The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wohlleben, G.
Right arrow Articles by Erb, K. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wohlleben, G.
Right arrow Articles by Erb, K. J.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Eosinophilic Disorders
The Journal of Immunology, 2003, 170: 4601-4611.
Copyright © 2003 by The American Association of Immunologists

Influenza A Virus Infection Inhibits the Efficient Recruitment of Th2 Cells into the Airways and the Development of Airway Eosinophilia1

Gisela Wohlleben*, Justus Müller{dagger}, Ursula Tatsch*, Christine Hambrecht*, Udo Herz{ddagger}, Harald Renz{ddagger}, Edgar Schmitt§, Heidrun Moll* and Klaus J. Erb2,*

* Center for Infectious Diseases and {dagger} Department of Pathology, University of Würzburg, Würzburg, Germany; {ddagger} Department of Clinical Chemistry and Molecular Diagnostics, Central Laboratory, Hospital of the Philipps University, Marburg, Germany; and § Department of Immunology, University of Mainz, Mainz, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8+ T cells secreting IFN-{gamma}, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-{gamma}- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-{gamma}, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma is an allergic disorder characterized by chronic airway inflammation and airway hyperreactivity (AHR)3 induced by allergen-specific Th2 cells secreting IL-4, IL-5, and IL-13 (1). The secretion of these cytokines leads to the production of allergen-specific IgE by B cells, the development of airway eosinophilia, the induction of airway smooth muscle contraction, and mucus production, respectively. Recent studies also indicate that chemokines such as eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, and MCP-4/CCL13 are also critically involved in the development of asthma (2, 3).

Although relatively much is known about the immunological mechanisms responsible for the development of asthma, its incidence, severity, and mortality rate have steadily increased over the past decades. The reason for this alarming development remains unknown. However, it appears that environmental factors may be responsible for this development, because the genetic predisposition of the affected population has not changed. One environmental factor believed to possibly have a strong impact on the development of asthma is the lack of exposure to infectious diseases early during infancy (4, 5). The rationale behind this hypothesis (also referred to as the hygiene hypothesis) is that children infected with viruses or bacteria early in life do not develop asthma because these infections establish a Th1-biased immunity leading to the inhibition of allergen-specific Th2 cell priming or Th2 effector function. It is believed that this effect is mediated by IFN-{gamma} secreted by Th1 cells, because it has been demonstrated that IFN-{gamma} can suppress the development of Th2 cells both in vitro and in vivo (6, 7). Supporting this view are numerous studies showing that the exposure of the lung to bacteria or bacterial products inhibits the development of asthma in animal models with the effect being associated with increased Th1 immune responses (8, 9, 10).

Infections with respiratory viruses such as rhinovirus, respiratory syncytial virus (RSV), and influenza A (flu) virus also induce Th1 responses. It is conceivable that infections with these viruses could also suppress the development of asthma. However, the opposite seems to be true, because most epidemiological studies show a clear association between infections with respiratory viruses and the exacerbation of asthma in humans (11, 12, 13, 14). Animal experiments addressing this issue have yielded somewhat conflicting results with some reports indicating that respiratory viral infections increase allergen-induced Th2 responses in the lung and others reporting no effect or a suppressive effect (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). For this reason, we investigated how an intranasal (i.n.) infection with flu virus influenced the development of allergen-induced Th2 responses in the lung of mice. In contrast to most previously published reports addressing similar questions (15, 22, 25, 26), we used an OVA immunization protocol where the initial allergen-specific Th2 response does not develop in the lung. The allergen-specific Th2 response is induced by an OVA/alum protocol, leading to the recruitment of Th2 cells into the airways and resulting in airway eosinophilia, mucus production, and AHR. Using this experimental protocol, we have previously reported that the application of mycobacteria into the lung of mice leads to an almost total lack of eosinophils and Th2 cells in the airways after i.n. allergen challenge (27). In this study, we report that, similar to the mycobacterial infection, flu virus infection suppressed the development of allergen-induced airway eosinophilia and the efficient recruitment of Th2 cells into the airways. However, in contrast to mycobacterial infection, allergen-induced AHR and goblet cell metaplasia were not inhibited by the flu virus infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

The experiments were performed with C57BL/6 mice or IFN-{gamma}- and IL-12 p35-deficient mice (both on C57BL/6 background). In the experiments using Th2 cells from mice transgenic for the OVA323–339-specific TCR-{alpha}{beta} (BALB/c genetic background), BALB/c mice were used as the recipients of the T cells. C57BL/6 and BALB/c mice were purchased from Charles River (Sulzfeld, Germany), and IFN-{gamma}-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). IL-12 p35-deficient mice were generously provided by M. Kopf (Swiss Federal Institute of Technology, Zurich, Switzerland). Mice transgenic for the OVA323–339-specific TCR-{alpha}{beta} were provided by E. Schmitt (University of Mainz, Mainz, Germany). All animals used for the experiments were between 5 and 7 wk of age and housed in a conventional animal facility.

OVA immunization protocol

Mice were injected i.p. with 2 µg of OVA (Sigma-Aldrich, St. Louis, MO) in 200 µl of alum adjuvant (Serva, Heidelberg, Germany) on day 0 and boosted again i.p. with 2 µg of OVA/alum on day 14. Ten days after the second i.p. immunization, mice were anesthetized by an i.p. injection of a mixture of ketamine and xylazine (Sigma-Aldrich) and treated i.n. with 50 µl of PBS containing 100 µg of OVA.

Infection of mice with flu virus

The HKx31 (H3N2) flu virus was grown in the allantoic fluid of 10-day-old embryonated eggs. Mice were anesthetized by i.p. inoculation with Avertin (2,2,2-tribromoethanol) and infected i.n. with 30 µl of PBS containing 2 x 105 50% egg infectious dose (EID50) of the HKx31 flu virus at the indicated time points before the i.n. application of OVA. For some experiments, mice were also infected with 1 x 106, 1 x 105, 1 x 104, or 1 x 103 EID50 of the HKx31 flu virus.

Infection of mice with Nippostrongylus brasiliensis

Naive mice and mice infected with flu virus were infected with 1000 L3 larvae of N. brasiliensis i.p. as described previously (8).

Bronchoalveolar lavage (BAL)

Six days after the i.n. OVA challenge, the mice of the different groups were sacrificed, the trachea were cannulated, and a BAL was performed by flushing lung and airways five times with 1 ml of PBS. BAL cells were counted and spun onto glass slides using a cytospin (Shandon Southern Products, Asmoor, U.K.) and afterward stained with Diff-Quik according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). Percentages of macrophages, lymphocytes, neutrophils, and eosinophils were determined microscopically using standard histological criteria.

Culture conditions of cells

Single-cell suspensions from the mediastinal lymph nodes (MLN; 2 x 106 cells/ml) of the different groups of mice were prepared and cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with sodium bicarbonate (3.024 g/l), 10 µg/ml streptomycin, 10 U/ml penicillin, 50 µM 2-ME, and 10% FCS. The cell preparations were stimulated with a mAb to CD3{epsilon} (145-2C11; 25 µg/ml) together with 200 U/ml recombinant human IL-2 (Novartis, Basel, Switzerland), 40 µg/ml OVA (Sigma-Aldrich), or medium. After 48 h, the culture supernatants were harvested and tested for the presence of cytokines by ELISA.

Cytokine and chemokine ELISA

For the detection of the different cytokines and chemokines, sandwich ELISAs were performed using the following Abs: biotinylated rat anti-mouse IL-4 (BVD4-1D11) and unconjugated rat anti-mouse IL-4 (BVD6-24GL); biotinylated rat anti-mouse IL-5 (TRFK4) and unconjugated rat anti-mouse IL-5 (TRFK5); biotinylated polyclonal goat anti-mouse IL-13 and unconjugated rat anti-mouse IL-13 (38213.11); biotinylated anti-mouse IFN-{gamma} (AN-18.17.24) and unconjugated rat anti-mouse IFN-{gamma} (R4-6A2); biotinylated polyclonal goat anti-mouse eotaxin/CCL11 and unconjugated polyclonal goat anti-mouse eotaxin/CCL11; and biotinylated polyclonal goat anti-mouse RANTES/CCL5 and unconjugated mAb rat anti-mouse RANTES/CCL5 (53433). For the detection of MCP-1/CCL2, the OptEIA set from BD PharMingen (San Diego, CA) was used. The Abs for the detection of IL-4, IL-5, and IFN-{gamma} were purchased from BD PharMingen, and the Abs for the detection of IL-13, eotaxin/CCL11, and RANTES/CCL5 were purchased from R&D Systems (Wiesbaden, Germany). The ELISA were performed in polyvinyl chloride microtiter plates (Dynatech, Denkendorf, Germany) according to the instructions of the manufacturer of the Abs. The binding reactions were visualized with a conjugate of peroxidase-labeled streptavidin (DAKO, Glostrup, Denmark) and the substrate ABTS (Sigma-Aldrich). The amounts of cytokines and chemokines present in the samples were determined by including serial dilutions of the different murine cytokines or murine chemokines (recombinant cytokines and chemokines were purchased from BD PharMingen). The BAL fluid was concentrated 3-fold on ultrafiltration membranes purchased from Sigma-Aldrich (Ultrafree-15 centrifuge filter units with Biomax-5K membrane; Millipore, Bedford, MA). Briefly, for 3-fold concentration, BAL fluid was centrifuged for 15 min in the indicated centrifuge filter units (2000 rpm) at 4°C.

OVA-specific IgE and IgG1

OVA-specific Ig levels were determined by coating microtiter plates with OVA (10 µg/ml) or anti-IgE mAb (1 µg/ml) and then blocking with 10% BSA for 60 min at room temperature (RT). Two-fold dilutions of serum were added and incubated for 2 h at RT. Appropriate dilutions of biotinylated rat anti-mouse IgG1 or OVA-labeled biotin (for the detection of OVA-specific IgE) were added for 2 h at RT. The binding reactions were visualized as described in Cytokine and chemokine ELISA. As a reference, the serum of untreated control mice was also included in the ELISA. OVA-specific IgG1 and IgE titers were defined as the serum dilution at which the OD was at least 2-fold higher than the OD measured when using a 1/10 dilution of serum from untreated control mice. All Abs were purchased from BD PharMingen.

Flow cytometric analysis

CD4+ and CD8+ T cells from the BAL producing IL-4 or IFN-{gamma} were detected by using two-color FACS analysis performed with a FACScan (BD Biosciences, Mountain View, CA). For this purpose, BAL cells from the different groups of mice were spun down and then resuspended in RPMI 1640 medium containing 10% FCS. The cells were then stimulated with phorbol ester (5 µg/ml) and calcium ionophore (0.5 µM) for 6 h (both reagents from Sigma-Aldrich). Brefeldin A (2 µg/ml; Sigma-Aldrich) was added for the last 2 h of the in vitro culture period. The stainings were performed according to the instructions from BD PharMingen. Briefly, after the 6 h stimulation, BAL cells were washed and stained with anti-CD4- or anti-CD8-FITC-labeled mAb. The cells were then fixed with 4% formalin in PBS for 20 min and later incubated with anti-CD16/CD32 mAb (2.4G2; Fc Block; 5 µg/ml). After 30 min, PE-labeled anti-IL-4 mAb (11B11) or PE-labeled anti-IFN-{gamma} was added. All Abs were purchased from BD PharMingen. No CD8+ T cells producing IL-4 above background staining (using isotype-matched mAb) could be detected in the BAL of any of the mice analyzed (data not shown).

Body plethysmography

AHR was assessed by head-out body plethysmography as described previously (28). Briefly, mice were placed in four body plethysmographs attached to a exposure chamber (Crown Glass, Somerville, NJ). Airflow was measured with a PTM 378/1.2 pneumotachograph (Hugo Sachs Electronics, March-Hugstetten, Germany) and a 8-T2 differential pressure transducer (Gaeltec, Dunvegan, U.K.). Airflow in response to various concentrations of methacholine (25, 50, 75, 100, and 150 mg/ml; 1 min) was delivered by a jet nebulizer (Pari-Boy; Pari-Werke, Starnberg, Germany). The concentration of methacholine that caused a 50% reduction in expiratory airflow (MCh50) was determined.

Generation of OVA-specific Th2 cells and adoptive transfers of MLN cells

CD4+MEL-14high T cells were isolated from spleen cells of mice transgenic for the OVA323–339-specific TCR{alpha}{beta} (DO11.10) on a BALB/c genetic background by positive selection using high-gradient magnetic cell separation in combination with MultiSort beads (MACS; Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions. The CD4 sort, as well as the MEL-14 sort, was performed twice. MEL-14highCD4+ T cells were enriched >99% and showed no proliferative response in the presence of Con A or soluble anti-{alpha}{beta}TCR mAb, which indicates negligible contamination with accessory cells. Primary stimulation was conducted by incubating 1 x 106 CD4+ T cells on anti-{beta} TCR clonotype-specific mAb (KJ1-26; 5 µg/ml)-coated 24-well plates in a total volume of 1.0 ml of culture medium with the addition of IL-4 (1000 U/ml) and anti-IFN-{gamma} mAb (XMG1.2; 10 µg/ml). After 96 h, the developing T cells were transferred to uncoated 24-well culture dishes, and 0.5 ml of IL-2-containing (human rIL-2; 1000 U/ml) culture medium was added. After an additional 48 h, the T cells were collected and transferred into BALB/c mice as indicated in Fig. 3. Simultaneously, aliquots of the generated Th2 cells were restimulated by immobilized anti-{alpha}{beta}TCR mAb (5 µg/ml) to determine their cytokine profile. After an additional 18 to 24 h, supernatants were collected and assayed for cytokines. Th2 cells secreted only IL-4 and IL-5 and no or very little IFN- {gamma} (data not shown). Flow cytometry revealed that all T cell populations used for restimulation consisted of >99% CD4+ Th cells. For the adoptive transfer experiments, BALB/c mice immunized with OVA were treated i.v. with 9 x 106 flu virus-immune MLN cells (n = 8) (from mice infected 2 wk with 2 x 105 EID50 flu virus) or 9 x 106 mesenteric lymph node (MESLN) cells from naive BALB/c mice as controls (n = 7) 1 wk before the i.n. application of OVA. Numbers of eosinophils were detected in the BAL fluid of the different groups of mice (6 days after the i.n. challenge with OVA) as described in Bronchoalveolar lavage.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 3. Intranasal infection with flu virus inhibits the recruitment of in vitro-generated OVA-specific CD4+ Th2 cells into the airways and reduces airway eosinophilia. A, OVA-specific Th2 cells derived from DO11.10 TCR transgenic mice (BALB/c background) were generated as described in Materials and Methods. OVA-transgenic T cells (5 x 106) were then i.v. injected into BALB/c control mice or BALB/c mice that had been infected with 2 x 105 EID50 of flu virus 1 wk before. One and 3 days after the i.v. application of the Th2 cells, the BALB/c mice were treated with OVA i.n. B, Three days after the last OVA airway challenge, a BAL was performed and the MLN cells were isolated. BAL and MLN cells were stained with Abs against CD4 and an Ab against the transgenic TCR. Shown are the FACS stainings of the BAL and MLN cells from one control and one flu-infected mouse that are representative for eight mice per group. The percentage of CD4+ T cells expressing the transgenic TCR is indicated. C, The total amount of CD4+ T cells expressing the transgenic TCR present in the BAL and MLN together with the total eosinophil numbers of individual mice are also shown.

 
Histological analysis

Tissues from flu virus-infected and/or OVA-immunized mice were fixed in 10% phosphate-buffered formalin for 24 h and embedded in paraffin wax. Sections (2–3 µm) were cut and stained using standard histological protocols with H&E and periodic acid-Schiff reaction. The stained sections were visualized by light microscopy.

Cutaneous anaphylaxis

Active cutaneous anaphylaxis was tested in flu virus/OVA-treated C57BL/6 mice (flu virus infection 3 wk before OVA airway challenge), OVA only-treated C57BL/6 mice, or C57BL/6 control mice. Ten days after OVA airway challenge, mice were injected i.v. with 200 µl of 0.5% Evans blue in PBS (Sigma-Aldrich). Subsequently, the skin of the belly was shaved, and 50 µl of PBS, 50 µl of PBS containing OVA (50 µg/ml), or compound 48/80 (5 mg/ml; Sigma-Aldrich) was injected intradermally into two premarked sites on the skin. After 15 min, mice were sacrificed. Positive reactions to OVA resulted in mast cell degranulation and fluid extravasation, which led to the formation of a blue patch around the injection site. Mast cell reactivity was considered positive when the wheals were >3 mm. Control mice (n = 6) not subjected to OVA immunization did not show any mast cell reactivity after the application of OVA. In contrast, all the mice immunized with OVA (n = 5), and almost all the mice infected with flu virus and immunized with OVA (five of six mice) showed cutaneous mast cell reactivity after the application of OVA. None of the mice had positive skin reactivity after the application of PBS, and all the mice reacted to compound 48/80.

Statistical analysis

Statistical significance was analyzed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Infection with flu virus suppresses the development of allergen-induced airway eosinophilia

To address the question of whether an infection with flu virus modulates the development of allergen-induced Th2 responses, C57BL/6 mice were infected once with the virus at different time points and subjected to an OVA immunization protocol (Fig. 1A). Fig. 1B shows that C57BL/6 mice infected with flu virus 9, 6, 3, or 1 wk before OVA airway challenge showed a strong decrease in eosinophil numbers in the airways in comparison to OVA only-immunized C57BL/6 mice. The strongest inhibitory effect was seen when the infection was performed 1 wk before OVA airway challenge. Furthermore, we also found that using 20- or 200-fold less flu virus for the i.n. infection 1 wk before i.n. OVA challenge also suppressed allergen-induced airway eosinophilia by 90 ± 3 or 77 ± 22%, respectively, in comparison to OVA only-immunized C57BL/6 mice (mean of six mice per group with SD). Histological examinations of lung tissue from C57BL/6 mice immunized with OVA and previously infected with flu virus 1, 3, or 6 wk before the i.n. application of OVA confirmed the strong reduction in lung eosinophil numbers in the airways (data not shown). Moreover, the quality of the inflammatory response also differed in the individual groups of C57BL/6 mice. OVA-sensitized C57BL/6 mice showed mostly eosinophil and lymphocyte infiltrations in the lung, whereas neutrophils, macrophages, and lymphocytes dominated in the flu virus-infected and flu virus-infected/OVA-immunized C57BL/6 mice. Similar results were obtained when the numbers of the different cell types were determined in the BAL fluid of the C57BL/6 mice analyzed (data not shown).



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 1. Intranasal infection with flu virus strongly inhibits the development of airway eosinophilia and reduces the amount of IL-5 secreted by the T cells from the lymph node of the lung. A, Experimental design used to investigate the influence of a flu infection on OVA-induced eosinophilia in the lung. B, C57BL/6 mice were subjected to the OVA immunization scheme and infected i.n. with 2 x 105 EID50 of flu virus 1, 3, 6, or 9 wk before i.n. OVA challenge. Six days following OVA airway challenge, BALs were performed, and the cells were counted and stained with H&E, and the different cell types were identified microscopically. Shown are the mean numbers of eosinophils ± SD present in the BALs of the different groups of mice (8–20 mice/group from one to three separate experiments). C, In parallel to the BALs, single-cell suspensions (2 x 105 cells/well) from MLN of the different groups of mice were prepared and stimulated in vitro for 48 h with anti-CD3/IL-2. Shown is the mean amount of IL-5 ± SD present in the MLN cultures from the different groups of mice. The experiments were repeated at least once with similar results. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared to OVA only-treated mice.

 
The observed reduction in eosinophil numbers correlated with significantly reduced amounts of IL-5 present in cultures of MLN cells stimulated in vitro from the groups of C57BL/6 mice infected 1, 3, or 6 wk, but not 9 wk before OVA i.n. challenge (Fig. 1C). No IL-4 could be detected in any of the cultures. MLN cells from the different groups of C57BL/6 mice incubated with medium alone and MLN cells from untreated C57BL/6 control mice stimulated in vitro produced no detectable IL-5 (data not shown).

Numbers of Th2 cells present in the airways after allergen airway challenge are strongly reduced by an infection with flu virus

The flu virus infection suppressed the development of airway eosinophilia more strongly than the amount of IL-5 secreted by T cells from the MLN. Furthermore, significant inhibition of airway eosinophilia did not correlate with reduced amounts of IL-5 secreted by MLN cells in vitro in C57BL/6 mice infected with flu virus 9 wk before OVA i.n. challenge. For this reason, we analyzed whether the strong suppression of airway eosinophilia in the flu virus-infected C57BL/6 mice could be explained by a stronger reduction of Th2 cell numbers in the airways. Table I shows that the amount of CD4+ Th2 cells present in the airways of flu virus-infected/OVA-treated C57BL/6 mice was strongly reduced in comparison to the amount of Th2 cells detected in the BAL fluid of C57BL/6 mice that were only immunized with OVA. Interestingly, suppression of allergen-induced eosinophilia and the reduction of Th2 cells present in the BAL fluid correlated with a significant increase in CD8+ but not CD4+ T cells producing IFN-{gamma} (Table I). Recently, it was reported that Th2 cells show impaired homing into Th1 cell-mediated inflamed sites (29, 30, 31). Therefore, it is possible that the flu virus infection reduced the numbers of Th2 cells in the airways by interfering with the efficient recruitment of Th2 cells. However, it is also possible that Th2 cell expansion in the airways may be inhibited by the flu virus infection. Figure 2 shows that CD4+ Th2 cells producing IL-4 could already be detected 1 day after OVA airway challenge in the BAL of OVA-sensitized C57BL/6 mice. The percentage of CD4+ T cells producing IL-4 gradually increased over the next 5 days (starting at 1.27% at day 1 and reaching 7.75% at day 6 after allergen challenge). No IL-4 producers were detectable in non-OVA-immunized C57BL/6 mice or C57BL/6 mice immunized i.p. with OVA/alum and i.n. challenged with PBS instead of OVA (data not shown). In contrast, the percentage of CD4+ T cells secreting IL-4 remained very low during the entire 6 days after OVA challenge in the flu virus-infected C57BL/6 mice (0.3–0.62% with an isotype background staining of 0.1–0.2%). The numbers of total CD4+ T cells did not significantly differ in the two groups of C57BL/6 mice (data not shown). These results suggest that the flu virus infection interfered with the efficient recruitment of Th2 cells into the airways. However, although likely, it is not clear whether the Th2 cells detected in the BAL fluid were OVA specific. For this reason, we generated OVA-specific CD4+ Th2 cells in vitro (derived from DO11.10 {alpha}{beta}TCR transgenic mice on BALB/c background) and injected the Th2 cells i.v. into BALB/c control mice or BALB/c mice that had been infected with flu virus 1 wk previously. The mice were then treated three times with OVA i.n. to induce the recruitment of the Th2 cells into the airways (Fig. 3A). Figure 3, B and C, shows that the percentage and the total numbers of CD4+ T cells expressing the transgenic TCR were similar in the MLN of OVA-treated and flu virus-infected/OVA-treated BALB/c mice. However, flu virus-infected/OVA-treated BALB/c mice showed a strong decrease in the numbers of {alpha}{beta}TCR transgenic CD4+ T cells in the BAL fluid in comparison with BALB/c mice treated only with OVA. Furthermore, airway eosinophilia induced by the transferred Th2 cells was also strongly reduced in the flu virus-infected BALB/c mice (Fig. 3C). The application of OVA alone without the transfer of Th2 cells did not lead to an increase in eosinophils in the airways (data not shown). Taken together, these data suggest that an infection with flu virus interferes with the efficient recruitment of Th2 cells into the airways after allergen challenge.


View this table:
[in this window]
[in a new window]
  		Table I. Flu virus infection leads to a strong reduction in the amount of CD4+ Th2 cells present in the airways of OVA-treated micea

 


View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 2. Flu virus infection inhibited the expansion or recruitment of Th2 cells in the airways of OVA-immunized mice after allergen airway challenge. OVA-immunized C57BL/6 mice were either infected i.n. with 2 x 105 EID50 of flu virus 1 wk before OVA airway challenge or left uninfected as an OVA control (Fig. 1A). A BAL was performed on both groups of mice 1, 2, 3, and 6 days following the OVA airway challenge. BAL cells were then stimulated, fixed, and stained for the expression of CD4 and IL-4 as described in Materials and Methods. Specificity of Ab binding was controlled by staining with irrelevant isotype-matched control Abs (data not shown). Shown are FACS stainings gated on CD4+ T cells representative of six mice per group. The percentage of CD4+ T cells producing IL-4 is indicated.

 
Flu virus-induced suppression of airway eosinophilia is associated with reduced amounts of IL-5 and increased amounts of IFN-{gamma}, eotaxin/CCL11, RANTES/CCL5, and MCP-1/CCL2 present in the airways

The results in the previous section suggest that the flu virus infection may suppress the development of airway eosinophilia by reducing the amount of Th2 cells secreting IL-5 in the lung, because IL-5 is necessary for the development and recruitment of eosinophils (32, 33). Supporting this view was the finding that the IL-5 levels in the BAL of flu virus-infected/OVA-treated C57BL/6 mice were significantly lower in comparison to the levels detected in the BAL fluid of OVA only-immunized C57BL/6 mice (Fig. 4A). However, a recent publication has shown that the production of eotaxin/CCL11 is also needed for the efficient recruitment of eosinophils into the airways (33). Therefore, it may be possible that the flu virus infection reduced the amounts of eotaxin/CCL11 present in the airways after allergen challenge, thereby suppressing the development of airway eosinophilia. Surprisingly, we found that the amount of eotaxin/CCL11 was increased in the BAL fluid of flu virus-infected/OVA-treated C57BL/6 mice in comparison to OVA only-immunized C57BL/6 mice (Fig. 4C). Interestingly, RANTES/CCL5, MCP-1/CCL2, and IFN-{gamma} were also strongly elevated in the BAL fluid of flu virus-infected/OVA-treated C57BL/6 mice (Fig. 4, B, D, and E). In a separate experiment, we found that C57BL/6 mice infected with flu virus for 2 wk also produced detectable levels of IFN-{gamma} (960 ± 420 pg/ml), eotaxin/CCL11 (160 ± 40 ng/ml), RANTES/CCL5 (320 ± 100 ng/ml), and MCP-1/CCL2 (87 ± 25 ng/ml) but no IL-5 (<0.2 U/ml) in the airways (mean values of eight mice ± SD). These results suggest that flu virus-induced suppression of airway eosinophilia may be due to increased IFN-{gamma} and decreased IL-5 levels but not eotaxin/CCL11, RANTES/CCL5, or MCP-1/CCL2 levels in the airways.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 4. The production of IL-5, IFN-{gamma}, eotaxin/CCL11, RANTES/CCL5, and MCP-1/CCL2 after allergen challenge in the lung is not suppressed by infection with flu virus. C57BL/6 mice were treated as described in Fig. 3. One day after OVA airway challenge, BALs were performed, and the BAL fluid was concentrated 3-fold. Shown are the amounts of IL-5, IFN-{gamma}, eotaxin/CCL11, RANTES/CCL5, and MCP-1/CCL2 in the BALs of six to seven C57BL/6 mice per group with SD, as determined by ELISA. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared to OVA control mice.

 
IFN-{gamma}-deficient mice do not show flu virus-induced suppression of airway eosinophilia

Table I and Figure 4 show that flu virus-induced suppression of airway eosinophilia correlated with increased amounts of IFN-{gamma} and CD8+ T cells secreting IFN-{gamma} in the BAL fluid of flu virus-infected/OVA-treated C57BL/6 mice in comparison to OVA only-immunized C57BL/6 mice. These findings suggest that Th1 immune responses may be responsible for the inhibition of allergen-induced Th2 responses in the airways of flu virus-infected C57BL/6 mice. Supporting this view are our results showing that flu virus infection-mediated suppression of airway eosinophilia was not detected in IFN-{gamma}-deficient mice (C57BL/6 background) (Fig. 5A). In addition, we found that the T cells from the MLN of IFN-{gamma} deficient mice infected with flu virus and immunized with OVA or only immunized with OVA secreted strongly elevated levels of IL-5 after in vitro stimulation in comparison with those of C57BL/6 control mice (Fig. 5B). Interestingly, Figure 5C shows that IFN-{gamma}-deficient mice (C57BL/6 background) infected with flu virus alone, but not C57BL/6 control mice, also developed airway eosinophilia 1 and 2 wk after infection. MLN cells from IFN-{gamma}-deficient mice infected with flu virus also showed increased secretion of IL-5 after in vitro stimulation with anti-CD3 (Fig. 5D) and flu virus Ag (data not shown) in comparison to MLN cells from C57BL/6 control mice infected with flu virus. These results suggest that IFN-{gamma} is not only responsible for flu virus-induced suppression of allergic Th2 responses but also for the weak Th2 responses normally observed in mice infected with flu virus. IL-12 p35 (C57BL/6 background)-deficient mice infected with flu virus 1 wk before OVA i.n. challenge also showed no flu virus-induced suppression of airway eosinophilia 6 days after OVA airway challenge in comparison with IL-12 p35-deficient mice only immunized with OVA (43.5 ± 7.9 vs 45.6 ± 19.5 eosinophils/ml BAL fluid x 104 (n = 4–6/group ± SD)). Taken together, these results clearly indicate that an infection with flu virus inhibits the development of airway eosinophilia by inducing Th1 immune responses.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 5. The suppression of allergen-induced eosinophilia after flu virus infection is dependent on IFN-{gamma}. OVA-immunized C57BL/6 control and IFN-{gamma}-deficient (C57BL/6 background) mice were either infected i.n. with flu virus 1 wk before OVA airway challenge or left uninfected as an OVA control (Fig. 1A). Six days following OVA airway challenge, BALs were performed, and the MLN cells were treated as described in Fig. 1. Shown are the mean numbers of eosinophils present in the BALs (A) and the amount of IL-5 detected in the MLN cell cultures stimulated in vitro for 48 h with anti-CD3/IL-2 (B) from the different groups of mice (mean from 8–10 mice/group with SD). In a separate experiment, IFN-{gamma}-deficient and C57BL/6 control mice were infected i.n. with 2 x 105 EID50 of flu virus. One and 2 wk later, BALs were performed, and the MLN cells were treated as described in Fig. 1. Shown are the mean amounts of eosinophils present in the BAL fluid (C) and the mean amount of IL-5 detected in the cell cultures stimulated with anti-CD3/IL-2 (D) of C57BL/6 control and IFN-{gamma}-deficient mice (mean from six mice per group with SD). The experiments shown in C and D were repeated once with similar results. *, p < 0.05 compared to OVA-Wt (A and B) or Flu-Wt (C and D).

 
The development of systemic allergen-specific Th2 responses, AHR, and goblet cell metaplasia are not suppressed by infection with flu virus

Our results thus far clearly show that the flu virus infection suppressed the development of allergen-specific Th2 responses in the lung. Because Th2 responses, in particular airway eosinophilia, are known to induce airway bronchoconstriction, we analyzed whether the flu virus infection inhibited the development of AHR in OVA-immunized C57BL/6 mice. Figure 6D shows that the flu virus infection 1 or 3 wk before OVA challenge exacerbated AHR in C57BL/6 mice immunized with OVA. In addition, we found that C57BL/6 mice infected with flu virus for 2 wk, but not 4 or 7 wk, also developed AHR (MCh50 ± SD: control mice, 91.1 ± 31.1 (n = 6); 2 wk flu-infected mice, 40.9 ± 25.6 (n = 7; p < 0.05, comparison between control and 2 wk-infected mice); 4 wk flu virus-infected mice, 71.6 ± 18.7 (n = 8); and 7 wk flu-infected mice, 91.2 ± 55.2 (n = 7)). The T cells from the MLN of C57BL/6 mice infected with flu virus for 2 wk secreted increased amounts of IL-13 after in vitro stimulation with anti-CD3 (560 ± 250 pg/ml) (mean of 7 mice per group with SD; the experiment was repeated once with similar results). The amount of IL-13 in the BAL fluid of infected C57BL/6 mice and in the cultures from MLN of noninfected C57BL/6 mice stimulated with anti-CD3 was below the detection level of 100 pg/ml of the IL-13 ELISA used.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 6. Infection with flu virus does not inhibit the development of OVA-induced systemic Th2 responses and leads to increased AHR in OVA-immunized mice. C57BL/6 mice were treated as described in Fig. 1. Serum and blood smears were prepared 6 days after OVA airway challenge. A, OVA-specific IgG1 and IgE Ab levels were determined by ELISA. Values represent serum Ab titers from between 12 and 16 mice per group with SD. B, Spleen cells were prepared and stimulated with a combination of anti-CD3/IL-2 for 48 h. Shown are the mean amounts of IL-5 detected in the cultures of six mice per group with SD. C, The mean percentage of eosinophils detected in the blood of the different groups of mice is shown (six mice per group with SD). D, In a separate experiment, C57BL/6 mice were treated with OVA and infected with flu virus as described in Fig. 1. Body plethysmography was assessed 6 days after the i.n. application of OVA. Controls received PBS i.n. only. Shown is the mean MCh50 with SD from 7–11 mice per group. *, p < 0.05 compared with the values obtained in OVA-immunized mice. *, p < 0.05; **, p < 0.01 compared to PBS control.

 
Next, we analyzed whether infection with flu virus had an effect on the development of systemic OVA-specific Th2 responses. Figure 6 shows that the flu virus infection did not influence OVA-specific IgG1 or IgE levels in the serum (Fig. 6A), the production of IL-5 by T cells from the spleen (Fig. 6B), or OVA immunization-induced blood eosinophilia (Fig. 6C). Figure 7 shows that the flu virus infection also did not suppress the development of allergen-induced goblet cell metaplasia and mucus production in the lung. Interestingly, we found that the flu virus infection directly induced goblet cell metaplasia (Fig. 7, C and E).



View larger version (175K):
[in this window]
[in a new window]
  		FIGURE 7. OVA-induced goblet cell metaplasia in the lung was not inhibited by infection with flu virus. C57BL/6 mice were treated as described in Fig. 1. Sections of the lung were subjected to a periodic acid-Schiff reaction staining. Shown are untreated C57BL/6 mice (A), OVA-immunized C57BL/6 mice (B), C57BL/6 mice infected with flu virus for 2 wk (C), C57BL/6 mice infected with flu virus 1 wk before OVA airway challenge (D), C57BL/6 mice infected with flu virus for 4 wk (E), and C57BL/6 mice infected with flu virus 3 wk before OVA airway challenge (F). Representative examples of five mice per group. Goblet cells are indicated by arrows (x200 magnification).

 
A recent publication by Walzl et al. (21) showed that flu virus-induced inhibition of RSV-specific Th2 responses in the lung could be transferred via flu virus-immune lymphocytes to naive mice, with the effect being long-lived. Using a similar approach, C57BL/6 mice immunized with OVA were treated i.v. with 9 x 106 flu virus-immune MLN (from C57BL/6 mice infected 2 wk with flu virus) or 9 x 106 MESLN cells from naive C57BL/6 mice as controls 1 wk before the i.n. application of OVA. There was no significant difference in the numbers of eosinophils detected in the BAL fluid of C57BL/6 mice treated with flu virus-immune MLN cells or MESLN cells from C57BL/6 control mice (48.7 ± 14.3 (n = 7) vs 43.1 ± 24 (n = 8) eosinophils/ml BAL x 104 ± SD). This result shows that flu virus infection-mediated reduction in airway eosinophilia cannot be transferred to naive C57BL/6 mice through the i.v. application of MLN cells from flu virus-infected C57BL/6 mice. Furthermore, C57BL/6 mice treated with MLN cells from flu virus-infected C57BL/6 mice and immunized with OVA developed allergic and not Th1-type inflammation in the lung (data not shown). Taken together, these findings suggest that IFN-{gamma}-induced Th1-type inflammation in the airways is needed for the suppression of allergen-induced airway eosinophilia. The discrepancy between our findings and those published by Walzl et al. (21) may be explained by activation (possibly by cross-priming) of flu virus-specific lymphocytes by an RSV infection but not by an immunization with OVA.

Helminth-induced Th2 responses in the lung are not affected by infection with flu virus

It was recently reported that an i.n. infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) suppressed the development of both allergen- and helminth-induced airway eosinophilia and reduced the amount of Th2 cell cytokines produced by the MLN cells of BCG- and OVA-immunized or BCG- and helminth-infected C57BL/6 mice (8). We felt it important to analyze whether, in addition to modulating an allergen-specific Th2 response, an infection with flu virus also reduced the development of a helminth-induced Th2 response in the lung. Figure 8 shows that a flu infection 1 or 3 wk before an infection with N. brasiliensis did not significantly alter the development of airway eosinophilia or the amount of IL-4 and IL-5 secreted by the MLN cells after in vitro stimulation in comparison with the values obtained in N. brasiliensis only-infected C57BL/6 mice. These results suggest that the flu virus infection did not inhibit the development of pulmonary Th2 responses induced by the helminth N. brasiliensis.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 8. Flu virus infection did not inhibit the development of airway eosinophilia in mice infected with the helminth N. brasiliensis. C57BL/6 mice were coinfected with flu virus 1 (A) or 3 (B) wk before an infection with N. brasiliensis as described in Materials and Methods. As controls, C57BL/6 mice were also only infected with N. brasiliensis. Ten days after infection with the helminths, BALs were performed, and the cells were counted and stained with H&E, and the different cell types were identified microscopically. In parallel to the BALs, single-cell suspensions (2 x 105cells/well) from MLN of the different groups of mice were prepared and stimulated in vitro for 48 h on anti-CD3-bound plates in the presence of IL-2. Shown are the mean numbers of eosinophils present in the BALs and the mean amounts of IL-4 and IL-5 present in the MLN cultures from six to eight mice (A) or five mice (B) per group with SD. The experiment shown in A was repeated once with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous studies have shown that Th1 immune responses leading to the production of IFN-{gamma} can inhibit the development of allergic Th2 responses (6, 7). However, although infections with respiratory viruses induce Th1 immune responses, most epidemiological studies indicate that viral infections are associated with the exacerbation and not the inhibition of asthma in humans (11, 13, 14). Furthermore, animal experiments addressing this issue have yielded somewhat conflicting results with some reports indicating that viral infections can both increase or suppress allergen-induced Th2 responses in the lung, depending upon the virus used and the time point of infection (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). The aim of our study was to analyze whether an infection with flu virus interfered with the development of Th2 responses in the lung induced by the recruitment of Th2 cells into the airways after the i.n. application of allergen. We found that infection with flu virus 1, 3, 6, or 9 wk before i.n. allergen challenge strongly inhibited the development of airway eosinophilia. The finding that blood eosinophil levels were not reduced by the flu virus infection suggests that the recruitment of eosinophils into the airways may have been impaired by the flu virus infection. One possibility is that the flu virus infection inhibited the allergen-induced production of MCP-1/CCL2, macrophage inflammatory protein (MIP)-1{alpha}/CCL3, RANTES/CCL5, MCP-3/CCL7, eotaxin/CCL11, or macrophage-derived chemokine/CCL22 in the lung, because these chemokines are associated with the recruitment of eosinophils into the airways (2, 34). However, our results clearly show that the production of MCP-1/CCL2, RANTES/CCL5, and eotaxin/CCL11 were not impaired in C57BL/6 mice immunized with OVA and infected with the flu virus. Interestingly, we found that the flu virus infection directly induced the production of these chemokines in the airways. Furthermore, recent publications have shown that infection of epithelial cells or macrophages with flu virus induced the production of RANTES/CCL5 and MCP-1/CCL2, or RANTES/CCL5, MIP-1{alpha}/CCL3, MCP-1/CCL2 and MCP-3/CCL7 by these cells, respectively (35, 36). Taken together, these results suggest that the inhibition of allergen-induced airway eosinophilia by infection with flu virus was not due to a reduction in the production of RANTES/CCL5, MIP-1{alpha}/CCL3, MCP-1/CCL2, MCP-3/CCL7, or eotaxin/CCL11 in the airways.

Recent publications indicate that, in addition to chemokines, adhesion molecules such as VCAM-1 or ICAM-1 expressed by endothelial cells may also be involved in the efficient accumulation of eosinophils into the airways (34). Therefore, it is possible that the flu virus infection reduced the expression of these or other adhesion molecules on the endothelial cells, thereby interfering with the homing of eosinophils into the airways. Supporting this view are the recently published results of Yang et al. (37). They showed that BCG infection-induced inhibition of established allergic inflammatory responses was associated with decreased VCAM-1 expression in the lung.

The suppression of allergen-induced airway eosinophilia observed in the flu-infected mice correlated with a strong reduction in the numbers of Th2 cells present in the airways. A possible explanation for this finding may be that the flu virus infection reduced the amount of total Th2 cells generated by the two i.p. applications of OVA/alum. However, we found that OVA-specific IgG1 and IgE serum levels, blood eosinophil levels, and IL-5 production by T cells in the spleen were not reduced by infection with flu virus. This result indicates that numbers of OVA-specific Th2 cells present in the periphery of OVA-treated C57BL/6 mice did not differ from the amount of Th2 cells present in the periphery of flu virus-infected/OVA-treated C57BL/6 mice. Furthermore, we also found that a flu virus infection strongly reduced the numbers of in vitro-generated and i.v.-injected OVA-specific Th2 cells detected in the airways of BALB/c mice challenged with OVA i.n. The eosinophilia induced by the OVA-specific Th2 cells was also strongly reduced in the flu virus-infected BALB/c mice in comparison to BALB/c control mice. Taken together, these results suggest that a flu virus infection interfered with the efficient recruitment of Th2 cells into the airways.

Reduced production of IL-5 in the airways due to the inhibition of Th2 cell recruitment to this site may also be the most likely explanation for the suppression of airway eosinophilia, because it has been shown that IL-5 is necessary for the efficient recruitment and survival of eosinophils in the airways (32, 33, 38). However, we cannot completely rule out the possibility that Th2 cells effectively home into the airways of flu-infected mice but can then no longer proliferate. Supporting this possibility is the finding of Lee et al. (39) showing that clonal expansion of T cells in the lung was impaired by Th1 cell-mediated inflammation. In contrast, Harris et al. (40) recently reported that activated T cells undergo clonal expansion in the LN but not in the lung after migrating to this site. Therefore, in our opinion, the most likely explanation for the strongly reduced numbers of Th2 cells in the airways of flu-infected mice is the impaired recruitment and not impaired clonal expansion of Th2 cells. This is further supported by our finding that 3-fold fewer numbers of Th2 cells were already detected 24 h after the i.n. application of OVA in flu virus-infected/OVA-treated C57BL/6 mice in comparison to OVA only-treated mice. However, this effect may be limited to the airways, because the numbers of adoptively transferred OVA-specific transgenic Th2 cells detected in the MLN were not affected by the flu virus infection. Furthermore, the amount of IL-5 secreted by the T cells from the MLN of flu virus-infected/OVA-treated C57BL/6 mice was also not strongly reduced in comparison to the amount of IL-5 secreted by the MLN of OVA-immunized mice after in vitro stimulation.

The mechanism of how a flu virus infection interferes with the recruitment of Th2 cells into the airways is not known. However, impaired Th2 cell recruitment may be due to a reduction in the expression of chemokines or adhesion molecules in the lung needed for the efficient recruitment of Th2 cells into the airways. Prospective candidates are the chemokines macrophage-derived chemokine/CCL22, T cell activation-3/CCL1, and thymus and activation-regulated chemokine/CCL17, and the adhesion molecules VCAM-1, fibronectin CS1, and ICAM-1, -2, and -3 (2, 41). We also cannot rule out the possibility that the flu virus infection also leads to a change in chemokine receptor expression or integrins on the surface of Th2 cells needed for homing into the airways.

Although it is not clear how an infection with flu virus inhibits the development of allergen-induced Th2 responses in the lung, our experiments using IFN-{gamma}- and IL-12 p35-deficient mice suggest that it is due to Th1 immune responses. Furthermore, the suppression of allergen-induced Th2 responses by the flu virus infection correlated with a significant increase in IFN-{gamma} levels and numbers of CD8+ but not CD4+ T cells producing IFN-{gamma}, suggesting that CD8+ T cells secreting IFN-{gamma} were responsible for the suppression of the allergen-induced Th2 responses. Previous studies have also shown that flu virus-induced suppression of Th2 responses was also due to the presence of IFN-{gamma} (25). However, in contrast to our finding, the inhibition of allergen-specific Th2 responses could be explained by the suppression of Th2 cell development by IFN-{gamma}. In our system, the development of allergen-specific Th2 cells was not inhibited by the flu virus infection. Our results point at an additional mechanism of how the presence of IFN-{gamma} may interfere with the development of allergic Th2 responses, namely, that the flu virus infection induces a strong IFN-{gamma}-mediated inflammatory response in the lung that results in the impaired recruitment of allergen-specific Th2 cells into the airways. Supporting this finding are results showing a similar effect during mycobacterial infection and showing that Th2 cells could not home efficiently into Th1 cell-mediated inflamed sites (29, 30, 42). However, this may not be a general phenomenon, because it was also reported that Th1 and Th2 responses can coexist at the same site (43). Furthermore, we also found that infection with flu virus did not decrease the development of Th2 responses in the lung after infection with the helminth N. brasiliensis. In contrast, previous studies have shown that an infection with BCG can reduce the development of N. brasiliensis-induced Th2 responses. The discrepancy between this finding and our results suggests that infections with mycobacteria are more efficient in inhibiting the development of Th2 responses than are infections with flu virus. This view is supported by the observation that mycobacterial infection was more efficient than flu virus infection in inhibiting the development of allergen-induced airway eosinophilia (8, 27).

An unexpected finding was that infection with flu virus not only exacerbated but also directly induced AHR in the absence of airway eosinophilia, because eosinophils have been shown to be necessary for the induction of AHR in some experimental systems (18). How can this occur? First, the flu virus infection may directly induce AHR by increasing airway smooth muscle contraction through the production of IL-1{beta}. Rhinovirus-induced IL-1{beta}-dependent increase in airway smooth-muscle contraction has been shown to occur in vitro (44), and it has been shown that flu virus infection leads to the production of IL-1{beta} in the lung (45). A second possibility is that the flu virus infection induced the production of IL-13 in the airways. IL-13 has also been shown to induce AHR independently of eosinophils (46). This would also explain why allergen-induced mucus production was not inhibited by the flu virus infection, because goblet cell metaplasia is also induced by the production of IL-13 (46). Supporting this view was our finding that T cells from the MLN of C57BL/6 mice infected with flu virus, but not from uninfected C57BL/6 control mice, produced IL-13 after in vitro stimulation with anti-CD3 and IL-2. A further source of IL-13 possibly inducing AHR are mast cells, because they can also produce and release IL-13 (47). In unison with this hypothesis is the report from Grunewald et al. (48), who showed that the application of flu virus Ag induced the degranulation of mast cells in the skin of mice previously infected with flu virus. Furthermore, a dissociation between airway eosinophilia and AHR has also been demonstrated in other recent reports. Gerhold et al. (49) reported that the application of LPS before allergen sensitization strongly reduced the development of OVA-induced airway eosinophilia but not AHR, with the effect being dependent on IL-12. In addition, Hansen et al. (50) also reported that the application of allergen-specific Th1 cells could suppress the development of Th2 cell-induced airway eosinophilia but not AHR. Our observation together with these findings suggest that Th1 immune responses may be more efficient in inhibiting the development of Th2 cell-induced eosinophilia than AHR. However, Coyle et al. (51) reported that IL-5-deficient mice infected with N. brasiliensis also developed AHR in the absence of airway eosinophilia, suggesting that the dissociation between airway eosinophilia and AHR may not only be associated with the induction of Th1 responses.

In conclusion, we found that a flu virus infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong reduction of allergen-induced airway eosinophilia. This effect correlated with a strong reduction in the recruitment of allergen-specific Th2 cells into the airways but not MLN of the lungs. The detailed mechanism of how a flu virus infection mediates these effects remains to be determined. However, our results suggest that it is dependent upon the induction of Th1 responses. Our experiments support the view that, generally, infections that induce strong Th1 responses leading to the production of IFN-{gamma} have the potential to suppress the development of local allergic Th2 cell-mediated responses in the lung of mice and possibly also humans. The mechanism may involve the inhibition of efficient Th2 cell homing into the airways. However, our results also indicate that a flu virus infection exacerbates allergen-induced AHR and does not inhibit the development of goblet cell metaplasia. Our findings may also have implications for human disease. Children infected with flu virus may directly develop AHR and thus virus-induced asthma. Furthermore, previous studies indicate that a flu virus infection during allergen sensitization may inhibit the development of tolerance and exacerbate allergic Th2 responses (15, 25). However, in the long term, a flu virus infection may contribute to the protection against asthma by reducing the development of eosinophilic inflammation in the airways.


   Footnotes
 
1 This work was supported by the Bundesministerium für Bildung und Forschung and the Bayerisches Staatsministerium für Wissenschaft, Forschung und Kunst. Back

2 Address correspondence and reprint requests to Dr. Klaus J. Erb, Center for Infectious Diseases, University of Würzburg, Röntgenring 11, 97070 Würzburg, Germany. E-mail address: klaus.erb{at}mail.uni-wuerzburg.de Back

3 Abbreviations used in this paper: AHR, airway hyperreactivity; CCL, CC chemokine ligand; MCP, monocyte chemoattractant protein; RSV, respiratory syncytial virus; flu, influenza A; i.n., intranasal; EID50, 50% egg infectious dose; BAL, bronchoalveolar lavage; MLN, mediastinal lymph node; RT, room temperature; MCh50, concentration of methacholine that causes a 50% reduction in expiratory airflow; MESLN, mesenteric lymph node; BCG, Mycobacterium bovis bacillus Calmette-Guérin; MIP, macrophage-inflammatory protein. Back

Received for publication December 9, 2002. Accepted for publication February 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Romagnani, S.. 2000. The role of lymphocytes in allergic disease. J. Allergy Clin. Immunol. 105:399.[Medline]
  2. Lukacs, N. W.. 2001. Role of chemokines in the pathogenesis of asthma. Nat. Rev. Immunol. 1:108.[Medline]
  3. Gutierrez-Ramos, J. C., C. Lloyd, M. L. Kapsenberg, J. A. Gonzalo, A. J. Coyle. 2000. Non-redundant functional groups of chemokines operate in a coordinate manner during the inflammatory response in the lung. Immunol. Rev. 177:31.[Medline]
  4. Erb, K., G. Wohlleben. 2002. Novel vaccines protecting against the development of allergic disorders: a double-edged sword?. Curr. Opin. Immunol. 14:633.[Medline]
  5. Herz, U., P. Lacy, H. Renz, K. Erb. 2000. The influence of infections on the development and severity of allergic disorders. Curr. Opin. Immunol. 12:632.[Medline]
  6. Gajewski, T. F., F. W. Fitch. 1988. Anti-proliferative effect of IFN-{gamma} in immune regulation. I. IFN-{gamma} inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J. Immunol. 140:4245.[Abstract]
  7. O’Garra, A.. 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8:275.[Medline]
  8. Erb, K. J., J. W. Holloway, A. Sobeck, H. Moll, G. Le Gros. 1998. Infection of mice with Mycobacterium bovis-bacillus Calmette-Guerin (BCG) suppresses allergen-induced airway eosinophilia. J. Exp. Med. 187:561.[Abstract/Free Full Text]
  9. Hansen, G., V. P. Yeung, G. Berry, D. T. Umetsu, R. H. DeKruyff. 2000. Vaccination with heat-killed Listeria as adjuvant reverses established allergen-induced airway hyperreactivity and inflammation: role of CD8+ T cells and IL-18. J. Immunol. 164:223.[Abstract/Free Full Text]
  10. Sur, S., J. S. Wild, B. K. Choudhury, N. Sur, R. Alam, D. M. Klinman. 1999. Long term prevention of allergic lung inflammation in a mouse model of asthma by CpG oligodeoxynucleotides. J. Immunol. 162:6284.[Abstract/Free Full Text]
  11. Sigurs, N., R. Bjarnason, F. Sigurbergsson, B. Kjellman, B. Bjorksten. 1995. Asthma and immunoglobulin E antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls. Pediatrics 95:500.[Abstract/Free Full Text]
  12. Sigurs, N., R. Bjarnason, F. Sigurbergsson, B. Kjellman. 2000. Respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7. Am. J. Respir. Crit. Care Med. 161:1501.[Abstract/Free Full Text]
  13. Eriksson, M., R. Bennet, A. Nilsson. 2000. Wheezing following lower respiratory tract infections with respiratory syncytial virus and influenza A in infancy. Pediatr. Allergy Immunol. 11:193.[Medline]
  14. Neuzil, K. M., P. F. Wright, E. F. J. Mitchel, M. R. Griffin. 2000. The burden of influenza illness in children with asthma and other chronic medical conditions. J. Pediatr. 137:856.[Medline]
  15. Suzuki, S., Y. Suzuki, N. Yamamoto, Y. Matsumoto, A. Shirai, T. Okubo. 1998. Influenza A virus infection increases IgE production and airway responsiveness in aerosolized antigen-exposed mice. J. Allergy Clin. Immunol. 102:732.[Medline]
  16. O’Donnell, D. R., P. J. Openshaw. 1998. Anaphylactic sensitization to aeroantigen during respiratory virus infection. Clin. Exp. Allergy 28:1501.[Medline]
  17. Schwarze, J., E. Hamelmann, K. L. Bradley, K. Takeda, E. W. Gelfand. 1997. Respiratory syncytial virus infection results in airway hyperresponsiveness and enhanced airway sensitization to allergen. J. Clin. Invest. 100:226.[Medline]
  18. Schwarze, J., G. Cieslewicz, E. Hamelmann, A. Joetham, L. D. Shultz, M. C. Lamers, E. W. Gelfand. 1999. IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection. J. Immunol. 162:2997.[Abstract/Free Full Text]
  19. Scheerens, J., G. Folkerts, H. Van Der Linde, P. J. Sterk, D. M. Conroy, T. J. Williams, F. P. Nijkamp. 1999. Eotaxin levels and eosinophils in guinea pig broncho-alveolar lavage fluid are increased at the onset of a viral respiratory infection. Clin. Exp. Allergy 29:74.
  20. Dakhama, A., A. M. Bramley, N. G. Chan, K. O. McKay, R. R. Schellenberg, R. G. Hegele. 1999. Effect of respiratory syncytial virus on subsequent allergic sensitization to ovalbumin in guinea-pigs. Eur. Respir. J. 13:976.[Abstract]
  21. Walzl, G., S. Tafuro, P. Moss, P. J. Openshaw, T. Hussell. 2000. Influenza virus lung infection protects from respiratory syncytial virus-induced immunopathology. J. Exp. Med. 192:1317.[Abstract/Free Full Text]
  22. Yamamoto, N., S. Suzuki, Y. Suzuki, A. Shirai, M. Nakazawa, M. Suzuki, T. Takamasu, Y. Nagashima, M. Minami, Y. Ishigatsubo. 2001. Immune response induced by airway sensitization after influenza A virus infection depends on timing of antigen exposure in mice. J. Virol. 75:499.[Abstract/Free Full Text]
  23. Suzuki, M., S. Suzuki, N. Yamamoto, S. Komatsu, S. Inoue, T. Hashiba, M. Nishikawa, Y. Ishigatsubo. 2000. Immune responses against replication-deficient adenovirus inhibit ovalbumin-specific allergic reactions in mice. Hum. Gene Ther. 11:827.[Medline]
  24. Adamko, D. J., B. L. Yost, G. J. Gleich, A. D. Fryer, D. B. Jacoby. 1999. Ovalbumin sensitization changes the inflammatory response to subsequent parainfluenza infection: eosinophils mediate airway hyperresponsiveness, m2 muscarinic receptor dysfunction, and antiviral effects. J. Exp. Med. 190:1465.[Abstract/Free Full Text]
  25. Tsitoura, D. C., S. Kim, K. Dabbagh, G. Berry, D. B. Lewis, D. T. Umetsu. 2000. Respiratory infection with influenza A virus interferes with the induction of tolerance to aeroallergens. J. Immunol. 165:3484.[Abstract/Free Full Text]
  26. Hurst, S. D., B. W. Seymour, T. Muchamuel, V. P. Kurup, R. L. Coffman. 2001. Modulation of inhaled antigen-induced IgE tolerance by ongoing Th2 responses in the lung. J. Immunol. 166:4922.[Abstract/Free Full Text]
  27. Major, T., G. Wohlleben, B. Reibetanz, K. J. Erb. 2002. Application of heat killed Mycobacterium bovis-BCG into the lung inhibits the development of allergen-induced Th2 responses. Vaccine 20:1532.[Medline]
  28. Neuhaus-Steinmetz, U., T. Glaab, A. Daser, A. Braun, M. Lommatzsch, U. Herz, J. Kips, Y. Alarie, H. Renz. 2000. Sequential development of airway hyperresponsiveness and acute airway obstruction in a mouse model of allergic inflammation. Int. Arch. Allergy Immunol. 121:57.[Medline]
  29. Austrup, F., D. Vestweber, E. Borges, M. Lohning, R. Brauer, U. Herz, H. Renz, R. Hallmann, A. Scheffold, A. Radbruch, A. Hamann. 1997. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues. Nature 385:81.[Medline]
  30. Lukacs, N. W.. 2000. Migration of helper T-lymphocyte subsets into inflamed tissues. J. Allergy Clin. Immunol. 106:264.
  31. Iezzi, G., D. Scheidegger, A. Lanzavecchia. 2001. Migration and function of antigen-primed nonpolarized T lymphocytes in vivo. J. Exp. Med. 193:987.[Abstract/Free Full Text]
  32. Mould, A. W., A. J. Ramsay, K. I. Matthaei, I. G. Young, M. E. Rothenberg, P. S. Foster. 2000. The effect of IL-5 and eotaxin expression in the lung on eosinophil trafficking and degranulation and the induction of bronchial hyperreactivity. J. Immunol. 164:2142.[Abstract/Free Full Text]
  33. Rothenberg, M. E., J. A. MacLean, E. Pearlman, A. D. Luster, P. Leder. 1997. Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia. J. Exp. Med. 185:785.[Abstract/Free Full Text]
  34. Tachimoto, H., M. Ebisawa, B. S. Bochner. 2002. Cross-talk between integrins and chemokines that influences eosinophil adhesion and migration. Int. Arch. Allergy Immunol. 128:(Suppl. 1):18.
  35. Matikainen, S., J. Pirhonen, M. Miettinen, A. Lehtonen, C. Govenius-Vintola, T. Sareneva, I. Julkunen. 2000. Influenza A and Sendai viruses induce differential chemokine gene expression and transcription factor activation in human macrophages. Virology 276:138.[Medline]
  36. Julkunen, I., K. Melen, M. Nyqvist, J. Pirhonen, T. Sareneva, S. Matikainen. 2000. Inflammatory responses in influenza A virus infection. Vaccine 19:(Suppl. 1):S32.
  37. Yang, X., Y. Fan, S. Wang, X. Han, J. Yang, L. Bilenki, L. Chen. 2002. Mycobacterial infection inhibits established allergic inflammatory responses via alteration of cytokine production and vascular cell adhesion molecule-1 expression. Immunology 105:336.[Medline]
  38. Dent, L. A., M. Strath, A. L. Mellor, C. J. Sanderson. 1990. Eosinophilia in transgenic mice expressing interleukin 5. J. Exp. Med. 172:1425.[Abstract/Free Full Text]
  39. Lee, S. C., Z. H. Jaffar, K. S. Wan, S. T. Holgate, K. Roberts. 1999. Regulation of pulmonary T cell responses to inhaled antigen: role in Th1- and Th2-mediated inflammation. J. Immunol. 162:6867.[Abstract/Free Full Text]
  40. Harris, N. L., V. Watt, F. Ronchese, G. Le Gros. 2002. Differential T cell function and fate in lymph node and nonlymphoid tissues. J. Exp. Med. 195:317.[Abstract/Free Full Text]
  41. Yusuf-Makagiansar, H., M. E. Anderson, T. V. Yakovleva, J. S. Murray, T. J. Siahaan. 2002. Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic approach to inflammation and autoimmune diseases. Med. Res. Rev. 22:146.[Medline]
  42. Erb, K. J., C. Trujillo, M. Fugate, H. Moll. 2002. Infection with the helminth Nippostrongylus brasiliensis does not interfere with efficient elimination of Mycobacterium bovis BCG from the lungs of mice. Clin. Diagn. Lab. Immunol. 9:727.[Abstract/Free Full Text]
  43. Li, L., Y. Xia, A. Nguyen, L. Feng, D. Lo. 1998. Th2-induced eotaxin expression and eosinophilia coexist with Th1 responses at the effector stage of lung inflammation. J. Immunol. 161:3128.[Abstract/Free Full Text]
  44. Hakonarson, H., C. Carter, N. Maskeri, R. Hodinka, M. M. Grunstein. 1999. Rhinovirus-mediated changes in airway smooth muscle responsiveness: induced autocrine role of interleukin-1{beta}. Am. J. Physiol. 277:13.
  45. Van Reeth, K., H. Nauwynck, M. Pensaert. 1998. Bronchoalveolar interferon-{alpha}, tumor necrosis factor-{alpha}, interleukin-1, and inflammation during acute influenza in pigs: a possible model for humans?. J. Infect. Dis. 177:1076.[Medline]
  46. Kuperman, D. A., X. Huang, L. L. Koth, G. H. Chang, G. M. Dolganov, Z. Zhu, J. A. Elias, D. Sheppard, D. J. Erle. 2002. Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat. Med. 8:885.[Medline]
  47. Kobayashi, H., Y. Okayama, T. Ishizuka, R. Pawankar, C. Ra, M. Mori. 1998. Production of IL-13 by human lung mast cells in response to Fc{epsilon} receptor cross-linkage. Clin. Exp. Allergy 28:1219.[Medline]
  48. Grunewald, S. M., C. Hahn, G. Wohlleben, M. Teufel, T. Major, H. Moll, E. B. Brocker, K. J. Erb. 2002. Infection with influenza A virus leads to flu antigen-induced cutaneous anaphylaxis in mice. J. Invest. Dermatol. 118:645.[Medline]
  49. Gerhold, K., K. Blumchen, A. Bock, C. Seib, P. Stock, T. Kallinich, M. Lohning, U. Wahn, E. Hamelmann. 2002. Endotoxins prevent murine IgE production, TH2 immune responses, and development of airway eosinophilia but not airway hyperreactivity. J. Allergy Clin. Immunol. 110:110.[Medline]
  50. Hansen, G., G. Berry, R. H. DeKruyff, D. T. Umets. 1999. Allergen-specific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation. J. Clin. Invest. 103:175.[Medline]
  51. Coyle, A. J., G. Kohler, S. Tsuyuki, F. Brombacher, M. Kopf. 1998. Eosinophils are not required to induce airway hyperresponsiveness after nematode infection. Eur. J. Immunol. 28:2640.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. A. Al-Garawi, R. Fattouh, T. D. Walker, E. B. Jamula, F. Botelho, S. Goncharova, J. Reed, M. R. Stampfli, P. M. O'Byrne, A. J. Coyle, et al.
Acute, but Not Resolved, Influenza A Infection Enhances Susceptibility to House Dust Mite-Induced Allergic Disease
J. Immunol., March 1, 2009; 182(5): 3095 - 3104.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
Q. Wu, R. J. Martin, J. G. Rino, S. Jeyaseelan, R. Breed, and H. W. Chu
A deficient TLR2 signaling promotes airway mucin production in Mycoplasma pneumoniae-infected allergic mice
Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1064 - L1072.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol PatholHome page
J. P. Buchweitz, J. R. Harkema, and N. E. Kaminski
Time-Dependent Airway Epithelial and Inflammatory Cell Responses Induced by Influenza Virus A/PR/8/34 in C57BL/6 Mice
Toxicol Pathol, April 1, 2007; 35(3): 424 - 435.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. F. Debes, M. E. Dahl, A. J. Mahiny, K. Bonhagen, D. J. Campbell, K. Siegmund, K. J. Erb, D. B. Lewis, T. Kamradt, and A. Hamann
Chemotactic Responses of IL-4-, IL-10-, and IFN-{gamma}-Producing CD4+ T Cells Depend on Tissue Origin and Microbial Stimulus
J. Immunol., January 1, 2006; 176(1): 557 - 566.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Keshavan, T. L. Deem, S. J. Schwemberger, G. F. Babcock, J. M. Cook-Mills, and S. D. Zucker
Unconjugated Bilirubin Inhibits VCAM-1-Mediated Transendothelial Leukocyte Migration
J. Immunol., March 15, 2005; 174(6): 3709 - 3718.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
A. E. Oran and H. L. Robinson
DNA Vaccines: Influenza Virus Challenge of a Th2/Tc2 Immune Response Results in a Th2/Tc1 Response in the Lung
J. Virol., April 15, 2004; 78(8): 4376 - 4380.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
G. Wohlleben, C. Trujillo, J. Muller, Y. Ritze, S. Grunewald, U. Tatsch, and K. J. Erb
Helminth infection modulates the development of allergen-induced airway inflammation
Int. Immunol., April 1, 2004; 16(4): 585 - 596.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wohlleben, G.
Right arrow Articles by Erb, K. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wohlleben, G.
Right arrow Articles by Erb, K. J.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Eosinophilic Disorders

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS