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Department of Respiratory Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College, London, United Kingdom
Correspondence and requests for reprints should be addressed to Peter J. M. Openshaw, M.D., Ph.D., Department of Respiratory Medicine, National Heart and Lung and Wright Fleming Institutes, Faculty of Medicine, Imperial College London, Paddington, London W2 1PG, UK. E-mail: p.openshaw{at}imperial.ac.uk
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ABSTRACT |
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 TOP ABSTRACT THE BURDEN OF RSV...HOST FACTORS AFFECTING...AUGMENTATION OF DISEASE BY...FI-RSV IN MICEDISSECTING IMMUNOPATHOGENESISSUMMARY OF IMMUNE MECHANISMS...CONCLUSIONS AND IMPLICATIONS FOR...REFERENCES |
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Key Words: animal models • asthma • bronchiolitis • viral disease
It is increasingly appreciated that symptoms and signs of many viral diseases are caused less by viral cytopathic effects than by the host's response to infection. The peak of viral infection often precedes the period of maximal illness, which coincides with cellular infiltration of infected tissues and the release of inflammatory mediators. The role of overexuberant immune responses is particularly clear in disease caused by respiratory syncytial virus (RSV).
Inflammation in RSV disease in seen during normal primary infection, but is highly variable in severity from one individual to another. It is increased by certain types of vaccination, a phenomenon remarkably reproducible in virtually all animal models. By comparing and contrasting the immunopathogenesis of primary bronchiolitis and vaccine-enhanced disease, it is possible to identify common mechanisms that are shared or distinct in these different situations. A more extensive version of this review may be found elsewhere (1).
It is well known that children who recover from RSV bronchiolitis are at increased risk of recurrent wheeze and asthma diagnosis in later childhood, but it is not clear if the relationship is causal. With recent evidence that RSV can persist, it is possible that low-level viral replication might drive chronic inflammation in some individuals with chronic lung disease.
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THE BURDEN OF RSV DISEASE |
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TOPABSTRACTTHE BURDEN OF RSV...HOST FACTORS AFFECTING...AUGMENTATION OF DISEASE BY...FI-RSV IN MICEDISSECTING IMMUNOPATHOGENESISSUMMARY OF IMMUNE MECHANISMS...CONCLUSIONS AND IMPLICATIONS FOR...REFERENCES |
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The impact of RSV in adults has been underappreciated, and it generally passes undiagnosed (Figure 1). RSV causes high morbidity and mortality in patients with underlying cardiopulmonary illnesses and the elderly (4) and in the immunosuppressed, particularly bone marrow transplant patients.
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In explaining the pathogenesis of RSV disease, it is important to recognize that only a small minority of RSV-infected children develop severe disease and that the disease in ventilated children (e.g., those from whom it is possible to obtain samples from the lung) may be very different from that in hospitalized nonventilated children. Virtually nothing is known about the pathogenesis of disease in the great majority of children who develop mild respiratory symptoms and are neither seen by doctors nor sent to hospital. Because only a few RSV-infected children get very ill, the pathways leading to severe disease only need to operate in a small minority or children predisposed by inherited or acquired factors.
Severe RSV infection in the first 6 months of life is often followed by recurrent childhood wheezing, an association that is lost by 11 to 13 years of age (6, 7). In the study by Sigurs (8), it was shown that children with severe RSV bronchiolitis in infancy had a significantly higher rate of asthma compared with age- and sex-matched control subjects (11% vs. 0%, at age 1; 23% vs. 1% at age 3; 23% vs. 2% at age 7.5). In this same cohort studied at 13 years of age, asthma had been diagnosed in 37% of the subjects with RSV bronchiolitis and 5.4% of the control group.
Accepting that RSV bronchiolitis and recurrent childhood wheeze are associated, the fundamental question is whether the association is causal. Does bronchiolitis act as a marker for an increased risk of allergy and wheezing illness due to genetic predisposition or impaired respiratory reserve, or does bronchiolitis lead to long-term changes in the lung that cause recurrent childhood wheezing (9)? Direct interventional studies demonstrating a causal relationship have not been published. However, administration of anti-RSV immune globulin administration to children at high risk of RSV disease seems to improve asthma scores and reduce atopy (10). It is possible that an anti-RSV neutralizing monoclonal antibody (e.g., palivizumab) also has long-term beneficial effects, but studies are yet to be published.
The mechanisms that could account for delayed effects of RSV infection are not clear, but could include immune "imprinting." A sustained increase in IL-2 receptor levels is seen after RSV infection, suggesting that inflammation may continue after the acute symptoms and signs have resolved (11), but direct histologic confirmation of persistent inflammation has not been possible. RSV has been shown to be persistent in in vitro macrophage-like cell lines in cattle (12), mice (13), and guinea pigs (14). If persistence occurs in humans, it could explain the apparent delayed effects and serve as a reservoir for future RSV outbreaks in infants.
Although severe RSV disease in infancy may cause recurrent wheezing, this is not the case with most viral infections. Uncomplicated common colds (without wheeze), type I herpetic stomatitis, chickenpox, and exanthema subitum seem to protect against wheeze up to 7 years of age. The risk of asthma diagnosis by this age is reduced by about 50% in children with two or more reported common colds by the age of 1 year (15). Viral infections typically induce T helper 1–type responses, characterized by high levels of IFN-
production; by contrast, asthma and atopy are typically characterized by T helper 2 cells producing interleukin (IL)-4 and IL-5 (Th2 cells). In contrast, analysis of nasal lavage and peripheral blood samples from RSV-infected children shows elevated IL-4/IFN- ratios in infants during the first week of acute bronchiolitis compared with infants with upper respiratory tract signs alone. These data are consistent with excessive type 2 and/or deficient type 1 immune responses in RSV bronchiolitis (16).
Whatever the role of RSV in the inception of asthma, it (and other viruses) can certainly lead to asthma exacerbations in older children and adults (17). Rhinoviruses are commonly found during acute exacerbations of chronic obstructive pulmonary disease, but there is intriguing preliminary data suggesting that RSV may also be present in some patients during remission (18). However, the role of RSV in the development or progression of chronic obstructive pulmonary disease (COPD) is unknown; it is possible that persistent infection leads to a shift of T cell function is such a way that inflammation is sustained, or that the local environment in COPD is favorable to viral survival.
RSV has been isolated from guinea pig and mouse lungs 100 days or more after experimental infection, and infectious virus can be recovered from mice with persistent RSV infection. Both in acute bronchiolitis and in chronic models of infection, the key sites for RSV-induced inflammation in the lung are the small airways; here epithelial damage and increased mucous production may result in small airway obstruction and hyperinflation. The small airways are the primary site for the persisting inflammation and airway obstruction, which is characteristic of COPD. Biopsies of subjects with COPD have demonstrated that the small airways are infiltrated with inflammatory cells, in particular CD8 cells, neutrophils, and airway macrophages. In particular, the presence of CD8 T cells and B lymphocytes organizing into follicles was associated with disease progression. Hogg and coworkers suggest that these cells are present in response to airway infection and bacterial colonization, which frequently occurs in patients with moderate to severe COPD (19). Indeed, it is possible to postulate that the presence of large numbers of CD8 T cells, characteristic of COPD airway biopsies and previously unexplained, may be due to persistent inflammatory stimulus of chronic viral infection.
Furthermore, bacteria and viruses may cooperate to produce cycles of destructive inflammation. In a study by Monick and colleagues, airway epithelial cells were unresponsive to bacterial LPS under normal conditions, but became highly responsive to LPS after RSV infection (20). This effect was ascribed to TLR4 upregulation, causing cellular sensitization to proinflammatory factors.
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HOST FACTORS AFFECTING PATHOGENESIS |
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TOPABSTRACTTHE BURDEN OF RSV...HOST FACTORS AFFECTING...AUGMENTATION OF DISEASE BY...FI-RSV IN MICEDISSECTING IMMUNOPATHOGENESISSUMMARY OF IMMUNE MECHANISMS...CONCLUSIONS AND IMPLICATIONS FOR...REFERENCES |
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In the mouse model, subjecting animals to primary infection up to 1 week of age leads to increased disease severity during adult reinfection with RSV. Mice that were infected with RSV neonatally are sicker and have greater cell recruitment to the lung, increased IL-4 production, and a mixture of lung eosinophilia and neutrophilia during adult RSV challenge (22). Therefore, in the mouse model at least, the timing of neonatal infection establishes and determines the subsequent "imprinted" pattern of T cell responses and, consequently, the nature and severity of disease during reinfection in adulthood. The practical implication of these studies is that delaying RSV infection beyond early infancy could have long-term benefits.
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AUGMENTATION OF DISEASE BY VACCINATION |
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TOPABSTRACTTHE BURDEN OF RSV...HOST FACTORS AFFECTING...AUGMENTATION OF DISEASE BY...FI-RSV IN MICEDISSECTING IMMUNOPATHOGENESISSUMMARY OF IMMUNE MECHANISMS...CONCLUSIONS AND IMPLICATIONS FOR...REFERENCES |
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Postmortem examinations showed bronchopneumonia with emphysema and pneumothorax. Microscopically, there was an intense inflammatory infiltrate, including mono- and polymorphonuclear cells and eosinophilia. These changes suggested an immunopathologic cause of enhanced disease. Analysis of serum from children immunized with FI-RSV shows that antibodies to the F and G proteins were generated, but were poorly neutralizing (24). The severity of illness was remarkably dependent on the age of the vaccinees, the younger the children suffering more severe symptoms. The reasons for this are not clear, but animal models suggest that there is an age-dependent factor as a major determinant of the pathogenic immune response (22).
Like FI-RSV, FI-measles virus can cause severe and generalized disease during subsequent natural infection. This "atypical" measles is seen after a delay of about 7 years, whereas enhanced RSV disease occurs within 2 years of FI-RSV administration (25). Therefore, a window of protection may be followed by a phase of disease enhancement (26), followed by a final period during which immune memory is still measurable but neither protection nor enhancement is seen.
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FI-RSV IN MICE |
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TOPABSTRACTTHE BURDEN OF RSV...HOST FACTORS AFFECTING...AUGMENTATION OF DISEASE BY...FI-RSV IN MICEDISSECTING IMMUNOPATHOGENESISSUMMARY OF IMMUNE MECHANISMS...CONCLUSIONS AND IMPLICATIONS FOR...REFERENCES |
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Murine studies have highlighted the vital importance of genetic background variation in both the virus and host, but have clearly demonstrated the role of T cells in augmented pathology. Connors and coworkers showed that CD4+ T cells are essential to production if FI-RSV vaccine augmented disease, and that RSV-specific antibodies (in the absence of CD4+ and CD8+ T cells) are not sufficient to cause enhancement (27). There is a marked increase in the expression of Th2-type cytokines (IL-5, IL-13, IL-10) and reduced expression of IL-12 in FI-RSV immunized mice, indicating a Th2 bias in the increased inflammation, and there is a correlation between the signal for IL-5 mRNA and the eosinophil infiltration. Skewing to a T helper 1 (Th1) pattern of cytokine production by priming with live RSV prevents subsequent enhanced disease (28).
Immune Priming with Individual RSV Antigens
Sensitization of BALB/c mice by dermal scarification with recombinant vaccinia viruses (rVV) expressing individual RSV proteins make it possible to dissect the contribution of different RSV antigens and T cell subsets to protection and pathology. The three most studied RSV proteins are the major surface glycoprotein (G), the fusion protein (F), and the transcription antiterminator (formerly, second matrix protein M2). Whereas rVV-G primes Th2 cells and leads to secondary RSV disease characterized by lung eosinophilia (29), rVV-F primes cytotoxic T lymphocytes (CTL) and a Th1 responses, resulting in secondary RSV disease with polymorphonuclear (PMN) efflux (30, 31), and rVV-M2 primes for a secondary RSV disease characterized by strong CTL (32).
Using deletion mutations of regions of the G protein, a site within G has been identified which seems of key importance to induction of Th2 cells. Deletion mutants of this region of G no longer prime an eosinophilic response to infection, but do prime the immune system to clear the virus more effectively (33). Sensitizing mice with recombinant vaccinia virus expressing the secreted soluble form of the G protein leads to a greater eosinophilic influx into the lungs after RSV challenge than mice sensitized with vaccinia expressing only the membrane-anchored form. Engineered recombinant RSV expressing only the membrane-bound form of G is immunogenic, but grows poorly in vivo and is unable to generate the eosinophilic response seen with nonrecombinant virus (34). Antibody depletion of cells bearing T1/ST2 (a marker of the Th2 cells) reduces eosinophilia in RSV-infected rVV-G–primed mice (35).
If an engineered, secreted form of the F protein is used to vaccinate, IL-4 and IL-5 production is increased but pulmonary eosinophilia after RSV challenge is not seen (36). The transcription antiterminator protein (M2) contains an immunodominant Kd-restricted peptide epitope (37) that can be used for mucosal vaccination in conjunction with the adjuvant LTK63 (38). M2 primes strong CTL responses and severe disease enhancement on RSV challenge. Crucially, this type of priming induces virtually no RSV-specific antibody, and transfer of CTLs to naive mice results in accelerated viral clearance but greater disease (39).
Immunization with vaccinia expressing individual RSV proteins gives valuable information about the possible pathways of RSV disease pathogenesis, but does not reproduce the pathology seen after FI-RSV vaccination. Lung eosinophilia is seen in RSV-challenged mice primed with FI-RSV or with rVV-G, but G protein is not necessary for formalin-inactivation enhanced disease (40). In another study, formalin-inactivated mutant RSV strains with truncated or deleted G or deleted SH induced lower protective antibody levels, but immunopathologic effects were still seen, with increased illness and eosinophilia (41). This suggests that immunity to G is important for protective immunity, but is not necessary for FI-RSV–enhanced disease. It is important to recognize that both situations may be characterized by lung eosinophilia, but that the pathways leading to eosinophilia are different.
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DISSECTING IMMUNOPATHOGENESIS |
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TOPABSTRACTTHE BURDEN OF RSV...HOST FACTORS AFFECTING...AUGMENTATION OF DISEASE BY...FI-RSV IN MICEDISSECTING IMMUNOPATHOGENESISSUMMARY OF IMMUNE MECHANISMS...CONCLUSIONS AND IMPLICATIONS FOR...REFERENCES |
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The Role of CD4 Helper T Cells
In many situations, murine disease severity is reduced by CD4 and/or CD8 T cell depletion (42). Depletion of CD4+ T cells (43) or transfer of CD8+ T cells modulates the eosinophilia seen in rVV-G–primed RSV-infected mice (44), while eosinophilia can be made to appear in strains that normally do not develop it if CD8 T cells are depleted or impaired in function (45).
This suggests that Th2 cells promote RSV-induced eosinophilia and that CD8 cells generally inhibit it. As described above, RSV G–induced pathology is mainly caused by the overactive Th2 CD4+ T cells (29). It has been shown that these CD4 T cells are oligoclonal, with approximately one-half of the cells expressing Vß14, and that Th2-like pulmonary injury can be abolished by elimination of this CD4+Vß14+ subpopulation (46). This intriguing finding suggests that this novel subset of CD4+ T cells is crucial to the development of pathology, akin to the situation after superantigen stimulation.
As described above, the effects of RSV are mediated through the cytokines and chemokines induced during infection. The immune background of the neonatal lung is different in the adult (47, 48), with a general bias toward Th2 responses. It may be that these factors in part account for enhanced disease severity in RSV-infected infants. Although murine FI-RSV and rVV-G vaccination models of disease augmentation have focused on the pathogenesis of lung eosinophilia and emphasized the role of Th2 cells, RSV immunopathology is certainly not solely Th2-related. During primary infection RSV, like most viral infections, tends to induce inflammation dominated by Th1 cytokine production even in BALB/c mice (which are prone to Th2 responses).
The Role of CD8 Cytotoxic T Cells
Cell transfer studies show that CD8 T cells can cause viral clearance, but also result in remarkable disease enhancement (39). If rVV-M2 is used to prime BALB/c mice, RSV infection results in extreme sickness due to a massive pulmonary influx of CD8+ CTL and pathology reminiscent of acute respiratory distress syndrome (ARDS). In a few cases, RSV bronchiolitis has also been associated with ARDS in humans, but the normal pathology of infantile bronchiolitis is very similar to that seen in mice with highly activated CD8 T cell responses.
The Effects of Virus-specific Antibody
When first observed, it was initially thought that FI-RSV pathology was antibody-mediated. This view went out of favor, but more recently gained support from studies in vitro and in primates, suggesting that antibody may increase viral replication. In addition to possible infection-enhancing antibody, antibody may enhance disease by forming immune complexes and activating complement. This has been shown to be important in both FI-RSV (40) and FI-measles virus (25). This effect appears to preferentially affect lung function, but possibly also affects Th2 differentiation.
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SUMMARY OF IMMUNE MECHANISMS OF RSV DISEASE |
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TOPABSTRACTTHE BURDEN OF RSV...HOST FACTORS AFFECTING...AUGMENTATION OF DISEASE BY...FI-RSV IN MICEDISSECTING IMMUNOPATHOGENESISSUMMARY OF IMMUNE MECHANISMS...CONCLUSIONS AND IMPLICATIONS FOR...REFERENCES |
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The incubation period of primary RSV is about 5 days; children hospitalized with bronchiolitis have usually already been ill for 3 to 6 days. Virus replicates to higher levels and remains detectable for longer during primary infection than after prior sensitization. It is therefore possible that "acquired" T and B cell responses are developing by this time, and contribute to disease. This situation is exacerbated by prior sensitization or in those otherwise predisposed to particular specific immune responses.
Age and host genetics certainly affect the balance of immune responses during primary infection, making first RSV infections sometimes resemble secondary disease seen in primed or sensitized hosts. In the neonatal period, Th1 responses are generally poor or short-lived and IL-12 production weak. One possibility is that both Th1 and Th2 responses are formed during the initial infection; however, there is specific IL-4–dependent apoptosis of Th1 cells in the neonatal environment. This may help explain why there is a natural tendency toward strong Th2 responses in neonates. An autocrine Th2 feedback loop driven by IL-4 might therefore tend to cause a cascade of events causing immune damage, or future skewing of responses to reinfection.
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CONCLUSIONS AND IMPLICATIONS FOR CHRONIC LUNG DISEASE |
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TOPABSTRACTTHE BURDEN OF RSV...HOST FACTORS AFFECTING...AUGMENTATION OF DISEASE BY...FI-RSV IN MICEDISSECTING IMMUNOPATHOGENESISSUMMARY OF IMMUNE MECHANISMS...CONCLUSIONS AND IMPLICATIONS FOR...REFERENCES |
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The immunopathogenesis of viral lung disease varies considerably from one individual to another and is affected by the postnatal age. Severe RSV bronchiolitis occurs only in a small minority of children, is usually transient and self-limiting. However, studies of disease augmentation by prior sensitization are capable of reproducing many features of the severe primary disease seen in susceptible individuals. In the case of RSV disease, it is evident that the severity of the host response is sometimes disproportionate to the threat posed by the pathogen.
A full appreciation of the role of infection in chronic lung disease was not possible before the advent of modern, sensitive methods of viral and bacterial detection. Studies of the mechanisms of inflammation in experimental models demonstrate clearly that the viral load is not necessarily an indicator of disease severity; indeed, low viral load and a strong host immune responses often go together. The difficulty of detecting viral agents in adult lung disease is therefore no indication of the importance of infection. With the introduction of novel antiviral and antibacterial agents, it is more important than ever to discover the role of infections in hitherto unexpected situations.
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FOOTNOTES |
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Conflict of Interest Statement: P.J.M.O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
(Received in original form April 7, 2005; accepted in final form April 27, 2005)
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TOPABSTRACTTHE BURDEN OF RSV...HOST FACTORS AFFECTING...AUGMENTATION OF DISEASE BY...FI-RSV IN MICEDISSECTING IMMUNOPATHOGENESISSUMMARY OF IMMUNE MECHANISMS...CONCLUSIONS AND IMPLICATIONS FOR...REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
