Division of Pulmonary and Critical Care Medicine, Department of Veterans Affairs Medical Center, University of Michigan, Ann Arbor, Michigan 48105
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ABSTRACT |
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We postulate that
intercellular adhesion molecule-1 (ICAM-1) on type I alveolar
epithelial cells (AEC) facilitates phagocytic activity of alveolar
macrophages (AM) in the alveolus. When wild-type and ICAM-1-deficient
mice were inoculated intratracheally with FITC-labeled microspheres, AM
phagocytosis of beads (after 1 and 4 h) was significantly reduced
in ICAM-1/
mice compared with controls. To focus on ICAM-1-mediated
interactions specifically involving AM and AEC, rat AM were placed in
culture with rat AEC treated with neutralizing anti-ICAM-1
F(ab')2 fragments. Blocking ICAM-1 significantly decreased
the AM phagocytosis of beads. Planar chemotaxis of AM over the surface
of AEC was also significantly impaired by neutralization of AEC ICAM-1.
ICAM-1 in rat AEC is associated with the actin cytoskeleton. Planar
chemotaxis of AM was also significantly reduced by pretreatment of the
AEC monolayer with cytochalasin B to disrupt the actin cytoskeleton.
These studies indicate that ICAM-1 on the AEC surface promotes mobility
of AM in the alveolus and is critically important for the efficient phagocytosis of particulates by AM.
pulmonary alveoli; inflammation; host defense; lung; intercellular adhesion molecule-1; alveolar epithelial cells
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INTRODUCTION |
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THE LUNG FORMS AN ENORMOUS INTERFACE of the internal milieu of the body with the outside environment. Even in the normal individual, the peripheral lung is constantly exposed to low-level entry of pathogens and other foreign material via inhalation and aspiration. The lung has a distinctive environment for the innate immune response, with its own special cells and noncellular constituents to deal with inhaled and aspirated organisms and nonmicrobial particulates. Alveolar macrophages (AM) are the resident inflammatory cells in the alveolar space (14). They are both effector cells that engulf and kill invading pathogens and sentinel cells that recognize danger signals and release inflammatory mediators to recruit and activate other phagocytic cells, especially neutrophils. Depletion experiments have confirmed that AM are an essential component of the pulmonary innate immune response to bacterial pathogens (5, 15).
AM reside in close contact with alveolar epithelial cells (AEC). Type I AEC are thin squamous cells that cover the great majority of the alveolar surface. Type I AEC express abundant intercellular adhesion molecule-1 (ICAM-1) on the cell surface, even in the normal, uninflamed lung (7, 10, 17). It appears that ICAM-1 expression is intimately related to the differentiation of the type I cell (4). Furthermore, a distinctive feature of ICAM-1 expression in type I AEC is intimate association with the actin cytoskeleton (3). On other cell types, ICAM-1 expression is important for leukocyte recruitment from the blood stream into the tissues and for lymphocyte activation (13). The function of ICAM-1 at the type I AEC surface has not been fully defined. We now demonstrate that ICAM-1 expression on AEC is important for AM phagocytosis of particles both in vivo and in vitro. Interactions of AM with ICAM-1 on AEC facilitate AM migration over the epithelial cell surface. Finally, we determined that disruption of the AEC cytoskeletal network also inhibits AM migration over AEC, suggesting that interactions with the AEC cytoskeleton are important for the role of ICAM-1 in the alveolar space. Thus these studies provide important new insights into the functional role of ICAM-1 on AEC and suggest a new mechanism that may contribute to the impaired innate immune response in the individual who has suffered acute lung injury.
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MATERIALS AND METHODS |
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Animals.
Wild-type C57BL/6 mice and mice deficient in ICAM-1 [(ICAM-1/
),
C57BL/6J-Icamtm1Bay (5); 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. Animals received food and water ad libitum.
Intrapulmonary phagocytosis of FITC-labeled microbeads. To measure the phagocytosis of microbeads in the lung in vivo, mice were first lightly anesthetized with pentobarbital sodium by intraperitoneal injection. The trachea was exposed, and 50 µl of FITC-labeled microbeads (5 µl of beads suspended in a total vol of 45 µl of PBS, for a total of 5 × 107 beads) were injected into the trachea under direct visualization. The latex microbeads (Fluorebrite carboxylate microspheres) averaged 1.7 µm in diameter and were obtained from Polysciences (Warrington, PA). The mice were allowed to recover from anesthesia, with their position changed frequently until ambulating. One and four hours after the intratracheal instillation of beads, mice were killed, and alveolar cells recovered by whole lung lavage. The percentage of AM that had engulfed beads and the phagocytic index were determined by microscopic counting of cytospin preparations, as described below.
Isolation of alveolar macrophages. Mice and 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 (rats) or serial 1-ml aliquots (mice) 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× balanced salt solution (BSS; 1× BSS is 140 mM NaCl, 5 mM KCl, 0.48 mM NaH2PO4, 2.02 mM Na2HPO4, 6 mM glucose, and 10 mM HEPES). The cell suspension was washed twice 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. In selected experiments, AM were fluorescently labeled according to the manufacturer's protocol using a Vybrant 5- (and -6) carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) cell tracer kit (Molecular Probes, Eugene, OR).
Isolation and culture of rat AEC. Rat type II AEC were isolated by elastase cell dispersion and IgG panning (12). Briefly, the rats were anesthetized, the trachea was cannulated, and the pulmonary circulation was perfused free of blood with BSS at 4°C. After multiple whole lung lavages with EGTA (1 mM) in BSS, 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 expressed high-level of ICAM-1 on the apical cell surface (10).
Phagocytosis of FITC-labeled microspheres. Rat type II AEC were allowed to spread in monolayer culture for 2 days. Murine anti-rat ICAM-1 F(ab')2 fragments (0.5 µg/ml; Seikagaku, Tokyo, Japan) or control F(ab')2 fragments at the same concentration [Chrompure mouse IgG F(ab')2; Jackson ImmunoResearch Laboratories] were then added to the cultures to block AEC ICAM-1. The monolayers were washed to remove unbound antibody fragments before rat AM were added (5 × 104 cells/well). FITC-labeled latex microspheres were added for 1 h before fixing. The cultures were then viewed on a Nikon Labphot 2 immunofluorescence microscope. The proportion of AM containing beads and the number of beads in each AM were determined by an observer blinded to the experimental conditions. Four replicate wells were evaluated for each condition. In each well, the fraction of cells containing labeled beads and the phagocytic index were determined by microscopic counting of at least 200 cells in random high-power fields. The phagocytic index was calculated as
PI = [proportion of AM with beads] × [mean number of beads per positive AM] To determine whether cross-linking ofAM planar chemotaxis. Monolayers of rat type II AEC were established in four-well Lab-Tek slides. After 2-3 days, when the cells had spread and expressed abundant ICAM-1 (10), murine anti-rat ICAM-1 F(ab')2 fragments or control F(ab')2 fragments were added. The monolayers were washed to remove unbound antibody fragments. The slides were tilted by ~20°, and rat AM were added to the dependent end of each well. The AM were allowed to adhere before the slide was returned to horizontal. To provide a chemotactic stimulus, recombinant murine GM-CSF (granulocyte/macrophage colony-stimulating factor; 1 ng/ml) was incorporated into a pellet made from Hydron casting solution (Interferon Sciences) (28). Aliquots of sterile solution were mixed with GM-CSF or saline and polymerized overnight under ultraviolet light. Hydron pellets (with or without GM-CSF) were added to each well at the end opposite to the AM. The slides were incubated at 37°C for 24 h and were fixed and viewed on a Nikon Labphot 2 immunofluorescence microscope. AM were identified by staining with FITC-conjugated Bandeiraea simplicifolia lectin 1 (Sigma). This lectin binds >95% of rodent AM and does not bind AEC (27). In some experiments, identical results were obtained using AM that had been fluorescently labeled using a Vybrant CFDA SE cell tracer kit (Molecular Probes; used according to the manufacturer's protocol). The slides were scored for the number of AM that had migrated 50 mm from the end of the well to which the AM had been adhered. Controls included Lab-Tek chambers with no Hydron pellet and chambers with Hydron pellets without GM-CSF.
Cytoskeletal disruption and phalloidin staining. Disruption of the actin cytoskeleton of AEC in primary cultures was accomplished by treatment of day 3 AEC monolayers with cytochalasin B (5 or 10 µg/ml; Sigma) for 1 h. Horizontal chemotaxis of AM over the surface of cytochalasin B-treated and control AEC monolayers was determined as described above. The AEC were washed extensively before the addition of AM to remove cytochalasin B. Collapse of the AEC actin cytoskeleton was confirmed by staining with rhodamine-labeled phalloidin (Molecular Probes) according to the manufacturer's recommendations. AEC monolayers remained intact after 24 h. An additional control experiment confirmed that carry-over of residual cytochalasin B did not directly impact AM chemotaxis. AEC were treated with cytochalasin B (10 µg/ml) or control medium, as above. After being washed ×3, fresh medium was placed on the AEC for 1 h. The media were then harvested. AM were placed in a Boyden chamber in AEC conditioned media, with GM-CSF added to the bottom well (10 ng/ml). AM migration was determined over 4 h (21).
Statistics. Data are expressed as means ± SE and compared with one-way analysis of variance with the Tukey-Kramer multiple comparisons test, using the InStat software program (version 3.01 for Windows 95; GraphPad Software, San Diego, CA). Data were considered statistically significant if P < 0.05.
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RESULTS |
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Phagocytosis of latex microspheres by AM in vivo and in vitro.
To determine the contribution of alveolar epithelial ICAM-1
to the phagocytic activity of macrophages within the alveolar space,
wild-type and ICAM-1/
mice were inoculated via the trachea with
FITC-labeled microspheres. After 1 and 4 h,
bronchoalveolar lavage was performed to recover AM. Previously, we
found that equivalent numbers of AM were recovered from wild-type and
ICAM-1
/
mice (20). Similarly, after inoculation with
FITC-labeled microspheres, the total number of AM returned did not
differ between the two groups of mice (1.82 ± 0.39 × 105 wild-type mice vs. 1.97 ± 0.35 × 105 ICAM-1
/
mice). However, the fraction of AM that had
engulfed beads and the phagocytic index were reduced significantly in
the ICAM-1
/
mice compared with the wild-type mice at both
time points (Fig. 1). Thus in
the absence of ICAM-1, efficiency of phagocytosis within the lung was
impaired.
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Effect of cross-linking AM 2-integrins on phagocytic
activity.
Enhanced phagocytosis in the presence of AEC ICAM-1 might be a
consequence of activation of the AM through ICAM-1-mediated ligation of
2-integrins on the AM surface. To explore this
possibility, AM in suspension were incubated with a primary anti-rat
2-integrin monoclonal antibody, followed by a
cross-linking polyclonal goat anti-murine secondary antibody. This
approach has been used previously to demonstrate priming of leukocytes
for respiratory burst activity through
2-integrins
(19, 26). FITC-labeled microbeads were added to the cell
suspensions, and AM phagocytic activity was determined after 1 h
by microscropic counting. Compared with untreated AM, cross-linking of
CD18 ligands on the AM increased neither the %AM containing
beads nor the phagocytic index (Table
1). In fact, in the presence of
either both antibodies or the secondary antibody alone, there were
modest but significant decreases in phagocytic activity. These data
suggest that the contribution of AEC ICAM-1 does not simply involve
cross-linking of receptors on the AM cell membrane but may specifically
involve localization of ICAM-1 on the AEC surface.
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Effect of inhibition of ICAM-1 on AM adhesion to AEC
monolayers.
We next examined the extent to which AM adherence to AEC was inhibited
by blocking AEC ICAM-1. AEC monolayers were exposed to
F(ab')2 fragments of control antibody or to anti-rat ICAM-1 F(ab)'2 fragments before the addition of AM. Neutralization
of AEC ICAM-1 led to only modest inhibition of AM adhesion to AEC (Fig.
3). Thus there are additional alternative
pathways for AM adhesion to AEC beyond those mediated by ICAM-1.
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Role of AEC ICAM-1 in planar chemotaxis of AM.
To investigate the interaction between AEC and AM during migration of
AM over the alveolar surface, we measured planar chemotaxis of AM (Fig.
4A). The chemotactic signal
chosen was GM-CSF, a potent AM chemotaxin in traditional Boyden chamber
experiments (21). There was no migration of AM over tissue
culture-treated plastic toward a pellet containing GM-CSF (data not
shown). Cross-linking AM 2-integrins did not allow
migration of AM over tissue culture-treated plastic in response to
GM-CSF. Nor was there migration of AM over AEC in the absence of
chemotactic stimulus when GM-CSF was omitted from the Hydron pellet or
when no pellet was placed in the culture. In contrast, there was
significant GM-CSF-induced migration of AM over AEC monolayers.
However, when AEC ICAM-1 had been blocked, AM planar chemotaxis in
response to GM-CSF was reduced significantly compared with monolayers
treated with control F(ab')2 fragments (Fig.
4B). Thus inhibition of ICAM-1-mediated interactions between AM and AEC resulted in reduced migration of AM over the AEC surface in
response to chemotactic signals.
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Effects of disruption of the AEC cytoskeleton on AM planar
chemotaxis over AEC monolayers.
We have found previously that ICAM-1 on the surface of AEC in primary
culture is associated with the actin cytoskeleton (3). To
determine the importance of the AEC cytoskeleton for AM planar chemotaxis, AEC monolayers were treated briefly with cytochalasin B to
disrupt the actin cytoskeleton and washed extensively before the
addition of AM. Staining with rhodamine-phalloidin confirmed that this
brief exposure to cytochalasin B was sufficient to influence the
cytoskeleton transiently without disrupting the monolayer (data not
shown). Migration of AM over the surface of cytochalasin B-pretreated
AEC monolayers was significantly impaired compared with migration over
control AEC (Fig. 5). We also determined
that AM chemotaxis in a Boyden chamber in response to GM-CSF
(21) was not impaired by exposure to the small amount of
residual cytochalasin B that might be left in the wells (data not
shown). Thus these data indicate that the actin cytoskeleton of AEC
makes an important contribution to AM movement over the AEC surface.
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DISCUSSION |
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There are three salient points from these experiments. First, phagocytosis of particulates by AM in vitro and in vivo is enhanced by interactions with ICAM-1 on the AEC surface. Second, ICAM-1 on AEC enhances mobility of AM over the epithelial surface. Finally, the cytoskeleton of the AEC makes an important contribution to AM planar chemotaxis. Together, these results provide new information to explain the role of ICAM-1 on the alveolar epithelial surface and expand the role of AEC in determining the activity of inflammatory cells in the lung.
ICAM-1 is expressed in abundance on type I AEC in the normal,
uninflamed lung. In many other cell types, ICAM-1 is induced only in
the setting of acute inflammation, following exposure to inflammatory
cytokines. In contrast, expression of ICAM-1 by type I AEC is
constitutive and is a function of cellular differentiation both in the
adult (10, 22) and during fetal development
(1). Previously, the significance of such high-level
expression of ICAM-1 on the alveolar surface has not been clear. Likely
explanations have focused on leukocyte recruitment or adhesion. Initial
studies with ICAM-1/
mice demonstrated impaired neutrophil
recruitment in response to chemical peritonitis and diminished
leukocyte recruitment to the skin, manifest as reduced contact
hypersensitivity (29). These investigators also found that
ICAM-1
/
splenocytes were ineffective stimulators in a mixed
leukocyte response (29). However, the situation in the
lung is less clear. ICAM-1-mediated interactions with endothelial cells
are necessary for optimal leukocyte recruitment in response to
gram-positive organisms in the lung (6). However, AEC
ICAM-1 is not essential for the recruitment of leukocytes to the lung
in the setting of gram-negative pneumonia (18, 20). Nor,
as we have shown, is ICAM-1 essential for the adherence of AM to AEC.
The present study indicates a different role for AEC ICAM-1 in the
alveolar space. By promoting planar mobility of AM over the AEC surface
to facilitate the pursuit of particulates that have entered the lung
AEC ICAM-1 is critical for the effector function of AM.
AM are the resident inflammatory cells in the peripheral lung. They play important roles as effector cells that engulf and kill invading microorganisms. They also serve as sentinel cells that secrete chemokines to recruit and activate other inflammatory cells in response to pathogen invasion. The alveolar space is an enormous site of interaction of the body with the outside world. It is constantly exposed to particulate antigens and microbes in the thousands of liters of air inhaled each day. The important role of AM for early responses to pathogens in the lung has been confirmed in experiments in which ablation of AM, followed by inoculation with pathogens, leads to more severe pneumonia and increased mortality (5, 15). Normal humans aspirate organisms in their oral secretions each night during sleep (16). When the number of organisms is small, or they are of no great virulence, resident AM alone are able to clear the microbes without recruiting other inflammatory cells and impairing the gas exchange function of the lungs. However, when there are increased numbers of pathogens or if the pathogens are particularly virulent, AM recruit other phagocytes, such as neutrophils, to control the infection (30).
These studies extend our previous observations, demonstrating increased
mortality of ICAM-1/
mice in the setting of pneumonia due to
Klebsiella pneumoniae (20). In the absence of
ICAM-1, there was increased growth of organisms within the lung and
diminished phagocytosis and killing of organisms by AM, leading to
decreased survival of the ICAM-1 mutant mice compared with wild-type
controls. In vitro experiments conducted as part of those studies were
consistent with a major effect on AM phagocytosis of organisms, leading
to diminished numbers of intracellular organisms available for killing. The present study suggests that impaired AM mobility in the absence of
AM-AEC interactions mediated by ICAM-1 is a primary defect that can
lead to impaired antimicrobial activity. This result suggests that AM
phagocytosis of a variety of different infectious agents will be
supported by AEC ICAM-1, although thus far, data are only available
concerning phagocytosis of latex microbeads and gram-negative
bacteria. It is also possible that ICAM-1 expressed on type I
AEC contributes to microbial killing in additional ways. Previous
studies have demonstrated that cross-linking of CD11b/CD18 on the
surface of phagocytes using a combination of its ligand (ICAM-1) and
anti-ICAM-1 antibodies primes phagocytes for increased respiratory
burst and release of reactive oxygen intermediates (19, 24,
26). Similarly, when rat AM were adhered to recombinant soluble
ICAM-1 immobilized on plastic dishes, there was increased activation of
nuclear factor-
B and increased production of tumor necrosis factor,
compared with cells in control dishes without immobilized ICAM-1
(25). Thus ICAM-1 is likely to promote killing of microbes
by phagocytes in the lung by multiple mechanisms.
To fulfill its roles as effector cells and sentinels, it is critical that AM be able to move to encounter pathogens. In the normal lung there are approximately the same number of AM as alveoli. Because there is approximately one AM for each alveolar unit, each macrophage must be responsible for an area considerably greater than itself (30) and must move not through tissue but across a surface that is covered with phospholipid (pulmonary surfactant). Our studies indicate that constitutively expressed ICAM-1 on the AEC surface is an important facilitator of AM mobility.
ICAM-1 is a transmembrane receptor with a short cytoplasmic tail that can interact with the actin cytoskeleton (9, 32). We have found previously that ICAM-1 expressed by rat AEC in primary culture is associated with the cytoskeleton and that this association is significantly greater than in rat pulmonary artery endothelial cells in culture (3). The physiological implications of this interaction in AEC have not been defined. Carpen et al. (8) measured the mobility of large granular lymphocytes over lipid bilayers in which they had incorporated either normal ICAM-1 (extending through the bilayer) or ICAM-1 bound to the bilayer via a glycosyl phosphatidylinositol (GPI) linkage and thus free to move within the lipid bilayer. They found diminished mobility of the lymphocytes when modified ICAM-1 was mobile within the lipid bilayer due to the GPI linkage. We now have extended these observations by demonstrating that AM planar chemotaxis over the surface of AEC is reduced by pretreatment of the AEC with cytochalasin B to collapse the actin cytoskeleton. Thus we propose a model in which ICAM-1 on AEC serves as a tether on which AM may pull as they migrate over the epithelial surface. Interactions of ICAM-1 with the AEC cytoskeleton serve to fix these footholds in place to allow more efficient migration.
These studies have important implications for innate immune defense in the setting of acute lung injury. After many insults, type I cells are lost preferentially (2). In the absence of type I cells, AM may well adhere avidly to the alveolar wall (either to fibrin or to the basement membrane). However, this substrate may not support efficient mobility of the AM across the injured alveolar surface. For instance, although AM adhered very well to tissue culture-treated plastic dishes, they could not move over this surface in response to a chemotactic signal. In contrast, AM were able to migrate over the surface of AEC. These observations suggest that loss of type I cell support for inflammatory cell migration within the alveolar space may lead to increased susceptibility to pneumonia of individuals with acute lung injury.
There are several important considerations concerning the model systems used for these experiments. Potential limitations include the use of latex microbeads rather than bacterial pathogens for the phagocytosis studies, the use of both mice and rats, and the use of primary cultures of type II cells instead of type I cells for the in vitro studies. The FITC-labeled latex microbeads used for phagocytosis assays both in vivo and in vitro are 1.7 µm in diameter. This particle size is within the range of particles that reach the alveolar space (11). The in vivo experiments were performed using mutant mice lacking the ICAM-1 gene. Thus it was guaranteed that there was no ICAM-1 activity and there were no confounding effects of antibodies on AM activity. However, multiple cells in the lung can express ICAM-1, including macrophages and endothelial cells. Our in vitro experiments confirmed that the mechanism explaining the importance of ICAM-1 for AM activity in the lung involved interactions between AM and AEC that are mediated by AEC ICAM-1. These experiments were performed using rat type II AEC that had been placed in primary culture. The behavior of these cells in culture and their expression of a series of type I alveolar epithelial cell characteristics over time in culture (23), including constitutive expression of abundant ICAM-1 (10), have been well described. Moreover, the interaction of ICAM-1 with the actin cytoskeleton in rat AEC in vitro has been characterized previously in these cells (3). Thus these cells are an appropriate model for the study of the effects of ICAM-1 expression on type I AEC.
In conclusion, we have found that phagocytosis of particulates by AM residing on the surface of AEC is impaired in the absence of AEC ICAM-1. Similarly, AM migration over the surface of AEC is impaired when ICAM-1 on the AEC is blocked or when the cytoskeleton of the AEC is disrupted. These observations help explain why ICAM-1 might be expressed in abundance on normal type I cells, why ICAM-1 should be associated with the cytoskeleton in these cells, and how loss of type I cells in the setting of acute lung injury leads to impaired host defense in the lung. They provide further evidence for the critical role of AEC as participants in the innate immune response in the normal lung.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. Jeffery L. Curtis and Bethany B. Moore for helpful suggestions after review of earlier versions of this manuscript.
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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, National Heart, Lung, and Blood Institute Grant HL-64558, Research Enhancement Award Program funds from the Department of Veterans Affairs, and by a Career Investigator Award from the American Lung Association (to R. Paine III).
Address for reprint requests and other correspondence: R. Paine III, Pulmonary Section (111G), Veterans Affairs Medical Center, 2215 Fuller Rd., Ann Arbor, MI 48105 (E-mail: rpaine{at}umich.edu).
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. Section 1734 solely to indicate this fact.
First published February 22, 2002;10.1152/ajplung.00430.2001
Received 2 November 2001; accepted in final form 15 February 2002.
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