1 Division of Pulmonary and
Critical Care Medicine, Intercellular adhesion molecule-1 (ICAM-1) is
expressed at high levels on type I alveolar epithelial cells (AEC) in
the normal alveolar space. We postulate that AEC ICAM-1 enhances the
antimicrobial activity of macrophages and neutrophils in the alveolar
space. Wild-type and mutant mice deficient in ICAM-1 were inoculated intratracheally with Klebsiella
pneumoniae. After 10 days, 43% of the ICAM-1 mutant
mice had died compared with 14% of the wild-type controls
(P = 0.003). Significantly more
bacteria were isolated from lungs of ICAM-1 mutant mice than controls
24 h after inoculation (log colony-forming units 5.14 ± 0.21 vs.
3.46 ± 0.16, P = 0.001). However,
neutrophil recruitment to the lung was not different. In similar
experiments in the rat, inhibition of alveolar ICAM-1 by intratracheal
administration of antibody resulted in significantly impaired clearance
of K. pneumoniae. The role of
phagocyte interactions with AEC ICAM-1 for antimicrobial activity was
investigated in vitro using primary cultures of rat AEC that express
abundant ICAM-1. Alveolar macrophage phagocytosis and killing of
K. pneumoniae were increased
significantly in the presence of AEC; these effects were inhibited
significantly (47.5 and 52%, respectively) when AEC ICAM-1 was
blocked. Similarly, neutrophil phagocytic activity for
K. pneumoniae in the presence of AEC
in vitro was decreased when ICAM-1 on the AEC surface was blocked. Thus
in the absence of ICAM-1, there is impaired ability to clear
K. pneumoniae from the lungs,
resulting in increased mortality. These studies indicate that AEC
ICAM-1 plays an important role in host defense against K. pneumoniae by determining the
antimicrobial activity of phagocytes within the lung.
lung; inflammation; adhesion molecules; infectious
immunity-bacteria
PNEUMONIA IS THE MAJOR cause of death due to infectious
diseases in the United States (36). Bacterial pathogens commonly enter
the lung via aspiration from the pharynx. The majority of normal
individuals aspirate oropharyngeal secretions each night during sleep
(20). In the face of this persistent exposure to microbial pathogens,
the lung has a complex group of protective mechanisms so that repeated
low-level entry of bacteria in the peripheral lung only rarely results
in pneumonia (17). In particular, when pathogens enter the peripheral
lung, they encounter alveolar macrophages (AM). These resident
phagocytes may themselves engulf and kill the organisms. If the number
of organisms is too great or the organisms are particularly virulent,
AM secrete early-response cytokines, such as tumor necrosis factor- The pulmonary alveolar space is the largest site of interaction of the
body with the external environment. Alveolar epithelial cells (AEC)
define this space. Type II AEC are cuboidal cells that produce
pulmonary surfactant and serve as stem cells for the alveolar
epithelium. Type I AEC are large thin cells that cover the vast
majority of the alveolar surface. Recent evidence indicates that AEC
may play important roles in the regulation of immune and inflammatory
responses in the lung (reviewed in Ref. 42). In particular, type I AEC
express abundant intercellular adhesion molecule-1 (ICAM-1) on the
apical cell surface in the normal, uninflamed lung. Type I cell
expression of ICAM-1 is regulated by factors controlling AEC
differentiation. The function of ICAM-1 expressed constitutively on
these epithelial cells has not yet been defined.
We have formed the hypothesis that ICAM-1 on the type I AEC surface
plays an important role in host defense through the enhancement of
inflammatory cell antimicrobial activity. To test this hypothesis, we
have used murine and rat models of pneumonia due to
Klebsiella pneumoniae. We now
demonstrate that transgenic mice deficient in ICAM-1 (ICAM-1 mutant)
have significantly increased mortality after intratracheal inoculation
with low numbers of K. pneumoniae compared with wild-type controls and that this reduced survival is not
a consequence of impaired neutrophil recruitment to the lung. To
address more directly the specific contribution of AEC ICAM-1 for host
defense against this organism, we have performed a series of studies in
the rat. When ICAM-1 in the alveolar space is blocked by the
intratracheal administration of a single dose of neutralizing
anti-ICAM-1 antibody, there is significant impairment of the clearance
of K. pneumoniae from the lungs, with
no decrease in neutrophil recruitment. Finally, in in vitro studies, we
have found that the antimicrobial activity of AM and neutrophils
against K. pneumoniae is enhanced
significantly in the presence of rat AEC that express
abundant ICAM-1 in primary culture and that this effect is largely
abrogated when AEC ICAM-1 is blocked with specific antibody.
Animals. Wild-type C57BL/6 mice and
mice deficient in ICAM-1
[C57BL/6J-Icamtm1Bay
(43); 6- to 12-wk-old males in each instance] were obtained from
Jackson Laboratories (Bar Harbor, ME). Specific pathogen-free Sprague-Dawley rats (150-g males) were obtained from Charles River Laboratory (Portage, MI). All animals were housed in individual isolator cages within the Animal Care Facilities at the University of
Michigan School of Medicine or the Veterans Affairs Research Laboratories until the day of experimentation. The experimental protocols were approved by the animal care committees at the University of Michigan or the Ann Arbor Veterans Affairs Medical Center. Animals
received food and water ad libitum.
Preparation of K. pneumoniae.
K. pneumoniae strain 43816, serotype 2 (American Type Culture Collection, Manassas, VA), was used for these
studies. This virulent strain induces an impressive inflammatory
response in mice (1, 18, 27). K. pneumoniae were grown in tryptic soy broth (Difco,
Detroit, MI) for 18 h at 37°C. The concentration of bacteria in
broth was determined by measuring the amount of absorbance at 600 nm.
Standard values based on known colony-forming units (CFU) were used to
calculate inoculum concentration. Bacteria were pelleted by
centrifugation at 10,000 rpm for 30 min, washed two times in saline,
and resuspended at the desired concentration.
Inoculation of mice and rats with K. pneumoniae. Mice were anesthetized with pentobarbital
sodium (1.8-2 mg/animal ip). The trachea was exposed, and
K. pneumoniae were administered via a sterile 26-gauge needle in a final volume of 30 µl made up in sterile
saline. Uninfected controls received an equal volume of sterile saline.
The skin incision was closed with surgical staples. For survival
studies, 1 × 103 CFU were
used. A lower dose (5 × 102
CFU) was used for all other studies. Rats were anesthetized with ketamine (20 mg/animal sc) and xylazine (1.0 mg/animal sc).
K. pneumoniae
(107 CFU in 200 µl) were
administered by the same technique as described for mice. Antibodies
were administered intratracheally simultaneously with the bacteria (250 µg antibody/animal).
F(ab')2 fragments of
antibodies were generated by pepsin digestion and protein A column
chromatography of intact antibody. Murine anti-ICAM-1 (1A29, IgG1) used in these studies was
the gift of Barbara Leone, Joseph Martin, and Donald C. Anderson of
Pharmacia & Upjohn Discovery Division (Kalamazoo, MI). The control
F(ab')2 IgG was obtained from Serotec. The F(ab')2
fragments were used to avoid paradoxically enhanced binding of AM to
ICAM-1 expressing AEC due to interactions of intact antibody with Fc
receptors on AM.
Lung harvest for histological
examination. Twenty-four hours after inoculation with
K. pneumoniae, mice were anesthetized with ether. After perfusion of the lungs via the right ventricle with
4% paraformaldehyde in PBS, the lungs and central airways were excised
en bloc. The lungs then were inflated with 1 ml of 4% paraformaldehyde
to improve resolution of anatomic relationships. After overnight
fixation in 4% paraformaldehyde, the lungs were dehydrated in ethanol,
embedded in paraffin, sectioned, and stained.
Differential cell counts in total lung
lavage. Bronchoalveolar lavage was performed 24 h after
inoculation with K. pneumoniae to
determine the number of lavageable leukocytes in the alveolar space of
infected mice. Mice were deeply anesthetized, and the trachea was
exposed and intubated with a 1.7-mm polyethylene catheter. Lung lavage
was performed using 1-ml aliquots of PBS with 5 mM EDTA. Total cell
numbers were determined using a hemocytometer. Cytospins were prepared
from bronchoalveolar lavage cells and stained with a modified
Wright-Giemsa stain (Diff-Quik; Baxter, McGaw Park, IL), and
differential counts were determined. From these stained cytospin
preparations, the percentages of AM and neutrophils containing
organisms were determined by microscopic counting.
Determination of lung CFU of K. pneumoniae. At appropriate time points, mice were
killed. The pulmonary vascular bed was perfused via the right ventricle
with 1 ml of PBS with EDTA (5 mM); the lungs then were removed
aseptically and placed in sterile saline (1.5 ml for mice and 3.0 ml
for rats). The tissues were homogenized with a tissue homogenizer under
a laminar flow hood. Lung homogenates were placed on ice, and serial
1:10 dilutions were made to
10 Determination of lung myeloperoxidase
assay. Lung myeloperoxidase (MPO) activity was used as
a quantitative index of the number of neutrophils in the lung (5, 28)
and was measured as described previously (39). Briefly, the lungs first
were perfused free of blood via the right ventricle with PBS with EDTA
(5 mM). The lungs were homogenized in 2 ml of 50 mM potassium
phosphate, pH 6.0, with 5% hexadecyltrimethylammonium bromide and 5 mM
EDTA. The resultant homogenate was sonicated and centrifuged at 12,000 g for 15 min. The supernatant was
mixed 1:15 with assay buffer and read at 490 nm. MPO units were
calculated as the change in absorbance over time.
Isolation and culture of AEC. Rat type
II AEC were isolated by elastase cell dispersion and IgG panning (13).
Briefly, the rats were anesthetized, the trachea was cannulated, and
the pulmonary circulation was perfused free of blood with a balanced
salt solution at 4°C. After multiple whole lung lavages with EGTA
(1 mM) in a balanced salt solution, porcine pancreatic elastase (4.3 U/ml; Worthington) was instilled via the trachea to release type II cells. Contaminating cells bearing Fc receptors were removed from the
cell suspension by panning on plates coated with rat IgG (Sigma, St.
Louis, MO). The cells were plated on tissue culture-treated plastic
dishes or in eight-chamber Lab-Tek slides (Nunc, Naperville, IL) at 2 × 105
cells/cm2 in DMEM supplemented
with penicillin-streptomycin (GIBCO, Grand Island, NY) and 10% newborn
calf serum (Sigma). Cells were cultured at 37°C in an atmosphere of
7.5% CO2 in air. The adherent
cells were consistently >92% epithelial cells by immunofluorescent
staining with anti-cytokeratin antibodies. After 2 days in culture,
these cells spread and express high-level ICAM-1 on the apical cell surface (11).
Isolation of AM. Rats were deeply
anesthetized with pentobarbital sodium and killed by exsanguination.
The trachea was cannulated with a blunted-end 16-gauge needle, and the
lungs were lavaged with 10 5-ml aliquots of cold PBS. The lavage
samples were pooled, and contaminating red blood cells were removed by
hypotonic lysis. Tonicity was restored by the addition of an equal
volume of 2× BSS [1× BSS is (in mM) 140 NaCl, 5 KCl,
0.48 NaH2PO4,
2.02 Na2HPO4, 6 glucose, and 10 HEPES]. The cell suspension was washed two
times in BSS and resuspended in DMEM at a concentration of 1 × 106 cells/ml. The recovered cells
were consistently >90% AM by microscopic examination of
cytocentrifuge preparations stained with Diff-Quik and were >95%
viable as determined by trypan blue exclusion.
Isolation of rat neutrophils. Newly
elicited neutrophils were obtained from the peritoneal cavities of
rats. The animals were anesthetized by the subcutaneous administration
of ketamine and xylazine. Glycogen (1% in sterile saline) was
administered via intraperitoneal injection. After 4 h, the rats again
were sedated with ketamine and xylazine and were killed by
exsanguination. The peritoneal cavity was lavaged with cold PBS. The
lavage aliquots were pooled, and the cells were collected by
centrifugation, washed two times in BSS, and resuspended in DMEM at a
concentration of 1 × 106
cells/ml. The isolated cells contained >90% neutrophils and were >95% viable as determined by trypan blue exclusion.
Phagocytosis and killing of K. pneumoniae by rat AM in
vitro. Rat type II AEC were placed in culture for 2 days in Labtek slide chambers. After the monolayers were washed
extensively with PBS, the medium was replaced with DMEM without
antibiotics. Rat AM were added to these wells at a ratio
of 1:2 AM-AEC or to control plastic wells without AEC. After 1 h,
K. pneumoniae were added to the wells.
Rat serum (final concentration 5%) was included as an opsonin. The
K. pneumoniae inoculum was 1:1
bacteria-AM (based on the number of AM plated). Preliminary studies
indicated that increased numbers of organisms had little effect on AM
phagocytosis. Parallel experiments to determine the phagocytic activity
of neutrophils for K. pneumoniae used
a ratio of 1:2 neutrophils-AEC, with a bacterial inoculum of 5:1
bacteria-neutrophil (based on the number of neutrophils plated). After
1 h, the cells were washed, fixed, and stained (Diff-Quik; Difco). In
each experiment, phagocytosis of K. pneumoniae by AM and neutrophils then was determined by microscopic counting in quadruplicate wells. Data shown are from a
representative experiment of at least three independent experiments using separate cell preparations.
In experiments to measure AM microbicidal activity for
K. pneumoniae in vitro, type II AEC
were isolated and cultured in 24-well dishes for 2 days. The monolayers
were washed extensively, and the medium was replaced with DMEM without
antibiotics. Preliminary studies determined that supernatants from AEC
cultured in this manner did not inhibit the growth of
K. pneumoniae. Rat AM were added to
quadruplicate wells or to control wells at a ratio of 1:2 AM-AEC.
K. pneumoniae were added after 1 h.
After 1 h of incubation, the monolayers were washed extensively with
PBS to remove extracellular organisms. The cells were then lysed in
water, the intracellular bacteria were recovered, serial dilutions were
carried out, and 10 µl of each dilution were plated on soy base blood
agar plates. After 18 h at 37°C, CFU were determined. Bacterial
killing was measured as the number of bacteria determined by
microscopic counting minus the number of CFU. Data shown are from a
representative experiment of at least three independent experiments
using separate cell preparations.
In selected experiments, AEC ICAM-1 was blocked by incubating the AEC
monolayer with F(ab')2
fragments of monoclonal antibody (MAb) 1A29 (murine monoclonal anti-rat
ICAM-1, 5 µg/ml; Seikagaku America) for 1 h, followed by extensive
washing before the addition of the phagocytes. Data shown are from a
representative experiment of at least three independent experiments
using separate cell preparations.
Statistical analysis. Survival data
were compared using Survival in K. pneumonia. To determine
the role of ICAM-1 in host defense against
Klebsiella in the lung, wild-type and
ICAM-1 mutant mice were inoculated intratracheally with
K. pneumoniae (103 CFU/animal,
n = 14). We chose this inoculum of
organisms because we wished to examine the role of ICAM-1 in the
response to moderate entry of bacteria in the lung rather than massive
aspiration. Preliminary studies indicated that this number of bacteria
would cause 10-20% mortality in wild-type (C57BL/6) control mice.
As shown in Fig. 1, a survival difference
between the ICAM-1 mutant mice and control animals was apparent within
3 days after inoculation. By day 10 postinoculation, 86% of the wild-type mice were still alive and only
57% of the ICAM-1 mutant mice were alive
(P < 0.05). Thus, in the absence of
ICAM-1, host defense against K. pneumoniae was significantly impaired.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TNF) or interleukin-1
(IL-1
), to promote the recruitment and
activation of additional leukocytes, including neutrophils from the
vascular space.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
8. Ten microliters of
each dilution were plated on soy base blood agar plates (Difco) and
incubated for 18 h at 37°C, and then colony counts for each animal
were determined.
2 analysis. All other data are
expressed as means ± SE and were compared using the InStat software
package from GraphPad Software (San Diego, CA). Data with comparable
SDs were evaluated using a two-tailed Student's
t-test, whereas data for which SDs
differed were compared using Welch's alternate
t-test. Differences were considered
statistically significant if P values
were <0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Survival after Klebsiella
pneumonia in wild-type and intercellular adhesion
molecule-1 (ICAM-1) mutant mice. ICAM-1 mutant mice and wild-type
controls were inoculated intratracheally with 1 × 103 colony-forming units (CFU)
K. pneumoniae on day
0, and the percentage of animals surviving over time
was determined. At 10 days, survival was decreased significantly in the
ICAM-1-deficient mice compared with infected wild-type controls
(n = 13 mice for wild type,
n = 14 mice for ICAM-1 mutant).
* P = 0.003 compared with
wild-type control mice.
Clearance of K. pneumoniae. Having
determined that ICAM-1 mutant mice were more susceptible to
K. pneumoniae than wild-type animals,
experiments were performed to examine in more detail the nature of the
defect in host defense. It was possible that the ICAM-1 mutant mice
failed to clear the organism from the lung, leading to increased
mortality. Early proliferation of the organisms within the lungs might
be greater in the ICAM-1 mutant animals due to impaired local
antimicrobial activity. Furthermore, mortality is a crude measure of
the host response. Therefore, experiments were performed in which the
burden of organisms in the lungs was determined 24 h after a low-dose
inoculum of K. pneumoniae was delivered to the lungs intratracheally. We chose this time point to
focus on the early events in the host response to K. pneumoniae in the lung. As shown in Fig.
2, there was almost a 200-fold increase in
the number of K. pneumoniae CFU
isolated from lung homogenates of ICAM-1 mutant mice compared with that
in wild-type controls (P = 0.001).
These data indicate that the early, local response to
K. pneumonia was impaired in the
ICAM-1-deficient animals.
|
Neutrophil recruitment to the lung.
One of the roles of ICAM-1 expressed on vascular endothelium is in
neutrophil recruitment to sites of inflammation. To determine whether
mice lacking ICAM-1 were unable to recruit neutrophils in the setting
of Klebsiella pneumonia, wild-type and
ICAM-1 mutant mice were inoculated with K. pneumoniae intratracheally. After 24 h, the lungs were
harvested, and the MPO content of the lungs was determined as a
reflection of total lung neutrophil accumulation. The baseline MPO
content in uninfected animals was low in the presence or absence of
ICAM-1. After infection with K. pneumoniae, lung MPO was increased in each group
(Fig. 3). Interestingly, ICAM-1 mutant mice
demonstrated 2.5-fold greater lung MPO activity when compared
with wild-type mice that had been inoculated with K. pneumoniae.
|
Although the pulmonary vasculature had been perfused free of blood
before determination of MPO content, it was possible that neutrophils
recruited to the lungs in the ICAM-1-deficient mice remained adherent
in the vascular space or in the interstitium and could not reach the
alveolus. Therefore, cell counts were determined on total lung lavage
24 h after inoculation with K. pneumoniae. As shown in Fig.
4, both wild-type and ICAM-1 mutant animals
had an increase in lung lavage leukocyte counts, with greater numbers
of alveolar leukocytes in the ICAM-1 mutant mice. In each group,
the increase in total leukocyte counts was due to increased
numbers of neutrophils. In the ICAM-1 mutant mice, there were
~2.4-fold more neutrophils in lung lavage compared with that in the
wild-type animals, although this trend did not achieve statistical
significance. Histological examination of lung sections demonstrated
patchy pneumonitis, with intra-alveolar accumulations of inflammatory
cells, especially neutrophils, and bacteria in both the wild-type and
ICAM-1 mutant mice (data not shown). There were no apparent qualitative
differences in histology between the wild-type and mutant mice. These
data demonstrate that mutant mice deficient in ICAM-1 recruit
neutrophils to the lung after intratracheal inoculation with
K. pneumoniae to at least as great a
degree as do wild-type control mice. Thus the defect in bacterial
clearance is not attributable to an impairment in neutrophil
recruitment to the lungs.
|
Effect of blocking intra-alveolar ICAM-1 on bacterial
clearance and neutrophil recruitment in the rat. We
have postulated that the increased susceptibility of the
ICAM-1-deficient mice to K. pneumoniae
is in part a reflection of the absence of ICAM-1 from the alveolar
epithelial surface. We have previously used a well-characterized system
involving primary culture of rat type II AEC for the in vitro study of
AEC ICAM-1 (3, 4, 11). For transition to in vitro studies in the rat
and to investigate the role of intra-alveolar ICAM-1 more specifically,
we next performed experiments in rats, using an MAb to block ICAM-1
binding. Rats were inoculated via the trachea with anti-ICAM-1 antibody
(1A29) or control antibody. At the same time, the mice received
K. pneumoniae via intratracheal
injection. After 24 h, the lungs were harvested, and CFU and MPO
content were determined (Fig. 5). The
number of viable bacteria obtained from the lungs of the animals that
had received anti-ICAM-1 antibody was ~10-fold greater compared with animals that had received control antibody. However, neutrophil recruitment to the lungs was not impaired by blocking intra-alveolar ICAM-1 compared with that in the controls. Thus these data extend the
observations concerning the role of ICAM-1 in host defense against
K. pneumoniae in the lung to the rat
and support a specific role for intra-alveolar ICAM-1 in host defense.
|
Phagocytosis and killing of K. pneumoniae by AM in
vitro in the presence of AEC. Because in vivo studies
demonstrated impaired host defense in the setting of pneumonia due to
K. pneumoniae and because normal type
I AEC express abundant ICAM-1, we conducted a series of experiments to
investigate the importance of interactions with AEC ICAM-1 for AM
antimicrobial activity against K. pneumoniae. Rat AM were exposed to K. pneumoniae for 1 h in control dishes or in dishes in
which rat type II AEC were allowed to spread in culture for 2 days to
express characteristics of the type I cell phenotype, including
high-level ICAM-1 expression. When alone in culture, rat AM
demonstrated poor phagocytic activity for K. pneumoniae. AEC alone had no phagocytic activity for
the bacteria. In contrast, when cultured with AEC, AM phagocytic
activity for K. pneumoniae in vitro
was enhanced significantly (Fig.
6A). In parallel experiments to evaluate the influence of interaction with AEC
on AM microbicidal activity against K. pneumoniae, bacterial killing was determined for AM in
control dishes and AM in the presence of AEC. Bacterial killing by AM
was increased significantly in the presence of AEC compared with AM
alone (Fig. 6B). Thus the increased
phagocytosis of K. pneumoniae in the
presence of AEC resulted in increased microbial killing.
|
Effect of blocking ICAM-1 on the AEC surface on AM
antimicrobial activity. To determine the mechanism by
which AEC increased AM activity against K. pneumoniae in vitro, experiments were performed to
define the contribution of ICAM-1 to this interaction. Rat AEC in
culture for 2 days were exposed to
F(ab')2 fragments of murine
anti-rat ICAM-1 or control antibody for 1 h. The monolayers then were
washed to remove any unbound antibody before the addition of AM, so
that ICAM-1 on AM would not be bound by antibody. AM phagocytosis and
killing of K. pneumoniae then were
determined (Fig. 7). Blocking ICAM-1 on the
AEC surface decreased AM phagocytosis of K. pneumoniae by a mean of 48.1 ± 4.7%. Similarly,
bacterial killing by AM cultured in the presence of AEC was decreased
50.2 ± 5.5% by blocking ICAM-1 on the AEC surface. These
data indicate that the enhanced activity of AM against
K. pneumoniae in the presence of AEC
was in large part attributable to AM interaction with ICAM-1 on the
surface of AEC.
|
Effect of blocking ICAM-1 on the AEC surface on
neutrophil phagocytic activity in vitro. Similar
experiments were performed to determine whether the interaction
with ICAM-1 on the surface of AEC might contribute to phagocytic
activity of neutrophils for K. pneumoniae in vitro. As shown in Fig.
8, the phagocytosis of K. pneumoniae by neutrophils in the presence of AEC was
decreased by 31% when ICAM-1 on the AEC surface had been blocked by
antibody compared with monolayers treated with control antibody. Thus
these in vitro studies in the rat extend the observations that
leukocyte phagocytosis of organisms within the alveolar space is
diminished in animals deficient in ICAM-1 compared with that in
ICAM-1-replete controls.
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DISCUSSION |
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This study demonstrates that mice deficient in the cell-surface adhesion molecule ICAM-1 are unable to deal effectively with a bacterial challenge. Compared with wild-type mice, the ICAM-1 mutant mice had a higher mortality from pneumonia after intratracheal inoculation with K. pneumoniae. After 24 h, the ICAM-1 mutant mice had significantly greater proliferation of bacteria in their lungs than did the control animals. Interestingly, this inability to control the infection was not a function of diminished numbers of leukocytes at the site of infection; neutrophil recruitment to the lungs in the setting of K. pneumoniae pneumonia was not impaired in the absence of ICAM-1. Furthermore, intratracheal administration of a single dose of neutralizing antibody to block ICAM-1 in the alveolar space in rats resulted in impaired clearance of K. pneumoniae, without decrement in inflammatory cell recruitment to the lungs. Phagocytosis and killing of K. pneumoniae by rat AM in vitro were enhanced greatly in the presence of AEC in primary culture. This increased antimicrobial activity was largely lost when AEC ICAM-1 was blocked with an MAb. Similarly, neutrophil phagocytic activity for K. pneumoniae in the presence of AEC was significantly reduced when AEC ICAM-1 was blocked with neutralizing antibody. Together, these studies indicate that ICAM-1 on the surface of AEC in the normal lung is likely to play an important role in host defense against K. pneumoniae.
We chose to use K. pneumoniae for these studies for several reasons. This gram-negative aerobic organism is an important cause of community-acquired pneumonia in individuals with impaired pulmonary defenses and is a major pathogen for nosocomial pneumonia (2, 25). After intratracheal inoculation with K. pneumoniae, both mice (18, 24) and rats (unpublished observations) develop pneumonia with histological features resembling human disease. There is a reproducible relationship between the size of the inoculum and the lethality of infection. Our goal was to study host defense in response to relatively modest numbers of organisms, as might occur in typical human disease, rather than in overwhelming aspiration. Therefore, the inoculum size in these experiments was carefully controlled. For survival experiments, an inoculum that was predicted to induce 10-20% mortality in the C57BL/6 wild-type controls was selected, whereas for studies of bacterial clearance and neutrophil recruitment, a still lower inoculum was chosen.
There are two novel aspects of our studies using the mice genetically deficient in ICAM-1. This is the first study to examine survival in mice deficient in ICAM-1 in the setting of bacterial pneumonia. These mutant mice have impaired neutrophil recruitment to the peritoneum in the setting of bacterial peritonitis and have impaired cutaneous inflammation in the setting of contact dermatitis (43). However, previous studies that have evaluated neutrophil recruitment during pneumonitis due to Streptococcus pneumoniae (7), Pseudomonas aeruginosa (37), or intratracheal inoculation with endotoxin (23) in mice deficient in ICAM-1 have found that neutrophil recruitment is similar to that in wild-type controls. Although alternative mRNA splicing has been described in these animals (22), Qin et al. (37) found no evidence that these ICAM-1 splice variants were involved in neutrophil recruitment in the mutant mice. In contrast, inhibition of ICAM-1 activity using monoclonal antibodies or antisense oligonucleotide probes has resulted in reduced neutrophil recruitment to the lungs, suggesting that the ICAM-1 mutant mice have developed additional mechanisms for leukocyte trafficking in pneumonia (23). Second, despite these presumed redundant pathways, we found significant differences in both survival and bacterial clearance between wild-type and ICAM-1 mutant animals. On the basis of studies described above comparing the effects of different approaches to the inhibition of ICAM-1 activity, it is likely that the experiments using ICAM-1 mutant mice underestimate the importance of ICAM-1 for host defense against K. pneumoniae in the lung.
In contrast to our findings with K. pneumoniae, Qin and colleagues (37) found that lung clearance of P. aeruginosa in these same ICAM-1 mutant mice was equivalent to that in wild-type controls. It is likely that this difference is a reflection of different requirements for innate immunity in the host defense against these two bacterial species. P. aeruginosa is an opportunistic pathogen found in patients who are neutropenic or who have received extensive therapy with broad-spectrum antibiotics. It is noteworthy that a much larger inoculum was used to induce pneumonia with P. aeruginosa than with K. pneumoniae. It is possible that neutrophil recruitment alone is adequate to deal with P. aeruginosa, whereas additional activating signals provided to leukocytes through ICAM-1 are involved in normal host defense against a more pathogenic organism such as K. pneumoniae. These results should be extrapolated with caution to other pathogenic organisms in the absence of direct experimental information.
Our data demonstrate that the increased susceptibility of mice deficient in ICAM-1 to pneumonia due to K. pneumoniae is not attributable to impaired neutrophil recruitment to the alveolar space. It is possible that this increased accumulation of neutrophils either is an indication of more severe infection (as might be anticipated with increased numbers of organisms in the lungs) or is a reflection of the fact that the infection cannot be controlled with the number of neutrophils recruited in the control animals due to the impaired activity of the neutrophils within the lung. Therefore, it is likely that the increased mortality and diminished bacterial clearance in ICAM-1 mutant mice is a function of impaired host response locally within the alveolar space. This hypothesis is supported by our finding that the phagocytosis and killing of K. pneumoniae by AM and neutrophils in vitro is diminished when phagocyte-AEC interactions mediated by ICAM-1 have been blocked with an MAb. In fact, in the absence of ICAM-1 activity, there was increased neutrophil accumulation in the lungs of infected animals (mice or rats) compared with that in intact controls. These results suggest that the absence of ICAM-1 on type I AEC, rather than loss of ICAM-1 induction on endothelial cells, played a critical role in rendering these mutant mice more vulnerable to pneumonia than their wild-type counterparts.
There are several important features to the studies in the rat in which alveolar ICAM-1 was blocked by intratracheal administration of an MAb. This experiment extends our observations in mice to the rat and supports the use of rat AEC in primary culture for further studies of the host defense mechanisms involved. Rat AEC are more accessible and have been characterized much more completely than their murine counterparts. In particular, the changes in expression of epithelial cell characteristics in cell culture have been defined in the rat (34). Furthermore, in contrast to the situation with mice genetically deficient in ICAM-1, by giving a single intratracheal dose of neutralizing antibody, it is possible to block ICAM-1 within the alveolar space, with little effect on ICAM-1 activity elsewhere (29). The reduction in clearance of K. pneumoniae from the lung 24 h after neutralization of alveolar ICAM-1 supports the focus on the interaction of high-level ICAM-1 expressed on the type I cell surface with phagocytes within the alveolar space.
Previous studies have confirmed that AM play a critical role in the
pulmonary inflammatory response to bacteria. Depletion of AM before the
introduction of bacteria into the lung leads to impaired bacterial
clearance and increased susceptibility to infection (6, 19). AM in lung
sections and AM harvested by bronchoalveolar lavage from animals
infected with K. pneumoniae demonstrate extensive phagocytosis of the organisms. However, both our
data and previous studies using murine AM (30-32) have shown that
AM in vitro demonstrate poor antimicrobial activity for gram-negative
organisms despite the presence of opsonizing antibodies. Thus AM are
clearly capable of engulfing and killing K. pneumoniae in vivo, although they do so poorly in
vitro. This difference suggests that the capacity of AM for
phagocytosis and killing of K. pneumoniae is significantly affected by the setting in
which the AM encounter the organisms and in particular by their interaction with the AEC that define the alveolar space. In in vitro
experiments to address the influence of ICAM-1 on neutrophil antimicrobial activity, we have used elicited peritoneal neutrophils. Although it is possible that pulmonary neutrophils may differ in
important ways from cells obtained from the peritoneum, at both sites
the cell neutrophils are freshly recruited from the vascular space and
express 2-integrins that are
counterreceptors for ICAM-1. Future studies may investigate the
potential differences in ICAM-1-mediated responses between neutrophils
from different sites.
Our in vitro data demonstrate that the phagocytosis and killing of
K. pneumoniae by rat AM and
neutrophils is enhanced significantly by the interaction with ICAM-1 on
AEC. ICAM-1 is expressed constitutively at high levels on type I AEC in
the normal lungs of pathogen-free animals (8, 11, 21). This is in
contrast to most parenchymal cells, which express ICAM-1 minimally, if
at all, constitutively, but in which ICAM-1 is induced by stimulation
with inflammatory mediators such as TNF, IL-1, and interferon- (4,
14). In the setting of bacterial pneumonia, type I cell ICAM-1 changes only little; ICAM-1 expression in the lung is increased overall, largely due to induction of ICAM-1 on endothelial cells and type II AEC
(8).
There are several mechanisms by which ICAM-1 expression on AEC may enhance the antimicrobial activity of phagocytes within the alveolar space. ICAM-1 is the counterreceptor for Mac-1 (CD11b/CD18) on macrophages and neutrophils (12). Binding to AEC ICAM-1 may facilitate AM antimicrobial activity through enhanced adhesion (15, 44) or lateral migration of the leukocytes over the alveolar surface (3, 9). Studies using blocking antibodies indicate that Mac-1 binding may amplify phagocytosis of microbes by neutrophils (16, 38). Cross-linking of Mac-1 also mediates the adhesion-dependent increase in the respiratory burst from phagocytes and thus may increase microbial killing (40, 41). It must be noted that these studies have investigated the effects of inhibition of ICAM-1 on numbers of CFU of bacteria in the lungs of infected mice and rats. Changes in the numbers of CFU are likely to be a reflection of microbial killing by host cells. Alternatively, effects on the rate of proliferation of K. pneumoniae in the lung also might contribute to the alteration in the numbers of bacteria present. Studies to elucidate further the signals delivered to the phagocyte through ICAM-1-Mac-1 binding, including studies of the effects of purified ICAM-1 on macrophage function, are ongoing in our laboratory.
Although previously considered passive bystanders, recent evidence indicates that AEC are active participants in the host response to pulmonary pathogens (42). In addition to high-level expression of ICAM-1, AEC express a number of molecules that are important in immune and inflammatory interactions and are likely to play a critical role in defining the immunological milieu of the alveolar space. The type II cell products surfactant proteins A and D are opsonins for bacterial pathogens with important roles in innate immunity in the lung (26, 35, 47-49). Granulocyte-macrophage colony-stimulating factor (10, 46) and monocyte chemoattractant protein-1 (33, 45) are cytokine products of AEC that have important effects on chemotaxis and activation of mononuclear phagocytes. Impaired AEC function in the setting of acute lung injury is likely to be a critical factor in the increased susceptibility of these patients to nosocomial pneumonia. Studies to define the contributions of these factors to innate immunity against K. pneumoniae in the lung are underway in our laboratories.
In summary, our studies demonstrate that the absence of ICAM-1 results in impaired ability to clear K. pneumoniae from their lungs, leading to increased mortality. This increased susceptibility to infection is not due to impaired neutrophil recruitment to the lungs. AM and neutrophil antimicrobial activity in vitro is enhanced through interactions with AEC that are mediated by ICAM-1. These studies indicate that alveolar cells play an important role in innate immunity in the lung through the expression of ICAM-1.
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ACKNOWLEDGEMENTS |
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We thank Barbara Leone, Joseph Martin, and Donald C. Anderson of Pharmacia & Upjohn Company Discovery Division for the gift of 1A29 murine anti-rat ICAM-1.
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FOOTNOTES |
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This work was supported by a Merit Review Award from the Medical Research Service; Department of Veterans Affairs (R. Paine); and National Institutes of Health Grants HL-50496, Specialized Center of Research in the Pathobiology of Fibrotic Lung Disease 1P50HL-56402 (R. Paine), HL-58200, HL-57243, and AA-10571 (T. J. Standiford). A. D. O'Brien was supported by Pulmonary Cellular and Molecular Biology Training Grant HL-07749. R. Paine is a Career Investigator of the American Lung Association.
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 and other correspondence: R. Paine III, Pulmonary Section (111G), VAMC, 2215 Fuller Rd., Ann Arbor, MI 48105 (E-mail: rpaine{at}umich.edu).
Received 29 June 1998; accepted in final form 26 February 1999.
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