Sustained increases in numbers of pulmonary dendritic cells after respiratory syncytial virus infection

Basic and clinical immunology Sustained increases in numbers of pulmonary dendritic cells after respiratory syncytial virus infection

Background: Respiratory syncytial virus (RSV) bronchiolitis in infants can lead to wheezing and early allergic sensitization. In mice, RSV infection enhances allergic airway inflammation and airway hyperresponsiveness. Dendritic cells are critical in inducing T-cell responses to both viruses and allergens and could be pivotal in regulating interactions between these. Objective: This study addresses the effects of RSV infection on phenotype and function of pulmonary dendritic cells. Methods: BALB/c mice were infected with RSV, and expression of CD11c, MHC II, and CD86 on lung and spleen cells was monitored by flow cytometry for 21 days after infection. CD11c+ cells were isolated to assess their phagocytic capacity and their ability to induce proliferation in allogenic T cells. Results: Numbers of pulmonary CD11c+ MHC IIhi cells increased 13-fold starting from day 6 after RSV infection. This was associated with increased CD86 expression, reduced phagocytosis, and increased allogenic T-cell stimulatory capacity in CD11c+ cells. These changes in the lung outlasted acute infection and were not observed in spleens. Conclusion: RSV infection results in sustained increases in numbers of mature dendritic cells in the lung. These might well contribute to the development of intense airway inflammation and airway hyperresponsiveness after RSV infection and to enhancement of subsequent responses to allergen exposure. (J Allergy Clin Immunol 2004;113:127-33.) Key words: Respiratory syncytial virus, antigen-presenting cells, dendritic cells, CD11c antigen, airway inflammation, mice, virusinduced asthma

In infants, respiratory syncytial virus (RSV) infection can result in intense inflammatory responses of the lower airways leading to bronchiolitis. RSV bronchiolitis increases the risk of asthma symptoms like recurrent

From the aKlinik für Kinder- und Jugendmedizin, St Josef-Hospital, Bochum, Germany, and bDepartment of Respiratory Medicine, NHLI, Imperial College London. Supported by grant 01GC9802 from Bundesministerium für Bildung und Forschung and grant AL 067454 from The Wellcome Trust. Received for publication July 7, 2003; revised October 16, 2003; accepted for publication October 28, 2003. Reprint requests: Jürgen Schwarze, MD, Department of Respiratory Medicine, National Heart and Lung Institute, Imperial College London, Faculty of Medicine, Norfolk Place, Paddington, London W2 1PG, United Kingdom. 0091-6749/$30.00 © 2004 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2004.10.057

Abbreviations used APC: Antigen-presenting cell DC: Dendritic cell FACS: Fluorescence-activated cell sorting FITC-DX: FITC-conjugated dextran MFI: Mean fluorescence intensity MNC: Mononuclear cell PMA: Phorbol 12-myristate 13-acetate RSV: Respiratory syncytial virus TCID50: Median tissue culture infective dose UV-RSV: RSV inactivated by ultraviolet light

wheezing,1,2 and some studies also report an increased risk of early allergen sensitization.2,3 In mouse models, intranasal infection with RSV results in viral replication in the lung and induces an inflammatory response with a transient alveolar lymphocytic influx followed by focal inflammation between bronchioles and small blood vessels comprising mononuclear cells (MNCs), neutrophils, and eosinophils.4 An early influx of cytotoxic natural killer cells to the lower airways is followed by an influx of T cells, primarily CD8+ cells, and an increase in cytotoxic T-cell activity.5 Both CD4+ and CD8+ T cells are not only involved in termination of RSV replication, but they also contribute to RSV-induced lung pathology.6 Associated with inflammation, mice develop transient airway hyperresponsiveness after RSV infection.7,8 T cells, in particular CD8+ T cells, are critical in RSVinduced airway hyperresponsiveness.9,10 In addition, RSV infection facilitates the development of allergic airway inflammation and airway hyperresponsiveness on subsequent allergen exposure via the airways.8 Primary T-cell responses are initiated by dendritic cells (DCs). Immature DCs are able to take up and process antigens. Contact with pathogens initiates maturation in DCs, which then migrate to regional lymph nodes. Here, DCs express not only CD11c, a marker shared with macrophages, but also high levels of peptide-MHC complexes and costimulatory molecules such as CD86. Pulmonary DCs have been studied in models of bacterial infection and allergic airway sensitization, but little is known about their role in respiratory viral infections. After infection with Sendai virus11 and influenza A virus,12 increases of DC numbers in the respiratory tract 127

Basic and clinical immunology

Marc Beyer, MD,a,b Holger Bartz, MD,a Katharina Hörner, MD,a Sandra Doths,a Cordula Koerner-Rettberg, MD,a and Jürgen Schwarze, MDa,b Bochum, Germany, and London, United Kingdom

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are only short lived. Virus-induced increases in pulmonary DC numbers could play a role in excessive inflammatory responses such as bronchiolitis and allergic inflammation. This hypothesis is supported by data from atopic patients who have increased numbers of airway DCs13 and by animal models in which increases in numbers of lung DCs facilitate allergic sensitization via the airways.14 This study aims to determine the effects of RSV infection on pulmonary DCs by monitoring their numbers, phenotype, and function.

tively. In some experiments CD8+ cells were depleted by using antiCD8–coated MACS-beads (Miltenyi) before positive selection of pulmonary CD11c+ cells.

Phagocytosis CD11c+ and CD11c– cells were incubated with FITC-DX (Sigma-Aldrich) at 37°C or 4°C for 1 hour, washed, and stained with anti-CD11c-PE-antibodies. Mean fluorescence intensity (MFI) (FITC) of CD11c+ or CD11c– cells was analyzed by FACS, and ∆MFI = MFI (37°C) – MFI (4°C) was calculated.

Mixed lymphocyte reaction METHODS Animals Female BALB/cAnNCrl mice, 8 to 12 weeks of age, from Charles River Laboratories (Sulzfeld, Germany) were kept under specific pathogen-free conditions. They were used under a protocol approved by Regierungspräsidium Arnsberg (NRW, Germany).

Virus

Basic and clinical immunology

Human RSV, type A (Long strain) from ATCC (Rockville, Md) free of chlamydia or mycoplasma contamination, was used. The virus was cultured and titrated by immunoplaque assay.15 Culture supernatants were used for infection. Supernatants from HEp-2 cell cultures free of RSV (mock) or RSV inactivated by ultraviolet light (UV-RSV) were used for sham infections. Virus was titrated in supernatants of lung homogenates by immunoplaque assay15 in HEp-2 cell cultures.

Experimental protocols

CD11c+ and CD11c– cells from lungs and spleens of BALB/c mice were incubated with mitomycin C, washed, and added to splenocytes (105 cells/well) from C57Bl/6 mice, enriched for T cells by nylon wool purification. Mixed cells were cultured for 5 days in RPMI 1640 supplemented with 2 mmol/L L-glutamine, 50 µmol/L β-mercaptoethanol, 1 mmol/L sodium pyruvate, 1 mmol/L nonessential amino acids, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FCS (all from Biochrom, Berlin, Germany). 3Hthymidine (Amersham Pharmacia, Freiburg, Germany) was added for the final 16 hours (0.5 µCi/well). Cells were harvested on paper filters, and incorporation of 3H-thymidine was measured as cpm by using an LKB 1209 Rackbeta liquid scintillation counter (LKB, Turku, Finland).

Statistical analysis Groups were compared by Student t test. P values for significance were set at .05. All values are expressed as the mean ± SD.

RESULTS

Mice were naïve, infected with RSV (3 × median tissue culture infective dose [TCID50] in 60 µL) or sham infected. At 4, 6, 10, 14, and 21 days after infection, lungs and spleens were harvested after lung lavage and perfusion of the pulmonary vasculature with cold PBS. Minced organs were incubated with collagenase I (Sigma-Aldrich, Taufkirchen, Germany), and EDTA was added to spleen fragments. Spleen cells were dispersed by nylon mesh and lung cells by vigorous pipetting. MNCs isolated by density gradient centrifugation were counted in a Neubauer chamber. For histologic examination lungs were inflation-fixed with formalin-saline. Inflated lungs were excised and embedded in paraffin wax, and sections were stained with hematoxylin-eosin. 105

Flow cytometry Cells were incubated with antibodies and mouse serum, washed, and suspended in staining buffer. A FACScan was used for data acquisition and Cell Quest software for analysis (both Becton Dickinson, Heidelberg, Germany). The following antibodies (all from Becton Dickinson except anti-F4/80) were used: anti-CD11c-FITC, antiCD3-FITC, anti-F4/80-FITC (Serotec, Oxford, United Kingdom); anti-CD3-PE, anti-CD4-PE, anti-CD8-PE, anti-CD86-PE, anti-I-APE (anti-MHC II), anti-CD11c-PE, and anti-IFNγ-PE; anti-CD8α CyChrome; rat IgG1, rat IgG2a/b, mouse IgG2b, and hamster IgG as isotype controls. For intracellular cytokine staining isolated CD11c+ cells were cultured with phorbol 12-myristate 13-acetate (PMA), ionomycin, and brefeldin A and prepared for analysis as described.16

Isolation of CD11c+ cells MNCs from lungs or spleens of 4 animals were pooled, incubated with anti-CD11c–coated MACS-beads, and sorted by AUTOMACS (Miltenyi, Bergisch-Gladbach, Germany). Purity of isolated CD11c+ cells assessed by fluorescence-activated cell sorting (FACS) was at least 85% and 90% for lungs and spleens, respec-

Sustained increases in numbers of lung DCs and CD11c+ CD8+ T cells after RSV infection Mice were infected intranasally with RSV or sham infected. On day 4 after inoculation, a 10-fold increase in RSV titers compared with day 2 after infection was detected by immunoplaque assays in all lungs of RSV-infected mice (RSV titer day 2: 867 ± 370 TCID50; day 4: 9635 ± 817 TCID50 per lung, P < .05, n = 6). No RSV was detectable 7 days after infection or in sham-infected animals. Monitoring disease by body weight, significant weight loss of 8.2% ± 4.5% (P < .05, n = 12) was observed on day 6 after RSV infection but not after UV-RSV inoculation. Histologic examination showed widespread perivascular and peribronchial inflammatory infiltrates on day 6 after RSV infection but not after inoculation with UV-RSV. On day 21 after infection inflammatory changes were largely resolved, but some isolated perivascular infiltrates remained (Fig 1). DC numbers in MNCs from spleens and lungs were monitored on days 4, 6, 10, 14, and 21 after infection, and expression of CD11c, MHC class II, and CD86 was assessed by FACS analysis. Cells expressing both CD11c and high levels of MHC II were regarded as DCs. During the time course analyzed, numbers of DCs in control animals inoculated with mock solution or UVRSV did not differ significantly from DC numbers in naïve mice (data not shown). After RSV infection, numbers of DCs increased from 1.0 ± 0.5 × 105 per lung on day 4 after infection to a maximum of 13.0 ± 3.9 × 105

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FIG 1. Low dose RSV infection results in peribronchial and perivascular inflammation in the lung. Mice were infected with RSV or UV-RSV and killed after 6 and 21 days. Lungs were inflated with formalin-saline, excised, and embedded in paraffin wax. Sections were stained with hematoxylin-eosin, and representative images were taken at ×100 magnification.

on day 6 after infection. Thereafter, numbers declined slowly but stayed significantly elevated until day 14 compared with control animals inoculated with UV-RSV. On day 21 DC numbers were still 3.8-fold higher than in control animals, but this difference did not reach statistical significance (Fig 2, A and B). Analysis of the percentage of pulmonary CD11c+ cells expressing MHC II and CD86 showed that in naïve mice 19.3% ± 2.8% of CD11c+ lung cells expressed MHC II at high levels (Fig 2, C). After UV-RSV inoculation, this percentage did not change significantly (data not shown). On days 6, 10, and 14 after RSV infection, the percentage of CD11c+ MHC IIhi cells increased significantly (P < .05, n = 4). On day 6, when the highest number of DCs were found in the lung, 52.4% ± 7.7% of CD11c+ cells expressed high levels of MHC II. The percentage of CD11c+ MHC IIhi cells increased further with a maximum of 69.7% ± 9.3% on day 10 after infection and a reduction to 47.1% ± 3.2% on day 21 after infection (Fig 2, C). Expression of CD86 was increased parallel to the upregulation of MHC II, albeit on a lower level. In mock infected animals, 8.0% ± 2.8% of CD11c+ cells expressed CD86. After RSV infection the percentage of CD11c+ CD86+ cells was increased from day 6 (17.25% ± 1.6%), peaked on day 10 (42.2% ± 2.8%), and remained elevated until day 14

FIG 2. MHC II and CD86 expression on pulmonary CD11c+ cells after RSV infection. Mice were naïve or infected intranasally with either RSV or UV-RSV. Lung MNCs from naïve mice and on days 4, 6, 10, 14, and 21 after infection were stained with antibodies to CD11c, MHC II, and CD86 and analyzed by FACS. A, Absolute numbers of CD11c+ MHC IIhi cells (DC) per lung after inoculation with either RSV (■) or UV-RSV (䊐) are expressed as mean ± SD (n = 4, *P < .05 vs UV-RSV). B, Expression of CD11c vs MHC II in pulmonary MNCs after RSV infection is presented in dot plots from a representative experiment. Numbers indicate the percentage of cells in the appropriate quadrant. C, Expression of CD86 (gray bars) and MHC IIhi (open bars) on pulmonary CD11c+ cells of naïve mice or after RSV infection are expressed as mean ± SD (n = 4, *MHC II, §CD86; P < .05 vs naïve).

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FIG 3. Flow cytometric analysis of isolated CD11c+ lung cells. CD11c+ lung cells isolated 10 days after mock or RSV infection were stained with anti-CD11c-PE and anti-F4/80-FITC or with antiCD11c-FITC and anti-CD4-PE or anti-CD8α-PE antibodies (open histogram) or isotype controls (shaded histogram). Histograms illustrate expression of these surface markers on cells gated for expression of CD11c from a representative experiment (n = 4, 4 mice pooled per experiment). Percentages of double positive cells are indicated.

(34.1% ± 1.3%). On day 21 after infection CD86 expression had returned almost to baseline (Fig 2, C). To assess effects of RSV infection on antigen-presenting cells (APCs) in the systemic compartment, splenocytes from infected mice and control animals were monitored for expression of CD11c, MHC II, and CD86. In naïve animals 63.3% ± 3.6% and 19.8% ± 4.2% of CD11c+ cells co-expressed MHC II or CD86, respectively. Neither total cell numbers of CD11c+ MHC IIhi or CD11c+ CD86+ nor their percentage within the CD11c+ population changed significantly during the course of RSV infection (data not shown). To analyze whether distinct subsets of APCs occur in the lung during RSV infection, we stained isolated CD11c+ cells for CD4, CD8α, and F4/80, surface markers detected on DCs in lymphoid organs.17,18 F4/80, which has been attributed to macrophages, is also expressed on pulmonary APCs.19,20 Although the majority of CD11c+ cells from sham-infected mice expressed F4/80, only a minority expressed this marker after RSV infection. CD4 and CD8α were virtually not expressed on APCs from noninfected mice. In contrast, on day 10 after infection a significant proportion of CD11c+ cells expressed CD8α (Fig 3). These cells were small in size, and 97.3% ± 0.6% co-expressed CD3. Furthermore, after stimulation with PMA/ionomycin intracellular IFN-γ was detectable in 67.0% ± 12.7% of CD11c+ CD8+ T cells, which thus appear to be cytotoxic T lymphocytes. Analysis in com-

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FIG 4. Phagocytosis by isolated pulmonary CD11c+ cells. Pulmonary CD11c+ cells were isolated on day 10 after mock (open bars) or RSV infection (grey bars). The remaining negative fraction was designated as CD11c– cells. Cells of each population were incubated with FITC-DX at 37°C or 4°C, and uptake was determined by FACS analysis as MFI. Values of ∆MFI (MFI-37°C – MFI4°C) are expressed as mean ± SD (n = 3, *P < .05 RSV vs mock).

partments of different cell size and granularity showed that CD11c+ MHC IIhi cells not only constituted almost the entire population of large granular cells, but that they also accounted for the majority (66%) of small CD11c+ cells that also comprised CD11c+ CD8+ T cells (34%). In summary, numbers of CD11c+ cells with a mature DC phenotype, co-expressing MHC II or CD86, increase in the lung after RSV infection. The highest numbers of these DCs are detected late in the course of infection. In addition, CD11c+ CD8α+ T cells accumulate in the lung, whereas the percentage of CD11c+ F4/80+ cells declines during RSV infection.

Decreased phagocytosis by pulmonary CD11c+ cells after RSV infection Phagocytosis is an important property of immature DCs, and the capacity for uptake of particles is downregulated during their maturation.21 We asked whether phenotypic maturation of DCs after RSV infection is accompanied by changes in phagocytosis. To this end, CD11c+ cells isolated from lungs and spleens 10 days after RSV or mock infection were incubated with FITC-conjugated dextran (FITC-DX). Uptake of FITC-DX was assessed by FACS, and ∆MFI was calculated. After RSV infection ∆MFI values of CD11c+ cells were significantly lower than after mock infection (P < .05, n = 3), demonstrating a decrease in the capacity to phagocytose FITC-DX (Fig 4). This suggests that RSV infection also induces functional maturation of pulmonary CD11c+ cells. Splenic CD11c+ cells, in contrast, did not phagocytose as efficiently as noninfected pulmonary CD11c+ cells, and they did not display any significant differences in FITC-DX uptake after RSV (∆MFI = 570 ± 141) or mock infection (∆MFI = 674 ± 194). CD11c– cells from lungs and spleens showed minimal uptake of FITC-DX only, indicating that phagocytosis in MNCs from these organs is restricted to CD11c+ cells.

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Enhanced allostimulatory capacity of lung CD11c+ cells after RSV infection

DISCUSSION This study aims to delineate the role of pulmonary DCs in experimental murine RSV infection. By using a comparatively low viral inoculum to induce mild disease with airway inflammation rather than severe disease with alveolar inflammation, we found that numbers of DCs in the lung increased during RSV infection and remained elevated even after resolution of infection. MHC II expression on CD11c+ cells was upregulated for at least 21 days after infection. In contrast to our results, infection of mice with a recombinant variant of RSV only led to significant increases in numbers of pulmonary DCs, if GM-CSF was encoded in this virus.22 These experiments differ from ours in that an attenuated, genetically modified strain of RSV was used, and, more importantly, in that FACS analysis was restricted to large granular cells. Our analysis included all viable MNCs and detected a considerable accumulation of DCs after RSV infection in a population of small cells with low granularity. This population has previously been shown to contain mature DCs.23 In addition to MHC II, expression of CD86 is

FIG 5. T-cell stimulatory capacity of isolated pulmonary CD11c+ cells. CD11c+ cells were isolated from lungs on day 10 after mock (䊐, 䊊) or RSV infection (■, 䊉); the remaining negative fraction was designated as CD11c– cells. Splenocytes of C57Bl/6 mice were enriched for T cells, and 105 cells were incubated alone or with increasing numbers of CD11c+ (䊐, ■) or CD11c– (䊊, 䊉) cells. T-cell proliferation was determined by 3H-thymidine incorporation after 5 days of culture. Cpm from a representative experiment are expressed as mean ± SD (n = 3, P < .05; *CD11c+ from RSV/mock vs T cells alone, §CD11c+ from RSV vs CD11c+ from mock and T cells alone).

also increased. CD86 expression has previously been detected on resting murine pulmonary DCs,24 and it was elevated in allergic airway inflammation.25 Overexpression of GM-CSF in the airways leads to increases in numbers of lung DCs and macrophages,14 and GM-CSF is produced by human bronchial epithelial cells after RSV infection.26 Thus, it is tempting to speculate that secretion of cytokines including GM-CSF by RSVinfected pulmonary epithelial cells results in enhanced recruitment of DCs to the lung or in proliferation and maturation of resident precursor cells. In the population of CD11c+ cells expanded by RSV infection we also detected apparently cytotoxic CD8+ T cells. Expression of CD11c has not been reported previously on pulmonary T cells in respiratory viral infections, but it has recently been observed on cytotoxic T cells after lymphocytic choriomeningitis virus infection.27 In human cytotoxic T-cell clones, CD11c seems to be critical for conjugate formation and target cell lysis,28 and increased numbers of CD11c+ T cells in bronchoalveolar lavage fluid after lung transplantation suggest a role for these cells in lung pathology.29 When functional changes in pulmonary CD11c+ cells after RSV infection were assessed, a significant decrease in FITC-DX uptake was noted. Rat and human pulmonary DCs are capable of efficient phagocytosis,30,31 and pulmonary as well as immature bone marrow– derived DCs downregulate phagocytic capacity during maturation.21,30 This suggests that CD11c+ cells underwent a maturation-induced reduction in phagocytic ability after RSV infection. Induction of increased T-cell proliferation by pulmonary CD11c+ cells from RSV-infected mice provides further evidence of functional maturation. The T-cell

Basic and clinical immunology

Mature DCs are able to induce proliferation of naïve T cells. We asked whether RSV-induced maturation also results in increased capacity of pulmonary CD11c+ cells to stimulate allogenic T cells. CD11c+ cells isolated from lungs and spleens of BALB/c mice 10 days after RSV or mock infection were cocultured with nylon wool purified T cells from naïve C57Bl/6 mice. Coculture of pulmonary CD11c+ cells from mock-infected mice with allogenic T cells yielded slight increases in T-cell proliferation, reaching statistical significance from an APC/Tcell ratio of 1:8 (Fig 5). After RSV infection pulmonary APCs induced significant T-cell proliferation already at an APC/T cell ratio of 1:16, and T-cell proliferation was up to 3-fold higher compared with coculture with APCs from mock-infected animals at equal ratios (Fig 5). Depletion of CD8+ cells before positive selection of CD11c+ cells did not have any significant effect on their allostimulatory capacity (data not shown). CD11c– cells did not induce significant T-cell proliferation at any concentration used. These findings indicate that pulmonary CD11c+ cells develop enhanced allostimulatory capacity after RSV infection. Interestingly, if splenic CD11c+ cells were cocultured under the same conditions, no difference between cells from mock-infected (32,067 ± 5,659 cpm) and RSV-infected (35,860 ± 13,956 cpm, n = 9) animals was detected at an APC/T-cell ratio of 1:2. However, splenic CD11c+ cells were about 10 times more efficient in stimulating T-cell proliferation than pulmonary CD11c+ cells even after RSV infection. These findings indicate that phenotypic maturation of pulmonary CD11c+ cells during RSV infection is associated with functional maturation, leading to an increased capacity to induce proliferation in allogenic T cells.

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stimulatory capacity of DCs can be enhanced both by GM-CSF secreted from cocultured alveolar epithelial cells32 and by exogenously added GM-CSF.30,33 In our study enhanced stimulatory capacity of APCs might well have been induced by secretion of GM-CSF by RSVinfected pulmonary epithelial cells. Interestingly, RSV infection did not alter T-cell stimulation by splenic CD11c+ cells or their phagocytic ability or phenotype, suggesting that RSV infection does not affect APCs in the systemic compartment. Pulmonary CD11c+ cells are less efficient in stimulating T cells than splenic DCs. This might reflect differences either in their maturational state or in the composition of populations isolated. Lungs contain high numbers of tissue macrophages, which can significantly inhibit Tcell stimulation by pulmonary DCs.34 The CD11c+ cell population after RSV infection likely contained F4/80+ macrophages, which might have reduced T-cell proliferative responses. Adherence steps to eliminate macrophages can induce maturation of DCs23 and were deliberately not included in our isolation protocol. Our findings demonstrate that RSV infection leads to sustained increases in pulmonary DC numbers. It has been reported that soluble IL-2–receptor levels in the blood are elevated for several months in infants after RSV bronchiolitis, whereas after acute measles and hemorrhagic dengue fever these levels return to baseline within 1 month.35 This suggests that in contrast to some other viral infections, long-lasting inflammatory responses persist after RSV bronchiolitis. Differences in the kinetics of DCs after infection with distinct respiratory viruses might influence subsequent immune and inflammatory responses in the lung. We have previously found that exposure to ovalbumin aerosol after the acute phase of RSV infection resulted in allergic airway inflammation and increased airway responsiveness.8 Enhanced effects of allergen sensitization in this model could be explained by RSVinduced increases in lung DC numbers. Elevated DC numbers can facilitate the development of allergic airway inflammation.14 In contrast, in influenza A virus infection numbers of pulmonary DCs were elevated during the acute phase of infection only,12 and sensitization and airway inflammation were induced exclusively if allergen was administered during this phase.36,37 In summary, our data provide evidence that RSV infection leads to increases in numbers of APCs in the lung, inducing their phenotypic and functional maturation. These changes occur locally only, last beyond acute infection, and might well contribute to enhanced immune and inflammatory responses subsequent to RSV infection. We thank S. Wythe for technical assistance, U. Schauer and C. Rieger for their support and helpful discussions, and P. J. M. Openshaw for critical review of the manuscript.

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Basic and clinical immunology

J ALLERGY CLIN IMMUNOL VOLUME 113, NUMBER 1