Modulation of airway inflammation and bacterial clearance by epithelial cell ICAM-1

Alicia L. Humlicek,1,* Liyi Pang,2,* and Dwight C. Look1

1Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242-1081; and 2Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110-1093

Submitted 4 March 2004 ; accepted in final form 20 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Many cell types in the airway express the adhesive glycoprotein for leukocytes intercellular adhesion molecule-1 (ICAM-1) constitutively and/or in response to inflammatory stimuli. In this study, we identified functions of ICAM-1 on airway epithelial cells in defense against infection with Haemophilus influenzae. Initial experiments using a mouse model of airway infection in which the bacterial inoculum was mixed with agar beads that localize inflammation in airways demonstrated that ICAM-1 expression was required for efficient clearance of H. influenzae. Airway epithelial cell ICAM-1 expression required few or no leukocytes, suggesting that epithelial cells could be activated directly by interaction with bacteria. Specific inhibition of ICAM-1 function on epithelial cells by orotracheal injection of blocking antibodies resulted in decreased leukocyte recruitment and H. influenzae clearance in the airway. Inhibition of endothelial cell ICAM-1 resulted in a similar decrease in leukocyte recruitment but did not affect bacterial clearance, indicating that epithelial cell ICAM-1 had an additional contribution to airway defense independent of effects on leukocyte migration. To assess this possibility, we used an in vitro model of neutrophil phagocytosis of bacteria and observed significantly greater engulfment of bacteria by neutrophils adherent to epithelial cells expressing ICAM-1 compared with nonadherent neutrophils. Furthermore, bacterial phagocytosis and killing by neutrophils after interaction with epithelial cells were decreased when a blocking antibody inhibited ICAM-1 function. The results indicate that epithelial cell ICAM-1 participates in neutrophil recruitment into the airway, but its most important role in clearance of H. influenzae may be assistance with neutrophil-dependent bacterial killing.

leukocyte adhesion; Haemophilus influenzae; mouse; neutrophils; phagocytosis


RESPIRATORY EPITHELIUM CONTAINS several constitutive innate mechanisms (e.g., mucociliary clearance, antibacterial molecules, resident macrophages) that constantly defend the airway against pathogens (10, 50). When bacteria overwhelm these constitutive defense mechanisms, an inflammatory response is activated in the airway that is critical for antibacterial defense and characterized by recruitment of additional leukocytes, particularly neutrophils, to sites of infection. Targeting leukocytes to bacterial locations requires both chemoattractant and adhesion molecules that direct leukocyte migration out of the vasculature through the endothelial cell layer, through intervening cell and connective tissue layers, and finally through the epithelial cell layer into the airway itself (25). Once in contact with bacteria in the airway, neutrophils participate in clearance of infection through processes that include bacterial phagocytosis, bacterial killing through antibacterial proteins and respiratory burst production of reactive oxygen intermediates, and postphagocytosis apoptosis (4). Multiple cell types and signals tightly orchestrate leukocyte recruitment and activation to efficiently control and eliminate infection yet minimize potential damage to the airway and lung. However, in some pulmonary disorders (e.g., cystic fibrosis, chronic bronchitis), the inflammatory response appears to be out of proportion with that needed to defend the airway and results in damage and dysfunction of the involved airways or lungs (2, 12). A better understanding of the coordination of factors that results in an effective response to bacteria in the airway may allow for new strategies to improve airway defense against bacteria, while limiting airway and lung damage.

Current evidence indicates that airway epithelial cells interact directly with bacteria in the environment and have the capability to activate, as well as participate, in inflammatory responses that defend the airway (14, 19). Increased cell surface expression of the adhesive glycoprotein for leukocytes intercellular adhesion molecule-1 (ICAM-1) is one mechanism for epithelial cell regulation of the airway response to bacterial pathogens (11). Expression of ICAM-1 by airway epithelial cells may be mediated both indirectly by epithelial cell communication with other cell types through cytokines and/or directly by epithelial cell interaction with bacteria (1, 11, 21, 49). Interaction of ICAM-1 with CD18/{beta}2-integrin-containing counterreceptors on leukocytes is a crucial mechanism for leukocyte adhesion to airway epithelial cells (21, 49). Under some conditions, ICAM-1 may also assist in leukocyte bacterial killing functions (20, 37, 40). However, ICAM-1 can be expressed on many cell types in addition to epithelial cells, including endothelial cells and fibroblasts (9, 16). Whether ICAM-1 expression serves similar functions on all cell types in the airway is unclear.

The present study focuses on defining the roles for epithelial cell ICAM-1 in defending the airway against bacteria. Initial experiments characterized a mouse model of pulmonary infection with the respiratory pathogen Haemophilus influenzae, which uses agar particles to impair neutrophil-dependent bacterial clearance and localize inflammation to airways in the lung. In this model, early airway neutrophil recruitment and efficient bacterial killing required ICAM-1 expression. Application of ICAM-1 blocking antibodies to epithelial cells by injection into the airway resulted in impairment of neutrophil recruitment that was similar to effects seen with inhibition of endothelial cell ICAM-1 function. However, airway injection of anti-ICAM-1 antibodies reduced bacterial clearance from the airway, suggesting an additional role for ICAM-1 on the epithelial cell surface. Using an in vitro epithelial cell culture system, we observed epithelial cell ICAM-1 augmentation of neutrophil phagocytosis and killing of bacteria. Together, our results support the concept that ICAM-1 on epithelial cells participates in neutrophil recruitment during airway infection, but its most important role in clearance of H. influenzae from the airway may be assistance with neutrophil-dependent bacterial killing.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bacteria preparation. Nontypable H. influenzae strain 12 was grown on chocolate blood agar supplemented with 1% Isovitalex (BD Scientific, Sparks, MD) or in brain-heart infusion broth supplemented with heme and NAD as described previously (11). Aerated, log-phase bacteria were prepared by inoculation of colonies from a fresh plate into 50 ml of broth, and the cultures were incubated with rotary shaking at 37°C until a turbidimetric estimate (optical density at 600 nm = 0.6–0.8) of ~3–4 x 109 colony-forming units (CFU)/ml was achieved (1.5–2 h). We quantitated bacteria by plating serial dilutions of broth cultures on chocolate blood agar solid medium.

Mouse airway infection model. Animal studies were performed under a protocol approved by the University of Iowa Institutional Animal Care and Use Committee. H. influenzae was incorporated into and mixed with amorphous agar particles (ranging in size from 30 to 200 µm), producing a suspension containing bacteria at ~107–10 CFU/ml as described previously (11, 24). Four-week-old C57BL/6J mice (Taconic, Germantown, NY), a strain highly susceptible to pulmonary bacterial infection (46), were housed under pathogen-free conditions. Each mouse was anesthetized with an intramuscular injection of 87.5 mg/kg of ketamine HCl and 12.5 mg/kg of xylazine HCl, placed in an intubation apparatus (13), and orotracheally intubated with a 22-gauge intravenous catheter. A 50-µl volume of PBS, control agar particles, or bacteria-agar particle suspension at 37°C (followed by 100 µl of air) was injected into the airway, and the mouse was allowed to recover. We performed the following experimental modifications: 1) we injected mice intravenously with 3 mg/kg nitrogen mustard (mechlorethamine hydrochloride; Sigma-Aldrich, St. Louis, MO) daily for the 2 days before infection or intraperitoneally (IP) with 4 ml/kg rabbit anti-mouse neutrophil serum (Accurate Chemical and Scientific, Westbury, NY) daily for the 2 days before and during infection, and we verified leukocyte depletion by quantitation of total blood leukocyte counts with a hemocytometer and differential counts by modified Wright's staining (Leukostat; Fisher Scientific, Pittsburgh, PA); 2) we examined same-strain wild-type and ICAM-1-null mice (a gift from J. Gutierrez-Ramos, Millennium Pharmaceuticals, Cambridge, MA) (51); and 3) we pretreated mice by IP or intratracheal (IT) injection of 10 mg/kg control or anti-ICAM-1 antibodies before bacterial inoculation.

Lung staining and immunohistochemistry. After a specified duration of infection, mice were anesthetized and then euthanized by cervical dislocation, the lungs were exposed, and the pulmonary vascular system was flushed via the right ventricle with sterile saline. For tissue staining, the trachea was cannulated with a 22-gauge intravenous catheter, and the right lung was inflated under 25 cmH2O pressure and fixed with 10% buffered formalin for 18 h. Paraffin-embedded 6-µm serial lung sections were stained with hematoxylin and eosin (to assess inflammatory cell influx) and were immunohistochemically stained for ICAM-1 expression with hamster MAb 3E2 (PharMingen, San Diego, CA) against mouse ICAM-1 (to assess cell-specific expression) as described previously (11). Slides were mounted for microscopy (Leitz Diaplan; Wild Leitz USA, Rockleigh, NJ), and images were acquired with a digital charge-coupled device camera system (SPOT; Diagnostic Instruments, Sterling Heights, MI) interfaced with SPOT software version 2.2.

Lung infection quantitation. One segment of the right lung was ligated at its root and removed under sterile conditions, minced with a razor blade in a petri dish with 0.2–0.5 ml of PBS, and homogenized with a glass tissue grinder (Kontes Glass, Vineland, NJ). Serial dilutions of the homogenate were then inoculated aseptically onto solid agar media for culture and quantification of lung bacterial load as a measure of pulmonary clearance. Preliminary experiments verified a homogeneous bacterial load in all segments of both lungs in our animal model.

Leukocyte recruitment assays. Airway leukocytes were isolated by bronchoalveolar lavage (BAL) of the left lung with 0.5-ml aliquots of PBS followed by quantitation of total leukocyte counts with a hemocytometer and differential counts by modified Wright's staining of cytospin preparations. Total lung neutrophil levels were assessed by harvest of two right lung lobes, determination of lung sample weight, and homogenization by tissue grinder at 4°C in 0.5 ml of 50 mM sodium phosphate, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide. Samples subsequently underwent multiple freezing and thawing and then sonication with a sonic dismembrator (Fisher Scientific, Pittsburgh, PA) set at 30%. Samples were cleared by centrifugation at 15,000 g for 15 min, and we determined neutrophil myeloperoxidase (MPO) activity by incubating 50 µl of homogenate with 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% H2O2 in a reaction volume of 1.5 ml as described previously (3). Absorbance at 460 nm was determined over time with activity determined by calculation of {Delta}absorbance·min–1·mg–1 of sample.

Antibody F(ab')2 and Fab preparations. Rat IgG2b MAb clone YN1/1.7.4 against mouse ICAM-1 (a gift from M. Dustin, New York University) (45) and isotype-matched control antibody were prepared from supernatants generated from hybridoma cells grown in DMEM with 10% FCS and were purified with a protein G-Sepharose column (Amersham Biosciences, San Francisco, CA). Antibodies were digested by incubation with porcine gastric mucosa pepsin (Roche Applied Science, Indianapolis, IN) in 50 mM sodium chloride and 70 mM sodium acetate, pH 4.0, at 37°C for 18 h. F(ab')2 fragments were purified by application to a Sephacryl S-200 column (Amersham Biosciences) driven by fast performance liquid chromatography with elution in PBS, pH 7.4. Fractions were analyzed by SDS-PAGE and Coomassie blue gel staining, and protein concentrations were determined by a bicinchoninic acid reaction method (Pierce, Rockford, IL). IP and IT injection of F(ab')2 fragments was performed to verify that antibodies did not elicit an inflammatory response. Mouse MAb clone R6.5 Fab fragments against human ICAM-1 were gifts from R. Rothlein and C. Wegner (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT) (44).

Neutrophil-bacteria interaction assays. Human tracheobronchial epithelial (hTBE) cells were obtained under a protocol approved by the University of Iowa Institutional Review Board. Epithelial cells were isolated from tracheal and bronchial mucosa by enzymatic dissociation and cultured in Laboratory of Carcinogenesis (LHC)-8e medium on 12-mm circular coverslips coated with collagen and albumin in 24-well tissue culture plates (1, 15, 22). Cells were incubated with 100 units/ml recombinant human IFN-{gamma} (a gift from Genentech, San Francisco, CA) for 24 h to induce ICAM-1 expression as described previously (1, 21). Neutrophils were isolated from human whole blood at >99% purity with Polymorphprep (Greiner Bio-One, Longwood, FL), and 7 x 105 neutrophils were left in suspension or added to epithelial cells in HBSS containing calcium and magnesium and supplemented with 0.15% dextrose, 1% BSA, 1% autologous serum, and 100 units/ml TNF-{alpha} (a gift from Genentech). After incubation for 30 min at 37°C to allow neutrophil adherence, 7 x 107 CFU Pseudomonas aeruginosa expressing green fluorescence protein (GFP, a gift from P. Singh and M. Parsek, University of Iowa) (7, 43) were incubated with neutrophils for 10 min. Samples were mounted for confocal microscopy (Zeiss 510; Carl Zeiss, Thornwood, NY), and images were acquired with integrated digital image capture interfaced with Laser Scanning Microscope 510 software version 3.2. The percentage of neutrophils interacting with bacteria was determined for >200 neutrophils in three samples. In experiments in which numbers of bacteria inside neutrophils were determined, epithelial cells were cultured on 96-well tissue culture plates, and adjustments for the difference in epithelial cell number included using 3 x 105 neutrophils and 3 x 107 CFU P. aeruginosa expressing GFP. In some experiments, epithelial cells were left untreated or incubated with IFN-{gamma}, or epithelial cells were incubated with 50 µg/ml of control or anti-ICAM-1 blocking antibodies before interaction with neutrophils. The number of bacteria inside >100 neutrophils/sample in five samples was counted under fluorescence microscopy (Leitz Diaplan, Wild Leitz USA), and results are expressed as the mean number of bacteria/neutrophil.

Neutrophil viability assay. We quantified dead and live neutrophil numbers by detection of plasma membrane permeability to ethidium homodimers in dead cells and intracellular esterase activity in live cells with a commercial fluorescence-based viability and cytotoxicity kit (Molecular Probes, Eugene, OR). At least five random low-power fields containing >100 adherent or nonadherent neutrophils were counted.

Neutrophil bacterial killing assay. Monolayers of hTBE cells in 96-well tissue culture plates coated with collagen and albumin were treated with 100 units/ml IFN-{gamma} for 24 h to induce ICAM-1 expression. Epithelial cells were incubated without or with 50 µg/ml antibodies for 30 min, and then 3 x 105 human neutrophils (isolated as outlined above) were added in HBSS containing calcium and magnesium and supplemented with 0.15% dextrose, 1% BSA, 1% autologous serum, and 100 units/ml TNF-{alpha}. In some experiments, unbound antibodies were removed from epithelial cells by washing before addition of neutrophils, whereas in other experiments antibodies were not removed during the subsequent adherence period (21, 31, 32). Plates underwent centrifugation at 350 g for 4 min followed by incubation for 30 min at 37°C to facilitate neutrophil adherence to epithelial cells. Approximately 103 H. influenzae were added to each well, and plates underwent centrifugation at 800 g for 4 min followed by incubation for predetermined durations to allow neutrophil killing of bacteria. Cells (but not bacteria) were then lysed by addition of one volume of 2% saponin for 10 min combined with scraping and mixing. Extracellular bacteria were not removed before determination of bacterial killing. Serial dilutions of the initial inoculum and cell lysates were inoculated aseptically onto solid agar media for culture and quantification of viable bacteria, and the percentage of bacteria killed based on the original inoculum was calculated as follows: (inoculum added – viable bacteria remaining)/inoculum added x 100.

Statistical analysis. For experiments involving comparison of two conditions, two-tailed unpaired Student's t-tests were used. Experiments with multiple comparisons were analyzed for statistical significance by one-way analysis of variance (ANOVA) for a factorial experimental design. The multicomparison significance level for the ANOVA was 0.05. If significance was achieved by one-way analysis, post-ANOVA comparison of means was performed by Scheffé's F-tests (52). Assays were performed two to four times to assure reproducible results, and P < 0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse model of airway infection with H. influenzae. To evaluate the in vivo pulmonary response to infection with H. influenzae, we examined a mouse model in which agar particles are combined with a bacterial inoculum and injected into the airway. In this model, bacterial infection results in induction of airway epithelial cell ICAM-1, increased expression of the CXC chemokines KC and macrophage inflammatory protein-2, and recruitment of leukocytes into the airway (11). Initial experiments confirmed that injection of H. influenzae into the airway resulted in recruitment of high levels of leukocytes (primarily neutrophils) into the lung (Fig. 1). However, pulmonary infection without agar particles resulted in predominant leukocyte recruitment into alveolar spaces, whereas the addition of agar particles localized inflammation primarily to areas in or near small airways. Furthermore, entrapment of agar particles in small airways impaired bacterial clearance at 24 and 48 h after inoculation (Fig. 2A), and chronic infection lasting for weeks could sometimes be established in animals. Impairment of bacterial clearance by agar particles was apparent after airway infection of mice with both large and smaller inoculums (Fig. 2B). On the basis of these results that establish the characteristics of this in vivo model of pulmonary infection with inflammation predominantly targeted to airways, we went on to use this system to examine the airway response to bacterial infection.



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Fig. 1. Agar airway particles affect inflammation location induced by Haemophilus influenzae. Nontypable H. influenzae strain 12 was suspended in PBS without or with agar particles, and ~108 colony-forming units (CFU) in 50 µl were inoculated into the trachea and bronchi of C57BL/6J mice. At 24 h after inoculation, the right lung was removed for histopathologic study, and lung sections were stained with hematoxylin and eosin (H & E) to visualize inflammation. Similar-sized airways were selected for photomicrographs to allow comparison. Bar size, 100 µm.

 


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Fig. 2. Agar airway particles affect pulmonary clearance of H. influenzae. H. influenzae was suspended in PBS without ({circ}) or with ({bullet}) agar particles, and ~108 CFU (A) or ~105 CFU (B) in 50 µl were inoculated into the trachea and bronchi of mice. At the indicated times after inoculation, the left lung was removed for bacterial quantitation to determine pulmonary bacterial clearance. Values are expressed as means ± SD (n = 4–8 in each group); *significant difference between animals infected without and with agar particles at the same time point.

 
Leukocyte requirement in airway defense against H. influenzae. To confirm a requirement for leukocytes in H. influenzae killing after airway infection, we induced neutropenia or leukopenia in mice before bacterial inoculation. Mice treated with nitrogen mustard developed >97% reduction in blood total leukocyte counts by the time of bacterial inoculation that persisted throughout the duration of infection. As a result of this treatment, total leukocytes (Fig. 3A) and neutrophils (Fig. 3B) in BAL fluid markedly decreased, and bacterial clearance was minimal (Fig. 3C). Under similar conditions, mice treated with anti-mouse neutrophil serum developed >85% reduction in blood neutrophil counts with little effect on other leukocyte subsets. This level of neutropenia resulted in a significant reduction in total leukocytes (Fig. 3A) and neutrophils (Fig. 3B) in BAL fluid and also inhibited bacterial clearance from the lung compared with animals treated with control antiserum (Fig. 3C). On the basis of previous observations in an isolated epithelial cell culture system in which we found direct ICAM-1 induction by bacteria-epithelial cell interaction (11), we also questioned whether leukocytes are required for airway epithelial cell ICAM-1 expression in response to bacteria. Using animals that had been treated with nitrogen mustard, we found that airway epithelial cell ICAM-1 expression was still detected in mice infected with H. influenzae (Fig. 4). Nitrogen mustard alone had no effect on pulmonary ICAM-1 expression (Pang and Look, unpublished observation). Thus leukocytes are required for bacterial clearance of H. influenzae in this model of bacteria-induced airway inflammation, but few or no leukocytes are required for airway epithelial cell ICAM-1 expression.



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Fig. 3. Leukocytes are required for bacterial clearance of H. influenzae. H. influenzae was suspended in PBS with agar particles, and ~108 CFU in 50 µl were inoculated into the trachea and bronchi of mice that had been pretreated with control antiserum ({bullet}), anti-mouse neutrophil serum to induce neutropenia ({square}), or nitrogen mustard to induce leukopenia ({circ}). At the indicated times after inoculation, the left lung underwent bronchoalveolar lavage (BAL) with quantification of total leukocytes (A) and neutrophils (B), and a portion of the right lung was removed for bacterial quantitation (C). In AC, values are expressed as means ± SD (n = 4 in each group); *significant difference from control animals at the same time point.

 


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Fig. 4. Leukocytes are not required for airway epithelial cell ICAM-1 expression. H. influenzae was suspended in PBS with agar particles, and ~108 CFU in 50 µl were inoculated into the trachea and bronchi of mice that had been pretreated with control diluent (left) or nitrogen mustard to induce leukopenia (right). A portion of the right lung was removed from animals (also used for results in Fig. 3) that were infected with 108 CFU of H. influenzae for 24 h. Serial lung sections were stained with H & E to visualize inflammation (top) or underwent immunochemical staining to visualize ICAM-1 (bottom). Similar-sized airways were selected for photomicrographs to allow for comparison. Bar size, 20 µm.

 
ICAM-1 requirement in airway defense against H. influenzae. To examine the role of ICAM-1 in airway clearance of bacterial infection, we infected the airways of mice without or with targeted deletion of the ICAM-1 gene with H. influenzae (51). These studies demonstrated that, compared with wild-type mice, mice without ICAM-1 had impaired clearance of H. influenzae that reached statistical significance after 96 h of infection (Fig. 5A). Other studies of respiratory infection suggest that decreased ICAM-1 expression or function may result in impaired leukocyte recruitment and/or antimicrobial activity, depending on the bacterial species and ICAM-1 manipulation method used (26, 27, 37, 39, 40). We worked to separate these possibilities by first assessing neutrophil recruitment into the airway that was induced by bacterial infection. Mice without ICAM-1 and infected with H. influenzae had significantly lower numbers of neutrophils in BAL fluid after 12 h of infection compared with wild-type animals (Fig. 5B). We assessed total lung neutrophil recruitment by determining whole lung MPO activity, and these results also indicate that lung neutrophil numbers were significantly decreased in ICAM-1 knockout mice after 12 h of infection (Fig. 5C). Indeed, the highest MPO activity in wild-type mice was detected at this time point and may reflect a critical time for pooling neutrophils into the lung for subsequent movement into the airway. Higher airway and lung neutrophil numbers were observed after 48 and 72 h of infection in ICAM-1 knockout mice (although difference in airway neutrophil numbers did not reach statistical significance), and this finding likely reflects a higher bacterial burden driving leukocyte recruitment into the lung through an ICAM-1-independent pathway at these time points. Differences in bacterial clearance appear to be independent of inoculum level because delayed clearance of H. influenzae was also seen after airway infection with lower numbers of bacteria (Fig. 5D). Although these results establish an important role for ICAM-1 in neutrophil recruitment and bacterial killing in the airway, they did not exclude ICAM-1 effects on leukocyte function. Furthermore, because ICAM-1 is expressed either constitutively or upon stimulation of many cells in the lung including vascular endothelial cells (21, 29, 31, 32, 44; and Humlicek and Look, unpublished observation), experiments in mice with gene deletion did not target the identification of a role for ICAM-1 on epithelial cells. Therefore, subsequent experiments focused on determining specific roles for ICAM-1 on epithelial cells in the airway response to bacterial infection.



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Fig. 5. ICAM-1 expression is required for efficient clearance of H. influenzae from the airway. H. influenzae was suspended in PBS with agar particles, and ~108 CFU in 50 µl were inoculated into the trachea and bronchi of C57BL/6J mice without or with homozygous targeted deletion in the gene for ICAM-1. At the indicated times after inoculation, a portion of the right lung was removed for bacterial quantitation to determine pulmonary bacterial clearance (A), the left lung underwent BAL with quantification of leukocyte subsets to determine neutrophil recruitment into the airway (B), and the rest of the right lung was removed for assay of myeloperoxidase (MPO) activity to assess neutrophil recruitment into the lung (C). A lower concentration of H. influenzae was also suspended in PBS with agar particles, and ~105 CFU in 50 µl were inoculated into the trachea and bronchi of C57BL/6J mice without or with homozygous targeted deletion in the gene for ICAM-1. At the indicated times after inoculation, a portion of the right lung was removed for bacterial quantitation (D). In AD, values are expressed as means ± SD (n = 4 in each group); *significant difference between ICAM-1 knockout mice ({circ} or open bars) compared with normal animals ({bullet} or solid bars) at the same time point.

 
Epithelial cell ICAM-1 assists with leukocyte recruitment and bacterial clearance. We used multiple experimental strategies to isolate effects of epithelial cell ICAM-1 on bacterial clearance that are independent of effects on leukocyte recruitment. The approach used in our mouse model of airway infection was based on previous reports in which IT antibody injection resulted in predominant effects on epithelial cells (29, 37). Immunohistochemical staining experiments of lung from infected animals that had been treated with anti-ICAM-1 antibodies confirmed that the predominant location of antibody binding was on endothelial cells after IP antibody injection and epithelial cells after IT injection (Fig. 6A). For subsequent experiments with the goal of selectively inhibiting epithelial cell ICAM-1 function, F(ab')2 fragments (rather than intact antibody) were used to avoid potential conflicting effects of binding of the antibody Fc portion to cells containing Fc receptors (particularly leukocytes such as neutrophils and macrophages) (29, 37). In addition, F(ab')2 fragments may be more effective than intact antibody for inhibition of ICAM-1 function (29, 30). Assessment of leukocyte recruitment into the airway after selective ICAM-1 inhibition revealed decreased BAL leukocytes in response to H. influenzae infection that was similar after IP or IT treatment with blocking antibodies to ICAM-1 (Fig. 6, B and C). However, IT treatment with anti-ICAM-1 resulted in significant impairment in bacterial clearance at 72 h compared with IP antibody treatment (Fig. 6, D and E). These results indicate that differences in leukocyte recruitment after inhibition of ICAM-1 with blocking antibodies do not account for differences in bacterial killing in our model system.



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Fig. 6. Epithelial cell ICAM-1 assists with bacterial clearance. A: H. influenzae was suspended in PBS with agar particles, and ~108 CFU in 50 µl were inoculated into the trachea and bronchi of mice that had been pretreated with intraperitoneal (IP) or intratracheal (IT) injection of PBS containing intact control or ICAM-1 antibodies (Ab). At 24 h after inoculation, the right lung was removed for histopathologic study. Lung sections were stained with hematoxylin and underwent immunochemical staining with anti-rat secondary antibody alone to localize primary Ab binding (arrowheads). Similar sized airways (a) and vessels (v) were selected for photomicrographs to allow for comparison. Bar size, 12 µm. H. influenzae was suspended in PBS with agar particles, and ~108 CFU in 50 µl were inoculated into the trachea and bronchi of mice that had been pretreated with IP (B) or IT (C) injection of control PBS or PBS containing control or ICAM-1 F(ab')2. At the indicated times after inoculation, the left lung underwent BAL with quantification of leukocytes to determine recruitment into the airway. D: using the animals presented in B with IP treatment, we removed a portion of the right lung for bacterial quantitation to determine pulmonary bacterial clearance. E: using the animals presented in C with IT treatment, we removed a portion of the right lung for bacterial quantitation to determine pulmonary bacterial clearance. In BE, labels are control PBS (black bar), control F(ab')2 (white bar), and ICAM-1 F(ab')2 (gray bar). Values are expressed as means ± SD (n = 4 in each group); *significant difference compared with animals treated with PBS alone at the same time point.

 
Airway epithelial cell ICAM-1 affects neutrophil antibacterial function. Previous reports indicate that constitutive alveolar epithelial cell ICAM-1 may function to allow phagocytosis of Klebsiella pneumoniae by macrophages and other leukocytes rather than, or in addition to, affecting recruitment to sites of infection (37, 38). The physical characteristics of the airway environment are very different from that inside alveoli, particularly with respect to constitutive expression of ICAM-1. Accordingly, we questioned whether local ICAM-1 induced on epithelial cells in the airway might function to modulate neutrophil antibacterial function against H. influenzae and other respiratory pathogens. To assess this possibility, we isolated human neutrophils, allowed them to adhere to monolayers of primary cultures of human airway epithelial cells that had been stimulated to express ICAM-1, and then exposed them to P. aeruginosa expressing high levels of GFP that allowed for bacterial tracking. During examination of cells under immunofluorescence microscopy, we noted that many bacteria colocalized with neutrophils that adhered to epithelial cells, whereas nonadherent neutrophils had few bacteria (Fig. 7A). We also observed few bacteria associated with epithelial cells, and confocal microscopy through the z-axis confirmed that most bacteria associated with neutrophils were at an intracellular location, indicating that phagocytosis had occurred (Humlicek and Look, unpublished observation). Quantitation of neutrophils confirmed there was a significantly higher percentage of adherent neutrophils containing bacteria compared with nonadherent neutrophils (Fig. 7B). The possibility that there were significant differences in neutrophil viability was excluded when we found that both nonadherent and adherent neutrophils were >92% viable as detected by ethidium homodimer exclusion and intracellular esterase activity. The use of airway epithelial cells that were not stimulated with IFN-{gamma} to increase ICAM-1 expression resulted in lower numbers of bacteria inside neutrophils (Fig. 7C). However, these experiments did not exclude the possibility that epithelial cells assisted with neutrophil antibacterial functions such as phagocytosis through ICAM-1-independent mechanisms. To more directly assess the role of airway epithelial cell ICAM-1 in this assay, we pretreated epithelial cells with blocking anti-ICAM-1 Fab fragments. We found that inhibition of ICAM-1 resulted in reduced numbers of bacteria inside neutrophils (Fig. 7D). To assess other neutrophil antibacterial functions, we allowed human neutrophils to adhere to airway epithelial cells in the presence of blocking anti-ICAM-1 Fab fragments, followed by addition of H. influenzae and assessment of bacterial killing. We found that inhibition of ICAM-1 resulted in significantly decreased killing of bacteria (Fig. 8). Bacteria added to epithelial cells alone resulted in no detectable bacterial killing. In addition, we have been unable to detect ICAM-1 on human neutrophils, and washing off unbound anti-ICAM-1 Fab fragments from epithelial cells before addition of neutrophils gave similar results (Humlicek and Look, unpublished observations), suggesting that blocking antibodies primarily inhibited epithelial cell ICAM-1. Together, the results suggest that epithelial cell ICAM-1 participates in neutrophil recruitment and antibacterial function during the airway inflammatory response to H. influenzae.



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Fig. 7. Epithelial cell ICAM-1 assists with neutrophil antibacterial functions. A: monolayers of human tracheobronchial epithelial (hTBE) cells were treated with 100 units/ml of IFN-{gamma} for 24 h, providing a surface with high-level ICAM-1 expression. Neutrophils were activated with 100 units/ml of TNF-{alpha} and either incubated alone or allowed to adhere to epithelial cells for 30 min. Cells were then incubated for 10 min with Pseudomonas aeruginosa that express high levels of green fluorescence protein (GFP), and confocal fluorescence combined with transmitted light photomicrographs of nonadherent and adherent neutrophils were obtained. Scale bar, 15 µm. B: percentages of adherent and nonadherent neutrophils associated with fluorescent bacteria as presented in A were quantified by counting. Each condition represents the mean ± SD for 3 samples with quantitation of >200 cells/sample; *significant difference between nonadherent and adherent cells. C: monolayers of hTBE cells were left untreated or incubated with 100 units/ml of IFN-{gamma} for 24 h. Neutrophils were activated with 100 units/ml of TNF-{alpha} and allowed to adhere to epithelial cells for 30 min. Cells were then incubated for 10 min with P. aeruginosa that express high levels of GFP, and the mean number of bacteria per neutrophil was quantified by counting. Each condition represents the mean ± SD for 5 samples with quantitation of >100 cells/sample; *significant difference between epithelial cells left untreated or treated with IFN-{gamma}. D: monolayers of hTBE cells were incubated with 100 units/ml of IFN-{gamma} for 24 h and then treated with control or anti-ICAM-1 blocking antibodies. Neutrophils were activated with 100 units/ml of TNF-{alpha} and allowed to adhere to epithelial cells for 30 min. Cells were then incubated for 10 min with P. aeruginosa that express high levels of GFP, and the mean number of bacteria per neutrophil was quantified by counting. Each condition represents the mean ± SD for 5 samples with quantitation of >100 cells/sample; *significant difference between epithelial cells treated with control or anti-ICAM-1 antibodies.

 


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Fig. 8. Epithelial cell ICAM-1 assists with neutrophil antibacterial functions. Monolayers of hTBE cells were incubated with 100 units/ml of IFN-{gamma} for 24 h and then left untreated (black bar) or treated with control (white bars) or anti-ICAM-1 blocking (gray bar) antibodies. Neutrophils were activated with 100 units/ml of TNF-{alpha} and allowed to adhere to epithelial cells for 30 min. Cells were then incubated with H. influenzae for the indicated duration and lysed, and viable bacteria were quantitated to determine bacterial killing. Values are expressed as means ± SD (n = 3); *significant difference compared with untreated cells at the same time point.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Leukocyte recruitment to sites of infection is a critical feature of host defense when constitutive antibacterial mechanisms are overwhelmed by the pathogen load in the airway. Leukocyte migration from the vasculature to the airway requires the expression and coordination of multiple chemoattractant and adhesion molecules, as leukocytes interact with multiple lung parenchymal cells and matrix molecules. For studies in this report, we focused on epithelial cells because they can initiate the inflammatory response to bacteria, compose the final cell layer for leukocyte transmigration before entering the airway lumen, and interact with leukocytes during bacterial killing at locations on the airway surface. The majority of experiments were done with H. influenzae, and our results confirm that efficient clearance of this organism from the respiratory system required neutrophils (47). To understand epithelial cell interaction with neutrophils in airway defense against bacteria, we focused on ICAM-1 because it is the predominant adhesion molecule for leukocytes on airway epithelial cells (21, 31, 48). Using a model system that results in inflammation located primarily inside the airway, we found that ICAM-1 participates in neutrophil recruitment for bacterial defense. As an additional role, airway epithelial cell ICAM-1 assists neutrophils with phagocytosis and killing of bacteria. Indeed, on the basis of the observation that impaired bacterial clearance was most prominent at later time points despite the presence of increased neutrophil numbers in ICAM-1-deficient mice compared with wild-type mice, we propose that ICAM-1 effects on neutrophil antibacterial activity are more important than effects on neutrophil recruitment in response to H. influenzae. Whether the findings in our studies of H. influenzae infection apply to other respiratory pathogens remains to be determined. However, this report extends our understanding of the airway epithelial cell contribution to neutrophil recruitment and function in defense against bacteria in the airway.

During pulmonary infection, ICAM-1 interaction with CD18/{beta}2-integrin counterreceptors on leukocytes is often required for efficient recruitment of leukocytes into the lung. However, previous studies focused on models of pneumonia in which infection and leukocyte recruitment were targeted to alveoli. In models in which bacteria such as P. aeruginosa and Escherichia coli or E. coli LPS alone are presumably localized in alveoli, leukocyte recruitment was impaired by inhibition of ICAM-1 using blocking antibodies or antisense oligonucleotides (18, 28, 39, 53). However, a significant portion of leukocyte recruitment can be independent of ICAM-1 expression in these models. Furthermore, leukocyte recruitment into air spaces appears not to require ICAM-1 in models of pneumonia induced by Streptococcus pneumoniae and K. pneumoniae (5, 18, 37, 39). Based on these observations, we questioned whether ICAM-1 participated in an inflammatory response localized in the airway.

Our results indicate that a portion of airway leukocyte recruitment in response to infection with H. influenzae requires ICAM-1 expression. These studies do not exclude the possibility that leukocytes originally enter air spaces through alveoli and then migrate into airways. Mice with a targeted deletion in the ICAM-1 gene (affecting expression on all cell types) had a delay in early neutrophil recruitment. In comparison, mice pretreated with blocking antibodies against ICAM-1 targeted to either epithelial or endothelial cells had reduced leukocyte recruitment at all time points. Differences in the effects of inhibiting ICAM-1 function using these two methods have been described previously in other pulmonary infection models and may be related to global vs. cell selective ICAM-1 inhibition, the persistence of alternate splice variants of ICAM-1 in knockout mice, antibody effects not related to inhibition of ICAM-1 with its counterreceptors, or the development of alternative pathways in genetically altered mice (17, 18, 39). Complete inhibition of leukocyte recruitment was not observed in our studies, even when higher concentrations of blocking antibodies were used (L. Pang and D. C. Look, unpublished observation). This result likely reflects the presence of an ICAM-1-independent mechanism for leukocyte recruitment in this model of pulmonary infection with H. influenzae.

Several neutrophil antibacterial activities are augmented by or require neutrophil adherence to specific surface biomolecules. For example, neutrophil release of reactive oxygen intermediates in response to TNF-{alpha}, granulocyte/macrophage-colony stimulating factor, and other mediators is greatly increased by interaction with surfaces containing matrix proteins such as fibronectin and laminin (8, 35, 36). Furthermore, neutrophil bactericidal activity is modulated by adherence to surfaces coated with matrix proteins (42). Effects of matrix proteins require neutrophil expression of CD18/{beta}2-integrins (the counterreceptors for ICAM-1) and result in several other biological effects on neutrophils, including decreased cAMP levels and actin cytoskeleton reorganization (6, 33, 34, 41). However, our mouse model system is characterized by neutrophil localization inside airways with intact epithelial cell layers and little apparent matrix protein exposure once leukocytes enter airway lumens. Therefore, we questioned whether airway epithelial cells might provide an adhesion surface for leukocyte antibacterial activities. This possibility is supported by the finding that adherence to endothelial cells increases neutrophil release of H2O2 in response to TNF-{alpha} (35). In addition, enhanced neutrophil respiratory burst and phagocytosis after neutrophil binding to ICAM-1 have been reported (23, 37, 40). Lastly, neutrophil and macrophage interaction with ICAM-1 expressed constitutively on alveolar epithelial cells promotes phagocytosis of K. pneumoniae, providing precedent for the possibility that epithelial cells assist leukocytes in defense of the respiratory system (37, 38).

Our results indicate that neutrophil interaction with the surface of epithelial cells promotes phagocytosis and killing of bacteria. This possibility was investigated because inhibition of epithelial ICAM-1 using blocking antibodies in our mouse model resulted in decreased bacterial clearance that was not seen with inhibition of endothelial cell ICAM-1, despite a similar inhibition of neutrophil migration into the airway. In addition, we were concerned that nonepithelial cells (e.g., macrophages) might also be affected by application of antibodies into the airways of mice. Thus in vitro experiments were performed that allowed for use of isolated airway epithelial cells and neutrophils. Phagocytosis assays were done with GFP-expressing P. aeruginosa to allow microscopic bacterial detection, as we were unable to find a detection method for H. influenzae in assays of interaction with neutrophils that had adequate sensitivity. In contrast, killing assays were performed with H. influenzae, allowing a correlation of in vitro and in vivo results. In these assays, epithelial cells were washed extensively before addition of neutrophils, and subsequent incubation periods were short, making it unlikely that antibacterial activities were due to epithelial cell release of soluble antibacterial factors or neutrophil activators before epithelial-neutrophil interaction (6, 8, 3335). Our results do not exclude the possibility that neutrophil adherence to epithelial cells triggered subsequent release of a factor that activates neutrophils for killing. However, because ICAM-1 is the dominant adhesive protein for leukocytes on airway epithelial cells and because bacterial killing required neutrophil adherence that was blocked by ICAM-1 antibodies (21, 31, 48), we conclude that expression of ICAM-1 on the apical epithelial cell surface provides adhesive sites for neutrophils at the site of infection that assist in antibacterial activity. On the basis of studies in our mouse model, ICAM-1 on the epithelial cell surface appears to be more important for neutrophil killing of H. influenzae compared with effects on neutrophil recruitment.

A paradigm for leukocyte antibacterial defense in the respiratory system is beginning to emerge based on leukocyte localization at sites of infection followed by leukocyte killing of pathogens that requires activator cytokines and a surface with suitable proteins for attachment. In alveoli, a constitutive system dependent on surface ICAM-1 expression on type I alveolar epithelial cells allows resident macrophage migration to and phagocytosis of inhaled bacteria (37, 38). When this system is overwhelmed, neutrophil recruitment is activated; this activation also uses ICAM-1 on alveolar epithelial cells for bacterial killing. In the airway where baseline expression of ICAM-1 on the surface of epithelial cells is low, constitutive soluble antibacterial activity and the mucociliary escalator appear to have primary responsibility for clearance of low levels of bacteria. When constitutive mechanisms are overwhelmed by infection in the airway, neutrophils are recruited to sites of infection by increased chemoattractant and cell adhesion molecule expression. We propose that airway epithelial cells have a central role in coordinating this response through expression of ICAM-1 that is induced by bacteria and/or inflammatory stimuli and provides a surface in the airway for neutrophil binding and bacterial killing. As a corollary, if airway epithelial cells are damaged exposing the underlying basement membrane, then matrix proteins may provide a similar surface for bacterial killing by neutrophils (8, 35, 36, 42). This multitiered system of lung defense allows efficient bacterial clearance with minimal host damage in the majority of pulmonary infections.


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This work was supported by National Heart, Lung, and Blood Institute Grant HL-65752 and the Cystic Fibrosis Foundation.


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge M. Dustin, Genentech, J. Gutierrez-Ramos, M. Parsek, R. Rothlein, P. Singh, and C. Wegner for generous gifts of reagents; M. Apicella, M. Monick, and G. Hunninghake for helpful discussion; and T. Broekelmann and M. Ramaswamy for technical assistance.

Present address for L. Pang: Louisiana State Univ. Health Science Center, Dept. of Obstetrics and Gynecology, 1542 Tulane Ave., New Orleans, LA 70112.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. C. Look, Univ. of Iowa Carver College of Medicine, Dept. of Internal Medicine, C33-GH, 200 Hawkins Dr., Iowa City, IA 52242-1081 (E-mail: dwight-look{at}uiowa.edu)

* A. L. Humlicek and L. Pang contributed equally to this work. Back

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.


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