90% reduced in lung, stomach, and colon of the LTC4S
/
mice. Bone marrow-derived mast cells (BMMC)
from the LTC4S
/
mice provided
no LTC4 in response to IgE-dependent
activation. Exocytosis and the generation of prostaglandin
D2, LTB4, and 5-hydroxyeicosatetraenoic acid by
BMMC from LTC4S
/
mice and
LTC4S +/+ mice were similar,
whereas the degraded product of LTA4,
6-trans-LTB4, was doubled in BMMC from
LTC4S
/
mice because of lack of
utilization. The zymosan-elicited intraperitoneal extravasation of
plasma protein and the IgE-mediated passive cutaneous anaphylaxis in
the ear were significantly diminished in the LTC4S
/
mice. These observations indicate that
LTC4S, but not microsomal or cytosolic glutathione
S-transferases, is the major LTC4-producing enzyme in
tissues and that its integrated function includes mediation of
increased vascular permeability in either innate or adaptive immune
host inflammatory responses.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-glutamyl transpeptidase and
dipeptidase to form LTD4 and LTE4, respectively
(17, 18). Two cysLT receptors, termed CysLT1 and
CysLT2 receptors, are presently known. Whereas the CysLT1 receptor has a marked preference for signal
activation by LTD4, the CysLT2 receptor has a
similar recognition of LTC4 and LTD4 with a
higher Kd value relative to that of the CysLT1 receptor (19, 20). Mast cells also metabolize the
released arachidonic acid to prostaglandin (PG) D2 by the
successive action of PG endoperoxide synthase-1 or PG endoperoxide
synthase-2 (21, 22) and hematopoietic PGD synthase (23, 24). Whereas a
PGD2 receptor, termed DP, is prominent on smooth muscle
such as airways and microvasculature, a recently identified
PGD2 receptor, termed CRTH2, is localized to hematopoietic
cells such as T helper type 2 (Th2) cells, basophils, and eosinophils
(25, 26).
/
mice. Finally, we determined whether the absence
of LTC4S in bone marrow-derived mast cells (BMMC) subjected
to IgE-dependent activation would lead to increased
non-enzymatic hydrolysis of the substrate LTA4 to
6-trans-LTB4 diastereoisomers or to the shunting
of LTA4 to LTA4 hydrolase to form more
LTB4.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
Mice--
A
5.7-kb 129/Ola mouse genomic fragment containing the
LTC4S gene (32) was subcloned into a pcDNA3
vector (Invitrogen). After the endogenous neomycin resistance gene
(neo) of the pcDNA3 and ~400 base pairs of the 5'
region of the 5.7-kb genomic fragment were removed, a neo
gene cassette from pMC1Neo Poly(A) (Stratagene) was inserted to replace
289 nucleotides of intron 1, exon II to exon IV, and 32 nucleotides of
intron 4 of the mouse LTC4S gene. The herpes simplex virus
thymidine kinase (TK) gene was inserted at the 3'-end of the
gene. The resultant targeting vector was linearized and electroporated
into the embryonic stem cell line, AB2.2 (Stratagene). The embryonic
stem cells were selected with G418 (200 µg/ml; Life Technologies,
Inc.) and ganciclovir (2 µM), and homologous
recombination was confirmed by Southern blot analysis of
EcoRI-digested genomic DNA from each embryonic stem cell
clone with the ~400-base pair 5' fragment as a probe (see Fig.
1A). The verified embryonic stem cell clones were
microinjected into blastocysts from C57BL/6 and BALB/c mice, and
chimeric mice were obtained. Chimeras were bred to C57BL/6 and BALB/c
mice, and offspring were genotyped by Southern blot analysis of tail
DNA as described above. Heterozygotes were backcrossed to a C57BL/6 or
BALB/c genetic background, and heterozygotes in the N2 or
N3 generation were intercrossed to obtain homozygotes. All
the experiments were performed with LTC4S
/
mice derived from a BALB/c background except for
the zymosan-induced peritonitis model in which mice from the C57BL/6
background were utilized. All procedures were approved by the Harvard
Medical Area Standing Committee on Animals.
80 °C with an intensifying screen. The probe was stripped,
and the blot was hybridized with a 32P-labeled mouse
glyceraldehyde-3-phosphate dehydrogenase cDNA probe.
-globulin as a standard. Enzyme activity was expressed as
pmol of LTC4-ME/mg/10 min.
-hexosaminidase (
-HEX) and eicosanoids. The cell pellets were
suspended in HBSA2+ and disrupted by repeated
freeze-thawing.
-HEX, a marker of mast cell degranulation, was
quantitated by spectrophotometric analysis of the hydrolysis of
p-nitrophenyl-
-D-2-acetamido-2-deoxyglucopyranoside (36). The percent release of
-HEX was calculated by the formula [S/(S+P)] × 100, where S and P are the
-HEX contents of equal portions of supernatant and cell pellet, respectively. PGD2
was measured by enzyme immunoassay according to the manufacturer's instructions (Cayman Chemical). Leukotrienes and
5-hydroxyeicosatetraenoic acid (5-HETE), the decay product of
5-hydroperoxyeicosatetraenoic acid, were measured by RP-HPLC as
described (37). Briefly, samples were applied to a C18 Ultrasphere RP
column (Beckman Instruments) equilibrated with a solvent of
methanol/acetonitrile/water/acetic acid (10:15:100:0.2, v/v), pH 6.0 (Solvent A). After injection of the sample, the column was eluted at a
flow rate of 1 ml/min with a programmed concave gradient to 55% of the
equilibrated Solvent A and 45% of Solvent B (100% methanol) over 2.5 min. After 5 min, Solvent B was increased linearly to 75% over 15 min
and was maintained at this level for an additional 15 min. The UV absorbance at 280 and 235 nm and the UV spectra were recorded simultaneously. The retention times for PGB2,
LTC4, 6-trans-LTB4, LTB4, LTE4, and 5-HETE were 21.1, 21.6, 23.2, 24.2, 25.0, and 30.7 min, respectively. LTC4,
6-trans-LTB4, LTB4,
LTE4, and 5-HETE were quantitated by calculating the ratio
of each peak area to the peak area of the internal standard
PGB2.
/
and LTC4S +/+
mice received intradermal injections of 25 ng of mouse monoclonal
anti-DNP IgE in 25 µl of saline in the right ear and 25 µl of
saline only in the left ear. After 20 h, mice were injected
intravenously with 100 µg of DNP-human serum albumin in 100 µl of
PBS. At 0, 15, 30, 45, 60, 120, and 240 min after the intravenous
injection, ear thickness was measured with calipers (Dyer
Company). The difference between the thickness of the right and
left ears at each time point reflects the extravasation of plasma
proteins because of the alteration in vascular permeability induced by
the activation of the local mast cells.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
Mice--
The
strategy to disrupt the LTC4S gene is shown in Fig.
1A. The neo
insertion interrupts the coding sequence in exons II, III, and IV,
including Arg-51 in exon II and Tyr-93 in exon IV, respectively, which
are the critical residues for LTC4S activity (38). Mouse
embryonic stem cells derived from the 129/Sv strain were transfected
with the linearized targeting vector and selected with G418 and
ganciclovir. Of 228 G418- and ganciclovir-resistant colonies isolated,
12 clones were identified as targeted clones by Southern blot analysis.
The verified embryonic stem cell clones were microinjected into
blastocysts from C57BL/6 and BALB/c mice for subsequent transfer to
pseudopregnant ICR female mice. 8 and 4 chimeric males with more than
50% chimerism were obtained from C57BL/6 and from BALB/c blastocysts,
respectively, and bred to C57BL/6 or BALB/c female mice. Of 12 chimeric
males, 10 were found to be fertile and provided only 2 male
heterozygotes from a total of 478 offspring, one from a C57BL/6 mother
and the other from a BALB/c mother, as genotyped by Southern blot
analysis. These heterozygotes were backcrossed to the respective
wild-type females to produce heterozygotes (N2 generation),
and N2 or N3 heterozygotes were interbred to
generate LTC4S
/
homozygous
mice. Southern blot analysis of EcoRI-digested DNA from the
N2F1 progeny demonstrated a 4.5-kilobase (kb)
band for the disrupted gene and a 5.8-kb band for the wild-type gene
and revealed that the ratio of wild-type, heterozygote, and homozygote offspring was 1:2:1 as illustrated for one litter (Fig. 1B).
The LTC4S
/
mice developed
normally without any apparent defects, and both genders were fertile.
Thus, we concluded that the disruption of LTC4S gene did
not cause embryonic lethality, developmental defects, or abnormalities
in fertility or parturition in the mouse.
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Fig. 1.
Generation of
LTC4S gene-disrupted
mice. A, genomic organization of the mouse
LTC4S gene (upper), structure of the
targeting vector (middle), and organization of the putative
recombinant LTC4S allele (lower). The
five exons are shown as boxes with the coding regions in
black. Restriction enzyme sites include BamHI
(B), EcoRI (E), HindIII
(H), PstI (P), PmlI
(Pm), and XmnI (X). The location of a
400-base pair fragment used for Southern blot analysis is shown as a
thick line. B, Southern blot analysis of
EcoRI-digested tail DNAs from LTC4S
/
, LTC4S +/
,
and LTC4S +/+ mice. C,
Northern blot analysis of total RNAs from BMMC of
LTC4S
/
and
LTC4S +/+ mice. Hybridizations were
performed with a 32P-labeled mouse LTC4S
cDNA probe (upper) and then with a
32P-labeled mouse glyceraldehyde-3-phosphate
(GAPDH) dehydrogenase cDNA probe (lower).
Molecular size markers are shown at left.
/
and
LTC4S +/+ mice revealed that the
mature LTC4S mRNA with a size of 0.8 kb present in BMMC
from the LTC4S +/+ mice was not
detected in BMMC from the LTC4S
/
mice (Fig. 1C). A 1.4-kb band
detected in BMMC from the LTC4S +/+
mice was considered to be an unspliced LTC4S mRNA.
Because we used a LTC4S cDNA probe that included exon
V, a 1.8-kb band detected in BMMC from the LTC4S
/
mice was considered to be an unstable transcript
that contained exon V and presumably the neo gene.
/
mice, whereas BMMC from
LTC4S +/
and
LTC4S +/+ mice showed
LTC4 biosynthesis of 2.6 ± 0.78 pmol/min/106 cells (n = 3) and 15.1 ± 0.77 pmol/min/106 cells (n = 3),
respectively. To examine the contribution of LTC4S to the
production of LTC4 in various mouse tissues, we measured the LTC4S activity by monitoring the conjugation of
LTA4-ME with glutathione in tissues from
LTC4S
/
, LTC4S
+/
, and LTC4S +/+
mice (Fig. 2). In the
LTC4S
/
mice, no GST activity
specific for LTA4-ME was detected in the tongue, spleen,
and brain, and only slight activity was detected in the lung (~3% of
the activity of wild-type), stomach (~10% of the activity of
wild-type), and colon (~3% of the activity of wild-type). These
results indicate that targeted disruption of the LTC4S gene
effectively eliminated the conjugation of LTA4-ME with
glutathione in these tissues and establish LTC4S as the
dominant enzyme for this reaction in these tissues.
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Fig. 2.
GST activity for LTA4-ME in
tissues isolated from LTC4S
/
, LTC4S
+/
, and
LTC4S +/+
mice. Tissues isolated from LTC4S
/
, LTC4S +/
,
and LTC4S +/+ mice were assayed for
conjugation of LTA4-ME with glutathione with quantitation
of LTC4-ME by RP-HPLC. Values represent means ± S.E.
(n = 3).
/
mice was essentially the
same as that of the LTC4S +/
and
LTC4S +/+ mice. The
LTC4-ME generated by the testis likely reflects the activity of MGST2, MGST3, and/or Mu-class GSTs (40, 41). However, it
appears that cysLTs are not involved in reproduction, because both
5-LO- and FLAP-deficient mice, which lack the cellular enzymatic capacity to generate the required LTA4 substrate, had
normal fertility and parturition (42-44).
RI) was used to demonstrate the absence of LTC4S in an integrated response of the 5-LO/LTC4S pathway and to
delineate the impact of that absence on arachidonic acid metabolism by
5-LO, by PG endoperoxide synthase-1, and by the terminal pathway
enzymes, LTA4 hydrolase and hematopoietic PGD synthase. As
shown in a representative RP-HPLC assay (Fig.
3), no LTC4 was detected
after activation of the BMMC from the LTC4S
/
mice; LTB4 was relatively
unaffected, and the 6-trans-LTB4
diastereoisomers were increased in this analysis at 280 nm. The data
for three independent experiments, including the quantitation of 5-HETE at 235 nm, are shown in Table I.
The BMMC from the LTC4S
/
mice
generated no LTC4, or its metabolites,
LTD4 and LTE4, produced 5-HETE, a decay product
of 5-hydroperoxyeicosatetraenoic acid, and LTB4, at the
same level as the BMMC from the LTC4S
+/+ mice but did exhibit a 2-fold increment in the
elaboration of 6-trans-LTB4 diastereoisomers,
the nonenzymatic breakdown products of LTA4. BMMC from
LTC4S
/
and
LTC4S +/+ mice released comparable
amounts of
-HEX, a marker for exocytosis (p = 0.2535), and generated comparable amounts of PGD2, another mast cell-derived eicosanoid (p = 0.3243). These
results indicate that LTA4, a common substrate for
LTA4 hydrolase and LTC4S, is not converted to
additional LTB4 but rather undergoes non-enzymatic hydrolysis to 6-trans-LTB4 diastereoisomers in
BMMC from the LTC4S
/
mice and
that the loss of LTC4S does not affect the IgE-mediated exocytosis or major cyclooxygenase pathway in BMMC from the
LTC4S
/
mice. We conclude that
LTC4S is the major LTC4-producing enzyme in
mouse mast cells and tissues and that the disruption of the LTC4S gene does not affect release of mast cell granules or
shunt substrate to other eicosanoid pathways of BMMC.
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Fig. 3.
HPLC analyses of the leukotrienes secreted
from BMMC of LTC4S
/
and
LTC4S +/+
mice. BMMC from LTC4S
/
and
LTC4S +/+ mice were stimulated with
IgE/anti-Ig, and the supernatants were analyzed by RP-HPLC. The results
are representative of the three independent experiments. Retention
times corresponding to the various eicosanoids are depicted and
labeled.
Release of -HEX and production of LTC4, 5-HETE, 6-trans
LTB4, LTB4, and PGD2 by BMMC from
LTC4S
/
and LTC4S+/+ mice
/
and
LTC4S +/+ mice to the
intraperitoneal injection of zymosan A by measurement of plasma protein
extravasation and of cysLTs and LTB4 concentrations in the
peritoneal lavage fluid. Protein extravasation in the
LTC4S
/
mice was significantly
reduced at 10, 30, and 60 min after zymosan injection as compared with
the LTC4S +/+ mice (Fig.
4A). RP-HPLC analyses of the
lavage fluids collected 120 min after zymosan injection revealed a
prominent peak of LTE4 (80.7 ± 4.1 ng/ml;
n = 4) as the only cysLT detected in the wild-type mice
but no LTE4 in the lavage fluid of the
LTC4S
/
mice (Fig. 4,
B and C). However, the LTB4 level in
the peritoneal lavage fluid at this time point was comparable for the
LTC4S
/
(1.34 ± 0.53 ng/ml; n = 4) and LTC4S
+/+ mice (0.82 ± 0.10 ng/ml; n = 4) (p = 0.3722) (Fig. 4C).
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Fig. 4.
Plasma protein extravasation in zymosan
A-induced peritoneal inflammation. A,
LTC4S /
and
LTC4S +/+ mice were injected
intraperitoneally with 1 ml of zymosan A in PBS (1 mg/ml) immediately after the intravenous injection of 0.5% Evans blue
dye. At 10, 30, 60, and 120 min, peritoneal lavage was performed. The
cells in the lavage fluid were removed by centrifugation, and the
absorbance of the lavage fluid supernatant at 610 nm was measured to
quantitate Evans blue dye extravasation. Error bars indicate
S.E. (n = 3-4). *, p < 0.05 as
compared with LTC4S +/+.
B, RP-HPLC analysis at 280 nm of the peritoneal lavage fluid
supernatants from LTC4S
/
(left) and LTC4S +/+
(right) 120 min after zymosan A injection. Arrows
depict the retention times for PGB2 and LTE4.
*, a peak detected at a retention time of about 25.2 min in the
LTC4S
/
lavage fluid was not a
cysLT as assessed by its UV spectrum. Results are representative of
four independent experiments summarized in panel C. C, leukotrienes in the peritoneal lavage fluid supernatants
120 min after zymosan injection of LTC4S
/
(white column) and
LTC4S +/+ (black column)
were resolved by RP-HPLC and quantitated for LTE4 by UV
absorbance at 280 nm relative to the PGB2 standard and for
LTB4 by enzyme immunoassay of the fraction eluting at the
correct time as defined by the [3H]LTB4.
Error bars indicate S.E. (n = 4).
/
mice compared with their controls and noted an
absence of LTE4, the most stable of the cysLTs (50). That
the cysLTs are major mediators of the increase in vascular permeability
induced by zymosan is supported by the previous findings of a similar
attenuation of this response in 5-LO-deficient and FLAP-deficient mice
(44, 49) but not in LTA4 hydrolase-deficient mice (49).
Thus, we conclude that the initial phase of zymosan-elicited plasma
protein extravasation in the peritoneal cavity is in large part because of the cysLTs and not because of LTB4 or even other 5-LO metabolites.
/
and LTC4S +/+
mice (Fig. 5). The intravenous injection
of DNP-human serum albumin elicited rapid ear swelling in both the
LTC4S
/
and wild-type mice that
peaked at 15 min, persisted to 45 min, and then declined with
resolution at 240 min. However, ear swelling was significantly reduced
from 15 to 60 min by ~50% in the LTC4S
/
mice. As BMMC from the LTC4S
/
mice had no capacity to generate
LTC4 in response to IgE-dependent activation
while being fully competent in secretory granule exocytosis and
generation of LTB4 and PGD2, we conclude that
cysLTs are major mediators of the ear swelling initiated in
situ by antigen activation of IgE-sensitized mast cells.
View larger version (17K):
[in a new window]
Fig. 5.
Passive cutaneous anaphylaxis in
LTC4S /
mice. LTC4S
/
and
LTC4S +/+ mice received intradermal
injections of 25 ng of mouse anti-DNP monoclonal IgE in 25 µl of
saline in the right ear and 25 µl of saline only in the left ear.
After 20 h, mice were injected intravenously with 100 µg of
DNP-human serum albumin in 100 µl of PBS, and ear thickness was
measured 0, 15, 30, 45, 60, 120, and 240 min later. The difference
between the thickness of the right and left ears at each time point is
expressed as the mean ± S.E. *, p < 0.05 as
compared with LTC4S +/+.
/
, FLAP
/
, or
LTA4 hydrolase
/
mice also
failed to show attenuated ear edema after passive systemic sensitization with monoclonal anti-DNP IgE followed in 24 h by antigen challenge (44, 49). In as much as there was a fall in body
temperature similar to that of the normal mice, these gene-disrupted
mice did experience systemic anaphylaxis. However, our results that
IgE-dependent passive cutaneous anaphylaxis was significantly reduced in the LTC4S
/
mice as compared with the wild-type littermates
indicate that the cysLTs play an important role in the increased
vascular permeability in the IgE-dependent allergic
reactions in the skin. Thus, the LTC4S
/
mice, generated by targeted disruption of the
gene, do have a discrete phenotype that becomes apparent during the
initial phase of altered vascular permeability that accompanies an
inflammatory reaction elicited by either an innate or a specific immune stimulus.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Arlene Sharpe and Lina Du (Brigham and Women's Hospital) for blastocyst injection of embryonic stem cells. We thank Dr. Kongyi Xu for technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants AI31599 and HL36110 and the Human Frontier Science Program (to Y. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Brigham and Women's
Hospital, Smith Bldg., Rm. 628, One Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1270; Fax: 617-525-1310; E-mail:
blam@rics.bwh.harvard.edu.
Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M103562200
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ABBREVIATIONS |
---|
The abbreviations used are:
cysLT(s), cysteinyl leukotriene(s);
LT, leukotriene;
5-LO, 5-lipoxygenase;
FLAP, 5-LO-activating protein;
LTC4S, LTC4 synthase;
PG, prostaglandin;
GST, glutathione S-transferase;
MGST(s), microsomal GST(s);
BMMC, bone marrow-derived mast cells;
5-HETE, 5-hydroxyeicosatetraenoic acid;
PBS, phosphate-buffered saline;
ME, methyl ester;
RP-HPLC, reverse phase high performance liquid
chromatography;
-HEX,
-hexosaminidase;
kb, kilobase;
DNP, dinitrophenyl;
HBSAa+, Hanks' balanced salt solution
containing bovine serum albumin.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Dahlen, S. E., Hedqvist, P., Hammarstrom, S., and Samuelsson, B. (1980) Nature 288, 484-486[Medline] [Order article via Infotrieve] |
2. | Lewis, R. A., and Austen, K. F. (1984) J. Clin. Invest. 73, 889-897[Medline] [Order article via Infotrieve] |
3. | Griffin, M., Weiss, J. W., Leitch, A. G., McFadden, E. R., Jr., Corey, E. J., Austen, K. F., and Drazen, J. M. (1983) N. Engl. J. Med. 308, 436-439[Medline] [Order article via Infotrieve] |
4. | Davidson, A. B., Lee, T. H., Scanlon, P. D., Solway, J., McFadden, E. R., Jr., Ingram, R. H., Jr., Corey, E. J., Austen, K. F., and Drazen, J. M. (1987) Am. Rev. Respir. Dis. 135, 333-337[Medline] [Order article via Infotrieve] |
5. | Soter, N. A., Lewis, R. A., Corey, E. J., and Austen, K. F. (1983) J. Invest. Dermatol. 80, 115-119[Abstract] |
6. | Laitinen, L. A., Laitinen, A., Haahtela, T., Vilkka, V., Spur, B. W., and Lee, T. H. (1993) Lancet 341, 989-990[Medline] [Order article via Infotrieve] |
7. | Israel, E., Dermarkarian, R., Rosenberg, M., Sperling, R., Taylor, G., Rubin, P., and Drazen, J. M. (1990) N. Engl. J. Med. 323, 1740-1744[Abstract] |
8. | Manning, P. J., Watson, R. M., Margolskee, D. J., Williams, V. C., Schwartz, J. I., and O'Byrne, P. M. (1990) N. Engl. J. Med. 323, 1736-1739[Abstract] |
9. | Clark, J. D., Milona, N., and Knopf, J. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7708-7712[Abstract] |
10. | Rouzer, C. A., and Samuelsson, B. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6040-6044[Abstract] |
11. | Miller, D. K., Gillard, J. W., Vickers, P. J., Sadowski, S., LeVeille, C., Mancini, J. A., Charleson, P., Dixon, R. A., Ford-Hutchinson, A. W., Fortin, R., Gauthier, J. Y., Rodkey, J., Rosen, R., Rouzer, C., Sigal, I., Strader, C., and Evans, J. F. (1990) Nature 343, 278-281[CrossRef][Medline] [Order article via Infotrieve] |
12. | Evans, J. F., Dupuis, P., and Ford-Hutchinson, A. W. (1985) Biochim. Biophys. Acta 840, 43-50[Medline] [Order article via Infotrieve] |
13. | Penrose, J. F., Gagnon, L., Goppelt-Struebe, M., Myers, P., Lam, B. K., Jack, R. M., Austen, K. F., and Soberman, R. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11603-11606[Abstract] |
14. | Lam, B. K., Penrose, J. F., Freeman, G. J., and Austen, K. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7663-7667[Abstract] |
15. |
Welsch, D. J.,
Creely, D. P.,
Hauser, S. D.,
Mathis, K. J.,
Krivi, G. G.,
and Isakson, P. C.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9745-9749 |
16. |
Lam, B. K.,
Owen, W. F., Jr.,
Austen, K. F.,
and Soberman, R. J.
(1989)
J. Biol. Chem.
264,
12885-12889 |
17. | Anderson, M. E., Allison, R. D., and Meister, A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1088-1091[Abstract] |
18. | Lee, C. W., Lewis, R. A., Corey, E. J., and Austen, K. F. (1983) Immunology 48, 27-35[Medline] [Order article via Infotrieve] |
19. | Lynch, K. R., O'Neill, G. P., Liu, Q., Im, D.-S., Sawyer, N., Metters, K. M., Coulombe, N., Abramovitz, M., Figueroa, D. J., Zeng, Z., Connolly, B. M., Bai, C., Austin, C. P., Chateauneuf, A., Stocco, R., Greig, G. M., Kargman, S., Hooks, S. B., Hosfield, E., Williams, D. L., Jr., Ford-Hutchinson, A. W., Caskey, C. T., and Evans, J. F. (1999) Nature 399, 789-793[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Heise, C. E.,
O'Dowd, B. F.,
Figueroa, D. J.,
Sawyer, N.,
Nguyen, T.,
Im, D.-S.,
Stocco, R.,
Bellefeuille, J. N.,
Abramovitz, M.,
Cheng, R.,
Williams, D. L., Jr.,
Zeng, Z.,
Liu, Q.,
Ma, L.,
Clements, M. K.,
Coulombe, N.,
Liu, Y.,
Austin, C. P.,
George, S. R.,
O'Neill, G. P.,
Metters, K. M.,
Lynch, K. R.,
and Evans, J. F.
(2000)
J. Biol. Chem.
275,
30531-30536 |
21. | DeWitt, D. L., and Smith, W. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1412-1416[Abstract] |
22. | O'Banion, M. K., Winn, V. D., and Young, D. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4888-4892[Abstract] |
23. |
Urade, Y.,
Ujihara, M.,
Horiguchi, Y.,
Igarashi, M.,
Nagata, A.,
Ikai, K.,
and Hayaishi, O.
(1990)
J. Biol. Chem.
265,
371-375 |
24. |
Murakami, M.,
Matsumoto, R.,
Austen, K. F.,
and Arm, J. P.
(1994)
J. Biol. Chem.
269,
22269-22275 |
25. |
Hirata, M.,
Kakizuka, A.,
Aizawa, M.,
Ushikubi, F.,
and Narumiya, S.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11192-11196 |
26. |
Hirai, H.,
Tanaka, K.,
Yoshie, O.,
Ogawa, K.,
Kenmotsu, K.,
Takamori, Y.,
Ichimasa, M.,
Sugamura, K.,
Nakamura, M.,
Takano, S.,
and Nagata, K.
(2001)
J. Exp. Med.
193,
255-262 |
27. | Jakobsson, P.-J., Morgenstern, R., Mancini, J., Ford-Hutchinson, A., and Persson, B. (1999) Protein Science 8, 689-692[Abstract] |
28. |
Jakobsson, P.-J.,
Mancini, J. A.,
and Ford-Hutchinson, A. W.
(1996)
J. Biol. Chem.
271,
22203-22210 |
29. |
Jakobsson, P.-J.,
Mancini, J. A.,
Riendeau, D.,
and Ford-Hutchinson, A. W.
(1997)
J. Biol. Chem.
272,
22934-22939 |
30. |
Scoggan, K. A.,
Jakobsson, P.-J.,
and Ford-Hutchinson, A. W.
(1997)
J. Biol. Chem.
272,
10182-10187 |
31. | Feinmark, S. J., and Cannon, P. J. (1987) Biochim. Biophys. Acta 922, 125-135[Medline] [Order article via Infotrieve] |
32. | Penrose, J. F., Baldasaro, M. H., Webster, M., Xu, K., Austen, K. F., and Lam, B. K. (1997) Eur. J. Biochem. 248, 807-813[Abstract] |
33. | Yokota, T., Lee, F., Rennick, D., Hall, C., Arai, N., Mosmann, T., Nabel, G., Cantor, H., and Arai, K. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1070-1074[Abstract] |
34. | Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene 108, 193-199[CrossRef][Medline] [Order article via Infotrieve] |
35. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
36. | Robinson, D., and Stirling, J. L. (1968) Biochem. J. 107, 321-327[Medline] [Order article via Infotrieve] |
37. |
Hsieh, F. H.,
Lam, B. K.,
Penrose, J. F.,
Austen, K. F.,
and Boyce, J. A.
(2001)
J. Exp. Med.
193,
123-133 |
38. |
Lam, B. K.,
Penrose, J. F.,
Xu, K.,
Baldasaro, M. H.,
and Austen, K. F.
(1997)
J. Biol. Chem.
272,
13923-13928 |
39. | Razin, E., Mencia-Huerta, J. M., Stevens, R. L., Lewis, R. A., Liu, F. T., Corey, E., and Austen, K. F. (1983) J. Exp. Med. 157, 189-201[Abstract] |
40. | Tsuchida, S., Izumi, T., Shimizu, T., Ishikawa, T., Hatayama, I., Satoh, K., and Satoh, K. (1987) Eur. J. Biochem. 170, 159-164[Abstract] |
41. | Ishikawa, T., Tsuchida, S., Satoh, K., and Satoh, K. (1988) Eur. J. Biochem. 176, 551-557[Abstract] |
42. | Chen, X.-S., Sheller, J. R., Johnson, E. N., and Funk, C. D. (1994) Nature 372, 179-182[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Goulet, J. L.,
Snouwaert, J. N.,
Latour, A. M.,
Coffman, T. M.,
and Koller, B. H.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12852-12856 |
44. |
Byrum, R. S.,
Goulet, J. L.,
Griffiths, R. J.,
and Koller, B. H.
(1997)
J. Exp. Med.
185,
1065-1075 |
45. | Doherty, N. S., Poubelle, P., Borgeat, P., Beaver, T. H., Westrich, G. L., and Schrader, N. L. (1985) Prostaglandins 30, 769-789[CrossRef][Medline] [Order article via Infotrieve] |
46. | Fearon, D. T., and Austen, K. F. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 1683-1687[Abstract] |
47. | Czop, J. K., and Austen, K. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2751-2755[Abstract] |
48. | Rao, T. S., Currie, J. L., Shaffer, A. F., and Isakson, P. C. (1994) J. Pharmacol. Exp. Ther. 269, 917-925[Abstract] |
49. |
Byrum, R. S.,
Goulet, J. L.,
Snouwaert, J. N.,
Griffiths, R. J.,
and Koller, B. H.
(1999)
J. Immunol.
163,
6810-6819 |
50. | Koller, M., Schonfeld, W., Knoller, J., Bremm, K. D., Konig, W., Spur, B., Crea, A., and Peters, W. (1985) Biochim. Biophys. Acta 833, 128-134[Medline] [Order article via Infotrieve] |
51. | Inagaki, N., Goto, S., Yamasaki, M., Nagai, H., and Koda, A. (1986) Int. Arch. Allergy Appl. Immunol. 80, 285-290[Medline] [Order article via Infotrieve] |
52. | Inagaki, N., Goto, S., Nagai, H., and Koda, A. (1985) Int. Archs Allergy Appl. Immunol. 78, 113-117[Medline] [Order article via Infotrieve] |
53. | Miura, T., Inagaki, N., Goto, S., Yoshida, K., Nagai, H., and Koda, A. (1992) Eur. J. Pharmacol. 221, 333-342[Medline] [Order article via Infotrieve] |