Molecular Basis of Cell and

Developmental Biology:
Targeted Gene Disruption Reveals the Role
of the Cysteinyl Leukotriene 2 Receptor in
Increased Vascular Permeability and in
Bleomycin-induced Pulmonary Fibrosis in
Mice

J. Biol. Chem. 2004, 279:46129-46134.

doi: 10.1074/jbc.M407057200 originally published online August 24, 2004

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Thomas C. Beller, Akiko Maekawa, Daniel S.

Friend, K. Frank Austen and Yoshihide
Kanaoka

THE JOURNAL OF BIOLOGICAL CHEMISTRY

2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 44, Issue of October 29, pp. 46129 46134, 2004
Printed in U.S.A.

Targeted Gene Disruption Reveals the Role of the Cysteinyl

Leukotriene 2 Receptor in Increased Vascular Permeability
and in Bleomycin-induced Pulmonary Fibrosis in Mice*
Received for publication, June 23, 2004, and in revised form, July 26, 2004
Published, JBC Papers in Press, August 24, 2004, DOI 10.1074/jbc.M407057200

Thomas C. Beller, Akiko Maekawa, Daniel S. Friend**, K. Frank Austen,

and Yoshihide Kanaoka
From the Departments of Medicine and Pathology, Harvard Medical School, and the Division of Rheumatology,
Immunology, and Allergy and the **Department of Pathology, Brigham and Womens Hospital,
Boston, Massachusetts 02115

The cysteinyl leukotrienes (cys-LTs),1 leukotriene (LT) C4,

LTD4, and LTE4, are lipid mediators (1, 2) that have been implicated in the pathobiology of allergic and asthmatic inflammatory
responses on the basis of the efficacy of specific intervention with
* This work was supported by National Institutes of Health Grants
AI-07306, AI-31599, and HL-36110. 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.
Both authors contributed equally to this work.
To whom correspondence should be addressed: Brigham and
Womens Hospital, Smith Bldg., Rm. 626C, One Jimmy Fund Way,
Boston, MA 02115. Tel.: 617-525-1263; Fax: 617-525-1310; E-mail:
ykanaoka@rics.bwh.harvard.edu.
1
The abbreviations used are: cys-LT, cysteinyl leukotriene; LT, leukotriene; PCA, passive cutaneous anaphylaxis; PBS, phosphate-buffered saline; BAL, bronchoalveolar lavage; CysLT1, cysteinyl leukotriene
1 receptor.
This paper is available on line at http://www.jbc.org

a biosynthetic inhibitor (3) or antagonists to a receptor subsequently shown to be the cysteinyl leukotriene 1 (CysLT1) receptor
(4). Two types of human receptors for cys-LTs, designated
CysLT1 receptor and CysLT2 receptor, belonging to the seventransmembrane, G protein-coupled receptor family, were cloned
and shown to be 38% homologous in their amino acid sequences
(59). The rank order of affinities of the cys-LTs for the CysLT1
and CysLT2 receptors is LTD4 LTC4 LTE4 LTB4 and
LTC4 LTD4 LTE4 LTB4, respectively. The CysLT1
receptor is expressed on airway smooth muscle, monocytes/
macrophages, eosinophils (5, 10, 11), a subset of neutrophils (11),
mast cells (12, 13), and endothelial cells (14). The CysLT2 receptor is expressed on monocyte/macrophages, airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood
leukocytes, brain cells (7), eosinophils (11, 15), mast cells (16),
and endothelial cells (17, 18). We and others have reported that
the mouse CysLT1 receptor can function as a receptor for LTD4 in
transfected cells with a ligand preference similar to that of the
human CysLT1 receptor (19 22). The mouse CysLT2 receptor
exhibits a ligand profile of LTC4 LTD4 LTE4 (22, 23). In
contrast to the extensive studies on the functions of the CysLT1
receptor (24, 25), the functional characterization of the CysLT2
receptor has been limited because of a lack of specific competitive
antagonists. In particular, any in vivo role for the CysLT2 receptor remains unknown.
LTC4 synthase is the pivotal enzyme for the generation of
LTC4, the parent compound of the cys-LTs (26 28). LTC4 synthase conjugates reduced glutathione to an epoxide intermediate, LTA4, generated from arachidonic acid by 5-lipoxygenase
in the presence of the 5-lipoxygenase-activating protein (29).
After carrier-mediated export (30), glutamic acid and glycine
are sequentially cleaved from the glutathione moiety of LTC4
by -glutamyl transpeptidase (31) or -glutamyl leukotrienase
(32) and dipeptidase (33) to form LTD4 and LTE4, respectively.
We have shown that the increased vascular permeability in
zymosan A-induced, monocyte/macrophage-mediated peritoneal inflammation and in IgE-dependent, mast cell-mediated
passive cutaneous anaphylaxis (PCA) is reduced by 50% in
LTC4 synthase-null mice as compared with their wild-type
littermates (34). We then demonstrated by targeted disruption
of the CysLT1 receptor in mice that this alteration of vascular
permeability is dominantly mediated through the CysLT1 receptor (35). Recently, we found that bleomycin-induced chronic
pulmonary inflammation and fibrosis are diminished in LTC4
synthase-null mice, thereby implicating the cys-LTs in this
pathobiology (36). However, CysLT1 receptor-null mice were
not protected, and this finding suggested a proinflammatory
role for the CysLT2 receptor in the chronic response. To define

46129

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The cysteinyl leukotrienes (cys-LTs) mediate both

acute and chronic inflammatory responses in mice, as
demonstrated by the attenuation of the IgE/antigen-mediated increase in microvascular permeability and of
bleomycin-induced pulmonary fibrosis, respectively, in
a strain with targeted disruption of leukotriene C4 synthase to prevent cys-LT synthesis. Our earlier finding
that the acute, but not the chronic, injury was attenuated in a strain with targeted disruption of the cysteinyl
leukotriene 1 (CysLT1) receptor suggested that the
chronic injury might be mediated through the CysLT2
receptor. Thus, we generated CysLT2 receptor-deficient
mice by targeted gene disruption. These mice developed
normally and were fertile. The increased vascular permeability associated with IgE-dependent passive cutaneous anaphylaxis was significantly reduced in CysLT2
receptor-null mice as compared with wild-type mice,
whereas plasma protein extravasation in response to
zymosan A-induced peritoneal inflammation was not altered. Alveolar septal thickening after intratracheal injection of bleomycin, characterized by interstitial infiltration with macrophages and fibroblasts and the
accumulation of collagen fibers, was significantly reduced in CysLT2 receptor-null mice as compared with
the wild-type mice. The amounts of cys-LTs in bronchoalveolar lavage fluid after bleomycin injection were
similar in the CysLT2 receptor-null mice and the wildtype mice. Thus, in response to a particular pathobiologic event the CysLT2 receptor can mediate an increase
in vascular permeability in some tissues or promote
chronic pulmonary inflammation with fibrosis.

46130

Targeted Gene Disruption of CysLT2 Receptor

the role of the CysLT2 receptor in vivo, we generated CysLT2

receptor-null mice by targeted gene disruption. We then examined plasma protein extravasation and neutrophil infiltration
in zymosan A-induced peritoneal inflammation, plasma protein
extravasation in IgE-dependent PCA, and chronic pulmonary
inflammation and fibrosis induced by intratracheal injection of
bleomycin.
EXPERIMENTAL PROCEDURES

RESULTS

Generation of CysLT2 Receptor Gene-disrupted MiceA targeting vector was designed so that the neo gene insertion interrupts 85% of the coding region of the mouse CysLT2 receptor
gene (Fig. 1A). Of 150 embryonic stem cell clones that survived
in the presence of G418 and ganciclovir, two were identified as
targeted clones by Southern blot analysis for the 2.6-kb band.
The embryonic stem cell clones were microinjected into BALB/c
mouse-derived blastocysts. Eight male chimeric mice (50%
chimerism by coat color) were obtained from each clone and
bred to C57BL/6 females. The heterozygotes, verified by Southern blot analysis, were intercrossed to obtain mice homozygous
for the CysLT2 receptor-null mutation. Southern blot analysis

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Generation of CysLT2 Receptor Gene-disrupted MiceBased on the

nucleotide sequences of the mouse CysLT2 receptor cDNA and genomic
DNA (22, 23), a 5-kb mouse genomic DNA fragment containing exon III
to 35 base pairs (bp) of exon VI was amplified by PCR with C57BL/6
mouse genomic DNA using a sense primer of 5-GACACTCAGCAACGGAGGAATTTT-3 and an antisense primer of 5-GACCTTGGTAGACATCAGGCATAC-3. The amplified fragment was subcloned into a pBluescriptII vector (Stratagene). A 1139-bp fragment containing part of
exon VI and the 3-flanking region was amplified by PCR with a sense
primer of 5-GGGGATGTGCTACATAAGGCC-3 and an antisense
primer of 5-GGTAGTATCTGTAGTCAGTTTCTG-3. A neo gene from a
pMC1Neo plasmid (Stratagene) and then the 1139-bp fragment were
inserted tandemly into the 3-side of the 5-kb fragment so that the neo
gene would disrupt the region encoding the N-terminal 264 amino acids
of the mouse CysLT2 receptor. Finally, the herpes simplex virus thymidine kinase (TK) gene was inserted into the 5-side of the 5-kb fragment
(Fig. 1A). The resultant targeting vector was linearized and electroporated into a C57BL/6 mouse embryonic stem cell line (Specialty Media,
Phillipsburg, NJ). The embryonic stem cells were selected with G418
(200 g/ml; Invitrogen) and ganciclovir (2 M; Sigma), and homologous
recombination was confirmed by Southern blot analysis of EcoRI-digested genomic DNA from each embryonic stem cell clone with a
1250-bp 3-fragment as a probe located outside the targeting vector
(Fig. 1A). The embryonic stem cell clones, verified by Southern blot
analysis, were microinjected into blastocysts from BALB/c mice; and
chimeric males were obtained. Chimeras were bred to C57BL/6 females,
and offspring were genotyped by Southern blot analysis of the tail DNA.
Heterozygotes were intercrossed to obtain CysLT2 receptor(/) mice.
All animal studies were approved by the Animal Care and Use Committee of the Dana-Farber Cancer Institute.
Northern Blot AnalysisTotal RNA from the lung and spleen tissue
of CysLT2 receptor-null and wild-type mice was isolated with TriReagent (Sigma) according to the manufacturers protocol. Poly(A)
RNA was then purified with Oligotex(dT) chromatography (Qiagen). A
4-g sample of the poly(A) RNA was resolved by electrophoresis on a
formaldehyde-denatured gel and transferred to a nylon membrane (Biodyne B, 0.45 m; Pall Corp., Ann Arbor, MI) with 20 SSC for 24 h. The
membrane was hybridized with 32P-labeled mouse CysLT2 receptor,
CysLT1 receptor, or glyceraldehyde-3-phosphate dehydrogenase coding
region at 42 C for 18 h; washed with 2 SSC, 0.1% SDS at room
temperature, and then with 0.2 SSC, 0.1% SDS at 60 C; and exposed
at 80 C to a Kodak X-AR film with an intensifying screen for 7 days
for the CysLT2 receptor and CysLT1 receptor, and for 1 day for glyceraldehyde-3-phosphate dehydrogenase.
Zymosan A-induced Peritoneal InflammationEach mouse received
an intravenous injection of 0.5% Evans blue dye (10 ml of dye solution/kg of body weight) in phosphate-buffered saline (PBS) immediately
before the intraperitoneal injection of 1 ml of zymosan A suspension (1
mg/ml in PBS; Sigma). Mice were killed by CO2 30, 60, 120, and 240 min
after the injection of zymosan A, and the peritoneal cavity of each
mouse was lavaged 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. The cell pellets were suspended in 100 l of PBS,
and the cells were counted. Cells (2 104) were cytospun onto a glass
slide, stained with Diff-Quik (Dade Behring, Deerfield, IL), and analyzed for cell types. The total neutrophils in the lavage fluid were
calculated from the percentage of the cell type and the total cell count.
PCAMice received intradermal injections of 25 ng of mouse monoclonal anti-dinitrophenyl IgE (Sigma) in 25 l of saline in the right ear
and 25 l of saline only in the left ear. After 20 h, mice were injected
intravenously with 100 g of dinitrophenyl-human serum albumin
(Sigma) and 1% Evans blue dye in 100 l of PBS. The mice were killed
by CO2 30 min after the intravenous injection, and a 6-mm diameter
disk of tissue was obtained from the center of each ear. Each disk was
incubated in 200 l of formamide at 55 C for 48 h. Extravasation of
Evans blue dye was quantitated by spectrophotometric analysis of the
formamide extracts at 610 nm, and vascular permeability enhancement

was calculated as the net difference between the sensitized and control
ears.
Intratracheal Injection of Bleomycin into MiceBleomycin sulfate
(2.5 units/kg, Sigma) dissolved in saline (2.5 l/g) or saline alone was
administered intratracheally to mice that had been anesthetized with
an intraperitoneal injection of pentobarbital (75 mg/kg).
Histologic Assessment of Lung TissueTwelve days after bleomycin
injection, the lower lobes of the lung were isolated, fixed for 4 h at room
temperature and overnight at 4 C in 4% paraformaldehyde in 0.1 M
sodium phosphate (pH 7.6), and washed twice with PBS. A portion of
each lobe was embedded in paraffin in a routine manner and stained
with hematoxylin and eosin. The majority of each lobe was dehydrated
and embedded in accordance with the JB-4 kit from Polysciences (Warrington, PA). Sections were cut on a Reichert-Jung Supracut microtome
(Leica, Deerfield, IL) with glass knives and picked up on glass slides.
Some of the sections were stained using the chloroacetate esterase
reaction with counterstaining by hematoxylin to depict neutrophils. To
assess extracellular matrix deposition, some sections were stained with
the Jones periodic acid-methenamine silver method (37) modified with
hematoxylin counterstain in the JB4-embedded lungs.
The degree of alveolar septal thickening was quantitated without
knowledge of the particular strain as described previously (36). Briefly,
5 pictures of randomly selected areas of the lower lobes of each lung at
low power (10 magnifications) stained with chloroacetate esterase and
hematoxylin were obtained, and the areas of septal thickening, defined
as macrophage proliferation in the alveolar septa accompanied by fibroblasts, giant cells, and increased extracellular matrix, were outlined.
After being converted to digital images with a flatbed computer scanner
(UMAX PowerLook III) and software (Adobe Photoshop 7.0), the number of pixels contained in the areas of the digital images that had septal
thickening and the number of pixels contained in the image of the entire
lung field were determined with the histogram function. The total
number of pixels outlined as septal thickening was divided by the total
number of pixels in the entire lung field and multiplied by 100 to
generate a percentage of area with septal thickening for each animal.
Bronchoalveolar Lavage Fluid Analysis in Mice with Bleomycininduced Pulmonary FibrosisTwelve days after treatment with bleomycin or saline, mice in each group were killed by intraperitoneal
injection of an overdose of pentobarbital. Bronchoalveolar lavage (BAL)
fluid was obtained by lavage with 2.25 ml of PBS with 1 mM EDTA (0.75
ml 3) through a 22-gauge intravenous canula. The fluid was centrifuged at 500 g for 5 min, and the supernatants were retained for
measurement of eicosanoids. The cell pellets were resuspended in 100
l of PBS, and total and differential cell counts were obtained as
described above.
Assays for eicosanoids were carried out on 2 ml of each BAL fluid
supernatant to which 10,000 cpm of [3H]LTC4 (PerkinElmer Life Sciences) was added to calculate percentage recovery. Proteins were precipitated from the supernatant by the addition of a 4 volume of
methanol and incubation on ice for 10 min. After centrifugation at
3,000 g for 10 min, samples were dried in a vacuum centrifuge,
resuspended in 50 mM sodium acetate (pH 4.0) with 10% methanol, and
subjected to C18 Sep-Pak cartridge purification (Waters Associates,
Milford, MA). The eluted samples obtained from each cartridge were
dried by vacuum centrifugation and resuspended in enzyme immunoassay buffer. The amounts of cys-LTs, LTB4, and prostaglandin E2 were
measured with enzyme immunoassay kits (Amersham Biosciences) according to the manufacturers instructions.
Statistical AnalysisResults were expressed as mean S.E. Students t test was used for the statistical analysis in cases in which the
variance was homogeneous, and Welchs test was used when the variance was heterogeneous. A value of p 0.05 was considered significant.

Targeted Gene Disruption of CysLT2 Receptor

46131

of EcoRI-digested DNA from the progeny demonstrated a

2.6-kb band for the disrupted allele and an 10-kb band for the
wild-type allele, respectively, as illustrated for one litter (Fig.
1B). Litter size from these intercrosses were normal (mean
6.6 offspring per litter; n 39 litters). Of 258 progeny genotyped from these matings, 62 were homozygotes (24.0%), 130
were heterozygotes (50.4%), and 66 were wild-type (25.6%),
consistent with the expected Mendelian frequency. In addition,
a similar number of male and female (29 and 33) homozygotes
were produced. The CysLT2 receptor-null mice that developed
normally (mean body weight 25.7 g at 8 weeks), were fertile,
and exhibited no apparent clinical abnormalities up to 10
months of age. There were no microscopic abnormalities in the
CysLT2 receptor-null mice (data not shown).
Because mouse CysLT2 receptor mRNA is most abundantly
expressed in the lung, spleen, and small intestine by Northern
blot analysis (22) and reverse transcription-PCR (23), we examined whether homologous recombination by the targeting
vector disrupted expression of the CysLT2 receptor mRNA in
these tissues. By Northern blot analysis with poly(A) RNAs, a
5.5-kb band was dominantly detected as previously reported
(22) and a 1.8-kb band that is considered to be the mature
transcript based on its size (23) was also faintly detected in
these tissues of the wild-type mice. In contrast, those bands
were not detected in the samples from CysLT2 receptor-null
mice, although a 9-kb band was detected in the spleen of
CysLT2 receptor-null mice (Fig. 1C). Because we used a CysLT2
receptor coding region probe, the 9-kb band was considered to
be an unstable transcript that contained part of exon VI. Expression of an aberrant and unstable transcript was also the
case for the LTC4 synthase-null mice (34). Importantly, there
was no change in the CysLT1 receptor mRNA expression in

these tissues by the disruption of the CysLT2 receptor gene

(Fig. 1C).
Role of the CysLT2 Receptor in Zymosan A-induced Peritoneal InflammationAlthough we previously demonstrated
that the early plasma protein extravasation in zymosan A-induced peritoneal inflammation is reduced by 50% in LTC4
synthase-null mice (34) and is similarly reduced in CysLT1
receptor-null mice (35), we performed the same study to determine whether there exists any role for the CysLT2 receptor.
Plasma protein extravasation assessed 30 to 240 min after
zymosan A injection in CysLT2 receptor-null mice was similar
at all time points compared with that of the wild-type mice
(Fig. 2A).
We also examined neutrophil infiltration, which generally
peaks 240 min after the injection of zymosan A (32, 38, 39). As
assessed with neutrophil counts 240 min after the injection,
there was no significant difference in neutrophil infiltration
between CysLT2 receptor-null and wild-type mice (Fig. 2B).
Role of the CysLT2 Receptor in Mast Cell-dependent
PCAWe previously demonstrated that the local edema induced by IgE-dependent PCA is reduced by 50% in the LTC4
synthase-null mice as compared with their wild-type littermates (34) and that this cys-LT-mediated response is dominantly through the CysLT1 receptor (35). Nonetheless, to determine whether the CysLT2 receptor is involved, we examined
plasma protein extravasation induced by IgE-dependent PCA
in CysLT2 receptor-null mice. Unexpectedly, Evans blue dye
extravasation in the ear of CysLT2 receptor-null mice was
reduced in a preliminary experiment. Thus, we included the
CysLT1 receptor-null mice in three more studies. Plasma protein extravasation was reduced by 65% 30 min after antigen
challenge as compared with the level in the wild-type litter-

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FIG. 1. Generation of CysLT2 receptor gene-disrupted mice. A, genomic organization of the mouse CysLT2 receptor gene (upper), structure
of the targeting vector (middle), and organization of the putative recombinant CysLT2 receptor allele (lower). Exons IIIVI are shown as boxes with
the coding regions in black. The location of the 1250-bp fragment used for Southern blot analysis is shown as a thick line. TK, herpes simplex virus
thymidine kinase. Restriction site E, EcoRI. B, Southern blot analysis of EcoRI-digested tail DNAs from the pups of a CysLT2 receptor (/) male
and a CysLT2 receptor (/) female mouse. C, Northern blot analysis of poly(A) RNA from the lung, spleen, and small intestine of CysLT2
receptor-null mice (/) and their wild-type littermates (/). Hybridizations were performed with a 32P-labeled mouse CysLT2 receptor cDNA
probe (upper), with a 32P-labeled mouse CysLT1 receptor cDNA probe (middle), and with a 32P-labeled mouse glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA probe (lower).

46132

Targeted Gene Disruption of CysLT2 Receptor

FIG. 3. PCA in CysLT2 receptor-null mice. CysLT2 receptor-null

mice (white bar), their wild-type littermates (black bar), and CysLT1
receptor-null mice (gray bar) received intradermal injections of 25 ng of
mouse anti-dinitrophenyl monoclonal IgE in 25 l of saline in the right
ear and 25 l of saline only in the left ear. After 20 h, mice were injected
intravenously with 100 g of dinitrophenyl-human serum albumin and
1% Evans blue dye in 100 l of PBS. A 6-mm diameter ear disk was
isolated 30 min after challenge, and Evans blue dye was extracted in
200 l of formamide at 55 C for 48 h. Extravasation of Evans blue dye
was quantitated by spectrophotometric analysis at 610 nm. The difference between the absorbance of the right and left ear extracts representing the net extravasation in the sensitized ear was expressed as the
mean S.E. Results are combined from three independent analyses
(n 10 mice per group). *, p 0.001.

FIG. 4. Histologic assessment of bleomycin-induced pulmonary fibrosis in CysLT2 receptor-null mice. The lower lobes of the
lungs were examined histologically 12 days after animals received an
intratracheal injection of saline or bleomycin. Sections of lung from
saline-treated wild-type mice (a, low power: 10 objective; d, high
power: 50 objective) and bleomycin-treated wild-type (b, low power; e,
high power) and CysLT2 receptor-null (c, low power; f, high power) mice
were stained with hematoxylin and eosin (a c), or by the chloroacetate
esterase reaction with counterstaining with hematoxylin (df). Sections
of lung from saline-treated wild-type mice (g, low power; j, high power)
and bleomycin-treated wild-type (h, low power; k, high power) and
CysLT2 receptor-null (i, low power; l, high power) mice were stained
with the Jones periodic acid-methenamine silver method with hematoxylin counterstain. Reticular fibers (type III collagen) and basement
membranes were stained dark brown to black.

mates, and the reduction was similar to that of CysLT1 receptor-null mice (Fig. 3).
Role of the CysLT2 Receptor in Bleomycin-induced Pulmonary FibrosisWe previously demonstrated that bleomycininduced pulmonary chronic inflammation and fibrosis are attenuated in LTC4 synthase-null mice but exacerbated in
CysLT1 receptor-null mice (36). To define a role for the CysLT2
receptor in the pathobiology of chronic inflammation with fibrosis, we injected bleomycin into the tracheas of CysLT2 receptor-null mice and their wild-type littermates and examined
lung histology and cys-LT levels in the BAL fluid 12 days after
the injection. The lung tissue of bleomycin-treated CysLT2
receptor-null mice had less septal thickening, with less accumulation of monocyte/macrophages, giant cells, fibroblasts,

and eosinophils and less extracellular matrix deposition than

that of their bleomycin-treated wild-type littermates (Fig. 4,
af). We used the Jones periodic acid-methenamine silver
method with modifications to assess the deposition of extracellular matrix proteins beneath the pleura, around the bronchi,
and in the alveolar septa. After bleomycin treatment, the
amount of black fibrillar material was less in the lung tissue of
the CysLT2 receptor-null mice as compared with lung tissue
from the wild-type mice (Fig. 4, gl).
To quantitate the degree of bleomycin-induced inflammation
and fibrosis, we analyzed the sections stained with chloroacetate esterase and hematoxylin by comparing digital images of
the areas of alveolar septal thickening and the total area of the
lower lung lobes of the mice 12 days after bleomycin injection.

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FIG. 2. Zymosan A-induced peritoneal inflammation in CysLT2

receptor-null mice. A, plasma protein extravasation. CysLT2 receptor-null mice (E) and their wild-type littermates () were injected
intraperitoneally with 1 ml of zymosan A in PBS (1 mg/ml) immediately
after an intravenous injection of 0.5% Evans blue dye. Peritoneal lavage
was performed 30, 60, 120, and 240 min after zymosan A injection, and
the absorbance of the lavage fluid supernatant was measured at 610 nm
to quantitate Evans blue dye extravasation. Values at each time point
are the mean S.E. (n 35 mice). Results are combined from two
independent experiments. B, neutrophil accumulation. Cell pellets 4 h
after zymosan A injection were assayed for total cell counts (black bars)
and neutrophil counts (gray bars) in each peritoneal lavage fluid from
CysLT2 receptor-null (right) and wild-type (left) mice. Values are the
mean S.E. Results are combined from two independent experiments
(n 35 mice per group).

46133

Targeted Gene Disruption of CysLT2 Receptor

TABLE I
Alveolar septal thickening and the amounts of cys-LTs, LTB4, and PGE2 in the BAL fluid in CysLT2 receptor-null mice and wild-type mice
12 days after treatment with bleomycin or saline
Alveolar septal thickening was quantitated by digital imaging as described under Experimental Procedures and the amounts of cys-LTs, LTB4,
and PGE2 in BAL fluid were measured by enzyme immunoassays. Data are combined from three independent experiments.
Mouse/treatment

Lung area with septal thickening

cys-LTs

Wild-type/saline (n 7)
CysLT2-null/saline (n 3)
Wild-type/bleomycin (n 18)
CysLT2-null/bleomycin (n 15)

0
0
39.7 3.9
15.4 2.7c

LTB4

PGE2

pg/BAL fluid, mean S.E.

10.1 5.6
50.7 27.0
777.6 153.6a
675.4 132.5d

170.8 41.5
217.2 4.7
100.3 20.3
95.1 19.0

253.2 27.7
289.5 56.7
350.2 27.8b
340.5 50.8

p 0.0017 versus. saline-treated wild-type group.

p 0.023 versus saline-treated wild-type group.
p 0.000018 versus bleomycin-treated wild-type group.
d
p 0.00075 versus saline-treated CysLT2-null group.
a
b
c

DISCUSSION

We generated CysLT2 receptor-null mice by targeted gene

disruption to seek in vivo evidence for the role of this receptor
in cys-LT-mediated pathobiology. Despite the extensive characterization of the CysLT2 receptor with transfected cells (79,
22, 23) and human umbilical vein endothelial cells (17, 18, 40),
little is known about the role of the second receptor in in vivo
inflammatory responses, because of the lack of specific antagonists for it. We have found that the CysLT2 receptor mediates
increased vascular permeability in IgE-dependent PCA and
macrophage and fibroblast accumulation and extracellular matrix protein deposition in response to bleomycin.
LTC4 and LTD4 directly elicit transient vasoconstriction of
small arterioles and then increased vascular permeability in
postcapillary venules (41, 42), where contraction of endothelial
cells is considered to be required for the plasma protein leakage
(43). Because increased vascular permeability induced by either intraperitoneal injection of zymosan A or IgE-dependent
PCA is attenuated in the CysLT1 receptor-null mice (35) to the
same extent as in the LTC4 synthase-null mice (34), we anticipated that the effect of CysLT2 receptor deficiency on increased vascular permeability in these acute inflammatory responses would be negligible. Surprisingly, increased vascular
permeability by IgE-dependent PCA, but not in zymosan-induced peritoneal inflammation, was attenuated in the CysLT2
receptor-null mice. Furthermore, this protection was equal to
that observed in the CysLT1 receptor-null mice in the same set
of experiments (Fig. 3). The lack of attenuation of the vascular
leak in the peritoneal cavity in the CysLT2 receptor-null mice
would imply heterogeneity of receptor distribution in the microvasculature across tissues (Fig. 2).
There was no impairment of mast cell degranulation by
morphologic analysis (data not shown), indicating that the
attenuation involved the microvasculature. The CysLT2 receptor mRNA is detected in endothelial cells of the mouse heart by
in situ hybridization (23) and on human umbilical vein endothelial cells in a 1000-fold excess to the CysLT1 receptor by

quantitative reverse transcription-PCR (17, 18). Moreover, the

calcium response of the human umbilical vein endothelial cells
to the cys-LTs was attributed to the CysLT2 receptor because
there was no inhibition by a selective CysLT1 receptor antagonist and some inhibition by a partial CysLT2 receptor agonist.
That the magnitude of the attenuation of the PCA was approximately equal in each of three independent experiments for the
CysLT2 receptor-null and CysLT1 receptor-null mice and for
the overall mean implies that the inhibition is linked, possibly
through CysLT2 and CysLT1 receptor heterodimers. Heterodimer formation has been reported for other G protein-coupled
receptors, such as opioid (44) and chemokine (45) receptors.
In this study, we have shown that CysLT2 is the receptor
responsible for mediating the contribution of the cys-LTs to
bleomycin-induced chronic pulmonary inflammation and fibrosis. The levels of cys-LTs in the BAL fluid of bleomycin-treated
CysLT2 receptor-null mice and wild-type mice remained comparable (Table I). Thus, either the lack of cys-LT production as
in the LTC4 synthase-null mice (36) or the lack of the CysLT2
receptor significantly attenuates the chronic injury (Fig. 4). In
contrast, the CysLT1 receptor-null mice were found to have
increased bleomycin-induced pulmonary inflammation and increased levels of cys-LTs in BAL fluid relative to their controls
(36). The source of cys-LTs in this response is most likely
alveolar and interstitial macrophages as cells of this lineage in
several species generate cys-LTs in response to a range of
ligands (46 49).
Mouse skin fibroblasts express both CysLT1 and CysLT2
receptors by in situ hybridization (22). Before the CysLT1 and
CysLT2 receptors were defined by their cloning, it was shown
that LTC4 and LTD4 could stimulate [3H]thymidine incorporation by in vitro cultured human skin fibroblasts in the presence
of indomethacin and that the effect of LTC4 was slightly superior to that of LTD4 (50). In addition, LTC4 and, to a lesser
extent, LTD4 could stimulate collagen synthesis of rat lung
fibroblasts in vitro and LTC4 could directly bind to the cells
(51). These earlier studies revealing a functional receptor for
cys-LTs on fibroblasts analyzed in vitro are compatible with a
CysLT2 receptor-mediated response based on the efficacy of
LTC4. Our findings that CysLT2 receptor-null mice have a
significant reduction in bleomycin-induced pulmonary fibrosis
could reflect a dominant role for this receptor in the fibroblast
component of the response.
We generated CysLT2 receptor-null mice and then demonstrated a role for the CysLT2 receptor in inflammatory responses
in vivo. Our studies highlight the CysLT2 receptor as a potential
therapeutic target for the treatment of idiopathic pulmonary
fibrosis, for which no efficient interventions are available (52).
AcknowledgmentsWe thank Mi Xiao Donovan for technical assistance and Dr. Arlene Sharpe and Lina Du (Transgenic Core Facility,

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The area of the lower lobes with alveolar septal thickening in

CysLT2 receptor-null mice was significantly reduced to only
39% that of wild-type mice (Table I). Bleomycin treatment
significantly increased the amounts of cys-LTs in the BAL fluid
in both wild-type mice and CysLT2 receptor-null mice. Despite
the attenuation of septal thickening, bleomycin-treated CysLT2
receptor-null mice had levels of cys-LTs comparable with those
of the bleomycin-treated wild-type mice (Table I). Bleomycin
treatment did not increase the amounts of LTB4 in either
wild-type mice or CysLT2 receptor-null mice. Although bleomycin treatment did increase the amount of prostaglandin E2 in
wild-type mice, but not in CysLT2 receptor-null mice (Table I),
the amounts of prostaglandin E2 recovered from both strains
with bleomycin injury were not different.

46134

Targeted Gene Disruption of CysLT2 Receptor

Brigham and Womens Hospital) for blastocyst injection of the embryonic stem cell clones.
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