Effects of RSV infection on pulmonary surfactant protein SP-A in cultured human type II cells: contrasting consequences on SP-A mRNA and protein
Joseph L. Alcorn,
James M. Stark,
Constance L. Chiappetta,
Gaye Jenkins, and
Giuseppe N. Colasurdo
Department of Pediatrics, The University of Texas Health Science Center at Houston, Houston, Texas
Submitted 22 November 2004
; accepted in final form 12 July 2005
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ABSTRACT
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Respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory illness in infants and children. Surfactant proteins A (SP-A) and D (SP-D) play critical roles in lung defense against RSV infections. Alterations in surfactant protein homeostasis in the lung may result from changes in production, metabolism, or uptake of the protein within the lung. We hypothesized that RSV infection of the type II cell, the primary source of surfactant protein, may alter surfactant protein gene expression. Human type II cells grown in primary culture possess lamellar bodies (a type II cell-specific organelle) and the ability to express surfactant protein mRNA. These cells were infected with RSV (by morphology and antibody binding). Surfactant protein mRNA levels determined by quantitative RT-PCR indicated a marked increase in SP-A mRNA levels (3-fold) 24 h after RSV exposure, whereas SP-D mRNA levels were unaffected. In contrast to mRNA levels, total SP-A protein levels (determined by Western blot analysis) were decreased 40% after RSV infection. The percentage of secreted SP-A was 43% of the total SP-A in the RSV-infected cells, whereas the percentage of secreted SP-A was 61% of the total SP-A in the uninfected cells. These changes in SP-A transcript levels and protein secretion in cultured human cells were recapitulated in RSV-infected mouse lung. Our findings suggest that type II cells are potentially important targets of RSV lower respiratory infection and that alterations in surfactant protein gene expression and SP-A protein homeostasis in the lung may arise via direct effects of RSV.
surfactant protein A; respiratory syncytial virus; alveolar type II cell
RESPIRATORY SYNCYTIAL VIRUS (RSV) is the most important cause of serious lower respiratory illness and viral lower respiratory tract disease in infants and children (55), primarily resulting in bronchiolitis and pneumonia, and often the distinctions between these two infections are blurred. Epidemiological studies have demonstrated the extent of infection by RSV: 50% of children will acquire RSV during the first year of life, 40% of these will develop lower respiratory tract disease, and 1% will require hospitalization. By 3 yr of age, every child will have been infected with RSV at least once (14, 15, 40, 41, 45). Lower respiratory infections (LRI) caused by respiratory syncytial virus are common occurrences in early childhood. Several epidemiological studies in children and recent laboratory investigations in animal models suggest an association between LRIs (including RSV infection) and allergic sensitization or persistent/recurrent respiratory obstruction (1, 35, 14, 15, 41, 46).
Viral LRIs appear to make infants and children more susceptible to superinfection by pathogenic bacteria (30, 31, 42). Increased frequencies of infection caused by Hemophilus influenzae (6, 53), meningococcus (52), and Staphylococcus aureus (6, 9) have been associated with recent RSV infection, causing otitis media, pneumonia, and meningitis. These data suggest that viral infection alters normal lung responses to a subsequent "challenge." The causative relationship between RSV LRI and bacterial superinfection has been demonstrated in animal models of cotton rats (46) and sheep (35, 8). These observations of RSV infection lead to the hypothesis that RSV infection perturbs the protective microenvironment in the lung, altering innate protection from acute secondary infection and potentially altering later adaptive responses to infection or allergic challenges. Although an association between RSV infection and subsequent secondary infection has been identified clinically and confirmed in animal models, the mechanism(s) underlying this association remains unclear and is key to the development of therapeutic interventions to prevent RSV-related bacterial superinfection.
The primary function of pulmonary surfactant, a complex surface-active lipoprotein, is to reduce surface tension at the alveolar air-liquid interface, thereby preventing alveolar collapse upon expiration and allowing normal breathing (10). Although the phospholipid portion of surfactant, which is composed largely of dipalmitoylphosphatidylcholine, acts directly to reduce surface tension, the major surfactant-associated proteins surfactant protein-B (SP-B) and surfactant protein-C (SP-C) also serve in this function of surfactant. Another important function of surfactant involves surfactant protein-A (SP-A) and surfactant protein-D (SP-D), which serve in host defense in the lung (21). SP-A and SP-D play critical roles in lung defense following infections by various pathogens, including RSV (11, 33). SP-A and SP-D are members of the collectin family of innate immune molecules. These proteins contain a collagen-like domain and a calcium-dependent lectin domain (carbohydrate recognition domain, or CRD; SP-A and SP-D bind to mannose and glucose) (12, 13, 50). They demonstrate reproducible differences in relative saccharide selectivity in vitro, although the biological significance of these differences is unknown. The carboxy-terminal domains of SP-A and SP-D are responsible for their lectin-like activity. The majority of biologically active surfactant proteins exist as multimers of trimeric subunits: SP-A as octadecamers (6 trimers) and SP-D as dodecamers (4 trimers). SP-A and SP-D also interact with lipids, including the lipid components of surfactant, and the lipids present in the bacterial cell wall. Their lectin binding and lipid binding characteristics predict a role for SP-A and D in defenses against various microbial, fungal, and viral infections (11, 34). Transgenic mice with targeted disruption of the SP-A or SP-D genes have demonstrated increased susceptibility to infection by a number of viral and bacterial pathogens (11, 34).
Pulmonary alveolar type II epithelial cells that are positioned in the corners of the alveoli carry out the highly specialized functions of synthesizing, secreting, and reutilizing surfactant (39, 49). Because alveolar type II cells are important sources of SP-A and SP-D in the lung, alteration of production of these proteins after injury (such as viral infection) would have important consequences on overall surfactant homeostasis and lung defense against bacterial pathogens. Human alveolar type II cells can be grown in primary culture and demonstrate lamellar bodies and production of all four surfactant proteins. To investigate the hypothesis that RSV infection of type II cells alters lung defense by altering SP-A and/or SP-D production, we infected primary cultures of human alveolar type II cells with purified RSV. These studies demonstrated that RSV actually increases levels of SP-A mRNA while having no effect on SP-D mRNA levels. However, despite the increase in SP-A mRNA, SP-A protein levels in the cell supernates were significantly decreased after RSV exposure. These studies indicate that RSV infection alters SP-A homeostasis in primary type II cultures and provide a clue to the mechanisms behind the enhanced susceptibility to secondary bacterial infections that occurs after viral infection in vivo (32, 38).
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MATERIALS AND METHODS
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Alveolar type II cell isolation and primary culture.
Alveolar type II epithelial cells were isolated from fetal lung explants as described previously (2). Lung explants from midtrimester human abortuses were obtained from Advanced Bioscience Resources (Alameda, CA) in accordance with protocols approved by The Committee for the Protection of Human Subjects of the University of Texas-Houston Health Science Center. Tissues were maintained in organ culture for 5 days in serum-free Waymouth's MB 752/1 medium (no. 11220, Invitrogen, Carlsbad, CA) in the presence of dibutyryl-cAMP (DBcAMP, 1 mM; no. 104396, Boehringer Mannheim, Indianapolis, IN) to increase the number of type II cells (43). Type II epithelial cells were isolated from the tissue by digestion with collagenase type I (0.5 mg/ml; no. C-0130, Sigma Chemical, St. Louis, MO) and collagenase type IA (0.5 mg/ml; no. C-9891, Sigma Chemical) for 15 min at 37°C with vigorous pipetting. The cell suspension was enriched for type II cells by incubation with DEAE-dextran (250 µg/ml; no. D9885, Sigma Chemical) and plated on 60-mm tissue culture dishes coated with extracellular matrix (ECM) prepared from Madin-Darby canine kidney cells (CRL 6253, American Type Culture Collection). The resulting human type II epithelial cells were cultured in serum-free Waymouth's MB 752/1 medium in the presence of DBcAMP (1 mM) in a humidified atmosphere of 95% air and 5% CO2.
Detection and visualization of lamellar bodies in type II cells.
Type II cells were incubated on ECM-coated coverslips in the presence of DBcAMP (1 mM). The cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS), treated with osmium tetroxide to stain the lamellar bodies as described previously by Mason et al. (37), and examined under light microscopy. For analysis of the ultrastructure of the cultured type II cells, transmission electron microscopy was performed. The cultured cells were scraped from the dishes, fixed in glutaraldehyde (2%), and processed for electron microscopy as described previously (2). In brief, the cells were dehydrated in graded alcohols, embedded in resin, sectioned, and stained with lead citrate and uranyl acetate. Sections were viewed and photographed with a 1200EX JEOL scanning electron microscope at the Electron Microscopy Laboratory (with a fee for service in the Department of Pathology, University of Texas-Houston Medical School).
RSV infection and propagation.
Human RSV, strain A2, used in all experiments, was sucrose purified as previously described (25). The virus stock of RSV was diluted in culture medium to a defined multiplicity of infection (MOI), which is defined as the number of plaque-forming units (pfu) per number of type II cells. Pulmonary alveolar type II cells in primary culture were exposed to RSV for 2 h and then washed and incubated. Cells and supernatant fluids were removed at various time points after RSV infection and stored at 80°C until assayed for the presence of SP-A. Control samples were prepared from uninfected cultures that were processed in the same manner. Cell viability was determined by Trypan blue exclusion as previously described (24).
Experimental animals and RSV infection.
BALB/c mice obtained from Harlan Sprague Dawley were used in all experiments. After being sedated with ketamine and xylazine, mice were infected by intranasal inoculation with 5 x 105 pfu of RSV A2. This experimental protocol has been described elsewhere (19, 25). RSV infection was confirmed by measuring titers in the lungs. All procedures used in this study were approved by the Animal Welfare Committee at the University of Texas Health Science Center-Houston and are compliant with federal guidelines.
Immunodetection of RSV-infected cells.
Isolated human type II cells in the primary culture were exposed to RSV at an MOI of 1.0. After 48 h, cells were fixed to a glass slide and stained for the presence of antigens expressed in the cytoplasm of RSV-infected cell proteins by using a direct immunofluorescence RSV detection kit (RSV Direct-IF 15'; catalog no. 20-042, Parc Technologique Delta SUD, Varilhes, France). The antibody recognizes the F0 and F1 subunit of the fusion protein of RSV expressed in the cytoplasm of infected cells.
Isolation of RNA.
RNA was isolated and purified from the cells and mouse lung using the Trizol reagent (no. 15596-026, Invitrogen). Trizol reagent is a ready-to-use reagent for the isolation of total RNA from cells and tissues that involves a single-step RNA isolation method. The concentration of the RNA was determined by measuring absorbance at 260 nm.
Real-time quantitative RT-PCR analysis of surfactant protein mRNA.
Quantitative real-time RT-PCR assays specific for the four human surfactant protein mRNAs were designed and performed at the Quantitative Genomics Core Laboratory (Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center-Houston), utilizing the 7700 Sequence Detector (Applied Biosystems, Foster City, CA) (16, 23). Specific quantitative assays for SP-A, SP-B, SP-C, and SP-D are shown in Table 1 and were developed using Primer Express software (Applied Biosystems) based on sequences from GenBank at the National Center for Biotechnology Information. (Note: the human SP-A assay was based on SP-A1 and SP-A2 sequences to allow quantitation of all SP-A mRNAs.) Fluorogenic primers are labeled with 6-carboxyfluorescein. Surfactant protein cDNAs were synthesized in 10-µl total volume by the addition of 6 µl/well RT master mix, consisting of 400 nM assay-specific reverse primer, 500 µM deoxynucleotides, Stratascript buffer, and 10 units of Stratascript reverse transcriptase (Stratagene, San Diego, CA), to a 96-well plate (ISC Bioexpress, Kaysville, UT) followed by a 4-µl volume of sample (25 ng/µl). Each sample was measured in triplicate plus a control without reverse transcriptase. Each plate also contained an assay-specific sDNA (synthetic amplicon oligo) standard spanning a 5-log range and a no template control. Each plate was covered with Biofilm A (MJR, Waltham, MA) and incubated in a thermocycler (MJR) for 30 min at 50°C, followed by 72°C for 10 min. Subsequently, 40 µl of a PCR master mix [400 nM forward and reverse primers (IDT, Coralville, IA), 100 nM fluorogenic probe (Biosource, Camarillo, CA), 5 mM MgCl2, and 200 µM deoxynucleotides, PCR buffer, and 1.25 units of Taq polymerase (Invitrogen)] were added directly to each well of the cDNA plate. RT master mixes and all RNA samples were pipetted using a Tecan Genesis RSP 100 robotic workstation (Tecan US, Research Triangle Park, NC); PCR master mixes were pipetted using a Biomek 2000 robotic workstation (Beckman, Fullerton, CA). Each assembled plate was then covered with an optically clear film (Applied Biosystems), and RT-PCR reactions were performed in the ABS 7700 with the following cycling conditions: 95°C, 1 min; 40 cycles of 95°C, 12 s; and 60°C, 30 s. The resulting data were analyzed using SDS 1.9.1 software (Applied Biosystems) with ROX. Synthetic DNA oligos used as standards (sDNA) encompassed the entire 5'3' amplicon for the assay (Eurogentec via VWR, Sugarland, TX). Oligo standards were diluted in 100 ng/µl yeast tRNA-H2O (Invitrogen) and spanned a 5-log range in 10-fold decrements starting at 0.8 pg/µl. Standards for housekeeping gene assays started 10-fold higher for abundant transcripts. It has been shown for several assays that in vitro transcribed RNA amplicon standards (sRNA) and sDNA standards have the same PCR efficiency when the reactions are performed as described above (Shipley GL, personal communication). Because of the inherent inaccuracies in quantifying total RNA by absorbance, the amount of RNA added to an RT-PCR from each sample was more accurately determined by measuring the
-actin transcript level in each sample. The final data were normalized and are presented as the molecules of transcript/molecules of normalizer transcript x 100 = %normalizer transcript.
Isolation of cellular proteins.
Proteins were isolated from human type II cells and mouse lung as described previously (59). The cells were scraped into 500-µl ice-cold lysis buffer (10 mM Tris, pH 7.6, 3 mM MgCl2, 40 mM KCl, 2 mM DTT, 5% glycerol, and 0.5% NP-40). After a freeze-thaw cycle and homogenization, the cells were centrifuged at 600 g for 10 min to remove debris, and the supernatant containing the cytosolic proteins was aliquoted, the protein concentration determined, and the aliquots stored at 80°C.
Immunoblot analysis of SP-A protein.
To prepare culture medium, we solubilized equal volumes (20 µl) in loading buffer (0.1 M Tris, pH 7.4, 50 µM DTT, 0.01% bromphenol blue, 2% SDS, and 10% glycerol) at 95°C for 5 min. To prepare cellular proteins, we solubilized isolated proteins (15 µl) in lysis buffer (0.05 M Tris·HCl buffer, pH 7.4, 2% SDS, 0.01% bromphenol blue, 0.7%
-mercaptoethanol, and 10% glycerol). Proteins were resolved on precast 420% Tris-glycine polyacrylamide gels (no. EC6025, Invitrogen). After protein resolution, gels were transferred to nitrocellulose paper and incubated with rabbit anti-human SP-A antibody (1:1,000 dilution) followed by peroxidase-conjugated goat anti-rabbit IgG (1:160,000 dilution; Sigma). The immunodetected protein was detected with chemiluminescence (ECL system; no. RPN2135, Amersham Biosciences, Piscataway, NJ) and recorded on high-performance chemiluminescence film. Quantification was performed using the imaging and quantitating abilities of the ChemiGenius2 imaging system (Syngene, Frederick, MD). In selected experiments, SP-A protein and steady-state mRNA levels were measured in vivo by using a well-established model of RSV infection (24). Briefly, BALB/c mice were infected with human RSV (5 x 105 pfu/mouse) or treated with uninfected culture medium. At 5 days postinfection, the lungs were lavaged and equal aliquots of bronchoalveolar lavage (BAL) fluid were collected, electrophoresed, and blotted. Relative concentrations of 36-kDa SP-A protein in the BAL was quantified as described above for immunoblot analysis of SP-A in the cell culture medium. SP-A mRNA levels from lung tissue were quantified using primers specific for murine SP-A mRNA.
Statistical analysis.
Statistical analysis of infected samples compared with uninfected samples was performed using the paired t-test.
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RESULTS
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Primary cultures of human type II epithelial cells express four major surfactant protein mRNAs.
Type II epithelial cells were isolated from fetal human lung in organ culture and incubated in primary culture as indicated in MATERIALS AND METHODS. The distinguishing features of alveolar type II cells are the expression of the four major surfactant proteins, SP-A, SP-B, SP-C, and SP-D (22), and the organelle involved in the storage, secretion, and reutilization of surfactant, the lamellar body (51). Shown in Fig. 1A are isolated human type II cells after 5 days of culture in serum-free medium containing DBcAMP on ECM-coated glass coverslips. The cells, which were stained for the presence of lamellar bodies using osmium tetroxide (37), contain numerous large osmiophilic granules indicative of the presence of lamellar bodies. Through visual criteria, these isolated cells have the characteristic cuboidal shape of type II cells. By electron microscopy, it can be seen that these granules have the morphological characteristics of lamellar bodies (Fig. 1B), mainly the typical multilamellated structure (44). The other definitive characteristic of alveolar type II cells is the expression of all major surfactant proteins. We therefore determined the levels of expression of the SP-A, SP-B, SP-C, and SP-D mRNAs, using quantitative real-time RT-PCR assays specific for the four human surfactant protein mRNAs. Shown in Fig. 2 are the steady-state levels of the mRNAs after 5 days of culture in serum-free medium containing DBcAMP. In our cells, the levels of SP-A mRNA were the highest (
100% of
-actin mRNA levels). SP-B mRNA levels were somewhat lower (
40%
-actin mRNA levels), followed by SP-C mRNA levels (only about 0.7% of
-actin mRNA levels) and SP-D levels (although much lower than SP-A mRNA levels,
0.1% of
-actin mRNA levels). These data demonstrate that mRNA of all four major surfactant proteins are transcribed under our culture conditions.

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Fig. 1. Isolated human type II cells in primary culture retain type II cell-specific morphology. A: light micrograph of type II epithelial cells after 5 days in primary culture stained with osmium tetroxide for presence of lamellar bodies. Numerous osmiophilic granules, which are shown as dark circular organelles, represent intracellular lamellar bodies. B: ultrastructure of our human type II cells in primary culture. An electron micrograph is shown of an isolated type II cells after 5 days in primary culture. The cells possess organelles with the typical lamellated structure of lamellar bodies (indicated by arrows).
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Fig. 2. Surfactant protein mRNA production in isolated human type II cells in primary culture. Type II cells were isolated and incubated in primary culture for 5 days, after which total RNA was isolated. The steady-state levels of surfactant protein (SP)-A, SP-B, SP-C, and SP-D mRNAs were quantified by quantitative RT-PCR using primers specific for human surfactant protein mRNAs. Values represent steady-state levels of surfactant protein mRNA relative to -actin mRNA (n = 3).
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RSV can infect primary cultures of human type II epithelial cells.
Type II epithelial cells were isolated from fetal human lung in organ culture and incubated in primary culture, as indicated in MATERIALS AND METHODS, on ECM-coated glass coverslips. The type II cells were infected with RSV and incubated for another 48 h. The cells were stained for the presence of antigens produced by replicating RSV. The results shown in Fig. 3 indicate that the type II cells are infected by RSV. Figure 3A demonstrates cytoplasmic staining of type II cells in primary culture, using the immunofluorescence method after RSV infection, whereas the uninfected cells shown are not reactive with the antibody to RSV F protein (Fig. 3B). In addition, examples of syncytia formation in the type II cells after RSV infection can be found on the coverslips. Shown in Fig. 3C is an example of a multinucleate cell compared with a group of uninfected cells. Presumably, the RSV infection of the cells results in the formation of syncytia, a typical cytopathological finding following RSV infection (7). The viability of the cells at 24 h postinfection, monitored using the Trypan blue exclusion method (24), was 84.7 ± 5.1% (n = 4) and 86.2 ± 7.5% (n = 4) for control and infected cells, respectively. These results indicate that RSV can infect human type II cells, isolated from fetal lung tissue, in primary culture and can elicit an expected response.

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Fig. 3. Isolated fetal human type II epithelial cells in primary culture can be infected by respiratory syncytial virus (RSV) and syncytialize. Isolated human type II cells in primary culture for 2 days were exposed to RSV at a multiplicity of infection (MOI) of 0.3. After 48 h, cells were fixed to a glass slide and stained for the presence of antigens that are expressed in the cytoplasm of RSV-infected cell proteins by using a direct immunofluorescence RSV detection kit as described in MATERIALS AND METHODS. A: cells exposed to RSV. B: uninfected control cells. C: type II cells infected with RSV at an MOI of 0.3. After 48 h, the cells were fixed to a glass slide and stained. Shown is a group of individual cells (left), whereas the arrow indicates a multinucleate cell, caused by syncytia formation between cells as a result of RSV infection and expression of RSV-F glycoprotein.
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RSV infection of type II cells alters steady-state levels of surfactant protein mRNAs.
Because primary alveolar type II cells grown in our culture system expressed the surfactant protein mRNAs, we then determined the effect of RSV infection of steady-state levels of these mRNAs. Beginning 1 h after 2-h exposure of the cells to RSV, the mRNA was collected at the indicated times (Fig. 4). The effects of the RSV on endogenous surfactant protein gene expression were determined using quantitative RT-PCR analysis of surfactant protein gene mRNA levels. Shown in Fig. 4 are the values that represent steady-state levels of surfactant protein mRNA relative to
-actin mRNA. At times of 1 or 6 h after infection of the cells, there was little change in the levels of surfactant protein mRNA steady-state levels. However, 24 h after RSV infection of type II cells, there was an approximate threefold increase in SP-A mRNA steady-state levels in the cells (P < 0.05). In contrast, the steady-state levels of SP-B were mildly elevated, and the levels of steady-state SP-C and SP-D mRNA levels were unaffected. The nature of this regulation of SP-A mRNA in not known at this time; we do not know whether the effect is directly due to viral replication or to factors produced by type II cells in response to RSV infection that may modulate SP-A gene expression.

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Fig. 4. RSV infection of human type II cells in primary culture results in a time-dependent increase in endogenous SP-A mRNA levels. Type II cells were isolated and incubated in primary culture for 2 days. The cells were then incubated in the absence or presence of RSV at an MOI of 0.3. At various times after infection, the cells were harvested and the RNA was quantified by RT-PCR using primers specific for human SP-A, SP-B, SP-C, and SP-D mRNA. Values represent steady-state levels of surfactant protein mRNA relative to -actin mRNA and are means (SD) of 2 separate experiments with 3 replicates.
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Steady-state levels of intracellular SP-A protein are decreased in RSV infected-type II cells.
Because we found that RSV infection of type II cells leads to an increase in steady-state SP-A mRNA levels, we then investigated the effects of infection of the virus on steady-state SP-A protein levels in the cells. Human type II cells in primary culture were exposed to RSV for 2 h, the medium was replaced with 2 ml of serum-free medium, and total cell protein and medium were collected 24 h later. The cells were lysed in 0.5 ml of lysis buffer, and 15 µl of cell lysate (3.33% of the total) and 20 µl of medium (1.00% of the total) were subjected to Western blot analysis. As shown in Fig. 5A, cellular SP-A protein was readily detected by immunoblot analysis of the cellular protein with the use of antibodies specific for SP-A protein. There was little change in the mobility of the SP-A protein, suggesting that posttranslational processing of the SP-A protein is unaffected by RSV infection. The effects of the RSV on endogenous SP-A protein levels were determined by quantification of the SP-A signal of the immunoblot. The SP-A signal from the various blots was used to determine the total amount of SP-A in the cells and medium by factoring in the fraction of the total loaded in each lane with the quantification of the SP-A signal in the lane. As shown in Fig. 5B, RSV infection of type II cells in primary culture led to a decrease in total cellular SP-A protein of
40%. It is unclear from the results whether this decrease is due to an effect of RSV on translation of the SP-A mRNA (increased in response to RSV infection, Fig. 4), proteolytic activity of the cell (processing), or increased uptake and degradation of the excreted SP-A protein.

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Fig. 5. SP-A protein production and secretion decrease in RSV infected-human type II cells in primary culture. A: type II cells were isolated and incubated in primary culture for 2 days. Type II cells were then infected with RSV (MOI of 1.0). After 24 h of incubation, cellular protein and medium were isolated. Intracellular and secreted SP-A protein levels in the cells were determined by immunoblot analysis, using polyclonal rabbit anti-SP-A antibodies as described in MATERIAL AND METHODS. B: quantitation of the immunodetected SP-A relative to total protein. Shown are means ± SE of 2 separate experiments with 3 replicates.
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Secretion of SP-A protein is reduced in RSV-infected type II cells.
The surprising results with regard to the effect of RSV infection to reduce SP-A protein levels in type II cells prompted us to determine the effect of RSV infection on secretion of SP-A protein. After infection of the cells with RSV, the amount of SP-A secreted into the media during a 24-h period was determined using immunoblot analysis. As shown in Fig. 5A, RSV infection of type II cells did not lead to protein processing changes (no alteration in molecular mobility of secreted SP-A protein from RSV-infected cells compared with SP-A protein isolated from uninfected cells). However, Fig. 5B demonstrates that RSV infection led to a decrease in the amount of secreted SP-A detectable in the medium. In uninfected cells, secreted SP-A was
35% higher than cellular SP-A, whereas secreted SP-A from RSV-infected cells was 14% lower. Relative to total SP-A, 61% of the SP-A in uninfected cells was secreted, whereas 43% of the SP-A in RSV-infected was secreted. It is unclear whether the changes in the ratio of cellular to secreted SP-A due to RSV infection are due to effects on the exocytosis of SP-A and/or effects on the uptake of secreted SP-A by the cells. Nevertheless, these results suggests that not only does RSV infection of type II cells lead to a decrease in overall cellular SP-A protein but also that secretion of the cellular protein is adversely affected as well. To further support our in vitro observation, we measured SP-A protein and steady-state SP-A mRNA in murine lungs obtained from control and RSV-infected BALB/c mice. As shown in Fig. 6A, the amount of SP-A detectable in the BAL fluid is
50% lower compared with control animals. In keeping with our in vitro observations, the reduction of SP-A protein was associated with increased levels of SP-A mRNA after RSV infection (Fig. 6B). Collectively, these data suggest that RSV disrupts SP-A homeostasis in vitro and in vivo by targeting type II pneumocytes.

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Fig. 6. SP-A protein and mRNA levels in the lungs of a murine model of RSV infection. BALB/c mice were infected with human RSV (5 x 105 plaque-forming units) or treated with uninfected culture medium. At 5 days postinfection, the lungs were lavaged with PBS and the bronchoalveolar lavage (BAL) fluid was collected. SP-A protein levels were determined by immunoblot analysis using polyclonal rabbit anti-SP-A antibodies. Relative concentrations of 36-kDa SP-A protein were blotted and then used for quantification. SP-A mRNA levels were quantified using primers specific for murine SP-A mRNA. Values are means ± SE from 3 separate animals. A: RSV infection decreased SP-A protein in the BAL fluid. B: RSV infection enhanced steady-state levels of SP-A mRNA relative to -actin.
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DISCUSSION
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In many ways it is quite remarkable that the lung has as little problem with infection as we observe clinically. The lung has an extensive surface area for gas transport and exchange (
150 m2, the size of a tennis court, in an adult) and handles about 15,000 liters of air each day (58), which contains an array of infectious agents that must be removed or neutralized by the lung's innate and adaptive defenses. The respiratory epithelium itself is more than a passive barrier to infection. It is metabolically active and produces many of the soluble components of the innate immunity in the lung. Despite defensive mechanisms within the lung, viral and bacterial infection still takes place. The lung collectins SP-A and SP-D have been demonstrated to have antimicrobial activity against a variety of bacterial and fungal agents. SP-A enhances uptake of gram-positive and gram-negative organisms, including S. aureus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, group B Streptococcus, H. influenzae, and Klebsiella pneumoniae, by lung phagocytes (11, 12, 28, 33, 34, 50). Although SP-A may not opsonize certain organisms (P. aeruginosa, E. coli) directly, it does enhance phagocytosis by direct interactions with alveolar macrophages. SP-D has been shown to interact with the lipopolysaccharide (LPS) core of a number of gram-negative organisms [E. coli, P. aeruginosa, and K. pneumoniae (11)]. Both SP-A and SP-D gene ablation (knockout) mice have been constructed and characterized (29, 60). Mice deficient in SP-A show defective clearance upon intratracheal inoculation with a number of bacteria. In addition, SP-A-deficient mice show defective killing of Mycoplasma pneumoniae. In contrast, although SP-D-deficient mice have decreased bacterial uptake upon challenge with group B Streptococcus or H. influenzae in vitro, in vivo killing of these organisms was unaffected.
Because alveolar type II cells are important sources of SP-A and SP-D in the lung (39, 49), alteration of production of these proteins following injury (such as viral infection) would have important consequences on overall surfactant homeostasis and lung defense against bacterial pathogens. However, little is known with regard to the effects of RSV infection of type II cells. To investigate the hypothesis that RSV infection of type II cells alters lung defense by altering SP-A and/or SP-D production, we had to infect primary cultures of human alveolar type II cells with purified RSV. Our culture system has previously been shown to allow for the culture human type II cells in primary culture and optimizes the expression of SP-A and SP-B mRNA (2). In this study, we have shown that these cells also express all of the four major surfactant proteins: SP-A, SP-B, SP-C, and SP-D. Our culture system also allows for the culture of type II cells that maintain the most prominent morphological feature of type II cells: the lamellar body, which is the organelle involved in the storage, secretion, and recycling of surfactant (10). In a slightly different culture system, Gonzales et al. (17, 18) have shown that isolated human alveolar type II cells in primary culture express the four surfactant protein mRNAs. Their pattern of expression, SP-A levels > SP-B levels > SP-C levels > SP-D levels, is similar to ours. The absolute numbers are different, possibly because of the use of dexamethasone in their culture techniques. However, the morphological features of the cells in their studies, lamellar bodies and cuboidal cell shape, are indicative of alveolar type II cells and are similar to ours.
We also have shown in this study that differentiated human type II cells in primary culture can be infected by RSV. Upon infection of type II cells with RSV, we found that RSV actually increases levels of SP-A mRNA threefold while having mild effects on SP-B mRNA levels and no effect on SP-D and SP-C mRNA levels. RSV infection of airway epithelial cells also results in changes in the activity of transcription factors, some of which may be involved in SP-A gene regulation. RSV infection of alveolar epithelial cells activates nuclear factor-
B (54). SP-A gene expression has been shown to be increased in response to nuclear factor-
B, and dominant negative mutants reduce expression (24). On the other hand, another group has suggested that nuclear factor-
B has no effect to regulate surfactant protein gene expression (47). Recent work from Kong et al. (27) has shown that in human A549 alveolar epithelial cells, RSV induced phosphorylation and nuclear translocation of signal transducer and activator of transcription (STAT)-1
. RSV also activated STAT-3 and IL-6. These findings may be applicable to these results, because others have suggested a potential interaction between STAT-1 and thyroid transcription factor (TTF)-1 due to close proximity of STAT-1 and TTF-1 binding sites in the Clara cell 10-kDa protein regulatory sequences (36). TTF-1 is the major regulatory factor influencing SP-A gene expression (24, 39). These data suggest that RSV may influence surfactant protein gene expression in alveolar type II cells by altering the transcription factors involved with SP-A transcription.
Our data demonstrate an apparent "disassociation" between SP-A protein and mRNA levels in the lung after RSV infection in vitro and in vivo. Despite the increase in SP-A mRNA described in this study, intracellular SP-A protein levels in the cell supernatants and secretion of SP-A from the type II cell were significantly decreased after RSV exposure. Although the decrease in SP-A protein levels in response to RSV was unexpected, two phenomena can explain it: decreased translational efficiency of existing SP-A mRNA or increased protease activity. There is no prior evidence that RSV decreases translational efficiency in infected cells, but protease activity in RSV-infected cells has been reported to increase in some systems. Ramaswamy et al. (48) has shown that RSV infection of human tracheobronchial epithelial cells results in modulation of the type I IFN JAK-STAT pathway that is mediated through proteasome-dependent degradation of STAT-2, suggesting that RSV infection induced proteasome-dependent inhibition of STAT-2 expression. Whether RSV is inducing increased proteasome activity in primary type II cells, leading to decreased SP-A levels, remains to be tested.
The other unexpected aspect of surfactant protein regulation in type II cells after RSV infection was the decreased secretion of SP-A from infected cells compared with secretion in uninfected cells. In retrospect, perhaps it is not surprising, because studies in mice have demonstrated that RSV infection alters SP-A levels in the lung (56, 57). In addition, clinical studies have provided similar findings in RSV-infected children; RSV infection reduces SP-A levels in the BAL fluid of infected children (20, 26). A recent study (35) reported an association between an allele of the human SP-A2 gene and susceptibility to severe RSV infection. There have been no studies to date attempting to measure whether this gene polymorphism may result in low SP-A levels or a gene product with altered function. The reduced SP-A in the BAL fluid in all these studies could be the result of increased uptake of SP-A after RSV infection by a number of cell types, including the respiratory epithelium and the alveolar macrophages. Again, there are no previous studies that address the mechanism of effect of RSV infection on secretion or processing of proteins. However, the RSV F and G glycoproteins mediate host specificity and attachment, and SP-A binds to the F glycoprotein (and possibly to G) of RSV (20). This could possibly explain the changes in exocytosis of SP-A through interference of the secretory pathways by the presence of the F glycoprotein. However, this hypothesis remains to be tested.
These studies indicate that RSV infection alters SP-A homeostasis in primary type II cultures and in a well-established mammalian model and provide a clue to the mechanisms behind the enhanced susceptibility to secondary bacterial infections that occurs following viral infection in vivo (32, 38). Our models could be useful to characterize the interactions between RSV and airway epithelium and the cellular regulation of SP-A. These studies may be key to understanding how RSV predisposes to bacterial infection.
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GRANTS
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This research was supported in part by National Institutes of Health (NIH) Grants R01-HL-68116 (to J. L. Alcorn) and R01-AI-46556 (to J. M. Stark), the Cystic Fibrosis Foundation (to J. M. Stark), and NIH Grant R21-E5012927 (to G. N. Colasurdo).
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ACKNOWLEDGMENTS
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We thank Dr. Pedro A. Piedra (Department of Virology and Microbiology, Baylor College of Medicine, Houston, TX) for generously providing the human RSV, strain A. We credit the Quantitative Genomics Core Laboratory, Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, for designing and performing the real-time quantitative RT-PCR assays for surfactant protein mRNAs. Finally, we are thankful for the assistance and guidance of Patricia Navarro in the Department of Pathology and Laboratory Medicine for expertise in obtaining electron microscope images.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. L. Alcorn, Jr., Dept. of Pediatrics, The Univ. of Texas-Houston Medical School, 6431 Fannin, Suite 3.222, Houston, TX 77030 (e-mail: joseph.l.alcorn{at}uth.tmc.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES
|
---|
- Abramson JS and Wheeler JG. Virus-induced neutrophil dysfunction: role in the pathogenesis of bacterial infections. Pediatr Infect Dis J 13: 643652, 1994.[ISI][Medline]
- Alcorn JL, Smith ME, Smith JF, Margraf LR, and Mendelson CR. Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses. Am J Respir Cell Mol Biol 17: 672682, 1997.[Abstract/Free Full Text]
- Al-Darraji AM, Cutlip RC, and Lehmkuhl HD. Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: immunofluorescent and electron microscopic studies. Am J Vet Res 43: 230235, 1982.[ISI][Medline]
- Al-Darraji AM, Cutlip RC, Lehmkuhl HD, and Graham DL. Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: pathologic studies. Am J Vet Res 43: 224229, 1982.[ISI][Medline]
- Al-Darraji AM, Cutlip RC, Lehmkuhl HD, Graham DL, Kluge JP, and Frank GH. Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: clinical and microbiologic studies. Am J Vet Res 43: 236240, 1982.[ISI][Medline]
- Andrade MA, Hoberman A, Glustein J, Paradise JL, and Wald ER. Acute otitis media in children with bronchiolitis. Pediatrics 101: 617619, 1998.[Abstract/Free Full Text]
- Blount RE Jr, Morris JA, and Savage RE. Recovery of cytopathogenic agent from chimpanzees with coryza. Proc Soc Exp Biol Med 92: 544549, 1956.
- Brogden KA, Lehmkuhl HD, and Cutlip RC. Pasteurella haemolytica complicated respiratory infections in sheep and goats. Vet Res 29: 233254, 1998.[ISI][Medline]
- Carfrae DC, Bell EJ, and Grist NR. Fatal haemorrhagic pneumonia in an adult due to respiratory syncytial virus and Staphylococcus aureus. J Infect 4: 7980, 1982.[CrossRef][ISI][Medline]
- Clements JA and King RJ. The Biochemical Basis of Pulmonary Function, edited by Crystal RG. New York: Dekker, 1976, p. 363387.
- Crouch E and Wright JR. Surfactant proteins a and d and pulmonary host defense. Annu Rev Physiol 63: 521554, 2001.[CrossRef][ISI][Medline]
- Crouch EC. Collectins and pulmonary host defense. Am J Respir Cell Mol Biol 19: 177201, 1998.[Abstract/Free Full Text]
- Crouch EC. Modulation of host-bacterial interactions by collectins. Am J Respir Cell Mol Biol 21: 558561, 1999.[Free Full Text]
- Denny FW and Clyde WA Jr. Acute lower respiratory tract infections in nonhospitalized children. J Pediatr 108: 635646, 1986.[ISI][Medline]
- Foy HM, Cooney MK, Maletzky AJ, and Grayston JT. Incidence and etiology of pneumonia, croup and bronchiolitis in preschool children belonging to a prepaid medical care group over a four-year period. Am J Epidemiol 97: 8092, 1973.[ISI][Medline]
- Gibson UE, Heid CA, and Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 6: 9951001, 1996.[Abstract]
- Gonzales LW, Angampalli S, Guttentag SH, Beers MF, Feinstein SI, Matlapudi A, and Ballard PL. Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic. Pediatr Pathol Mol Med 20: 387412, 2001.[CrossRef][ISI][Medline]
- Gonzales LW, Guttentag SH, Wade KC, Postle AD, and Ballard PL. Differentiation of human pulmonary type II cells in vitro by glucocorticoid plus cAMP. Am J Physiol Lung Cell Mol Physiol 283: L940L951, 2002.[Abstract/Free Full Text]
- Graham BS, Perkins MD, Wright PF, and Karzon DT. Primary respiratory syncytial virus infection in mice. J Med Virol 26: 153162, 1988.[ISI][Medline]
- Griese M. Respiratory syncytial virus and pulmonary surfactant. Viral Immunol 15: 357363, 2002.[CrossRef][ISI][Medline]
- Haagsman HP and Diemel RV. Surfactant-associated proteins: functions and structural variation. Comp Biochem Physiol A Mol Integr Physiol 129: 91108, 2001.[CrossRef][ISI][Medline]
- Hawgood S and Shiffer K. Structures and properties of the surfactant-associated proteins. Annu Rev Physiol 53: 375394, 1991.[CrossRef][ISI][Medline]
- Heid CA, Stevens J, Livak KJ, and Williams PM. Real time quantitative PCR. Genome Res 6: 986994, 1996.[Abstract]
- Islam KN and Mendelson CR. Potential role of nuclear factor
B and reactive oxygen species in cAMP and cytokine regulation of surfactant protein-A gene expression in lung type II cells. Mol Endocrinol 16: 14281440, 2002.[Abstract/Free Full Text]
- Kao YJ, Piedra PA, Larsen GL, and Colasurdo GN. Induction and regulation of nitric oxide synthase in airway epithelial cells by respiratory syncytial virus. Am J Respir Crit Care Med 163: 532539, 2001.[Abstract/Free Full Text]
- Kerr MH and Paton JY. Surfactant protein levels in severe respiratory syncytial virus infection. Am J Respir Crit Care Med 159: 11151118, 1999.[Abstract/Free Full Text]
- Kong X, San Juan H, Kumar M, Behera AK, Mohapatra A, Hellermann GR, Mane S, Lockey RF, and Mohapatra SS. Respiratory syncytial virus infection activates STAT signaling in human epithelial cells. Biochem Biophys Res Commun 306: 616622, 2003.[CrossRef][ISI][Medline]
- Korfhagen TR. Surfactant protein A (SP-A)-mediated bacterial clearance: SP-A and cystic fibrosis. Am J Respir Cell Mol Biol 25: 668672, 2001.[Free Full Text]
- Korfhagen TR, LeVine AM, and Whitsett JA. Surfactant protein A (SP-A) gene targeted mice. Biochim Biophys Acta 1408: 296302, 1998.[ISI][Medline]
- Korppi M, Koskela M, Jalonen E, and Leinonen M. Serologically indicated pneumococcal respiratory infection in children. Scand J Infect Dis 24: 437443, 1992.[ISI][Medline]
- Korppi M, Leinonen M, Koskela M, Makela PH, and Launiala K. Bacterial coinfection in children hospitalized with respiratory syncytial virus infections. Pediatr Infect Dis J 8: 687692, 1989.[ISI][Medline]
- LeVine AM, Koeningsknecht V, and Stark JM. Decreased pulmonary clearance of S. pneumoniae following influenza A infection in mice. J Virol Methods 94: 173186, 2001.[CrossRef][ISI][Medline]
- LeVine AM and Whitsett JA. Pulmonary collectins and innate host defense of the lung. Microbes Infect 3: 161166, 2001.[CrossRef][ISI][Medline]
- LeVine AM, Whitsett JA, Gwozdz JA, Richardson TR, Fisher JH, Burhans MS, and Korfhagen TR. Distinct effects of surfactant protein A or D deficiency during bacterial infection on the lung. J Immunol 165: 39343940, 2000.[Abstract/Free Full Text]
- Lofgren J, Ramet M, Renko M, Marttila R, and Hallman M. Association between surfactant protein A gene locus and severe respiratory syncytial virus infection in infants. J Infect Dis 185: 283289, 2002.[CrossRef][ISI][Medline]
- Magdaleno SM, Wang G, Jackson KJ, Ray MK, Welty S, Costa RH, and DeMayo FJ. Interferon-
regulation of Clara cell gene expression: in vivo and in vitro. Am J Physiol Lung Cell Mol Physiol 272: L1142L1151, 1997.[Abstract/Free Full Text]
- Mason RJ, Walker SR, Shields BA, Henson JE, and Williams MC. Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain. Am Rev Respir Dis 131: 786788, 1985.[ISI][Medline]
- McCullers JA and Rehg JE. Lethal synergism between influenza virus and Streptococcus pneumoniae: characterization of a mouse model and the role of platelet-activating factor receptor. J Infect Dis 186: 341350, 2002.[CrossRef][ISI][Medline]
- Mendelson CR. Role of transcription factors in fetal lung development and surfactant protein gene expression. Annu Rev Physiol 62: 875915, 2000.[CrossRef][ISI][Medline]
- Mufson MA, Krause HE, Mocega HE, and Dawson FW. Viruses, Mycoplasma pneumoniae and bacteria associated with lower respiratory tract disease among infants. Am J Epidemiol 91: 192202, 1970.[ISI][Medline]
- Mufson MA, Levine HD, Wasil RE, Mocega-Gonzalez HE, and Krause HE. Epidemiology of respiratory syncytial virus infection among infants and children in Chicago. Am J Epidemiol 98: 8895, 1973.[ISI][Medline]
- Nohynek H, Eskola J, Laine E, Halonen P, Ruutu P, Saikku P, Kleemola M, and Leinonen M. The causes of hospital-treated acute lower respiratory tract infection in children. Am J Dis Child 145: 618622, 1991.[ISI][Medline]
- Odom MJ, Snyder JM, and Mendelson CR. Adenosine 3',5'-monophosphate analogs and
-adrenergic agonists induce the synthesis of the major surfactant apoprotein in human fetal lung in vitro. Endocrinology 121: 11551163, 1987.[Abstract]
- Okazaki T, Johnston JM, and Snyder JM. Morphogenesis of the lamellar body in fetal lung tissue in vitro. Biochim Biophys Acta 712: 283291, 1982.[ISI][Medline]
- Parrott RH, Kim HW, Arrobio JO, Hodes DS, Murphy BR, Brandt CD, Camargo E, and Chanock RM. Epidemiology of respiratory syncytial virus infection in Washington, DC. II. Infection and disease with respect to age, immunologic status, race and sex. Am J Epidemiol 98: 289300, 1973.[ISI][Medline]
- Patel J, Faden H, Sharma S, and Ogra PL. Effect of respiratory syncytial virus on adherence, colonization and immunity of non-typable Haemophilus influenzae: implications for otitis media. Int J Pediatr Otorhinolaryngol 23: 1523, 1992.[CrossRef][ISI][Medline]
- Pryhuber GS, Khalak R, and Zhao Q. Regulation of surfactant proteins A and B by TNF-
and phorbol ester independent of NF-
B. Am J Physiol Lung Cell Mol Physiol 274: L289L295, 1998.[Abstract/Free Full Text]
- Ramaswamy M, Shi L, Monick MM, Hunninghake GW, and Look DC. Specific inhibition of type I interferon signal transduction by respiratory syncytial virus. Am J Respir Cell Mol Biol 30: 893900, 2004.[Abstract/Free Full Text]
- Rooney SA. Regulation of surfactant secretion. Comp Biochem Physiol A Mol Integr Physiol 129: 233243, 2001.[CrossRef][ISI][Medline]
- Shepherd VL. Distinct roles for lung collectins in pulmonary host defense. Am J Respir Cell Mol Biol 26: 257260, 2002.[Free Full Text]
- Stratton CJ. Morphology of surfactant producing cells and the alveolar lining. In: Pulmonary Surfactant, edited by Robertson B, Golde LMGV, and Batenburg JJ. Amsterdam: Elsevier Science, 1984, p. 67118.
- Stuart JM, Cartwright K, and Andrews NJ. Respiratory syncytial virus infection and meningococcal disease. Epidemiol Infect 117: 107111, 1996.[ISI][Medline]
- Takala AK, Meurman O, Kleemola M, Kela E, Ronnberg PR, Eskola J, and Makela PH. Preceding respiratory infection predisposing for primary and secondary invasive Haemophilus influenzae type b disease. Pediatr Infect Dis J 12: 189195, 1993.[ISI][Medline]
- Tian B, Zhang Y, Luxon BA, Garofalo RP, Casola A, Sinha M, and Brasier AR. Identification of NF-
B-dependent gene networks in respiratory syncytial virus-infected cells. J Virol 76: 68006814, 2002.[Abstract/Free Full Text]
- Tripp RA. Pathogenesis of respiratory syncytial virus infection. Viral Immunol 17: 165181, 2004.[CrossRef][ISI][Medline]
- Van Schaik SM, Enhorning G, Vargas I, and Welliver RC. Respiratory syncytial virus affects pulmonary function in BALB/c mice. J Infect Dis 177: 269276, 1998.[ISI][Medline]
- Van Schaik SM, Vargas I, Welliver RC, and Enhorning G. Surfactant dysfunction develops in BALB/c mice infected with respiratory syncytial virus. Pediatr Res 42: 169173, 1997.[Abstract]
- Wilmott RW, Khurana-Hershey G, and Stark JM. Current concepts on pulmonary host defense mechanisms in children. Curr Opin Pediatr 12: 187193, 2000.[CrossRef][ISI][Medline]
- Xu N, Loflin P, Chen CY, and Shyu AB. A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy. Nucleic Acids Res 26: 558565, 1998.[Abstract/Free Full Text]
- Zhang L, Ikegami M, Dey CR, Korfhagen TR, and Whitsett JA. Reversibility of pulmonary abnormalities by conditional replacement of surfactant protein D (SP-D) in vivo. J Biol Chem 277: 3870938713, 2002.[Abstract/Free Full Text]
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