RAPID COMMUNICATION
Cloning of rat eotaxin: ozone inhalation increases mRNA and protein
expression in lungs of Brown Norway rats
Yukio
Ishii1,
Manabu
Shirato1,
Akihiro
Nomura1,
Tohru
Sakamoto1,
Yoshiyuki
Uchida1,
Morio
Ohtsuka1,
Masaru
Sagai2, and
Shizuo
Hasegawa1
1 Department of Respiratory
Medicine, Institute of Clinical Medical Sciences, University of
Tsukuba, and 2 National Institute
for Environmental Studies, Tsukuba, Ibaraki 305, Japan
 |
ABSTRACT |
The C-C chemokine eotaxin is thought to be
important in the selective recruitment of eosinophils to the site of
inflammation in guinea pigs, mice, and humans. We isolated the rat
eotaxin gene to determine whether a similar molecule might play a role in the pulmonary infiltration of eosinophils during acute inflammation in the rat. The cDNA for rat eotaxin encoded a 97-amino acid protein containing a 74-amino acid mature eotaxin protein with 97.3% identity to mouse eotaxin. The recombinant protein encoded by this gene displayed specific chemotactic activity for eosinophils when analyzed with a microchemotactic chamber. The expression of eotaxin mRNA increased ~1.6-fold immediately after exposure to ozone and was 4-fold higher after 20 h. The number of lavageable eosinophils at the
same time points were 3- and 15-fold greater, respectively, than
control eosinophils. Immunocytochemistry revealed that alveolar macrophages and bronchial epithelial cells were positive for eotaxin. These results suggest that eotaxin may be involved in the recruitment of eosinophils into the air spaces during certain inflammatory conditions in rats.
messenger ribonucleic acid; chemokine; cytokine; inflammation; eosinophils
 |
INTRODUCTION |
THE INFILTRATION of inflammatory cells into the airways
is a pathological characteristic of pulmonary inflammation. Among these
cells are the eosinophils, which are activated at the site of
inflammation to release preformed cationic proteins, oxygen radicals,
and lipid mediators (7, 26). This leads to hyperresponsiveness of the
airway to certain stimuli and to damage to the airway epithelium (3,
25). Although eosinophils constitute only a minority of the circulating
leukocytes, they are recruited in large numbers into tissue sites
during inflammation, suggesting that specific chemotactic factors are
involved in their recruitment.
The mechanism underlying the migration of eosinophils into tissue sites
is not completely understood. It has been reported that the chemotactic
cytokines, especially the C-C chemokines, are important (1, 5, 20).
Eotaxin, a novel member of the C-C chemokine family, was recently
identified, and the eotaxin genes from guinea pigs (13, 22), mice (8,
21), and humans (6, 18) have been cloned. Eotaxin displays potent and
specific chemotactic activity for eosinophils in these three species
both in vivo and in vitro. The expression of eotaxin mRNA was shown to
be induced in the lungs during allergic inflammation. Moreover, the
levels of eotaxin mRNA paralleled the kinetics of eosinophil accumulation during such inflammation (8).
To determine whether a similar molecule might be involved in the
infiltration of eosinophils into the lungs during acute inflammation in
rats, we molecularly cloned the rat homologue of the eotaxin gene and
analyzed the expression of its mRNA and protein products during
eosinophilic inflammation of the lungs in Brown Norway (BN) rats
exposed to ozone. The role of eotaxin in the recruitment of eosinophils
into the lung was investigated.
 |
MATERIALS AND METHODS |
Animals and exposure to ozone. Male BN
rats (Charles River Laboratories, Kanagawa, Japan), 200-250 g,
were exposed for 6 h to 1.2 parts/million (ppm) ozone in a chamber 1.68 m3. Ozone was generated from pure
oxygen by an Arc generator and was continuously monitored with an Ox
analyzer (model 806, Kimoto-Denshi, Tokyo, Japan).
Bronchoalveolar lavage. The lungs of
anesthetized rats (50 mg/kg body wt ip of pentobarbital sodium) were
lavaged with a total volume of 48 ml of phosphate-buffered saline (PBS)
containing 3 mM EDTA before, immediately after, and 20 h after exposure
to ozone. At each time point, lavaged cells were combined and
resuspended in 5 ml of PBS. Cell viability was determined by trypan
blue exclusion. Differential cell counts were performed by standard
light-microscopic techniques based on staining with Diff-Quik (American
Scientific Products, McGaw Park, IL). Results are expressed as the
total number of cells recovered per lung.
Polymerase chain reaction cloning.
Total RNA was extracted by the guanidinium thiocyanate method (4) from
the lungs of BN rats 20 h after exposure to ozone. RNA (1 µg) was
reverse transcribed with oligo(dT), and 5 µl of the resulting cDNA
were amplified with a DNA thermal cycler (Perkin-Elmer Cetus,
Branchburg, NJ) with the oligonucleotide primers
5'-CCTCCACCATGCAGAGCTCC and 5'-AGGCTCTGGGTTAGTGTCAA, corresponding to base pairs (bp) 40-59 and 355-374,
respectively, of mouse eotaxin cDNA (8). Polymerase chain reaction
(PCR) conditions were 30 cycles of denaturation at 95°C for 45 s,
annealing at 45°C for 60 s, and extension at 72°C for 90 s. The
amplified PCR products were subcloned directly into a pGEM-T vector (TA vector, Promega, Madison, WI) and sequenced by the dideoxy chain termination method with an autosequencer (ABI PRISM-310, Perkin-Elmer).
5' Rapid amplification of cDNA
ends. With the use of total RNA from the lungs of BN
rats 20 h after exposure to ozone, first-strand cDNA was synthesized
with a Marathon cDNA amplification kit (Clontech Laboratories, Palo
Alto, CA). After second-strand synthesis, the cDNA adaptor was ligated
with T4 DNA ligase, and rapid amplification of cDNA ends (RACE) PCR was
performed with the oligonucleotide primers
5'-CCATCCTAATACGACTCACTATAGGGC and 5'-AGGCTCTGGGTTAGTGTCAA. PCR conditions were 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 68°C for 4 min.
The amplified PCR products were subcloned directly into a pCR3.1-Uni expression vector (TA vector, Invitrogen, San Diego, CA) and sequenced.
Production of recombinant eotaxin.
Eotaxin cDNA subcloned into a pCR3.1-Uni expression vector was
transfected into COS cells with LipofectAMINE (GIBCO BRL, Gaithersburg,
MD). After 72 h of culture, the supernatants of eotaxin-transfected or
mock-transfected (vector alone) COS cells were concentrated by
ultrafiltration in a Centricon-3 (Amicon, Danvers, MA). The recombinant
protein was characterized by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis under reducing conditions on
15% gels and stained with Coomassie blue or was transferred to
polyvinylidene difluoride membranes (Nippon Bio-Rad Laboratories,
Tokyo, Japan) for Western blot analysis with anti-murine eotaxin
antibody (Pepro Tech, London, UK).
Chemotaxis. Normal rat granulocytes
were obtained from peripheral blood via the abdominal aorta with
two-step Ficoll-Hypaque (Sigma Chemical, St. Louis, MO) density
gradient centrifugation. Isolated cells were washed two times with
Hanks' balanced salt solution (HBSS), and the residual red blood cells
were removed by hypotonic lysis. The chemotactic assay was performed
with a 48-well modified Boyden chamber (Neuro Probe, Cabin John, MD). The cells were resuspended to a concentration of 1.5 × 106 cells/ml in HBSS containing
2% bovine serum albumin, and 50 µl (7.5 × 104 cells) were placed in the top
of the chamber. The lower wells of the chambers were filled with
various concentrations of COS cell supernatants. The chambers were
incubated at 37°C for 30 min; cells that crossed the polycarbonate
filter (3-µm pore; Neuro Probe) and adhered to the bottom were
stained with Diff-Quik and counted in a high-performance field
(×1,000). The results are presented as the total number of cells
per 5 high-performance field.
RNA analysis. Total RNA was extracted
from the lungs of anesthetized rats that were removed at the same time
as bronchoalveolar lavage (BAL) was performed. RNA (20 µg/lane) was
electrophoresed on formaldehyde-agarose gels and transferred to nylon
membranes (Hybond-N+, Amersham).
Blots were prehybridized at 68°C for 30 min in ExpressHyb hybridization solution (Clontech) and hybridized with a
[
-32P]dCTP-labeled
0.3-kilobase (kb) rat eotaxin cDNA PCR fragment at 68°C for 60 min
in the same solution. After removal of the probe, the blots were
hybridized with a
[
-32P]dCTP-labeled
0.47-kb rat
-actin cDNA fragment (17). The blots were washed with
2× saline-sodium citrate (SSC)-0.05% SDS at room temperature for
30 min, followed by 0.1× SSC-0.1% SDS at 50°C for 30 min.
Autoradiograms were made with a bioimaging analyzer (BAS5000, Fuji
Photo Film, Tokyo, Japan).
Immunocytochemistry. At the same time
as BAL, the rats were anesthetized and perfusion fixed first with
saline and then with 4% paraformaldehyde-PBS via the pulmonary artery.
The lungs were removed and immersed in the same fixative for 2 h at
4°C, washed with PBS three times for 5 min each, transferred to PBS
containing 30% sucrose for 18 h at 4°C, and embedded in optimum
cutting temperature compound (Miles, Elkhart, IN). Cryostat sections (8 µm) were cut and mounted on
poly-L-lysine-coated glass
slides. After incubation with 2% normal goat serum in 0.01 M phosphate
buffer containing 0.5 M NaCl and 0.1% Tween 20 (Sigma Chemical) for 20 min at room temperature, the sections were incubated for 1 h at room
temperature with a 1:125 dilution of rabbit anti-mouse eotaxin antibody
(Pepro Tech); as a control, nonimmune rabbit serum was used. After the sections were washed, they were incubated with biotinylated goat anti-rabbit immunoglobulin G and then with avidin-biotinylated peroxidase complex (ABC Kit, Vector Laboratories, Burlingame, CA).
Reactions were visualized with 3,3'-diaminobenzidine
tetrahydrochloride in the presence of
H2O2.
Statistics. Data are presented as
means ± SD and were analyzed by standard one-way analysis of
variance in combination with Duncan's multiple comparison test. A
level of P < 0.05 was accepted as
statistically significant.
 |
RESULTS |
Cloning of rat eotaxin cDNA. To
identify the rat homologue of the eotaxin gene, we utilized PCR primers
corresponding to highly conserved regions of the mouse and human
eotaxin genes (8). After amplification, a single PCR product of ~330
bp was obtained; its size resembled that of the mouse eotaxin cDNA
fragment (334 bp) obtained with these primers. The 5' sequence of
this cDNA fragment was extended by RACE, and the product was cloned and sequenced.
The coding region of cDNA consisted of an open reading frame of 291 bases encoding a 97-amino acid protein that contained a 23-amino acid
signal peptide (Fig.
1A).
By homology with the NH2 terminus
of the mature mouse protein, signal peptide cleavage was predicted to
occur between alanine and histidine, resulting in a 74-amino acid
mature protein. It is a member of the C-C chemokine family as indicated
by the cysteine pair at amino acids 9 and 10 (Fig.
1A). As with other eotaxins, rat
eotaxin is characterized by the presence of a two-amino acid gap
located before the second proline of the putative mature protein as
well as a highly conserved domain near the carboxy terminus
(ICADPKKKWVQD; Fig. 1B). Comparison of the predicted amino acid sequence of rat eotaxin with those of other
eotaxins revealed a high degree of homology to the mouse protein
(97.3%) but a lesser homology to guinea pig (63.5%) and human
(62.2%) eotaxins (Fig. 1B). Rat
eotaxin showed lower amino acid homology to other rat C-C chemokines
such as regulated on activation normal T cell expressed and secreted
(RANTES) (31.8%), macrophage inflammatory protein (MIP)-1
(31.9%),
and MIP-1
(39.1%).

View larger version (29K):
[in this window]
[in a new window]

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
A: nucleotide sequence and deduced
amino acid sequence of rat eotaxin cDNA coding region. Underlined amino
acids correspond to predicted signal sequence. Arrowhead, signal
peptidase cleavage site predicted by homology with
NH2 terminus of mature mouse
eotaxin. B: amino acid sequence
alignment of rat eotaxin (Eot) with mouse, guinea pig (GP), and human
eotaxins and other rat C-C chemokines. Amino acids are numbered from
predicted mature NH2 terminus
after signal peptidase cleavage. Asterisks indicate 4 cysteine residues
characteristic of C-C chemokine family. RANTES, regulated on activation
normal T cell expressed and secreted; MIP, macrophage inflammatory
protein. These sequence data are available from GenBank under accession
no. U96637.
|
|
Expression and eosinophil chemotactic activity of
recombinant eotaxin. To evaluate the molecular size and
functional chemotactic properties of the protein encoded by cloned rat
eotaxin cDNA, the latter was subcloned into an expression vector and
used for transient transfection of COS cells. The supernatant from
these cells was concentrated 10-fold and analyzed by Western blotting with an anti-mouse eotaxin antibody. A single of ~8.5 kDa reacted with this antibody (Fig.
2A).
This molecular mass was consistent with that calculated
from the deduced amino acid sequence (~8.4 kDa).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
A: Western blot analysis of
supernatants from COS cells transfected with expression vector alone
(lane 1) or with rat eotaxin cDNA in
expression vector (lane 2). Both
samples were stained with anti-mouse eotaxin antibody. Nos. on
left, molecular mass markers in kDa.
B: chemotaxis of rat eosinophils
(solid bars) and neutrophils (open bars) to a fivefold concentrated
supernatant (sup) from COS cells transfected with expression vector
alone (moc/sup ×5) and unconcentrated (eot/sup) or fivefold
concentrated (eot/sup ×5) supernatant from eotaxin-transfected
COS cells. Results are total number of cells in 5 high-performance
field (hpf; ×1,000). C:
proportion of eosinophils and neutrophils in cell suspension medium
after density gradient centrifugation (upper chamber) and in cells
attached to filter as a result of chemotactic assay (migrated).
|
|
The chemotactic activity of the recombinant protein toward rat
granulocytes was assayed in vitro in a microchemotaxis chamber. The
granulocytes were obtained from the peripheral blood of normal rats,
with a ratio of eosinophils to neutrophils of ~1:9 (Fig. 2C). The recombinant protein
demonstrated chemotactic activity toward eosinophils, whereas it lacked
activity toward neutrophils (Fig.
2B); 90.2% of the cells that had
migrated toward a fivefold concentrate of eotaxin cDNA supernatant were
eosinophils (Fig. 2C). No
chemotactic activity on eosinophils was observed in supernatant from
cells transfected with vector alone (Fig.
2B).
Expression of eotaxin mRNA and protein in lung
tissues. We next determined by Northern blot analysis
whether the expression of rat eotaxin mRNA was correlated with the
degree of eosinophil infiltration in the acute pulmonary inflammation
induced by exposure to ozone. The size of the rat eotaxin message
(~900 bp) was found to be consistent with those of mature mouse (8,
21) and human (6, 18) eotaxin mRNAs. Although a small amount of eotaxin mRNA was constitutively expressed in rat lungs before exposure to
ozone, its expression was increased after ozone exposure (Fig. 3). Analysis of the density ratios of the
eotaxin and
-actin messages demonstrated that the expression of
eotaxin mRNA was enhanced ~1.6-fold immediately after exposure to
ozone and 4-fold 20 h later. The number of eosinophils in BAL fluid at
the same time points was 3-fold and 15-fold greater, respectively, than the control number (Fig. 3).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 3.
Top: expression of rat eotaxin mRNA in
lungs of Brown Norway rats and hybridization of this blot with a
-actin probe. Lane 1, before
exposure to ozone; lane 2, immediately
after 6-h exposure to 1.2 parts/million ozone; lane
3, 20 h after exposure to ozone. Blots are
representative of each group. Bottom:
no. of eosinophils recovered by bronchoalveolar lavage in ozone-exposed
Brown Norway rats. Results are means ± SD per lung for 4 animals.
* Significantly different compared with rats before exposure to
ozone (air), P < 0.01.
|
|
We assayed protein expression and tissue localization of eotaxin by
immunocytochemistry. Eotaxin staining was localized most strongly in
alveolar macrophages 20 h after exposure to ozone (Fig.
4C) as
well as in a fraction of the bronchial epithelial cells (Fig.
4D). We observed only weak
immunoreactivity to eotaxin on alveolar macrophages immediately after
exposure to ozone (Fig. 4B) but none
before ozone exposure (Fig. 4A). No
immunoreactivity to any cell type was detected with nonimmune rabbit
serum at any time.
 |
DISCUSSION |
Eotaxin was first identified as a potent eosinophil chemoattractant in
BAL fluid that was obtained after challenging sensitized guinea pigs
with allergen (9, 14). Since then, several in vitro and in vivo studies
have shown eotaxin to be a potent and selective eosinophil
chemoattractant in humans (6, 18) and mice (8, 21). The novel rat C-C
chemokine we identified here is selectively chemotactic for
eosinophils. On the basis of sequence similarity and function, this
chemokine can be considered a rat equivalent of eotaxins.
In agreement with the findings in guinea pigs (13, 22) and humans (6),
eotaxin mRNA was expressed in rat lungs even before the exposure to
ozone. It has been reported that eosinophils mainly reside in the
tissue, inasmuch as there are normally several hundred times as many
eosinophils in the tissue as in the blood (24). Eosinophils, like
neutrophils, are often present in the walls of blood vessels or the
interstitium of the lung as marginated and/or interstitial
pools. Therefore, it is likely that the constitutive expression of
eotaxin mRNA we observed in unexposed rat lungs was associated with
baseline eosinophil homing to the marginated and/or
interstitial pools. However, we were unable to detect eotaxin protein
immunocytochemically in unexposed lungs. One possible explanation is
that the amount of eotaxin protein in these samples may be below the
level of detection. Alternatively, eotaxin mRNA may be expressed, but
not translated, before the exposure to ozone.
After exposure to ozone, we observed that the number of BAL-recovered
eosinophils was increased, suggesting that these cells were recruited
in the airways or alveolar spaces by ozone stimulation. Interestingly,
eotaxin mRNA and protein expression were also enhanced in the lungs,
suggesting that the translation of eotaxin mRNA occurs within a short
time. The localization of eotaxin protein at luminal sites such as
alveolar macrophages and bronchial epithelial cells during
ozone-induced pulmonary inflammation suggests that the additional
expression of eotaxin may be involved in the migration of eosinophils
from the marginated and/or interstitial pools into the air
spaces. Similar results have also been observed in mice, in which
eotaxin expression was found to parallel the accumulation of
eosinophils during allergic inflammation (8).
Although we have shown that rat eotaxin acts as a selective
chemoattractant of eosinophils in vitro and that its expression can be
correlated with the number of BAL-recovered eosinophils, it is not
clear whether eotaxin contributes to the ozone-induced eosinophilic
inflammation in vivo. Indeed, it has been reported that several
chemotactic factors such as MIP-2 (11, 12), cytokine-induced neutrophil
chemoattractant (10, 12), and leukotriene (LT) B4 (15, 16, 23) are induced by
exposure to ozone; among these,
LTB4 is chemotactic for
eosinophils (19). However, the epithelial cell response was detected
only after high in vitro exposure (~4 ppm ozone), and increases in
LTB4 have not been observed in
vivo (15, 16, 23). Thus, among the ozone-inducible chemoattractants, only eotaxin is upregulated by near-ambient levels of ozone, suggesting a specific contribution of eotaxin to the recruitment of eosinophils into the lungs of rats exposed to ozone in vivo. Our findings that
eotaxin mRNA was not expressed in the lungs of Sprague-Dawley rats 20 h
after a 6-h exposure to 1.2 ppm ozone (data not shown) do not
contradict this hypothesis, inasmuch as ozone exposure leads to the
accumulation of neutrophils in the lung of Sprague-Dawley rats (2, 12).
Although the mechanisms that underlie the influx of inflammatory cells
into tissue sites are not well known, it is likely that the appearance
in tissues of different types of inflammatory cells during acute
inflammation is regulated, at least in part, by the differential
expression of chemokines. The rat eotaxin cDNA we have cloned may be an
important tool for exploring the molecular mechanisms for eosinophil
traffic during inflammation.
 |
ACKNOWLEDGEMENTS |
This study was supported in part by Ministry of Education of Japan
Research Grant 08770425 and by a research grant from the Kanae
Foundation of Research for New Medicine.
 |
FOOTNOTES |
Address for reprint requests: Y. Ishii, Dept. of Respiratory Medicine,
Institute of Clinical Medical Sciences, Univ. of Tsukuba, Tsukuba,
Ibaraki 305, Japan.
Received 6 May 1997; accepted in final form 8 October 1997.
 |
REFERENCES |
1.
Baggiolini, M.,
and
C. A. Dahinden.
CC chemokines in allergic inflammation.
Immunol. Today
15:
127-133,
1994[Medline].
2.
Bassett, D. J. P.,
C. L. Elbon,
S. S. Reichenbaugh,
G. A. Boswell,
T. M. Stevens,
M. C. McGowan,
and
J. S. Kerr.
Pretreatment with EDU decreases rat lung cellular responses to ozone.
Toxicol. Appl. Pharmacol.
100:
32-40,
1989[Medline].
3.
Bousquet, J.,
P. Chanez,
J. Y. Lacoste,
G. Barneon,
N. Ghavanian,
I. Enander,
P. Venge,
S. Ahlstedt,
J. Simony-Lafontaine,
and
P. Godard.
Eosinophilic inflammation in asthma.
N. Engl. J. Med.
323:
1033-1039,
1990[Abstract].
4.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
5.
Dahinden, C. A.,
T. Geiser,
T. Brunner,
V. Von Tscharner,
D. Caput,
P. Ferrara,
A. Minty,
and
M. Baggiolini.
Monocyte chemotactic protein 3 is a most effective basophil- and eosinophil-activating chemokine.
J. Exp. Med.
179:
751-756,
1994[Abstract].
6.
Garcia-Zepeda, E. A.,
M. E. Rothenberg,
R. T. Ownbey,
J. Celestin,
P. Leder,
and
A. D. Luster.
Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia.
Nat. Med.
2:
449-456,
1996[Medline].
7.
Gleich, G. J.,
and
C. R. Adolphson.
The eosinophilic leukocytes: structure and function.
Adv. Immunol.
39:
177-253,
1986[Medline].
8.
Gonzalo, J. A.,
G. Q. Jia,
V. Aguirre,
D. Friend,
A. J. Coyle,
N. A. Jenkins,
G. S. Lin,
H. Katz,
A. Lichtman,
N. Copeland,
M. Kopf,
and
J. C. Gutierrez-Ramos.
Mouse eotaxin expression parallels eosinophil accumulation during lung allergic inflammation but it is not restricted to a Th2-type response.
Immunity
4:
1-14,
1996[Medline].
9.
Griffiths-Johnson, D. A.,
P. D. Collins,
A. G. Rossi,
P. J. Jose,
and
T. J. Williams.
The chemokine, eotaxin, activates guinea pig eosinophils in vitro and causes their accumulation into the lung in vivo.
Biochem. Biophys. Res. Commun.
197:
1167-1172,
1993[Medline].
10.
Haddad, E. B.,
M. Salmon,
H. Koto,
P. J. Barnes,
I. Adcock,
and
K. F. Chung.
Ozone induction of cytokine-induced neutrophil chemoattractant (CINC) and nuclear factor-
b in rat lung: inhibition by corticosteroids.
FEBS Lett.
379:
265-268,
1996[Medline].
11.
Haddad, E. B.,
M. Salmon,
J. Sun,
S. Liu,
A. Das,
I. Adcock,
P. J. Barnes,
and
K. F. Chung.
Dexamethasone inhibits ozone-induced gene expression of macrophage inflammatory protein-2 in rat lung.
FEBS Lett.
363:
285-288,
1995[Medline].
12.
Ishii, Y., H. Yang, T. Sakamoto, A. Nomura, S. Hasegawa, F. Hirata, and D. J. P. Bassett. Rat alveolar macrophage
cytokine production and regulation of neutrophil recruitment following
acute ozone exposure. Toxicol. Appl.
Pharmacol. In press.
13.
Jose, P. J.,
I. M. Adcock,
D. A. Griffiths-Johnson,
N. Berkman,
T. N. C. Wells,
T. J. Williams,
and
C. A. Power.
Cloning of an eosinophil chemoattractant cytokine and increased mRNA expression in allergen-challenged guinea pig lungs.
Biochem. Biophys. Res. Commun.
205:
788-794,
1994[Medline].
14.
Jose, P. J.,
D. A. Griffiths-Johnson,
P. D. Collins,
D. T. Walsh,
R. Moqbel,
N. F. Totty,
O. Truong,
J. J. Hsuan,
and
T. J. Williams.
Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation.
J. Exp. Med.
179:
881-887,
1994[Abstract].
15.
Koren, H. S.,
R. B. Devlin,
D. E. Graham,
R. Mann,
M. P. McGee,
D. H. Horstman,
W. J. Kozumbo,
S. Becker,
D. E. House,
W. F. McDonnell,
and
P. A. Bromberg.
Ozone-induced inflammation in the lower airways of human subjects.
Am. Rev. Respir. Dis.
139:
407-415,
1989[Medline].
16.
Leikauf, G. D.,
K. E. Driscoll,
and
H. E. Wey.
Ozone-induced augmentation of eicosanoid metabolism in epithelial cells from bovine trachea.
Am. Rev. Respir. Dis.
137:
435-442,
1988[Medline].
17.
Nudel, U.,
M. Zakut,
S. Neuman,
Z. Levy,
and
D. Yaffe.
The nucleotide sequence of the rat cytoplasmic
-actin gene.
Nucleic Acids Res.
11:
1769-1771,
1983.
18.
Ponath, P. D.,
S. Qin,
D. J. Ringler,
I. C. Lewis,
J. Wang,
N. Kassam,
H. Smith,
X. Shi,
J. A. Gonzalo,
W. Newman,
J. C. G. Ramos,
and
C. R. Mackay.
Cloning of the human eosinophil chemoattractant, eotaxin.
J. Clin. Invest.
97:
604-612,
1996[Abstract/Free Full Text].
19.
Resnick, M. B.,
and
P. F. Weller.
Mechanisms of eosinophil recruitment.
Am. J. Respir. Cell Mol. Biol.
8:
349-355,
1993[Medline].
20.
Rot, A.,
M. Krieger,
T. Brunner,
S. C. Bischoff,
T. J. Schall,
and
C. A. Dahinden.
RANTES and macrophage inflammatory protein 1 alpha induce the migration and activation of normal human eosinophil granulocytes.
J. Exp. Med.
176:
1489-1495,
1992[Abstract].
21.
Rothenberg, M. E.,
A. D. Luster,
and
P. Leder.
Mouse eotaxin: an eosinophil chemoattractant inducible in endothelial cells and in interleukin 4-induced tumor suppression.
Proc. Natl. Acad. Sci. USA
92:
8960-8964,
1995[Abstract].
22.
Rothenberg, M. E.,
A. D. Luster,
C. M. Lilly,
J. M. Drazen,
and
P. Leder.
Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung.
J. Exp. Med.
181:
1211-1216,
1995[Abstract].
23.
Seltzer, J.,
B. G. Bigby,
M. Stulberg,
M. J. Holtzman,
J. A. Nadel,
I. F. Ueki,
G. D. Leikauf,
E. J. Geotzl,
and
H. A. Boushey.
O3-induced change in bronchial reactivity to methacholine and airway inflammation in humans.
J. Appl. Physiol.
60:
1321-1326,
1986[Abstract/Free Full Text].
24.
Spry, C. J.
Mechanism of eosinophilia. V. Kinetics of normal and accelerated eosinophilpoiesis.
Cell Tissue Kinet.
4:
351-364,
1971[Medline].
25.
Wardlaw, A. J.,
S. Dunnette,
G. J. Gleich,
J. V. Collins,
and
A. B. Kay.
Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma. Relationship to bronchial hyperreactivity.
Am. Rev. Respir. Dis.
137:
62-69,
1988.
26.
Weller, P. F.
The immunobiology of eosinophils.
N. Engl. J. Med.
324:
1110-1118,
1991[Medline].
AJP Lung Cell Mol Physiol 274(1):L171-L176
1040-0605/98 $5.00
Copyright © 1998 the American Physiological Society