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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3912-3923
Antigen-Induced Eosinophilic Lung Inflammation Develops in Mice
Deficient in Chemokine Eotaxin
By
Yi Yang,
James Loy,
Rolf-Peter Ryseck,
Daniel Carrasco, and
Rodrigo Bravo
From the Department of Oncology and Experimental Pathology,
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton,
NJ.
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ABSTRACT |
The mechanisms that regulate the selective infiltration of
eosinophils in certain allergic diseases are still poorly understood. The CC chemokine eotaxin is a potent chemoattractant, highly specific for eosinophils. Recent studies have implicated that eotaxin plays an
important role in the recruitment of eosinophils in different inflammation processes. A number of other chemokines, cytokines, and
chemoattractants also have chemotactic activities for eosinophils and
some of them present high selectivity for eosinophils. To further study
the role of eotaxin in inflammation, we generated mutant mice with the
eotaxin gene disrupted and replaced by the Escherichia coli
 -galactosidase gene. These mice developed normally and had no
histologic or hematopoietic abnormalities. Furthermore, our studies
showed that the lack of eotaxin did not affect the recruitment of
eosinophils in the inflammation models induced by Sephadex beads and
thioglycollate, as well as in an experimental lung eosinophilia model
induced by ovalbumin aerosol challenge, even at the onset of the
inflammatory response. The replacement of the eotaxin gene by the
-galactosidase gene provided a useful marker to monitor the activity
of the eotaxin promoter under normal conditions and after antigen
challenges. Immunohistochemical staining suggested that endothelial
cells were the major sources of eotaxin expression.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE CHEMOKINES ARE a family of small
secreted proteins that have chemotactic activities for leukocytes and
play a key role in leukocyte trafficking.1-5 They are also
important in processes such as hematopoiesis, anaphylaxis,
angiogenesis, and malignancy.1,2,5 The importance of
chemokines and their receptors in inflammatory and infectious diseases,
especially in the infection by human immunodeficiency virus-type 1 (HIV-1), has recently been described.6,7 There
are two major subfamilies of chemokines, designated as CXC (or  ) and
CC (or ), based on the conserved cysteine motif near the amino
terminus of the protein.4,5 The CXC chemokines, which
include interleukin-8 (IL-8), Gro-, -, and - ,
IP10, and SDF-1, attract neutrophils and T cells in general. The
largest subfamily, the CC chemokines, attract mainly mononuclear cells
and also lymphocytes, eosinophils, and basophils. These include
monocyte chemotactic proteins (MCP-1, -2, -3, -4, and -5), macrophage
inflammatory proteins (MIP-1 and MIP-1), RANTES (regulated upon
activation, normal T cell expressed and secreted), and eotaxin. There
are also two single-member subfamilies, one of which is the C (or )
subfamily with lymphotactin, which has only one conserved cysteine
residue at the N-terminal and selectively attracts
lymphocytes.8 The other subfamily, with the chemokine named
fractalkine or neurotactin, contains a CX3C motif that
exists in either membrane-bound or secreted forms.9,10 The
receptors for the different chemokines are a family of
seven-transmembrane G-protein-coupled receptors. So far, there are
approximately 20 such receptors cloned and most of them have identified
ligands.5,6 These include the CXCR receptors (CXCR1-4) for
CXC chemokines and the CCR receptors (CCR1-8) for CC chemokines. The
relationship between chemokines and receptors is complicated, because
most chemokines can bind to more than one receptor and most receptors have more than one ligand.6
The CC chemokine eotaxin was originally discovered in the
bronchoalveolar lavage (BAL) fluid of guinea pigs sensitized and challenged with ovalbumin aerosol.11 This protein, and its
homolog found later in mice and humans,12-16 is a potent
and specific chemoattractant for eosinophils. This is in contrast to
most other CC chemokines, which have the capacity to attract more than
one type of leukocyte.17 Eotaxin specifically binds to the
CCR3 receptor, which is expressed selectively and at high levels on
eosinophils.16,18,19 Other chemokines with potent
chemotactic activities on eosinophils include eotaxin-2,20
MCP-4,21,22 RANTES,23 MCP-3,24,25
and, to a lesser extent, MIP-1,23 MCP-2,25
and, possibly, MCP-5.26 Most of these chemokines also use
the CCR3 receptor when they are active on eosinophils.17,27
One exception is MIP-1, which uses another CC receptor also
expressed on eosinophils, CCR1.19,28 Unlike eotaxin, most
of these chemokines also bind to other receptors and are active on
other leukocytes, such as monocytes, lymphocytes, and basophils. Some
classical chemoattractants, such as C5a, leukotriene B4 (LTB4), and
platelet-activating factor (PAF), have also been found to be active on
eosinophils.17,29,30
The accumulation of eosinophils is characteristic of allergic diseases
such as asthma and some parasitic infections.31,32 The
mechanisms that regulate the infiltration of eosinophils during these
processes are being elucidated. Recent studies indicate that eotaxin
may play an important role in the recruitment of eosinophils in
different inflammation models.12-14,33 Models of antigen-induced airway allergic inflammation and lung eosinophilia have
been developed in both guinea pig and mouse.11,13,34 The
eotaxin mRNA in lung is induced in response to antigen challenge and
parasitic infection.13,34 Prechallenge treatment of mice with anti-CD3 monoclonal antibody (MoAb) inhibits the induced eotaxin
mRNA expression and significantly reduces eosinophil infiltration in
the lung.34 Administration of antibodies specific for
eotaxin in vivo reduce the accumulation of eosinophils.35
On the other hand, eotaxin expression is found constitutively in some
tissues, such as thymus, lymph nodes, skin, and heart, where there is
no eosinophil infiltration.12-14 It has been shown that in
vitro eotaxin can inhibit the infection of certain HIV-1 strains that
use CCR3 as coreceptor.36 Recent reports showed that in
vitro eotaxin is also a potent chemoattractant for human
basophils.20,37 All of these results suggest that eotaxin
may have some unknown functions besides chemotaxis for eosinophils.
The generation of mutant mice with a loss of eotaxin function could
provide a definitive answer to the role of this chemokine in the
recruitment of eosinophils in inflammation and, possibly, other
biological processes. A recent report on the targeted disruption of the
mouse eotaxin gene indicates a partially reduced eosinophil recruitment
in the experimental lung allergic inflammation model and in a model of
parasite-derived antigen-induced stromal keratitis.38 We
report here a different approach to generate eotaxin knockout mice by
replacing the eotaxin gene with the Escherichia coli
-galactosidase gene, allowing the identification of the cells that
express eotaxin.
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MATERIALS AND METHODS |
Construction of targeting vector.
Mouse genomic DNA containing the eotaxin gene was cloned from a
mouse library prepared from D3 (129/sv) embryonic stem (ES) cell DNA.
The pPNT vector containing the neo (neomycin) and tk (thymidine kinase) genes was used to construct the targeting vector. A
3.5-kb mouse genomic DNA containing exon 3 and the 3
untranslated region of the eotaxin gene was cloned into the
Xba I-Kpn I sites of pPNT vector to generate pPNT-Ex3.
A 3.2-kb BamHI-Sac I mouse genomic DNA containing the
5 untranslated region and the first four amino acids of eotaxin
precursor protein was fused in-frame to the E coli
-galactosidase gene, and this fragment was then subcloned into the
Not I-Xho I sites of pPNT-Ex3 to generate the targeting
vector pPNT-Ex.
Generation of knockout mice.
The targeting vector (25 µg) was linearized by Not I
digestion and transfected into 1 × 107 129/Sv-derived
ES cells (CJ7) by electroporation using a Bio-Rad gene pulser (Bio-Rad,
Hercules, CA). The ES cells were grown under positive-negative selection in G418 for neomycin-resistant and fialuridine (FIAU) for thymidine kinase-negative clones. About 400 double-resistant clones were screened by Southern blot analysis using a
320-bp external probe (immediately outside the 5 arm of the
targeting vector) generated by polymerase chain reaction (PCR), and
three true recombinant clones were obtained. All three clones were
injected into blastocysts of ICR strain of mice and the resulting male
chimeras were mated with female mice of ICR strain for germline
transmission of the targeted allele. Heterozygous offsprings were
interbred to generate homozygous mice that had two alleles of the
eotaxin gene disrupted. Siblings from heterozygote matings were used to
control for strain background effects. Genotyping was performed by
Southern blot analysis of mouse tail/toe DNA using the 320-bp probe.
Routinely, genotyping was performed by PCR analysis using a pair of
primers specific for the neo gene to identify the targeted
allele and a pair of primers to amplify a 400-bp genomic DNA that is
deleted in knockout mice. The sequence for the neo-specific
primer pair used is as follows: sense,
5-CGGCCACAGTCGATGAATCCAGAAA-3, and antisense,
5-CCATTCGACCACCAAGCGAAACATC-3. The sequence for the
eotaxin-specific primer pair used is as follows: sense,
5-AGTCCATCCCCAAGTTAGAG-3, and antisense,
5-ACATTGTAGGAATGGCATGGCT-3. PCR was performed with 25 µmol/L of each of the sense and antisense primers under the following
amplification conditions: 1 minute at 94°C, 1 minute at 55°C,
and 1.5 minutes at 72°C for 30 cycles.
Southern and Northern blots and reverse transcription-PCR (RT-PCR).
Mouse genomic DNA was digested with Spe I (New
England BioLabs, Beverly, MA), electrophoresed, and transferred to a
GeneScreen Plus nylon membrane (DuPont, Wilmington, DE). The membrane
was hybridized with the 32P-labeled 320-bp probe overnight
at 65°C in a hybridization solution (5× SSPE/0.1%
pyrophosphate [PPiESS], 1× Denhardt's solution, 1% sodium
dodecyl sulfate [SDS]), washed once for 15 minutes at room
temperature, and washed twice at 65°C for 20 minutes in 0.2× PPiESS with 0.5% SDS.
RNA was extracted from different tissues with Trizol reagent (GIBCO
BRL, Gaithersburg, MD) as per the supplier's instructions. Ten
micrograms of RNA was electrophoresed on a 1% agarose gel containing
0.7% formaldehyde and then transferred in 20× SSC to a
GeneScreen Plus nylon membrane and hybridized with a
32P-labeled mouse eotaxin full-length cDNA or a probe
specific for GAPDH. The hybridization was performed as in the Southern
blot, except that the hybridization solution contained 100 µg/mL of salmon sperm DNA.
For RT-PCR, first-strand cDNAs were generated by reverse transcription
with poly(dT) primers from 5 µg of total RNA using the SuperScript
reverse transcriptase kit (GIBCO BRL). Five percent of the product was
then used as the template for PCR amplification using primers specific
for mouse eotaxin, fic, or -actin.
Experimental lung inflammation model.
The ovalbumin (OVA) challenge of mice was performed as described
before.34 Briefly, 10 µg of OVA (Sigma Chemical Co, St Louis, MO) in aluminum hydroxide (5 mg/mL in
phosphate-buffered saline [PBS]) was injected into mice
intraperitoneally on days 0, 7, and 14. Sham-immunized mice were
injected with aluminum hydroxide only. On any day between days 21 and
24, mice were restrained in a plexiglass box and challenged once with
aerosolized OVA (50 mg/mL in PBS) generated by an ultrasonic nebulizer
(DeVilbiss Co, Somerset, PA) for 20 minutes. Control mice
(sham-immunized) were also challenged with aerosolized OVA. At 6, 12, 18, 24, and 48 hours after challenge, mice were anesthetized with 0.6 mL of avertin (200 mg/mL of tribromoethanol; Aldrich,
Milwaukee, WI) by intraperitoneal injection and perfused
with 2% paraformaldehyde. Tissues were harvested, postfixed in the
same perfusion solution for 1 hour, and then stored in 30% sucrose/PBS
solution overnight. The tissues were sectioned at 30 to 100 µm and
stained with hematoxylin and eosin (H&E). For paraffin sections,
tissues were postfixed in 10% neutral-buffered formalin, embedded in
paraffin, sectioned at 4 to 6 µm, and stained with H&E.
BAL was performed at 18 hours after ovalbumin aerosol challenge. Mice
were anesthetized and the trachea was surgically exposed. The lungs
were lavaged via the trachea with 0.5-mL aliquots of PBS containing 0.6 mmol/L EDTA (8 × 0.5 mL at 37°C). The total cells were
determined with a hemacytometer. Fifty thousand cells were collected by
cytospin and stained with Diff-Quick stain set (Dade Diagnostic,
Newark, DE). The percentages of eosinophils, neutrophils,
macrophages, and lymphocytes were determined from a count of six
different high-power (100× objective) views for each mouse. These
percentages were multiplied by total cell number to obtain the number
of different cell populations.
Inflammation induced by thioglycollate and Sephadex.
Mice were injected intraperitoneally with 3 mL of 3% Brewer's
thioglycollate (DIFCO, Detroit, MI) in PBS, and 24 hours postinjection, peritoneal cells were lavaged with Dulbecco's modified Eagle's medium
(DMEM)/5% fetal bovine serum (FBS), and total cell
numbers were determined with a hemacytometer. Cells were collected by centrifugation and resuspended at 1 × 106 cells/mL.
Fifty thousand cells were collected by cytospin and stained with
Diff-Quick stain set (Dade Diagnostic). Different type of cells were
counted at six different high-power views for each mouse. The number of
different cell populations was calculated by multiplying the percentage
by total cell counts.
Sephadex G50 beads (superfine; Sigma Chemical Co) were suspended in
PBS. Each mouse received 0.3 mg of these beads by intravenous injection
of the tail vein. At 6 or 24 hours after injection, mice were killed
and perfused with 2% paraformaldehyde. Lungs were harvested, postfixed
in the same perfusion solution for 1 hour, and then stored in 30%
sucrose/PBS solution overnight. Then tissues were processed as
described above.
-Galactosidase and immunohistochemical staining.
To determine -galactosidase activity, frozen sections were stained
in a solution containing 1 mg/mL of X-gal (GIBCO BRL) overnight and
then counter-stained with eosin. For immunohistochemical analysis,
frozen tissue sections were first stained in X-gal solution as
described above and then treated in 0.03% hydrogen peroxide in PBS,
blocked with Vector Avidin/Biotin blocking kit SP-2001 (Vector
Laboratories, Burlingame, CA), and blocked with 10% normal rabbit
serum. The primary anti-CD34 antibody (Pharmigen, San Diego, CA; 1:100
dilution) was added and incubated for 90 minutes at room temperature.
After washing in PBS, the sections were treated with a secondary
antibody Vector biotinylated antirat (1:100) for 30 minutes and rinsed
in PBS. The sections were then treated with the Vector ABC Elite
Peroxidase (PK6100) and Vector DAB (SK-4100) kits (Vector
Laboratories). Finally, the sections were counter-stained with eosin.
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RESULTS |
Eotaxin-deficient mice show no phenotypic abnormalities.
Like most chemokine genes, eotaxin consists of three exons and
two introns.12 To knockout eotaxin, we constructed
a targeting vector by disrupting the first two exons
(Fig 1A). As a marker for the activity of
the eotaxin promoter, we fused the E coli -galactosidase gene in-frame to the first four amino acids of the
leader sequence of the eotaxin precursor. The targeted allele disrupts
the eotaxin gene by deleting most of exon 1 and all of exon 2 with the
insertion of the E coli -galactosidase gene and the
neomycin-resistance gene. The ES cell clones that underwent homologous
recombination were screened by Southern blot analysis using a 5
external probe. After Spe I digestion, the targeted allele
generated a 10-kb fragment, whereas the wild-type allele generated a
14-kb fragment (Fig 1B). The positive clones were further confirmed by
a 3 external probe that distinguishes the Spe I
digestion by 6-kb and 14-kb fragments for the targeted and wild-type
alleles, respectively (data not shown). Positive ES cell clones were
injected into blastocysts, and the male chimeras obtained were used to
generate heterozygous animals. These were interbred to generate
homozygous mice for the disrupted eotaxin allele. Figure 1C
shows the result of Southern blot analysis of the Spe
I-digested genomic DNA from wild-type (+/+), heterozygous (+/ ),
and homozygous (/) mice. Because eotaxin mRNA is
abundant in lymph nodes,12 we performed Northern blot
analysis of total RNA from lymph nodes of wild-type and mutant mice
with a probe that is specific for eotaxin. As expected, there
is no eotaxin mRNA expression in knockout mice (Fig 1D).
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| Fig 1.
Generation of eotaxin null mice. (A) Strategy for
generating eotaxin knockout mice by homologous recombination.
The three-exon and two-intron genomic structure of the eotaxin
gene is shown on the top. The targeting vector is shown in the middle.
The 5 arm contains 3.2 kb of mouse genomic DNA with the sequence
encoding the first 4 amino acids of eotaxin fused in-frame to the E
coli -galactosidase gene. The 3 arm of 3.5 kb of mouse
genomic DNA containing exon 3 and the 3 untranslated sequence of
the eotaxin gene is inserted between the neomycin-resistance gene (neo)
and the herpes simplex virus thymidine kinase (tk) gene. The targeted
allele after homologous recombination and the screening strategy are
shown at the bottom. The black boxes represent the exons, with their
respective number above. Southern blot analysis of Spe
I-digested genomic DNA from ES cells (B) and from wild-type (+/+),
heterozygous (+/), and knockout (/) mice (C). The upper band
(14 kb) shows the wild-type allele, and the lower band (10 kb) shows
the targeted allele. (D) Northern blot of total RNA (10 µg) from
lymph nodes of wild-type (+/+), heterozygous (+/), and
knockout (/) mice. The blot was probed with
32P-labeled mouse eotaxin cDNA (upper panel),
stripped, and tested with a probe specific for mouse GAPDH (bottom).
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The eotaxin / mice appear healthy and
normal. All tissues examined of 6-week-old animals were unremarkable. A
total number of 206 mice born from heterozygous matings showed normal
1:2:1 Mendelian ratio with 46 wild-type (22.3%), 112 heterozygous
(54.4%), and 48 knockout (23.3%) mice. Fluorescence-activated cell
sorting (FACS) analysis of cell surface markers for T
cells, B cells, granulocytes, and macrophages from lymphoid organs,
including thymus, lymph nodes, spleen, and bone marrow, showed no
differences between wild-type and knockout mice (data not shown). The
markers examined included CD4, CD8, TCR, and CD25 in thymus; CD4,
CD8, TCR, CD25, Thy-1.2, and B220 in lymph nodes; CD4, CD8,
TCR, CD25, Thy-1.2, B220, Ter-119, and Mac-1 in spleen; and
Ter-119, Mac-1, B220, and IgM in bone marrow. Therefore, it appears
that mice deficient for eotaxin have no hematopoietic abnormalities.
The expression of -galactosidase in eotaxin heterozygous and null
mice.
Because the targeted allele contained the E coli
-galactosidase gene, the activity of the eotaxin promoter could be
examined in heterozygous and knockout mice by X-gal staining to detect the -galactosidase activity. Sections of different tissues from wild-type and heterozygous mice were stained with X-gal as described in
Materials and Methods. Tissues such as lymph nodes, thymus, intestine,
and stomach were found to contain cells with high -galactosidase activity as detected by the X-gal blue staining
(Fig 2A). In the small intestine, the
staining localized to the vasculature in the lamina propria surrounding
the intestinal crypts. The -galactosidase staining in lymph nodes
localized to vessels and was most prominent in parafollicular areas in
the cortex with less prominent staining of medullary sinuses. In
thymus, the -galactosidase staining was localized only in the
medullary region and observed in scattered large (20 to 25 µm) cells
with a centrally located nucleus and angular cytoplasmic profiles. The
histomorphology of the cells in the thymic medulla was most consistent
with dendritic cells. Most of the blue staining in lymph nodes and
small intestine localized to cells with the morphology of endothelial
cells. Furthermore, the -galactosidase staining in lymph nodes and
small intestine colocalized with immunohistochemical staining for CD34,
a marker for endothelial cells (Fig 2C). Several markers specific for
T, B, and monocyte/macrophage cells showed no colocalization with -galactosidase staining. These results suggest that, in lymph nodes
and small intestine, eotaxin is expressed by endothelial cells. Other
tissues had either very weak or no detectable X-gal staining (data not
shown). Wild-type tissues showed no background in staining, as shown in
Fig 2A, with the exception of the stomach, which had a slight
background staining (not shown). Eotaxin-deficient mice with two copies
of the -galactosidase gene have the same pattern of X-gal staining
in different tissues (data not shown).
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| Fig 2.
Expression of -galactosidase and eotaxin in
different tissues in eotaxin+/ mice. (A)
Expression of -galactosidase detected by X-gal staining. Frozen
tissue sections from wild-type (+/+) and heterozygous (+/)
mice were stained with X-gal overnight and counter-stained with eosin.
Shown are the tissues with strong -galactosidase staining. (B)
Expression of eotaxin and fic in different tissues
detected by RT-PCR. Total RNA was isolated from eotaxin heterozygous
and null mice and amplified by RT-PCR with primers specific for
mouse eotaxin, fic, or -actin. (C) Costaining of
X-gal with CD34 markers in lymph nodes and small intestine. Frozen
sections were stained with X-gal overnight, followed by
immunohistochemical staining with anti-CD34 antibody and then
counter-stained with eosin as described in Materials and Methods. The
blue staining illustrates the -galactosidase activity and the brown
staining is for CD34 marker.
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It has been shown that eotaxin mRNA is abundant in mouse
tissues such as skin and skeletal muscle as well as in thymus, lymph node, and intestine.12 However, different reports showed
different mRNA expression results in some mouse tissues such as skin
and intestine.12,13 In humans, eotaxin mRNA is
abundant in the heart.14 We did not detect
-galactosidase expression by X-gal staining in the skin, skeletal
muscle, or heart, where abundant eotaxin mRNA was previously
reported. To examine whether this may be due to differences in mouse
genetic background, we performed RT-PCR to check the eotaxin
expression in our animals. Figure 2B shows that the expression of
eotaxin detected by RT-PCR in the thymus, lymph node, and
intestine of heterozygous mice is consistent with the -galactosidase
expression detected by X-gal staining. We also detected abundant
eotaxin expression in the heart (results from 2 different mice
are shown), but in the heart tissue of eotaxin heterozygous and null
mice, there was no detectable -galactosidase expression (data not
shown). Because the other chemokine gene fic
(fibroblast-induced cytokine), the mouse homolog of MCP-3, is adjacent
to eotaxin (unpublished data), we examined whether the replacement of the eotaxin gene with the
-galactosidase and neomycin genes could affect the expression of
fic. As shown in Fig 2B, the expression of fic in
thymus and intestine is not affected in the eotaxin null mice.
Eosinophil recruitment into lung tissue after ovalbumin challenge.
Eotaxin was originally purified from BAL fluid of guinea pig sensitized
and challenged with aerosol ovalbumin.11 In a similar mouse
model, eotaxin mRNA in lung was induced rapidly and eosinophils infiltrated the lung after ovalbumin aerosol
challenge.13,34 Prechallenge treatment of mice with
anti-CD3 MoAb inhibited the induced eotaxin mRNA expression and
significantly reduced eosinophil infiltration in the
lung.34 These results implicate that eotaxin may be
responsible for the recruitment of eosinophils into the lung in this
antigen-induced lung inflammation model. A recent report using
eotaxin-deficient mice generated by an approach different from ours
indicated that, at 18 hours after ovalbumin challenge, the number of
eosinophils in the BAL was reduced by 70% in the eotaxin/ mice compared with wild-type
mice.38 However, at a later time (48 hours) after ovalbumin
challenge, there was no difference in eosinophil numbers between
wild-type and eotaxin null mice. This result suggests that eotaxin is
important in the early recruitment of eosinophils in this
antigen-induced lung eosinophilia model.
To further determine the role of eotaxin in the early recruitment of
eosinophils in this model, we examined our eotaxin null mice for
eosinophil infiltration in lung tissues at different time points after
the challenge with ovalbumin aerosol. In this inflammation model,
eotaxin mRNA in lung was induced rapidly after ovalbumin
aerosol challenge (peaking at 6 hours) and a significant number of
eosinophils were detected in lung tissues at 6 hours postchallenge.34 Therefore, we examined lung tissues
starting at 6 hours after ovalbumin challenge. Lung tissues from
wild-type and eotaxin knockout mice were analyzed at 6, 12, 24, and 48 hours after ovalbumin aerosol challenge. Lung inflammation and
eosinophil numbers were examined without knowledge of the genotype of
each mouse. Inflammation was characterized by aggregates of granular leukocytes (neutrophils and eosinophils) in the peribronchial adventitia with lesser number of macrophages and lymphocytes. Inflammation severity scores were highest in the 24- and 48-hour groups
as compared with the 6- and 12-hour groups
(Fig 3A). There was no significant
difference in the severity of inflammation at different time points
between wild-type and knockout mice. As controls, sham-immunized mice
show no or minor inflammation at all time points (data not shown). The
number of eosinophils in the lung tissues of wild-type and eotaxin null
mice was determined microscopically. The numbers of infiltrated
eosinophils at different time points, even at the early time point of 6 hours after ovalbumin challenge, between wild-type and knockout mice
were similar (Fig 3B). As a direct comparison to the result of
Rothenberg et al,38 lung lavage was performed and the
number of different cells in the BAL fluid was assessed at 18 hours
after the ovalbumin aerosol challenge. As shown in Fig 3C, no
significant differences in the number of total cells, neutrophils,
macrophages, and lymphocytes as well as eosinophils were detectable
between wild-type and eotaxin null mice. The eosinophil counts were 2.9 ± 1.1 × 105 and 3.1 ± 1.2 × 105 for wild-type and eotaxin null mice, respectively. We
noticed a slight but not significant increase (about 2-fold) in the
number of lymphocytes in eotaxin null mice (Fig 3C). A representative result of the staining of the cells from the BAL is shown in
Fig 4. The majority of the infiltrated
cells in the lung tissues at 6 and 12 hours and in the BAL fluid at 18 hours after challenge were neutrophils (Figs 3C and 4). In a similar
model in guinea pigs, using an assay to measure eosinophil peroxidase
(EPO), Humbles et al39 observed that eosinophils appeared
in BAL mainly in the late phase (24 hours after OVA challenge),
whereas, in lung tissues, eosinophil accumulation elevated at 6 hours
and remained high up to 24 hours. We also estimated the number of
eosinophils in the BAL fluid using an assay that specifically detects
mouse eosinophil peroxidase40 and found no difference in
the level of eosinophil peroxidase between wild-type and eotaxin null
mice (data not shown). Furthermore, we examined eosinophil
degranulation and found no difference between wild-type and eotaxin
null mice (data not shown). To determine that eotaxin is expressed in
wild-type mice after ovalbumin challenge, we performed Northern blot
analysis of total lung RNA from wild-type mice at 6 and 18 hours after ovalbumin challenge. As shown in Fig 3D, the eotaxin mRNA was induced
in agreement with previous reports using the same
model.34,38
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| Fig 3.
Normal infiltration of eosinophils in lung tissues of
eotaxin/ mice challenged with ovalbumin. (A)
Inflammation scores of lung tissues of wild-type (+/+) and eotaxin
null (/) mice at different time points after ovalbumin challenge.
Inflammation severity was scored to account for different infiltrated
cell types with 0 as the least and 4 as marked inflammation. (B)
Quantification of eosinophils in lung tissues of wild-type (+/+)
and eotaxin null (/) mice at different time points after aerosol
challenge. Data represent the mean ± SEM of eosinophils from 6 to 8 high-power fields for each mouse; n = 7 for both +/+ and /
at 6 hours; n = 4 for both +/+ and / at 12 hours; n = 6 for +/+ and n = 8 for / at 24 hours. (C) Quantification of
different cells recovered from the BAL fluid of wild-type (+/+) and
eotaxin null (/) mice at 18 hours after OVA aerosol challenge.
Data represent the mean ± SEM of cells; n = 5 for wild-type and n
= 6 for eotaxin null mice. (D) Northern blot analysis of total lung
RNA from untreated and OVA-challenged wild-type mice. The blot was
probed with 32P-labeled mouse eotaxin cDNA (upper panel),
stripped, and tested with a probe specific for mouse GAPDH (bottom).
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| Fig 4.
Staining of eosinophils from the BAL fluid of wild-type
(+/+) and eotaxin null (/) mice. Lung lavage was performed at
18 hours after OVA aerosol challenge and the cells were stained by
Diff-Quick staining after cytospin. Representative pictures are shown
for each genotype in lower (upper panel) and higher (lower panel)
magnification. Arrowheads indicate eosinophils.
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The expression of -galactosidase in eotaxin null mice
after ovalbumin challenge.
With the replacement of eotaxin by the -galactosidase gene
in our knockout mice, eotaxin expression could be examined indirectly by detecting the -galactosidase activity after ovalbumin aerosol challenge of eotaxin null mice. Previous reports suggested that resident epithelial cells and alveolar macrophages appear to be the
primary source of eotaxin in response to ovalbumin
challenge.13,35 The fact that -galactosidase is not
secreted after expression facilitated the determination of the cell
types responsible for eotaxin expression during inflammation.
We examined the expression of -galactosidase in different tissues
from eotaxin null mice at different time points after ovalbumin
challenge. Table 1 shows the level of
-galactosidase activity detected by X-gal staining in different
tissue sections. At 6 and 12 hours after the ovalbumin challenge, no
-galactosidase staining was detected in the lung tissues. The
-galactosidase staining began to appear in the lung tissues at 18 hours after challenge. The staining was more evident in the lung
tissues of eotaxin null mice at 24 and 48 hours after aerosol challenge
(Fig 5). The positive staining localized to the peribronchial and perivascular connective tissue and was present in
cells with histomorphologic features of macrophages or stromal cells
(fibroblasts/fibrocytes). In addition, peripheral nerves surrounding
the lungs of eotaxin null mice at 24 and 48 hours after aerosol
challenge had positive staining in cells with histomorphologic features
of Schwann cells. In thymus and lymph nodes, there was no significant
difference in the -galactosidase staining in eotaxin null mice at 6, 12, and 18 hours after ovalbumin challenge compared with untreated mice
(Table 1). There was a slight increase in -galactosidase staining in
thymus and a strong increase in lymph nodes in mice 24 and 48 hours
after ovalbumin challenge (Table 1 and Fig 5). In addition to the cells
with histomorphological features of dendritic cells, positive staining
was also observed in vessels of the thymic medulla. Besides endothelial
cells, macrophages in the subcapsular sinuses of lymph nodes also
stained positively for -galactosidase. As control, the
-galactosidase staining in the intestine (positive) and spleen
(negative) in eotaxin null mice did not change after ovalbumin
challenge (Table 1).
View this table:
[in this window]
[in a new window]
|
Table 1.
The Expression of -Galactosidase in Various
Tissues at Different Time Points After Ovalbumin Challenge of Eotaxin
Null Mice
|
|
View larger version (127K):
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[in a new window]
| Fig 5.
The expression of galactosidase in lung, thymus,
and lymph nodes 24 hours after ovalbumin aerosol challenge. Frozen
sections of lung, thymus, and lymph nodes from untreated or ovalbumin
aerosol-challenged (OVA 24 hours) mice were stained with X-gal
overnight followed by counter-stain with eosin. Shown are results from
a representative experiment.
|
|
Eosinophil recruitment in other inflammation models.
Lung eosinophilia induced by Sephadex beads has been developed in rats
and guinea pigs.41,42 Eosinophils corresponded to more than
40% of the cell population in BAL fluid in Sephadex bead-treated
animals.42 In guinea pigs treated with Sephadex beads,
eotaxin protein was shown to be induced in the BAL fluid and paralleled
the eosinophil infiltration in lung tissue and the infiltration of
eosinophils was significantly inhibited by a polyclonal antieotaxin
antibody.43 Because this type of experiment has not been
reported in mice, we examined the role of eotaxin in this inflammation
model using eotaxin/ mice. Lung
tissues from wild-type and mutant mice were collected at 6 and 24 hours
after Sephadex G50 injection. At both time points, there was mild to
moderate interstitial inflammation in lung tissue with intralesional
foreign material. The inflammation was more severe at the 24-hour time
point as compared with the 6-hour time point. However, the inflammation
was composed predominantly of neutrophils, with lesser numbers of
macrophages, lymphocytes, and eosinophils (data not shown). In most
areas, eosinophils constituted less than 5% to 10% of the
inflammatory cell population, and there was no difference in the
severity or composition of the inflammation in eotaxin null mice as
compared with wild-type controls. Figure 6A
shows the result of the eosinophil count in lung tissues of wild-type
and eotaxin null mice 6 and 24 hours after Sephadex bead injection. As
shown, there are very few eosinophils and no significant differences
between wild-type and knockout mice.
View larger version (18K):
[in this window]
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| Fig 6.
Infiltration of eosinophils and other leukocytes after
Sephadex beads (A) and thioglycollate injections (B). (A) Lung tissues
of wild-type (+/+) and eotaxin null (/) mice were examined
for eosinophils at 6 and 24 hours after mice received Sephadex G50
(superfine) beads by intravenous injection. Data represent the mean ± SEM from three fields for each mouse and from 5 mice of each group. (B)
Cells were collected by peritoneal lavage 24 hours after
intraperitoneal injection of thioglycollate into mice and stained by
Diff-Quick staining. Data represent the mean ± SEM from at
least six high-power fields from 6 mice of each phenotype.
|
|
In a nonspecific inflammation model induced in peritoneal cavity by
thioglycollate, eosinophils comprised 15% to 20% of the recruited
cells. To examine the role of eotaxin in this model, wild-type and
eotaxin-deficient mice were injected with thioglycollate. At 24 hours
after injection, cells recovered from the peritoneal lavage were
examined for different cell populations. At this time point,
neutrophils are the major population of infiltrated cells followed by
macrophages and eosinophils and a minor population of lymphocytes and
other types of cells. As shown in Fig 6B, compared with wild-type,
there is no difference in the number of infiltrated neutrophils,
macrophages, and eosinophils in eotaxin knockout mice.
| |
DISCUSSION |
Some chemokines have been found to be active on eosinophils, among
which eotaxin and the recently identified eotaxin-2 are the most
selective as well as most effective for eosinophil
chemotaxis.17,20 The eosinophil growth factor IL-5 has also
been found to selectively promote eosinophil chemotaxis, as well as
eosinophil growth, differentiation, and activation.44-46
IL-5 has been shown to play an essential role in the mediation of
eosinophil infiltration in a mouse asthma model, because, in
IL-5-deficient mice, the lung eosinophilia and airway hyperactivity
resulting from ovalbumin aerosol challenge were
abolished.46 Like IL-5, eotaxin expression was induced in
models of allergic inflammation and other conditions characterized by
eosinophil accumulation.12,13,47 The role of eotaxin in the
recruitment of eosinophils in these inflammation models was complicated
by the fact that the expression of other chemokines, such as RANTES,
MCP-4, MCP-3/FIC (fibroblast-induced cytokine), MIP-1, and MCP-5,
which are also active on eosinophils, paralleled with the infiltration
of eosinophils.26,34,35,48,49 To determine the role of
eotaxin in the recruitment of eosinophils during inflammation, and
possible other functions of eotaxin, we generated eotaxin-deficient mice. These mice develop normally with no histologic or hematopoietic defect compared with their wild-type littermates. These results are
consistent with another line of eotaxin knockout mice generated by
Rothenberg et al38 with a different targeting strategy and different genetic background. Mice deficient for CC chemokine MIP-1
and CC chemokine receptor CCR1 also had no abnormalities in
development, histology, and hematopoiesis.50,51 In
contrast, mice deficient for the CXC chemokine SDF-1 showed a perinatal lethal phenotype,52 and mice deficient for the murine IL-8
receptor homolog CXCR2 showed histologic defects in spleen and lymph
nodes and disordered hematopoiesis.53
Although with no apparent phenotype, mice deficient for MIP-1 or
CCR1 had impaired inflammatory responses to microbial or antigen
challenge.50,51 The eotaxin knockout mice generated by
Rothenberg et al38 showed 50% to 70% reduction in the
recruitment of eosinophils at early time points after challenge with
parasite-derived antigen (1 day) or ovalbumin aerosol (18 hours).
However, at a later time point after parasite-derived antigen (8 days)
or ovalbumin aerosol (48 hours) challenge, there is no difference in
eosinophil infiltration between eotaxin null mice and wild-type mice.
These results led them to suggest that eotaxin is important in the
early recruitment of eosinophils in these inflammatory processes.
However, in the ovalbumin-induced lung inflammation model, our eotaxin knockout mice did not show any significant differences in the number of
infiltrated eosinophils in lung tissues compared with wild-type mice,
even at the time points of 6 and 12 hours after ovalbumin aerosol
challenge (Fig 3). As a direct comparison, there is no difference in
the number of eosinophils in the BAL fluid at 18 hours after ovalbumin
aerosol challenge in our eotaxin null mice compared with wild-type (Fig
3C). Eotaxin mRNA in lung was shown to be rapidly induced after
ovalbumin challenge with levels detectable by 3 hours and peaking by 6 hours.34 The difference seen between the knockout mice
might be due to the different genetic background of the mouse strains.
There have been reports that the phenotypes of some knockout mice are
dependent on genetic background.54,55 Indeed, our
mice showed some differences from those of Rothenberg et
al12,38 in the expression of eotaxin in tissues like
skin, where eotaxin mRNA was not detectable in our mice but abundant in
those of Rothenberg et al12,38 (Fig 2B). To make sure that
eotaxin is also induced after ovalbumin challenge in our strain, we
checked eotaxin mRNA expression in wild-type mice and found it was
indeed induced in the lung at 6 and 18 hours after ovalbumin challenge
(Fig 3D). On the other hand, Rothenberg et al38 also
observed a significant reduction (~70%) of total eosinophil count in
the peripheral blood in their knockout mice compared with wild-type. We
did not observe significant differences in the blood eosinophil count
between our knockout and wild-type mice (wild-type and knockout mice
had 44 ± 16 [mean ± SEM, n = 10] and 36 ± 7 [n = 12]
eosinophils/µL of blood, respectively; P = .61). Thus, the
reduced eosinophil infiltration in eotaxin-deficient mice at early time
points after ovalbumin challenge observed by Rothenberg et
al38 could be accounted for by the reduced
level of blood eosinophils in their knockout mice. In response to
ovalbumin challenge, the eosinophil pool in the bone marrow is
mobilized by the signal provided by IL-5.44-46,56
Therefore, at later time points after ovalbumin challenge, there is no
difference in eosinophil infiltration between eotaxin null and
wild-type mice. The absence of eotaxin may be compensated by other
chemokines such as RANTES, MIP-1, and MCP-3/FIC. A chemokine named
eotaxin-2 with very similar function to eotaxin has recently been
identified in humans.20 It will be interesting to determine
whether there is a mouse homolog of eotaxin-2 and, if there is one,
whether it is involved in this type of allergic inflammation. Contrary
to the general notion, there have been recent reports that eosinophilic
airway inflammation develops normally in mice deficient of IgE, B
cells, and mast cells.57-60 These results, together with
ours, are consistent with the existence of parallel pathways of
eosinophil recruitment to the airways after antigen challenge.
Our experiments with thioglycollate- and Sephadex G50-induced
inflammation in which the infiltrated cells are predominantly neutrophils indicated that eotaxin null mice had no defect in the
recruitment of neutrophils, macrophages, lymphocytes, and eosinophils
(Fig 6). Rothenberg et al38 also examined the
thioglycollate model at a later time point (48 hours), when macrophages
were the predominant infiltrated cells, and found no defect in their knockout mice. Thus, it appears that eotaxin does not play a role in
the recruitment of eosinophils in the inflammatory response induced by
thioglycollate. In rats and guinea pigs, Sephadex bead injection
induced inflammatory response with predominantly eosinophil infiltration.41,42 In guinea pigs, the expression of
eotaxin correlates with the infiltration of eosinophils in lung after Sephadex bead injection and the infiltration of eosinophils was inhibited by antieotaxin antibodies.43 However, in both
wild-type and eotaxin/ mice, the
inflammatory response induced by Sephadex bead injection is
characterized predominantly by neutrophils with less macrophages, lymphocytes, and eosinophils (Fig 6A). It seems that mice respond to
Sephadex beads differently from rats and guinea pigs, probably due to
the different number of circulating eosinophils in these animals.
The replacement of the eotaxin gene with E coli
-galactosidase gene provided a marker for the expression of eotaxin
in different tissues under normal conditions or after challenge with
antigen. The E coli lacZ reporter gene has been used to
target into mouse genome to replace other genes and, in several cases,
faithfully reproduced the expression pattern of the targeted
genes.61-64 However, the shortcoming of this technique is
that the expression of the -galactosidase may not always faithfully
represent the expression of the targeted gene protein product. The
expression of many genes is regulated by posttranscription (ie, RNA
half-life and/or translation control), which could be altered
by gene replacement. In addition, the lacZ mRNA may be
differentially regulated and translated in different tissues. Our X-gal
staining results are consistent with the expression pattern of eotaxin
mRNA in different tissues, with the exception of the heart (Fig 2). We
detected high levels of eotaxin expression in the heart by RT-PCR.
Another group also reported a high level of eotaxin expression in the
human heart but not in the blood cells detected by Northern
blot.14 However, in the heart tissue sections of eotaxin
null or heterozygous mice, we did not observe any detectable
-galactosidase expression (data not shown). The mRNA level in the
heart is comparable to that in the thymus, lymph nodes, and intestine,
where the -galactosidase activity was easily detected in eotaxin
null and heterozygous mice (Fig 2B). Thus, the lack of X-gal staining
in heart in eotaxin null and heterozygous mice cannot be due to the
sensitivity of the assay but rather may be due to the different
regulation of the eotaxin mRNA in wild-type mice and the lacZ
mRNA in eotaxin+/ and
eotaxin/ mice.
After ovalbumin challenge, -galactosidase staining was detectable in
the lung of eotaxin null and heterozygous mice at 18, 24, and 48 hours
but not at 6 or 12 hours after aerosol challenge (Table 1 and Fig 5).
This pattern of staining does not parallel the induced eotaxin
mRNA expression in the lung, which peaks at 6 hours.34 The
protein activity of eotaxin detected in guinea pig BAL fluid also
peaked around 3 to 6 hours after ovalbumin challenge.11
Because we could not detect -galactosidase activity in the lung
tissue of eotaxin null mice at 6 and 12 hours after ovalbumin
challenge, the induced eotaxin expression at early time points may be
mainly in the BAL fluid and only at a later time in lung tissue. Our
results suggest that eotaxin might also play a role in the later phase
of the inflammatory response after ovalbumin challenge. The
-galactosidase staining in lung tissue suggested that, in the late
phase of the inflammation, stromal cells (fibroblasts/fibrocytes) and
infiltrated macrophages could be the sources for eotaxin expression. Previous reports with in situ hybridization and eotaxin antibody staining indicated that resident lung epithelial cells and alveolar macrophages were the principal cells producing
eotaxin.13,35 However, we did not detect any
-galactosidase staining in lung epithelial cells after ovalbumin
challenge. The difference may be due to variations in the protocols
used to challenge mice. In our experiments, we challenged mice with
ovalbumin aerosol only once, whereas mice were repeatedly challenged
with ovalbumin aerosol several times in the above-mentioned
reports.13,35 Therefore, the level of the induced
expression may be quite different.
At the late phase of the ovalbumin-induced inflammation, we also found
that there was increased -galactosidase staining in thymus and lymph
nodes (Fig 5). This finding suggested that eotaxin expression was
increased in these tissues. However, we did not observe significant
infiltration of eosinophils in these tissues (data not shown). In a
recent report, a dense infiltration of eosinophils was observed in the
tracheobronchial lymph nodes, especially pronounced in the
B-cell-deficient mice challenged with ovalbumin aerosol.58
It was suggested that these infiltrated eosinophils might be important
for T-cell activation in response to antigen challenge. The
investigation of the strong induced expression of eotaxin in thymus and
lymph nodes may provide clues to other physiological functions of
eotaxin. The presence of -galactosidase staining of Schwann cells in
nerves surrounding the lungs after ovalbumin challenge was an
intriguing finding. This finding suggested that eotaxin might be
expressed by Schwann cells during the inflammation processes in lung.
The primary function of Schwann cells is to secrete and maintain the
myelin sheath around axons in peripheral nerves. However, Schwann cells
are capable of expressing MHC-II molecules and produce various
cytokines, including IL-10, IL-12, and nitrite, under certain
stimulatory conditions.65-67 Stimulation of the vagus nerve
by factors released by mast cells leads to bronchoconstriction and is
an important factor in the pathophysiology of asthma. Production of
eotaxin by Schwann cells may provide another mechanism whereby the
nervous system modifies the immune response in asthma.
| |
ACKNOWLEDGMENT |
The authors thank S. Lira, M. Swerdel, and A. Lee in the Transgenic
Unit and all the staff in Veterinary Sciences at BMS for generating and
maintaining the mice; A. Lewin, D. Barton, M. French, C. Rizzo, C. Ryan
and J. Stevens for excellent technical assistance; C. Raventos-Suarez
and K. Class for flow cytometry; and N. Thompson and T. Nelson for
assistance with DNA sequencing. We thank Drs Y. Zhou, V. Iotsova, and
D. Dambach for helpful suggestions.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted July 9, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Rodrigo Bravo, PhD, Department
of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, PO
Box 4000, Princeton, NJ 08543.
| |
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Abstract
Introduction