Virus-induced Airway Hyperresponsiveness and Asthma

GERT FOLKERTS, WILLIAM W. BUSSE, FRANS P. NIJKAMP, RONALD SORKNESS, and JAMES E. GERN

University of Utrecht, Utrecht, The Netherlands; and University of Wisconsin, Madison, Wisconsin

	CONTENTS

Epidemiology of Viral Infections and Asthma

The Effects of Viral Infection on Airway Obstruction   and Asthma

  Do Viruses That Trigger Asthma Infect the   Lower Airway?

  The Effect of Respiratory Viral Infections on   Airway Responsiveness

  Alterations in Neural Control of the Airways

  Changes in Small Airway Function

The Effect of Respiratory Viruses on Airway Inflammation

  Epithelial Cells

  Endothelial Cells

  Granulocytes

  Macrophages and Monocytes

  T Cells

  Interactions among Viruses, Allergy, and Preexisting   Airway Inflammation

Summary and Therapeutic Implications

	EPIDEMIOLOGY OF VIRAL INFECTIONS AND ASTHMA

Wheezing is a common complication of respiratory infections (Table 1). In infancy, infections with respiratory viruses, particularly respiratory syncytial virus (RSV) and parainfluenza virus (PIV), cause wheezing and bronchiolitis, which can be recurrent. Moreover, infants with reduced lung function and/ or exposure to tobacco smoke are at greatest risk for developing wheezing with RSV infection (1), but only a subset of those who wheeze in infancy subsequently develop persistent asthma. For those who develop persistent wheezing, atopy, as indicated by elevated total immunoglobin E (IgE) and the development of allergen-specific IgE, is a major risk factor in this process (2).

There is now evidence that viral infections in early childhood may also act on the immune system to modify the subsequent risk of allergen sensitization and/or asthma (3). For example, several studies have shown that the odds of allergen sensitization are inversely related to the number of older siblings in the family, which presumably determines the exposures to infectious diseases in early childhood (4). In addition, data from Africa indicate that measles infection in early childhood reduces the risk of allergen sensitization (7). Some bacterial infections may have similar effects: Japanese schoolchildren who develop a strong positive tuberculin skin test after bacillus Calmette-Guèrin (BCG) vaccination, possibly signifying exposure to tuberculosis, also have reduced rates of allergy and asthma (8). Vaccination with either BCG or measles virus, however, is not associated with a reduced risk of atopy (8, 9).

In contrast to the implications of these studies, however, there are data to indicate that severe infections with RSV may enhance allergen sensitization and the risk of developing asthma (10). Although not all studies have found RSV infections to increase the risk of allergy (11), these findings suggest that the effects of infections on the subsequent risk of developing allergies or asthma may depend on which pathogen infects the host early in immune development.

These studies have generated more controversy than definitive answers regarding the relationships between predetermined patterns of immune response, infections in childhood, and the development of allergy and asthma; nonetheless, they have helped to frame some basic questions regarding the development of the immune system in childhood. Additional prospective studies are needed to determine whether childhood infections can cause lasting effects on the immune system to modulate the subsequent risk of allergy and asthma. Alternately, there may be immune factors, perhaps genetically determined, that regulate both the immune response to infections and the risk of developing allergies and/or asthma.

In children and/or adults with existing asthma, respiratory viruses are frequent causes of exacerbations of asthma (14- 17). This association was first demonstrated more than two decades ago by detecting viruses using either serology or culture during wheezing episodes (15, 18). However, many respiratory viruses, particularly rhinovirus (RV), are difficult to detect by standard virologic methods, and thus these initial studies likely underestimated the impact of respiratory viral infections on wheezing. Recently, the use of reverse transcription polymerase chain reaction (RT-PCR) assays, which are much more sensitive in detecting RV than standard techniques (19, 20), has established the importance of RV in causing exacerbations of asthma. Using RT-PCR in addition to standard techniques to identify respiratory viruses, Johnston and colleagues (16) determined that 80-85% of school-aged children with wheezing episodes tested positive for a virus, and the virus most commonly detected was RV. Moreover, Nicholson and colleagues (17) used similar techniques to demonstrate that approximately half of exacerbations in adults with asthma were associated with viral infection. Virus-induced asthma may be severe: indeed, seasonal patterns of upper respiratory virus prevalence correlate closely with hospital admissions for asthma, especially in children (21). Together, current studies indicate that RV infections are the most common cause of asthma exacerbations in children, and also contribute substantially to the morbidity of asthma in adults.

In addition to epidemiologic studies that link respiratory viral infections to exacerbations of asthma, there is clinical evidence that respiratory allergies are a risk factor for wheezing during infections with common cold viruses (14, 15). Duff and colleagues (22) evaluated infants and children who presented to a hospital emergency department with wheezing. Wheezing children older than 2 yr of age were more likely to have respiratory allergies (odds ratio [OR] = 4.5) or have a confirmed viral infection (OR = 3.7), when compared with children without wheezing. Children with the greatest risk (OR = 10.8) for wheezing were those who had both respiratory allergies and a viral infection. These findings imply that viral infections and respiratory allergies may have synergistic effects on lower airway physiology that greatly increase the likelihood of wheezing.

Collectively, it is known that respiratory viral infections frequently cause an increase in airway obstruction in patients with asthma, which suggests that there may be specific interactions between respiratory allergies and viral infections that either initiate or exacerbate underlying asthma. Since the degree of airflow obstruction in asthma is a composite of airway inflammation, plugging due to mucus secretion and cellular debris, and smooth muscle contraction, the effects of viral infections on these factors are of importance to understanding virus-induced asthma. This review will explore potential mechanisms by which respiratory viruses enhance airway responsiveness and/or inflammation in the context of asthma.

	THE EFFECTS OF VIRAL INFECTION ON AIRWAY OBSTRUCTION AND ASTHMA

A number of mechanisms have been identified that may contribute to the development of airway obstruction and airway hyperresponsiveness (AHR) during acute respiratory viral illness (23) (Table 2). Evidence relevant to each of these potential mechanisms is presented in the following sections.

Do Viruses That Trigger Asthma Infect the Lower Airway?

There is little doubt that some respiratory viruses, e.g., influenza, RSV, and parainfluenza virus (PIV), can infect lower airway tissues and cause tissue inflammation and lower airway obstruction. It remains to be established, however, whether RV infections cause wheezing by directly infecting the lower airway or whether there are alternate mechanisms through which upper airway viral infections can cause changes in lower airway function, leading to bronchoconstriction and airway obstruction. Several epidemiologic studies suggest that RV can be a lower airway pathogen. For example, some (26), but not all (30, 31), studies have linked RV with lower airway syndromes such as bronchitis, bronchiolitis, and pneumonia. There are also well-documented reports of fatal RV pneumonia in children, including a case in which RV was recovered from lung tissue at autopsy (32). Rhinovirus infections are associated with increased inflammation in lower airway biopsies, including increased submucosal lymphocytes and epithelial eosinophils (33). Finally, although RV has proved difficult to culture from lower airway secretions obtained via bronchoalveolar lavage (BAL), RV RNA was detected in BAL cells from volunteers after experimental inoculation with RV16 (34). Interpretation of these findings is limited, however, by the necessity of passing the bronchoscope through the upper airway in order to get lower airway samples. Nevertheless, these data suggest that, at least under some conditions, RV and other viral infections extend into the lower airway and induce bronchial inflammation that could contribute to virus-induced exacerbations of asthma.

The Effect of Respiratory Viral Infections on Airway Responsiveness

Viral respiratory infections can cause increased airway responsiveness in humans and in animals. Increased airway responsiveness usually begins early during the acute viral infection and can be observed in response to inhalation of histamine, methacholine, citric acid, or allergen (33, 35). The use of experimentally induced infection of volunteers with RV or influenza viruses has enabled longitudinal examination of lung physiology before, during, and after infections. Cheung and colleagues (42) inoculated 14 subjects with mild asthma with either RV16 or placebo and found that airway responsiveness transiently increased during the acute infection, and returned to baseline levels by 1 wk after the inoculation. The maximal response to inhaled methacholine was significantly greater during the acute RV16 infection and, in contrast to changes in airway responsiveness, remained elevated up to 15 d after the acute infection. Thus, viral infections can enhance both the reactivity of the lower airway and the magnitude of bronchoconstriction in response to inhaled contractile substances in asthma, and the latter effect can persist for weeks after the acute infection.

Some studies of normal and allergic volunteers have not found changes in lower airway physiology after experimental infection, and it has become apparent that not all of the viruses used to inoculate volunteers are equally likely to affect lower airway physiology. Although RV16 consistently has produced changes in the lower airway in several laboratories (33, 39, 40, 42), RV2, RV-Hanks, and RV39 usually do not (46). For example, Halperin and colleagues (48) infected 19 asthmatic volunteers with either RV39 or RV-Hanks and found increased airway responsiveness to inhaled histamine in only four subjects. Interestingly, the four subjects who developed increased bronchial responsiveness during RV infection also had significant drops (> 10%) in FEV₁, indicating that increases in bronchial responsiveness during viral infection may relate to significant changes in airway obstruction.

Since clinical studies of naturally acquired infections have noted that severe colds are more likely to produce lower airway effects (51, 52), attenuation of viruses used for experimental inoculations could contribute to failure to produce lower airway changes in some studies. Picornaviruses such as RV can become attenuated quickly during passage in tissue culture, because of the high rate of spontaneous mutations during viral replication (53). It is unlikely that only a few serotypes of RV (e.g., RV16) cause asthma exacerbations during natural infection, since over 30 serotypes have been cultured from wheezing children and adults to date (18).

Host factors can also influence the effects of viral infections on lower airway function. For example, in studies in which viral infections induce AHR, allergic individuals experience greater changes in airway responsiveness compared with nonallergic control subjects (40, 54). Furthermore, subjects having FEV₁ in the lower range of normal tend to have greater changes in airway responsiveness during viral illness (54). Since asthma is usually associated with both lower FEV₁ values and allergy, especially in children, these preexisting conditions may promote increased airway responsiveness, and thus increased lower respiratory symptoms, during viral respiratory infections in patients prone to virus-induced exacerbations of asthma.

Together, studies of naturally acquired and experimentally induced viral infections demonstrate that viral respiratory infections can cause increased airway responsiveness, although effects on the lower airway physiology are greatly influenced by characteristics of both the host and the virus. It seems logical to assume that virus-mediated enhancement of airway responsiveness contributes to increased symptoms of asthma, but additional studies are needed to determine whether this is a major mechanism involved in the pathogenesis of asthma exacerbations.

Alterations in Neural Control of the Airways

Although neural mechanisms of virus-induced bronchoconstriction and airway obstruction are of great interest, they are particularly difficult to study in humans, because definitive experiments often require the disruption of neural tissue. Consequently, much of our understanding has been gained through the use of animal models. Viral infections could potentially cause bronchoconstriction and increased airway responsiveness by enhancing parasympathetic bronchoconstrictive responses, by stimulating reflex bronchospasm or neuropeptide release from sensory C fibers, or by interfering with the function of nonadrenergic, noncholinergic neurons, which produce the potent bronchodilator nitric oxide. Each of these mechanisms has been explored (Figure 1).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Effects of viral infections on neural regulation of airway function. Virus-mediated damage to the epithelial layer can expose sensory nerves, which increases stimulation of sensory nerves by inhaled particles or inflammatory mediators. Sensory nerves can directly release neuropeptides that induce contraction of smooth muscle cells, or can trigger reflex bronchoconstriction by activating parasympathetic nerves. This reflex bronchoconstriction (BC) causes release of acetylcholine and contraction of smooth muscle cells via stimulation of muscarinic M3 receptors. Normally, acetylcholine inhibits acetylcholine release via stimulation of muscarinic M2 receptors on the presynapse of the postganglionic parasympathetic nerve (auto-inhibitory feedback). Viruses and inflammatory mediators can block this auto-inhibitory feedback, thus enhancing acetylcholine release and potentiating bronchoconstriction. Abbreviation: CNS = central nervous system.

Parasympathetic effects. Altered airway neural control mechanisms were observed first in nonasthmatic human subjects who had transient increases in airway responsiveness to inhaled histamine or citric acid during viral upper respiratory illness (35). Normalization of the responses to airway challenges after atropine treatment suggested that reflex bronchoconstriction was responsible for virus-associated hyperresponsiveness, and a concomitantly increased sensitivity of the cough reflex suggested that the sensory, rather than the efferent, limb of airway reflexes was the likely site of abnormality (35). In agreement with this hypothesis, studies of rodents infected with PIV have demonstrated reduced airway responses to intravenous agonists after vagotomy, confirming a reflex bronchoconstriction component of virus-induced hyperresponsiveness (55). Further studies in guinea pigs, rats and cats demonstrated that the efferent, parasympathetic bronchoconstrictor limb of the reflex is enhanced during acute viral illness (55, 57).

Dysfunction of prejunctional M₂-muscarinic inhibitory autoreceptors, which normally limit acetylcholine release from parasympathetic fibers innervating airway smooth muscle, may contribute to the enhanced response to parasympathetic stimulation (57, 58). Viral illness may interfere with M₂ receptor function through the effects of viral neuraminidase (60) or by inducing inflammatory cell products such as eosinophil cationic proteins (61). Effects on M₂ receptor function are transient, however, as inhibitory M₂ function is restored shortly after virus is cleared from the airways (57). In rats infected with Sendai virus, the M₂ receptor mechanism does not fully account for virus-associated parasympathetic facilitation, because the bronchoconstrictor response to parasympathetic nerve stimulation continues to be enhanced for at least 2 wk after the M₂ function has normalized (57). Although a number of inflammatory mediators are known to have facilitory effects on airway parasympathetic fibers (62), the M₂ receptor-independent factors responsible for virus-induced parasympathetic hyperresponsiveness have not yet been identified.

Sensory C fibers. Respiratory viral illness could provoke asthma by stimulating airway sensory nerve fibers through at least two mechanisms: sensory C fibers in the airways could initiate bronchoconstrictor reflexes via the brainstem and/or cause airway edema and smooth muscle contraction via release of neuropeptides such as substance P and neurokinin A (63, 64). In addition to their direct effects on airway smooth muscle and vascular tissue, neuropeptides may contribute to airway obstruction and hyperresponsiveness by causing increased leukotriene synthesis (65), release of mast cell mediators (66, 67), facilitation of airway parasympathetic output (68), and airway mucus secretion (69). There is evidence that viral infections can potentiate the release of neuropeptides and/or sensitivity to these potent inflammatory mediators. For example, the hyperresponsiveness to histamine and cholinergic challenge of tracheas from bovine PI-3 infected guinea pigs is blocked by depleting neuropeptides from C fibers with capsaicin prior to viral inoculation (70). Furthermore, an increased airway contractile response to neuropeptides has been observed in influenza A-infected ferret tracheas and in guinea pigs infected with PIV (70). Finally, rats with current or recent viral illnesses have markedly enhanced plasma extravasation from airway postcapillary venules after stimulation of neuropeptide release from airway C fibers (74).

Virus-mediated epithelial damage can contribute to increased airway responsiveness in a number of ways. If the epithelial barrier is compromised, then exposed sensory nerves may be more easily stimulated by inhaled particles and inflammatory mediators. One possible explanation for the enhanced effects of neuropeptides in infected airways is that epithelial cell damage leads to a reduction in the activity of airway neutral endopeptidase, an enzyme that inactivates substance P and neurokinin A (71, 73, 74, 78).

Despite the considerable amount of information obtained from these animal models, there is still a lack of consensus regarding the contributions of sensory C fibers and tachykinins to virus-induced asthma exacerbations in humans. Reflex bronchospasm was implicated as an important component of virus-induced hyperresponsiveness to aerosolized histamine and citric acid in nonasthmatic human subjects (35). In contrast, patients with mild asthma had no change in the reflex bronchoconstrictor response to aerosolized bradykinin during experimental RV16 infections (79). Although the asthmatic subjects did not develop the tachyphylaxis to bradykinin that was observed in the normal control subjects, this study does not support reflex bronchoconstriction as a prominent component of virus-induced asthma.

Nonadrenergic, noncholinergic effects. Finally, viral infection also may affect mechanisms that normally serve to oppose airway smooth muscle contraction. The nonadrenergic inhibitory neural response is defective in tracheal smooth muscle from RSV-infected cotton rats (80), and the release of the putative mediator, nitric oxide, is decreased in hyperresponsive tracheas from infected guinea pigs (81).

Summary. There is relatively little information regarding the interface between virus-induced airway inflammation and neural dysregulation, and more research is needed to define these pathways. Animal models have been key to defining potential neural pathways for virus-induced airway dysfunction, but interpretation of this information can be limited by the potential of interspecies differences in airway physiology. Further advances in the understanding of virus effects on neural control of the airways await the development of new specific antagonists of neural pathways or the development of new experimental techniques.

Changes in Small Airway Function

While much has been learned regarding virus-induced changes in the airways, proximal airways have received the most attention. Changes in small airway structure and function, though difficult to measure, may contribute substantially to the hyperinflation and gas exchange abnormalities that are components of severe, acute asthma. Patients with mild asthma exhibit an increase in the maximal airway response to methacholine during a cold, consistent with excessive airway narrowing due to airway wall thickening or airway/parenchymal uncoupling (42). Animals with acute viral illness have morphologic evidence of bronchiolar wall edema and inflammatory cell infiltration, epithelial hyperplasia, folding, and sloughing, which, along with increased debris in the airway lumen, produce marked bronchiolar narrowing or plugging (82). Rats with modest increases in pulmonary resistance and methacholine sensitivity during acute viral illness have air trapping and ventilation/ perfusion mismatches (57); these findings suggest that virus- induced changes in peripheral airways may have serious functional consequences, even in the absence of marked changes in the usual measures of airway obstruction and responsiveness.

	THE EFFECT OF RESPIRATORY VIRUSES ON AIRWAY INFLAMMATION

Because respiratory viruses can cause inflammation and injury in healthy airways, it is reasonable to assume that they can modulate inflammation and worsen injury in diseased airways. The mechanisms by which RV infection of bronchial epithelium increases airway inflammation are not established but do not necessarily involve extensive destruction of airway epithelium. In general, biopsies of nasal mucosa during acute RV infections have shown little cytopathology (86), although some studies have detected increased epithelial detachment or sloughing (89). These findings have led to the hypothesis that it is the immune response to common cold viruses that is responsible for triggering virus-induced exacerbations of asthma. Components of virus-induced immune response that could contribute to airway inflammation and asthma include: (1) early nonspecific responses to viral infection mediated by epithelial cells, endothelial cells, phagocytes, and mononuclear cells; and (2) adaptive immune responses mediated by T cells. The potential contributions of these immune responses to airway inflammation and asthma, and possible interactions between pre-existing airway inflammation and virus-induced responses, will be reviewed in the following sections.

Epithelial Cells

Epithelial cells are the principal hosts for respiratory viruses, which cause varying degrees of damage during replication. Rhinovirus and coronaviruses produce little or no cellular damage in vivo or in monolayers of cultured nasal epithelial cells (87, 92). In contrast, influenza and RSV cause marked cytopathic effects in tissue culture and can cause widespread damage to bronchial epithelium in vivo. Damage to epithelial cells could adversely affect airway homeostasis, since the epithelial layer can synthesize and release smooth muscle relaxing factors like nitric oxide. Indeed, the production of nitric oxide is decreased in guinea pig airways after a viral infection and is associated with hyperresponsiveness (81). The degree of virus-induced cytopathology may also depend on the local immune responses and the anatomic source of the epithelial cell. For example, RSV is more infectious to primary nasal than to bronchial epithelial cells in culture, and cytopathic effects develop later in bronchial epithelial cells (93).

In addition to serving as a barrier and producing enzymes and mediators that contribute to normal airway homeostasis, epithelial cells help to trigger the initial immune response to respiratory viral infections. Respiratory viruses such as RSV, influenza, and RV rapidly stimulate epithelial cells to secrete a wide range of pro-inflammatory cytokines and chemokines, including interleukin-6 (IL-6), IL-8, IL-11, granulocyte/macrophage colony-stimulating factor (GM-CSF), and RANTES (94). For example, nasal secretions of children with virus-induced asthma contain elevated levels of IL-8, which is a potent neutrophil chemoattractant, and IL-8 levels in the nasal secretions correlate with neutrophil myeloperoxidase levels, indicative of neutrophil activation (101). RANTES is a chemoattractant for eosinophils (102), and GM-CSF is a potent activator of eosinophil survival and adhesion molecule expression, and is a cofactor for eosinophil superoxide production and degranulation (103). Interleukin-11 is secreted in very large amounts after epithelial cells are infected with RV or RSV in vitro and may have direct effects on bronchial hyperresponsiveness (106, 107). Molecular mechanisms for the activation of cytokine genes in epithelial cells are now being elucidated. For example, nuclear factor-kappa B activation has been shown to be important factor in the virus-induced transcriptional regulation of IL-6 (108) and may be a key transcription factor to induce the synthesis of a broad array of other inflammatory cytokines (109).

Infection of epithelial cells with several respiratory viruses induces the expression of intercellular adhesion molecule-1 (ICAM-1) (99, 110). Conversely, interferon gamma (IFN-), IL-1, and tumor necrosis factor alpha (TNF-) are released by inflammatory cells or the epithelial layer itself (94) and can induce the expression of adhesion molecules, i.e., ICAM-1, on human airway epithelial and endothelial cells (113, 114). The increase in adhesion molecule expression, together with virus-induced chemokines, recruits leukocytes to the airway and could increase binding of neutrophils and eosinophils to epithelial cells (111, 113, 115). These effects probably potentiate antiviral activity, but could also increase the chances that airway epithelial cells will be damaged by granulocyte mediators.

Endothelial Cells

Endothelial cells are likely to contribute to airway dysfunction during respiratory illnesses through at least two pathways. First, endothelial cells could have a major impact on airway inflammation and respiratory symptoms via their role in recruiting leukocytes to the airway during RV infections. Second, an early event during a clinical cold is transudation of plasma proteins from the vascular tissue of the nasal mucosa leading to increased nasal secretions and congestion, two hallmarks of RV infections (116). Levels of albumin and IgG, markers of serum transudation, reach peak values 2-4 d after virus inoculation and correspond with the time of maximum cold symptoms (116) and increases in bronchial responsiveness (42). Activation of kinins has been suggested as a possible mechanism for the changes in endothelial permeability that lead to transudation (117, 118); however, clinical trials have so far failed to confirm this hypothesis. For example, the bradykinin antagonist NPC 567 did not improve cold symptoms (119). Likewise, oral prednisone therapy decreased kinin levels in nasal secretions, but cold symptoms were not diminished (120). Thus, studies of nasal secretions indicate that increased nasal permeability contributes to cold symptoms and raise the possibility that these mechanisms could also produce lower airway obstruction and increased symptoms of asthma. Mechanisms by which viral infections affect endothelial cells to increase vascular permeability are as yet unclear.

Granulocytes

Neutrophils are the main cells recruited to the airway during the acute stages of viral respiratory infections (87, 121), and this is likely to be in response to chemotactic factors, such as IL-8 and leukotriene B₄ (LTB₄), released during the initial stages of the viral infection (45, 94, 100, 101, 124, 125). Respiratory viruses can activate neutrophil inflammatory functions, as indicated by enhancement of superoxide responses, chemotaxis, and adhesion (126, 127). In addition, complexes of RSV and antibody induce IL-6 and IL-8 secretion by neutrophils (100): this pattern of cytokine production could further enhance neutrophil and eosinophil recruitment to the lung.

Although the role of the neutrophil in asthma has yet to be clearly defined, there is evidence to implicate neutrophils in the pathogenesis of virus-induced asthma exacerbations. Grünberg and colleagues (45) experimentally inoculated 35 subjects with atopic asthma with either RV16 or placebo and measured effects on AHR, peripheral blood leukocyte counts, and IL-8 levels in nasal secretions. Neutrophil counts in the peripheral blood correlated with cold and asthma symptom scores and cold-induced changes in AHR.

Evidence that respiratory viral infections can activate eosinophils comes from several sources. For example, eosinophil granular proteins and leukotriene C₄ (LTC₄) have been detected in the nasal secretions of infants and children with wheezing illnesses (128). Increases in sputum eosinophil cationic protein (ECP) during the acute phase of experimentally induced RV infection correlated with increased airway responsiveness in a group of adults with asthma after experimental inoculation with RV16 (132). Parainfluenza-infected guinea pigs develop airway eosinophilia and AHR (133), but pretreatment of guinea pigs with IL-5-specific neutralizing antibody reduced the number of eosinophils in blood and BAL and prevented the development of AHR after the viral respiratory tract infection (134).

Mechanisms by which respiratory viruses activate granulocytes are under investigation. It has been demonstrated that RSV can directly cause eosinophil LTC₄ release (135), but RV has little effect on eosinophil activation in vitro (136). It is likely that mediators and cytokines (e.g., IL-8, GM-CSF) secreted by other virus-activated cells in the lung contribute substantially to the activation of granulocytes, mast cells, and basophils.

Macrophages and Monocytes

Airway macrophages are likely to be involved in the early immune response to respiratory viruses through the secretion of mediators and cytokines with antiviral properties and/or pro-inflammatory effects. During viral upper respiratory infections, nasal secretions contain IL-1, TNF-, and IL-8 (45, 101, 137, 138), and these same cytokines have been detected when monocytes or macrophages are cultured with respiratory viruses in vitro (94, 139). These acute phase cytokines can cause systemic symptoms such as fever and malaise that are commonly associated with viral respiratory infections, and IL-1 and TNF- can also increase cell recruitment into the airway through effects on adhesion molecule expression on endothelial cells. Probably as a result of effects on ICAM-1, TNF- increases the susceptibility of an epithelial cell line (BEAS2B) to become infected with major group RV (99). Furthermore, TNF- has been closely associated with wheezing illnesses in infancy (142) and the development of the late-phase allergic reaction and asthma (143, 144). Finally, the appearance of interferon in the nasal secretions during viral infection has been associated with the onset of the recovery process, and Roberts and colleagues (145) demonstrated that monocytes and macrophages generate interferon upon virus incubation in vitro.

Besides secreting cytokines, macrophages incubated with RSV or PIV produce lipid mediators such as prostaglandin E₂, platelet-activating factor, and thromboxane B₂ (146), and also generate reactive oxygen species (133, 148); each of these effects could produce increased airway inflammation. Conversely, there is evidence that viral infections can interfere with certain macrophage activities, such as antigen presentation. For example, influenza, RV, and RSV inhibit T-cell proliferative responses in vitro, in part due to effects on antigen-presenting cells, such as the macrophage (149). Indeed, RSV and influenza can replicate in cultured alveolar macrophages (133, 153), although RV do not (139).

T Cells

Respiratory viral infections induce specific and nonspecific T-cell activation, and there is evidence linking these T-cell responses to asthma disease severity during viral infections (154). Rhinovirus infection is usually accompanied by peripheral blood lymphopenia, and the degree of lymphopenia correlates with severity of cold symptoms (155) and changes in airway responsiveness (33, 42). Moreover, lymphocytes are increased in the nasal secretions and lower airway epithelium during acute RV infection, coincident with increases in airway responsiveness (33, 122).

Natural killer cells, CD4+ T cells, and CD8+ T cells all participate in antiviral immune responses. Cytotoxic CD8+ T cells are a major source of IFN- during viral infections and exert potent antiviral activity by lysing virus-infected cells. CD4+ T cells have less cytotoxic potential but can direct immune responses through the secretion of cytokines. Studies of mice have shown that synthesis of IFN-, IL-4, IL-5, and IL-10 is increased in the lung during respiratory viral infection (156, 157), indicating that viral infections induce a broad array of cytokines. The balance of T helper cells, types 1 and 2 (Th1 and Th2)-like responses is likely to be important in coordinating both cytotoxic and antibody responses to viral infection, which provide antiviral activity and immunity to reinfection, respectively.

There is evidence to indicate that Th2-like responses have relatively weak antiviral activity, and under some conditions can modify and possibly hinder antiviral responses. For example, administration of IL-4 to influenza-infected mice delays viral clearance and suppresses the generation of cytotoxic T-cell precursors (158). Some viruses may have evolved mechanisms to induce Th2-type responses, and thereby increase the chances of successful viral replication (159). For example, Alwan and colleagues found that CD4+ T-cell lines specific for the RSV attachment protein (protein G) secrete IL-3, IL-4, and IL-5 but little IL-2 (i.e., a Th2 pattern) when stimulated with antigen (160), and similar findings have been reported in humans (161). In addition, mice that are sequentially given protein G- specific T cells followed by an RSV infection develop pulmonary eosinophilia and more severe lung disease (162). These findings raise the possibility that Th2-like cytokines induced by viral infection could potentiate preexisting allergic inflammation and increase morbidity associated with viral infections.

Virus-specific T-cell responses are generally not detectable in the airway or blood until 7-10 d after inoculation with virus. This time delay raises questions about whether virus-specific T cells contribute to virus-induced asthma symptoms, which often begin during the first few days of the respiratory infection. With this in mind, there are three possible scenarios to describe T-cell involvement in the pathogenesis of virus-induced exacerbations of asthma: (1) T cells help to clear viral infections but do not contribute to asthma symptoms; (2) virus-specific T-cell responses contribute to asthma symptoms but only during the latter stages of viral infections; or (3) viral infections rapidly activate T cells, which contribute to airway inflammation and symptoms during the entire illness.

Experiments in mice indicate that the majority of T cells attracted to the lung during an acute viral infection are not virus-specific (163), and there is evidence that experimentally induced RV infection produces both antigen-dependent and antigen-independent T-cell activation (164). These observations suggest that viral infections can activate a broad range of T cells, and raise the possibility that this effect could contribute to airway dysfunction. Several mechanisms for the rapid activation of T cells have been proposed. For example, T-cell responses induced by either influenza or RV can be cross- reactive among different viral serotypes (165). Since both of these viruses cause recurrent infections with different serotypes, part of the T-cell response could be a memory response, which would be expected to have faster kinetics and potentially be of a greater magnitude than a primary T-cell response.

Considering the large number of cytokines and mediators generated during a viral infection, it is likely that lung T cells are activated in an antigen-independent manner through "bystander" effects, and there is evidence to support this hypothesis. For example, when RV is incubated with peripheral blood mononuclear cells, the early activation marker CD69 is expressed on 25-50% of T cells, and IFN- mRNA and protein is induced (168). This T-cell activation is caused by soluble factors secreted by RV-activated monocytes. Antigen-independent T-cell activation has also been demonstrated in mice with a systemic viral infection [lymphocytic choriomeningitis virus (LCMV)] (169). In this model, viral infection induced nonspecific, limited proliferation of CD44^hi (memory phenotype) T cells, and this effect was mediated by IFN-. Finally, high concentrations of RANTES, a chemokine induced by respiratory viruses, can also cause antigen-independent T-cell activation (170). Together, these studies indicate that viruses can cause nonspecific T-cell recruitment and activation early during the course of infection, and suggest that this effect could significantly augment airway inflammation and thus contribute to respiratory symptoms.

Interactions among Viruses, Allergy, and Preexisting Airway Inflammation

Techniques of experimentally inducing viral infections in human volunteers have helped to define the effects of colds on lung function and to evaluate separate and combined effects of viruses and allergens on lung physiology. For example, Lemanske and colleagues (39) evaluated the effects of RV infection on the pulmonary response to inhaled allergen in a group of subjects with allergic rhinitis. Rhinovirus infection increased airway responsiveness to both histamine and allergen inhalation and also increased the probability of a late-phase allergic response 4-6 h after allergen challenge. Adding to these observations, Calhoun and colleagues (44) found that during an acute RV infection, subjects with allergic rhinitis, but not control subjects, had an increase in the immediate release of histamine after segmental allergen challenge. Furthermore, RV16 infection enhanced allergen-induced eosinophil recruitment into the lower airway in allergic subjects. Increased allergen-induced histamine release, eosinophil recruitment, and late-phase allergic responses was still noted 4-6 wk after the allergen challenge (39, 44).

There are limited data on the effects of respiratory viruses associated with asthma on lower airway histology in humans. Fraenkel and associates (33) found an increase in submucosal lymphocytes in bronchial biopsies during experimental RV infection, which was accompanied by increased histamine responsiveness. During the acute cold there was also an increase in epithelial eosinophils, and in asthma this inflammatory change persisted into convalescence. Trigg and colleagues (41) found evidence of lower airway inflammation in normal and atopic subjects during natural colds. Neutrophils were increased in bronchial washings, and bronchial biopsies revealed increased epithelial eosinophils and CD8+ T cells.

These studies provide evidence that infections with common cold viruses can cause inflammation of the lower airway. Furthermore, there is evidence that infection with RV may intensify both the immediate and late responses to allergen challenge. The increased recruitment of eosinophils to the lower airway may explain the enhanced likelihood of a late allergic response to antigen seen during RV16 infection.

Interactions between respiratory viruses and mast cells, basophils, and IgE are of interest in evaluating potential mechanisms of virus-induced asthma. Interferon and other factors released by peripheral blood cells incubated with virus promote basophil histamine release (171), although there is no evidence that respiratory viruses activate circulating basophils in vivo (174). Additional studies are needed to define mechanisms of virus-induced mast cell and basophil activation under conditions that more closely resemble the environment of the airway.

Viral infections can produce short-term increases in total IgE. Skoner and colleagues (175) found that experimental infection with RV39 increases total IgE levels in atopic subjects but not in control subjects. In addition to effects on total IgE, RSV and PIV can induce virus-specific IgE, the presence of which correlates with wheezing lower respiratory infections and hypoxia (176). Finally, allergen sensitization in rodents is enhanced if the exposure to antigen occurs during the acute phase of infection with influenza virus (179) or RSV (180). Potential mechanisms for this effect include enhanced allergen absorption due to increased epithelial permeability (181) or viral interference with the development of tolerance (182).

Finally, Coyle and colleagues (183) found that allergic sensitization with ovalbumin before infection with virus (lymphocytic choriomeningitis virus) increased secretion of IL-5 and decreased secretion of IFN- by virus-specific T cells. These changes in the virus-specific immune responses were reproduced by incubating virus-specific CD8+ T cells with viral peptide in the presence of IL-4. These data suggest that the immune response to viral infections can be altered by environmental influences such as allergen sensitization, and that this effect may be caused by the composition of the cytokine milieu at the time of viral infection.

	SUMMARY AND THERAPEUTIC IMPLICATIONS

Viral respiratory infections exert considerable influence on airway function and asthma in all age groups. In infancy, respiratory viruses such as RSV cause episodes of wheezing that may be recurrent but are largely transient. In addition, there are indications that early viral infections may be able to affect the development of the immune system and modify the subsequent risk of allergy and asthma. Finally, in children and adults with established asthma, common cold viruses such as RV frequently trigger acute symptoms of asthma.

Antiviral agents would seem to be an obvious solution to virus-induced respiratory problems. However, current antiviral medications (e.g., rimantadine, ribavirin) have limited efficacy and must be started very early in the disease course to be effective. For prevention of RV infections, prophylactic use of topical IFN-, soluble ICAM-1, or capsid-binding agents that prevent viral binding or uncoating has proven efficacy (184- 188). The clinical utility of these medications is limited, however, since they are generally ineffective if they are started after the onset of symptoms (189). Furthermore, IFN- administered to patients with chronic respiratory diseases (including asthma) after close contact with people with upper respiratory symptoms does not prevent cold-related lower respiratory symptoms (190). Finally, the capsid-binding agents and soluble ICAM-1 specifically target RV, which account for about two-thirds of virus-induced asthma episodes, but have no effect on other viruses that can trigger asthma symptoms. Finding a broad spectrum antiviral agent with low cost and toxicity continues to present a major obstacle.

There is now evidence that the immune response to respiratory viral infections, though critical to clear virus from the airway, also contributes to airway obstruction and respiratory symptoms. The mechanisms by which these changes occur appear to be associated with the ability of respiratory viruses to induce the production of pro-inflammatory cytokines and mediators. Some of these cellular and cytokine responses have been correlated with upper respiratory cold symptoms, changes in airway responsiveness, or lower airway symptoms (Table 3), although causal relationships have yet to be proven. Current data suggest that viral infections coordinately activate epithelial cells, endothelial cells, and leukocytes to cause airway edema, obstruction, and increased responsiveness (Figure 2). The epithelial cell is the host cell for respiratory viruses and is also the sentinel cell to initiate antiviral immune responses through the secretion of a broad array of cytokines, chemokines, and mediators. This early activation of epithelial cells and other resident airway cells stimulates changes in endothelial cell physiology: increased adhesion molecule expression to increase leukocyte recruitment, and increased vascular permeability, leading to edema and increased secretions. Granulocytes, macrophages, and T cells are activated by viruses and/or virus-induced cytokines, enhancing airway inflammation and obstruction and disrupting normal neural physiology in the lung to increase airway responsiveness. Finally, processes involved in the resolution of airway inflammation after viral infection include the lysis of virus-infected cells by cytotoxic lymphocytes, suppression of inflammation, mediated in part by cytokines such as transforming growth factor beta and IL-10, and the repair of airway structures with consequent airway remodeling. These processes may determine the nature of persistent changes in airway function after viral infections.

View larger version (52K): [in this window] [in a new window]   Figure 2.   Cellular inflammatory responses to respiratory viral infection. (a) Respiratory viral infections are initiated when a virus enters a host epithelial cell, and viral replication causes the release of new infectious particles and activates secretion of cytokines, chemokines, and mediators by the epithelial cells. (b) Viral particles released into the airway cause activation of resident cells in the airway, including macrophages, lymphocytes, and granulocytes. Cytokines from epithelial cells and other resident airway cells increase adhesion molecule expression and increase airway responsiveness. (c) The combination of chemokine secretion and increased adhesion molecule expression causes recruitment and activation of additional leukocytes, which further add to airway inflammation and increased responsiveness. (d ) Cytotoxic T cells kill virus-infected cells, and cytokines such as TGF- and IL-10 are likely to play a role in the downregulation of airway inflammation after viral infection. Increased numbers of airway lymphocytes and eosinophils may persist for weeks after the viral infection. The steps illustrated in this diagram can occur sequentially and/or in parallel, and the timing depends on factors related to both the host and virus. Abbreviations: EOS = eosinophil; PMN = polymorphonuclear leukocyte; mac = macrophage; lymph = lymphocyte; CTL = cytotoxic lymphocyte; O2 = superoxide; AHR = airway hyperresponsiveness.

This model suggests several opportunities, and a number of challenges, for the design of new therapeutic interventions for virus-induced asthma. The most effective treatment now used for virus-induced exacerbations of asthma is to prescribe a short "burst" of oral corticosteroid early during the course of a viral respiratory infection (191). Despite proven efficacy in lessening lower airway consequences of viral respiratory infections in patients with asthma, systemic corticosteroids have little effect on common cold symptoms (120, 192), indicating that the mechanisms that cause upper versus lower respiratory symptoms associated with RV infection may be distinct. Use of systemic corticosteroids may be associated with side effects, and it is clearly desirable to develop more specific treatments that inhibit the pro-inflammatory effects of viral infections, while avoiding immunosuppression and corticosteroid-induced morbidity.

In any case, since many of the cells, cytokines, and mediators unleashed by viral infections have overlapping inflammatory effects, it seems unlikely that therapeutic approaches aimed at inhibiting any one factor will reverse viral effects on airway function. With this in mind, several teams of investigators are working to define the intracellular mechanisms by which viruses activate cytokine genes, with the hope that viruses may trigger a common signaling pathway(s) that leads to the activation of multiple pro-inflammatory genes. If this is true, new therapies for virus-induced asthma could target this pathway.

Another critical challenge is to determine why individuals with allergy and lower airway inflammation are so susceptible to the effects of respiratory viruses, such as RV, that cause mild disease in normal individuals. Is there a fundamental difference in the immune response to viruses in the presence of atopy? Alternatively, are the more severe clinical manifestations of respiratory viral infections in asthma a result of the different types of cells, or perhaps heightened activation state of the cells, in the asthmatic airway? With the answers to these questions will come a better appreciation of how airway inflammation is regulated in asthma and how best to treat this process when it is augmented by viral infection.

	Footnotes

Correspondence and requests for reprints should be addressed to William W. Busse, M.D., H6/360 CSC, University of Wisconsin Hospital, Madison, WI 53792-3244. E-mail: wwb{at}medicine.wisc.edu

(Received in original form July 31, 1997 and in revised form February 5, 1998).

Acknowledgments: The authors thank Robert F. Lemanske, Jr., M.D., for reviewing the manuscript and providing many helpful suggestions.

Supported by NIH grants AI26609, AI34891, AI40685, and HL44098.

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