Respiratory Syncytial Virus (RSV) is a member of the pneumovirus genus of the paramyxoviridae, and has a negative-sense, non-segmented, single-stranded RNA genome. It is the most common cause of lower respiratory tract disease in infants and children worldwide, is a frequent initiator of acute asthma exacerbations in young children, and has a disease impact comparable to that of non-pandemic influenza A in the elderly.  In the United States, 50 to 70% of infants are infected with RSV in the first year of life and approximately 2-3% of all cases of RSV bronchiolitis result in severe hypoxia, or a need for parenteral fluid supplementation that necessitates hospitalization.

Vectorial Na⁺ transport across the distal lung epithelium

For gas exchange to occur optimally, the alveoli of the adult mammalian lungs must remain open and free from fluid.  In utero, gas exchange occurs across the placenta and the fetal lung is filled with fluid which enters the trachea and distal lung epithelia because of the osmotic gradient created by the active secretion of chloride (Cl^-) ions.  Although this fluid is essential for lung growth and development, it must be rapidly removed shortly after birth to allow for gas exchange to occur.  Various studies in rats, sheep, guinea pigs and other species established that just prior to birth, the distal alveolar epithelium converts from Cl^- secretion to sodium (Na⁺) absorption and this active transport of Na⁺ ions across distal lung epithelial cells is essential for the clearance  of fetal fluid.   Studies showing reabsorption of intratracheally instilled isotonic fluid or plasma from the alveolar spaces of adult anesthetized animals and resected human lungs, and its near complete  inhibition by  elimination of the Na,K-ATPase or by replacement of Na⁺ ions in the alveolar epithelial fluid by large cations, indicate that  adult alveolar epithelial cells are capable of actively transporting sodium (Na⁺) ions. 

Based on the results of a large number of studies on anesthetized and conscious animals, human lungs as well as isolated epithelial cells, we know that the distal lung epithelial cells (Clara, alveolar type I and II cells) actively transport Na⁺ ions in a vectorial fashion from the alveolar to the interstitial sides (Figure 1).  Na⁺ ions diffuse passively down their electrochemical gradient (created by the action of the basolaterally located Na,K-ATPase) into alveolar epithelial cells through apically located amiloride-sensitive channels (ENaC) or cyclic nucleotide gated ion channels  and are extruded across the basolateral cell membranes by the ouabain-sensitive Na⁺,K⁺-ATPase. Movement of Na⁺ ions from the alveolar to the interstitial space necessitates the simultaneous movement of an anion (such as Cl^-) to preserve electro-neutrality.  A number of in vivo studies suggested the movement of Cl^- ion occurs via both trans-cellular Cl^- channels (such as CFTR) as well as  para-cellular pathways.   More recent studies have provided convincing evidence of the presence of functional  CFTR in both fetal  and adult ATII cells and showed that functional CFTR was necessary for the increase of vectorial Na⁺ transport by β₂-agonists.   Thus in summary, although the vectorial movement of Na⁺ ions across epithelial cells requires the presence of both passive (channels) and energy consuming (Na,K-ATPAse) basolateral transporters, ion channels constitute the rate-limiting step in this process, offering more than 90% of the resistance to trans-cellular Na⁺ transport.

Importance of Active Na⁺ transport in Lung Fluid Balance in the Adult Lung

While it remains unclear whether active Na⁺ transport plays an important role in keeping alveolar spaces free of fluid in the normal lung, a variety of studies have clearly established that active Na⁺ transport plays an important role in limiting the degree of alveolar edema in adult mammalian lungs following acute or chronic injury to the alveolar epithelium.  For example, intratracheal instillation of a Na⁺ channel blocker in rats exposed to hyperoxia,  increased the amount of extravascular lung water. Conversely, intratracheal instillation of adenoviral vectors expressing the Na⁺,K⁺-ATPase genes increased survival of rats exposed to hyperoxia.  Moreover, patients with acute lung injury who are still able to concentrate alveolar protein (as a result of active Na⁺ reabsorption) have a better prognosis than those that cannot.  Finally, decreased Na⁺ reabsorption predisposes mountaineers to pulmonary edema. In addition, abnormalities in ENaC channel number and opening have been linked to the pathogenesis of cystic fibrosis and Liddle’s syndrome.

We hypothesized that inhibition of epithelial Na⁺ transport by RSV may contribute significantly to the pathogenesis of RSV bronchiolitis.  Inhibition of active Na⁺ transport can result in formation of an excessive volume of ALF, impairment of gas exchange, narrowing of airway lumens and dilution of the surface-active materials that stabilize small airways. The resultant small airway obstruction, which would be exacerbated by any intercurrent inflammatory process, such as that occurring during RSV bronchiolitis, would be predicted to be most severe in infancy and early childhood when airway diameter is lowest.  

 SUMMARY OF PUBLISHED RESULTS

In our first series of experiments we investigated the effects of intranasal instillation of RSV on alveolar fluid clearance (AFC) and nasal potential differences (NPD) of BALB/c mice. We chose these mice because C57BL/6 mice are not as easily infected with RSV. Mice were infected intranasally (I.N.), drop-wise, with 10⁶ PFU of RSV strain A2 (in 100 ml, with 50 ml administered to each nostril) under light anesthesia. Mock-infected mice received an equal volume of supernatant from uninfected Hep2 cells. Uninfected animals received no instillate. In certain experiments, mice were infected with 100 μl of UV-inactivated RSV A2. Mice were placed in the lateral recumbent position, allowed to recover, and returned to their cage. In some experiments, mice were individually marked and subsequently weighed on a daily basis.

(i) Infection of Balb/c mice with RSV decreased Na⁺-dependent alveolar fluid clearance (AFC), which resulted in mild systemic hypoxemia.  These effects occur in the absence of detectable cytopathicity of the virus for the respiratory epithelium, and require replication-competent virus;

Davis IC, Sullender WM, Hickman-Davis JM, Lindsey JR, Matalon S.  Nucleotide-mediated inhibition of alveolar fluid clearance in BALB/c mice after respiratory syncytial virus infection. Am J Physiol Lung Cell Mol Physiol. 2004 Jan;286(1):L112-20. Epub 2003 Aug 29.

(ii) RSV infection results in higher levels of pyrimidines and purines in the alveolar space.  Intratracheal administration of UTP or UDP (in concentrations similar to those found in the epithelial lining fluid after RSV infection) also decreased AFC Davis IC, Sullender WM, Hickman-Davis JM, Lindsey JR, Matalon S.  Am J Physiol Lung Cell Mol Physiol. 2004 Jan;286(1):L112-20.

(iii)  the effects of RSV on AFC and oxygen saturation of Balb/c mice are reversed by intraalveolar administration of antagonists of P2Y nucleotide receptors (XAMR0721), enzymes that enhance the breakdown of pyrimidines (UDP-glucose pyrophosphoryrase and apyrase) and by inhibitors of volume regulated chloride ion channels (such as fluoxetine, nifumic acid etc); Davis IC, Sullender WM, Hickman-Davis JM, Lindsey JR, Matalon S.  Am J Physiol Lung Cell Mol Physiol. 2004 Jan;286(1):L112-20. Epub 2003 Aug 29. ; Davis IC, Lazarowski ER, Hickman-Davis JM, Fortenberry JA, Chen FP, Zhao X, Sorscher E, Graves LM, Sullender WM, Matalon S.  Am J Respir Crit Care Med. 2006 Mar 15;173(6):673-82.

(iv) the effects of RSV on AFC and oxygen saturation of Balb/c mice are both prevented and reversed by systemic or intranasal administration of inhibitors of the de-novo pathway of pyrimidine synthesis (such as leflunomide or A77-1726, its active metabolite).  Furthermore the protective effects of most leflunomide and A77 are obviate by concommitant administration of uridine which stimulates UDP and UTP synthesis via the salvage pathway; Davis IC, Lazarowski ER, Hickman-Davis JM, Fortenberry JA, Chen FP, Zhao X, Sorscher E, Graves LM, Sullender WM, Matalon S.  Am J Respir Crit Care Med. 2006 Mar 15;173(6):673-82; Davis IC, Lazarowski ER, Chen FP, Hickman-Davis JM, Sullender WM, Matalon S. Am J Respir Cell Mol Biol. 2007 Oct;37(4):379-86. 

(v) infection of Balb/c mice with RSV, stimulates production of keratinocyte cytokine which activates a sequence of events involving PKCz and GRK2 leading to dissociation of  G proteins from b₂ receptors.  These events render the distal epithelial cells of these mice insensitive to b-agonists; Davis IC, Xu A, Gao Z, Hickman-Davis JM, Factor P, Sullender WM, Matalon S. Respiratory syncytial virus induces insensitivity to beta-adrenergic agonists in mouse lung epithelium in vivo. Am J Physiol Lung Cell Mol Physiol. 2007 Aug;293(2):L281-9. Epub 2007 Apr 13.

These results establish for the first time that the RSV virus, the most common cause of bronchiolitis and pneumonia among infants under 1 year of age and a common cause of pneumonia among the elderly and immunocompromised patients, decreases alveolar fluid clearance to levels known to cause significant morbidity in patients with ARDS.  Furthermore, and most important, the results of these studies have led to the development of a new potential treatment for RSV. 

Currently there is no treatment for RSV.    We have been granted US Provisional Patent Application #60/573,558: “Methods for using pyrimidine synthesis inhibitors to increase airway epithelial cell fluid uptake.”  (Filed May 21, 2004; Inventors: Dr. Ian C. Davis, Dr. Wayne Sullender and Dr. Sadis Matalon; Converted to International PCT application (#PCT/US2005/017939); May 2005) for the use of pyrimidine synthesis for the treatment of RSV (click here for additional information).  We are truly excited by the fact that we were able to test a new paradigm which formed the rational basis for the development of a potentially new treatment for the most common pediatric viral infection. 

Co-Investigators

Ian C. Davis, DVM, PhD
Research Assistant Professor of Anesthesiology
Current Position: Assistant Professor, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA

Wayne Sullender, MD
Professor of Pediatrics and Microbiology
University of Alabama at Birmingham

Eduardo R. Lazarowski, PhD
Departments of Medicine and Pharmacology,
UNC, Chapel Hill, NC, USA

Weifeng Song, MD, PhD
Research Associate
Department of Anesthesiology
University of Alabama at Birmingham

Lan Chen, MD
Research Associate
Department of Anesthesiology
University of Alabama at Birmingham

Kedar Shrestha, PhD
Research Associate
Department of Anesthesiology
University of Alabama at Birmingham

Grant Support