help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haeberle, H. A.
Right arrow Articles by Garofalo, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haeberle, H. A.
Right arrow Articles by Garofalo, R. P.
American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1433-1438, (2002)
© 2002 American Thoracic Society

Original Article

Perflubron Reduces Lung Inflammation in Respiratory Syncytial Virus Infection by Inhibiting Chemokine Expression and Nuclear Factor–{kappa}B Activation

Helene A. Haeberle, Frances Nesti, Hans-Juergen Dieterich, Zoran Gatalica and Roberto P. Garofalo

Departments of Pediatrics, Pathology, Microbiology, and Immunology, University of Texas Medical Branch, Galveston, Texas; and Department of Anesthesiology, University Hospital, Tuebingen, Germany

Correspondence and requests for reprints should be addressed to Roberto P. Garofalo, M.D., Dept. of Pediatrics, Division of Immunology/Allergy/Rheumatology, University of Texas Medical Branch of Galveston, 301 University Boulevard, Galveston, TX 77555-0369. E-mail: rpgarofa{at}utmb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Airway mucosa inflammation plays a critical role in the pathogenesis of lower respiratory tract infections caused by respiratory syncytial virus (RSV), the major etiologic agent of bronchiolitis in infancy. Type and intensity of cellular infiltration are dictated by inflammatory chemokines, which are rapidly and abundantly induced in lung tissue by RSV. This process is, to a large extent, transcriptionally regulated by RSV-mediated activation of the nuclear factor–{kappa}B. The administration of a perfluorocarbon (PFC) liquid, such as perflubron, during partial liquid ventilation improves lung function and also reduces inflammation. In this study we demonstrate that treatment of BALB/c mice with perflubron intranasally 6 hours after RSV infection significantly inhibited lung cellular inflammation as well as the expression of the chemokines RANTES, MIP-1{alpha}, MIP-1ß, and MIP-2, compared with phosphate-buffered saline–treated control mice. However, perflubron treatment did not affect RSV replication. Strikingly, treatment with perflubron abrogated nuclear factor–{kappa}B activation in lung of RSV-infected mice. These results demonstrate a novel mechanism by which PFC may exert antiinflammatory activity and suggest that partial liquid ventilation with PFC may be considered in future clinical trials for infants with severe RSV infections requiring mechanical ventilation.

Key Words: respiratory syncytial virus • perflubron • nuclear factor–{kappa}B • inflammation • chemokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Respiratory syncytial virus (RSV) is the major etiologic agent of serious epidemic lower respiratory tract disease in infancy (1). More than 50% of all infants are infected with RSV during the first year of life, and by the age of 2 more than 90% of children have been infected (2). In addition, severe lower respiratory tract infections caused by RSV may occur at any age in patients with compromised cardiac, pulmonary, or immune systems and in the elderly (3). Hospitalization is required in up to 3% of infants less than 1 year old with RSV infection (4), with mortality rates ranging from less than 1% in industrialized countries to 7% in developing countries (5). Bronchiolitis, the most severe form of RSV infection, is characterized clinically by wheezing, dyspnea, respiratory distress, and radiologic evidence of hyperinflation of the lung (6). Hypoxemia and hypercapnia may develop in more severe cases, leading to respiratory insufficiency. Mechanical ventilation is required in 7–21% of infants hospitalized with RSV infection (7). Autopsy studies of fatal disease have identified necrosis and sloughing of epithelial cells of the small airways, edema, and increased mucus secretion causing airflow obstruction. An intense peribronchial infiltration of mononuclear cells has been described as a hallmark of this infection (3), and neutrophil-rich exudates detected by bronchoalveolar lavage (BAL) (8). Finally, the presence of eosinophil-specific proteins and histamine in respiratory secretions at concentrations that correlate with disease severity suggests the participation of eosinophils and basophils in the pathology of RSV infection (9–11).

Although all these clinical and histopathologic features underscore the critical role played by mucosal inflammation in the pathogenesis of severe RSV infection, attempts to find effective antiinflammatory therapies for this condition have so far been largely unsuccessful (12). Indeed, the mechanisms that regulate selective recruitment of inflammatory cells to the airways and their activation following RSV infection are not fully understood. Chemokines are chemotactic cytokines that direct leukocyte transendothelial migration and movement through the extracellular matrix and have emerged as central regulatory molecules in inflammatory, immune, and infectious processes of the lung (13). Recent studies have suggested that chemokines may be critically involved in the pathogenesis of RSV-induced bronchiolitis in human infants as well as in animal models (10, 14–16). Studies in vitro have also demonstrated that RSV is among the most potent biologic stimuli to induce chemokine and cytokine production by respiratory epithelial cells, a process that is largely controlled by virus-mediated activation of the transcription factor nuclear factor (NF)-{kappa}B (17–20). NF-{kappa}B comprises a family of inducible transcription factors that include the potent Rel A (p65) transactivator, Rel B, c-Rel, NF-{kappa}B1 (p50), and NF-{kappa}B2 (p52) subunits. Inducible NF-{kappa}B subunits interact with cytoplasmic inhibitors, collectively known as I{kappa}B, through motifs contained within a conserved NH2-terminal Rel homology domain. Extracellular stimuli initiate a signaling cascade that leads to rapid phosphorylation of I{kappa}B{alpha}, coupled to the rapid ubiquitination and proteolytic degradation of phosphorylated I{kappa}B{alpha} through the 26S proteasome, thereby exposing the nuclear localization signal and freeing NF-{kappa}B to translocate to the nucleus, where it binds and activate target genes (reviewed in Ref. 21).

Perflubron (perfluorooctylbromide, C8F17Br) is a perfluorocarbon (PFC) that easily disperses throughout the lung. Because of its physical characteristics, including the high solubility of oxygen and CO2 in it and the low surface tension, perflubron has been used in clinical trials of partial liquid ventilation in adult patients and premature infants with acute respiratory distress syndrome (22, 23). Perflubron not only enhances gas exchange and improves lung compliance (24), but also, despite its biochemical inert structure, displays antiinflammatory effects (25–27). A few recent reports have shown for example that the use of perflubron in partial liquid ventilation resulted in significant reduction in the number of inflammatory cells in BAL samples of patients treated for acute respiratory distress syndrome. The decrease in cell infiltration was accompanied by lower concentrations of inflammatory and immunomodulatory cytokines, compared with patients ventilated without perflubron (28). The molecular mechanisms involved in the antiinflammatory activity of perflubron remain largely unknown. Therefore, using a BALB/c mouse model of experimental RSV infection, we tested in this study the effect of perflubron on airway pathology, chemokine expression, and NF-{kappa}B activation in lung tissue. We show that intranasal administration of perflubron significantly reduces RSV-induced lung inflammation in parallel with the inhibition of inducible chemokine expression. Moreover, we provide novel evidence that perflubron may exert its antiinflammatory activity by blocking the activation of the potent transcription factor NF-{kappa}B.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
RSV Preparation
The long strain of RSV (A2) was grown in HEp-2 cells (ATCC, Rockville, MD) and sucrose-gradient purified as described elsewhere (29). The virus titer was determined by a methylcellulose plaque assay (30).

Mice and Infection Protocol
Female, 3- to 4-week-old BALB/c mice were purchased from Harlan (Houston, TX) and housed under pathogen-free conditions in the animal research facility of the University Texas Medical Branch (UTMB, Galveston, TX) in accordance with the National Institutes of Health and UTMB institutional guidelines for animal care. Under light anesthesia, mice were infected intranasally with 50 µl of purified RSV diluted in phosphate-buffered saline (PBS) (final administered dose: 1 x 107 plaque-forming units) or were inoculated with PBS alone (sham infection). The 50-µl volume was selected to allow infection of the mice with a high titer of purified RSV and distribution of the inoculum mainly in the lung tissue (31). Six hours later, 50 µl of perflubron (Imagent; Alliance, San Diego, CA) or control PBS were gently inoculated intranasally (25 µl in each nostril) using a micropipette. Twenty-four hours after infection, the mice were killed and the lungs were removed for histopathology and for extraction of RNA and nuclear proteins.

Pulmonary Histopathology
Lungs were perfused and fixed in 10% buffered formalin and embedded in paraffin. Multiple 4-µm sagittal sections of whole lung were stained with hematoxylin and eosin (H&E). Slides were analyzed and scored for cellular inflammation under light microscopy by an expert pathologist who was blind to the treatment groups, as previously described (16, 32). Briefly, inflammatory infiltrates were scored enumerating the layers of inflammatory cells surrounding the vessels and bronchioles. Zero to three layers of inflammatory cells was considered "normal." Moderate to abundant infiltrate (more than three layers of inflammatory cells surrounding 50% or more of the circumference of the vessel or bronchioles) was considered "abnormal." The number of abnormal perivascular and peribronchial spaces divided by the total perivascular and peribronchial spaces was the percentage reported as the pathology score. A total of approximately 15 perivascular and peribronchial spaces per lung was counted for each animal.

Chemokine mRNA Expression by RNAse Protection Assay
Chemokine mRNA expression was determined by RNAse protection assay (RPA) as described previously (16). Lungs were quick frozen in liquid nitrogen and stored at -80°C until total RNA was isolated by the thiocyanate–phenol–chloroform method. Chemokine mRNA expression was determined by a multiprobe RPA using the RiboQuant kit (Pharmingen, San Diego, CA). The probe was labeled with {alpha}-[32P]-UTP (3,000 Ci/mmol, 10 µCi/µl; Du Pont NEN Research Products, Boston, MA) using a T7 polymerase. After overnight hybridization with 5 µg of total RNA and RNA digestion, the samples were treated with proteinase K–sodium dodecyl sulfate mixture, extracted by phenol–chloroform and precipitated in the presence of ammonium acetate. The samples were finally loaded on a QuickPoint sequence gel (Novex, San Diego, CA), exposed to an XAR film (Eastman Kodak, Rochester, NY), and developed at -70°C. The identity of each protected fragment was established by analyzing its migration distance against a standard curve of the migration distance versus the log nucleotide length for each undigested probe. The quantity of each mRNA species in the original RNA sample was then determined on the basis of the signal intensity (measured using AlphaImager 2,200 optical densitometer; Alpha Innotech Corp., San Leandro, CA) given by the appropriately sized, protected probe fragment bands. Sample loading was normalized to the housekeeping gene L32, included in each template set.

Extraction of Lung Nuclear Proteins and Electrophoretic Mobility Shift Assay
For extraction of nuclear proteins, lungs were quick frozen in liquid nitrogen immediately after removal from the thoracic cavity. Nuclear proteins were isolated from the lung tissue using a modified method described by Bohrer and colleagues (33). Lung tissue was homogenized in 5 ml ice-cold Buffer A (10 mM 2-hydroxyethyl-piperazine N'-2-ethanesulfonic acid [Hepes]–KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol [DTT], 0.2 mM phemylmethyl sulfonyl fluoride [PMSF], 0.6% nonident P40 [NP-40]) before centrifugation (350 x g, 4°C for 30 seconds). The supernatant was kept on ice for 5 minutes and centrifuged for 5 minutes at 6,000 x g at 4°C, and the pellet was resuspended in 200 µl Buffer B (10 mM Hepes–KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1.2 M sucrose, 0.5 mM DTT, 0.2 mM PMSF). After centrifugation (13,000 x g, 4°C, 30 minutes), the pellet was resuspended in 100 µl Buffer C (20 mM Hepes–KOH, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM ethylenediamine-tetraacetic acid, 0.5 mM DTT, 0.2 mM PMSF, 2 mM benzamidine, 5 µg/ml leupeptin, 25% glycerol), incubated on ice for 20 minutes, and centrifuged (6,000 x g, 4°C, 2 minutes). The supernatant was quick frozen in aliquots at –80°C. For electrophoretic mobility shift assay (EMSA), nuclear proteins were normalized by protein assay (Protein Reagent; Bio-Rad, Hercules, CA) and used to bind to duplex oligonucleotides corresponding to the RANTES NF-{kappa}B binding site, under conditions described previously (17, 19). Nuclear proteins were incubated with the probe for 20 minutes at room temperature before they were fractionated on a 6% nondenaturing polyacrylamide gel in tris-borate-EDTA (TBE) buffer. Gels were dried and exposed for autoradiography at -70°C overnight. NF-{kappa}B–binding complexes were quantified by optical densitometry.

RSV Titration in Lung Tissue
For virus titration, lungs were weighted and homogenized in Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum in a 10% ratio (wt/vol). Homogenized samples were centrifuged at 2,000 x g for 10 minutes. Serial dilutions of the supernatants were tested on Hep2 cells in a methylcellulose plaque assay (16, 30). The final virus titer is expressed as plaque-forming units per gram of lung tissue.

Statistical Analysis
Statistical analysis was performed using the SigmaStat 3.0 program (Jandel Corp., San Rafael, CA). The data were analyzed by the Wilcoxon test and the Kruskal–Wallis One-Way Analysis for variance on ranks and by analysis of variance for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The BALB/c mouse model shows close similarity to the pathogenesis of RSV-induced lower airway disease in humans (34). We have recently established that experimental infection of BALB/c mice with highly purified preparations of RSV A, at a dose of 107 plaque-forming units, induces a severe inflammatory response in lung tissue as early as 24 hours following intranasal inoculation. Lung inflammation was characterized by an excess of monocytes/macrophages, lymphocytes, and to a lesser extent, neutrophils, surrounding bronchioles and vessels, with evidence of involvement of alveolar spaces (16). Therefore, to determine the effect of PFC on RSV-induced lung inflammation, 4- to 6-week-old BALB/c mice were inoculated intranasally with perflubron 6 hours after infection. Previous studies have shown that intranasal application results in efficient and homogeneous distribution in all lung segments (35). Twenty-four hours after virus inoculation, the mice (n = 9 animals in each group) were killed, and lung paraffin sections were stained with H&E. As shown in Figure 1 , no inflammation could be detected in sham-infected animals. On the other hand, mice infected with RSV had evident foci of perivascular/peribronchial mononuclear cell infiltration. Strikingly, mice infected with RSV and treated 6 hours later with perflubron had a significant reduction in lung inflammation (pathology score: 18%) compared with PBS-treated control mice (pathology score: 31%). To assess if perflubron treatment interfered with the ability of RSV to replicate, viral titer was determined in lung tissue of RSV-infected mice treated with perflubron or treated with PBS control. RSV titer in mouse lung tissue cannot be correctly determined until Day 3–5 after infection because the virus undergoes an "eclipse phase" following inoculation (16, 31, 34). Therefore, we measured lung RSV titers 4 days after infection and found that they were not significantly different in animals that had been treated with perflubron or PBS (RSV + perflubron, 5.3 ± 0.05 plaque-forming units/g versus RSV + PBS, 4.9 ± 0.2 plaque-forming units/g; n = 3 mice/group). This finding indicates that reduced lung inflammation is not due to interference with RSV replication or to a potential antiviral activity per se of perflubron.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. Lung inflammation in RSV-infected mice. Groups of BALB/c mice were sham-inoculated (Sham) or infected with RSV, followed 6 hours after infection by intranasal perflubron (RSV + perflubron) or PBS (RSV). Twenty-four hours after infection, H&E-stained sagittal sections of the lung were scored for cellular inflammation. The experiment was repeated three times. The figure presents data (mean ± SEM) of nine mice per group. *p < 0.05.

 
We have previously shown that RSV-mediated lung inflammation is temporally associated with inducible expression of several proinflammatory chemokines (16). Thus, to determine if antiinflammatory effect of perflubron in RSV infection may be seen in the context of chemokine inhibition, total RNA was isolated from the lung tissue and analyzed by RPA with a multiprobe containing the DNA templates for eight murine chemokines. In sham-infected mice, only a protected band specific for RANTES mRNA was visible. In RSV-infected mice, on the other hand, we consistently observed the upregulation of RANTES mRNA, as well as the appearance of mRNA for eotaxin, MIP-1ß, MIP-1{alpha}, MIP-2, and MCP-1 (Figure 2A) . Overall, the expression of all RSV-inducible chemokines was weaker in mice that were treated with perflubron 6 hours postinoculation compared with PBS-treated control mice. Detailed quantitation by densitometry analysis showed that lung mRNA for the CC chemokines RANTES (p = 0.02), MIP-1{alpha} (p = 0.03), MIP-1ß (p = 0.03), and MIP-2 (p = 0.04) were significantly reduced in RSV-infected animals that received perflubron compared with those treated with control PBS (Figure 2B).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 2. Chemokine mRNA expression in lung tissue of RSV-infected mice. Expression of mRNA for murine chemokines was investigated by RPA (24 hours after infection). (A) Representative result of three independent experiments: RNA was isolated from lung tissue and hybridized with a 32P-labeled RiboQuant MultiProbe (Pharmingen) containing DNA templates for eight murine chemokines (Ltn, RANTES, eotaxin, MIP-1ß, MIP-1{alpha}, MIP-2, MCP-1, and TCA-3) and the housekeeping gene L32 (ribosomal RNA). After RNAse treatment and purification, protected probes were run on a sequence gel, exposed to an XAR film, and developed. Each lane (1 through 9) is an individual animal: 1–3, sham-infected mice; 4–6, RSV-infected mice treated with PBS; 7–9, RSV-infected mice treated with perflubron. (B) The quantity of each mRNA species in the original RNA sample was determined on the basis of the signal intensity (by optical densitometry) given by the appropriately sized, protected probe fragment band. Sample loading was normalized to the L32 gene included in each template set. Density of each chemokine mRNA is expressed relative to that of L32. The bar graph presents the results (mean ± SEM) of three mice per group. * p < 0.05.

 
Although the antiinflammatory properties of PFC have been previously reported, the molecular mechanisms that are involved remain largely unknown. We have recently reported in detailed studies that RSV infection in mice strongly induces activation of NF-{kappa}B in lung tissue. Inhibition of NF-{kappa}B nuclear translocation significantly blocked lung inflammation and chemokine expression (Haeberle and coworkers, manuscript submitted). Thus, we hypothesized that the effect of perflubron observed in RSV-infected mice might be due to its ability to inhibit NF-{kappa}B. In the following studies, groups of BALB/c mice were treated as described previously, and lung nuclear proteins were isolated for EMSA (24 hours). A low degree of constitutive NF-{kappa}B DNA-binding activity was detected in lung nuclear extracts of sham-infected animals (Figure 3) . Sequence specificity of the nuclear protein–DNA complex was extensively confirmed by competition using unlabeled oligonucletide probes (Haeberle and coworkers, manuscript submitted). Nuclear activity of NF-{kappa}B greatly increased in the lungs of RSV-infected mice. Strikingly, treatment with perflubron abrogated RSV-induced NF-{kappa}B activation in all the animals tested (Figure 3). Perflubron alone had no influence on NF-{kappa}B activation (Figure 3, lane 7).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. EMSA of NF-{kappa}B–binding complexes in RSV-infected mice. (A) Autoradiogram of a representative experiment. Groups of BALB/c mice were sham-inoculated (lanes 1 and 7) or infected with RSV, followed 6 hours after infection by intranasal PBS (lane 2) or perflubron (lanes 3–7). Twenty-four hours after infection, the mice were killed, and nuclear proteins extracted from the lungs were used for EMSA (with double-stranded RANTES NF-{kappa}B oligonucleotide probe). Arrows indicate the specific NF-{kappa}B DNA complexes. (B) The experiment was repeated three times. The bar graph presents the results (mean ± SEM) of nine mice per/group. * p < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Lower respiratory tract infections caused by RSV are characterized by profound cellular inflammation of the airway mucosa, which contributes to the disease manifestations, including air flow limitation, lung atelectasis/emphysema, and hypoxemia (36). A number of recent studies in infants suggest that inflammatory chemokines, including RANTES, eotaxin, MCP-1, and MIP-1{alpha}, play a critical role in the pathogenesis of severe lower respiratory tract infections caused by RSV (10, 14, 15, 29, 37). Indeed, the profile of the RSV-inducible chemokines closely reflects the type of cellular infiltration, which is characteristic of the lower respiratory infections, caused by this virus. We have previously described that experimental infection of mice with RSV induces a rapid dose-dependent expression of several inflammatory chemokines 24 hours after infection. Genetically altered mice with a selective deletion of the MIP-1{alpha} gene had a striking reduction in lung inflammation following RSV infection, compared with control littermates (16). Our observations that treatment of mice with perflubron significantly reduced airway inflammation and profoundly inhibited the expression of chemokine genes, including RANTES, MCP-1, and MIP-1{alpha}, further argue in favour of an essential role of these molecules in RSV-mediated lung pathology. Moreover, because NF-{kappa}B is required for RSV-induced transcription of chemokine genes, we suggest that the effect of perflubron on RSV-induced lung chemokines is due to the dramatic inhibitory activity of perflubron on NF-{kappa}B activation that we report in this study. Additional support for this hypothesis comes from the detailed studies reported elsewhere in which we showed that RSV-infected mice treated with specific inhibitors of NF-{kappa}B had significant reduction of lung pathology and chemokine production compared with control animals (Haeberle and coworkers, submitted). Others have reported that mice deficient in the NF-{kappa}B p50 subunit are incapable of mounting eosinophilic airway inflammation and are deficient in the production of eotaxin, MIP-1{alpha}, and MIP-1ß (38), whereas mice deficient in the c-Rel subunit have decreased expression of MCP-1 in lung tissue following allergen challenge compared with wild-type control mice (39). Both p50 and c-Rel subunits are present in the RSV-inducible NF-{kappa}B heterodimer complexes (Haeberle and coworkers, submitted).

PFC are currently under investigation for various medical uses, such as use as intrapulmonary agents in partial liquid ventilation (also known as perflurocarbon-associated gas exchange [24]), as contrast agents for enhancement of ultrasound images, and as blood substitutes for intravascular oxygen carrier (40–44). Recent reports have also demonstrated the efficacy of PFC liquids as a delivery vehicle for pulmonary administration of biologic agents, including antibiotics and gene vectors (45). Largely based on the histologic evidence of reduced inflammatory responses in lungs treated with PFC, and perflubron in particular (25, 46), a number of in vitro studies have focused on the identification of cellular and molecular mechanisms that might explain this property. Thus, PFC or perflubron has been shown to inhibit activation and chemotaxis of neutrophils (47–49), to attenuate production of reactive oxygen species by alveolar macrophages (50), and to decrease production of the inflammatory cytokines TNF-{alpha}, IL-6, IL-1, and IL-8 by macrophages and epithelial cells (51–53). Our study shows for the first time that perflubron also exerts a profound effect on the expression of lung chemokines, both of the CXC type (with activity on neutrophils) and the CC type (with activity on monocytes, T cells, basophils, and eosinophils). Two possible mechanisms have been suggested to explain these in vitro effects of PFC on the inducible expression of cytokine gene and inhibition of inflammatory mediator release: (1) In macrophages, and other phagocytic cells, ingestion of PFCs particles may result in alterations of cell membrane receptor–ligand binding, thus affecting cellular responses dependent on membrane-bound activation mechanisms, and/or may disrupt intracellular signaling mechanisms (50). In this regard, it has been recently shown that perflubron decreases neutrophil function by globally inhibiting cellular tyrosine phosphorylation and the Syk pathway in particular (48). (2) In airway epithelial cells, the physical properties of PFC may play an important role by providing a physical barrier that prevents binding of stimulatory molecules to their cellular receptors (53). For that reason and to avoid possible confounding effects of PFC on RSV interaction with the lung mucosa (i.e., its effect on viral inoculum distribution or its physical barrier effect), we have administered perflubron 6 hours after RSV inoculation. This time point falls well beyond the time required for an intranasal viral inoculum to reach the lung and for attachment and entry of RSV in susceptible cells (54, 55).

In the present studies, we provide evidence for a novel mechanism by which perflubron may regulate gene expression in vivo, i.e., by inhibiting the binding activity of the "master switch," NF-{kappa}B. Although the exact cell population involved cannot be ruled out at the moment, there is strong experimental evidence that both alveolar macrophages/monocytes and respiratory epithelial cells play a major role as initiators of the inflammatory and immune responses in the lung following RSV infection (reviewed in Ref. 56). Previous studies in vitro have demonstrated that RSV infection induced increased NF-{kappa}B DNA-binding activity in cultured lung epithelial cells, an activity that leads to the transcriptional induction of several inflammatory chemokines (17–20, 57–59). Direct interaction of RSV with macrophages/monocytes as well results in the transcriptional induction of a number of NF-{kappa}B–dependent inflammatory gene products, including IL-6, IL-1, and TNF-{alpha}, which in the course of an in vivo infection may regulate the release of chemokines via autocrine/paracrine mechanisms (60–62). Thus, the inhibitory activity of perflubron on NF-{kappa}B may affect the local production of chemokines directly by impairing chemokine gene transcription or indirectly by blocking the production of potent cytokines that are known to regulate chemokine production (62–66). As a result, inhibition of chemokine release in the airways is indeed reflected in the significant reduction of lung pathology that we have observed in RSV-infected animals treated with perflubron.

In summary, we show in this study that the PFC perflubron decreases RSV-induced lung inflammation in mice by a mechanism that involves the inhibition of NF-{kappa}B–dependent production of inducible chemokines in the lung. Previous clinical trials have shown controversial results regarding the beneficial effect of antiinflammatory therapy (corticosteroids) in lower respiratory tract infections caused by RSV (reviewed in Ref. 12). Recently, five randomized controlled trials studied the effect of corticosteroids in the treatment of RSV-proven bronchiolitis. In four of these trials, no beneficial effect of corticosteroids could be demonstrated in terms of severity score, oxygen saturation and oxygen use, lung function, or length of hospital stay. However, in all these four studies, patients with sever bronchiolitis and patients who needed mechanical ventilation were excluded. On the other hand, a recent randomized placebo-controlled trial in RSV-infected infants requiring mechanical ventilation has shown that the use of corticosteroids (prednisolone) resulted in a significantly shorter duration of hospitalization compared with placebo-treated control infants (67). These findings suggest that antiinflammatory therapies for RSV lower respiratory tract infections may prove to be beneficial in the setting of a more severe disease, i.e., when mechanical ventilation is required. Thus, as previously shown in premature infants with severe acute respiratory distress syndrome (22), partial liquid ventilation with perflubron might be considered in future for treatment of infants with severe RSV infections requiring mechanical ventilation.


   Acknowledgments
 
The authors thank Esther Tamayo for assistance in the preparation of purified RSV and Prof. Klaus Unertl for the valuable discussions and support.

This work was supported by grants AI 15939 and P01 AI 46004 from the National Institute of Allergy and Infectious Diseases, 644-0-0 from the Fortune Program of the University of Tuebingen, Germany, and a grant of the John Sealy Memorial Endowment Fund for Biomedical Research at UTMB.

Received in original form September 21, 2001; accepted in final form March 4, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Heilman CA. From the National Institute of Allergy and Infectious Diseases and the World Health Organization: respiratory syncytial and parainfluenza viruses. J Infect Dis 1990;161:402–406.[Medline]
  2. Glezen WP, Taber LH, Frank AL, Kasel JA. Risk of primary infection and reinfection with respiratory syncytial virus. Am J Dis Child 1986; 140:543–546.[Abstract/Free Full Text]
  3. Hall CB. Respiratory syncytial virus and parainfluenza virus. N Engl J Med 2001;344:1917–1928.[Free Full Text]
  4. Shay DK, Holman RC, Newman RD, Liu LL, Stout JW, Anderson LJ. Bronchiolitis-associated hospitalizations among US children, 1980–1996. JAMA 1999;282:1440–1446.[Abstract/Free Full Text]
  5. John TJ, Cherian T, Steinhoff MC, Simoes EA, John M. Etiology of acute respiratory infections in children in tropical southern India. Rev Infect Dis 1991;13:S463–S469.
  6. Ruuskanen O, Ogra PL. Respiratory syncytial virus. Curr Probl Pediatr 1993;23:50–79.[CrossRef][Medline]
  7. Outwater KM, Crone RK. Management of respiratory failure in infants with acute viral bronchiolitis. Am J Dis Child 1984;138:1071–1075.[Abstract/Free Full Text]
  8. Everard ML, Swarbrick A, Wrightham M, McIntyre J, Dunkley C, James PD, Sewell HF, Milner AD. Analysis of cells obtained by bronchial lavage of infants with respiratory syncytial virus infection. Arch Dis Child 1994;71:428–432.[Abstract/Free Full Text]
  9. Garofalo R, Kimpen JL, Welliver RC, Ogra PL. Eosinophil degranulation in the respiratory tract during naturally acquired respiratory syncytial virus infection. J Pediatr 1992;120:28–32.[CrossRef][Medline]
  10. Harrison AM, Bonville CA, Rosenberg HF, Domachowske JB. Respiratory syncytical virus-induced chemokine expression in the lower airways: eosinophil recruitment and degranulation. Am J Respir Crit Care Med 1999;159:1918–1924.[Abstract/Free Full Text]
  11. Welliver RC, Wong DT, Sun M, Middleton E Jr, Vaughan RS, Ogra PL. The developement of respiratory syncytial virus-specific IgE and the release of histamine in nasopharyngeal secretion after infection. N Engl J Med 1981;841–846.
  12. van Woensel J, Kimpen J. Therapy for respiratory tract infections caused by respiratory syncytial virus. Eur J Pediatr 2000;159:391–398.[CrossRef][Medline]
  13. Oppenheim JJ, Zachariae CO, Mukaida N, Matsushima K. Properties of the novel proinflammatory supergene "intercrine" cytokine family. Annu Rev Immunol 1991;9:617–648.[Medline]
  14. Sheeran P, Jafri H, Carubelli C, Saavedra J, Johnson C, Krisher K, Sanchez PJ, Ramilo O. Elevated cytokine concentrations in the nasopharyngeal and tracheal secretions of children with respiratory syncytial virus disease. Pediatr Infect Dis J 1999;18:115–122.[CrossRef][Medline]
  15. Garofalo RP, Patti J, Hintz KA, Hill V, Ogra PL, Welliver RC. Macrophage inflammatory protein-1alpha (not T helper type 2 cytokines) is associated with severe forms of respiratory syncytial virus bronchiolitis. J Infect Dis 2001;184:393–399.[CrossRef][Medline]
  16. Haeberle HA, Kuziel WA, Dieterich HJ, Casola A, Gatalica Z, Garofalo RP. Inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of MIP-1alpha in lung pathology. J Virol 2001;75:878–890.[Abstract/Free Full Text]
  17. Garofalo R, Sabry M, Jamaluddin M, Yu RK, Casola A, Ogra PL, Brasier AR. Transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the RelA transcription factor as a mechanism producing airway mucosal inflammation. J Virol 1996;70:8773–8781.[Abstract/Free Full Text]
  18. Thomas LH, Friedland JS, Sharland M, Becker S. Respiratory syncytial virus-induced RANTES production from human bronchial epithelial cells is dependent on nuclear factor-kappa B nuclear binding and is inhibited by adenovirus-mediated expression of inhibitor of kappa B alpha. J Immunol 1998;161:1007–1016.[Abstract/Free Full Text]
  19. Casola A, Garofalo RP, Haeberle H, Elliott TF, Lin R, Jamaluddin M, Brasier AR. Multiple cis regulatory elements control RANTES promoter activity in alveolar epithelial cells infected with respiratory syncytial virus. J Virol 2001;75:6428–6439.[Abstract/Free Full Text]
  20. Mastronarde JG, He B, Monick MM, Mukaida N, Matsushima K, Hunninghake GW. Induction of interleukin (IL)-8 gene expression by respiratory syncytial virus involves activation of nuclear factor (NF)-kappa B and NF-IL-6. J Infect Dis 1996;174:262–267.[Medline]
  21. Karin M, Delhase M. The I kappa B kinase (IKK) and NF-kappa B: key elements of proinflammatory signalling. Semin Immunol 2000;12:85–98.[CrossRef][Medline]
  22. Leach CL, Greenspan JS, Rubenstein SD, Shaffer TH, Wolfson MR, Jackson JC, DeLemos R, Fuhrman BP. Partial liquid ventilation with perflubron in premature infants with severe respiratory distress syndrome: the LiquiVent Study Group. N Engl J Med 1996;335:761–767.[Abstract/Free Full Text]
  23. Hirschl RB, Conrad S, Kaiser R, Zwischenberger JB, Bartlett RH, Booth F, Cardenas V. Partial liquid ventilation in adult patients with ARDS: a multicenter phase I–II trial. Adult PLV Study Group. Ann Surg 1998;228:692–700.
  24. Fuhrman BP, Paczan PR, DeFrancisis M. Perfluorocarbon-associated gas exchange. Crit Care Med 1991;19:712–722.[Medline]
  25. Nesti FD, Fuhrman BP, Steinhorn DM, Papo MC, Hernan LJ, Duffy LC, Fisher JE, Leach CL, Paczan PR, Burak BA. Perfluorocarbon-associated gas exchange in gastric aspiration. Crit Care Med 1994;22:1445–1452.[Medline]
  26. Leach CL, Fuhrman BP, Morin FC III, Rath MG. Perfluorocarbon-associated gas exchange (partial liquid ventilation) in respiratory distress syndrome: a prospective, randomized, controlled study. Crit Care Med 1993;21:1270–1278.[Medline]
  27. Papo MC, Paczan PR, Fuhrman BP, Steinhorn DM, Hernan LJ, Leach CL, Holm BA, Fisher JE, Kahn BA. Perfluorocarbon-associated gas exchange improves oxygenation, lung mechanics, and survival in a model of adult respiratory distress syndrome. Crit Care Med 1996;24: 466–474.[CrossRef][Medline]
  28. Croce MA, Fabian TC, Patton JH Jr, Melton SM, Moore M, Trenthem LL. Partial liquid ventilation decreases the inflammatory response in the alveolar environment of trauma patients. J Trauma 1998;45:273–280.[Medline]
  29. Olszewska PB, Casola A, Saito T, Alam R, Crowe SE, Mei F, Ogra PL, Garofalo RP. Cell-specific expression of RANTES, MCP-1, and MIP-1alpha by lower airway epithelial cells and eosinophils infected with respiratory syncytial virus. J Virol 1998;72:4756–4764.[Abstract/Free Full Text]
  30. Kisch AL, Johnson KM. A plaque assay for respiratory syncytial virus. Proc Soc Exp Biol Med 1963;112:583–588.
  31. Graham BS, Perkins MD, Wright PF, Karzon DT. Primary respiratory syncytial virus infection in mice. J Med Virol 1988;26:153–162.[Medline]
  32. Murphy BR, Sotnikov A, Paradiso PR, Hildreth SW, Jenson AB, Baggs RB, Lawrence L, Zubak JJ, Chanock RM, Beeler JA, et al. Immunization of cotton rats with the fusion (F) and large (G) glycoproteins of respiratory syncytial virus (RSV) protects against RSV challenge without potentiating RSV disease. Vaccine 1989;7:533–540.[CrossRef][Medline]
  33. Bohrer H, Qiu F, Zimmermann T, Zhang Y, Jllmer T, Mannel D, Bottiger BW, Stern DM, Waldherr R, Saeger HD, et al. Role of NFkappaB in the mortality of sepsis. J Clin Invest 1997;100:972–985.[Medline]
  34. Taylor G, Stott EJ, Hughes M, Collins AP. Respiratory syncytial virus infection in mice. Infect Immun 1984;43:649–655.[Abstract/Free Full Text]
  35. Eyles JE, Spiers ID, Williamson ED, Alpar HO. Tissue distribution of radioactivity following intranasal administration of radioactive microspheres. J Pharm Pharmacol 2001;53:601–607.[CrossRef][Medline]
  36. Simoes EA. Respiratory syncytial virus infection. Lancet 1999;354:847–852.[Medline]
  37. Hornsleth A, Loland L, Larsen LB. Cytokines and chemokines in respiratory secretion and severity of disease in infants with respiratory syncytial virus (RSV) infection. J Clin Virol 2001;21:163–170.[CrossRef][Medline]
  38. Yang L, Cohn L, Zhang DH, Homer R, Ray A, Ray P. Essential role of nuclear factor kappaB in the induction of eosinophilia in allergic airway inflammation. J Exp Med 1998;188:1739–1750.[Abstract/Free Full Text]
  39. Donovan CE, Mark DA, He HZ, Liou HC, Kobzik L, Wang Y, De Sanctis GT, Perkins DL, Finn PW. NF-kappa B/Rel transcription factors: c-Rel promotes airway hyperresponsiveness and allergic pulmonary inflammation. J Immunol 1999;163:6827–6833.[Abstract/Free Full Text]
  40. Habler OP, Messmer KF. Tissue perfusion and oxygenation with blood substitutes. Adv Drug Deliv Rev 2000;40:171–184.[CrossRef][Medline]
  41. Day SE, Gedeit RG. Liquid ventilation. Clin Perinatol 1998;25:711–722.[Medline]
  42. Yoxall CW, Subhedar NV, Shaw NJ. Liquid ventilation in the preterm neonate. Thorax 1997;52:S3–S8.[Free Full Text]
  43. Lane TA. Perfluorochemical-based artificial oxygen carrying red cell substitutes. Transfus Sci 1995;16:19–31.[CrossRef][Medline]
  44. Goldberg BB. Ultrasound contrast agents. Clin Diagn Ultrasound 1993; 28:35–45.[Medline]
  45. Cox CA, Cullen AB, Wolfson MR, Shaffer TH. Intratracheal administration of perfluorochemical-gentamicin suspension: a comparison to intravenous administration in normal and injured lungs. Pediatr Pulmonol 2001;32:142–151.[CrossRef][Medline]
  46. Van Eeden SF, Klut ME, Leal MA, Alexander J, Zonis Z, Skippen P. Partial liquid ventilation with perfluorocarbon in acute lung injury: light and transmission electron microscopy studies. Am J Respir Cell Mol Biol 2000;22:441–450.[Abstract/Free Full Text]
  47. Virmani R, Fink LM, Gunter K, English D. Effect of perfluorochemical blood substitutes on human neutrophil function. Transfusion 1984;24: 343–347.[CrossRef][Medline]
  48. Fernandez R, Sarma V, Younkin E, Hirschl RB, Ward PA, Younger JG. Exposure to perflubron is associated with decreased Syk phosphorylation in human neutrophils. J Appl Physiol 2001;91:1941–1947.[Abstract/Free Full Text]
  49. Rossman JE, Caty MG, Rich GA, Karamanoukian HL, Azizkhan RG. Neutrophil activation and chemotaxis after in vitro treatment with perfluorocarbon. J Pediatr Surg 1996;31:1147–1150.[CrossRef][Medline]
  50. Smith TM, Steinhorn DM, Thusu K, Fuhrman BP, Dandona P. A liquid perfluorochemical decreases the in vitro production of reactive oxygen species by alveolar macrophages. Crit Care Med 1995;23:1533–1539.[CrossRef][Medline]
  51. Koch T, Ragaller M, Haufe D, Hofer A, Grosser M, Albrecht DM, Kotzsch M, Luther T. Perfluorohexane attenuates proinflammatory and procoagulatory response of activated monocytes and alveolar macrophages. Anesthesiology 2001;94:101–109.[CrossRef][Medline]
  52. Thomassen MJ, Buhrow LT, Wiedemann HP. Perflubron decreases inflammatory cytokine production by human alveolar macrophages. Crit Care Med 1997;25:2045–2047.[CrossRef][Medline]
  53. Baba A, Kim YK, Zhang H, Liu M, Slutsky AS. Perfluorocarbon blocks tumor necrosis factor-alpha-induced interleukin- 8 release from alveolar epithelial cells in vitro. Crit Care Med 2000;28:1113–1118. [see comments][CrossRef][Medline]
  54. Bennett CR, Hamre D. Growth and serological characteristics of respiratory syncytial virus. J Infect Dis 1962;110:8–16.
  55. Levine S, Hamilton R. Kinetics of respiratory syncytial virus growth cycle in HeLa cells. Arch Ges Virusforsch 1969;28:122–132.
  56. Graham BS, Johnson TR, Peebles RS. Immune-mediated disease pathogenesis in respiratory syncytial virus infection. Immunopharmacology 2000;48:237–247.[CrossRef][Medline]
  57. Jamaluddin M, Casola A, Garofalo RP, Han Y, Elliott T, Ogra PL, Brasier AR. The major component of IkappaBalpha proteolysis occurs independently of the proteasome pathway in respiratory syncytial virus-infected pulmonary epithelial cells. J Virol 1998;72:4849–4857.[Abstract/Free Full Text]
  58. Bitko V, Velazquez A, Yang L, Yang YC, Barik S. Transcriptional induction of multiple cytokines by human respiratory syncytial virus requires activation of NF-kappa B and is inhibited by sodium salicylate and aspirin. Virology 1997;232:369–378.[CrossRef][Medline]
  59. Fiedler MA, Wernke-Dollries K. Incomplete regulation of NF-kappaB by IkappaBalpha during respiratory syncytial virus infection in A549 cells. J Virol 1999;73:4502–4507.[Abstract/Free Full Text]
  60. Becker S, Quay J, Soukup J. Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophages. J Immunol 1991;147:4307–4312.[Abstract]
  61. Matsuda K, Tsutsumi H, Sone S, Yoto Y, Oya K, Okamoto Y, Ogra PL, Chiba S. Characteristics of IL-6 and TNF-alpha production by respiratory syncytial virus-infected macrophages in the neonate. J Med Virol 1996;48:199–203.[CrossRef][Medline]
  62. Thomas LH, Wickremasinghe MI, Sharland M, Friedland JS. Synergistic upregulation of interleukin-8 secretion from pulmonary epithelial cells by direct and monocyte-dependent effects of respiratory syncytial virus infection. J Virol 2000;74:8425–8433.[Abstract/Free Full Text]
  63. McManus CM, Liu JS, Hahn MT, Hua LL, Brosnan CF, Berman JW, Lee SC. Differential induction of chemokines in human microglia by type I and II interferons. Glia 2000;29:273–280.[CrossRef][Medline]
  64. Stellato C, Beck LA, Gorgone GA, Proud D, Schall TJ, Ono SJ, Lichtenstein LM, Schleimer RP. Expression of the chemokine RANTES by a human bronchial epithelial cell line: modulation by cytokines and glucocorticoids. J Immunol 1995;155:410–418.[Abstract]
  65. Lilly CM, Nakamura H, Kesselman H, Nagler AC, Asano K, Garcia ZE, Rothenberg ME, Drazen JM, Luster AD. Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids. J Clin Invest 1997;99:1767–1773.[Medline]
  66. Standiford TJ, Kunkel SL, Phan SH, Rollins BJ, Strieter RM. Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells. J Biol Chem 1991;266:9912–9918.[Abstract/Free Full Text]
  67. van Woensel JB, Wolfs TF, van Aalderen WM, Brand PL, Kimpen JL. Randomised double blind placebo controlled trial of prednisolone in children admitted to hospital with respiratory syncytial virus bronchiolitis. Thorax 1997;52:634–637.[Abstract]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Hansdottir, M. M. Monick, N. Lovan, L. Powers, A. Gerke, and G. W. Hunninghake
Vitamin D Decreases Respiratory Syncytial Virus Induction of NF-{kappa}B-Linked Chemokines and Cytokines in Airway Epithelium While Maintaining the Antiviral State
J. Immunol., January 15, 2010; 184(2): 965 - 974.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. M. Castro, A. Guerrero-Plata, G. Suarez-Real, P. A. Adegboyega, G. N. Colasurdo, A. M. Khan, R. P. Garofalo, and A. Casola
Antioxidant Treatment Ameliorates Respiratory Syncytial Virus-induced Disease and Lung Inflammation
Am. J. Respir. Crit. Care Med., December 15, 2006; 174(12): 1361 - 1369.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
M. Hubler, A. R. Heller, J. U. Bleyl, M. G. de Abreu, T. Kroll, T. Rossel, and T. Koch
Perfluorohexane Vapor Has Only Minor Effects on Spatial Pulmonary Blood Flow Distribution in Isolated Rabbit Lungs
Anesth. Analg., April 1, 2005; 100(4): 1122 - 1128.
[Abstract] [Full Text] [PDF]

Home page
 Br J AnaesthHome page
U. Kaisers, K. P. Kelly, and T. Busch
Liquid ventilation
Br. J. Anaesth., July 1, 2003; 91(1): 143 - 151.
[Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. J. Tobin
Critical Care Medicine in AJRCCM 2002
Am. J. Respir. Crit. Care Med., February 1, 2003; 167(3): 294 - 305.
[Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. J. Tobin
Pediatrics, Surfactant, and Cystic Fibrosis in AJRCCM 2002
Am. J. Respir. Crit. Care Med., February 1, 2003; 167(3): 333 - 344.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haeberle, H. A.
Right arrow Articles by Garofalo, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haeberle, H. A.
Right arrow Articles by Garofalo, R. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2002 American Thoracic Society