Role of the type 1 TNF receptor in lung inflammation after
inhalation of endotoxin or Pseudomonas
aeruginosa
Shawn J.
Skerrett1,2,
Thomas R.
Martin1,2,
Emil Y.
Chi3,
Jacques J.
Peschon4,
Kendall M.
Mohler4, and
Christopher B.
Wilson5,6
Departments of 1 Medicine,
3 Pathology,
5 Immunology, and
6 Pediatrics, University of
Washington School of Medicine;
4 Immunex Corporation; and
2 Veterans Affairs Puget Sound
Health Care System, Seattle, Washington 98108
 |
ABSTRACT |
To determine the
roles of the type 1 tumor necrosis factor (TNF) receptor (TNFR1) in
lung inflammation and antibacterial defense, we exposed transgenic mice
lacking TNFR1 [TNFR1(
/
)] and wild-type control mice to aerosolized lipopolysaccharide or
Pseudomonas aeruginosa. After LPS,
bronchoalveolar lavage fluid (BALF) from TNFR1(
/
) mice
contained fewer neutrophils and less macrophage inflammatory protein-2
than BALF from control mice. TNF-
, interleukin-1
, and total protein levels in BALF as well as tissue intercellular adhesion molecule-1 expression did not differ between the two groups.
In contrast, lung inflammation and bacterial clearance after infection
were augmented in TNFR1(
/
) mice. BALF from infected TNFR1(
/
) mice contained more neutrophils and
TNF-
and less interleukin-1
and macrophage inflammatory protein-2
than that from control mice, but protein levels were similarly elevated in both groups. Lung inflammation and bacterial clearance were also
augmented in mice lacking both TNF receptors. Thus TNFR1 facilitates
neutrophil recruitment after inhalation of lipopolysaccharide, in part
by augmenting chemokine induction. In contrast, TNFR1 attenuates lung
inflammation in response to live bacteria but does not contribute to
increased lung permeability and is not required for the elimination of
P. aeruginosa.
tumor necrosis factor; pneumonia; lung injury; lipopolysaccharide; cytokines; interleukin-1
 |
INTRODUCTION |
THE PRESENCE OF BACTERIAL ENDOTOXIN or live
gram-negative bacteria in the distal air spaces of the lungs elicits an
acute inflammatory response composed mainly of neutrophils. This
requires the upregulation of adhesion molecules on circulating
leukocytes and the pulmonary vascular endothelium and the expression of
endogenous chemotactic factors that draw the marginated leukocytes
across the endothelial and epithelial barriers into the air spaces
(56). The inflammatory response elicited by endotoxin and gram-negative bacteria has important consequences for the host. On one hand, inflammation plays an essential role in host defense because
neutrophils are required for the elimination of gram-negative bacteria
from the lungs (45). On the other hand, inflammation can cause tissue injury, manifested by an alteration in capillary-endothelial
permeability, pulmonary edema, and epithelial damage.
Tumor necrosis factor (TNF)-
is a pleiotropic cytokine that can
amplify pulmonary inflammatory responses by stimulating the release of
chemotactic factors from alveolar macrophages and airway epithelial
cells and by upregulating the expression of leukocyte and endothelial
adhesion molecules (56). Instillation of recombinant TNF-
(rTNF-
)
into the tracheobronchial tree induces chemokine release in
bronchoalveolar lining fluid (23) and upregulation of intercellular
adhesion molecule-1 (ICAM-1) on pulmonary vascular endothelium (37).
Exposure to aerosolized or endotracheally administered rTNF-
has
been associated with margination of leukocytes in the pulmonary
vasculature (8, 17) and neutrophilic infiltration of the interstitium
or alveolar septae (17, 68). Some investigators (23, 61, 64) have
found that airway administration of rTNF-
results in an
accumulation of neutrophils in the alveolar spaces, but others (8, 17,
37, 70) have found that the recruited neutrophils remain subepithelial.
Similarly, some authors (23, 37, 68) have reported that airway
challenge with rTNF-
increases the permeability of the
alveolar-capillary membrane, but others (62, 70) have found no effect
of rTNF-
on lung permeability.
TNF-
is induced in the lungs in response to airway challenge with
lipopolysaccharide (LPS) (38, 40, 64, 70), but its role in mediating
lung inflammation and injury in this setting is unclear. Administration
of TNF-
inhibitors has been reported to reduce LPS-induced lung
inflammation in some models (24, 66) but not in others (58).
The role of TNF-
in mediating lung inflammation in the course of
gram-negative bacterial pneumonia also is incompletely defined. TNF-
is induced in the lung in response to gram-negative bacillary infection
(15, 18, 20, 29, 46, 52, 60), but conflicting data have emerged
regarding its contribution to host defense. Some reports (18, 24, 29,
52) suggested that TNF-
facilitates the clearance of gram-negative
bacteria from the lungs, but other studies (18, 46) have found
otherwise. Furthermore, inhibition of TNF-
was shown to reduce
neutrophil recruitment to the lungs in response to
Klebsiella pneumoniae infection (29)
but not in response to Pseudomonas
aeruginosa or Legionella
pneumophila infection (18, 46, 52).
The effects of TNF-
are mediated through two distinct cell surface
receptors, the 55-kDa type 1 TNF receptor (TNFR1) and the 75-kDa type 2 TNF receptor (TNFR2). TNFR1 mediates most proinflammatory and cytotoxic
effects of TNF-
, including shock and tissue injury induced by
endotoxin (7, 13, 41, 42, 47). Transgenic mice with targeted
disruptions in the TNFR1 gene [TNFR1(
/
)] are
resistant to shock and tissue injury induced by endotoxin and
D-galactosamine or rTNF-
but
are more susceptible to infection with intracellular bacteria such as
Listeria monocytogenes and Mycobacterium tuberculosis (14, 41,
42, 47). TNFR1(
/
) mice also exhibit diminished tissue
inflammation in response to systemic or local administration of
rTNF-
, a defect associated with diminished upregulation of
endothelial adhesion molecules (25, 39). TNFR1(
/
) mice
thus provide a focused model in which to test the importance of TNF-
in pulmonary inflammatory responses to different stimuli.
We compared the responses of TNFR1(
/
) and wild-type
[TNFR1(+/+)] mice to aerosolized LPS and live
P. aeruginosa to better define the
role of TNF-
in lung inflammation and host defense and to identify
the specific roles of TNFR1 in these responses. We hypothesized that
TNFR1-deficient animals would have diminished inflammatory responses to
LPS and live bacteria and would exhibit defective clearance of
P. aeruginosa from the lungs.
 |
MATERIALS AND METHODS |
Animals. C57BL/6
TNFR1(
/
) mice and mice lacking both TNFRs
[TNFR1(
/
) and
TNFR2(
/
)] were bred at the Animal Care
Facility of the University of Washington (Seattle, WA) from breeding
pairs generated at Immunex Research and Development Corporation
(Seattle) (41). Genotypes were confirmed from tail-snip DNA by the
polymerase chain reaction with primers specific for the wild-type and
disrupted genes for TNFR1 and TNFR2. Specific pathogen-free
C57BL/6 wild-type control mice were purchased from Charles River
(Wilmington, MA). The animals were housed in sterile microisolator
cages in the vivarium of the Seattle Division of the Puget Sound
Veterans Affairs Health Care System and permitted free access to
sterile food and water. The mice were 7-12 wk of age at the time
of study. Male and female animals were used in approximately equal
numbers. The experiments were approved by the Animal Studies Committee
of the Puget Sound Veterans Affairs Health Care System.
Endotoxin.
Escherichia coli 011:B4 LPS was
purchased from Sigma (St. Louis, MO), reconstituted to 10 mg/ml in
sterile saline for injection, divided into aliquots, and stored at
70°C. The same stock was used for all experiments. For each
experiment, an aliquot was thawed, briefly sonicated, and diluted in
sterile saline to 10, 33, or 100 µg/ml for injection.
Bacteria. P. aeruginosa PAK was a generous gift from Steve Lory
(University of Washington). Several colonies from an agar plate were
inoculated into Luria broth (LB) and grown overnight at
37°C in a shaking incubator. The broth was diluted to 30%
glycerol, divided into aliquots, and flash-frozen in
ethanol-dry ice before storage at
70°C. For each experiment,
an aliquot was thawed, diluted 1:100 in LB, and then incubated for 6 h
at 37°C in a shaking incubator. Then 2.5 ml of this suspension were
transferred to each of four flasks containing 250 ml of LB, which were
incubated for 16 h at 37°C in a shaking incubator. The bacteria
then were pelleted, washed twice in phosphate-buffered saline (PBS)
containing 10 mM magnesium chloride, and then resuspended in 20 ml of
the same buffer.
Exposure to aerosolized LPS or
bacteria. Mice were placed in wire mesh cages within a
sealed 55-liter Plexiglas chamber connected to a separate aerosol
chamber as previously described (53). Airflow through the system was
established at 20 l/min by negative pressure. Aerosols were generated
by twin jet nebulizers (Marquest Medical Products, Englewood, CO), each
containing 8 ml of suspended LPS or bacteria and driven by forced air
at 15 psi. Exposures were continued for 30 min.
Processing of tissues after exposure to
LPS. In each experiment, 4 and 24 h after exposure to
aerosolized LPS, four to six TNFR1(+/+) and four to six
TNFR1(
/
) mice were anesthetized with intraperitoneal
pentobarbital sodium and exsanguinated by cardiac puncture. The trachea
of each animal was exposed and cannulated with a 22-gauge polyethylene
catheter. In some experiments, the lungs were lavaged four times with
1-ml volumes of 0.85% sodium chloride containing 0.6 mM EDTA and
prewarmed to 37°C. Bronchoalveolar lavage specimens were
centrifuged at 300 g, and the
supernatants were aspirated, divided into aliquots, and stored at
70°C. The cell pellets were resuspended in RPMI 1640 medium
(BioWhittaker, Walkersville, MD) containing 10% heat-inactivated fetal
calf serum (Hyclone Laboratories, Logan, UT). Cell counts were
performed with a hemocytometer, and differential counts were determined from cytocentrifuged specimens prepared with a modified Wright-Giemsa stain (Diff-Quik, American Scientific Products, McGaw Park, IL). In
other experiments, one lung was tied off and the other was inflated to
15 cmH2O pressure
with a fixative (10% Formalin in PBS or 4% paraformaldehyde) and then
immersed in the same fixative. The fixed tissues were embedded in
paraffin, and coronal sections were stained with hematoxylin and eosin.
Processing of tissues after exposure to P. aeruginosa. Immediately after exposure to aerosolized
bacteria, four wild-type mice were anesthetized with pentobarbital
sodium and exsanguinated by cardiac puncture to determine bacterial
deposition in each experiment. The left lung and the spleen were
harvested, homogenized in
PBS-MgCl2, serially diluted in the
same buffer, and quantitatively cultured by spreading 0.1-ml aliquots
on LB agar in duplicate. The colonies were counted after an overnight
incubation at 37°C in 5%
CO2-humidified air. Bacterial
clearance was determined by similarly processing lung and spleen
tissues of wild-type and TNFR-deficient mice 4 and 24 h after
infection. At the 4- and 24-h time points, the right lung of each mouse
was lavaged or fixed in Formalin as described in
Processing of tissues after exposure to
LPS.
Tissue expression of ICAM-1 by
immunohistochemistry. Coronal sections of
paraformaldehyde-fixed lung tissue were predigested with 0.01% Pronase
in PBS, then blocked with 5% nonfat dry milk in PBS. The sections were
incubated with hamster anti-mouse ICAM-1 (Endogen, Woburn, MA) diluted
1:100 in 5% nonfat dry milk in PBS (final concentration of antibody
10.8 µg/ml), hamster IgG (Jackson ImmunoResearch Laboratories, West
Grove, PA) diluted 1:1,000 in 5% nonfat dry milk in PBS (final
concentration 10 µg/ml), or 5% nonfat dry milk in PBS for 90 min at
room temperature. The sections were washed, incubated with biotinylated
goat anti-hamster IgG (Jackson ImmunoResearch Laboratories) diluted
1:100 in 5% nonfat dry milk with 1% goat serum for 60 min at room
temperature, and then developed with avidin-alkaline phosphatase
(Vector Laboratories, Burlingame, CA) before being counterstained with
methylene blue.
Measurement of cytokines and total protein in
bronchoalveolar lavage fluid supernatants.
Immunoreactive TNF-
and interleukin (IL)-1
were measured in
sandwich ELISAs, as previously described (54), with mouse-specific
antibodies and standards obtained from Genzyme (Cambridge, MA). The
C-X-C chemokines macrophage inflammatory protein (MIP)-2 and KC were
measured by ELISA with reagents purchased from R&D Systems
(Minneapolis, MN). TNF-
bioactivity was measured in an L929
cytotoxicity assay as previously described (36). The specificity of the
cytopathic effect was confirmed in parallel plates by its abolition in
the presence of anti-mouse TNF-
(Genzyme). Total protein
concentration was measured with the bicinchoninic acid method (BCA
assay, Pierce, Rockford, IL).
Measurement of chemotactic activity.
The chemotactic activity for neutrophils that were present in
bronchoalveolar lavage fluid (BALF) was measured in microchemotaxis
chambers with nitrocellulose filters with 3-µm pores as previously
described (34, 36). Peritoneal exudate neutrophils were elicited from
wild-type mice by instilling 2 ml of 0.1% oyster glycogen (Sigma)
intraperitoneally. Cells were harvested 4 h later by peritoneal lavage
with 0.85% saline containing 0.6 mM EDTA, washed twice in Hanks'
balanced salt solution (GIBCO BRL, Grand Island, NY), and suspended to a concentration of 3 × 106/ml in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal calf serum, 10 mM HEPES
(BioWhittaker), 100 U/ml of penicillin, and 100 µg/ml of streptomycin
(GIBCO BRL). The exudate cells were >85% neutrophils and >90%
viable by the exclusion of trypan blue. The bronchoalveolar lavage
supernatants were added to the lower wells of 48-well microchemotaxis
chambers in triplicate, and the neutrophil suspension was added to the
upper wells. Control lower wells contained 10% zymosan-activated serum
or PBS. Zymosan-activated mouse serum was generated by suspending
zymosan particles (Sigma) at 0.5 mg/ml in fresh mouse serum and
incubating at 37°C for 1 h, followed by 56°C for 30 min. The
particles were pelleted by centrifugation, and the supernatant was used
fresh in the chemotaxis assay. The chambers were incubated for 2 h at
37°C in 5% CO2-humidified air, then the nitrocellulose filters separating the upper and lower
wells were removed, stained with Diff-Quik, and mounted on microscope
slides. The total number of neutrophils that had migrated through each
filter were counted in 10 consecutive high-power fields (×540)
with the aid of an eyepiece grid.
Data analysis. Data are expressed as
means ± SE. Comparisons between two groups of animals were made
with independent two-tailed t-tests. A
P value
0.05 was considered significant.
 |
RESULTS |
Leukocyte recruitment after inhalation of
LPS. As shown in Fig.
1A,
exposure of mice to aerosolized LPS resulted in a dose-related increase
in the number of neutrophils present in bronchoalveolar lavage
specimens 24 h later. At each of the three concentrations of LPS that
were tested, there were significantly fewer neutrophils in the
bronchoalveolar lavage specimens in mice lacking TNFR1 than in
wild-type control mice. After exposure to aerosolized LPS at 10, 33, or
100 µg/ml, there were 54, 61, and 38% fewer neutrophils,
respectively, in the bronchoalveolar lavage specimens of
TNFR1(
/
) mice than in samples from control mice. There
also was a dose-related increase in mononuclear cells (mainly
macrophages) recovered in bronchoalveolar lavage samples in response to
aerosolized LPS (Fig. 1B). The
number of mononuclear cells in bronchoalveolar lavage samples did not
differ between the two groups of animals.

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Fig. 1.
Bronchoalveolar lavage (BAL) cells in type 1 tumor necrosis factor
(TNF) receptor (TNFR1) wild-type [TNFR1(+/+)] and
TNFR1-deficient [TNFR1( / )] mice 24 h after
inhalation of lipopolysaccharide (LPS).
A: total number of polymorphonuclear
leukocytes (PMN) harvested from lavage of both lungs from each animal.
B: total number of mononuclear cells
(MN) harvested from lavage of both lungs from each animal. Data are
means ± SE of combined results from 3 separate experiments;
n, no. of mice (nos. in parentheses).
* P < 0.05 compared
with TNFR1(+/+) mice.
|
|
Figure 2 shows the time course of leukocyte
recruitment to the lungs after inhalation of LPS (100 µg/ml). In both
groups of animals, the peak number of neutrophils was recovered 4 h
after exposure to LPS. In the TNFR1(
/
) mice, there were
24, 36, and 38% fewer neutrophils in BALF 4, 12, and 24 h,
respectively, after inhalation of LPS. The differences were significant
at the latter two time points. In contrast to neutrophils, the peak
number of mononuclear cells was recovered 24 h after exposure
to LPS. There were significantly more mononuclear cells in the
bronchoalveolar lavage samples from TNFR1(
/
) mice 4 h
after inhalation of LPS but no differences at the later time points.
Thus neutrophil recruitment to the lungs after inhalation of LPS could
occur without normal expression of TNFR1, but sustained neutrophilic
inflammation in response to LPS was significantly diminished in mice
lacking TNFR1. In contrast, early expansion of the bronchoalveolar
mononuclear cell population after inhalation of LPS was augmented in
mice lacking TNFR1.

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Fig. 2.
BAL cells in TNFR1(+/+) and TNFR1( / ) mice at intervals
after inhalation of LPS (100 µg/ml).
A: total number of PMN harvested by
lavage of both lungs from each animal.
B: total number of MN harvested by
lavage of both lungs from each animal. Data are means ± SE of
combined results from 4 separate experiments;
n, no. of mice (nos. in parentheses).
* P < 0.05 compared with
TNFR1(+/+) mice.
|
|
Lung histopathology after inhalation of
LPS. Histological changes were more evident 4 h after
inhalation of endotoxin than after 24 h. At the 4-h time point in
TNFR1(+/+) mice, there were patchy alveolar infiltrates composed of
mononuclear and polymorphonuclear leukocytes, red blood cells, and an
eosinophilic exudate (Fig. 3A). In
other areas, there were scattered interstitial and alveolar neutrophils, with mild interstitial thickening. There was intense submucosal neutrophilic infiltration of bronchi and bronchioles, but
margination of leukocytes in the pulmonary vasculature was not evident.
In TNFR1(
/
) mice, the same pattern of peribronchial neutrophil infiltration was present, and scattered interstitial and
alveolar neutrophils could be found, but there were fewer intra-alveolar cells and no interstitial thickening (Fig.
3C). Inflammation was less evident
24 h after endotoxin exposure in both strains of mice (Fig. 3,
B and
D). In TNFR1(
/
) mice,
the cellularity was homogeneous, with occasional interstitial and alveolar neutrophils; peribronchial inflammation was less intense than
at the earlier time point. The histological findings 24 h after
endotoxin were very similar in the TNFR1(
/
) mice, but peribronchial neutrophil infiltration was less prominent than in the
wild-type animals.

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Fig. 3.
Light micrographs of hematoxylin and eosin-stained lung tissue after
inhalation of LPS (×120). Lung tissue was harvested from
TNFR1(+/+) (A and
B) and TNFR1( / )
(C and
D) mice 4 (A and
C) and 24 h
(B and
D) after exposure to aerosolized LPS
(100 µg/ml).
|
|
Lung tissue expression of ICAM-1 after inhalation of
LPS. In sections of lung tissue from wild-type and
TNFR1(
/
) mice that were unexposed to LPS, incubation with
anti-ICAM-1 yielded generalized immunoperoxidase staining of alveolar
septae, weak and inconsistent staining of venular endothelium, and no
staining of bronchial epithelium or arteriolar endothelium. A similar
pattern of staining was observed in sections of lung tissue harvested 4 or 24 h after inhalation of LPS; no change in the distribution or
intensity of ICAM-1 expression in response to inhaled LPS was evident.
There were no differences in lung tissue expression of ICAM-1 between the two strains of mice at either time point after exposure to LPS or
among unexposed animals. Sections incubated with control immunoglobulin
or nonfat milk-PBS were uniformly negative for any immunoperoxidase staining.
Chemotactic activity and chemokine levels in BALF
after inhalation of LPS. As shown in Fig.
4, no increase in chemotactic activity for
neutrophils was detectable in the BALF supernatants of wild-type mice
at any time after exposure to aerosolized LPS in comparison with the
small amount of activity present in normal BALF. However, a significant
increase in chemotactic activity was present in the BALFs of
TNFR1(
/
) mice harvested 4 and 12 h after exposure to
aerosolized endotoxin. By 24 h, the chemotactic activity had returned
to background levels. In contrast to total chemotactic activity,
chemokine levels in the BALF after inhalation of LPS were lower in
TNFR1(
/
) mice than in wild-type control mice. The
concentration of immunoreactive MIP-2 in BALF 4 h after inhalation of
LPS was 295.5 ± 60.8 pg/ml in TNFR1(+/+) mice
(n = 4), but MIP-2 was detectable in
lavage fluid from only one of four TNFR1(
/
) mice (29.0 ± 21.0 pg/ml if the lower limit of the assay of 8 pg/ml is
substituted for in the samples with undetectable MIP-2;
P = 0.017). Similarly, KC was
detectable in the BALF from two of four TNFR1(+/+) mice 4 h after LPS
exposure but was undetectable from all four specimens from
TNFR1(
/
) mice. Thus the impaired accumulation of
bronchoalveolar neutrophils in TNFR1(
/
) mice after
inhalation of LPS was associated with diminished production of C-X-C
chemokines but increased total chemotactic activity in BALF.

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Fig. 4.
Chemotactic activity for PMN in BAL fluid (BALF) supernatants harvested
from TNFR1(+/+) and TNFR1( / ) mice after no treatment (Nl
BALF) or after exposure to aerosolized LPS (100 µg/ml). Chemotactic
activity was measured in microchemotaxis chambers with peritoneal
exudate PMN harvested from normal mice. Chemotactic activity in
positive control (10% zymosan-activated serum) was 288 PMN/10
high-power fields (hpf), and chemotactic activity in negative control
(PBS) was 3 PMN/hpf. Data are means ± SE;
n, no. of mice (nos. in parentheses).
* P < 0.05 compared with
TNFR1(+/+) mice.
|
|
Cytokines in BALF after inhalation of
LPS. The concentrations of immunoreactive TNF-
in
BALF 4 h after inhalation of LPS were 2,349.1 ± 603.2 and
2,625.6 ± 499.8 pg/ml in the TNFR1(+/+) and TNFR1(
/
)
mice, respectively (n = 12/group; not
significant). The concentrations of bioactive TNF-
were 16,368.1 ± 5,303.2 and 8,181 ± 4,898.4 pg/ml in the TNFR1(+/+)
(n = 12) and TNFR1(
/
) (n = 11) mice, respectively (not
significant). Neither immunoreactive nor bioactive TNF-
was detected
in any specimen of BALF harvested 12 or 24 h after inhalation of LPS.
Immunoreactive IL-1
at levels of 53-76 pg/ml was detectable in
three of eight samples of BALF harvested from TNFR1(
/
)
mice 4 h after inhalation of LPS but from none of eight samples from
TNFR1(+/+) mice. Thus the transient secretion of TNF-
induced by
inhalation of LPS was not affected by the absence of TNFR1, but the
levels of LPS-induced IL-1
were more often detectable in the
TNFR1(
/
) mice.
Protein concentrations in BALF after inhalation of
LPS. As shown in Fig. 5,
the protein concentrations in bronchoalveolar lavage supernatants did
not change significantly after inhalation of endotoxin in either group
of mice. Thus the lung inflammation resulting from the inhalation of
endotoxin was not associated with a consistent change in permeability
detectable by the measurement of total protein.

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Fig. 5.
Total protein concentration in BALF supernatants harvested from
TNFR1(+/+) and TNFR1( / ) mice after no treatment and at
intervals after inhalation of LPS (100 µg/ml). Data are means ± SE; n, no. of mice (nos. in
parentheses).
|
|
Inflammatory cell recruitment after inhalation of
P. aeruginosa. As shown in Fig.
6A, the
inhalation of virulent P. aeruginosa was followed by a brisk influx of neutrophils to the lungs in both
groups of mice. Four hours after infection, there were significantly more neutrophils in bronchoalveolar lavage samples from
TNFR1(
/
) mice than in the wild-type control mice; after
24 h, the neutrophil accumulation was nearly identical in the two
groups. Similarly, there was a marked increase in the number of
mononuclear cells in bronchoalveolar lavage samples from infected
animals (Fig. 6B). Significantly
more mononuclear cells were recovered from the lungs of
TNFR1(
/
) mice than from the TNFR1(+/+) control mice at
24-h time points. Thus the accumulation of inflammatory cells in the
lungs in response to the inhalation of P. aeruginosa was augmented in animals lacking TNFR1.

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Fig. 6.
BAL cells harvested from TNFR1(+/+) and TNFR1( / ) mice
after inhalation of live Pseudomonas
aeruginosa. A: total
number of PMN harvested by lavage of 1 lung from each animal.
B: total number of MN harvested by
lavage of 1 lung from each animal. Data are means ± SE of combined
results from 3 separate experiments; n = 10-12 mice/value. * P < 0.05 compared with TNFR1(+/+) mice.
|
|
Lung histopathology after inhalation of P. aeruginosa. Four hours after infection, there was
diffuse inflammation in both TNFR1(
/
) and wild-type
animals (Fig. 7). In the TNFR1(+/+) mice, there was a generalized increase in cellularity, with patchy areas of
consolidation (Fig. 7A).
Interstitial and alveolar infiltration with neutrophils was evident at
high power, and red blood cells were present in many alveoli.
Subepithelial neutrophilic inflammation of the conducting airways was
most apparent in the terminal bronchioles. In the
TNFR1(
/
) mice, there was diffuse, nearly
confluent inflammation (Fig. 7C).
Sheets of neutrophils were present through the interstitium and
alveolar spaces, with generalized alveolar hemorrhage. Subepithelial and transepithelial infiltration with neutrophils was evident along
conducting airways of all sizes. There were prominent perivascular cuffs composed of mononuclear and polymorphonuclear leukocytes that
were not apparent in the wild-type animals. Twenty-four hours after
infection, the lungs of TNFR1(+/+) mice exhibited diffuse, predominantly neutrophilic inflammation, with focal areas of dense consolidation (Fig. 7B).
Peribronchial infiltration with neutrophils remained evident, and small
perivascular cuffs of mixed cellularity could be discerned. In the
TNFR1(
/
) mice, the pattern was very similar to that in
the wild-type animals, but perivascular cuffs of mononuclear and
polymorphonuclear leukocytes were more prominent (Fig.
7D).

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Fig. 7.
Light micrographs of hematoxylin and eosin-stained lung tissue after
inhalation of P. aeruginosa
(×120). Lung tissue was harvested from TNFR1(+/+)
(A and
B) and TNFR1( / )
(C and
D) mice 4 (A and
C) and 24 h
(B and
D) after infection.
|
|
Chemotactic activity and chemokine levels in BALF
after inhalation of P. aeruginosa. Chemotactic activity
for neutrophils was detected in BALF harvested 4 and 24 h after
inhalation of P. aeruginosa in both
TNFR1(+/+) and TNFR1(
/
) mice (Fig.
8). There was a trend toward more
chemotactic activity in the BALF harvested from wild-type mice 4 h
after infection, but there were no significant differences between the
two groups of animals at either time point. Paralleling the trend in
total chemotactic activity, the levels of MIP-2 and KC in BALF 4 h
after exposure to P. aeruginosa were
approximately twofold higher in TNFR1(+/+) mice than in
TNFR1(
/
) animals (Fig. 9),
although only the difference in MIP-2 concentration reached
significance. No chemokines were detected in the lavage fluid from
uninfected animals. Thus the greater number of bronchoalveolar
neutrophils in TNFR1-deficient mice 4 h after infection did not result
from an increased production of chemotactic factors.

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Fig. 8.
Chemotactic activity for PMN in BALF supernatants harvested from
TNFR1(+/+) and TNFR1( / ) mice after no treatment or after
inhalation of live P. aeruginosa (PA).
Chemotactic activity was measured in microchemotaxis chambers with
peritoneal exudate PMN harvested from normal mice. Chemotactic activity
in positive control (10% zymosan-activated serum) was 181.3 PMN/10
hpf, and chemotactic activity in negative control (PBS) was 3.3 PMN/hpf. Data are means ± SE; n = 5 normal control mice and 10/group of infected mice.
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Fig. 9.
Chemokine levels in BALF 4 h after inhalation of P. aeruginosa. Macrophage inflammatory protein (MIP)-2 and
KC were measured by ELISA. Data are means ± SE;
n = 10 mice for MIP-2 and 5 mice for
KC. * P < 0.05 compared with
TNFR1(+/+) mice.
|
|
Cytokines in BALF after inhalation of P. aeruginosa. Levels of both TNF-
and IL-1
in BALF
were highest 4 h after infection but remained detectable at
24 h in both groups of mice (Fig. 10). Immunoreactive TNF-
concentrations were significantly higher in
TNFR1(
/
) than in wild-type animals 4 h after infection. A similar trend was observed in levels of bioactive TNF-
, which did
not reach significance (P = 0.085). In
contrast, bronchoalveolar levels of IL-1
were significantly lower in
TNFR1(
/
) mice at both time points.

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|
Fig. 10.
Cytokine levels in BALF at intervals after inhalation of
P. aeruginosa.
Immunoreactive (Immuno) TNF- (A)
and interleukin (IL)-1 (C) were
measured by ELISA. Bioactive TNF-
(B) was measured as lysis of L929
cells. Data are means ± SE; n = 5 normal control mice and 10/group of infected mice.
* P < 0.05 compared with
TNFR1(+/+) mice.
|
|
Total protein concentration in BALF after inhalation
of P. aeruginosa. As shown in Fig.
11, there was a marked increase in the
total protein concentration in BALFs harvested from both groups of
animals at both time points after infection with P. aeruginosa. There were no differences between the
TNFR1(+/+) and TNFR1(
/
) mice. Thus TNFR1 did not
contribute to the protein leak in this model.

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|
Fig. 11.
Total protein concentration in BALF supernatants harvested from
TNFR1(+/+) and TNFR1( / ) mice after no treatment and at
intervals after inhalation of live P. aeruginosa. Data are means ± SE;
n = 7 normal control mice and
n = 10/group of infected mice.
|
|
Clearance of aerosolized P. aeruginosa from the
lungs. Inhalation of P. aeruginosa was followed by a gradual net bacterial clearance by 24 h after infection in both TNFR1(+/+) and
TNFR1(
/
) mice (Fig. 12). At
the 4-h time point, there were significantly fewer bacteria remaining
in the lungs of TNFR1(
/
) mice than in the wild-type
control mice. However, by 24 h after infection, both groups had
achieved equivalent net clearance. Thus the early clearance of
P. aeruginosa from the lungs was
augmented in the absence of TNFR1.

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|
Fig. 12.
Clearance of P. aeruginosa from lungs
of TNFR1( / ) and TNFR1(+/+) mice. One lung from each mouse
was homogenized and quantitatively cultured to determine colony-forming
units (CFU)/lung. Data are means ± SE of combined results from 2 experiments; n = 7-8
mice/measurement. * P < 0.05 compared with TNFR1(+/+) mice.
|
|
Spleen cultures. There were no
differences between TNFR1(
/
) and TNFR1(+/+) mice in the
systemic dissemination of infection after inhalation of
P. aeruginosa. Four hours after
infection, 5 of 12 spleen cultures were positive in
TNFR1(
/
) mice vs. 3 of 9 spleen cultures in TNFR1(+/+)
mice. After 24 h, 3 of 12 spleen cultures were positive in each group.
Inflammatory cell recruitment, chemokine levels, and
bacterial clearance in TNFR1(
/
) and
TNFR2(
/
) mice. The unexpected finding of
increased lung inflammation and accelerated bacterial clearance in the
TNFR1(
/
) mice, in contrast to the impaired neutrophil
recruitment to inhaled LPS observed in these animals, raised the
possibility that the inflammatory response to P. aeruginosa might be mediated, in part, through TNFR2.
To investigate this possibility, we conducted a single experiment with
mice lacking both TNFRs. As shown in Fig.
13, there were significantly fewer bacteria in the lungs of TNFR1(
/
) and
TNFR2(
/
) mice 4 and 24 h after infection than in
wild-type control mice. The augmented bacterial clearance was
associated with a trend toward increased early neutrophil accumulation
in the TNFR-deficient mice. Four hours after infection, there were 1.7 ± 0.2 × 106 and 6.9 ± 3.6 × 106
bronchoalveolar neutrophils/lung in wild-type and
TNFR1(
/
) and TNFR2(
/
) mice, respectively
(n = 3/group). After 24 h, there were
7.4 ± 1.1 × 106 and 7.7 ± 1.5 × 106
neutrophils/lung in the control and TNFR1(
/
) and
TNFR2(
/
) mice, respectively
(n = 4/group). Despite the robust
inflammation, chemokine levels were diminished in the TNFR-deficient
mice. The concentrations of MIP-2 in BALF 4 h after infection were 10.2 ± 1.8 and 3.1 ± 1.8 ng/ml in the wild-type and
TNFR1(
/
) and TNFR2(
/
) mice, respectively
(P = 0.03;
n = 4). The levels of KC were
7.4 ± 1.5 and 1.6 ± 0.6 ng/ml in the wild-type and
knockout mice, respectively (P = 0.01;
n = 4). No chemokines were detected in
the lavage fluid from uninfected animals. Thus mice lacking both TNF
receptors responded to aerosolized P. aeruginosa with the same pattern of accelerated
bacterial clearance and increased neutrophil accumulation in the lung
despite a blunted chemokine response that was observed in mice lacking
only TNFR1.

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Fig. 13.
Clearance of P. aeruginosa from lungs
of mice deficient in both TNFR1 and type 2 TNFR ( / ) and
of wild-type mice (+/+). One lung from each mouse was homogenized and
quantitatively cultured to determine CFU/lung. Data are means ± SE;
n = 4 mice/measurement.
* P < 0.05 compared with
wild-type mice.
|
|
 |
DISCUSSION |
The major finding of this study is that the role of TNFR1 in regulating
pulmonary inflammation differed with the inciting stimulus. After
exposure to aerosolized LPS, there was a persistent reduction in
neutrophil recruitment to the air spaces of the lungs in
TNFR1-deficient mice, in comparison with wild-type animals, that was
associated with depressed chemokine levels in BALF. In contrast, early
neutrophil influx and bacterial clearance after exposure to aerosolized
P. aeruginosa were augmented
in mice lacking TNFR1, as well as in mice deficient in both TNFRs,
despite diminished bronchoalveolar concentrations of chemokines. These
data suggest that TNFR1 facilitates neutrophilic lung inflammation in
response to inhaled LPS but serves to downregulate acute inflammatory
responses to virulent P. aeruginosa.
Our observations support a role for TNF-
in promoting lung
inflammation in response to inhaled LPS and suggest that this effect is
mediated, at least in part, through TNFR1. Several investigators (9,
73) have reported that neutralization of TNF-
reduces lung
inflammation after systemic challenge with endotoxin, and there is
evidence that neutrophilic infiltration of the lungs in response to
systemic TNF-
requires TNFR1 (39). However, previous efforts to
define the role of TNF-
in mediating neutrophil recruitment to the
lung in response to airway challenge with LPS have been inconclusive.
Ulich et al. (66) found that coadministration of human soluble TNFR1
reduced by 50-60% the number of bronchoalveolar neutrophils
recovered 6 h after intratracheal injection of LPS in rats
but did not affect the number of neutrophils 4 or 12 h after LPS
challenge. Ulich et al. (65) also reported that
coadministration of soluble human TNFR2 with intratracheal LPS reduced
neutrophil recruitment by up to 40% 6 h later, whereas coinjection of
a dimeric construct of human TNFR2 linked to the Fc fragment of IgG did not influence neutrophil recruitment in response to intratracheal LPS.
Interestingly, administration of soluble TNFR1 or TNFR2 in these
studies (65, 66), which reduced neutrophil recruitment in response to
LPS, was associated with increased TNF-
bioactivity in BALF. In
contrast, intratracheal injection of dimeric TNFR2, which did not
influence neutrophil recruitment, was associated with reduced TNF-
bioactivity in BALF (65). Kolls et al. (24) reported that systemic or
intratracheal injection of mice with an adenoviral vector encoding for
the extracellular domain of TNFR1 linked to mouse IgG heavy chain
neutralized TNF-
bioactivity and reduced the number of neutrophils
in BALF 3 h after intratracheal injection of LPS. Tang et al. (58)
found that coadministration of anti-TNF-
neutralized bronchoalveolar
TNF-
bioactivity induced by intratracheal LPS but did not affect
neutrophil recruitment. The use of transgenic mice lacking TNFR1
provides a more stable defect in TNF-
signaling than the
administration of soluble receptors or antibodies, which may have
incomplete or transient effects. Peschon et al. (41) found that
TNFR1-deficient mice exhibited a marked defect in neutrophil
recruitment to the lungs after intranasal deposition of
Micropolyspora faeni but did not
observe any differences from control mice in bronchoalveolar lavage
cell populations 2 and 24 h after nasal instillation of LPS. In
contrast, we identified a defect in neutrophil recruitment in
TNFR1-deficient animals that was sustained over 24 h and was evident at
three different concentrations of aerosolized LPS.
Although we observed a consistent reduction in bronchoalveolar
neutrophils after inhalation of LPS in TNFR1-deficient animals, histological sections demonstrated an inflammatory response in these
animals, indicating that signaling through TNFR1 is not required for
neutrophil emigration into the air spaces of the lungs. TNF-
is
known to stimulate its own synthesis and release in vitro, (43),
raising the possibility of a blunted TNF-
response to LPS in mice
lacking TNFR1. However, we found no deficiency in bronchoalveolar
TNF-
induction in TNFR1(
/
) mice, indicating that
positive feedback through TNFR1 is not required for maximal TNF-
release in response to inhaled LPS. Similarly, other investigators (41,
47) found that serum TNF-
responses to systemic LPS challenge were
not impaired in TNFR1(
/
) mice. It is possible that
TNF-
contributes to the inflammatory response in
TNFR1(
/
) mice via TNFR2. TNFR2 can mediate nuclear
factor-
B activation in some cell lines in the absence of TNFR1 (48)
and thus may potentially stimulate nuclear factor-
B-dependent
proinflammatory gene transcription (1). Indeed, transgenic mice that
overexpress TNFR2 are more susceptible to lethal challenge with LPS or
rTNF-
and exhibit chronic tissue inflammation that is independent of TNF-
or TNFR1 (12). However, TNF-
-mediated upregulation of endothelial and epithelial adhesion molecule expression appears to be
controlled exclusively by TNFR1 (25, 39), and neutrophilic lung
inflammation in response to M. faeni
is accentuated in TNFR2-deficient mice (41). It is likely that other
inflammatory mediators with overlapping functions can partially fulfill
the proinflammatory activities of TNF-
in TNFR1-deficient mice.
IL-1
has many of the same effects as TNF-
, including stimulation
of chemokine release and upregulation of adhesion molecules (10), and
we found that IL-1
levels were more readily detectable in
TNFR1-deficient mice than in wild-type control mice after LPS challenge.
One mechanism by which TNF-
amplifies lung inflammation is by
augmenting chemokine production. TNF-
stimulates chemokine release
by macrophages and by cells that do not respond directly to LPS in
vitro, such as epithelial cells and fibroblasts (56). TNF-
also
induces bronchoalveolar chemokine release in vivo (23). Chemokines are
major components of the chemotactic activity of BALF in
endotoxin-exposed animals (16), and chemokine depletion impairs
neutrophil recruitment in response to LPS or gram-negative infection
(2, 16, 19, 49, 59, 63). We found that levels of MIP-2 and KC in BALF
harvested 4 h after inhalation of LPS were lower in TNFR1-deficient
mice than in wild-type control mice, suggesting that TNFR1 is required
for optimal chemokine induction in vivo in response to inhaled LPS.
However, we also found that BALF from LPS-exposed TNFR1-deficient mice
contained more chemotactic activity for neutrophils than lavage fluid
from LPS-exposed wild-type animals. It is possible that the lavage fluid from TNFR1(
/
) mice contained additional, unmeasured
chemotactic factors or lacked inhibitors of chemotaxis in comparison
with lavage fluid from control animals. Alternatively, the peritoneal exudate neutrophils used in the chemotaxis assay may have responded to
the net chemotactic activity differently from circulating neutrophils in vivo. Nevertheless, it seems likely that the diminished
bronchoalveolar chemokine response of TNFR1(
/
) mice
contributed to the impaired neutrophil recruitment observed in
these animals in response to aerosolized LPS.
Another mechanism by which TNF-
promotes acute lung inflammation is
by upregulating the expression of adhesion molecules on endothelial
cells and circulating leukocytes (56). Both E. coli LPS and live P. aeruginosa elicit neutrophil emigration to the alveolar
spaces by mechanisms dependent on ICAM-1 and CD18 in normal mammals
(11, 27, 28, 44). It has been reported that pulmonary endothelial
ICAM-1 expression is upregulated in response to gram-negative pneumonia
or airway deposition of LPS (4, 44, 59) and that TNF-
is an
important mediator of pulmonary vascular ICAM-1 expression in vivo (31,
37). Furthermore, TNF-
-induced endothelial adhesion molecule
expression is mediated by TNFR1 (39). Thus it is attractive to
hypothesize that diminished endothelial ICAM-1 expression may have
contributed to the impaired neutrophil recruitment in
TNFR1(
/
) mice exposed to aerosolized LPS. However, we
were unable to detect any change in ICAM-1 expression after inhalation
of LPS because of high baseline expression of ICAM-1 in the alveolar
interstitium. Other investigators (4, 39) also have found it difficult
to detect changes in pulmonary ICAM-1 expression by
immunohistochemistry because of high constitutive expression.
Furthermore, there are ICAM-1- and CD18-independent mechanisms of
neutrophil recruitment into the alveolar spaces in response to
bacterial stimuli (11, 27, 28, 44) that may be more developed in
TNFR-1-deficient transgenic animals.
A key finding in our studies is that inflammatory cell recruitment to
the lungs of TNFR1-deficient mice was augmented after inhalation of
live P. aeruginosa, in contrast to the
impaired response to aerosolized LPS. Bronchoalveolar levels of
chemokines were depressed in TNFR1(
/
) mice in response to
both stimuli. One potential explanation for these divergent effects is
that live bacteria elicit neutrophils directly as well as indirectly. Whereas inflammatory responses to LPS are dependent on host-derived mediators such as chemokines and complement fragments, neutrophil recruitment in response to gram-negative infection involves
bacterial-derived chemotactic factors such as formylated peptides, as
well as endogenous mediators stimulated by LPS and other bacterial
products (32, 51). As a result, neutrophil recruitment to the lung in
gram-negative pneumonia is only partly dependent on endogenous
cytokines (19, 20) and is not reduced in the absence of C5a receptors
(21). Moreover, some strains of P. aeruginosa are directly toxic to the alveolar
epithelium and induce a bidirectional protein leak across the
epithelial and endothelial barriers (26, 46, 72), in contrast to the
compartmentalized response to airway challenge with LPS (38, 72). The
exudation of serum proteins such as LPS binding protein and soluble
CD14 into the air spaces amplifies cytokine responses to bacterial
products (33, 35, 72). Furthermore, contamination of the bloodstream
with live bacteria and bacterial products leads to endothelial and
circulating leukocyte activation that primes the host for greater
responsiveness to tissue chemotactic signals (60, 72).
Our observations argue strongly that the dominant role of TNFR1 in
response to inhaled P. aeruginosa is
to attenuate lung inflammation. One mechanism by which this might occur
is suggested by the evidence that soluble TNFRs shed from the surface
of cells can bind free TNF-
and thereby inhibit peak TNF-
activity (67). Thus if any signaling occurs through TNFR2 in
TNFR1(
/
) mice, then the absence of soluble TNFR1 could
result in increased stimulation from free TNF-
by way of TNFR2.
However, it is unlikely that this is the explanation for the augmented
inflammatory response to infection in TNFR1(
/
) mice for
two reasons. First, the ratios of bioactive to immunoreactive TNF-
in postinfection BALF were ~5:1 in both TNFR1(
/
) and
TNFR1(+/+) mice (Fig. 10), rendering improbable any differences in
soluble inhibitors of TNF-
. Second, we observed an increased
inflammatory response to P. aeruginosa in mice lacking both TNFR1 and TNFR2, indicating that signaling through
TNFR2 did not account for the exaggerated response in TNFR1-deficient
mice. Another pathway by which TNFR1 may contribute to the
downmodulation of inflammatory responses is by signaling the induction
of anti-inflammatory mediators. For example, TNF-
stimulates the
secretion of IL-10 (6), which inhibits pulmonary inflammation (5, 50).
It is possible that an altered balance of pro- and anti-inflammatory
mediators led to the more robust early neutrophil response after
inhalation of P. aeruginosa in mice
lacking TNFR1.
Although the increased numbers of neutrophils in the lungs of
TNFR1-deficient mice with early P. aeruginosa infection may reflect increased cellular
recruitment, an alternative explanation is that elicited neutrophils
survive longer in the air spaces of animals lacking TNFR1. Neutrophils
are short-lived cells and ordinarily undergo apoptosis within hours of
their entry into tissues (55), after which they undergo endocytosis and
are cleared by macrophages. Exposure to TNF-
and ingestion of
gram-negative bacteria accelerate neutrophil apoptosis (57, 69).
Because TNFR1 can mediate apoptosis (7), it is possible that in the absence of TNFR1, apoptosis of neutrophils was delayed, leading to
greater accumulation of these cells in the lungs. Prolonged neutrophil
survival might improve microbial killing, perhaps accounting for the
accelerated clearance of P. aeruginosa
in TNFR1(
/
) mice.
We found no evidence that TNFR1 plays a role in increasing lung
permeability in response to LPS or P. aeruginosa. The protein concentration in BALF from
LPS-exposed animals did not differ from that in control animals,
indicating that inhalation of LPS resulted in little, if any,
alteration in epithelial permeability despite the influx of
neutrophils. Other investigators (22, 72) have made similar
observations, but some (30, 40, 71) have reported that intratracheal
injection of LPS or exposure to high concentrations of aerosolized LPS
in rats causes an increase in epithelial permeability. In contrast to
the results with aerosolized LPS, we found that the BALF protein
concentration was markedly elevated in infected mice whether or not
TNFR1 was present. These observations indicate that the mechanism of
increased epithelial permeability in these animals is not dependent on
TNFR1. Similarly, Rezaiguia et al. (46) observed that TNF-
depletion
did not influence epithelial protein permeability in rats with
P. aeruginosa pneumonia but
did reduce the increase in alveolar fluid clearance in infected
animals. It is likely that bacterial-derived factors and host mediators
other than TNF-
are important in altering epithelial permeability
(26, 72).
We were surprised to find that pulmonary clearance of
P. aeruginosa was augmented in
TNFR1(
/
) and TNFR1(
/
) and
TNFR2(
/
) mice because most investigations have supported
an important role for TNF-
in host resistance to gram-negative
bacteria. Buret et al. (3) reported that intratracheal injection of
rTNF-
improved pulmonary clearance of P. aeruginosa in rats, apparently by stimulating the
phagocytosis of bacteria by bronchoalveolar neutrophils. Kolls et al.
(24) found that systemic administration of an adenoviral vector
encoding a soluble inhibitor of TNF-
resulted in impaired clearance
of aerosolized P. aeruginosa in BALB/c
mice. Similarly, Laichalk et al. (29) reported that systemic administration of human soluble TNFR2 linked to the Fc fragment of
human IgG resulted in impaired neutrophil recruitment and bacterial clearance after intratracheal challenge of CBA/J mice with
K. pneumoniae. However, Rezaiguia et
al. (46) reported that antibody-mediated depletion of TNF-
in rats
did not affect pulmonary neutrophil recruitment or clearance of
P. aeruginosa (46). The study by Gosselin et al. (18), using a model of chronic P. aeruginosa airway infection in mice, suggested that the
role of TNF-
is host dependent: anti-TNF-
impaired pulmonary
bacterial clearance in BALB/c mice without influencing neutrophil
recruitment but had no effect in DBA/2 mice. Our finding of accelerated
bacterial clearance in TNFR-deficient mice may be related to the
greater early accumulation of neutrophils in these animals. These data suggest that TNF-
is not required for resistance to
P. aeruginosa in C57BL/6 mice.
In summary, we found that TNFR1-deficient mice exhibited impaired
pulmonary inflammatory responses to inhaled LPS but augmented responses
to live P. aeruginosa
despite impaired chemokine responses to both stimuli. The absence of
TNFR1 did not affect the protein leak associated with gram-negative
pneumonia nor did it impair bacterial clearance from the lungs. These
findings support a complex role for TNFR1 in regulating pulmonary
inflammation that varies with the nature of the inciting stimulus.
Elucidation of the mechanisms underlying augmented neutrophil
recruitment and accelerated bacterial clearance in TNFR1-deficient mice
challenged with P. aeruginosa will
require further investigation.
 |
ACKNOWLEDGEMENTS |
We thank Sandra Guidotti, Tamara Rogers, Catherine Brissette, Venus
Wong, John Ruzinski, and Mechthild Jonas for technical assistance.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Specialized Center of Research Grant HL-30542.
Address for reprint requests and other correspondence: S. J. Skerrett,
Veterans Affairs Puget Sound Health Care System (151L), 1660 South
Columbian Way, Seattle, WA 98108 (E-mail:
shawn{at}u.washington.edu).
Received 24 October 1997; accepted in final form 25 January 1999.
 |
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