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INTRODUCTION |
The surfactant proteins compose a group of molecules with diverse
biological functions. Surfactant protein
(SP)1-A and SP-D are
calcium-dependent lectins that have primary roles in host
defense by serving as regulators for innate immune responses (1, 2).
SP-B and SP-C are primarily involved in maintaining physiological
responses specifically in the lung by reducing lung surface tension
thereby promoting proper respiration. In support of the critical
function of surfactant proteins, a deficiency or dysregulation of
surfactant proteins can have profound effects. For example, surfactant
deficiency is responsible for the respiratory distress syndrome of
infancy, and surfactant replacement is an effective therapy for this
disorder. Furthermore, reduced levels of surfactant constituents are
found in the lungs of patients with adult respiratory distress
syndrome. Experimental disruption of SP-A, SP-B, or SP-D in mice
results in various lung-specific problems including impairment in
pulmonary host responses to infection (SP-A and SP-D) and neonatal
respiratory failure (SP-B) (3-6). Hence, normal pulmonary function
depends upon proper expression and function of surfactant proteins. The
pleiotropic and fundamental properties of surfactant proteins
underscore the importance of elucidating the role of surfactant
proteins in various lung disorders.
Asthma is an airway inflammatory disorder whose prevalence is on the
rise worldwide despite increased availability of therapeutic options
(7). An extensive amount of experimentation in humans and rodents using
models of allergen-induced airway inflammation has demonstrated an
essential role for Th2 lymphocytes, eosinophils, and various cytokines
(e.g. IL-4, IL-5, and IL-13) (8, 9), but the specific role
of surfactant proteins has not been extensively examined. Because
surfactant lipids and proteins are primary molecules involved in
maintaining airway patency, an impaired process in patients with
asthma, they are likely to be involved, at least in part, in the
pathophysiology of asthma. Consistent with this, segmental allergen
challenge in sensitized animals and humans results in loss of
surfactant activity (10-12). Furthermore, although no difference in
bronchoalveolar lavage fluid (BALF) phospholipid levels are seen
between patients with asthma and controls, following allergen
challenge, only patients with asthma have a change in the ratio between
phosphatidylcholine and phosphatidylglycerol, the major lipid
constituents of surfactant (13).
In the present investigation, we were interested in testing the
hypothesis that allergic airway inflammation regulates the expression
of hSP-C, a major gene product of alveolar type II epithelial cells. We
chose to utilize an established murine model of asthma and an
established transgenic system to facilitate direct analysis of human
SP-C expression. Specific transcription of the hSP-C gene in pulmonary
epithelial cells is regulated by the first 3.7 kb of the SP-C promoter
(14). This region confers all of the necessary elements to direct
specific expression of transgenes in the distal respiratory epithelium
and has been instrumental for experimental analysis of lung development
and disease (15). We therefore examined the effect of allergic airway
inflammation on the expression of transgenes under the control of the
hSP-C 3.7-kb promoter.
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MATERIALS AND METHODS |
Mice--
Specific pathogen-free FVB mice, 8-10 weeks old were
obtained from the NCI, Frederick, MD. Eotaxin or SP-B transgenic mice were generated by inserting the mouse eotaxin cDNA coding region (20-328 base pairs) or the coding region of the human SP-B cDNA (10-837 base pairs) (16) into the BamHI/EcoRI
site of the hSP-C 3.7 transgenic cassette (construct I and
II, respectively in Fig. 1). This transgenic construct
utilizes the simian virus 40 (SV40) polyadenylation signal that is used
as a probe for identifying transgenic mice by Southern blot analysis of
tail DNA. In other experiments, transgenic mice containing a
"mammalianized" SP-C 3.7 promoter followed by noncoding 5' exons
from the rabbit
-globin gene, the hSP-B cDNA, and the 3'
polyadenylation signal from bovine growth hormone were examined
(construct III in Fig. 1) (17). All mice (FVB/N) were housed
under pathogen-free conditions and examined between 6 and 16 weeks of age.
Allergen Treatment of Mice--
A mouse model of allergic lung
disease was established using methods described previously (18). In
brief, mice were lightly anesthetized with Metofane inhalation
(methoxyfluorane; Pittman-Moore, Mundelein, IL), and 100 µg (50 µl
of saline) of Aspergillus fumigatus (Bayer Pharmaceuticals,
Spokane, WA) or 50 µl of normal saline alone was applied to the nares
using a micropipette with the mouse held in the supine position. After
instillation, mice were held upright until alert. After three
treatments per week for 3 weeks, mice were sacrificed between 18 and
20 h after the last intranasal challenge. In some experiments,
mice were treated with neutralizing doses (1.0 mg) of anti-IL-5
(TRFK-5) or control IgG1 (GL113 (anti-Escherichia coli
-galactosidase); both kindly provided by Dr. Fred Finkelman, University of Cincinnati and Robert Coffman, DNAX, Palo Alto, CA) by
intraperitoneal injection each week prior to the 1st weekly allergen
challenge. In other experiments, a mouse model of allergic lung disease
was established using intranasal ovalbumin (OVA). In brief, mice were
sensitized by intraperitoneal injection with OVA (50 µg)/alum (1 mg)
in 0.9% sterile saline on day 0 and day 12. Mice were then challenged
intranasally with 50 µl containing 150 µg of OVA or saline on days
20, 21, 22, and 23 and then sacrificed 18-20 h after the last OVA challenge.
BALF Analysis--
Mice were euthanized by CO2
inhalation and exsanguinated, and the trachea was cannulated and
lavaged with 2 replicate volumes (0.8 ml) of normal saline containing
0.2 mM EDTA to obtain BALF. Lavage fluid was centrifuged at
250 × g for 10 min at 4 °C, and the cell pellet was
resuspended in PBS and analyzed for total cell count and differential
analysis. Aliquots of the BALF supernatant were taken for total protein
analysis by bicinchoninic acid analysis (Pierce).
Quantitation of Eotaxin in BALF--
A sandwich enzyme-linked
immunosorbent assay (ELISA) was performed to measure eotaxin protein
levels in BALF. The wells of ELISA plates (DYNEX Technologies,
Chantilly, VA) were first coated with 40 ng (100 µl) of diluted mouse
eotaxin-specific goat IgG (AF-420NA; R & D Systems, Minneapolis, MN) in
PBS and incubated overnight at 4 °C. Following the incubation, the
plate was washed with phosphate-buffered saline with 0.05% Tween 20 (PBST). Nonspecific binding sites were blocked with 300 µl of PBS
containing 1% bovine serum albumin, 5% sucrose, and 0.05%
NaN3 and incubated at room temperature for 1 h. Mouse
eotaxin (kindly provided by PeproTech, Rocky Hill, NJ) was used as a
quantitation standard. The eotaxin standard and BALF samples were
diluted in the blocking solution and added to each well and incubated
for 2 h at room temperature. Wells were washed three times with
PBST and then further incubated with 20 ng (100 µl) of mouse eotaxin
affinity-purified polyclonal secondary antibody (BAF-420, R & D
Systems) for 2 h at room temperature followed by three washes with
PBST. Fifty nanograms (100 µl) of streptavidin-horseradish peroxidase
(Immunotech, Marseilles, France) was added and incubated for 1 h
at room temperature. Finally, ABTS peroxidase substrate solution that
contains 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonate) at a
concentration of 0.3 g/liter in a glycine/citric acid buffer and 0.01%
H2O2 (Kirkegaard & Perry, Gaithersburg, MD) was
used to complete the reaction. The substrate reaction was stopped by adding 50 µl/well of 1 M H2SO4
and quantitated by an ELISA plate reader at 495 nm.
Northern Blot Analysis--
RNA was extracted from the lung
tissue using the Trizol reagent (Life Technologies, Inc.) following the
manufacturer's protocol. Twenty micrograms of total RNA from each
sample were electrophoresed on 1.5% formaldehyde agarose gels and
transferred to GeneScreen hybridization membrane (PerkinElmer Life
Sciences) with 10× SSC. The membrane was UV cross-linked and
prehybridized at 42 °C for 1 h in a 50% formamide buffer (pH
7.5), containing 10% dextran sulfate, 5× SSC, 1× Denhardt's
solution, 1% SDS, 100 µg/ml of herring sperm DNA, and 20 mM Tris. The 32P-labeled probes were prepared
for mouse eotaxin cDNA (19), SV40 polyadenylation signal sequence,
and mouse surfactant protein cDNAs (SP-A, SP-B, SP-C, and SP-D)
(kindly provided by Dr. Jeffrey Whitsett) as described previously (20,
21) and hybridized overnight at 42 °C using 1-2 × 106 dpm/ml of the respective probes (22, 23). The membranes
were washed for 20 min at 42 °C, 20 min at 50 °C, 20 min at
60 °C in 2× SSC, 0.1% SDS and 20 min in 0.1× SSC, 0.1% SDS.
Western Blot Analysis--
Surfactant proteins (SP-A, SP-B,
SP-C, and SP-D) in the BALF were examined by Western blot analysis.
Mice were lavaged with five 1.0-ml washes of PBS (pH 7.4 containing 1%
FBS and 0.2 mM EDTA). The recovered BALF (~4.6 ml) was
centrifuged at 18,000 × g for 20 min, and the pellet
was resuspended in 100 µl of distilled water. Total protein in each
samples was determined by bicinchoninic acid analysis (Pierce). For
SP-A, SP-B, and SP-D, 10 µg of total protein was electrophoresed in
4-20% Tris glycine SDS-polyacrylamide gels (NOVEX, San Digeo, CA). In
some experiments, an equal volume of BALF (100 µl) was reduced to 10 µl by centrifugation under negative pressure and subjected to
electrophoresis. Protein was transferred to polyvinylidene difluoride
membranes (Bio-Rad), and equal loading was verified by staining with
Ponceau S (Sigma). To reduce nonspecific binding, membranes were
blocked with 5% nonfat dry milk in Tris-buffered saline with 0.05%
Tween 20 (TBST). Immunoblotting was performed utilizing 1:5000 dilution
of guinea pig anti-rat SP-A, rabbit anti-bovine SP-B, or rabbit
anti-murine SP-D (kindly provided by Jeffrey Whitsett, University of
Cincinnati) (24-26). For SP-C detection, 10-20% Tricine gels (NOVEX)
were employed using an upper buffer composed of 0.1 M
Tris-Tricine with 0.1% SDS and a lower buffer composed of 0.2 M Tris, pH 8.9. Rabbit anti-recombinant human SP-C serum
(kindly provided by Jeffrey Whitsett) was diluted 1:25,000 in TBST and
incubated with the membranes overnight at 4 °C. Horseradish
peroxidase-conjugated secondary antibody (Calbiochem) was diluted
1:5000 in TBST. Surfactant protein standards (5 ng) (kindly provided by
Jeffrey Whitsett) were also electrophoresed as positive controls. The
signal was developed using enhanced chemiluminescence (Amersham
Pharmacia Biotech) according to manufacturer's instructions.
Statistical Analysis--
Data are expressed as mean ± S.E. Statistical significance comparing different sets of mice was
determined by Student's t test.
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RESULTS |
Generation of hSP-C Eotaxin Transgenic Mice--
The 3.7-kb
5'-flanking region of the hSP-C gene contains all of the necessary
elements to promote specific expression of transgenes into pulmonary
type II cells (27). We initially generated transgenic mice that
utilized the hSP-C promoter to direct expression of eotaxin, an
eosinophil-selective chemokine (28, 29), in type II cells of mice to
examine the consequences of eotaxin overexpression in the lungs (see
transgenic construct I in Fig.
1). Three different eotaxin transgenic
founder lines were established that had variable but increased
expression of eotaxin lung mRNA (Fig.
2A) and protein in the BALF
(Fig. 2B). For example, wild-type mice had 345 ± 205 pg/ml eotaxin in the BALF, and the transgenic lines had increased levels ranging from 687 ± 61 to 3587 ± 223 pg/ml. The
specificity of the ELISA for eotaxin was determined by demonstrating
undetectable eotaxin in mice genetically deficient in eotaxin (Fig.
2B). Interestingly, the base-line level of eosinophils in
the BALF and lungs was not different between wild-type and eotaxin
transgenic mice (data not shown). This prompted investigation of the
effect of the eotaxin transgene during allergic airway inflammation,
since it was hypothesized that eotaxin would cooperate with other
signals generated during allergic airway inflammation (e.g.
IL-5) to promote eosinophil recruitment into the lung.

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Fig. 1.
SP-C promoter transgenic constructs. The
hSP-C promoter containing transgenic constructs employed in this study
are schematically diagrammed. The hSP-C promoter is located 5' of all
constructs and is illustrated in the shaded rectangles. The
inserted cDNAs are murine eotaxin (designated mEotaxin)
in construct I and hSP-B in constructs II and III, respectively. The
polyadenylation signal (abbreviated polyadenyl) is derived
from the SV40 small t intron and exon (designated I/E)
(constructs I and II) or from the bovine growth
hormone exon (construct III). In construct III, noncoding
exons 2 and 3 from the rabbit -globin gene (designated
r -Glob E2/3) have been inserted between the hSP-C
promoter and the hSP-B cDNA. The relative proportions of the DNA
components are not drawn to scale.
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Fig. 2.
Transgene expression in hSP-C-driven eotaxin
transgenic mice. A, the level of transgene mRNA was
determined by Northern blot analysis of total RNA isolated from
wild-type (WT) and three different eotaxin transgenes lines,
designated ET 1.2, ET 1.5, and ET 2.4. The expression of transgene was
determined by hybridization to the transgene-specific probe from the
SV40 polyadenylation sequence. The total amount of RNA was visualized
by ethidium bromide (EtBr) staining of the gel. Each lane
represents a separate mouse. B, the level of eotaxin protein
in the BALF was determined by ELISA. The specificity of the ELISA was
determined by analysis of BALF from eotaxin-deficient mice
(Eotaxin / ). The results of this experiment
are expressed as mean ± S.E., n = 7-8
mice.
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Aspergillus-induced Airway Inflammation in Eotaxin Transgenic
Mice--
Allergic airway inflammation (asthma) can be triggered in
mice by repeated exposure of the airways to antigens. For example, exposure of naive mice to A. fumigatus antigens 3 doses/week
for 3 weeks results in marked inflammatory responses. We therefore subjected eotaxin transgenic mice to Aspergillus-induced
allergic airway inflammation to determine if the eotaxin transgene
would enhance eosinophil recruitment. In wild-type mice, 18 h
after the last allergen exposure, there was ~100-fold increase in the total BALF cells with the largest increase being in the eosinophil lineage (4 ± 2 × 103 to 1.9 ± 0.16 × 106) followed by the neutrophil lineage (1.3 ± 1.2 × 104 to 5.6 ± 2.4 × 105). We next examined the effect of the airway challenge
on the number of eosinophils in the eotaxin transgenic mice.
Surprisingly, we did not observe a significant difference in eosinophil
levels between wild-type and eotaxin transgenic mice (Fig.
3). Although a highly significant
increase in eosinophil numbers was observed in both groups when
compared with their respective saline control mice, the number of
eosinophils in the allergen-challenged wild-type and eotaxin transgenic
mice was comparable (1.39 ± 1.06 × 106 and
3.2 ± 1.5 × 106, respectively). Similarly,
levels of eosinophils in the lung tissue did not differ between
wild-type and transgenic mice (data not shown).

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Fig. 3.
Eosinophil levels in the BALF following
allergen or saline challenge. Wild-type and hSP-C
eotaxin-transgenic mice were challenged with saline or A. fumigatus allergen, and the level of eosinophils in the BALF was
determined. Each circle represents a separate animal. The
horizontal line is the mean.
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Allergen Exposure Induces Down-regulation of the hSP-C
Transgene--
Since we expected the eotaxin transgene to cooperate
with allergen-induced signals in the lung thereby promoting increased eosinophil levels, we hypothesized that allergen challenge might be
inhibiting the expression of the transgene. We therefore examined the
effect of airway inflammation on the transgene expression. Interestingly, whereas eotaxin transgenic mice had readily detectable transgene expression following saline treatment, the transgene mRNA
was barely detectable following allergen challenge (Fig. 4A). The decreased expression
of the transgene was seen in all hSP-C eotaxin transgenic lines (data
not shown). We also subjected sensitized mice to allergen challenge
with OVA to determine if the allergen-induced SP-C down-regulation
was applicable to other allergens. Similar to the effects of
challenge with A. fumigatus, OVA exposure also
down-regulated the level of the eotaxin transgene (Fig.
4B).

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Fig. 4.
Effect of allergen challenge on the hSP-C
transgene mRNA level. A, the level of hSP-C-driven
eotaxin transgene mRNA was determined by Northern blot analysis of
total RNA isolated from mice challenged with saline or A. fumigatus allergen. The expression of the transgene was determined
by hybridization to transgene-specific probe from the SV40
polyadenylation sequence. B, hSP-C-driven eotaxin transgenic
mice were subjected to allergen challenge with OVA or control saline.
The total amount of RNA was visualized with ethidium bromide
(EtBr) staining of the gel. Each lane represents a separate
mouse. Quantitative analysis of the relative mRNA signal was
performed by comparison of the intensity of the transgene mRNA to
the 28 S ribosomal protein RNA signal and revealed 7.2 ± 1.9- and 7.3 ± 2.3 (mean ± S.D.)-fold decreases following
allergen challenge in A and B,
respectively.
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Effect of Allergic Airway Inflammation on the Expression of Other
Related Genes--
We were next interested in determining if allergic
airway inflammation was a nonspecific phenomenon associated with the
down-regulation of other epithelial genes. We therefore examined the
expression of the endogenous murine surfactant proteins and eotaxin. Of
note, the mRNA for SP-D, and to a lesser extent SP-A, was increased following allergen challenge compared with saline challenge (Fig. 5A). Additionally, the protein
level of SP-D, and to a lesser extent SP-A, was also increased.
Interestingly, whereas the endogenous SP-C mRNA was not altered at
this time point, the level of the SP-C protein was substantially
reduced (Fig. 5B). We also examined the expression of the
endogenous eotaxin mRNA. As expected, allergen challenge markedly
increased the level of eotaxin in the lung. Taken together, these
results establish that the down-regulation of the hSP-C transgene by
allergen was a specific event, not likely related to generalized
epithelial cell toxicity.

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Fig. 5.
The effect of allergen challenge on the
expression of endogenous eotaxin and surfactant proteins.
A, the expression of the mRNA for the endogenous SP-A,
-B, -C, and -D, as well as eotaxin, was determined in wild-type mice
subjected to control saline or A. fumigatus allergen
exposure. The level of the mRNA was determined by Northern blot
analysis using specific cDNA probes. The total amount of RNA was
visualized by ethidium bromide (EtBr) staining of the gel.
Quantitative analysis of the relative mRNA signal was performed by
comparison of the intensity of the transgene mRNA to the 28 S
ribosomal protein RNA signal and revealed 1.68 ± 0.08-, 1.05 ± 0.07-, 1.03 ± 0.05-, and 7.23 ± 0.38 (mean ± S.D.)-fold increases for SP-A, SP-B, SP-C, and SP-D, respectively.
B, the surfactant protein levels present in 10 µg of total
protein in the BALF from saline and A. fumigatus
allergen-treated wild-type mice were analyzed by Western blot analysis.
When normalization was performed with an equal volume of BALF, the SP-C
protein level remained reduced following allergen challenge (data not
shown). Each lane represents a separate mouse.
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Allergen-induced Down-regulation of the hSP-C Transgene Is
Independent of Eotaxin--
We were next interested in ruling out the
possibility that the mechanism for the down-regulation of the transgene
by allergen was dependent upon the presence of the eotaxin cDNA in
the genetic construct. Although unlikely, it remained possible that the
inhibition of the transgene was dependent upon sequences in the eotaxin
cDNA or that the overexpressed eotaxin protein was inhibiting the
transgene. To test these possibilities, we examined the effect of
allergen challenge on an independent hSP-C transgenic mouse line. We
examined transgenic mice that contained the hSP-B cDNA under the
control of the same 3.7-kb hSP-C promoter used in the eotaxin
transgenic mice (construct II in Fig. 1) (16).
Interestingly, down-regulation of the hSP-C-SP-B transgene in response
to allergen was observed in all mice when compared with their
respective saline-treated controls (Fig.
6).

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Fig. 6.
The effect of allergen challenge on the
expression of hSP-C transgenes containing the SP-B reporter
cDNA. The level of the hSP-C driven-SP-B transgene mRNA
was determined by Northern blot analysis of total RNA isolated from
mice challenged with saline or A. fumigatus allergen. The
expression of transgene was determined by hybridization to the
transgene-specific probe from the SV40 polyadenylation sequence. The
total amount of RNA was visualized by ethidium bromide
(EtBr) staining of the gel. Each lane represents a separate
mouse. Quantitative analysis of the relative mRNA signal was
performed by comparison of the intensity of the transgene mRNA to
the 28 S ribosomal protein RNA signal and revealed 11.0 ± 2.7 (mean ± S.D.)-fold decrease following allergen challenge.
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Allergen-induced Down-regulation of the hSP-C Transgene Is
Independent of the 3'-Untranslated Region (UTR) of the
Construct--
It remained possible that the ability of allergen
exposure to decrease the level of transgenes regulated by the hSP-C
promoter was dependent upon the 3'-UTR of the transgenic construct,
since 3'-UTR have been shown to be involved in mRNA stability (30). Since the hSP-C transgenic construct contained a viral polyadenylation signal (from SV40) (14), we were interested in examining the effect of
allergen challenge in hSP-C transgenic mice that utilized the same
3.7-kb hSP-C promoter but a different 3'-UTR. To test this, we employed
transgenic mice that contained a mammalian-derived 3'-UTR composed of
the bovine growth hormone polyadenylation sequence (construct
III in Fig. 1) (17). Interestingly, we observed down-regulation of
the hSP-C-transgene in these mice in response to allergen when compared
with saline-treated controls (Fig. 7).
Taken together, these results demonstrate that allergen-induced
inflammation down-regulates the hSP-C transgene by directly affecting
the hSP-C promoter.

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Fig. 7.
The effect of allergen challenge on the
expression of the hSP-C transgene containing the bovine growth hormone
3'-UTR. The level of hSP-C driven-SP-B transgene mRNA was
determined by Northern blot analysis of total RNA isolated from mice
challenged with saline or A. fumigatus allergen. The
expression of the transgene was determined by hybridization to the
transgene-specific cDNA probe from the bovine growth hormone
polyadenylation sequence. The total amount of RNA was visualized with
ethidium bromide (EtBr) staining of the gel. Each lane
represents a separate mouse. Quantitative analysis of the relative
mRNA signal was performed by comparison of the intensity of the
transgene mRNA to the 28 S ribosomal protein RNA signal and
revealed 12.0 ± 2.1 (mean ± S.D.)-fold decrease following
allergen challenge.
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Allergen-induced Down-regulation of the hSP-C Transgene Is Blocked
by Anti-IL-5--
We hypothesized that the influx of inflammatory
cells during allergic airway inflammation was involved in
down-regulating the SP-C transgene. Since eosinophils are the
predominant inflammatory cell in A. fumigatus-induced
allergic airway inflammation, we were interested in determining if
these cells could directly down-regulate the SP-C transgene. To test
this hypothesis, we treated mice with neutralizing doses of anti-IL-5,
a well accepted approach for attenuating allergen-induced eosinophil
recruitment to the lungs (31). For example, eosinophils were 1.8 ± 0.4 × 106 and 1.0 ± 0.3 × 104 in the BALF of allergen-challenged mice following
nonimmune control and anti-IL-5 serum, respectively. Interestingly,
mice treated with anti-IL-5 (but not isotype matched control antiserum)
were resistant to allergen-induced SP-C inhibition. Northern blot
analysis of lung RNA revealed readily detectable transgene mRNA
levels in mice treated with saline and in allergen-challenged mice
pretreated with anti-IL-5 (Fig.
8A). In contrast,
allergen-challenged mice treated with control antiserum had a markedly
reduced level of transgene mRNA. Quantitative analysis of the
transgene mRNA level, performed by normalization of the mRNA
signal to the 28 S ribosomal protein mRNA, revealed no significant
inhibition of transgene mRNA in mice treated with anti-IL-5 (Fig.
8B). These data suggest that the allergen-induced
eosinophilic inflammation was responsible for the inhibition of the
hSP-C promoter.

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Fig. 8.
The effect of anti-IL-5 on allergen-induced
inhibition of the hSP-C eotaxin transgene. A, mice were
subjected to treatment with saline ( ) or allergen (+) (A. fumigatus), and the level of the hSP-C eotaxin transgene was
determined. Allergen-challenged mice were pretreated with anti-IL-5 or
isotype-matched control antiserum. The level of the mRNA was
determined by Northern blot analysis using a probe encoding for the
SV40 polyadenylation sequence. Each lane represents a separate mouse.
B, quantitative analysis (by PhosphorImager computation) of
the relative mRNA signal from the bands in A was
performed by comparison of the intensity of the transgene mRNA to
the 28 S ribosomal protein mRNA signals. The results of this
experiment are expressed as mean ± S.E.
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 |
DISCUSSION |
Surfactant proteins have been recognized as essential molecules
involved in two main critical processes in the lung, maintenance of
alveolar surfactant function and host defense. As such, they have the
potential to be critically involved in the pathogenesis of asthma, an
allergic inflammatory disorder of the lungs characterized by airway
obstruction. To examine this process, we have subjected mice to
experimental models of asthma (antigen-induced allergic airway
inflammation) and examined the effect on transgenes regulated by the
hSP-C promoter, as well as on the level of endogenous surfactant proteins. We have found the following: 1) allergen challenge inhibits the human SP-C promoter; 2) that allergen-induced inhibition of the
hSP-C promoter is dependent on IL-5, suggesting a critical role for
eosinophilic inflammation in this process; and 3) that allergen
challenge increases the surfactant proteins known to be involved in
innate immune responses (SP-A and SP-D). Previous studies on the
expression of surfactant proteins in patients with asthma have been
limited to analysis of surfactant activity in BALF rather than
surfactant protein mRNA levels, since surfactant is synthesized by
small airway and alveolar cells that reside in regions that are
difficult to sample experimentally in humans. Several recent clinical
studies have shown that the BALF of patients with asthma at base line
or following segmental allergen challenge contains dysfunctional
surfactant (10, 11). Additionally, experimental dissection in a guinea
pig model of asthma has revealed that allergen challenge decreases the
surfactant activity in the BALF (32). Additionally, significant changes
in surfactant levels have been reported in patients with interstitial
pulmonary fibrosis and adult respiratory distress syndrome (1).
In the present study, we have analyzed the effect of experimental
allergen challenge on the expression of the hSP-C promoter. We have
chosen to focus primarily on transgenic mice expressing reporter genes
under the control of the hSP-C 3.7-kb promoter. Although this region
may have distinct regulatory elements compared with the mouse or human
SP-C promoter, it is sufficient for directing specific gene expression
in type II pneumocytes, the cells that normally synthesize SP-C. This
promoter construct has been used to generate over 100 different
transgenic mouse lines for extensive analysis of lung development and
function (15, 27). Our initial results were limited to hSP-C transgenic
mice overexpressing murine eotaxin cDNA and demonstrated that
allergen challenge markedly inhibited the expression of this transgene.
Experimental dissection of hSP-C transgenic mice that were generated
with distinct reporter cDNAs (e.g. SP-B instead of
eotaxin) and 3'-UTRs (e.g. bovine growth hormone instead of
the SV40 polyadenylation signal) revealed that allergen challenge was
affecting the hSP-C promoter, rather than other elements present in the
transgenic construct. Most experimental allergen challenges were
conducted utilizing A. fumigatus as an antigen. It should be
noted that this antigen was selected because it represents a model
antigen that has already been extensively analyzed (33-35). To prove
that the results were not strictly related to this allergen, an
unrelated antigen (OVA) was used to demonstrate that the effect was not
allergen-specific. Additionally, airway inflammation itself, and not
allergen challenge, was demonstrated to be responsible for inhibition
of the hSP-C promoter. In particular, depletion of allergen-induced
airway eosinophilia with antiserum against IL-5 resulted in abolishment
of the inhibitory effect of allergen challenge. These data suggested
that eosinophil influx into the lung was responsible for inhibition of
the hSP-C promoter. Eosinophils are known to be a rich source of
pleiotropic molecules including lipid mediators (e.g.
leukotrienes and platelet-activating factor), cationic granule proteins
(e.g. major basic protein), and cytokines (e.g.
IL-1 and transforming growth factor-
/
) (36-38). Furthermore,
pulmonary epithelial cells express receptors for several of these
products including tumor necrosis factor-
and transforming growth
factor-
/
. Interestingly, transcription of the SP-C promoter is
inhibited by certain proinflammatory triggers such as tumor necrosis
factor-
(39). In the mouse SP-C gene, the tumor necrosis
factor-
-sensitive elements are located 320 base pairs 5' from the
start of transcription (39). However, it should be pointed out that
although the 3.7-kb hSP-C promoter has been extensively characterized
and appears to function similar to the endogenous promoter, it remains
possible that promoter elements not included in the 3.7-kb region may
be differentially affected by allergen challenge. This concern may
explain the finding that the endogenous mouse SPC mRNA is not
substantially reduced by allergen challenge. However, since the SPC
protein level is reduced by allergen challenge, we favor the view that
the mouse SPC mRNA is down-regulated at earlier points in the
allergen challenge regime. Alternatively, the hSPC promoter may be
distinctly inhibited by allergic airway inflammation compared with the
mouse SPC promoter. Taken together, our data suggest that allergen
exposure induces eosinophilic lung inflammation which inhibits the
transcription of the hSP-C promoter.
In contrast to the inhibition of the hSP-C promoter by allergen
challenge, we have found that allergic lung inflammation increases the
levels of SP-A and SP-D (but not SP-B). These proteins are known to be
critically involved in innate immune responses. Interestingly, the
binding of SP-A and SP-D to A. fumigatus enhances the
phagocytosis of this organism by human neutrophils and alveolar
macrophages (40, 41). Furthermore, these proteins can inhibit specific IgE binding to A. fumigatus allergens and block
allergen-induced histamine release from human basophils (42). Thus, the
ability of allergen challenge to increase the level of the surfactant proteins involved in immune responses indicates that these proteins are
indeed likely to be involved in the host response to allergens.
Finally, our results have several biological conclusions concerning
eosinophil recruitment to the lung. We demonstrate that overexpression
of eotaxin in the lung is not sufficient for eosinophil recruitment to
this organ. This is consistent with the finding that eotaxin is
constitutively produced by epithelial cells in the murine lung but not
associated with substantial levels of pulmonary eosinophils (43). In
contrast, in other organs such as the small intestine, eotaxin
expression is critical for base-line eosinophil tissue homing (23). We
also demonstrate that overexpression of eotaxin is not associated with
eosinophil desensitization, as has been reported with other chemokine
transgenic mice (44). In particular, we found no significant difference
in the level of allergen-induced eosinophil recruitment into the lung
of transgenic mice compared with wild-type mice (Fig. 3).
In conclusion, these findings indicate that attention should be focused
on the level of expression of transgenes regulated by the hSP-C
promoter when hSP-C transgenic mice are subjected to inflammatory
triggers. Additionally, these results suggest that surfactant protein
dysfunction in patients with asthma is likely to be caused, at least in
part, by allergen-induced and IL-5-dependent decreases in
the level of SP-C.