Adenoviral E3-14.7K protein in LPS-induced lung inflammation

Kevin S. Harrod, Amber D. Mounday, and Jeffrey A. Whitsett

Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The adenoviral E3-14.7K protein is a cytoplasmic protein synthesized after adenoviral infection. To assess the contribution of E3-14.7K-sensitive pathways in the modulation of inflammation by the respiratory epithelium, inflammatory responses to intratracheal lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha were assessed in transgenic mice bearing the adenoviral E3-14.7K gene under the direction of the surfactant protein (SP) C promoter. When E3-14.7K transgenic mice were administered LPS intratracheally, lung inflammation as indicated by macrophage and neutrophil accumulation in bronchoalveolar lavage fluid was decreased compared with wild-type control mice. Lung inflammation and epithelial cell injury were decreased in E3-14.7K mice 24 and 48 h after LPS administration. Intracellular staining for surfactant proprotein (proSP) B, proSP-C, and SP-B was decreased and extracellular staining was markedly increased in wild-type mice after LPS administration, consistent with LPS-induced lung injury. In contrast, intense intracellular staining of proSP-B, proSP-C, and SP-B persisted in type II cells of E3-14.7K mice, whereas extracellular staining of proSP-B and proSP-C was absent. Inhibitory effects of intratracheal LPS on SP-C mRNA were ameliorated by expression of the E3-14.7K gene. Similar to the response to LPS, lung inflammation after intratracheal administration of TNF-alpha was decreased in E3-14.7K transgenic mice. Levels of TNF-alpha after LPS administration were similar in wild-type and E3-14.7K-bearing mice. Cell-selective expression of E3-14.7K in the respiratory epithelium inhibited LPS- and TNF-alpha -mediated lung inflammation, demonstrating the critical role of respiratory epithelial cells in LPS- and TNF-alpha -induced lung inflammation.

lipopolysaccharide; tumor necrosis factor-alpha ; adenovirus


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A CASSETTE OF ADENOVIRAL GENES located within the E3 gene region encodes a number of proteins that modulate host responses to the virus during infection (9, 37). The adenoviral E3-14.7K gene encodes a 14.7-kDa, nonsecreted cytoplasmic protein that blocks tumor necrosis factor (TNF)-alpha -mediated cytotoxicity after adenoviral infection in vitro (10, 11, 15, 38). Mutant adenovirus deficient in the E3-14.7K gene produces increased inflammation when administered to the lungs of rodents (27). Likewise, an in vivo study (13) demonstrated that expression of the E3-14.7K gene in the distal respiratory epithelium of transgenic mice blunted lung inflammation and epithelial cell turnover after administration of E1- to E3-deleted adenoviral vectors to the lung. These studies suggest that the E3-14.7K protein may interfere with signaling in respiratory epithelial cells, thereby modulating lung inflammation and injury after adenoviral infection.

Acute respiratory distress syndrome is an often fatal respiratory disease seen after infection or injury. Proinflammatory cytokines such as TNF-alpha mediate the infiltration of inflammatory cells into the pulmonary air spaces through the induction of adhesion and chemotactic molecules. Because a number of distinct cell types are responsive to TNF-alpha , it is presently unclear whether inflammatory signals arise from the respiratory epithelium or from infiltrating cells. Pulmonary inflammation is also associated with increased alveolar permeability, causing a leak into the alveolar spaces of serum proteins that inhibit pulmonary surfactant function (12, 23, 24). Surfactant protein (SP) B and SP-C enhance surfactant spreading and stability and protect surfactant against inactivation by serum proteins (3). SPs are decreased in the bronchoalveolar lavage fluid (BALF) of acute respiratory distress syndrome patients (12). Intratracheal administration of TNF-alpha into laboratory animals caused increased lung inflammation, pulmonary edema, and pulmonary dysfunction (21, 31, 33). Previous work (2, 25) from this laboratory demonstrated that SP-B and SP-C mRNAs were decreased in the lungs of mice after intratracheal TNF-alpha administration and that these effects are mediated, at least in part, at the transcriptional level.

Pulmonary response to infection is a complex inflammatory event involving integrated interactions between pathogens and a number of cell types in the lungs. To assess the contribution of signaling through the respiratory epithelium during acute lung injury, transgenic mice that express E3-14.7K, a nonsecreted, cytosolic protein, specifically in the distal lung epithelium were administered lipopolysaccharide (LPS) or TNF-alpha intratracheally. LPS administration to the lungs of mice is a well-recognized model for studying the role of TNF-alpha -mediated signaling events during pulmonary inflammation and injury. Lung inflammation and SP dysregulation after LPS or TNF-alpha administration were attenuated in the lungs of E3-14.7K mice. These results suggest that signaling through E3-14.7K-sensitive pathways in respiratory epithelial cells contributes to lung inflammation and altered SP homeostasis in vivo after LPS-induced lung injury.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intratracheal LPS and TNF-alpha administration. Six- to twelve-week-old SP-C/E3-14.7K and FVB/N wild-type control mice (n = 6-12 mice/group) were used. Briefly, mice were anesthetized with methoxyflurane vapor, and an anterior midline incision was made to expose the trachea. Intratracheal inoculation of 30 µg of LPS (Salmonella typhymurium) or 50 µg of recombinant mouse TNF-alpha (Endogen, Woburn, MA) in 100 µl of delivery vehicle (PBS, pH 7.4) was performed with a bent, 27-gauge tuberculin syringe (Monoject, St. Louis, MO). The incision was closed with one drop of Nexaband liquid, and the mice were allowed to recover. Mice recovered rapidly and remained active after the procedure. At 24 or 48 h after administration, mice were killed by injection of pentobarbital sodium. A midline incision was made in the abdomen. Exsanguination was accomplished by transection of the inferior vena cava to reduce hemorrhage in the lung. For histological examination, the left lung lobe was inflated with 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) and fixed overnight. For studies of cell counts and cell differential analysis, BALF was collected after intratracheal instillation of 1 ml of PBS. Samples from three installations per mouse were pooled for analysis.

TNF-alpha and granulocyte-macrophage colony-stimulating factor mRNA analysis. Abundance of TNF-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA was determined by RT-PCR analysis of whole lung total RNA. Briefly, whole lung total RNA was isolated by phenol-chloroform extraction and precipitation with isopropanol with the Phase-Lock protocol (5 Prime right-arrow 3 Prime, Boulder, CO). Total RNA quantitation was confirmed by gel electrophoresis. Total RNA was converted to cDNA by the RT reaction (GIBCO BRL, Life Technologies, Gaithersburg, MD). PCR for TNF-alpha and beta -actin cDNAs was performed as described previously (1) with the following primer tandems: beta -actin primer 1, 5'-GTG GGC CGC TCT AGG CAC CAA-3'; beta -actin primer 2, 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3'; TNF-alpha primer 1, 5'-CCA GAC CCT CAC ACT CAG AT-3'; and TNF-alpha primer 2, 5'-AAC ACC CAT TCC CTT CAC AG-3'. PCR with OptiPrime reagents (Stratagene, La Jolla, CA) was performed for 25 cycles on a Perkin-Elmer 2400 Gene Amp System thermal cycler with the following parameters: initiation at 94°C for 30 s, annealing at 59°C for 30 s, and elongation of 72°C for 30 s. Ethidium bromide staining of 2% agarose gel electrophoresis was used to visualize PCR products. Primers used for analysis of GM-CSF mRNA were reported previously (39).

Inflammatory cell populations. Inflammatory cell numbers and percentages were evaluated 24 and 48 h after LPS administration. BALF (n = 6 mice/group) was obtained by intratracheal instillation of 1 ml of PBS into the exposed lungs that were maintained within the thoracic cavity. Lavage fluid was reinfused twice into the lung before final collection. BALF cells were isolated by centrifugation at 500 g and resuspended in 500 µl; 100 µl of cell suspension were mixed with 100 µl of 0.4% trypan blue (GIBCO BRL, Life Technologies, Grand Island, NY), and the cells were counted with a hemocytometer. To determine inflammatory cell types in BALF, 50 × 104 cells were mounted on slides by cytospin centrifugation in 100 µl of PBS at 600 rpm for 3 min. Cell types were identified and counted by differential staining microscopy with Diff-Quik (Baxter Healthcare, Miami, FL). Cell populations were determined by counting 100 cells, and a percentage was calculated based on five sample sets from three animals per group.

Histology and immunohistochemistry. Histopathological changes and surfactant proprotein (proSP) B and proSP-C and mature SP-B immunohistochemical staining were evaluated 24 and 48 h after LPS administration in the inflation-fixed mouse lung. Inflation-fixed lungs were washed in PBS three times and processed in serial alcohol washes. Paraffin-embedded lungs were sectioned at 5 µm and stained with hematoxylin and eosin for morphological analysis. Pathological assessment of lung inflammation was graded blindly on a scale of 0-4, and a score was determined from the mean of six animals.

ProSP-B, proSP-C, and SP-B staining was performed on paraffin-embedded lung sections as described previously (35). Sections were blocked with 2% normal goat serum for 1-2 h at room temperature and incubated overnight at 4°C with the appropriate murine SP antibody at various dilutions. Sections were rinsed five times with 0.2% Triton X-100 in PBS and incubated for 1 h at room temperature with the peroxidase-conjugated IgG secondary antibody at a dilution of 1:100 or 1:200. Sections were rinsed five times with 0.2% Triton X-100 in PBS, and peroxidase staining was detected with the ABC Elite kit (Vector Laboratories, Burlingame, CA). SP staining was observed by light microscopy with a Nikon microscope.

In situ hybridization for SP-C mRNA. In situ hybridization for SP-C mRNA was performed as described previously (36). Radiolabeled riboprobes were generated by in vitro transcription of linearized full-length SP-C cDNA fragments. Paraffin-embedded lung sections (5 µm) were air-dried on RNase-free silanated glass slides, rehydrated, and treated with proteinase K for 5 min at room temperature. Riboprobes were applied to the sections and hybridized for 16 h at 55°C. RNA-RNA hybrids were washed at high stringency with 50% formamide, 2× saline-sodium citrate (SSC), and 10 mM dithiothreitol at 65°C for 30 min and treated with RNase A (20 µg/ml) for 30 min at 37°C. Lung sections were rinsed again at high stringency, followed by lower stringency washes in 2× SSC and 0.1× SSC. All slides were dehydrated through a graded series of alcohol washes followed by xylene and dipped in 50% Ilford K5 nuclear track photographic emulsion. Slides were developed at 2 or 7 days in D-19 developer for 2 min at 16°C and fixed with Kodak fixer. Selected samples were photographed with dark-field illumination and then counterstained with hematoxylin and eosin for bright-field examination and photomicroscopy.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pulmonary inflammation after LPS administration. Cell counts in BALF of wild-type mice were increased 24 and 48 h after intratracheal administration of LPS compared with those in untreated wild-type mice (Fig. 1A). LPS-induced increase in BALF cellularity was significantly decreased after 24 and 48 h in the lungs of E3-14.7K mice compared with that in wild-type mice. Cell counts in BALF of E3-14.7K mice were only modestly increased compared with those in untreated mice. There were no differences in cell counts in BALF of wild-type and E3-14.7K mice before LPS administration (data not shown).


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Fig. 1.   E3-14.7K blocks inflammatory cell influx in lungs after treatment with lipopolysaccharide (LPS; A) and tumor necrosis factor (TNF)-alpha (B). Bronchoalveolar lavage fluid (BALF) was isolated from wild-type and E3-14.7K mice after LPS or TNF-alpha administration, and cell counts were determined with a hemocytometer and trypan blue exclusion. Normal cell counts in BALF from untreated wild-type mice are shown for comparison. A: after LPS administration, cell counts in BALF from E3-14.7K mice were decreased compared with those in wild-type mice after 24 and 48 h. Values are means ± SE; n = 10-15 mice/group. Cell counts in BALF from untreated wild-type or untreated E3-14.7K mice were not different (data not shown). * Significant difference from untreated, P <=  0.05 (by ANOVA). B: cell counts in BALF from E3-14.7K mice were decreased 24 h after TNF-alpha exposure compared with those in wild-type mice. Values are means ± SE; n = 5 mice/group. * Significant difference from untreated, P <=  0.05 (by Student's t-test).

Inflammatory cells from BALF of LPS-treated wild-type and E3-14.7K mice were analyzed by differential staining and light microscopy. Cells in BALF from untreated mice consisted primarily of alveolar macrophages. Both macrophages and neutrophils were increased 24 and 48 h after LPS treatment in wild-type mice (Table 1). After intratracheal LPS, the number of cells in BALF from E3-14.7K mice was moderately increased and consisted of both alveolar macrophages and neutrophils.

                              
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Table 1.   No. of macrophages and neutrophils from wild-type and E3-14.7K mice after exposure to LPS

Pulmonary inflammation after TNF-alpha administration. Cell counts in BALF from wild-type mice were increased 24 h after intratracheal administration of recombinant mouse TNF-alpha compared with those in untreated wild-type mice (Fig. 1B). In contrast, cell counts in BALF from E3-14.7K mice 24 h after intratracheal TNF-alpha were not increased and were similar to those in BALF from untreated wild-type mice. Cell counts in BALF from untreated wild-type and untreated E3-14.7K mice were not different (data not shown).

TNF-alpha mRNA expression in the lungs of E3-14.7K mice after LPS and TNF-alpha administration. Because LPS-induced lung injury is thought to be mediated, in part, by TNF-alpha , the effect of E3-14.7K in the respiratory epithelium on TNF-alpha expression was assessed in lung tissue after intratracheal LPS. TNF-alpha mRNA was measured by RT-PCR of total RNA from lung homogenates of wild-type and E3-14.7K mice 24 h after LPS administration. TNF-alpha mRNA was not detected in the lungs of untreated mice (Fig. 2). However, TNF-alpha mRNA was readily detectable in lung homogenates of the lungs of both wild-type and E3-14.7K mice after LPS administration. No difference in the level of TNF-alpha mRNA was noted in wild-type compared with E3-14.7K mice after LPS treatment. Likewise, GM-CSF mRNA was increased in lungs of LPS-treated mice, and the levels were similar in wild-type and E3-14.7K transgenic mice (data not shown).


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Fig. 2.   TNF-alpha mRNA expression is not altered in lungs of E3-14.7K mice after LPS administration. TNF-alpha mRNA abundance was determined by RT-PCR and ethidium-stained gel electrophoresis of total RNA from lung homogenates of wild-type (wt) and E3-14.7K mice (n = 6-8/group). TNF-alpha mRNA abundance increased but was not different in lungs of E3-14.7K mice compared with that of wt mice 24 h after LPS administration. Abundance of beta -actin mRNA was not altered between groups or after treatment. Lane M, size markers; -, lung RNA from untreated mice; +, positive control for TNF-alpha mRNA expression in lung total RNA.

Lung histology of E3-14.7K mice after LPS administration. Paraffin-embedded lung sections from wild-type and E3-14.7K mouse lungs after LPS administration were assessed by hematoxylin and eosin staining and light microscopy (Fig. 3). Increased pulmonary infiltration of macrophages and neutrophils was readily observed in the lungs of wild-type mice 24 and 48 h after intratracheal LPS. Increased septal wall thickening and cellular sloughing of the respiratory epithelium were observed. In contrast, cellular infiltrates were decreased in the lungs of LPS-treated E3-14.7K mice. In addition, alveolar septal wall thickening and cellular sloughing were decreased in lungs from E3-14.7K mice.


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Fig. 3.   Lung inflammation is decreased in E3-14.7K mice after LPS administration. Lung inflammation was assessed by hematoxylin and eosin staining of lung sections 24 h after LPS treatment. Lungs of wt mice contained increased numbers of macrophages (arrows) and neutrophils (arrowhead) as well as increased septal wall thickening compared with lungs of E3-14.7K mice after LPS exposure. Data represent findings from 10-15 mice/group. Original magnification, ×230.

E3-14.7K attenuates LPS-mediated SP dysregulation. To assess the role of E3-14.7K signaling pathways in SP expression after LPS injury, proSP-B, proSP-C, and mature SP-B were assessed by immunohistochemistry in wild-type and E3-14.7K mice. ProSP-B staining in the lungs of untreated wild-type mice was observed in type II cells, bronchi, and the bronchiolar epithelium and was not observed in alveolar macrophages or in the alveolar air spaces (Fig. 4). ProSP-B staining was decreased in alveolar type II cells of wild-type mice 48 h after treatment of LPS and was readily detected in the alveolar air spaces. ProSP-B staining in the alveolar air spaces of wild-type mice after LPS treatment was confined to inflammatory cells. In contrast, treatment of E3-14.7K mice with LPS was associated with proSP-B staining in alveolar and bronchiolar epithelial cells. ProSP-B staining was not noted in the air spaces, similar to the staining pattern of proSP-B in untreated wild-type mice. Thus after LPS exposure, pathological changes in proSP-B staining induced by LPS were ameliorated in E3-14.7K mice compared with wild-type mice.


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Fig. 4.   Aberrant surfactant proprotein (proSP) B immunostaining 48 h after LPS treatment is attenuated in lungs of E3-14.7K mice. Immunohistochemical staining of proSP-B protein in lung sections from untreated wt mice was observed in alveolar type II cells (top, arrowhead) and not in air spaces (arrows). After LPS administration, staining of proSP-B was decreased in alveolar type II cells (middle, arrowheads), and increased staining was observed in alveolar air spaces (middle, arrows). Immunostaining of proSP-B in wt mice after LPS treatment was often associated with inflammatory cells in alveolar air spaces. In contrast, lungs of E3-14.7K mice 48 h after LPS treatment had increased immunostaining of proSP-B in alveolar type II cells (bottom, arrowheads) and decreased staining within inflammatory cells in alveolar air spaces (bottom, arrows). Data represent findings from 10-15 mice/group. Original magnification, ×230.

Immunostaining for the active SP-B peptide was assessed in E3-14.7K mice and wild-type mice after LPS administration (Fig. 5). In untreated wild-type mice, SP-B staining was confined to alveolar type II cells and was rarely detected in the alveolar air spaces. In contrast, large aggregates of SP-B staining material were observed in the alveolar air spaces of wild-type mice after LPS treatment. Likewise, SP-B staining was observed in inflammatory cells within the alveolar air spaces. After LPS treatment, SP-B staining in E3-14.7K mice was confined to alveolar type II cells and was rarely detected in the air spaces or associated with inflammatory cells, a pattern similar to SP-B staining in untreated wild-type mice. The intensity of SP-B protein staining in mononuclear cells was decreased in the lungs of E3-14.7K mice compared with that in wild-type mice after LPS administration.


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Fig. 5.   Aberrant immunostaining of surfactant protein (SP) B after LPS treatment is attenuated in lungs of E3-14.7K mice. Immunohistochemical staining of mature (mat) SP-B protein in lung sections from untreated wt mice was observed in alveolar type II cells (top, arrowhead). After LPS administration to wt mice, SP-B staining was increased markedly in alveolar air spaces; SP-B staining was associated with inflammatory cells within alveolar air spaces (middle, arrow). In contrast, in E3-14.7K mice treated with LPS, SP-B staining was deceased in alveolar air spaces and inflammatory cells (bottom, arrow) and only observed in alveolar type II cells (bottom, arrowhead). Data represent findings from 10-15 mice/group. Original magnification, ×230.

ProSP-C was also assessed by immunostaining in E3-14.7K and wild-type mice after LPS administration (Fig. 6). In untreated wild-type mice, proSP-C staining was observed in alveolar type II cells, whereas no staining was observed in alveolar macrophages. After LPS administration to wild-type mice, proSP-C was decreased in alveolar type II cells and readily detectable in mononuclear cells within the alveoli. In the lungs of LPS-treated E3-14.7K mice, proSP-C staining was detected in alveolar type II cells, with little or no staining observed in alveolar macrophages, findings similar to those in untreated wild-type mice.


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Fig. 6.   Aberrant immunostaining of proSP-C after LPS is attenuated in lungs of E3-14.7K mice. Immunohistochemical staining of proSP-C protein in lung sections from untreated wt mice was observed in alveolar type II cells (top, arrowheads), with no staining observed in alveolar air spaces (top, arrow). In wt mice after LPS administration, proSP-C immunostaining was decreased in alveolar type II cells (middle, arrowheads), and increased proSP-C immunostaining was observed in alveolar air spaces; some proSP-C immunostaining was associated with inflammatory cells in alveolar air spaces (middle, arrows). In contrast, proSP-C immunostaining in E3-14.7K mice after LPS administration was detected in alveolar type II cells (bottom, arrowheads), although proSP-C staining was not detected in inflammatory cells in alveolar air spaces (bottom, arrows). Data represent findings from 10-15 mice/group. Original magnification, ×230.

Effects of LPS on SP-C mRNA in E3-14.7K mice. To assess the role of E3-14.7K-regulated pathways in SP-C mRNA, the lungs of wild-type and E3-14.7K mice were analyzed by in situ hybridization for SP-C mRNA expression after LPS treatment. In untreated wild-type mice, SP-C mRNA was readily detected in alveolar type II cells (Fig. 7). SP-C mRNA was markedly decreased in the lungs of wild-type mice after LPS administration. Decreased SP-C mRNA expression in the lungs of wild-type mice was localized to regions of increased inflammatory cells and alveolar septal wall thickening. In contrast, SP-C mRNA hybridization signal was maintained in the lungs of LPS-treated E3-14.7K mice. The intensity and number of cells expressing SP-C mRNA in the lungs of E3-14.7K mice after LPS resembled the pattern and intensity of SP-C mRNA in the lungs of untreated wild-type mice.


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Fig. 7.   SP-C mRNA is maintained in E3-14.7K mice after LPS treatment. SP-C mRNA was assessed by in situ hybridization of lung sections from untreated wt mice and wt or E3-14.7K mice 24 h after LPS administration. SP-C mRNA was observed in alveolar type II cells of untreated mice. Twenty-four hours after LPS treatment, SP-C mRNA was markedly decreased in lungs of wt mice. In contrast, SP-C mRNA was maintained in lungs of E3-14.7K mice after LPS administration. Data represent findings from 8 mice/group. Original magnification, ×175.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates the role of E3-14.7K-sensitive pathways in the modulation of lung inflammation and SP dysregulation after LPS- and TNF-alpha -induced injury. LPS administration to the lungs of wild-type mice caused acute lung inflammation and SP dysregulation, findings similar to those reported for TNF-alpha in vitro and in vivo (2, 19, 22, 25). The expression of E3-14.7K in the respiratory epithelium of mice decreased acute lung inflammation and attenuated the inhibitory effects of LPS on SP expression in alveolar type II cells. E3-14.7K did not alter TNF-alpha mRNA abundance in the lungs of mice, consistent with the concept that E3-14.7K inhibits TNF-alpha signaling pathways and not TNF-alpha production. Taken together, these findings suggest that LPS- and TNF-alpha -induced lung inflammation and LPS-induced SP dysregulation occur through E3-14.7K-regulated pathways in respiratory epithelial cells and that signaling through the respiratory epithelium plays a critical role in the inflammatory response during LPS-induced lung injury.

In the present study, the infiltration of inflammatory cells into the alveolar air spaces after acute LPS injury was attenuated by the expression of E3-14.7K in the respiratory epithelium. E3-14.7K protein, a nonsecreted, cytoplasmic protein, was expressed selectively in respiratory epithelial cells in SP-C/E3-14.7K transgenic mice (13). The finding that lung inflammation after intratracheal LPS or TNF-alpha administration was abrogated by selective expression of E3-14.7K in respiratory epithelial cells supports the concept that E3-14.7K-sensitive pathways in respiratory epithelial cells are required for the inflammatory responses to TNF-alpha or LPS in vivo. Intratracheal LPS administration in rodents causes an acute lung injury that is mediated, at least in part, through TNF-alpha -regulated cellular events in the lung (22, 26, 28, 32). Intratracheal administration of LPS to rodents induces TNF-alpha to peak levels within 3 h after administration (22), and the stimulation of inflammatory cell migration, including neutrophils and monocytic cell types into the lung air spaces and lung parenchyma, occurs at time points that coincide with TNF-alpha induction (28, 31). Indeed, the administration of LPS and the subsequent activation of TNF-alpha are known to induce the expression of intracellular adhesion molecule-1 and facilitate inflammatory cell migration into the lung in animal models (28). Chronic TNF-alpha exposure through the constitutive expression of TNF-alpha specifically in the lungs of transgenic mice causes progressive lung inflammation and alveolitis, with marked lung damage and remodeling (20). Interestingly, the induction of TNF-alpha mRNA by LPS was not altered in the lungs of E3-14.7K mice. Likewise, abundance of GM-CSF mRNA was not changed in the lungs of E3-14.7K mice compared with that in wild-type mice after LPS administration (data not shown), suggesting that E3-14.7K protein in respiratory epithelial cells did not influence lung inflammation via GM-CSF signaling in alveolar macrophages. The findings in this study are consistent with the concept that signaling events in the respiratory epithelium per se are important determinants of pulmonary inflammation after LPS administration.

The loss of SP expression after intratracheal LPS treatment was inhibited by the expression of E3-14.7K in respiratory epithelial cells. The decreased proSP-B and proSP-C staining in alveolar type II cells and the appearance of proSPs in the alveolar air spaces in the lungs of wild-type mice after LPS injury is consistent with LPS-induced type II cell injury or aberrant secretion from type II cells. For instance, alveolar type II cell turnover after LPS-induced lung injury and subsequent phagocytosis of cellular debris, including proSPs may explain the presence of proSPs in the alveolar air spaces. Likewise, SP secretion by alveolar type II cells may also occur after lung injury. The difficulty in precise identification of alveolar cell types in light of the decreased expression of proSPs should be noted. In the present study, E3-14.7K expression in respiratory epithelial cells inhibited the LPS-induced appearance of proSPs in the alveolar air spaces. Indeed, E3-14.7K has been shown to alter cell turnover after TNF-alpha treatment in vitro (11) and in the lungs of transgenic mice after adenoviral infection in vivo (13).

In the present study, the expression of E3-14.7K in the distal lung epithelium corrected the LPS-induced decrease in SP-C mRNA. SP-C mRNA was decreased both in vivo and in cultured cell studies after exposure to TNF-alpha (2). The reduction in SP-C mRNA abundance by TNF-alpha appears to occur, in part, at the level of gene transcription (2). A 320-bp promoter element for the SP-C gene was sufficient to mediate the inhibitory effects of TNF-alpha on SP-C expression in a lung epithelial cell culture system (2). Likewise, the intratracheal administration of human or mouse recombinant TNF-alpha reduced SP-C mRNA abundance and SP-C promoter activity in the lungs of transgenic mice (2). The results from the present study suggest that the presence of E3-14.7K protein in the respiratory epithelium is capable of attenuating the downregulation of SP-C promoter-driven transgene expression in vivo. The expression of E3-14.7K in the lungs of transgenic mice attenuated the loss of SP-C mRNA expression during LPS-mediated lung injury, providing further evidence that signaling through E3-14.7K-sensitive pathways in alveolar epithelial cells may play an important role in the SP dysregulation during acute lung injury.

The adenoviral E3-14.7K protein is a 14.7-kDa cytosolic, nonsecreted protein that blocks TNF-alpha -induced cell death in vitro (10, 11, 14). The adenoviral E3 gene region encodes a number of viral proteins that likely allow adenovirus to escape host immunosurveillance or attenuate host immune responses that mediate viral clearance (37). After adenoviral infection in vitro, the expression of the adenoviral transcriptional regulator gene E1A sensitizes infected cells to cell lysis after exposure to TNF-alpha (4, 6). The expression of E3-14.7K, either encoded by the infecting virion or through transfection of the gene in vitro, blocks E1A-sensitized cell lysis by TNF-alpha (10, 14). Cytotoxicity of sensitized cells by TNF-alpha is dependent on the activation of cytosolic phospholipase A2 (15, 29) and likely occurs through an apoptosis-mediated mechanism (16, 34). E3-14.7K inhibits both cytosolic phospholipase A2 activation and induction of intracellular proteases after TNF-alpha in sensitized cells (15, 34). The ectopic expression of E3-14.7K in vivo blocks inflammation in a number of animal models, including the lung (7, 8, 13, 30). The precise molecular interaction of E3-14.7K is not known; however, recent studies (5, 17, 18) suggest that E3-14.7K may bind proteins involved in apoptotic pathways. The current study supports the concept that E3-14.7K-sensitive pathways within the respiratory epithelium are critical in the initiation of inflammation and SP dysregulation during LPS-induced acute lung injury.


    ACKNOWLEDGEMENTS

We thank Dr. William S. M. Wold for assistance in providing the E3-14.7K cDNA and for thoughtful discussion. We also thank Timothy Cho, Seema Bhatt, and Lorie Stuart for technical assistance.


    FOOTNOTES

Present address of K. S. Harrod: Asthma and Immunology Group, Lovelace Respiratory Research Institute, PO Box 5890, Albuquerque, NM 87185.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. A. Whitsett, Div. of Neonatology and Pulmonary Biology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: whitj0{at}chmcc.org).

Received 30 July 1999; accepted in final form 3 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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