Abstract
To examine the effects of acid exposure on the adherence of Streptococcus pneumoniae to cultured human tracheal epithelial cells, cells were exposed to acid at various pH levels, and various concentrations of S. pneumoniae were added to the culture medium. The number of S. pneumoniae adhering to cultured human tracheal epithelial cells increased after acid exposure. Y-24180, a specific inhibitor of the receptor for the platelet-activating factor (PAF) and PAF itself decreased the number of S. pneumoniae adhering to cultured human tracheal epithelial cells after acid exposure. Acid exposure increased the activation of transcription factor nuclear factor (NF)- κ B and the expression of protein and messenger RNA (mRNA) of the PAF receptor. The pyrrolidine derivative of dithiocarbamate (PDTC), an inhibitor of NF- κ B, also decreased the number of S. pneumoniae adhering to the cultured human tracheal epithelial cells after acid exposure. Acid exposure increased the content of interleukin (IL)-1 α and IL-1 β in the culture supernatants, but monoclonal antibodies to IL-1 α and IL-1 β failed to inhibit the increased number of S. pneumoniae adhering to cultured human tracheal epithelial cells after acid exposure. These findings suggest that acid exposure stimulates the adherence of S. pneumoniae to the airway epithelial cells via increases in PAF receptors. Increases in PAF receptor expression may be, in part, mediated via activation of transcription factors and subsequent PAF receptor mRNA expression by acid exposure. Increased adherence of S. pneumoniae may be one of the reasons why pneumonia develops after gastric juice aspiration.
Respiratory aspiration of gastric content occurs in patients with a variety of neurologic disorders and under general anesthesia, and is a common problem for clinicians (1). Determining the bacteriologic flora of aspiration pneumonia showed infection of various microorganisms, including Streptococcus pneumoniae (2). Infection of S. pneumoniae after aspiration of gastric and oropharyngeal contents often causes life-threatening pneumonia and complicates sepsis and acute respiratory distress syndrome (3). Bacteriologic flora examination revealed the presence of S. pneumoniae in secretions in the upper respiratory tract of healthy subjects and hospitalized patients (4, 5). Furthermore, Vallés and coworkers (5) showed the correlation between the microorganisms, including S. pneumoniae, of subglottic secretions and aspiration pneumonia. Because gastric contents damage the ciliated and nonciliated airway epithelial cells (6, 7), impairment of mucociliary transport by aspirated gastric contents is suggested to cause the colonization of microorganisms in airways (8). However, the effects of gastric contents on the development of pneumonia caused by infection of S. pneumoniae have not been examined.
The adherence of S. pneumoniae to epithelial cells is an important step in the development of respiratory tract infection of this bacterium (9). S. pneumoniae adheres to the airway epithelial cells and vascular endothelial cells via binding to either a receptor for the G-protein–coupled platelet-activating factor (PAF receptor), N-acetylgalactosamine β-1-4-galactose, or N-acetylgalactosamine β-1-3-galactose (9-14). Of the ligands of S. pneumoniae, inflammatory activation of human epithelial and endothelial cells by treatment with inflammatory cytokines interleukin (IL)-1α and tumor necrosis factor (TNF)-α induces the adherence of this bacteria to the PAF receptor (13, 14). Acid aspiration induces not only lung injury but also the production of an inflammatory cytokine IL-8 in alveolar and bronchoalveolar lavage fluid (15). Likewise, PAF is suggested to be involved as a potent inflammatory mediator in the acute lung injury caused by acid aspiration in mice (16). Based on these findings, we hypothesized that acid exposure may stimulate airway epithelial cells to induce the production of the PAF receptor, thereby increasing susceptibility to infection by S. pneumoniae.
In the present study, we investigated whether S. pneumoniae adheres to cultured human tracheal epithelial cells via binding to the PAF receptor, and the mechanisms of adherence of S. pneumoniae to the epithelial cells induced by acid exposure.
Reagents for cell culture media were obtained as follows: Eagle's minimum essential medium, Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, phospate-buffered saline (PBS), and fetal calf serum (FCS) were from GIBCO-BRL Life Technologies (Palo Alto, CA); fluorescein isothiocyanate (FITC), trypsin, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), PAF, Sigma type XIV protease, human placental collagen, penicillin, streptomycin, gentamicin, and amphotericin B were from Sigma Chemical (St. Louis, MO); Ultroser G serum substitute (USG) was from BioSepra (Marlborough, MA); pyrrolidine derivative of dithiocarbamate (PDTC) was from Calbiochem-Novabiochem (San Diego, CA).
Tracheas for cell cultures were obtained 3 to 6 h after death from 57 patients (age, 65 ± 5 yr; 26 female, 31 male) under a protocol passed by the Tohoku University Ethics Committee. Nineteen of the patients were smokers. None had a respiratory illness, including bronchial asthma, and they died of acute myocardial infarction (n = 13), congestive heart failure (n = 6), malignant tumor other than lung cancer (n = 15), rupture of aortic aneurysm (n = 3), liver cirrhosis (n = 3), renal failure (n = 4), leukemia (n = 4), malignant lymphoma (n = 2), cerebral bleeding (n = 5), and cerebral infarction (n = 2).
The isolation and culture of the human tracheal surface epithelial cells were performed as described previously (17). In brief, the surface epithelium was scored into longitudinal strips and pulled off the submucosa. The tracheal surface epithelial cells were isolated by digestion with protease (0.4 mg/ml; Sigma type XIV), dissolved in PBS at 4°C overnight. Cells were pelleted (200 × g for 10 min) and suspended in DMEM/Ham's F-12 containing 5% FCS (50/50, vol/ vol). Cell counts were made using a hemocytometer, and estimates of viability were done using trypan blue and by measuring the amount of lactate dehydrogenase (LDH) in the medium as previously reported (18). The cells were then plated at 1 × 106 viable cells/ml in 6-well tissue culture plates (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) coated with human placental collagen (17). This medium was replaced by DMEM/Ham's F-12 containing 2% USG on the first day after plating. Cell culture media were supplemented with 105 U/liter penicillin, 100 mg/liter streptomycin, 50 mg/liter gentamicin, and 2.5 mg/liter amphotericin B.
We confirmed cilia beating on the epithelial cells and the absence of fibroblasts in 6-well plates using an inverted microscope (MIT-2; Olympus, Tokyo, Japan). Furthermore, to determine whether cultured cells can form tight junctions, we observed whether the human tracheal epithelial cells made a dome formation to confirm that cells on 6-well plates support tight junctions. We found that the human tracheal epithelial cells made a dome formation when cells made confluent cell sheets as described by Widdicombe and associates (19).
S. pneumoniae R6 (unencapsulated) was isolated from expectrated sputum obtained from patients with acute bronchopneumonia. The isolated bacteria were seeded on blood-agar plates consisting of Müller Hinton broth (Difco, Detroit, MI) with 5% sheep blood and 0.5% agar (20). Colonies were gathered, resuspended in PBS to a concentration of 108 CFU/ml, and then sonically dispersed into a single cell.
Bacteria (108 CFU/ml) were mixed for 1 h at 4°C with FITC (1 mg/ ml) dissolved in a buffer containing 0.05 M Na2CO3 and 0.1 M NaCl (21). Subsequently, the bacteria were washed three times with PBS and resuspended to a final concentration of 108 CFU/ml. In the preliminary experiments, the numbers of S. pneumoniae detected by gram stain and by fluorescent labeling were equivalent. We also found in the preliminary experiments that S. pneumoniae labeled with FITC can be grown in the growth medium described previously.
The number of S. pneumoniae adhering to the human tracheal epithelial cells were counted with methods described previously (13, 21). To examine the effects of acid exposure on the adherence of S. pneumoniae to human tracheal epithelial cells, the epithelial monolayers in the 6-well plates were further incubated in control DMEM/Ham's F-12 medium containing 2% USG for 4 h after acid exposure at various pH levels for various periods, except as otherwise described. The epithelial cell monolayers were then rinsed twice with PBS, and FITC-labeled S. pneumoniae (106 CFU/ml; 20 μl/well) suspended in 2 ml of DMEM/Ham's F-12 containing 2% USG were added to the epithelial cells in 6-well tissue culture plates. The mixtures were incubated for 30 min at 37°C and subsequently rinsed five times with fresh DMEM/ Ham's F-12 containing 2% USG to remove nonadherent bacteria. Adherent FITC-labeled S. pneumoniae were counted visually with an inverted microscope (IX70; Olympus) equipped for fluorescence with a DM500 filter (Olympus). Adherence was expressed as the number of bacteria adhering per 100 human tracheal epithelial cells counted in a ×200 field. During the bacteria adherence experiments, the cell culture medium was not supplemented with any antibiotic.
To examine the effects of incubation periods on the adherence of S. pneumoniae to human tracheal epithelial cells, FITC-labeled S. pneumoniae (106 CFU/ml) were added to the human tracheal epithelial cells and coincubated with the epithelial cells for periods ranging from 0 to 6 h at 37°C in 5% CO2/95% air.
To examine the concentration-response curve for adherence of the S. pneumoniae, FITC-labeled S. pneumoniae were added to the human tracheal epithelial cells at concentrations ranging from 104 to 108 CFU/ml and coincubated with the epithelial cells for 30 min at 37°C in 5% CO2/95% air.
To examine the time course of the number of S. pneumoniae adhering to human tracheal epithelial cells after acid exposure, the pH of the DMEM/Ham's F-12 containing 2% USG was adjusted to 2.5 by the addition of a hydrochloric acid solution. The human tracheal epithelial cells were exposed to acid at pH 2.5 for 5 min and rinsed with PBS. FITC-labeled S. pneumoniae (106 CFU/ml) were added to the fresh DMEM/Ham's F-12 containing 2% USG and coincubated with human tracheal epithelial cells for 0.5, 1, 2, 3, or 4 h at 37°C in 5% CO2/95% air.
To examine the effects of exposure periods to acid at various pH on the number of S. pneumoniae adhering to cultured human tracheal epithelial cells, the pH of the DMEM/Ham's F-12 containing 2% USG was adjusted to 1.5, 2.5, 3.0, or 4.0 by the addition of a hydrochloric acid solution. The human tracheal epithelial cells were treated with acid at various pH levels for 1, 5, 10, 20, or 30 min at 37°C and rinsed with PBS. The human tracheal epithelial cells were then incubated for 4 h in fresh DMEM/Ham's F-12 containing 2% USG, and FITC-labeled S. pneumoniae (106 CFU/ ml) were added to the medium and coincubated with human tracheal epithelial cells for 30 min at 37°C in 5% CO2/95% air.
In the preliminary experiments, we found that acid exposure increased the content of IL-1α (50 to 70 pg/ml) and IL-1β (5 to 7 pg/ml) in culture supernatants of human tracheal epithelial cells. IL-1 is reported to increase the adherence of S. pneumoniae to human lung epithelial cells (13). Therefore, to examine the effects of IL-1α and IL-1β produced after acid exposure on the adherence of S. pneumoniae to cultured human tracheal epithelial cells, human tracheal epithelial cells were cultured in DMEM/ Ham's F-12 containing 2% USG supplemented with either IL-1α (65 pg/ml; Genzyme, Cambridge, MA), IL-1β (6 pg/ml; Genzyme), or both IL-1α (65 pg/ml) and IL-1β (6 pg/ml) for 4 h (13). The concentration of each cytokine was matched to a net increase in the culture medium after acid exposure. Furthermore, human tracheal epithelial cells were treated with either IL-1α (10 ng/ml) or TNF-α (10 ng/ml) for 4 h because IL-1α and TNF-α at this concentration increase adherence of S. pneumoniae to human lung epithelial cells (13). We also studied the effects of IL-1β (10 ng/ml, 4 h) on the adherence of S. pneumoniae. The epithelial cells were then coincubated with FITC-labeled S. pneumoniae (106 CFU/ml) for 30 min at 37°C in 6-well plates.
In the preliminary experiments, we found that the osmolarity of the acidic medium at pH 1.5 and pH 2.5, measured with ion- selective electrodes (7), was higher than that of the control medium. Therefore, to examine the effects of osmolarity on the acid exposure–induced increases in the number of S. pneumoniae adhering to the cultured human tracheal epithelial cells, the osmolarity of each acidic medium was matched to that of control medium by adding distilled water, and the acidic medium at pH 1.5 and pH 2.5 was exposed to the epithelial cells for 5 min. Furthermore, to delete the influence of the Cl concentration in the acidic medium on the acid exposure–induced increases in the number of S. pneumoniae adhering to the cultured human tracheal epithelial cells, the pH of the DMEM/Ham's F-12 containing 2% USG was adjusted to 1.5, 2.5, 3.0, or 4.0 by the addition of a glacial acetic acid solution instead of a hydrochloric acid solution. The human tracheal epithelial cells were treated with acid at various pH levels (pH 1.5, 1 min; pH 2.5, 5 min; pH 3.0, 5 min; and pH 4.0, 30 min) at 37°C and rinsed with PBS.
To examine an inhibitor of the PAF receptor Y-24180 (22) on S. pneumoniae adherence to human tracheal epithelial cells induced by acid exposure, the human tracheal epithelial cells were exposed to acid (pH 1.5, 1 min; pH 2.5, 5 min; pH 3.0, 5 min; and pH 4.0, 30 min) and further cultured for 4 h in fresh DMEM/ Ham's F-12 containing 2% USG. The human tracheal epithelial cell monolayers were then pretreated with Y-24180 (10−5 M) for 30 min (22) and coincubated with FITC-labeled S. pneumoniae (106 CFU/ml) for 30 min in 6-well plates in DMEM/Ham's F-12 containing 2% USG. We also studied the effects of Y-24180 on the adherence of S. pneumoniae induced by treatment with either IL-1α (10 ng/ml, 4 h), IL-1β (10 ng/ml, 4 h), or TNF-α (10 ng/ml, 4 h). During the bacteria adherence experiments, cell culture medium was not supplemented with any antibiotic.
To examine whether S. pneumoniae binds near the active site of the PAF receptor on the human tracheal epithelial cells after acid exposure, the human tracheal epithelial cells were exposed to acid (pH 1.5, 1 min; pH 2.5, 5 min; pH 3.0, 5 min; and pH 4.0, 30 min) and further cultured for 4 h in fresh DMEM/Ham's F-12 containing 2% USG. The human tracheal epithelial cell monolayers were then pretreated with PAF (10−5 M, 10 min) (13) and coincubated with FITC-labeled S. pneumoniae (106 CFU/ml) for 30 min in 6-well plates in DMEM/Ham's F-12 containing 2% USG.
In the preliminary experiments, we found that acid exposure increased nuclear factor (NF)-κB activation. Therefore, to examine the relation between NF-κB activation and the adherence of S. pneumoniae to the cultured human tracheal epithelial cells after acid exposure, we studied the effects of a specific NF-κB inhibitor, PDTC (23), on the adherence of S. pneumoniae. Human tracheal epithelial cells were pretreated with PDTC (10−4 M) in DMEM/Ham's F-12 containing 2% USG and exposed to acid (pH 1.5, 1 min; pH 2.5, 5 min; pH 3.0, 5 min; and pH 4.0, 30 min). PDTC was dissolved in distilled water and then diluted in the culture medium. The epithelial cells were further cultured in fresh DMEM/Ham's F-12 containing 2% USG for 4 h. The human tracheal epithelial cell monolayers were then coincubated with FITC-labeled S. pneumoniae (106 CFU/ml) for 30 min in 6-well plates in DMEM/Ham's F-12 containing 2% USG.
To determine the role of endogenous IL-1α and IL-1β in the adherence of S. pneumoniae, confluent human tracheal epithelial cells were preincubated using the monoclonal mouse antihuman IL-1α (10 μg/ml; Genzyme), antihuman IL-1β (10 μg/ml; Genzyme), or isotype-matched mouse immunoglobulin (Ig) G1 control monoclonal antibody (10 μg/ml; Chemicon International, Temecula, CA) for 4 h after acid exposure.
Because cytokines IL-1α and TNF-α are reported to increase the expression of the PAF receptor in epithelial and endothelial cells (13, 24), we measured IL-1α and TNF-α by specific enzyme-linked immunosorbent assays (ELISAs). Furthermore, we measured the content of IL-1β in the supernatants because human tracheal epithelial cells produce IL-1β in the supernatants (25). Sensitivities of the assays were 10 pg/ml for IL-1α and IL-1β ELISA kit (Ohtsuka, Tokushima, Japan) and 4 pg/ml for TNF-α ELISA kit (Ohtsuka), respectively. In preliminary experiments, we found that the concentrations of IL-1β and TNF-α in the culture medium were low (0 to 10 pg/ml). Therefore, we concentrated the culture medium by freeze-dry methods with a centrifugal vaporizer (Tokyo Rikakikai, Tokyo, Japan) before measuring the concentration of IL-1β and TNF-α. After 1 ml of the culture medium was freeze-dried, the pellet was dissolved in 200 μl of water and the concentrations of IL-1β and TNF-α were measured. The value was normalized according to the medium volume.
We used an average value of replicate cultures from the same trachea (n = 3) for the analysis of cytokine production.
Messenger RNA (mRNA) was isolated from monolayers of human tracheal epithelial cells cultured in 6-well plates as previously described (26) using a Fast Trak 2.0 Kit (Invitrogen, Carlsbad, CA) according to the instruction manual. The amounts of mRNA were determined spectrophotometrically and stored at −80°C.
Northern blot analysis was done as previously described (25, 27, 28). Equal amounts of mRNA (2 μg) extracted from human tracheal epithelial cells were subjected to electrophoresis on a 1% agarose-formaldehyde gel. The gel was then transferred via capillary action onto a nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was hybridized with [α32P]deoxycytidine triphosphate (3,000 Ci/mmol; Amersham)-labeled human PAF receptor complementary DNA (1-kb EcoRI and SmaI fragment; Riken Gene Bank, Tsukuba, Japan) with a random-primer labeling kit (Takara, Osaka, Japan). Hybridization with a radiolabeled probe was performed overnight at 42°C. After a high-stringency washing was performed (1× standard saline sodium citrate/0.1% sodium dodecyl sulfate at room temperature for 30 min and 0.2× standard saline sodium citrate/0.1% sodium dodecyl sulfate at room temperature for 60 min), autoradiographic detection of the hybridized probe was performed by exposure to Kodak Scientific Imaging film (Eastman Kodak, Rochester, NY) for 48 to 72 h at −70°C. Quantification of autoradiographic bands was accomplished with an image analyzer (Bioimaging Analyzer BAS-2000; Fuji Photo Film Co., Tokyo, Japan).
The effects of acid exposure on the PAF receptor protein expression in human tracheal epithelial cells were assayed by flow cytometry analysis as described previously (29, 30). Cells were removed from the 24-well tissue culture plate by incubating with Cell Dissociation Solution (Sigma) at 37°C for 10 min, collected, and washed with cold PBS. The cells were then incubated with an anti-PAF receptor monoclonal antibody at 1:200 dilution (1 mg/ml; Cayman Chemical, Ann Arbor, MI) at 4°C for 30 min. For negative control, mouse serum (1:200, DAKO, Santa Barbara, CA) was used instead of the first antibody. The cells were then washed twice and incubated with 1:25 diluted FITC-conjugated goat antimouse IgG (DAKO) at 4°C for 20 min, extensively washed, resuspended in ice-cold PBS at a cell concentration of 106/ml, and analyzed by a flow cytometer (FACS Calibur; Becton Dickinson, Franklin Lakes, NJ). A total of 10,000 cells was analyzed by gating on a uniform cell population on a two-parameter histogram of forward-versus-side scatter. The fluorescence histograms were overlaid to determine significant differences from negative control antibodies. Expression of the PAF receptor was determined by subtracting the mean fluorescence intensity measured with the control monoclonal antibody from that measured with the antibody to the PAF receptor.
To examine the effects of acid exposure on PAF receptor expression on the cultured human tracheal epithelial cells, cells were exposed to acid (pH 2.5) for 5 min and cultured for 4 h in fresh DMEM/Ham's F-12 medium containing 2% USG. As a positive control, we studied PAF receptor expression in the cells treated with IL-1β (10 ng/ml) for 4 h.
Nuclear extracts were prepared using the methods described previously (31, 32). For isolation of nuclear extracts, all procedures were performed on ice. The human tracheal epithelial cells in 6-well dishes were washed with ice-cold PBS, harvested by scraping into FCS, and pelleted in a 1.5-ml microfuge tube at 1,850 × g for 5 min. After repeating this procedure once more, the pellet was suspended in one packed cell volume of lysis buffer (10 mM N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid [Hepes] pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) and incubated for 15 min. Membrane lysis was accomplished by adding 25 μl of 10% Nonidet P-40 followed by vigorous agitation. The nuclei were then collected by centrifugation, resuspended in 50 μl of extract buffer (20 mM Hepes, 420 mM NaCl, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF), and agitated vigorously at 4°C for 15 min. After removal of debris by centrifugation, the protein concentration of the nuclear extract was determined. The nuclear extracts were then stored at −70°C until use.
Electrophoretic mobility shift assays (EMSAs) were performed as described previously (31, 32). Radiolabeled double-stranded oligonucleotide probes for the NF-κB or the promoter-specific transcription factor (SP-1) site were prepared by annealing complementary oligonucleotides and by end labeling with [γ-32P]adenosine triphosphate and T4 polynucleotide kinase. The radiolabeled probes used for NF-κB and SP-1 were composed of the following sequences, respectively: 5′-GATCGAGGGGACTTTCCCTAGC-3′ for NF-κB (33, 34) (Stratagene, La Jolla, CA) and 5′-AATTACCGGGCGGGCGGGCTACCGGGCGGGCT-3′ for SP-1 (Stratagene) (35). The labeled probes were purified by Chroma Spin + TE-10 (Clontech, Palo Alto, CA) and diluted with buffer (10 mM Tris-HCl, 1 mM EDTA) to the desired concentration. Equivalent amounts of nuclear protein were incubated with 2 μg of salmon sperm DNA and 2 to 5 fmol (20,000 dpm) of the radiolabeled probe for 20 min at room temperature in 20 μl of a buffer containing 10 mM Hepes (pH 7.9), 50 mM KCl, 2 mM MgCl2, 0.25 mM DTT, 0.25 mM PMSF, 0.1 mM EDTA, and 10% glycerol. Resolution was accomplished by electrophoresing 12 μl of reaction solution on 4% nondenaturing polyacrylamide gels in a TBE buffer (89 mM Tris-HCl, 89 mM boric acid, and 2 mM EDTA, pH 8.0) for 60 min at 150 V at room temperature. Autoradiographic detection of the hybridized probe was performed by exposing Kodak Scientific Imaging film for 48 to 72 h at −70°C.
To study the effects of acid exposure on the activation of NF-κB and SP-1, nuclear extracts from human tracheal epithelial cells were isolated before, 0.5, and 1 h after acid exposure (pH 2.5, 5 min). Because we found in the preliminary experiments that all of IL-1α (10 ng/ml), IL-1β (10 ng/ml), and TNF-α (10 ng/ml) increased the number of S. pneumoniae adhering to epithelial cells, we also studied the effects of IL-1α (10 ng/ml), IL-1β (10 ng/ml), or TNF-α (10 ng/ml) on the activation of NF-κB and SP-1 as positive control.
Supershift assays were used to determine which members of the NF-κB family were involved in acid exposure–induced NF-κB– DNA binding. In these studies, EMSAs were performed as described previously except that rabbit polyclonal antibodies against the NF-κB subunit proteins p65, p50, c-Rel, and Rel B (Santa Cruz Biotechnology, Santa Cruz, CA) were included in the 1-h radiolabeled probe-extract binding reaction at 4°C (31, 32). Preimmune serum (Santa Cruz Biotechnology) was used to control for any nonspecific effects of these antisera.
Results are expressed as mean ± standard error of the mean (SEM). Within group comparisons were tested using Wilcoxon's matched pairs test, and between group comparisons were tested using the Mann-Whitney U test. Significance was accepted at P < 0.05. The value of n refers to the number of donors from which cultured epithelial cells were used.
Adherence of S. pneumoniae to the cultured human tracheal epithelial cells was observed consistently. The number of epithelial cells observed in each ×200 field was 696 ± 61 (n = 196) and did not differ significantly among the tracheas (n = 28, P > 0.20). Measurement of the number of S. pneumoniae adhering to human tracheal epithelial cells at differing times after the addition of S. pneumoniae into the medium revealed no significant numbers of S. pneumoniae at 10 min after the addition of S. pneumoniae. Adhered S. pneumoniae were observed at 20 min, and the number of adhering S. pneumoniae progressively increased between 20 and 150 min after the addition of S. pneumoniae (Figure 1A). The adherence of S. pneumoniae to human tracheal epithelial cells was constant, and the coefficient of variation of the number of adherent S. pneumoniae at 30 min was small (7.9%; n = 20). The number of S. pneumoniae adhering to human tracheal epithelial cells increased significantly with time (P < 0.05) (Figure 1A).
Fig. 1. (A) Effects of incubation periods on the number of S. pneumoniae adhering to cultured human tracheal epithelial cells. Adherence is expressed as the number of S. pneumoniae per 100 human tracheal epithelial cells. Results are reported as mean ± SEM from seven samples. (B) Concentration-response curve of coincubated S. pneumoniae in the adherence of S. pneumoniae to cultured human tracheal epithelial cells. Human tracheal epithelial cells were incubated with increasing concentrations of S. pneumoniae for 30 min. Adherence is expressed as the number of adhered S. pneumoniae per 100 human tracheal epithelial cells. Results are reported as mean ± SEM from seven samples.
[More] [Minimize]Likewise, the measurement of the number of S. pneumoniae adhering to human tracheal epithelial cells at differing concentrations of S. pneumoniae revealed no significant number of S. pneumoniae at 104 CFU/ml. Adherence of S. pneumoniae was observed at 105 CFU/ml, and the number of adhering S. pneumoniae increased progressively between additions of 105 and 108 CFU/ml of S. pneumoniae into the culture medium (Figure 1B). The number of S. pneumoniae adhering to human tracheal epithelial cells increased in a concentration-dependent fashion (P < 0.05).
Treatment of cells with acid significantly increased the number of S. pneumoniae adhering to human tracheal epithelial cells. Measurement of the number of S. pneumoniae adhering to human tracheal epithelial cells at differing times after acid exposure revealed no significant increase in the number of S. pneumoniae at 0.5 and 1 h after 5 min of acid exposure at pH 2.5 (Figure 2). An increase in the number of S. pneumoniae was observed at 2 h, and the number of adhering S. pneumoniae increased progressively between 2 and 4 h after acid exposure (Figure 2). Likewise, the effects of acid exposure on the number of S. pneumoniae adhering to human tracheal epithelial cells were related to pH and exposure period of acid (Figure 3). Maximal effects on the adherence of S. pneumoniae at each pH level were obtained at 1 min of exposure at pH 1.5, at 5 min of exposure at pH 2.5 and pH 3.0, and at 30 min of exposure at pH 4.0 (Figure 3). In all acid-exposure conditions, the maximal effects of acid exposure on the number of S. pneumoniae adhering to human tracheal epithelial cells were obtained at 5 min of exposure period at pH 2.5 (Figure 3). The osmolarity of acidic medium at pH 1.5 (410 ± 12 mOsm/liter, P < 0.01, n = 5) and at pH 2.5 (346 ± 9 mOsm/liter, P < 0.05, n = 5) was higher than that of control medium (315 ± 6 mOsm/liter, n = 5), at pH 3.0 (318 ± 7 mOsm/liter, n = 5), and at pH 4.0 (316 ± 6 mOsm/liter, n = 5). When the osmolarity of acidic medium was matched to that of the control medium by adding distilled water, the number of S. pneumoniae after exposure to acidic medium at pH 1.5 (91 ± 5/100 cells, P < 0.01, n = 7) and at pH 2.5 (96 ± 8/100 cells, P < 0.01, n = 7) was significantly higher than that in the control medium (44 ± 5/100 cells, n = 7). When the acidic medium was made by adding glacial acetic acid instead of HCl, the number of S. pneumoniae adhering to the cultured human tracheal epithelial cells also increased after exposure to the acidic medium. The number of S. pneumoniae after exposure to acidic medium at each pH was 93 ± 7/100 cells at pH 1.5 (P < 0.01, n = 7), 98 ± 8/100 cells at pH 2.5 (P < 0.01, n = 7), 81 ± 7/100 cells at pH 3.0 (P < 0.05, n = 7), and 61 ± 5/100 cells at pH 4.0 (P < 0.05, n = 7), and was significantly higher than 43 ± 3/100 cells in the control medium (n = 7).
Fig. 2. The number of S. pneumoniae adhering to the human tracheal epithelial cells after acid (pH 2.5, 5 min, solid columns) or sham (open columns) exposure. S. pneumoniae were added to the culture medium at various times after acid or sham exposure, and S. pneumoniae were coincubated with human tracheal epithelial cells for 30 min. Adherence is expressed as the number of adhered S. pneumoniae per 100 human tracheal epithelial cells. Results are reported as mean ± SEM from seven samples. Significant differences from sham exposure (control) are indicated by *P < 0.05 and **P < 0.01.
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Fig. 3. Effects of exposure periods to acid at various pH levels on the number of S. pneumoniae adhering to cultured human tracheal epithelial cells. The epithelial cells were incubated in control DMEM/Ham's F-12 medium with 2% USG for 4 h after acid exposure, and S. pneumoniae were then coincubated with human tracheal epithelial cells for 30 min. Results are reported as mean ± SEM from seven samples. Significant differences from sham exposure (control) are indicated by *P < 0.05 or **P < 0.01.
[More] [Minimize]Human tracheal cell viability, assessed by the exclusion of trypan blue, was consistently > 96% when the cells were briefly exposed to acid at pH 1.5 for 1 min, at pH 2.5 for 5 min, at pH 3.0 for 5 min, or at pH 4.0 for 30 min. Short periods of acid exposure at pH 2.5 for 5 min, at pH 3.0 for 5 min, and at pH 4.0 for 30 min did not alter the amount of LDH in the supernatants released during 24 h after acid exposure (12 ± 2 IU/liter at pH 2.5, 10 ± 2 IU/liter at pH 3.0, and 12 ± 2 IU/liter at pH 4.0 versus 11 ± 2 IU/liter in control medium, P > 0.20, n = 7), although the amount of LDH in the supernatants increased 24 h after acid exposure at pH 1.5 for 1 min (45 ± 3 IU/liter, P < 0.05, n = 7). Short periods of acid exposure (pH 1.5, 1 min; pH 2.5, 5 min; pH 3.0, 5 min; pH 4.0, 30 min) had no effect on viable cell numbers when acid exposure was performed after cells had made confluent sheets in 6-well dishes. Cell counts 24 h after acid exposure (4.3 ± 0.3 × 106 at pH 1.5; 4.4 ± 0.4 × 106 at pH 2.5; 4.5 ± 0.3 × 106 at pH 3.0; and 4.2 ± 0.3 × 106 at pH 4.0, P > 0.50, n = 7) were not significantly different from control (4.4 ± 0.4 × 106, n = 7).
A PAF receptor inhibitor, Y-24180 (10−5 M) (Figure 4A), or PAF (10−5 M, 10 min) (Figure 4B) alone did not affect the adherence of S. pneumoniae to the cultured human tracheal epithelial cells in the control condition. However, both Y-24180 (10−5 M) (Figure 4A) and PAF (10−5 M, 10 min) (Figure 4B) significantly inhibited adherence of S. pneumoniae induced by acid exposure.
Fig. 4. Effects of an inhibitor of the PAF receptor, Y-24180 (10−5 M) (solid columns) (A), and pretreatment with PAF (10−5 M) (hatched columns) (B) on the number of S. pneumoniae adhering to cultured human tracheal epithelial cells after acid exposure at various pH levels for 1 min (pH 1.5), 5 min (pH 2.5 and pH 3.0), or 30 min (pH 4.0). The epithelial cells were incubated in control DMEM/Ham's F-12 medium with 2% USG for 4 h after acid exposure and treated with either Y-24180 (30 min) or PAF (10 min), and S. pneumoniae were then co-incubated with human tracheal epithelial cells for 30 min. Adherence is expressed as the number of adhered S. pneumoniae per 100 human tracheal epithelial cells. Results are reported as mean ± SEM from seven samples. Significant differences from medium alone (control, open columns) are indicated by *P < 0.05 or **P < 0.01. Significant differences from exposure to acid are indicated by + P < 0.05.
[More] [Minimize]All of IL-1α (10 ng/ml, 4 h), IL-1β (10 ng/ml, 4 h), and TNF-α (10 ng/ml, 4 h) (13) increased the number of S. pneumoniae adhering to the cultured human tracheal epithelial cells (63 ± 5/100 cells for IL-1α, P < 0.05; 67 ± 5/100 cells for IL-1β, P < 0.05; and 59 ± 5/100 cells for TNF-α, P < 0.05, n = 7) compared with that in medium alone (46 ± 4/ 100 cells, n = 7). Y-24180 (10−5 M) reduced the number of S. pneumoniae induced by these cytokines (42 ± 2/100 cells for IL-1α plus Y-24180, P < 0.01; 42 ± 3/100 cells for IL-1β plus Y-24180, P < 0.01; and 41 ± 2/100 cells for TNF-α plus Y-24180, P < 0.05; n = 7) compared with that for each cytokine alone.
Because acid exposure did not alter cell numbers (see previous text), all cytokine values are reported in picograms per milliliter of supernatant. The contents of IL-1α and IL-1β in the supernatant increased 4 h after acid exposure (pH 2.5, 5 min) compared with those 4 h after sham exposure (65 ± 5 pg/ml in acid exposure versus 28 ± 4 pg/ml in sham exposure for IL-1α, P < 0.05, n = 7; and 5.6 ± 0.4 pg/ml in acid exposure versus 3.0 ± 0.3 pg/ml in sham exposure for IL-1β, P < 0.05, n = 7). In contrast, TNF-α was under the limit of detection of the assay in the supernatants from cells after acid or sham exposure.
Acid exposure increased the content of IL-1α (50 to 70 pg/ml) and IL-1β (5 to 7 pg/ml) in culture supernatants of human tracheal epithelial cells. However, neither the preincubation with IL-1α (65 pg/ml, 4 h) or IL-1β (6 pg/ml, 4 h) nor the combination of IL-1α (65 pg/ml) and IL-1β (6 pg/ml) for 4 h increased the number of S. pneumoniae adhering to the cultured human tracheal epithelial cells (Figure 5A).
Fig. 5. (A) Effects of IL-1α (65 pg/ml, 4 h), IL-1β (6 pg/ml, 4 h), and the combination of IL-1α (65 pg/ml, 4 h) and IL-1β (6 pg/ml, 4 h) (IL-1α + IL-1β) on the number of S. pneumoniae adhering to cultured human tracheal epithelial cells. Adherence is expressed as the number of adhered S. pneumoniae per 100 human tracheal epithelial cells. Results are reported as mean ± SEM from seven samples. (B) The number of S. pneumoniae adhering to cultured human tracheal epithelial cells after acid exposure (Acid; pH 2.5, 5 min) in the presence of monoclonal antibodies to IL-1α (a-IL-1α), IL-1β (a-IL-1β), the combination of monoclonal antibodies to IL-1α and IL-1β (a-IL-1α + a-IL-1β), mouse purified immunoglobulin G1 monoclonal antibody (IgG1), or absence of an antibody. Adherence is expressed as the number of adhered S. pneumoniae per 100 human tracheal epithelial cells. Results are reported as mean ± SEM from seven samples. Significant differences from medium alone (control) are indicated by **P < 0.01.
[More] [Minimize]Likewise, the number of S. pneumoniae adhering to human tracheal epithelial cells 4 h after acid exposure (pH 2.5, 5 min) in the presence of either the monoclonal antihuman IL-1α (10 μg/ml), the monoclonal antihuman IL-1β (10 μg/ml), or the combination of both antibodies did not differ from that after acid exposure in the presence or absence of the mouse IgG1 control monoclonal antibody (Figure 5B).
The baseline expression of PAF receptor mRNA was constant in confluent human tracheal epithelial cell sheets, and the coefficient of variation was small (7.6%; n = 7). Neither smoking nor cause of death influenced the baseline expression of PAF receptor mRNA. Exposure of the cells to acidic medium (pH 2.5, 5 min) caused increases in PAF receptor mRNA (Figure 6A). Human tracheal epithelial cells 4 h after acid exposure were shown to overexpress PAF receptor mRNA twofold compared with those 4 h after exposure to control medium (Figure 6B, control).
Fig. 6. (A) Northern blot analysis demonstrating increases in PAF receptor (PAF-R) mRNA levels of human tracheal epithelial cells 4 h after acid exposure (acid; pH 2.5, 5 min) compared with exposure to medium alone (control). β-Actin was used as a housekeeping gene. (B) Expression of PAF receptor (PAF-R) mRNA in human tracheal epithelial cells 4 h after acid exposure (acid; pH 2.5, 5 min; solid column). Open column, exposure to control medium (control). PAF receptor mRNA is normalized to a constitutive expression of β-actin mRNA. The epithelial cells were incubated in control DMEM/Ham's F-12 medium with 2% USG for 4 h after acid exposure, and PAF receptor mRNA expression was examined. Results are reported as mean ± SEM from seven samples. Significant difference from control is indicated by *P < 0.05.
[More] [Minimize]Expression of the PAF receptor was also assayed by flow cytometric analysis. Human tracheal epithelial cells 4 h after acid exposure (pH 2.5, 5 min) were shown to increase PAF receptor–specific fluorescence intensity compared with those after sham exposure (Figures 7A, 7B, and 7D). Likewise, IL-1β (10 ng/ml, 4 h) increased PAF receptor– specific fluorescence intensity in the human tracheal epithelial cells (Figures 7C and 7D).
Fig. 7. Flow cytometry analysis demonstrating increases in the PAF receptor expression of human tracheal epithelial cells 4 h after acid exposure (pH 2.5, 5 min) (B) or 4 h after treatment with IL-1β (10 ng/ml) (C) compared with sham exposure (A). (D) PAF receptor fluorescence intensity in human tracheal epithelial cells 4 h after acid exposure (acid; pH 2.5, 5 min) and 4 h after treatment with IL-1β (10 ng/ml). Results are reported as mean ± SEM from seven samples. Significant differences from sham exposure (control) are indicated by *P < 0.05.
[More] [Minimize]Nuclear extracts from the human tracheal epithelial cells after acid exposure or sham exposure contained NF-κB, as demonstrated by the presence of a complex consisting of protein bound to a DNA fragment carrying the NF-κB (Figure 8A). The baseline intensity of NF-κB binding activity was constant, and increased activation of NF-κB was present in cells at 1 h after acid exposure (Figure 8A). Likewise, all of IL-1α (10 ng/ml), IL-1β (10 ng/ml), and TNF-α (10 ng/ml) increased activation of NF-κB at 0.5 and 1 h (data not shown). Specificity of the NF-κB binding was confirmed by supershift EMSA in which antibodies to the p50 or p65 subunit of NF-κB ablated NF-κB bands (data not shown). The supershifting of the NF-κB band with the antibody to the p50 or p65 subunit of NF-κB was constantly observed at all times of the cell culture. However, the supershifting of the NF-κB band was not observed with either antibody to p52, c-Rel, Rel B, or preimmune antiserum (data not shown). In contrast, acid exposure (pH 2.5, 5 min) did not increase the activation of SP-1 at any time (Figure 8B). Likewise, IL-1α (10 ng/ml), IL-1β (10 ng/ml), and TNF-α (10 ng/ml) did not increase the activation of SP-1 at any time (data not shown).
Fig. 8. EMSA demonstrating the time course of NF-κB (A) and SP-1 (B) DNA binding activity of human tracheal epithelial cells before (0), 30, and 60 min after exposure to acid (pH 2.5, 5 min). NF-κB and SP-1 DNA binding activity are highlighted by the arrowheads. Data are representative of three different experiments.
[More] [Minimize]A specific NF-κB inhibitor, PDTC (10−4 M) alone, did not affect the adherence of S. pneumoniae to cultured human tracheal epithelial cells in the control condition (45 ± 4/ 100 cells in NF-κB inhibitor versus 44 ± 4/100 in medium alone, P > 0.50, n = 7). However, PDTC (10−4 M) significantly inhibited adherence of S. pneumoniae induced by acid exposure at pH 1.5 (61 ± 5/100 cells in PDTC versus 92 ± 6/100 in acidic medium alone, P < 0.05, n = 7), at pH 2.5 (65 ± 5/100 cells in PDTC versus 97 ± 3/100 in acidic medium alone, P < 0.05, n = 7), and at pH 3.0 (59 ± 3/100 cells in PDTC versus 81 ± 5/100 in acidic medium alone, P < 0.05, n = 7) without inhibitory effects at pH 4.0 (60 ± 2/100 cells in PDTC versus 67 ± 6/100 in acidic medium alone, P > 0.20, n = 7).
The present study suggests that acid exposure increases the adherence of S. pneumoniae by inducing surface expression of the PAF receptor, a receptor for S. pneumoniae (9, 13), in cultured human tracheal epithelial cells. These conclusions are based on the observations that acid exposure increased the number of S. pneumoniae adhering to the human tracheal epithelial cells and increased the expression of the mRNA and protein of PAF receptor in the human tracheal epithelial cells. A specific inhibitor of the PAF receptor, Y-24180, reduced the number of adhered S. pneumoniae after acid exposure. In the absence of the acid exposure, time course and concentration dependency on the number of adhered S. pneumoniae to human tracheal epithelial cells were consistent with a previous report in resting human lung cells (13). Because Y-24180 was without effect on the number of S. pneumoniae adhering to the epithelial cells in the culture medium alone, S. pneumoniae may adhere to the nonactivated human tracheal epithelial cells via binding to receptors other than the PAF receptor, such as N-acetylgalactosamine β-1-4-galactose and N-acetylgalactosamine β-1-3-galactose, as shown in the resting lung cells (9-13). In contrast, inflammatory activation induced by acid exposure may enhance the adherence of S. pneumoniae to human tracheal epithelial cells through the upregulation of the PAF receptor expression (9, 13). Because pretreatment of PAF also inhibited the acid exposure-induced adherence of S. pneumoniae, S. pneumoniae may bind near the active site of the PAF receptor as shown in the vascular endothelial cells and lung cells (13).
Acid exposure–induced increases in the adherence of S. pneumoniae to the epithelial cells might not depend on alterations in the osmolarity or Cl concentration of acidic medium because adjusting osmolarity and adding acetic acid instead of HCl failed to change the acid exposure- induced effects. Therefore, the changes in acidity itself could have stimulatory effects on the adherence of S. pneumoniae.
The present study did not observe the location of S. pneumoniae in the cultured human tracheal epithelial cells. However, in previous reports (13, 21), 2 to 3% of S. pneumoniae were internalized into the cultured endothelial cells within the 30 min of incubation periods. Therefore, S. pneumoniae might begin to be internalized into the cultured human tracheal epithelial cells because we cocultured S. pneumoniae with the epithelial cells for 30 min after acid exposure.
Acid exposure increased the production of IL-1α and IL-1β in the culture supernatants in the present study. However, antibodies to human IL-1α and IL-1β failed to inhibit increased adherence of S. pneumoniae to the human tracheal epithelial cells. Furthermore, adding IL-1α (65 pg/ml), IL-1β (6 pg/ml), or combinations of IL-1α (65 pg/ml) and IL-1β (6 pg/ml), at the concentrations observed in the culture supernatants after acid exposure, failed to increase the number of S. pneumoniae adhering to the human tracheal epithelial cells. Therefore, endogenous IL-1α and IL-1β produced in the human tracheal epithelial cells might not have a sufficient effect in stimulating the adherence of S. pneumoniae after acid exposure.
On the other hand, acid exposure increased activation of NF-κB at 1 h after exposure in the present study. Two different promoters, transcripts 1 and 2, have been demonstrated to express mRNA of the PAF receptor, and both transcripts have been shown in the lung (36). Transcript 1 has consensus sequences for transcription factor NF-κB and SP-1. In contrast, transcript 2 contains consensus sequences for transcription factor activator protein (AP)-1, AP-2, and SP-1 (36). In the present study, 10 ng/ml of IL-1α, IL-1β, and TNF-α increased the activation of NF-κB and the number of S. pneumoniae adhering to the human tracheal epithelial cells as shown previously in the human lung cells (13). A specific inhibitor of the PAF receptor, Y-24180, reduced the number of S. pneumoniae after treatment with IL-1α, IL-1β, TNF-α, or acid exposure. These findings suggest that the increased activation of NF-κB in transcript 1 (36) might be, in part, associated with the upregulation of the PAF receptor expression in the human tracheal epithelial cells after acid exposure. This statement is also supported by the present results that a specific NF-κB inhibitor, PDTC, inhibited the increases in the number of S. pneumoniae after acid exposure. In contrast, SP-1 in transcript 1 (36) might not relate to the PAF receptor expression after acid exposure because acid exposure, IL-1α, IL-1β, and TNF-α did not increase activation of SP-1 in the present study. Because acid exposure increased the production of mRNA and protein of the PAF receptor, acid exposure might cause PAF receptor protein production through an increase in mRNA associated with NF-κB activation.
Hydrochloric acid and gastric juice induce tracheal epithelial damage in mice and humans, including desquamation of ciliated and nonciliated cells (6), and increased epithelial permeability (7). Intratracheal inoculation of hydrochloric acid reduces the clearance of S. pneumoniae (37). Thus, the aspiration of gastric secretions is suggested to affect the airway mucociliary clearance and local antibacterial defenses, resulting in the development of bacterial proliferation (37). Furthermore, acid aspiration induces the production of an inflammatory cytokine IL-8 in alveolar and bronchoalveolar lavage fluid (15). PAF is involved in the acute lung injury caused by acid aspiration in mice as an inflammatory mediator (16). Therefore, acid aspiration may not only damage the lung cells but also cause the production of inflammatory mediators. S. pneumoniae are suggested to adhere to the activated lung cells by inflammatory cytokines via binding to the PAF receptor (14). In the present study, transient exposure to acid increased the PAF receptor expression in the human tracheal epithelial cells. Therefore, increased adherence of S. pneumoniae via the binding of the PAF receptor may be one reason for the airway pneumococcal infection after aspiration of gastric juice.
S. pneumoniae were isolated from transtracheal aspiration, thoracentesis, or endotracheal secretions in aspiration-induced pulmonary infections (2, 3). Bacteriologic flora examination revealed the presence of S. pneumoniae in secretions in the upper respiratory tract of healthy subjects and hospitalized patients (4, 5) whereas S. pneumoniae were not isolated from gastric content in patients with gastrointestinal disease (38). Vallés and coworkers (5) showed the correlation between the microorganisms, including S. pneumoniae of subglottic secretions and aspiration pneumonia. Furthermore, aspiration of oropharynx bacterial pathogens to the lower respiratory tract is suggested to be an important risk factor for nosocomial pneumonia (39). Therefore, it is possible that S. pneumoniae in aspired oral and nasopharyngeal secretions adhere to the lower respiratory tract epithelium, and adherence of S. pneumoniae is promoted by acid aspiration.
In summary, we have demonstrated that the expression of the PAF receptor increased in human tracheal epithelial cells after acid exposure, and a specific inhibitor of the PAF receptor, Y-24180, and pretreatment with PAF inhibited adherence of S. pneumoniae to human tracheal epithelial cells induced by acid exposure. Therefore, transient exposure of acid may induce adherence of S. pneumoniae to human tracheal epithelial cells via binding to the PAF receptor. In addition to the impairment of bacterial clearance (37) caused by airway epithelial damage (6, 7), increased adherence of S. pneumoniae to the airway epithelial cells through binding to the PAF receptor may be important in the development of aspiration pneumonia. A specific inhibitor for the PAF receptor may have protective effects against infection by S. pneumoniae after gastric juice aspiration.
The authors thank Mr. Grant Crittenden for the English correction.
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Abbreviations: colony-forming unit, CFU; Dulbecco's modified Eagle's medium, DMEM; dithiothreitol, DTT; ethylenediaminetetraacetic acid, EDTA; enzyme-linked immunosorbent assay, ELISA; electrophoretic mobility shift assay, EMSA; fluorescein isothiocyanate, FITC; N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid, Hepes; immunoglobulin, Ig; interleukin, IL; lactate dehydrogenase, LDH; messenger RNA, mRNA; nuclear factor, NF; platelet-activating factor, PAF; phosphate-buffered saline, PBS; pyrrolidine derivative of dithiocarbamate, PDTC; phenylmethylsulfonyl fluoride, PMSF; standard error of the mean, SEM; tumor necrosis factor, TNF; ultroser G serum substitute, USG.
