Role for Innate IFNs in Determining Respiratory Syncytial Virus Immunopathology 1
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免疫学杂志 2005年第11期
Abstract
Respiratory syncytial virus (RSV) is the major cause of severe lower airway disease in infa nts and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccine development were recognized in an early vaccine trial, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection. Subsequent research in animal models has shown that the vaccine-enhanced disease is mediated at least in part by memory cells producing Th2 cytokines. Previously we had observed enhanced, eosinophilic lung pathology during primary infection of IFN-deficient STAT1–/– mice that are incapable of generating Th1 CD4+ cells. To determine whether these effects depended only on Th2 cytokine secretion or involved other aspects of IFN signaling, we infected a series of 129SvEv knockout mice lacking the IFN-R (IFN-R–/–), the IFN-R (IFN-R–/–), or both receptors (IFN-R–/–). Although both the IFN-R–/– and the IFN-R–/– animals generated strong Th2 responses to RSV-F protein epitopes, predominantly eosinophilic lung disease was limited to mice lacking both IFNRs. Although the absolute numbers of eosinophils in BAL fluids were similar between the strains, very few CD8+ T cells could be detected in lungs of IFN-R–/– animals, leaving eosinophils as the predominant leukocyte. Thus, although CD4+ Th2 cell differentiation is necessary for the development of allergic-type inflammation after infection and appears to be unaffected by type I IFNs, innate IFNs clearly have an important role in determining the nature and severity of RSV disease.
Introduction
Respiratory syncytial virus (RSV) 3 is the major cause of severe lower airway disease in infants and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccination became evident in early vaccine trials, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection (1, 2). Subsequent studies in animal models have shown that unlike natural infection, formalin-inactivated RSV priming leads to the development of a Th2 memory response, which is thought to mediate the immunopathology seen after virus challenge (3, 4, 5). This is of particular concern in light of recent studies showing a link between severe RSV disease in childhood and the development of asthma later in life (6). To test this hypothesis, in previous studies we examined whether mice lacking the ability to develop a Th1 response would develop eosinophilic airway disease after primary RSV infection (7). For that purpose we studied mice lacking STAT1 (8), the major mediator of IFN- and IFN- signaling. Th1 differentiation of naive CD4+ cells depends on the presence of the transcription factor T-bet, the induction of which is STAT1 dependent (9, 10). In fact, RSV-infected STAT1–/– mice did develop Th2 cytokine responses and severe eosinophilic lung disease without priming, and this correlated with high levels of IL-13 production by virus-specific CD4+ T cells.
Although these results clearly demonstrated the link between the development of Th2 cells and enhanced disease, we were struck by the fact that IFN-–/– animals studied in parallel with STAT1–/– mice were much less affected. This result was unanticipated, because we had assumed that mice lacking IFN- would also default to a Th2 CD4+ T cell differentiation pathway. We concluded that although CD4+ T cells producing Th2 cytokines were necessary for an allergic-type, eosinophilic response to develop, other factors could modulate this effect. Because STAT1 mediates signaling through both the IFN- and IFN- receptors (11), we wondered whether type I () IFNs were acting to diminish the pathology seen in STAT1 animals. We hypothesized that either IFN- was affecting CD4+ T cell differentiation, or other effects of type I IFN were acting to limit eosinophilic disease.
For a more rigorous approach to these questions, we have compared the innate and adaptive responses to acute RSV infection in a series of 129SvEv knockout mice lacking the IFN-R, the IFN-R, both receptors, or STAT1–/–. In this study we have observed that both mouse strains lacking the IFN-R produce virus-specific Th2 lymphocytes in response to RSV infection regardless of IFN- signaling. Nonetheless, significant differences remain between mice lacking both IFNRs and mice that retain the IFN-R. As found previously in the BALB/c mouse, inflammatory infiltrates from infected mice deficient in IFN- signaling remained primarily lymphocytic, whereas bronchoalveolar lavage (BAL) specimens from IFN-R–/– (IFN-R–/–) mice consisted primarily of eosinophils, with a notable lack of CD8+ T cells. The degree of eosinophilia seen in the lungs of IFN-R–/– mice therefore reflects a paucity of responding CTLs as well as a large number of eosinophils presumably elicited by IL-5- and IL-13-producing CD4+ cells. This study demonstrates that although Th2 cytokines are essential for the development of an allergic-type inflammatory response, they are not the sole determinant of virally induced immune pathogenesis.
Materials and Methods
Statistical analysis
Data are expressed as the mean ± SEM. Comparisons between groups were based on ANOVA, calculated using SigmaStat software (SAS Institute). Dunnett’s procedure, which adjusts for multiple comparisons against a predefined group, was applied only when there was a significant overall difference between groups. A value of p < 0.05 was considered statistically significant.
Results
Control of RSV replication is STAT1 dependent
We have previously observed a strain-dependent increase in virus production after i.n. infection of STAT1-deficient mice (7). In the current study we examined the relative RSV susceptibilities of additional strains of knockout animals (wild-type, IFN-R–/–, IFN-R–/–, IFN-R–/–, and STAT1–/–), all on a 129SvEv background. Groups of 10 animals from each genotype were inoculated i.n. with 1 x 107 PFU of RSV. The titers shown in Fig. 1 are derived from lungs taken on day 5 after infection, the peak of virus production, and on day 8 when virus would normally be cleared. In vivo, both IFN- and IFN- are induced by virus infection, albeit with somewhat different kinetics, and we expected that either cytokine alone would induce an antiviral state (17). This prediction was borne out; mice lacking either the IFN-R or the IFN-R demonstrated titers similar to those in wild-type mice, although titers from STAT1–/– animals were increased 100-fold. By day 8 after infection, no virus could be detected in lungs of any strain (data not shown). The surprising finding was that mice unable to respond to either type of IFN showed no detectable increase in virus production relative to wild-type controls. This nonequivalence of the IFN-R–/– and STAT1–/– mutations was not entirely unexpected, because STAT1 is essential for many, but not all, IFN signaling pathways (8, 18). What was surprising, and unlike other viruses, was the relative STAT1 dependence and IFN independence of RSV replication in vivo (19).
Altered inflammatory responses in lungs of IFN-deficient mice
Along with the increased viral load there was an exacerbated inflammatory response in the lungs of STAT1–/– mice (Fig. 2). There were no significant differences in the numbers of BAL leukocytes recovered from wild-type, IFN-R–/–, IFN-R–/–, or IFN-R–/– mice on days 5 and 8 after infection. However, samples from STAT1–/– mice showed 15- and 5-fold greater numbers of inflammatory cells at 5 and 8 days after infection, respectively (p < 0.001 on days 5 and 8). Although we have also found an enhanced inflammatory response to RSV by STAT1–/– mice on the BALB/c background, where differences in viral titers are much smaller (7), the 100-fold increase in virus found in the STAT1–/– 129SvEv animals (Fig. 1) undoubtedly influences many immune parameters. Because we wanted to determine the effects of IFN, rather than virus load, on the outcome of RSV infection, we omitted STAT1–/– mice from the following studies and focused instead on comparing the responses of mice lacking either or both IFNRs to those of strain-matched controls.
We had previously described pronounced eosinophilic lung disease after primary RSV infection of STAT1–/– and IFN-R–/–, but not IFN-R–/–, IFN-R–/–, or IFN-–/–, mice, as judged by microscopic examination of infected tissues (7). For a more quantitative picture, we collected BALs from mice of each genotype at 5 days (Fig. 3A) and 8 days (Fig. 3B) after i.n. RSV inoculation and performed differential cell counts on Wright-Giemsa-stained cytospins made from individual animals (four to six mice per group). Lavage fluids obtained from wild-type mice consisted almost entirely of monocytes/macrophages and lymphocytes, with the proportion of lymphocytes increasing by day 8 when tissue inflammation was well developed. In IFN-R–/–, IFN-R–/–, and IFN-R–/– mice, neutrophils were present on day 5, but decreased to near background levels by day 8. Eosinophils were present only in the lungs of IFN-R–/– and IFN-R–/– animals. In the double-receptor knockout mice, eosinophils appeared earlier and consistently made up a significantly larger proportion of the total infiltrate. These differences were consistent over three similar experiments, one of which is pictured in Fig. 3, A and B. When these data were analyzed by ANOVA, all three experiments (Fig. 3C) showed significant differences between the IFN-R–/– and IFN-R–/– strains (p < 0.05). Another significant and consistent difference among the different genotypes was the relative dearth of lung lymphocytes in IFN-R–/– animals on day 8 (p < 0.05), shown in Fig. 3B.
Photomicrographs corresponding to these BAL data are shown in Fig. 4. H&E-stained lung sections from infected wild-type (A and D), IFN-R–/– (B and E), and IFN-R–/– (C and F) mice 8 days after inoculation are pictured. As predicted from the equivalent BAL cell numbers (Fig. 2), the overall extent of inflammation was similar in all four strains. This can be seen at the lower (x200) magnification. All sections show pronounced perivascular infiltrates with foci of peribronchial, peribronchiolar, and alveolar inflammation. There was little evidence of cytopathic effect or epithelial cell necrosis. Appreciable differences between strains were apparent only at higher (x400) magnification, where the very different characters of the infiltrates are evident. Consistent with the data in Fig. 3, the inflammatory process is entirely lymphocytic in wild-type mice (Fig. 4D) and predominantly lymphocytic in IFN-R–/– mice (Fig. 4E), but is overwhelmingly eosinophilic in IFN-R–/– animals (Fig. 4F). This relates to both the relative increase in eosinophils and the relative decrease in lymphocytes seen in the double-knockout animals. Lung sections from IFN-R–/– mice (not pictured in this figure) were similar to those from wild-type mice, but with an overall decrease in lymphocytic inflammation.
On day 5 after infection, the total number of lymphocytes present in the lungs of all animals was quite small (data not shown), but by day 8 we observed a large expansion of T cells in wild-type and IFN-R–/– mice. This increase was primarily due to the expanding CD8+ cell population, as calculated from cell counts, BAL differentials, and flow cytometry data (Fig. 5A). Interestingly, this expansion was very limited in mice lacking the IFN-R, although the difference in CD8+ T cell number was statistically significant (p < 0.05) only for the IFN-R–/– animals. This observation is consistent with data from other laboratories showing a role for type I IFNs in the activation and survival of CTLs (20, 21). There were no significant differences in numbers of BAL CD4+ T cells among strains. Fig. 5B shows the number of eosinophils present in BALs taken from each strain on day 8 postinfection. These were very similar in IFN-R–/– and IFN-R–/– animals, leading us to conclude that the eosinophilic predominance in IFN-R–/– mice reflects primarily a lack of CD8+ T cells.
CD4+ T cell differentiation in IFN-deficient knockout mice
Having determined that there were significant qualitative and quantitative differences between RSV lung disease in IFN-R–/– and IFN-R–/– mice, we sought to determine the role of IFNs in CD4+ T cell differentiation. It has been established that STAT1-mediated induction of the transcription factor T-bet is required for the development of Th1 lymphocytes (10). Consistent with this finding, we have observed Th2 cytokine production by STAT1-deficient CD4+ T cells responding to an RSV-F protein epitope that stimulates primarily IFN- in wild-type mice (7). Although a large body of evidence demonstrates the importance of IFN- in Th1 differentiation in vitro, in vivo studies using mice deficient in IFN- signaling have not shown a clear Th2 predisposition (22, 23, 24). In light of the uncertainty regarding the role of IFN- in Th1 differentiation, and with the differing disease patterns observed in IFN-–/– or IFN-R–/– and STAT1–/– mice infected with RSV (7) or influenza (25), it was important to learn whether virus-specific CD4+ T cells from RSV-immune IFN-R–/–, IFN-R–/–, and IFN-R–/– mice were primarily Th1 or Th2.
To perform this experiment in the 129SvEv animals, a dominant viral epitope recognized by RSV-immune CD4+ lymphocytes in this strain was required. Overlapping peptides corresponding to the RSV-F protein sequence were mixed to make five pools of 10 peptides and one pool of seven peptides, and these peptide pools were assayed for their ability to stimulate the proliferation of immune splenocytes. Because pool 1 stimulated the most robust proliferation response (data not shown), we next screened peptides 1–10 individually (Fig. 6). From this secondary screen, peptide 9, corresponding to aa 81–100 (QELDKYKNAVTELQLLMQNT), was chosen for additional characterization. An IFN- ELISPOT assay performed with undepleted or CD4+ T cell-depleted immune splenocytes demonstrated that CD4+ T cells were required for an IFN- response to this peptide. Undepleted cultures produced an average of 85 spots/106 splenocytes compared with an average of 1 spot/106 splenocytes in CD4+ T cell-depleted samples. Depletion of CD8+ T cells had no effect on IFN- production (data not shown).
Cytokine production by immune CD4+ T cells from mice of each genotype was assessed by quantitation of cytokine gene transcripts after peptide stimulation of cultured splenocytes. Cells were harvested from two or three mice of each strain that had been inoculated i.n. with 106 PFU of RSV 28 days previously. After 7 days of culture with peptide 9, the splenocytes were washed and restimulated for a 6-h period before harvesting. Equal amounts of RNA from each sample were analyzed in a multi-probe RNAP, and signals were quantitated using the Storm PhosphorImager and ImageQuant software. Relative levels of cytokine transcripts made by each strain are shown in Fig. 7. We concluded from this set of experiments that although RSV does not appear to generate a strong Th1 response to F protein in the wild-type mouse, mice lacking the IFN-R alone or lacking both IFNRs default to a Th2 differentiation pathway in our system.
Role of IFNs in lung cytokine and chemokine production
The earliest response to virus is initiated by infected lung epithelial cells. A number of cytokines and chemokines are induced by RSV (26, 27, 28), and these inflammatory mediators directly affect the trafficking of lymphocytes and granulocytes to the site of infection. Chemokines promoting both Th1 and Th2 cell migration are produced by the infected epithelium in vitro, but in vivo, their synthesis is also affected by the presence of other cytokines. Many of the chemokines attracting Th1 cells (IFN-inducible T cell chemoattractant (I-TAC), IP-10, monokine induced by IFN- (MIG)) are induced by IFNs (29, 30, 31, 32), and IL-13 is known to increase the synthesis of eotaxin (33, 34), a major eosinophil attractant. We wondered whether the altered inflammatory responses in the absence of IFN- and/or IFN- signaling correlated with an altered chemokine profile. To answer this question, BAL supernatants from infected mice were collected 5 days after i.n. inoculation with 107 PFU of RSV and were assayed for the presence of IP-10. IP-10 was chosen because it can be induced by either type I or type II IFNs (29, 32). Fig. 8 shows the data obtained for both eotaxin and IP-10 assayed at two time points in all four strains of mice. Although no IP-10 could be detected in mice lacking both IFNRs (IFN-R–/–), mice retaining either the IFN-R ((IFN-R–/–) or the IFN-R (IFN-R–/–) continued to produce IP-10. Eotaxin was induced in all strains, but eosinophilia occurred only in those lacking virus-specific Th1 cells, that is, the IFN-R–/– and IFN-R–/– mice. In addition, eotaxin levels remained significantly elevated on day 8 only in mice lacking both IFNRs (p < 0.001).
These same BAL supernatants from each animal were also assayed for the presence of Th2 cytokines. Levels of IFN-, IL-5, and IL-13 in lavage samples collected at 5 and 8 days after inoculation were determined by ELISA or by the Luminex100 system (Fig. 9). Although there were increased levels of IL-5 and IL-13 in lungs of IFN-R–/– and IFN-R–/– mice, these differences were statistically significant only for IL-5. IFN- levels were similarly high for all strains on day 5, probably reflecting production by NK as well as T cells. By day 8, these levels had dropped, most precipitously in the IFN-R–/– animals. Thus, it appears that chemokine synthesis is affected by early IFN production, and this suggests that chemokine levels may be influenced by the presence of Th2 lymphocytes.
Discussion
This conclusion was inconsistent with the absence of this pathology in infected mice lacking IFN-, a cytokine known to activate STAT1 and drive Th1 differentiation of naive CD4+ T cells. In vivo experiments to determine whether IFN- itself is essential for Th1 development have been complicated by the importance of IFN- in establishing IL-12 responsiveness. Current models of naive CD4+ cell activation postulate that STAT1 activation is required for Th1 commitment, but that the STAT4-mediated IL-12 signal is still necessary for IFN- production in response to Ag (10, 38). Because IFN- up-regulates IL-12R expression, a robust Th1 response should require IFN-, and we would expect mice lacking this cytokine to mount a Th2 response to virus infection. Nonetheless, in our experiments with either IFN-–/– BALB/c or 129SvEv IFN-R–/– mice, eosinophils were present in infiltrates surrounding vessels and airways of infected lungs, but only at low levels. This observation has been made by others (24, 39), who concluded, as we did, that RSV pathogenesis in IFN--deficient animals is similar to that in wild-type animals. We therefore wondered whether, in the absence of IFN-, IFN- could act in a compensatory manner to inhibit Th2 lymphocyte development and eosinophilic disease (7). To test this hypothesis we needed to determine 1) whether Th2 differentiation was blunted in IFN-R–/– mice, and 2) whether mice lacking both IFNRs (IFN-R–/–) responded differently from those lacking one or the other.
We approached the question of T cell differentiation in infected IFN--deficient mice by comparing cytokine responses of four mouse strains to a single MHC class II restricted viral peptide. The peptide used (F9), corresponding to aa 81–100 of the RSV-F protein, specifically stimulated the proliferation of and IFN- production by CD4+ T cells from RSV-immune wild-type 129SvEv mice. Using an RNAP to survey simultaneously for Th1 and Th2 cytokine production, we observed that although peptide-stimulated T cells from IFN-R–/– and IFN-R–/– animals produced levels of IFN- transcript similar to those in wild-type animals, IL-13 production was increased 10- to 20-fold (Fig. 7). The elevated amounts of IL-13 as well as IL-5 induced by peptide F9 in the absence of the IFN-R demonstrated that the IFN- pathway is essential for inhibiting the Th2 response to RSV infection.
Having found that both the IFN-R–/– and the IFN-R–/– animals default to a Th2 lymphocyte response after infection, an alternate explanation for the significant differences in lung pathology between these animals was required. Characterization of BAL samples from mice of each genotype at two time points suggested that the major difference between these strains was not an increase in eosinophils, but, rather, an absence of CD8+ lymphocytes in both strains of mice lacking the IFN-R–/–. In wild-type and IFN-R–/– mice, there was a large expansion in the number of lymphocytes present in the lungs by day 8 after inoculation. In IFN-R–/– animals, this expansion occurred in the presence of eosinophils and neutrophils (Fig. 3). In contrast, mice lacking both IFNRs showed little increase in lymphocyte numbers as the infection progressed, and eosinophils were the predominant cell type on day 8. This pattern was seen consistently in three replicate experiments and was significant in each (p < 0.05). Evaluation of tissue sections showed a similar picture (Fig. 4), with largely lymphocytic infiltrates in infected IFN-R–/– mice and predominantly eosinophilic inflammation in lungs of IFN-R–/– animals.
We have observed that Th2 cytokine production by CD4+ T cells is necessary, but not sufficient, for the development of eosinophilic lung inflammation after RSV infection. Other factors contribute to pulmonary eosinophilia during primary infection in mice. It has been shown that a large number of genes are induced by RSV infection of respiratory epithelial cells; among these are chemokines that regulate inflammatory cell trafficking (26, 27, 40, 41). A number of innate immune mediators will impact relative levels of chemokine production by these cells (31), and among these are the IFNs. Both IFN- and IFN- are produced early in infection: IFN- by the virus-infected cells and innate IFN- by activated NK cells (17). A number of the chemokines responsible for Th1 and CTL trafficking, among them the CXCR3 ligands I-TAC, MIG, and IP-10, are IFN-inducible genes (31, 32, 41). We wondered whether IP-10, which is known to be induced by both type I and type II IFNs, would be detectable in mice lacking either or both IFNRs and whether differences in chemokine production could be contributing to the differences in pathology. As shown in Fig. 9, IP-10 was detectable in BALs from all animals on day 5, except in those lacking both IFNRs. Eotaxin, another RSV-induced chemokine, is a chemoattractant for activated Th2 cells and eosinophils (42). Although eotaxin was present in BALs from mice of all genotypes at early times, levels were significantly increased only in IFN-R–/– mice on day 8 (p < 0.001; Fig. 9A). Allergic inflammation is a self-amplifying process, and the elevated eotaxin levels in animals with eosinophilic disease may reflect this situation. If activated Th2 cells and eosinophils secreting IL-13 (43) traffic to the lung, increasing IL-13 concentrations will up-regulate eotaxin production by infected epithelium (34) (see Fig. 10).
In summary, our data suggest that although Th2 lymphocytes are necessary for the development of an eosinophilic response to primary RSV infection, they are not sufficient. In this system it is the IFN- and IFN- produced during the innate immune response, rather than lymphocyte IFN-, that determines disease outcome. RSV lung pathology in IFN-R–/– animals with an intact type I IFN signaling pathway is similar to that seen in wild-type controls. CD8+ T cells, rather than eosinophils, continue to dominate the process. In the absence of both IFNRs, disease is markedly eosinophilic and without the usual expansion of the lung lymphocyte compartment (44, 45). This is consistent with a report from Hussell et al. (46) showing a correlation between the magnitude of the CD8+ T cell response and the propensity of a mouse strain to develop eosinophilia. Therefore, the elevated eotaxin levels, the absence of IP-10 induction, and the lack of CTLs in IFN-R–/– animals work together to create differences between two strains of animals that both generate Th2 responses. Fig. 10 depicts a model showing how the summation of these influences will determine the composition of the inflammatory infiltrate.
We propose that innate IFN- and - affect local chemokine production by increasing levels of the Th1 and Tc1 chemoattractants (I-TAC, IP-10, and MIG) as well as stimulating CD8+ T cell proliferation and activation. An important role for IFN- in CTL survival and IFN- production has also been shown in other systems (20, 21, 47). If virus-specific Th1 cells and CTLs are present, they will be drawn to the site of infection and augment local IFN- production. If activated Th2 lymphocytes are present, they will preferentially accumulate in response to eotaxin, increasing local IL-13 and eotaxin production and thereby amplifying an allergic-type inflammatory response. In animals lacking the IFN-R–/–, it appears that the presence of type I IFNs does not block eosinophil accumulation, but can still inhibit a primarily allergic response by promoting CD8+ T cell accumulation. It has been suggested that in addition to their roles as CXCR3 agonists, I-TAC, IP-10, and MIG may also act to block Th2 cell and eosinophil accumulation by binding the CCR3 expressed by those cell types and acting as antagonists (48). If this is a biologically significant process, this antagonism could also play a role in shaping the inflammatory response. Overall, our findings in the mouse model suggest that although the link between RSV and asthma (6) is probably due to the ability of this virus to promote Th2 lymphocyte differentiation, it may be exacerbated by the limited type I IFN induction by RSV infection in vivo (49, 50).
Acknowledgments
We thank Michel Aguet and Bob Schreiber for providing IFN-R–/–, IFN-–/–, and IFN-R–/– mice; Tom Hamilton for anti-IP-10 antiserum; and Philip R. Johnson for RSV F protein peptides. We also thank Chris Walker, Naglaa Shoukry, Andrew Cawthon, and Christine Biron for helpful discussions, and Karen Watkins for manuscript preparation.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grant AI47226 from the National Institutes of Health (to J.E.D.).
2 Address correspondence and reprint requests to Dr. Joan E. Durbin, Department of Pediatrics, Ohio State University College of Medicine and Public Health, Columbus Children’s Research Institute, 700 Children’s Drive, Room WA4014, Columbus, OH 43205-2696. E-mail address: durbinj{at}pediatrics.ohio-state.edu
3 Abbreviations used in this paper: RSV, respiratory syncytial virus; BAL, bronchoalveolar lavage; i.n., intranasally; IP-10, IFN-induced protein 10; I-TAC, IFN-induced T cell chemoattractant; MIG, monokine induced by IFN-; RNAP, RNase protection assay.
Received for publication December 3, 2004. Accepted for publication March 24, 2005.
References
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Respiratory syncytial virus (RSV) is the major cause of severe lower airway disease in infa nts and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccine development were recognized in an early vaccine trial, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection. Subsequent research in animal models has shown that the vaccine-enhanced disease is mediated at least in part by memory cells producing Th2 cytokines. Previously we had observed enhanced, eosinophilic lung pathology during primary infection of IFN-deficient STAT1–/– mice that are incapable of generating Th1 CD4+ cells. To determine whether these effects depended only on Th2 cytokine secretion or involved other aspects of IFN signaling, we infected a series of 129SvEv knockout mice lacking the IFN-R (IFN-R–/–), the IFN-R (IFN-R–/–), or both receptors (IFN-R–/–). Although both the IFN-R–/– and the IFN-R–/– animals generated strong Th2 responses to RSV-F protein epitopes, predominantly eosinophilic lung disease was limited to mice lacking both IFNRs. Although the absolute numbers of eosinophils in BAL fluids were similar between the strains, very few CD8+ T cells could be detected in lungs of IFN-R–/– animals, leaving eosinophils as the predominant leukocyte. Thus, although CD4+ Th2 cell differentiation is necessary for the development of allergic-type inflammation after infection and appears to be unaffected by type I IFNs, innate IFNs clearly have an important role in determining the nature and severity of RSV disease.
Introduction
Respiratory syncytial virus (RSV) 3 is the major cause of severe lower airway disease in infants and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccination became evident in early vaccine trials, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection (1, 2). Subsequent studies in animal models have shown that unlike natural infection, formalin-inactivated RSV priming leads to the development of a Th2 memory response, which is thought to mediate the immunopathology seen after virus challenge (3, 4, 5). This is of particular concern in light of recent studies showing a link between severe RSV disease in childhood and the development of asthma later in life (6). To test this hypothesis, in previous studies we examined whether mice lacking the ability to develop a Th1 response would develop eosinophilic airway disease after primary RSV infection (7). For that purpose we studied mice lacking STAT1 (8), the major mediator of IFN- and IFN- signaling. Th1 differentiation of naive CD4+ cells depends on the presence of the transcription factor T-bet, the induction of which is STAT1 dependent (9, 10). In fact, RSV-infected STAT1–/– mice did develop Th2 cytokine responses and severe eosinophilic lung disease without priming, and this correlated with high levels of IL-13 production by virus-specific CD4+ T cells.
Although these results clearly demonstrated the link between the development of Th2 cells and enhanced disease, we were struck by the fact that IFN-–/– animals studied in parallel with STAT1–/– mice were much less affected. This result was unanticipated, because we had assumed that mice lacking IFN- would also default to a Th2 CD4+ T cell differentiation pathway. We concluded that although CD4+ T cells producing Th2 cytokines were necessary for an allergic-type, eosinophilic response to develop, other factors could modulate this effect. Because STAT1 mediates signaling through both the IFN- and IFN- receptors (11), we wondered whether type I () IFNs were acting to diminish the pathology seen in STAT1 animals. We hypothesized that either IFN- was affecting CD4+ T cell differentiation, or other effects of type I IFN were acting to limit eosinophilic disease.
For a more rigorous approach to these questions, we have compared the innate and adaptive responses to acute RSV infection in a series of 129SvEv knockout mice lacking the IFN-R, the IFN-R, both receptors, or STAT1–/–. In this study we have observed that both mouse strains lacking the IFN-R produce virus-specific Th2 lymphocytes in response to RSV infection regardless of IFN- signaling. Nonetheless, significant differences remain between mice lacking both IFNRs and mice that retain the IFN-R. As found previously in the BALB/c mouse, inflammatory infiltrates from infected mice deficient in IFN- signaling remained primarily lymphocytic, whereas bronchoalveolar lavage (BAL) specimens from IFN-R–/– (IFN-R–/–) mice consisted primarily of eosinophils, with a notable lack of CD8+ T cells. The degree of eosinophilia seen in the lungs of IFN-R–/– mice therefore reflects a paucity of responding CTLs as well as a large number of eosinophils presumably elicited by IL-5- and IL-13-producing CD4+ cells. This study demonstrates that although Th2 cytokines are essential for the development of an allergic-type inflammatory response, they are not the sole determinant of virally induced immune pathogenesis.
Materials and Methods
Statistical analysis
Data are expressed as the mean ± SEM. Comparisons between groups were based on ANOVA, calculated using SigmaStat software (SAS Institute). Dunnett’s procedure, which adjusts for multiple comparisons against a predefined group, was applied only when there was a significant overall difference between groups. A value of p < 0.05 was considered statistically significant.
Results
Control of RSV replication is STAT1 dependent
We have previously observed a strain-dependent increase in virus production after i.n. infection of STAT1-deficient mice (7). In the current study we examined the relative RSV susceptibilities of additional strains of knockout animals (wild-type, IFN-R–/–, IFN-R–/–, IFN-R–/–, and STAT1–/–), all on a 129SvEv background. Groups of 10 animals from each genotype were inoculated i.n. with 1 x 107 PFU of RSV. The titers shown in Fig. 1 are derived from lungs taken on day 5 after infection, the peak of virus production, and on day 8 when virus would normally be cleared. In vivo, both IFN- and IFN- are induced by virus infection, albeit with somewhat different kinetics, and we expected that either cytokine alone would induce an antiviral state (17). This prediction was borne out; mice lacking either the IFN-R or the IFN-R demonstrated titers similar to those in wild-type mice, although titers from STAT1–/– animals were increased 100-fold. By day 8 after infection, no virus could be detected in lungs of any strain (data not shown). The surprising finding was that mice unable to respond to either type of IFN showed no detectable increase in virus production relative to wild-type controls. This nonequivalence of the IFN-R–/– and STAT1–/– mutations was not entirely unexpected, because STAT1 is essential for many, but not all, IFN signaling pathways (8, 18). What was surprising, and unlike other viruses, was the relative STAT1 dependence and IFN independence of RSV replication in vivo (19).
Altered inflammatory responses in lungs of IFN-deficient mice
Along with the increased viral load there was an exacerbated inflammatory response in the lungs of STAT1–/– mice (Fig. 2). There were no significant differences in the numbers of BAL leukocytes recovered from wild-type, IFN-R–/–, IFN-R–/–, or IFN-R–/– mice on days 5 and 8 after infection. However, samples from STAT1–/– mice showed 15- and 5-fold greater numbers of inflammatory cells at 5 and 8 days after infection, respectively (p < 0.001 on days 5 and 8). Although we have also found an enhanced inflammatory response to RSV by STAT1–/– mice on the BALB/c background, where differences in viral titers are much smaller (7), the 100-fold increase in virus found in the STAT1–/– 129SvEv animals (Fig. 1) undoubtedly influences many immune parameters. Because we wanted to determine the effects of IFN, rather than virus load, on the outcome of RSV infection, we omitted STAT1–/– mice from the following studies and focused instead on comparing the responses of mice lacking either or both IFNRs to those of strain-matched controls.
We had previously described pronounced eosinophilic lung disease after primary RSV infection of STAT1–/– and IFN-R–/–, but not IFN-R–/–, IFN-R–/–, or IFN-–/–, mice, as judged by microscopic examination of infected tissues (7). For a more quantitative picture, we collected BALs from mice of each genotype at 5 days (Fig. 3A) and 8 days (Fig. 3B) after i.n. RSV inoculation and performed differential cell counts on Wright-Giemsa-stained cytospins made from individual animals (four to six mice per group). Lavage fluids obtained from wild-type mice consisted almost entirely of monocytes/macrophages and lymphocytes, with the proportion of lymphocytes increasing by day 8 when tissue inflammation was well developed. In IFN-R–/–, IFN-R–/–, and IFN-R–/– mice, neutrophils were present on day 5, but decreased to near background levels by day 8. Eosinophils were present only in the lungs of IFN-R–/– and IFN-R–/– animals. In the double-receptor knockout mice, eosinophils appeared earlier and consistently made up a significantly larger proportion of the total infiltrate. These differences were consistent over three similar experiments, one of which is pictured in Fig. 3, A and B. When these data were analyzed by ANOVA, all three experiments (Fig. 3C) showed significant differences between the IFN-R–/– and IFN-R–/– strains (p < 0.05). Another significant and consistent difference among the different genotypes was the relative dearth of lung lymphocytes in IFN-R–/– animals on day 8 (p < 0.05), shown in Fig. 3B.
Photomicrographs corresponding to these BAL data are shown in Fig. 4. H&E-stained lung sections from infected wild-type (A and D), IFN-R–/– (B and E), and IFN-R–/– (C and F) mice 8 days after inoculation are pictured. As predicted from the equivalent BAL cell numbers (Fig. 2), the overall extent of inflammation was similar in all four strains. This can be seen at the lower (x200) magnification. All sections show pronounced perivascular infiltrates with foci of peribronchial, peribronchiolar, and alveolar inflammation. There was little evidence of cytopathic effect or epithelial cell necrosis. Appreciable differences between strains were apparent only at higher (x400) magnification, where the very different characters of the infiltrates are evident. Consistent with the data in Fig. 3, the inflammatory process is entirely lymphocytic in wild-type mice (Fig. 4D) and predominantly lymphocytic in IFN-R–/– mice (Fig. 4E), but is overwhelmingly eosinophilic in IFN-R–/– animals (Fig. 4F). This relates to both the relative increase in eosinophils and the relative decrease in lymphocytes seen in the double-knockout animals. Lung sections from IFN-R–/– mice (not pictured in this figure) were similar to those from wild-type mice, but with an overall decrease in lymphocytic inflammation.
On day 5 after infection, the total number of lymphocytes present in the lungs of all animals was quite small (data not shown), but by day 8 we observed a large expansion of T cells in wild-type and IFN-R–/– mice. This increase was primarily due to the expanding CD8+ cell population, as calculated from cell counts, BAL differentials, and flow cytometry data (Fig. 5A). Interestingly, this expansion was very limited in mice lacking the IFN-R, although the difference in CD8+ T cell number was statistically significant (p < 0.05) only for the IFN-R–/– animals. This observation is consistent with data from other laboratories showing a role for type I IFNs in the activation and survival of CTLs (20, 21). There were no significant differences in numbers of BAL CD4+ T cells among strains. Fig. 5B shows the number of eosinophils present in BALs taken from each strain on day 8 postinfection. These were very similar in IFN-R–/– and IFN-R–/– animals, leading us to conclude that the eosinophilic predominance in IFN-R–/– mice reflects primarily a lack of CD8+ T cells.
CD4+ T cell differentiation in IFN-deficient knockout mice
Having determined that there were significant qualitative and quantitative differences between RSV lung disease in IFN-R–/– and IFN-R–/– mice, we sought to determine the role of IFNs in CD4+ T cell differentiation. It has been established that STAT1-mediated induction of the transcription factor T-bet is required for the development of Th1 lymphocytes (10). Consistent with this finding, we have observed Th2 cytokine production by STAT1-deficient CD4+ T cells responding to an RSV-F protein epitope that stimulates primarily IFN- in wild-type mice (7). Although a large body of evidence demonstrates the importance of IFN- in Th1 differentiation in vitro, in vivo studies using mice deficient in IFN- signaling have not shown a clear Th2 predisposition (22, 23, 24). In light of the uncertainty regarding the role of IFN- in Th1 differentiation, and with the differing disease patterns observed in IFN-–/– or IFN-R–/– and STAT1–/– mice infected with RSV (7) or influenza (25), it was important to learn whether virus-specific CD4+ T cells from RSV-immune IFN-R–/–, IFN-R–/–, and IFN-R–/– mice were primarily Th1 or Th2.
To perform this experiment in the 129SvEv animals, a dominant viral epitope recognized by RSV-immune CD4+ lymphocytes in this strain was required. Overlapping peptides corresponding to the RSV-F protein sequence were mixed to make five pools of 10 peptides and one pool of seven peptides, and these peptide pools were assayed for their ability to stimulate the proliferation of immune splenocytes. Because pool 1 stimulated the most robust proliferation response (data not shown), we next screened peptides 1–10 individually (Fig. 6). From this secondary screen, peptide 9, corresponding to aa 81–100 (QELDKYKNAVTELQLLMQNT), was chosen for additional characterization. An IFN- ELISPOT assay performed with undepleted or CD4+ T cell-depleted immune splenocytes demonstrated that CD4+ T cells were required for an IFN- response to this peptide. Undepleted cultures produced an average of 85 spots/106 splenocytes compared with an average of 1 spot/106 splenocytes in CD4+ T cell-depleted samples. Depletion of CD8+ T cells had no effect on IFN- production (data not shown).
Cytokine production by immune CD4+ T cells from mice of each genotype was assessed by quantitation of cytokine gene transcripts after peptide stimulation of cultured splenocytes. Cells were harvested from two or three mice of each strain that had been inoculated i.n. with 106 PFU of RSV 28 days previously. After 7 days of culture with peptide 9, the splenocytes were washed and restimulated for a 6-h period before harvesting. Equal amounts of RNA from each sample were analyzed in a multi-probe RNAP, and signals were quantitated using the Storm PhosphorImager and ImageQuant software. Relative levels of cytokine transcripts made by each strain are shown in Fig. 7. We concluded from this set of experiments that although RSV does not appear to generate a strong Th1 response to F protein in the wild-type mouse, mice lacking the IFN-R alone or lacking both IFNRs default to a Th2 differentiation pathway in our system.
Role of IFNs in lung cytokine and chemokine production
The earliest response to virus is initiated by infected lung epithelial cells. A number of cytokines and chemokines are induced by RSV (26, 27, 28), and these inflammatory mediators directly affect the trafficking of lymphocytes and granulocytes to the site of infection. Chemokines promoting both Th1 and Th2 cell migration are produced by the infected epithelium in vitro, but in vivo, their synthesis is also affected by the presence of other cytokines. Many of the chemokines attracting Th1 cells (IFN-inducible T cell chemoattractant (I-TAC), IP-10, monokine induced by IFN- (MIG)) are induced by IFNs (29, 30, 31, 32), and IL-13 is known to increase the synthesis of eotaxin (33, 34), a major eosinophil attractant. We wondered whether the altered inflammatory responses in the absence of IFN- and/or IFN- signaling correlated with an altered chemokine profile. To answer this question, BAL supernatants from infected mice were collected 5 days after i.n. inoculation with 107 PFU of RSV and were assayed for the presence of IP-10. IP-10 was chosen because it can be induced by either type I or type II IFNs (29, 32). Fig. 8 shows the data obtained for both eotaxin and IP-10 assayed at two time points in all four strains of mice. Although no IP-10 could be detected in mice lacking both IFNRs (IFN-R–/–), mice retaining either the IFN-R ((IFN-R–/–) or the IFN-R (IFN-R–/–) continued to produce IP-10. Eotaxin was induced in all strains, but eosinophilia occurred only in those lacking virus-specific Th1 cells, that is, the IFN-R–/– and IFN-R–/– mice. In addition, eotaxin levels remained significantly elevated on day 8 only in mice lacking both IFNRs (p < 0.001).
These same BAL supernatants from each animal were also assayed for the presence of Th2 cytokines. Levels of IFN-, IL-5, and IL-13 in lavage samples collected at 5 and 8 days after inoculation were determined by ELISA or by the Luminex100 system (Fig. 9). Although there were increased levels of IL-5 and IL-13 in lungs of IFN-R–/– and IFN-R–/– mice, these differences were statistically significant only for IL-5. IFN- levels were similarly high for all strains on day 5, probably reflecting production by NK as well as T cells. By day 8, these levels had dropped, most precipitously in the IFN-R–/– animals. Thus, it appears that chemokine synthesis is affected by early IFN production, and this suggests that chemokine levels may be influenced by the presence of Th2 lymphocytes.
Discussion
This conclusion was inconsistent with the absence of this pathology in infected mice lacking IFN-, a cytokine known to activate STAT1 and drive Th1 differentiation of naive CD4+ T cells. In vivo experiments to determine whether IFN- itself is essential for Th1 development have been complicated by the importance of IFN- in establishing IL-12 responsiveness. Current models of naive CD4+ cell activation postulate that STAT1 activation is required for Th1 commitment, but that the STAT4-mediated IL-12 signal is still necessary for IFN- production in response to Ag (10, 38). Because IFN- up-regulates IL-12R expression, a robust Th1 response should require IFN-, and we would expect mice lacking this cytokine to mount a Th2 response to virus infection. Nonetheless, in our experiments with either IFN-–/– BALB/c or 129SvEv IFN-R–/– mice, eosinophils were present in infiltrates surrounding vessels and airways of infected lungs, but only at low levels. This observation has been made by others (24, 39), who concluded, as we did, that RSV pathogenesis in IFN--deficient animals is similar to that in wild-type animals. We therefore wondered whether, in the absence of IFN-, IFN- could act in a compensatory manner to inhibit Th2 lymphocyte development and eosinophilic disease (7). To test this hypothesis we needed to determine 1) whether Th2 differentiation was blunted in IFN-R–/– mice, and 2) whether mice lacking both IFNRs (IFN-R–/–) responded differently from those lacking one or the other.
We approached the question of T cell differentiation in infected IFN--deficient mice by comparing cytokine responses of four mouse strains to a single MHC class II restricted viral peptide. The peptide used (F9), corresponding to aa 81–100 of the RSV-F protein, specifically stimulated the proliferation of and IFN- production by CD4+ T cells from RSV-immune wild-type 129SvEv mice. Using an RNAP to survey simultaneously for Th1 and Th2 cytokine production, we observed that although peptide-stimulated T cells from IFN-R–/– and IFN-R–/– animals produced levels of IFN- transcript similar to those in wild-type animals, IL-13 production was increased 10- to 20-fold (Fig. 7). The elevated amounts of IL-13 as well as IL-5 induced by peptide F9 in the absence of the IFN-R demonstrated that the IFN- pathway is essential for inhibiting the Th2 response to RSV infection.
Having found that both the IFN-R–/– and the IFN-R–/– animals default to a Th2 lymphocyte response after infection, an alternate explanation for the significant differences in lung pathology between these animals was required. Characterization of BAL samples from mice of each genotype at two time points suggested that the major difference between these strains was not an increase in eosinophils, but, rather, an absence of CD8+ lymphocytes in both strains of mice lacking the IFN-R–/–. In wild-type and IFN-R–/– mice, there was a large expansion in the number of lymphocytes present in the lungs by day 8 after inoculation. In IFN-R–/– animals, this expansion occurred in the presence of eosinophils and neutrophils (Fig. 3). In contrast, mice lacking both IFNRs showed little increase in lymphocyte numbers as the infection progressed, and eosinophils were the predominant cell type on day 8. This pattern was seen consistently in three replicate experiments and was significant in each (p < 0.05). Evaluation of tissue sections showed a similar picture (Fig. 4), with largely lymphocytic infiltrates in infected IFN-R–/– mice and predominantly eosinophilic inflammation in lungs of IFN-R–/– animals.
We have observed that Th2 cytokine production by CD4+ T cells is necessary, but not sufficient, for the development of eosinophilic lung inflammation after RSV infection. Other factors contribute to pulmonary eosinophilia during primary infection in mice. It has been shown that a large number of genes are induced by RSV infection of respiratory epithelial cells; among these are chemokines that regulate inflammatory cell trafficking (26, 27, 40, 41). A number of innate immune mediators will impact relative levels of chemokine production by these cells (31), and among these are the IFNs. Both IFN- and IFN- are produced early in infection: IFN- by the virus-infected cells and innate IFN- by activated NK cells (17). A number of the chemokines responsible for Th1 and CTL trafficking, among them the CXCR3 ligands I-TAC, MIG, and IP-10, are IFN-inducible genes (31, 32, 41). We wondered whether IP-10, which is known to be induced by both type I and type II IFNs, would be detectable in mice lacking either or both IFNRs and whether differences in chemokine production could be contributing to the differences in pathology. As shown in Fig. 9, IP-10 was detectable in BALs from all animals on day 5, except in those lacking both IFNRs. Eotaxin, another RSV-induced chemokine, is a chemoattractant for activated Th2 cells and eosinophils (42). Although eotaxin was present in BALs from mice of all genotypes at early times, levels were significantly increased only in IFN-R–/– mice on day 8 (p < 0.001; Fig. 9A). Allergic inflammation is a self-amplifying process, and the elevated eotaxin levels in animals with eosinophilic disease may reflect this situation. If activated Th2 cells and eosinophils secreting IL-13 (43) traffic to the lung, increasing IL-13 concentrations will up-regulate eotaxin production by infected epithelium (34) (see Fig. 10).
In summary, our data suggest that although Th2 lymphocytes are necessary for the development of an eosinophilic response to primary RSV infection, they are not sufficient. In this system it is the IFN- and IFN- produced during the innate immune response, rather than lymphocyte IFN-, that determines disease outcome. RSV lung pathology in IFN-R–/– animals with an intact type I IFN signaling pathway is similar to that seen in wild-type controls. CD8+ T cells, rather than eosinophils, continue to dominate the process. In the absence of both IFNRs, disease is markedly eosinophilic and without the usual expansion of the lung lymphocyte compartment (44, 45). This is consistent with a report from Hussell et al. (46) showing a correlation between the magnitude of the CD8+ T cell response and the propensity of a mouse strain to develop eosinophilia. Therefore, the elevated eotaxin levels, the absence of IP-10 induction, and the lack of CTLs in IFN-R–/– animals work together to create differences between two strains of animals that both generate Th2 responses. Fig. 10 depicts a model showing how the summation of these influences will determine the composition of the inflammatory infiltrate.
We propose that innate IFN- and - affect local chemokine production by increasing levels of the Th1 and Tc1 chemoattractants (I-TAC, IP-10, and MIG) as well as stimulating CD8+ T cell proliferation and activation. An important role for IFN- in CTL survival and IFN- production has also been shown in other systems (20, 21, 47). If virus-specific Th1 cells and CTLs are present, they will be drawn to the site of infection and augment local IFN- production. If activated Th2 lymphocytes are present, they will preferentially accumulate in response to eotaxin, increasing local IL-13 and eotaxin production and thereby amplifying an allergic-type inflammatory response. In animals lacking the IFN-R–/–, it appears that the presence of type I IFNs does not block eosinophil accumulation, but can still inhibit a primarily allergic response by promoting CD8+ T cell accumulation. It has been suggested that in addition to their roles as CXCR3 agonists, I-TAC, IP-10, and MIG may also act to block Th2 cell and eosinophil accumulation by binding the CCR3 expressed by those cell types and acting as antagonists (48). If this is a biologically significant process, this antagonism could also play a role in shaping the inflammatory response. Overall, our findings in the mouse model suggest that although the link between RSV and asthma (6) is probably due to the ability of this virus to promote Th2 lymphocyte differentiation, it may be exacerbated by the limited type I IFN induction by RSV infection in vivo (49, 50).
Acknowledgments
We thank Michel Aguet and Bob Schreiber for providing IFN-R–/–, IFN-–/–, and IFN-R–/– mice; Tom Hamilton for anti-IP-10 antiserum; and Philip R. Johnson for RSV F protein peptides. We also thank Chris Walker, Naglaa Shoukry, Andrew Cawthon, and Christine Biron for helpful discussions, and Karen Watkins for manuscript preparation.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grant AI47226 from the National Institutes of Health (to J.E.D.).
2 Address correspondence and reprint requests to Dr. Joan E. Durbin, Department of Pediatrics, Ohio State University College of Medicine and Public Health, Columbus Children’s Research Institute, 700 Children’s Drive, Room WA4014, Columbus, OH 43205-2696. E-mail address: durbinj{at}pediatrics.ohio-state.edu
3 Abbreviations used in this paper: RSV, respiratory syncytial virus; BAL, bronchoalveolar lavage; i.n., intranasally; IP-10, IFN-induced protein 10; I-TAC, IFN-induced T cell chemoattractant; MIG, monokine induced by IFN-; RNAP, RNase protection assay.
Received for publication December 3, 2004. Accepted for publication March 24, 2005.
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