Neural Mechanisms of Respiratory Syncytial Virus-induced Inflammation and Prevention of Respiratory Syncytial Virus Sequelae

GIOVANNI PIEDIMONTE

Departments of Pediatrics, Medicine, and Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida

	INTRODUCTION

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Respiratory syncytial virus (RSV) belongs to the paramyxovirus family and is made of a single strand of RNA encoding 10 proteins, the most important of which in terms of infectivity are the two surface glycoproteins, G and F. The G protein mediates attachment of the virus to host cells, while the F (fusion) protein mediates viral penetration and subsequent spread to adjacent respiratory epithelial cells, with fusion of cell membranes and formation of syncytia (1). The resulting infection is an extremely common, self-limited disease among infants. What is most intriguing about RSV lower respiratory tract infection, however, is the risk of persistent, recurrent wheezing, especially during the first years after the original infection. Although the frequency of this postbronchiolitic wheezing declines with age, a significant proportion of children continue to experience recurrent episodes of wheezing up to 7 to 11 yr later (2, 3). Beyond the first decade of life there no longer appears to be a significant relationship between RSV bronchiolitis and recurrent wheezing (2). Therefore, although RSV bronchiolitis may not cause lifelong respiratory problems, it appears to be associated with prolonged respiratory morbidity during the developmental years of life.

RSV bronchiolitis in infancy is also associated with changes in pulmonary function. Because these changes are reversible, it is likely that they reflect a transient dysfunction of the airway control mechanisms rather than permanent remodeling. Some authors have proposed that the respiratory dysfunction following RSV bronchiolitis may be, at least in part, the result of changes in the neural pathways to the airways (4). Others believe that there is an alteration of the local immune response (5). It is possible that these two potential mechanisms are linked, with combined neuroimmune responses leading to persistent airway inflammation and hyperreactivity.

	NEURAL CONTROL OF THE AIRWAYS

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Airway patency depends on the degree of smooth muscle constriction, mucosal edema, and mucous secretion in bronchi and bronchioles, which are controlled by three peripheral neural pathways: adrenergic, cholinergic, and nonadrenergic-noncholinergic (NANC). The adrenergic neural component induces bronchorelaxation and is mediated by catecholamine neurotransmitters, but few adrenergic fibers directly innervate the airway smooth muscle. The cholinergic neural component predominantly induces bronchoconstriction and is mediated by the neurotransmitter acetylcholine. The NANC component provides an important neural influence on airway patency in many mammals, including humans, and consists of two distinct subsystems, inhibitory (NANCi) and excitatory (NANCe), which act in a ying/yang type of equilibrium on the airways. The NANCi system primarily induces relaxation of airway smooth muscle, mediated mainly by the neurotransmitter vasoactive intestinal peptide (VIP) but also by nitric oxide, both of which are released by efferent parasympathetic fibers. The NANCe system primarily induces contraction of airway smooth muscle, mediated by small peptide neurotransmitters called tachykinins, which include the 11-amino acid neuropeptide substance P, and the neurokinins A and B.

	NANCe AND SUBSTANCE P

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In the airways, a dense network of unmyelinated (C-type) sensory nerve fibers forming the NANCe pathway is strategically located just below the respiratory epithelial surface, where it is accessible to physical and chemical changes in the inspired air and in the bronchial environment. Such changes can trigger action potentials at the terminal varicosities of these fibers (6), causing the local release of tachykinins, in particular substance P, which then diffuse into the bronchial and bronchiolar submucosa and smooth muscle.

Substance P has been studied extensively as a bronchoconstrictor. Larsen and Colasurdo (7), for example, demonstrated that contractile responses to nerve stimulation were increased in 4- and 8-wk-old ferrets infected with RSV as compared with pathogen-free controls, but this difference resolved by 24 wk. NANCi responses were absent in all 4-wk-old ferrets and significantly decreased in 8-wk-old ferrets infected with RSV, and this difference persisted at 24 wk.

However, substance P, independent of its activity as a bronchoconstrictor, is also one of the most potent and versatile inflammatory mediators known. It greatly increases endothelial permeability and blood flow in postcapillary venules, causing airway mucosal edema; stimulates T and B lymphocyte proliferation and activation; attracts leukocytes to the vascular endothelium; causes degranulation of mast cells with release of other mediators of inflammation; and primes and activates monocytes and macrophages to release a variety of cytokines such as tumor necrosis factor  and interleukin 6. Receptors for substance P have been identified in all these cell types in humans.

Lundberg and Saria (8) showed that functional ablation of substance P-producing neurons by capsaicin, the pungent principle of plants of the Capsicum family, markedly inhibited the noxious effects on the lung of a variety of irritants, suggesting that this tachykinin mediates neurogenic inflammation. Capsaicin activates the opening of a cation channel at the terminals of unmyelinated sensory nerves, which allows the release of multiple neuropeptides, including substance P. Depending on its dosage and modalities of administration, capsaicin can transiently activate the nerve fibers or permanently delete them.

Substance P acts at three receptor subtypes (NK-1, NK-2, and NK-3) that have a typical rhodopsin-like structure, with seven transmembrane-spanning domains, a G protein-coupled intracellular domain, and an extracellular domain that binds the amino terminal of tachykinin peptides. The NK-1 receptor subtype has a particularly high affinity for substance P and mediates its proinflammatory and immunomodulatory effects. Bozic and coworkers (9) showed that targeted disruption of the NK-1 receptor gene in mice protected the lung from immune complex-mediated injury, suggesting that the substance P-NK-1 interaction is an important early event in the inflammatory cascade and may amplify other inflammatory pathways downstream.

Most of the target cells containing the NK-1 receptor express a series of peptidasesparticularly neutral endopeptidase and kininase II (angiotensin-converting enzyme)which cleave the carboxyl-terminal dipeptide of substance P, thereby attenuating its biologic effects. In the airways, neutral endopeptidase is expressed at high concentrations on the basal cells of the epithelium near the substance P-containing nerve fibers, whereas kininase II is expressed primarily in the vascular endothelium. Piedimonte and colleagues (10) showed that glucocorticoids prevent a neurogenically mediated increase in vascular permeability in the airways. They also found (11) that this antiinflammatory effect of glucocorticoids is due to increased peptidase activity in the airway mucosa and is completely reversed when neutral endopeptidase and kininase II activities are simultaneously inhibited.

	EFFECT OF RSV ON THE NANCe NEUROGENIC CASCADE

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Studies of animal models have shown that infections due to respiratory viruses, including parainfluenza virus (12), adenovirus (13), and RSV (14), can cause profound changes in the NANCe pathways innervating the airways. Of these viral pathogens, RSV in particular appears to promote long-term alterations in airway function. Piedimonte and associates (14) reported the results of a study designed to determine whether RSV infection caused increased inflammatory responses to substance P, either released by sensory nerves stimulated with capsaicin or injected into the circulation. These studies were performed in Fischer F-344 rats, a strain that has been shown in previous work by Lemanske (5) to develop a strong helper T cell type 1 immune response against parainfluenza virus and to exhibit no structural changes after infection. Another important characteristic of this F-344 rat model is that no evidence of viral antigens can be found in the lungs 30 d after inoculation of RSV. Thus, the time course of RSV infection in adult F-344 rats resembles the typical course in humans, in contrast to other animal models (e.g., guinea pigs) that are unable to clear this virus even after several months. Therefore, this model seems ideal to study the short- and long-term physiologic abnormalities caused by a transient lower respiratory tract infection with RSV.

Evans blue was used as a tracer of albumin extravasation in the airways of F-344 rats inoculated endotracheally with RSV suspensions or with virus-free medium. Five days after inoculation, the extravasation of Evans blue-labeled albumin from the airway microvasculature after infusion of capsaicin or substance P was significantly greater in RSV-infected airways than in pathogen-free controls (Figure 1). Interestingly, the intrinsic permeability of the vessels was similar between RSV-infected rats and pathogen-free controls (Figure 1, left columns). The difference in extravasation of Evans blue-labeled albumin reflected a difference in the reactivity of the blood vessels to sensory neuropeptides. The fact that substance P reproduced the response to capsaicin indicates that this neuropeptide is released from the unmyelinated sensory fibers and mediates their inflammatory effects.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Potentiation of airway neurogenic inflammation 5 d after the endotracheal administration of respiratory syncytial virus (RSV) or virus-free medium measured in the extrapulmonary airways of Fischer F-344 rats. In rats injected with vehicle (left columns), RSV by itself caused only minimal increase in vascular permeability compared with pathogen-free controls. However, the increase in vascular permeability elicited in the extrapulmonary airways by capsaicin (center columns) and by exogenous substance P (right columns) was significantly larger in RSV-inoculated rats than in pathogen-free rats. ***p < 0.001 versus pathogen-free rats. (Reproduced by permission from Piedimonte G, Rodriguez MM, King KA, McLean S, Jiang X. Respiratory syncytial virus upregulates expression of the substance P receptor in rat lungs. Am J Physiol 1999;277:L831-L840.)

In addition to increased airway vascular permeability in virus-infected rats, sensory nerve stimulation is followed by vasodilation and increased adhesion of leukocytes to the vascular endothelium because of increased expression of adhesion molecules. The neurogenically mediated inflammatory responses occur predominantly in the extrapulmonary airways (i.e., trachea and mainstem bronchi) of RSV-infected adult rats and predominantly in the intrapulmonary airways of RSV-infected weanling rats (15). This suggests a developmental modification in the anatomic distribution of NANCe fibers across the respiratory tract. It is possible that these maturational changes are in part responsible for the age-related differences in the clinical manifestation of acute RSV infection. They may also parallel the long-term decline in bronchial obstructive symptoms that occurs in children after RSV bronchiolitis.

Neutral endopeptidase enzymatic activity is markedly reduced in the airways of rodents infected with influenza or parainfluenza viruses, whereas it is not affected by RSV (14). Therefore, RSV-induced potentiation of neurogenic inflammation cannot be explained by decreased catabolism of substance P after being released from nerve fibers. Rather, the increase in microvascular permeability elicited by the stimulation of unmyelinated sensory nerves during RSV infection appeared to be exaggerated as the result of upregulation of the NK-1 receptor. In fact, a high density of substance P-binding sites was found in the airway epithelium, subepithelium, and endothelium in the lungs of RSV-infected rats. This upregulation presumably occurs at the gene expression level, as NK-1 receptor mRNA levels increase approximately 5-fold as a result of RSV infection (Figure 2), which explains at least in part the changes in the neurogenic cascade.

View larger version (26K): [in this window] [in a new window]   Figure 2.   NK-1 receptor expression in lung tissues of Fischer F-344 rats 5 d after endotracheal inoculation of respiratory syncytial virus (RSV) or virus-free medium. Densitometry analysis of RT-PCR bands normalized to the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) revealed an approximately 5-fold increase in NK-1 receptor mRNA from the lungs of RSV-infected rats compared with pathogen-free controls. (+) = amplification product of the full-length rat SP (NK-1) receptor cDNA, used as a positive control. Each band was obtained from the lungs of a different animal (six pathogen-free rats and five RSV-infected rats). ***p < 0.001 versus pathogen-free rats. O.D. = optical density. (Reproduced by permission from Piedimonte G, Rodriguez MM, King KA, McLean S, Jiang X. Respiratory syncytial virus upregulates expression of the substance P receptor in rat lungs. Am J Physiol 1999;277:L831- L840.)

An exaggerated neurogenic inflammatory response persists long after RSV has been cleared from the lung (16), suggesting that the underlying molecular mechanisms may become independent from the presence of replicating virus. Romaguera and coworkers (17) found that T lymphocyte subpopulations within the bronchial-associated lymphoid tissue of RSV-infected rats expand and express high levels of the NK-1 receptor. As a result, substance P released from unmyelinated sensory fibers after stimulation by any inhaled irritant may cause a new cycle of inflammation mediated by these NK-1-expressing T lymphocytes. Auais and associates (18) provided evidence for this hypothesis by showing that the number of lymphocytes recovered by bronchoalveolar lavage from the capsaicin-stimulated lungs of RSV-infected rats is much higher than that recovered from pathogen-free controls.

Figure 3 illustrates how RSV can change the magnitude of neurogenic inflammatory responses by upregulating the NK-1 receptor. The resulting neuroimmune interactions may underlie long-term airway dysfunction following the initial RSV infection and predispose to recurrent cycles of inflammation and hyperreactivity triggered by irritants entering the airways.

View larger version (23K): [in this window] [in a new window]   Figure 3.   Respiratory syncytial virus (RSV) changes the magnitude of neurogenic inflammatory responses by upregulating the NK-1 receptor. The resulting neuroimmune interactions may underlie long-term airway dysfunction after the initial RSV infection and predispose to recurrent cycles of inflammation and hyperreactivity triggered by irritants entering the airways.

	EFFECT OF PALIVIZUMAB ON RSV-INDUCED, NANCe-MEDIATED INFLAMMATION

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Palivizumab, a humanized monoclonal antibody against the F protein of RSV, was approved by the U.S. Food and Drug Administration in 1998 for passive immunoprophylaxis against RSV infection. It has been shown to reduce hospitalization from RSV infection in high-risk infants (19).

Piedimonte and colleagues (20) attempted to determine whether treatment with antibodies against RSV would prevent the exaggerated, neurogenically mediated inflammation of the lower respiratory tract in the rat model described above. Five days after intranasal or endotracheal inoculation of RSV, the increase in vascular permeability in response to sensory nerve stimulation with capsaicin was measured with Evans blue tracer injected into the circulation of F-344 rats treated with palivizumab. Palivizumab given 24 h before intranasal inoculation of RSV reduced neurogenic inflammation to the same level as that of pathogen-free controls (Figure 4). Moreover, palivizumab suppressed neurogenic inflammation when administered 72 h after RSV was inoculated intranasally, before infection is established in the lower respiratory tract. Interestingly, King and colleagues found a significantly lower body weight in rats acutely infected with RSV compared with pathogen-free controls, and this difference was not prevented by palivizumab (15). Furthermore, the difference in weight remained significant even when the animals were killed 30 d after inoculation, suggesting that RSV may have other, nonrespiratory effects interfering with growth and that these effects may be long-lasting.

View larger version (46K): [in this window] [in a new window]   Figure 4.   Comparison of the inhibitory effect in rats of palivizumab injected either 24 h before or 72 h after intranasal inoculation of respiratory syncytial virus (RSV) or virus-free medium. Capsaicin-induced extravasation of Evans blue-labeled albumin was significantly inhibited by both treatment modalities in RSV-infected rats, although the magnitude of the inhibitory effect was greater when the antibody was administered before the virus was inoculated. ***p < 0.001 versus RSV-infected rats pretreated with vehicle only. [Reproduced by permission from Piedimonte G, King KA, Holmgren NL, Bertrand PJ, Rodriguez MM, Hirsch RL. A humanized monoclonal antibody against respiratory syncytial virus (palivizumab) inhibits RSV-induced neurogenic-mediated inflammation in rat airways. Pediatr Res 2000;47:351-356.]

The protective effect of palivizumab suggests that if the RSV infection remains limited to the upper airways, or if viral penetration is inhibited in the early phase of lower airway infection, the infection is not associated with abnormal neurogenic inflammatory responses in the lower airways. Thus, RSV infections limited to the upper airways should have no pulmonary consequences caused by neural reflexes or other indirect mechanisms, which may be involved in the pathogenesis of wheezing in patients infected with other respiratory viruses (e.g., rhinoviruses) thought to be unable to replicate well at the temperature of the lower respiratory tract. It can also be hypothesized that palivizumab given before or in the early phase of RSV infection may reduce the risk of recurrent wheezing during the first decade of life associated with this infection.

	CONCLUSION

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RSV appears to make the airways abnormally susceptible to the proinflammatory effects of substance P by upregulating NK-1 receptor gene expression and thereby increasing the density of these substance P receptors on target cells. Among these targets are important cellular mediators of inflammatory and immune responses such as endothelial cells, lymphocytes, macrophages, and mast cells. Thus, this effect may establish important neuroimmune interactions that result in long-term dysregulation following RSV infection and may predispose to persistent airway inflammation and hyperreactivity. The neurogenic inflammatory reaction observed in animal models supports this hypothesis and could be a target for the treatment and prevention of RSV disease and its sequelae. Studies of humans are necessary to confirm the validity of these relationships.

	DISCUSSION

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Openshaw: Can you explain why weight loss is seen even if you block the RSV infection with palivizumab?

Dr. Piedimonte: With palivizumab given 24 h before infection, most of the airway inflammation is prevented, yet the body weight of RSV-infected rats is still significantly lower compared with that of the controls inoculated with virus-free medium, and it is similar to that of the RSV-infected rats that did not receive palivizumab. We suspect that in the early stages of the infectious process RSV may release factors interfering with growth, but additional studies are needed to clarify the exact mechanisms of this effect. In any case, it would be interesting to follow the growth pattern of children who develop severe RSV disease early in life.

Cohen: Could the maturational changes in the terminal airways of children as they develop into adolescents and adults, characterized by the reduced density of type C fibers and the substance P that they traffic, parallel what we see in those children who have had RSV infections but subsequently have no postbronchiolitic symptoms associated with it?

Dr. Piedimonte: That is my hypothesis. I believe that the lower respiratory tract is susceptible to postbronchiolitic hyperreactivity early, but as a series of inflammatory pathways, such as neurogenic-mediated inflammation, undergo maturation, the child no longer responds that way.

Cohen: So by extension, any effort to limit exposure or severity of response to RSV or to any other respiratory virus or irritant early in life may reduce the number of receptors and neurotransmitters trafficking in the lower airway, so there is no response to subsequent triggering of the system.

Dr. Piedimonte: Absolutely. I believe there is a global process of dynamic neural remodeling in children that continues with age and can be affected by exogenous factors such as respiratory viruses. The mismatch of peptidergic nerves and peptide receptors found in the lungs and in other organs (e.g., the brain) may reflect areas that earlier in life had these nerve fibers. The fibers regressed, but the receptors are still expressed or can be expressed. I think we need to stress the concept that the lungs and many other organs of children are not simply scaled-down versions of adult organs.

DaBaets: Have you any idea how long the up-regulation of substance P receptors lasts if there is no subsequent triggering of the system?

Dr. Piedimonte: At the molecular level, this lasts in the airways for only a limited period of time, but exaggerated neurogenic inflammatory responses persist long after RSV has been cleared from the lung. One possible explanation is the persistence of NK-1 receptor-bearing lymphocyte subpopulations. However, other peptides are also upregulated during RSV infection. Some of these peptides are extremely potent growth factors, such as nerve growth factor, which controls the distribution and reactivity of sensory nerves and may mediate the second phase of this process, which is the chronic phase.

	Footnotes

Correspondence and requests for reprints should be addressed to Giovanni Piedimonte, M.D., Department of Pediatrics, Division of Pulmonology, University of Miami School of Medicine, 1601 NW 12th Avenue (D-820), Miami, FL 33136. E-mail: gpiedimo{at}med.miami.edu.

	References

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20. Piedimonte G, King KA, Holmgren NL, Bertrand PJ, Rodriguez MM, Hirsch RL. A humanized monoclonal antibody against respiratory syncytial virus (palivizumab) inhibits RSV-induced neurogenic-mediated inflammation in rat airways. Pediatr Res 2000; 47: 351-356 [Medline].