1 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229; and 2 Department of Environmental Medicine, University of Rochester, Rochester, New York 14642
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ABSTRACT |
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Clara cell secretory protein (CCSP) is an
abundant 10-kDa polypeptide synthesized and secreted primarily by
nonciliated bronchiolar epithelial cells in the mammalian lung. To
determine the potential role of CCSP in pulmonary inflammation after
acute viral infection, CCSP gene-targeted {CCSP-deficient
[CCSP(/
)]} mice were exposed to a
recombinant E1- and E3-deficient adenoviral vector, Av1Luc1, intratracheally. Lung inflammation was markedly increased in
CCSP(
/
) mice compared with wild-type control mice and was
associated with an increased number of polymorphonuclear cell
infiltrates and epithelial cell injury in both conducting airways and
alveolar regions. Histological evidence of pulmonary inflammation in
CCSP(
/
) mice was associated with increased production of
cytokine (interleukin-1
and -6 and tumor necrosis factor-
) mRNA
and protein, as well as chemokine (macrophage inflammatory protein-1
and -2 and monocyte chemoattractant protein-1) mRNA expression within
the lung in response to adenoviral infection. Adenoviral-mediated gene
transfer was decreased in CCSP(
/
) mice relative to
wild-type mice as measured by luciferase enzyme activity in lung
homogenates. The present study suggests that CCSP is involved in
modulating lung inflammation during viral infection and supports a role
for CCSP in lung host defense.
lung epithelium; pulmonary infection; viral pathogenesis
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INTRODUCTION |
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CLARA CELL SECRETORY PROTEIN (CCSP) is a 10-kDa dimeric
protein synthesized primarily by nonciliated bronchiolar epithelial cells (11). CCSP is one of the most abundant soluble proteins within
the epithelial lining fluid of the lung (11). Nonciliated bronchiolar
epithelial cells (also termed Clara cells) lining the conducting
airways synthesize and secrete CCSP and a variety of proteins
implicated in host defense, including surfactant protein (SP) A, SP-B,
and SP-D (17). CCSP is structurally similar to the rabbit uteroglobin
(UG) protein, which is known to bind polychlorinated biphenyls (12, 13)
and inhibit secretory phospholipases
A2 (6) and chemotaxis of
inflammatory cells in uterine tissues (15). The physiological function
of CCSP in vivo as well as the role of CCSP in lung infection and
injury is not well understood. To delineate the function of CCSP, CCSP
gene-targeted {CCSP-deficient [CCSP(/
)]} mice were generated (14).
Pulmonary accumulation of polychlorinated biphenyls after
intraperitoneal administration was markedly decreased in
CCSP(
/
) mice, suggesting that CCSP is a determinant of
lipophilic toxin accumulation within the lung (14). After hyperoxic
lung injury, pulmonary edema was exacerbated and proinflammatory
cytokine gene expression was increased in CCSP(
/
) mice,
providing support for the concept that CCSP plays a role in host
responses to lung injury (5).
Lung inflammation after viral infection with adenoviruses and
adenoviral vectors has been well characterized. Adenoviruses are
ubiquitous viral pathogens that cause respiratory, gastrointestinal, and genitourinary infections (16). Adenoviruses usually cause acute
respiratory pathology; however, adenoviruses can also persist as
asymptomatic infections of the respiratory tract (16). Acutely, adenoviral infection causes lung infiltration of macrophages and neutrophils in the alveolar air spaces, generally observed 2-3 days after infection (2). Concentrations of the cytokines tumor necrosis factor (TNF)-, interleukin (IL)-1, and IL-6 are also increased in pulmonary tissues after adenoviral infection, coinciding with the appearance of macrophages in alveolar regions (2). Because of
their tropism for airway epithelial cells, adenoviruses have been
utilized as vectors for gene transfer to the lung. The efficiency and
duration of gene expression with recombinant adenoviruses, however, are
limited by host inflammatory and immune responses (1, 18, 19, 22). Thus
lung inflammation after infection with adenoviral vectors has been well
characterized and provides a useful model for studying viral-induced
inflammation in the lung.
Although the role of CCSP in infectious injury in the lung has not been
addressed, this report tests whether CCSP modulates host responses to
viral infection in the lung. CCSP(/
) mice were infected
with a replication-deficient adenoviral vector, and lung inflammation
and acute immune responses were measured. The deficiency of CCSP in
mice increases lung inflammation after intratracheal administration of
an adenovirus.
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MATERIALS AND METHODS |
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Mice. CCSP(/
) (129J
Ola/129J hybrid) and wild-type 129J (Taconic Farms, Germantown, NY)
mice were housed under pathogen-free conditions in the Children's
Hospital (Cincinnati, OH) Research Foundation vivarium as required by
American Association for Accreditation of Laboratory Animal Care
guidelines.
Intratracheal administration of
adenovirus. Eight- to twelve-week-old
CCSP(/
) and 129J wild-type control mice
(n = 6-12 mice/group) were used.
The procedure for intratracheal administration of adenoviral vectors
was previously described by Zsengeller et al. (22). Briefly, mice were
anesthetized with methoxyflurane vapor, and a ventral midline incision
was made to expose the trachea. Intratracheal inoculation of 1 × 109 plaque-forming units of
Av1Luc1, an E1- and E3-deleted adenoviral vector expressing firefly
luciferase from the Rous sarcoma virus promoter, in 100 µl of
delivery vehicle (10 mM Tris, 1 mM
MgCl2, and 10% glycerol, pH 7.4)
was performed with a bent, 27-gauge tuberculin syringe (Monoject, St.
Louis, MO). The incision was closed with one drop of Nexaband liquid,
and the mice were allowed to recover. Mice recover rapidly and remain
active after the procedure. At a predetermined time of biological
analysis, the mice were killed by a lethal injection of
pentobarbital sodium. A midline incision was made in the abdomen.
Exsanguination was accomplished by transection of the inferior vena
cava to reduce hemorrhage in the lung. For histological studies, RNA
and protein analyses, the right upper, middle, and lower lobes of the
lung were clamped with a hemostat and removed for measurement of
luciferase activity, RNA, and protein. The left lung lobe was inflated
with 4% paraformaldehyde (Electron Microscopy Sciences, Ft.
Washington, PA) and fixed overnight for histological examination.
Evaluation of acute inflammatory cell infiltrates. Inflammatory cell numbers and percentages were evaluated at 4 and 24 h after adenoviral vector administration. Bronchoalveolar lavage (BAL; n = 6 mice/group) fluid was obtained by intratracheal instillation of 1 ml of PBS into the lung while it was maintained within the thoracic cavity. The lavage was reinfused into the lung two times before final collection. BAL cells were isolated by centrifugation at 500 g and resuspended in 500 µl of PBS, 100 µl of the cell suspension were mixed in 100 µl of 0.4% trypan blue (GIBCO BRL, Grand Island, NY), and the cells were counted with a hemocytometer. To determine inflammatory cell types in BAL, 5 × 104 cells were mounted on slides by cytospin centrifugation in 100 µl of PBS at 600 rpm for 3 min. Cell types were identified and counted by differential staining microscopy with Diff-Quik (Baxter Healthcare, Miami, FL). Inflammatory cell populations were determined by counting 100 cells, and a percentage was calculated based on five sample sets from three animals per group.
Cytokine analysis. Cytokine mRNA
abundance was determined by RT-PCR analysis of whole lung total RNA.
Briefly, whole lung total RNA was isolated by phenol-chloroform
extraction and precipitation with isopropanol with the Phase-Lock
protocol (5 Prime 3 Prime, Boulder, CO). Total RNA
quantitation was confirmed by gel electrophoresis. Total RNA was
converted to cDNA by the RT reaction (GIBCO BRL, Gaithersburg, MD). PCR
for cytokine cDNA was performed with the following primer tandems:
-actin primer 1,
5'-GTGGGCCGCTCTAGGCACCAA-3' and primer
2,
5'-CTCTTTGATGTCACGCACGATTTC-3'; IL-6
primer 1,
5'-TTGCCTTCTTGGGACTGATGCT-3' and
primer 2,
5'-GTATCTCTCTGAAGGACTCTGG-3'; TNF-
primer 1,
5'-CCAGACCTCACACTCAGAT-3' and primer
2, 5'-AACACCCATTCCCTTCACAG-3'; macrophage
inflammatory protein (MIP)-1
primer
1, 5'-ACTGCCCTTGCTGTTCTTCTCT-3' and
primer 2,
5'-AGGCATTCAGTTCCAGGTCAGT-3'; MIP-2 primer
1, 5'-ATGGCCCCTCCCACCTGC-3' and
primer 2,
5'-TCAGTTAGCCTTGCCTTTGTT-3'; and monocyte chemoattractant protein (MCP)-1 primer 1,
5'-ATGCAGGTCCCTGTCATGCTT-3' and primer 2, 5'-CTAGTTCACTGTCACACTGGT-3'. PCR with
the OptiPrime reagents (Stratagene, La Jolla, CA) was performed for 25 cycles on a Perkin-Elmer 2400 Gene Amp System thermal cycler by the
following parameters: initiation at 94°C for 30 s, annealing
temperature at 59°C for 30 s, and elongation temperature of
72°C for 30 s. Ethidium bromide staining of 2% agarose gel
electrophoresis was used to visualize PCR products.
Cytokine concentrations were assessed in lung homogenates by ELISA according to the manufacturer's recommendations (Endogen, Woburn, MA). Standard curves were calculated for known standards to verify linearity of analysis and for calculation of cytokine concentration.
Adenoviral-mediated transgene expression. To detect adenoviral-mediated luciferase activity, the right upper lobe was removed and immediately homogenized in 800 µl of lysis buffer as previously described (22). The luciferase reaction was initiated by injection of 100 µl of 1 mM luciferin, and data were collected for 10 s at 25°C with a Monolight 2010 luminometer (Analytical Luminescence Laboratories, San Diego, CA). Light units were normalized to total lung homogenate protein as measured by the Lowry assay. Luciferase activity in the mouse lung is expressed as relative light units per microgram of lung protein.
Pulmonary histopathology. Histopathological changes were evaluated at 7 and 14 days after Av1Luc1 administration in the inflation-fixed mouse lung. Inflation-fixed lungs were washed in PBS three times and divided in half for preparation of paraffin embedding. Paraffin-embedded lungs were sectioned at 5 µm and stained with hematoxylin and eosin for morphological analysis. Pathological assessment of lung inflammation was graded blindly on a scale of 0-4, and a score was determined from the mean (±SE) of six animals.
Statistical analysis. Statistical
analysis for multiple groups was determined by ANOVA with StatWorks
computer software. All data are presented as means ± SE.
Differences were considered significant at
P 0.05.
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RESULTS |
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BAL cell counts are increased in CCSP(/
)
mice. To assess the role of CCSP in modulating lung
inflammatory cell infiltrates after adenoviral infection, adult
CCSP(
/
) mice (in 129J strain background) and 129J
wild-type mice (7-15 wk of age) were intratracheally injected with
a recombinant, replication-deficient (E1- and E3-deleted) adenoviral
vector, Av1Luc1. Inflammatory cells in BAL fluid were assessed at 4 and
24 h postinfection for cell counts in BAL fluid. Both wild-type (129J)
and CCSP(
/
) mice tolerated
109 plaque-forming units of
adenovirus and were responsive and active within 1 h of the surgical
procedure. No mortality was observed after administration of the virus
to either group of mice. At 4 h after adenoviral infection, cell counts
in BAL fluid from control 129J mice were not different from cell counts
in uninfected mice (Fig.
1A).
Four hours after Av1Luc1 administration, cell counts in BAL fluid from
CCSP(
/
) mice were increased threefold compared with those
in wild-type mice. Twenty-four hours after adenoviral infection, cell
counts in BAL fluid from wild-type mice were increased compared with
cell counts in BAL fluid from uninfected wild-type mice. Cell counts in
BAL fluid from infected CCSP(
/
) mice at 24 h were
increased 3.5-fold compared with those in infected wild-type mice. Cell
counts in BAL fluid from uninfected mice did not vary in either group.
The number of cells obtained from BAL in CCSP(
/
) and
wild-type mice were similar before infection.
|
To determine whether individual inflammatory cell populations were
altered in BAL fluid from CCSP(/
) mice, BAL fluid was obtained from adenoviral-infected wild-type and CCSP(
/
)
mice and examined after cytospin centrifugation and differential cell staining. Four hours after adenoviral infection, inflammatory cells in
BAL fluid from wild-type mice were predominantly macrophages and were
not different from those in BAL fluid from uninfected mice (Fig.
1B, Table
1). Four hours after adenoviral infection of CCSP(
/
) mice, BAL fluid contained primarily
macrophages, with the notable appearance of neutrophils. Twenty-four
hours after adenoviral infection, numerous macrophages and increased numbers of neutrophils were noted in wild-type mice. Increased macrophages and neutrophils were detected in BAL fluid from
CCSP(
/
) mice 24 h after adenoviral infection compared
with those from wild-type control mice.
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Increased cytokine and chemokine responses after
adenoviral infection in CCSP(/
) mice.
Cytokine and chemokine mRNAs were assessed from lung homogenates of
CCSP(
/
) and control mice 4 and 24 h after adenoviral
infection. The proinflammatory cytokine TNF-
and IL-6 as well as the
neutrophilic chemokine MIP-2 and MIP-1
mRNAs were unchanged 4 h
after infection in either wild-type or CCSP(
/
) mice (Fig.
2). After 4 h, the monocytic chemokine MCP-1 was increased in the lungs of CCSP(
/
) mice but was
not detected in wild-type mice. At 24 h after adenoviral
administration, proinflammatory cytokine TNF-
and IL-6 mRNAs as well
as chemokine MIP-2 and MIP-1
mRNAs were increased in the lungs of
CCSP(
/
) mice compared with wild-type mice. MCP-1 mRNA was
also increased in the lungs of CCSP(
/
) mice at 24 h after
infection compared with wild-type mice.
|
Concentrations of the proinflammatory cytokines TNF-, IL-6, and
IL-1
were measured in lung homogenates from CCSP(
/
)
and wild-type mice by ELISA. Four hours after infection, IL-6, IL-1
, and TNF-
were unchanged in either wild-type or CCSP(
/
)
mice (data not shown). However, 24 h after infection, the concentration of IL-6 was increased in wild-type mice compared with uninfected control mice (Fig. 3). In CCSP(
/
) mice 24 h after infection, the concentration of IL-6 was increased threefold
compared with that in wild-type mice. Concentrations of IL-1
and
TNF-
were also increased to a greater extent in
CCSP(
/
) mice compared with wild-type mice 24 h after
adenoviral infection.
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Adenoviral gene expression is reduced in the lungs of
CCSP(/
) mice. The adenoviral vector
Av1Luc1 used in these studies encodes the luciferase reporter gene
under the control of the Rous sarcoma virus promoter region. To
determine whether viral gene expression is altered in the lungs of
CCSP(
/
) mice after adenoviral infection, luciferase
activity was measured in lung homogenates of control and
CCSP(
/
) mice 7 and 14 days after adenoviral infection.
Luciferase activity in control mice was higher 7 days after adenoviral
infection and was decreased 14 days after infection. Luciferase
activity in the lungs of CCSP(
/
) mice was significantly
decreased compared with that in the lungs of wild-type mice 7 and 14 days after adenoviral administration (Fig.
4). Luciferase activity in the
lungs of CCSP(
/
) mice was higher at 7 days after
infection and decreased at 14 days after adenoviral administration.
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Increased lung inflammation in CCSP(/
)
mice after adenoviral infection. To determine whether
adenoviral-mediated lung inflammation was influenced by CCSP, lung
histology was assessed after administration of the adenovirus.
Pulmonary infiltrates were observed in the lungs of all mice receiving
adenovirus at 7 and 14 days after infection. In wild-type mice 7 days
after infection, lung inflammation consisted of focal alveolar
infiltrates composed primarily of mononuclear cells, with occasional
neutrophils (Fig. 5). In CCSP(
/
) mice 7 days after infection, alveolar inflammation consisted of large areas of
consolidation. Lung inflammation in CCSP(
/
) mice was more
extensive and involved more regions of the lung. Lung inflammation was
increased 14 days after infection in both CCSP(
/
) and
wild-type mice. Severe lung inflammation persisted in
CCSP(
/
) mice and included alveolar septal thickening,
with extensive regions of consolidation noted in the lung parenchyma.
Lung inflammation in the CCSP(
/
) mice was increased
compared with that in wild-type mice 7 and 14 days after administration
of the virus.
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DISCUSSION |
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The present study demonstrates increased inflammatory responses in CCSP
gene-targeted mice after intratracheal administration of Av1Luc1, an
E1- and E3-deleted recombinant adenoviral vector. Inflammatory cells in
BAL fluid were increased in CCSP(/
) mice, and neutrophils
appeared earlier in the course of infection. Expression of the
proinflammatory cytokines IL-6, IL-1
, and TNF-
as well as the
neutrophilic chemokines MIP-1
and MIP-2 and the monocytic chemokine
MCP-1 were increased in the lungs of CCSP(
/
) mice after
infection. Lung inflammation was increased and luciferase activity, as
a measure of viral gene expression, was decreased in the lungs of
CCSP(
/
) mice. These results indicate that CCSP deficiency
exacerbates the early host responses and inflammation to adenoviral
infection in the lung.
In a previous study (2) using the mouse model of adenoviral pneumonia,
lung infiltrates appeared in the lung parenchyma 2 days after infection
and were monocytic or neutrophilic in appearance. In the present study,
total inflammatory cells in BAL fluid and neutrophilic infiltration
increased earlier in CCSP(/
) mice than in wild-type mice
and were associated with earlier expression of cytokines and chemokines
in the CCSP(
/
) mice. The induction of inflammatory
responses and cytokine production after adenoviral exposure in
wild-type mice is consistent with previous findings regarding host
inflammatory responses after adenoviral infection (2). The findings in
the present study that inflammatory cell influx and proinflammatory
cytokines and chemokines are increased in the lungs of
CCSP(
/
) mice are consistent with the concept that CCSP
plays a role in limiting alveolar influx of macrophages and neutrophils
and cytokine responses early during the course of adenoviral
infection.
Adenoviral infection is associated with acute and chronic cytopathic
effects that disrupt the respiratory epithelium (2, 22). After
adenoviral vector administration in mice, respiratory epithelial cell
proliferation is increased in both normal and immunodeficient mice,
suggesting that adenoviral infection per se has cytopathic effects
independent of viral clearance by immune cells (22). Surfactant protein
homeostasis was also disrupted in both immunocompetent and
immunodeficient mice after adenoviral vector infection (21). In the
present study, the increase in lung inflammation observed in
CCSP(/
) mice may reflect a possible role for CCSP in
cytoprotection of the lung epithelium after injury. In support of this
concept, CCSP(
/
) mice succumbed to lung injury earlier
than wild-type mice during oxygen exposure (5).
In the present study, monocytic and neutrophilic infiltration was more
extensive in the lung parenchyma of CCSP(/
) mice after
adenoviral infection. The early enhancement of cytokine expression in
CCSP(
/
) mice likely contributes to the increase in lung
inflammation seen later in the course of infection. The proinflammatory
cytokines IL-6 and TNF-
initiate both acute and chronic inflammatory
events, including the activation of adhesion molecules and chemokines
(10). TNF-
also exerts important cytopathic effects on virally
infected cells (8, 9). Increased expression of the chemokines MIP-1
,
MIP-2, and MCP-1 in adenoviral-infected CCSP(
/
) mice may
contribute to increased lung inflammation by induction of inflammatory
cell chemotaxis. The findings in the present study suggest that CCSP
deficiency increases the expression of important inflammatory mediators
after adenoviral infection and may influence later lung inflammatory
events.
Host immune responses to adenoviral vectors limit the efficiency and
duration of viral gene expression. Adenoviral vector administration to
immunodeficient mouse models (1, 18, 19, 22) or the use of
immunomodulatory therapies (3, 22) limits host immune responses and
extends the duration and level of adenoviral-mediated gene expression.
The loss of adenoviral transgene expression has been attributed, at
least in part, to the rapid loss of viral DNA in infected cells (22). A
number of mechanisms may explain the loss of adenoviral DNA, including
the cytopathic effects of virus infection, the uptake and depletion of
viral particles by macrophages and other inflammatory cells, and T
cell-mediated cytolytic killing during the later phase (7-14 days)
of infection. In the present study, adenoviral gene expression in
CCSP(/
) mice was decreased and associated with increased
pulmonary inflammation. Thus the present study is consistent with
previous findings that host immune and inflammatory responses to
adenoviral infection limit adenoviral vector gene expression in vivo.
Although the function of CCSP has not been clearly defined, there is
increasing evidence that CCSP plays an important role in the modulation
of various inflammatory responses (4, 5, 7, 11, 20). In a hyperoxic
lung injury model, survival of CCSP(/
) mice was reduced
compared with control mice (5). Likewise, the onset of lung edema
occurred earlier in CCSP(
/
) mice. Expression of the
proinflammatory cytokines IL-3, IL-6, and IL-1
was increased in the
lungs of CCSP(
/
) mice, and in the case of IL-1
,
increased expression was localized to the lung parenchyma (5). Lung
inflammation and injury in hyperoxic CCSP(
/
) mice were
not limited to the bronchiolar epithelium but also involved the
alveolar epithelium, suggesting that CCSP plays a role in limiting lung
injury and inflammation in both the alveolar and bronchiolar regions of
the lung. In the present study, lung inflammation in
CCSP(
/
) mice was increased markedly in the lung
parenchyma compared with that in wild-type mice. Whether CCSP produced
by conducting airway cells traffics to alveolar regions of the lung is
not known at present. It is also possible that CCSP itself modulates
inflammation in the lung parenchyma by events mediated by its action in
the conducting airways.
The findings in the present study suggest that the lack of CCSP
increases the host response to viral infection in the lung. Alternately, altered Clara cell function may also contribute to the
observed increase in lung inflammatory responses in
CCSP(/
) mice. Ultrastructural analysis demonstrated that
secretory granules in Clara cells of CCSP(
/
) mice were
abnormal or absent (14). Thus it is possible that altered inflammatory
responses to adenoviral infection in the present study resulted from
disrupted Clara cell function rather than the lack of CCSP. Clara cells
secrete a number of host defense molecules, including SP-A and SP-D
(13). Both SP-A and SP-D are likely important in host defense after
lung infection (17). Thus the production and secretion of important immunomodulatory factors by the lung epithelium may play an important role in lung injury after infection.
Despite the relative abundance of CCSP in the BAL fluid, the
physiological function of CCSP is not understood. CCSP (also termed
CC10 and UG) has been shown in vitro to act as an immunosuppressant mediated, in part, by its ability to inhibit secretory phospholipases A2. Recently, another
gene-targeted mouse model of CCSP called the UG gene-targeted
[UG(/
)] mouse model was described (20). UG(
/
) mice spontaneously developed severe renal fibrosis,
with extensive deposition of fibronectin and collagen. As in the
present study involving the CCSP(
/
) mice, no lung
pathology was reported in the UG(
/
) mouse model. The
CCSP(
/
) mice used in the present study have no apparent
renal pathology. The discrepancy in phenotype between the two
CCSP(
/
) mice may be related to differences in vivarium
conditions or genetic strains.
The present findings support the concept that CCSP functions to
modulate the host responses during viral-induced lung inflammation. CCSP deficiency exacerbates early inflammatory responses to viral infection, suggesting that CCSP plays a role in innate immunity to
infectious agents. Cytokines and chemokines likely play a role in the
transition of early nonspecific immune responses to more specific
adaptive immune mechanisms. In the present study, the enhanced
induction of cytokines and chemokines in the CCSP(/
) mice
after adenoviral infection may explain the increased lung inflammation
later during the course of infection and supports the concept that
secretory products of lung epithelial cells are important modulators of
lung inflammation and injury. The precise molecular mechanisms by which
CCSP limits lung inflammation in vivo remain to be discerned. However,
the present findings support the potential utility of CCSP as a
therapeutic strategy to influence inflammation after lung injury and
infection.
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ACKNOWLEDGEMENTS |
---|
We thank Shilpa Jain-Vora and Michael Jones for preparation of the cytokine primers, Nannette Mittereder and Bruce Trapnell for preparation of the adenoviral vector, and Anne Marie Levine and Thomas Korfhagen for thoughtful discussion.
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FOOTNOTES |
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This work was supported by the Cystic Fibrosis Foundation; National Heart, Lung, and Blood Institute Grants HL-41496 (to J. A. Whitsett) and HL-51376 (to B. R. Stripp); and the Parker B. Francis Foundation (K. S. Harrod).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. A. Whitsett, Children's Hospital Medical Center, Div. of Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
Received 20 March 1998; accepted in final form 14 August 1998.
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