PERSPECTIVE
Epithelial Regulation of Innate Immunity to Respiratory Syncytial Virus

Roberto P. Garofalo and Helene Haeberle

Departments of Pediatrics, and Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas

In the year 2001, we will mark the 45th anniversary of research on respiratory syncytial virus (RSV), the agent that was discovered as the cause of a "nosocomial outbreak" of colds in chimpanzees (1). Despite almost a half century of active clinical and basic investigations, an effective vaccine is still not available, and each year approximately 100,000 infants are hospitalized with RSV disease in the United States only (2). Moreover, the long-term morbidity that is associated with severe RSV infections results in health care burden and costs that are disproportionally higher than those estimated based only on hospitalization costs to treat acute primary infections. RSV vaccines with immunogenic, protective, and nonreactogenic properties are currently under investigation, but a significant obstacle to their development is still our poor understanding of the pathogenetic mechanisms that determine the severity of lower respiratory tract infections caused by the virus. Paradoxically, intense research efforts boosted by the unfortunate experience with the first RSV formalin-inactivated vaccine (3) has significantly contributed to our understanding of the immune-mediated mechanisms responsible for the enhanced disease that occurred in a subset of vaccinated infants, but has incompletely identified the immunopathogenic components in naturally acquired primary infection. Nonetheless, mounting experimental evidence suggests that early inflammatory and immune events characteristic of the "innate" host response may be crucial in determining the outcome of acute RSV infection as well as its long-term consequences (asthma and recurrent wheezing).

The epithelium of the respiratory mucosa, the main function of which is to provide a protective physical barrier against injurious inhaled stimuli, is the main target of RSV. Following inhalation or self-inoculation of the virus into the nasal mucosa and infection of the local respiratory epithelium, RSV spreading along the respiratory tract occurs mainly by cell-to-cell transfer of the virus along the intracytoplasmic bridges (4). A number of molecules that are produced by human epithelial cells as a consequence of RSV infection have been described. Among them are potent immunomodulatory and inflammatory mediators, including cytokines (interleukin [IL]-1, tumor necrosis factor [TNF]-, IL-6, IL-11), chemokines (IL-8, GRO, MCP-1, MIP-1, RANTES), interferons (IFN-/) and growth factors (GM-CSF, G-CSF) (reviewed in [5]). Some of these molecules appear to be selectively expressed only in the lower airway respiratory tract, following RSV infection (6, 7). In addition, type II alveolar epithelial cells are able to produce opsonins such as complement (8) and surfactant proteins (9) responsible for serum-independent phagocytosis of pathogens by neutrophils, monocytes, and macrophages. Therefore, respiratory epithelial cells appear to be ideally located and armed to function as initiators of host defense mechanisms by regulating the prototypic cellular elements of the innate immune system.

In this Perspective we will briefly review some of the components of the innate immune system known to be involved in RSV infection, in particular surfactant protein-A (SP-A), alveolar macrophages, eosinophils, neutrophils, and NK cells, all of which may be significantly affected by the infected respiratory mucosa epithelium.

	SP-A and Alveolar Macrophages

SP-A is a member of the collectin family of mammalian polypeptides and is abundantly expressed by lung epithelial cells (9). Collectins bind complex carbohydrates, thereby acting as opsonins for microorganisms, including bacteria and viruses. For this reason, collectins are considered part of an innate response of the lung to acute injury, where they modulate local inflammatory and immune responses (10). SP-A has also an important function for lung homeostasis, mainly protecting surfactant from inactivation by serum proteins. Alveolar macrophages, a major component of the innate-effector immune system in the lung, are susceptible to RSV, although the infection is abortive after few replication cycles (11). Both in vitro and in vivo studies demonstrate that alveolar macrophages coexpress RSV-specific proteins and human leukocyte-associated antigen-DR (HLA-DR) molecules, thus may serve as effective antigen presenting cells (12) and secrete potent inflammatory and immunomodulatory cytokines (IL-6, TNF-, IL-10) in response to RSV infection (11, 13).

Barr and colleagues present the results of a study in vitro that examines the effect of SP-A on RSV uptake by peripheral blood mononuclear cells (PBMC) and the macrophage-like cell line U937 (14). The authors show that SP-A enhances uptake of fluorescein isothiocyanate (FITC)- labeled RSV by both cell types. As a result of this event, production of TNF- and IL-10 is altered. In both cells, opsonization of RSV by SP-A increases TNF- and decreases IL-10 levels compared to RSV alone. The study confirms work by others demonstrating the important role that airway epithelial collectins play in viral clearance of RSV, both in vitro (15, 16) and in mice with targeted inactivation of the SP-A gene (17) or mice to which recombinant surfactant protein-D was administered intranasally (16). Following RSV infection, viral titers and number of BAL inflammatory cells (mainly neutrophils) are increased in SP-A/ mice compared to SP-A+/+ controls. Although the reason for the impaired viral clearance in SP-A/ mice is not fully understood, it appears that reduced alveolar macrophage oxygen radical production and viral killing might be involved. These results are consistent with the observation of an enhanced RSV phagocytosis, in the presence of SP-A, by human macrophages shown in the study by Barr and coworkers. TNF- has been claimed to have antiviral activity against RSV (18). However, viral clearance is impaired in SP-A/ mice compared to SP-A+/+ animals, despite increased levels of TNF- in lung tissue of SP-A/ mice (17). This finding is also partially in contrast with the increased production of TNF- by RSV/SP-A-exposed monocytes and U937 cells compared to those exposed to nonopsonized RSV reported in Barr's study. An explanation for this discrepancy is that airway epithelium may represent, alone or in combination with macrophages, the source of lung TNF-, IL-6, and MIP-2, all of which were found increased in SP-A deficient mice compared to controls. If this is the case, the mechanism(s) for enhanced cytokine/chemokine production by epithelial cells in absence of SP-A remains to be defined. Nevertheless, the SP-A/ mouse model provides convincing evidence against the hypothesis of an antiviral activity of TNF- in RSV infection.

The observation made by Barr and colleagues that IL-10 production is significantly reduced by monocytes and macrophages incubated with SP-A-opsonized RSV deserves attention also. The role of IL-10 in primary RSV infection is unknown. Studies in progress in our laboratory indicate that IL-10 may function as a potent suppressor of early inflammatory events in RSV-infected mice, without exhibiting antiviral activity (19). On the other hand, the well known immunoregulatory properties of this pleiotropic cytokine should be considered as we interpret the recently reported association of IL-10 with recurrent wheezing in RSV-infected children (20).

	Eosinophils

Although eosinophils have been universally recognized as cells involved only in the innate immune response against helminthic parasites, this view has been challenged by recent studies of RSV pathogenesis. Several groups have shown that during RSV infection eosinophils are recruited to and degranulate into the airway mucosa (21, 22). Significantly higher concentration of eosinophil cationic protein (ECP) has been shown to be present in NPS obtained from subjects affected by bronchiolitis (i.e., wheezing) compared to those with localized upper respiratory tract illness (21); thus, ECP is currently used as a marker of RSV disease severity. RSV-infected respiratory epithelial cells, by secreting a number of chemokines, cytokines, and growth factors, appear to regulate the multistep process of eosinophil transendothelial migration, chemotaxis, and degranulation (reviewed in [5]). Epithelial-derived RANTES, for example, displays strong chemotactic activity for eosinophils in vitro (7, 23). Moreover, eosinophil degranulation can be effectively induced by coculture of eosinophils with RSV-infected lung alveolar epithelial cells, a process mediated by the upregulation of the beta integrin CD11b driven by epithelial-secreted GM-CSF (24). In addition, by providing an appropriate milieu of inflammatory mediators, RSV-infected epithelial cells can also activate eosinophils and uninfected epithelial cells, thus triggering further eosinophil degranulation (24). These observations have important implication to understand "early" immunity as well as proinflammatory/pathogenetic events that follow RSV infection of epithelial cells. Indeed, the killing and eradication of infected cells by cytotoxic eosinophil products may positively impact the outcome of RSV-infection. On the other hand, eosinophil-mediated cytotoxicity against bystander uninfected epithelial cells may significantly contribute to the enhanced pathology observed in certain RSV infections.

Similar considerations should be made when trying to understand another aspect of the relationship between RSV infection and eosinophils: the direct interaction of the virus with eosinophils. Our laboratory has described that culture of highly purified preparations of blood eosinophils with RSV (A strain) results in expression of RSV F glycoprotein in the cells, as detected by typical intracytoplasmic granular fluorescence immunoreactivity (7). Moreover, RSV viral particles were seen by transmission electron microscopy in phagocytic vacuoles at the periphery of eosinophil cytoplasm (25). These studies suggest that RSV is able to infect human eosinophils, although the productive or abortive nature of this infection, as well as the potential receptor or receptor-like structure used for viral entry, remains to be determined. By infecting eosinophils, RSV is able to induce the production of oxygen radicals, to prime eosinophils for enhanced superoxyde generation and leukotriene C4 release, and to trigger intracellular signaling and transcriptional events that lead to the production of the chemokines RANTES and MIP-1 (7, 26). On the other hand, a recent study has shown that eosinophils can mediate the destruction of RSV virions in vitro, an effect that directly depends on the action of the secreted ribonuclease, eosinophil-derived neurotoxin (EDN) (27). Therefore, in RSV infection eosinophils may function as a prototypic cell of the innate immune system, capable of uptake and phagocytosis of virions, antigen processing, and presentation to specific T cells receptors (28), as well as direct viral inactivation. However, as discussed for eosinophil-mediated killing of infected epithelial cells, this process may result in detrimental inflammatory damage to the airway mucosa.

	Neutrophils

Early after RSV infection, neutrophils are a sizable component of the inflammatory response in the airway mucosa, both in infants and in animal models (29, 30). Epithelial-derived IL-8 is the main neutrophil chemoattractant secreted after RSV infection (5). Studies in vitro have shown that neutrophils avidly adhere to RSV-infected epithelial cells (31). This process appears to be dependent on epithelial ICAM-1, the expression of which is mediated via the autocrine effect of RSV-induced IL-1 (32), and the granulocyte CD18 integrin (33). As a result of this interaction, effector functions of neutrophils are activated, resulting in cytotoxicity against epithelial cells (34). Neutrophil-mediated epithelial cell damage is further enhanced in the presence of activated components of the complement pathway (35). Although serum is the major source of complement, epithelial cells are able to secrete complement proteins (8). In this regard, RSV strongly induces release of the C3 component by infected type II epithelial cells (R. P. Garofalo, personal observation). Direct stimulatory effect of RSV on neutrophils has also been reported. Following in vitro exposure to RSV, peripheral blood neutrophils secrete the chemokines IL-8, MIP-1, MIP-1, and granule-associated myeloperoxidase (MPO) (36). This observation is supported by studies in children with RSV infection, where higher levels of MPO and neutrophil elastase in nasopharyngeal secretions have been demonstrated in subjects with bronchiolitis compared with other milder forms of disease (37, 38). In the BALB/c mouse model, early objective evidence of respiratory illness (increased respiratory rate) occurs at Day 1-2 following RSV infection, concurrently with a sharp increase in percentage of neutrohilic granulocytes in BAL fluids and when a T cell-specific immune response has not yet developed (30).

	Natural Killer Cells

Natural killer cells (NK) cells are important effectors in the innate immune response and play a key role in the first line of host defense against viral infections. NK cells mediate significant levels of cytotoxicity that is activated whenever viral interferon (IFN-/) is induced. Thus, early after a viral infection, cytokines produced by cells of the innate immune system and/or nonimmune cells can elicit responses mediated by NK cells. Other key cytokines produced in the course of viral infection known to activate NK cell function, including cytotoxicity, proliferation, and migration are IL-12, IL-15, interferon--inducing factor, and the chemokines, MIP-1, MIP-1, MCP-1,2,3, and RANTES (39). The role of NK cells in the context of RSV infection is not fully known. Infection of airway epithelial cells by RSV is a potent stimulus for production of IFN-, followed by autocrine upregulation of MHC-I molecules by these cells (40). Therefore, epithelial-derived IFN- has the potential to mediate immunoregulatory functions affecting both innate and adaptive responses to RSV infection. Studies in mice have shown that the majority of lymphocytes recovered from BAL fluids during the first days of primary RSV infection have phenotypic characteristics of NK cells (41), and NK activity peaks around this time (42). Recently, investigations have been performed in the mouse model using a cold-adapted temperature-sensitive strain of RSV that has been evaluated as an RSV vaccine candidate (strain CP52). CP52 has a deletion of both the viral SH and G genes, and was compared with the parent strain (B1). The results of this important study show that the absence of the G and SH protein in CP52 markedly increases the number of BAL NK cells after both primary and secondary infections, compared to the B1 strain (43). We must note that, in mouse models of vaccine-enhanced disease, the RSV G protein has been linked to pulmonary eosinophilia, enhanced disease, and increased expression of Th2 cytokines (44). Therefore, it appears that the G protein and/or the SH protein inhibits trafficing of NK cells to the lung: the mechanism of this inhibition is currently unknown, but as suggested by the authors of this study it may explained by a possible effect of these proteins on the profile or magnitude of chemokines produced in the inflammatory response to RSV. Among them, MIP-1 is of particular interest since it induces focal NK tissue migration (39) and is produced in vivo during RSV infection and in culture by infected lung epithelial cells (7). Mice with targeted deletion of the MIP-1 gene show a striking reduction of focal inflammation in response to RSV infection, although viral peak titers are not affected (45).

	Concluding Remarks

Infection of respiratory epithelial cells is the first event occurring after RSV in inhalation or inoculation into the nasal mucosa. This is rapidly followed by the induction of a network of epithelial cell gene products that have profound immune and inflammatory regulatory functions. The cascade of subsequent events involves mediators and cellular elements that belong to the innate immune system, including interferons, chemokines, surfactanct proteins, phagocytes, and NK cells. These early elements of the host response to RSV are major determinants of the elimination or the progression of the infection, significantly affect airway mucosa inflammation, and ultimately may dictate the nature of the specific adaptive immune response to the virus (Figure 1). Although significant achievements have been made in our understanding of the players involved in RSV immunopathology, three major areas remain a challenge for future research: (1) the biological characterization of virus-specific gene products that mediate airway inflammation/pathology; (2) the dissection of the role of epithelial chemokines in orchestrating innate and adaptive immune responses to RSV; and (3) the identification of host-specific and genetically-determined signaling pathways that regulate gene expression in respiratory epithelial cells (46). Progress in these areas will be fundamental to achieve the goals of a rationale design of both protective, nonreactogenic mucosal RSV vaccines and novel pharmacologic strategies that could be used to specifically target the airway epithelium early after infection. Ultimately, interfering with the harmful component of the airway inflammatory response may lead to a substantial reduction in the development of RSV-induced asthma and recurrent wheezing.

View larger version (39K): [in this window] [in a new window]   Figure 1.   A schematic representation of immunomodulatory mediators produced by RSV-infected epithelium that regulate the function of cells of the innate immune system. Further, IFN-/ and IL-1 upregulate epithelial expression of mayor histocompatibility complex I (MHC-I) and intercellular adhesion molecule-1 (ICAM-1), respectively. These elements determine elimination of the virus and/or inflammation and tissue damage of the airway mucosa. Third component of complement, C3; surfactant protein-A, SP-A.

	Footnotes

Address correspondence to: Roberto P. Garofalo, M.D., Department of Pediatrics, Division of Immunology/Allergy/Rheumatology, 301 University Blvd., Galveston, TX 77555-0369. E-mail: rpgarofa{at}utmb.edu

(Received in original form October 11, 2000).

Acknowledgments: This work was supported in part by grants AI 15939 and P01 AI46004-01 from the National Institutes of Health and by grant 644-0-0 of the Fortune Program of the University of Tuebingen, Germany. The authors thank Dr. Louis Bont and Dr. Antonella Casola for the helpful discussions.

Abbreviations ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; FITC, fluorescein isothiocyanate; HLA-DR, human leukocyte- associated antigen-DR; IL, interleukin; MPO, myeloperoxidase; NK, natural killer; PBMC, peripheral blood mononuclear cells; RSV, respiratory syncytial virus; SP-A, surfactant protein-A; TNF, tumor necrosis factor.

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