Role of alveolar epithelial cell intercellular adhesion molecule-1 in host defense against Klebsiella pneumoniae

Aidan D. O'Brien1,2, Theodore J. Standiford1, Kathy A. Bucknell1, Steven E. Wilcoxen1, and Robert Paine III1,2

1 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor 48109; and 2 Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48105


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF) or interleukin-1beta (IL-1beta ), to promote the recruitment and activation of additional leukocytes, including neutrophils from the vascular space.

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-8. 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.

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 chi 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

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.


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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.


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Fig. 2.   Numbers of K. pneumoniae in the lungs of wild-type and ICAM-1 mutant mice. ICAM-1 mutant and wild-type mice were inoculated intratracheally with 5 × 102 CFU K. pneumoniae. After 24 h, the animals were killed, and K. pneumoniae CFU were determined in lung homogenates. Data are expressed as log CFU/animal (means ± SE, n = 14 mice in each group). * P = 0.001 compared with wild-type mice infected with K. pneumoniae.

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.


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Fig. 3.   Lung myeloperoxidase (MPO) activity in ICAM-1-deficient mice with Klebsiella pneumonia. ICAM-1 mutant and wild-type mice were inoculated intratracheally with 5 × 102 CFU K. pneumoniae or with sterile saline. MPO activity was measured in total lung homogenates 24 h after challenge. Data are expressed as mean MPO activity ± SE (n = 14 mice in infected groups and n = 3 mice in saline control groups). MPO activity in lung homogenates of uninfected wild-type and ICAM-1 mutant mice was 6.98 ± 0.9 and 4.99 ± 0.9, respectively. * P = 0.002 for infected ICAM-1 mutant mice compared with infected controls.

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.


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Fig. 4.   Cell counts in bronchoalveolar lavage of ICAM-1-deficient mice with Klebsiella pneumonia. ICAM-1 mutant and wild-type mice were inoculated intratracheally with 5 × 102 CFU K. pneumoniae or with sterile saline. After 24 h, the animals were killed, and bronchoalveolar lavage was performed. Total number of lavageable cells was determined, and numbers of alveolar macrophages (AM) and neutrophils were calculated from Diff-Quik-stained cytospins (n = 5 mice/ infected group and n = 2 mice/saline control group). Data are expressed as means ± SE.

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.


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Fig. 5.   Effect of neutralization of intra-alveolar ICAM-1 on lung bacterial growth and MPO activity in rats inoculated intratracheally with K. pneumoniae. Sprague-Dawley rats were inoculated intratracheally with K. pneumoniae (107 CFU/animal) mixed with antibodies (250 µg/animal) in a total of 200 µl. After 24 h, the animals were killed, and bacterial CFU (A) and MPO activity (B) were determined in lung homogenates as described in MATERIALS AND METHODS. Control animals received F(ab')2 control antibody, whereas anti-ICAM animals received F(ab')2 fragments of monoclonal antibody (MAb) 1A29 specific for rat ICAM-1. Data are means ± SE; n = 7 animals in each group. * P = 0.029. ** P = 0.156.

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.


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Fig. 6.   Effect of alveolar epithelial cells (AEC) on AM antimicrobial activity in vitro. Rat AM were cultured in control plastic wells or in wells containing rat AEC monolayers for 1 h. K. pneumoniae then were added. After 1-h further incubation, phagocytosis (A) and killing (B) of K. pneumoniae were determined as described in MATERIALS AND METHODS. Data are expressed as means ± SE from 6 separate experiments for phagocytosis and 4 separate experiments for killing. * P < 0.0001 compared with phagocytosis of AM alone in the absence of AEC. ** P = 0.01 compared with AM bacterial killing in the absence of AEC.

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.


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Fig. 7.   Effect of blocking of AEC ICAM-1 on AM antimicrobial activity in vitro. AEC monolayers were exposed to F(ab')2 fragments of murine anti-rat ICAM-1 (MAb 1A29) or to control MAb for 1 h. Monolayers were washed to remove unbound antibody before the addition of AM and K. pneumoniae. AM phagocytosis (A) and killing (B) of K. pneumoniae were determined as described in MATERIALS AND METHODS. Data are expressed as means ± SE from 4 independent experiments. * P < 0.0001 compared with AM + AEC in the presence of control IgG. ** P = 0.025 compared with AM with AEC in the presence of control IgG.

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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Fig. 8.   Effect of blocking of AEC ICAM-1 on neutrophil antimicrobial activity in vitro. AEC monolayers were exposed to F(ab')2 fragments of murine anti-rat ICAM-1 (MAb 1A29) or to control MAb for 1 h. Monolayers were washed to remove unbound antibody before the addition of neutrophils and K. pneumoniae. Neutrophil phagocytosis of K. pneumoniae was determined as described in MATERIALS AND METHODS. Data are presented as means ± SE of 4 wells in an experiment, representative of 3 independent experiments. * P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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-gamma (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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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