help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on October 30, 2003, doi:10.1165/rcmb.2003-0181OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0181OCv1
30/5/702    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rosenthal, L. A.
Right arrow Articles by Lemanske, R. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rosenthal, L. A.
Right arrow Articles by Lemanske, R. F., Jr.
American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 702-709, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0181OC

Attenuated Innate Mechanisms of Interferon-{gamma} Production in Rats Susceptible to Postviral Airway Dysfunction

Louis A. Rosenthal, Lance D. Mikus, Amjad Tuffaha, Anne G. Mosser, Ronald L. Sorkness and Robert F. Lemanske, Jr.

Morris Institute for Respiratory Research, and Departments of Medicine and Pediatrics, University of Wisconsin Medical School; and School of Pharmacy, University of Wisconsin–Madison, Madison, Wisconsin

Address correspondence to: Louis A. Rosenthal, Ph.D., University of Wisconsin Medical School, K4-948 CSC-9988, 600 Highland Avenue, Madison, WI 53792. E-mail: lar{at}medicine.wisc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
After Sendai virus (SeV)-induced bronchiolitis as weanlings, BN, but not F344, rats develop a postbronchiolitis asthma-like phenotype, which can be prevented by supplemental interferon (IFN)-{gamma} treatment. We have shown that splenocytes from BN weanlings, compared with those from F344 weanlings, have a markedly reduced capacity for IFN-{gamma} production. We hypothesized that SeV-induced IFN-{gamma} production occurs via innate mechanisms that are attenuated in BN weanlings. Therefore, we investigated potential mechanisms of SeV-induced IFN-{gamma} production in BN and F344 weanlings. SeV-stimulated splenocytes secreted the IFN-{gamma}–inducing cytokines, interleukin (IL)-12 and IL-18. BN splenocytes produced significantly less IL-12 (P = 0.001) and IL-18 (P < 0.001) than did F344 splenocytes. Depletion studies demonstrated that natural killer cells were the primary source of SeV-induced IFN-{gamma} production. Anti–IL-12 antibody, IL-12 p40 homodimer, and IL-18 binding protein each inhibited SeV-induced IFN-{gamma} production by 82–94%, and the combination of IL-12 p40 homodimer and IL-18 binding protein abolished SeV-induced IFN-{gamma} production, demonstrating synergism between IL-12 and IL-18. Therefore, SeV-induced IFN-{gamma} production occurred via innate IL-12–, IL-18–, and natural killer cell–dependent mechanisms, which were attenuated in BN weanlings. Attenuation of innate IFN-{gamma}–producing responses to SeV in BN weanlings may be a critical factor in their susceptibility to postbronchiolitis chronic airway dysfunction.

Abbreviations: analysis of variance, ANOVA • enzyme-linked immunosorbent assay, ELISA • interferon, IFN • interleukin, IL • chimeric protein linking IL-18 binding protein a and Fc region of IgG1, IL-18BPa/Fc • natural killer, NK • peripheral blood mononuclear cells, PBMC • plaque-forming units, pfu • respiratory syncytial virus, RSV • Sendai virus, SeV


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Current evidence supports the hypothesis that genetic, environmental, and developmental factors contribute to the inception of asthma in childhood, but the identity and complex interplay of these factors remain to be elucidated (1). Respiratory syncytial virus (RSV)-induced bronchiolitis has been identified as an important risk factor for the development of asthma in children (2–4). Although it is unclear why some children are susceptible to the development of a postbronchiolitis asthmatic phenotype whereas others experience no chronic airway sequelae, decreased interferon (IFN)-{gamma} production by peripheral blood mononuclear cells (PBMC) from infants during virus-induced bronchiolitis has been associated with susceptibility to subsequent persistent lower respiratory tract symptoms (5).

A rat model that parallels the early childhood asthmatic phenotype in several respects has been generated to investigate the potential roles of cytokine dysregulation and respiratory viral infection in the development of asthma. Following parainfluenza type 1 (Sendai) virus (SeV)-induced bronchiolitis as weanlings (3–4 wk of age), high IgE producer "atopic" BN rats, but not low IgE producer "nonatopic" F344 rats, develop an asthma-like phenotype characterized by episodic reversible airway obstruction, airway hyperresponsiveness to methacholine, chronic airway inflammation, and airway wall remodeling (6–8). The development of this postbronchiolitis asthma-like phenotype resembles asthma inception in children in that genetic (rat strain), environmental (paramyxovirus infection), and developmental (age at time of respiratory viral illness) factors interact, in ways that remain to be determined, to induce chronic airway dysfunction (9).

Several lines of evidence indicate that susceptibility to the development of postbronchiolitis chronic airway dysfunction is associated with differences between BN and F344 rats with regard to host responses to SeV infection. There are no strain-related differences in lung viral titers during the acute SeV infection or in the subsequent persistence of viral RNA in the lungs and peribronchial lymph nodes (10). Therefore, differences in the host response to the infection, rather than differences in the magnitude or rate of clearance of the infection, might be the principal explanation for differential susceptibility to chronic airway sequelae. The observed strain-related differences in IFN-{gamma} production in response to SeV support this view. For example, during the acute SeV infection, peak bronchoalveolar lavage fluid IFN-{gamma} levels are significantly lower in BN weanlings than in F344 weanlings (11). In addition, splenocytes from uninfected BN weanlings, compared with those from uninfected F344 weanlings, exhibit significantly lower levels of SeV-induced IFN-{gamma} production, and in response to interleukin (IL)-12, natural killer (NK) cells from BN weanlings secrete significantly less IFN-{gamma} than do those from F344 weanlings (11). Supplementation of endogenous IFN-{gamma} production by treatment of BN weanlings with aerosolized IFN-{gamma} during the acute infection prevents the development of the postbronchiolitis chronic airway sequelae (8). Furthermore, treatment of F344 weanlings with anti–IFN-{gamma} antibody before SeV inoculation markedly reduces peak bronchoalveolar lavage fluid IFN-{gamma} levels and renders these rats susceptible to the development of postbronchiolitis chronic airway dysfunction, including increased airway obstruction and premature airway closure (12). Therefore, the host's capacity to mount an IFN-{gamma} response during the acute viral bronchiolitis appears to be a critical factor regulating the ability of the host to resolve this early life airway injury.

Given these results, we hypothesized that the production of IFN-{gamma} during the host's initial exposure to SeV occurs via innate mechanisms, which are attenuated in BN weanlings. Consequently, we investigated mechanisms regulating SeV-induced IFN-{gamma} production in splenocytes from uninfected weanlings of both strains. Our results demonstrate that innate mechanisms, which are dependent on IL-12, IL-18, and NK cells, are responsible for SeV-induced IFN-{gamma} production in these cultures, and that these innate responses are markedly attenuated in BN weanlings.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Specific pathogen–free inbred BN/CrlBR and CDF (F344)/CrlBR male rats were purchased as 3- to 4-wk-old weanlings from Charles River Laboratories (Wilmington, MA). The animals were housed in an American Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited facility, and all procedures were approved by the University of Wisconsin Animal Care and Use Committee.

Virus
Parainfluenza 1 (Sendai) virus strain P3193 was propagated in the allantoic fluid of 9- to 11-d-old embryonated chicken eggs. Inoculated eggs were incubated at 37°C for 48 h, and the allantoic fluid was collected aseptically. Pooled allantoic fluid was centrifuged for 20 min at 500 x g. The supernate was frozen at –80°C overnight, rapidly thawed, centrifuged, and stored in small aliquots at –80°C. No bacterial growth was observed when samples of the supernate were cultured in tryptic soy broth and thioglycollate broth. Allantoic fluid, which served as a vehicle control, was collected aseptically from uninfected eggs and stored at –80°C. Viral titers were determined by plaque assay as described by Castleman and colleagues (13). The SeV stock preparation had a titer of 1.2 x 109 plaque-forming units (pfu). To obtain ultraviolet-inactivated SeV, the virus stock, 3 ml in a 60-mm tissue culture dish, was exposed for 60 min to a germicidal ultraviolet lamp at a distance of 10 cm. The preparation was reconstituted to the original volume with allantoic fluid and frozen at –80°C. A plaque assay showed no detectable infectious virus in this ultraviolet-inactivated virus preparation. For other studies, virus stock was added to Microcon centrifugal filter devices with a molecular weight cutoff of 100,000 (Millipore, Bedford, MA) and centrifuged for up to 14 min at 14,000 x g. The filtrate, which contained no detectable infectious virus by plaque assay, was aliquotted and stored at –80°C.

Cells
Single cell suspensions of spleen cells were prepared from uninfected BN and F344 weanling rats as previously described (11). For experiments requiring depletion of either NKR-P1A+ (a marker of NK cells) or CD3+ cells from spleen cell suspensions, splenocytes were incubated with FITC-conjugated mAbs to either NKR-P1A (10/78; mouse IgG1) or CD3 (G4.18; mouse IgG3) (BD Pharmingen, San Diego, CA), respectively, and then with an anti-FITC mAb which is coupled to super-paramagnetic microbeads (Miltenyi Biotec, Auburn, CA). The spleen cell suspensions were then depleted of either NKR-P1A+ or CD3+ cells by negative selection using an automated magnetic cell sorting system (autoMACS; Miltenyi Biotec) according to the manufacturer's instructions. The resulting cell fractions were analyzed by flow cytometry, as previously described (11), to confirm that they were essentially free of FITC-labeled cells. Unfractionated splenocytes or splenocytes depleted of either NKR-P1A+ or CD3+ cells (5 x 105 cells/well) were incubated as triplicate cultures for 24 h in 96-well round bottom plates at 37°C in 5% CO2. The cells were cultured, at a final volume of 200 µl, in complete medium containing RPMI 1640 (Mediatech, Herndon, VA) with 10% fetal calf serum (Hyclone, Logan, UT), 4 mM L-glutamine, 10 mM Hepes, 100 U/ml penicillin, 100 µg/ml streptomycin (Mediatech), and 50 µM 2-mercaptoethanol (Sigma, St. Louis, MO). Cells were incubated with either SeV (5 x 105 or 5 x 106 pfu/well, which represents a multiplicity of infection of 1 or 10, respectively), ultraviolet-inactivated SeV (5 x 105 or 5 x 106 pfu equivalents/well), 100,000 molecular weight cutoff filtrate from SeV stock preparation, 2 ng/ml of recombinant murine IL-12 (R&D Systems, Minneapolis, MN), or 10 µg/ml of Con A (Sigma). Control wells contained cells incubated with either medium or an appropriate concentration of allantoic fluid, the vehicle for the SeV. In some experiments, either neutralizing, affinity-purified goat anti-murine IL-12 IgG (R&D Systems), control goat IgG (Sigma), a recombinant chimeric protein linking human IL-18 binding protein a with the Fc region of human IgG1 (IL-18BPa/Fc; R&D Systems), or control myeloma-derived human IgG1 (Sigma) were added to wells at a final concentration of 1 µg/ml, which yielded optimal inhibition in preliminary experiments. In other experiments, recombinant murine IL-12 p40 homodimer (R&D Systems) was added to wells at final concentrations of 0.2, 2, or 20 ng/ml. Polymyxin B (Sigma) was added to some cultures at a final concentration of 5 µg/ml. After the 24-h incubation period, the supernates were harvested, centrifuged to remove residual cells, and stored at –80°C.

Splenic Macrophages
To prepare cultures of splenic macrophages, 1.5 x 107 spleen cells in 1.5 ml of complete medium were added to each well of 6-well tissue culture plates and incubated for 1 h at 37°C. Nonadherent cells were removed by washing with warm RPMI 1640, and the adherent cells were incubated in complete medium overnight at 37°C. After this incubation, the cell monolayers were washed extensively with warm RPMI 1640. Adherent cell monolayers were incubated in a final volume of 1 ml complete medium with either SeV (2.5 x 107 pfu/well, which yielded the same final concentration of virus as the higher viral dose in the splenocyte experiments) or an equivalent volume of allantoic fluid for 24 h at 37°C. The supernates were harvested, centrifuged to remove residual cell debris, and stored at –80°C. To confirm that the monolayers had equivalent numbers of cells, the wells were washed thoroughly with phosphate-buffered saline before the adherent cells were lysed by the addition of 1 ml of 1% Triton X-100 (Pierce, Rockford, IL) to each well. The protein concentrations of the lysates were determined using a Coomassie protein assay reagent (Pierce) according to the manufacturer's instructions.

Virus Replication
Splenic macrophage cultures were prepared in 6-well tissue culture plates as described above, except that 3 x 107 spleen cells in 1 ml of complete medium were added to each well and incubated for 1.5 h at 37°C. Adherent cell monolayers were incubated with SeV (2.5 x 107 pfu/well) in 1 ml of RPMI 1640 for 1 h at 37°C. After thorough washing with RPMI 1640, 1 ml of serum-free Macrophage-SFM medium (Invitrogen, Carlsbad, CA) was added to each well. The plates were incubated for an additional 2, 24, 48, or 96 h (one well per time point in each experiment) at 37°C and then stored at –80°C. Viral titers in the supernates were determined by plaque assays (13), which were performed in triplicate. The macrophages were cultured under serum-free conditions to prevent interference with the trypsin-dependent step in the plaque assay (13).

Enzyme-Linked Immunosorbent Assay
The levels of cytokines in the supernates were measured by enzyme-linked immunosorbent assay (ELISA). Rat IFN-{gamma}–specific ELISA kits, with a sensitivity of 13 or 16 pg/ml, were purchased from Biosource International (Camarillo, CA) or BD Pharmingen, respectively. Rat IL-12 p70 heterodimer– and rat IL-18–specific ELISA kits, with sensitivities of 2.5 and 4 pg/ml, respectively, were obtained from Biosource International. ELISA kits that recognize both the p70 heterodimer and p40 subunit of rat IL-12 were acquired from Biosource International and had a sensitivity of 5 pg/ml.

Data Analyses
If log-transformed data conformed to the assumptions for parametric tests, a randomized block (where each block represented an individual source of splenocytes) ANOVA was performed, and Fischer's least significant difference test was used for planned post hoc pairwise comparisons. A residual analysis was employed to test the adequacy of the ANOVA models. For other comparisons, a repeated measures ANOVA was performed using paired treatment groups for each batch of splenocytes. In other instances, paired t tests were performed, and Bonferroni adjustments were used to account for multiple planned comparisons. Data that did not conform to parametric assumptions were analyzed in an analogous manner using Kruskal-Wallis and Mann-Whitney tests. SYSTAT version 10 software (SPSS, Chicago, IL) was used for analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SeV-Induced IL-12 Production by Splenocytes
To evaluate the role of IL-12 in the innate responses of weanling rats to SeV, we examined whether IL-12 production is induced by SeV and whether BN and F344 splenocytes differ with respect to their capacity for SeV-induced IL-12 production. Detectable levels of IL-12 p70 heterodimer (the active form of IL-12) were measured in the supernates of SeV-stimulated splenocyte cultures from all eight F344 and seven of eight BN weanling rats (Figure 1) . However, BN splenocytes produced significantly less IL-12 in response to both doses of SeV than did F344 splenocytes (Figure 1; P = 0.001). At virus doses of 5 x 105 and 5 x 106 pfu, the median levels of IL-12 secreted by BN splenocytes (4.2 and 6.2 pg/ml, respectively) were 3.6- and 5-fold lower, respectively, than those secreted by F344 splenocytes (15 and 31 pg/ml, respectively). Supernates from BN and F344 splenocytes that had been incubated with allantoic fluid (the vehicle for the SeV) contained little or no IL-12. In other experiments, using an ELISA that detects both IL-12 p70 and the IL-12 p40 subunit, BN splenocytes also produced significantly less IL-12 p70/p40 in response to both doses of SeV than did F344 splenocytes (n = 5 rats per strain; P = 0.009, Mann-Whitney test). At virus doses of 5 x 105 and 5 x 106 pfu, the median levels of IL-12 p70/p40 were, respectively, 5 and 6 pg/ml for BN splenocytes and 45 and 111 pg/ml for F344 splenocytes, and there was no IL-12 p70/p40 detected in cultures incubated with allantoic fluid.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. IL-12 production by splenocytes from weanling BN and F344 rats in response to stimulation with SeV. Splenocytes from weanling BN (open circles) and F344 (filled circles) rats were incubated for 24 h with SeV (5 x 105 or 5 x 106 pfu/well), and IL-12 p70 levels were measured in the supernates by ELISA. Data represent mean values for triplicate cultures from individual rats of each strain (n = 8). Bars indicate medians. Dotted line represents the lower limit of detection of the IL-12 p70 ELISA (2.5 pg/ml). IL-12 p70 was not detected in splenocyte cultures incubated with allantoic fluid, with the exception of two F344 cultures, which had values of 3.4 and 4.4 pg/ml. Asterisks indicate a significant difference between BN and F344 responses to the same dose of virus (P = 0.001; Mann-Whitney test).

 
Role of IL-12 in SeV-Induced IFN-{gamma} Production by Splenocytes
Given the observation that IL-12 secretion was induced by incubation of splenocytes with SeV, we examined whether IL-12 was an important mediator of SeV-induced IFN-{gamma} production in these cultures. Consistent with our previous findings (11), BN splenocytes produced significantly less IFN-{gamma} in response to SeV than did F344 splenocytes (Figure 2 ; P < 0.001). The median levels of IFN-{gamma} secreted by BN splenocytes, at virus doses of 5 x 105 and 5 x 106 pfu, were almost 8- and 11-fold lower, respectively, than those secreted by F344 splenocytes. Incubation of BN or F344 splenocytes with allantoic fluid resulted in no detectable IFN-{gamma} production. The addition of neutralizing anti–IL-12 antibody to either BN or F344 splenocyte cultures significantly reduced the levels of SeV-induced IFN-{gamma} production compared with addition of either no antibody or a control antibody to the cultures (Figure 2; P < 0.001). IFN-{gamma} values for cultures treated with the control antibody did not significantly differ from those that were not treated with antibody. The median percent reductions in SeV-induced IFN-{gamma} levels in BN and F344 splenocyte cultures containing anti–IL-12 antibody, compared with parallel cultures that received no antibody, were 94% and 82%, respectively, at a virus dose of 5 x 105 pfu, and 92% and 86%, respectively, at a virus dose of 5 x 106 pfu, which demonstrated that this IFN-{gamma} production was markedly dependent on IL-12.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 2. Effect of neutralizing antibody to IL-12 on SeV-induced IFN-{gamma} production by splenocytes from weanling BN and F344 rats. Splenocytes from weanling BN (open boxes) and F344 (filled boxes) rats were incubated for 24 h with either (A) 5 x 105 or (B) 5 x 106 pfu/well of SeV in the presence of either no antibody (No Ab), a control antibody (Ctrl Ab; 1 µg/ml), or a neutralizing antibody to IL-12 ({alpha}–IL-12; 1 µg/ml), and IFN-{gamma} levels were measured in the supernates by ELISA. Data represent mean values for triplicate cultures from individual rats of each strain (n = 3) and are shown as box plots. Dotted line represents the lower limit of detection of the IFN-{gamma} ELISA (16 pg/ml). IFN-{gamma} was not detected in supernates from BN and F344 splenocytes incubated with allantoic fluid. There was a significant difference between BN and F344 responses to SeV (P < 0.001; ANOVA). Asterisks indicate a significant difference between responses of cultures treated with either no antibody or control antibody and those treated with anti–IL-12 antibody (P < 0.001; ANOVA).

 
Cell Source of SeV- and IL-12–Induced IFN-{gamma} Production
To determine the cell source(s) of SeV- and IL-12–induced IFN-{gamma} production, we obtained spleen cells from uninfected weanling BN and F344 rats, and used mAbs to either NKR-P1A or CD3 along with magnetic cell sorting to deplete the cell suspensions of either NK cells or T cells, respectively. Unfractionated weanling BN and F344 splenocytes as well as spleen cells depleted of either NKR-P1A+ or CD3+ cells were incubated for 24 h with either SeV (5 x 105 pfu), IL-12, or Con A (a T cell mitogen), and IFN-{gamma} levels were measured in the supernates (Figure 3) . The levels of SeV- and IL-12–induced IFN-{gamma} in BN and F344 spleen cell cultures without NKR-P1A+ cells were significantly lower than those in unfractionated spleen cell cultures (P < 0.003); depletion of NKR-P1A+ cells abrogated IFN-{gamma} production by weanling BN and F344 spleen cells (Figures 3A and 3B). However, Con A–stimulated IFN-{gamma} production by spleen cells depleted of NKR-P1A+ cells was comparable to that of unfractionated splenocytes, which indicated that CD3+ T cells were present and functional (Figure 3C). In contrast, SeV- and IL-12–stimulated IFN-{gamma} production by spleen cells depleted of CD3+ cells was comparable to that of unfractionated splenocytes, and depletion of CD3+ cells from splenocytes inhibited Con A–stimulated IFN-{gamma} production by 97–100% (Figure 3). Supernates from spleen cells incubated with either allantoic fluid or medium alone contained no detectable IFN-{gamma}. Two independent experiments using the 5 x 106 pfu dose of SeV yielded comparable results. These results demonstrated that NK (NKR-P1A+ CD3–) cells were the primary source of SeV- and IL-12–induced IFN-{gamma} production in weanling BN and F344 splenocyte cultures.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Effect of depletion of either NK (NKR-P1A+) cells or T (CD3+) cells on SeV-, IL-12-, and Con A–induced IFN-{gamma} production by splenocytes from weanling BN and F344 rats. Unfractionated splenocytes (open boxes) from weanling BN and F344 rats or splenocytes that had been depleted of either NKR-P1A+ (filled boxes) or CD3+ cells (filled triangles) were incubated for 24 h with either (A) SeV (5 x 105 pfu/well), (B) IL-12 (2 ng/ml), or (C) Con A (4 µg/ml), and IFN-{gamma} levels were measured in the supernates by ELISA. Data represent mean values for triplicate cultures from two to three independent experiments. Dotted line represents the lower limit of detection of the IFN-{gamma} ELISA (13 pg/ml). IFN-{gamma} was not detected in supernates from BN and F344 splenocytes incubated with medium or allantoic fluid. For both rat strains, there was a significant difference between unfractionated splenocytes and splenocytes depleted of NKR-P1A+ cells with regard to responses to either SeV or IL-12 (P < 0.003; repeated measures ANOVA).

 
SeV-Induced IL-12 Production by Splenic Macrophages
We evaluated splenic macrophages to determine if these cells could be a source of the IL-12 that mediates IFN-{gamma} production by SeV-stimulated splenocytes. Consistent with the results in Figure 1, macrophages from BN weanlings, compared with those from F344 weanlings, produced significantly less IL-12 p70 in response to SeV (Figure 4 ; P < 0.001). Little or no IL-12 was detected in macrophage cultures that had been incubated with allantoic fluid. Although IL-12 p70 was not detected in most of the SeV-stimulated BN macrophage cultures (Figure 4), in other experiments, SeV-stimulated macrophages from all of the BN rats tested produced measurable levels of IL-12 p70/p40. Similar to the data in Figure 4, BN macrophages produced significantly less SeV-induced IL-12 p70/p40 than did F344 macrophages, with median levels of 11 and 80 pg/ml, respectively (n = 5 rats per strain; P = 0.009, Mann-Whitney test). Lysates of the BN and F344 macrophage monolayers had equivalent protein concentrations, which indicated that equivalent numbers of cells had been present (data not shown).



View larger version (11K):
[in this window]
[in a new window]
  		Figure 4. IL-12 production by splenic macrophages from weanling BN and F344 rats in response to stimulation with SeV. Splenic macrophages from weanling BN (open circles) and F344 (filled circles) rats were incubated for 24 h with SeV (2.5 x 107 pfu/well), and IL-12 p70 levels were measured in the supernates by ELISA. Data represent values for cultures from individual rats of each strain (n = 8). Bars indicate medians. Dotted line represents the lower limit of detection of the IL-12 p70 ELISA (2.5 pg/ml). A Mann-Whitney test was used to compare BN and F344 responses. IL-12 p70 was not detected in macrophage cultures incubated with allantoic fluid, with the exception of one F344 culture, which had a value of 2.6 pg/ml.

 
SeV-Induced IFN-{gamma} and IL-12 Production: Role of Viral Infectivity
To determine whether active, infectious SeV was required for cytokine induction, splenocytes from uninfected weanling BN and F344 rats were incubated for 24 h with either active, infectious SeV or ultraviolet-inactivated, noninfectious SeV. Ultraviolet-inactivated virus was less potent than active virus with regard to the stimulation of IFN-{gamma} and IL-12 p70 production by BN and F344 splenocytes (Figures 5A and 5B , respectively). The reduction in potency was most pronounced at the lower viral dose. At the lower dose of 5 x 105 pfu, ultraviolet inactivation of SeV almost completely abrogated IFN-{gamma} (P < 0.002) and IL-12 (P < 0.01) production by BN and F344 splenocytes, with median percent reductions of 95–100%. However, at the higher dose of 5 x 106 pfu, ultraviolet inactivation of SeV resulted in a significant, but sometimes incomplete, reduction in IFN-{gamma} and IL-12 production by BN and F344 splenocytes (P < 0.002).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. IFN-{gamma} and IL-12 production by BN and F344 splenocytes in response to ultraviolet-inactivated SeV, and SeV replication in splenic macrophages. Splenocytes from weanling BN (open boxes) and F344 (filled boxes) rats were incubated for 24 h with either SeV (5 x 105 or 5 x 106 pfu/well) or an equivalent amount of ultraviolet-inactivated SeV (UV-SeV), and (A) IFN-{gamma} and (B) IL-12 p70 levels were measured in the supernates by ELISA. Data represent mean values for triplicate cultures from three independent experiments and are shown as box plots. Dotted lines represent the lower limits of detection of the IFN-{gamma} (16 pg/ml) and IL-12 p70 (2.5 pg/ml) ELISAs. After incubation with allantoic fluid, IFN-{gamma} and IL-12 p70 were not detected in supernates from BN and F344 splenocytes. Significant differences were observed between responses to SeV and those to ultraviolet-inactivated SeV (*P < 0.002, repeated measures ANOVA; **P < 0.01, paired t test). (C) Splenic macrophage cultures from BN (open boxes) and F344 (filled boxes) weanlings were inoculated with SeV (2.5 x 107 pfu/well) and incubated for the indicated times. Viral titers were determined by plaque assay. Data represent mean values for triplicate plaque assay determinations for each culture. Data from three to four experiments are shown as box plots. There was a significant difference in viral titers between BN and F344 cultures (P < 0.001; ANOVA). *P = 0.034, **P < 0.007, ANOVA indicate a significant difference compared with 2 h BN cultures. {dagger}Significant difference compared with 2 h F344 cultures (P < 0.002; ANOVA).

 
Because ultraviolet inactivation led to only partial inhibition of IFN-{gamma} production at the higher virus dose, we examined whether virus particles were required for cytokine production by adding a 100,000 molecular weight–cutoff filtrate of the SeV preparation, which contained no infectious SeV, to weanling BN and F344 splenocytes and measuring IFN-{gamma} and IL-12 production. No IFN-{gamma} or IL-12 production was detected in these cultures, which demonstrated that all of the cytokine-inducing activity of the SeV preparation resided in the fraction with a molecular weight > 100,000. In addition, the SeV preparation induced comparable levels of IFN-{gamma} production in either the presence or absence of polymyxin B (an antibiotic that binds to and neutralizes bacterial endotoxin), which demonstrated that bacterial contamination of the virus preparation was not responsible for the observed results (data not shown).

To more directly determine whether productive infection of cell sources of SeV-induced IL-12 production occurred, we inoculated splenic macrophage cultures from weanlings with SeV and monitored viral replication by plaque assay (Figure 5C). There were significant differences in viral titers between BN and F344 macrophage cultures (P < 0.001). Compared with BN cultures at 2 h after inoculation, significant increases in viral titers were observed in BN cultures at 24 (P = 0.034), 48, and 96 h (P < 0.007) after inoculation, indicating active viral replication. In contrast, in F344 macrophage cultures, viral titers, which were not different at 2 and 24 h after inoculation, significantly decreased at 48 and 96 h after inoculation (P < 0.002).

SeV-Induced IL-18 Production by Splenocytes
Because IL-18 can act in a synergistic manner with IL-12 to induce IFN-{gamma} production by NK cells (11, 14), we examined whether IL-18 secretion was also induced in splenocyte cultures by incubation with SeV. For splenocytes of both strains, spontaneous IL-18 secretion was observed in the absence of SeV, but there was a significant (1.5-fold) increase in the median level of IL-18 production after stimulation with SeV (Figure 6A ; P < 0.001). The levels of spontaneous and SeV-induced IL-18 secretion from BN splenocytes were significantly lower than those from F344 splenocytes (Figure 6A; P < 0.001), representing a 2.6- or 2.7-fold difference in median levels of IL-18 in the presence or absence of SeV, respectively. As with SeV-induced IFN-{gamma} and IL-12 production at this higher viral dose (5 x 106 pfu), ultraviolet inactivation of SeV resulted in a significant, but partial, inhibition of IL-18 production by BN and F344 splenocytes (Figure 6A; P < 0.001). The levels of spontaneous IL-18 secretion did not differ between splenocytes cultured with allantoic fluid and those cultured with medium alone.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 6. IL-18 production by splenocytes and splenic macrophages from weanling BN and F344 rats in response to stimulation with SeV. (A) Splenocytes from weanling BN (open boxes) and F344 (filled boxes) rats were incubated for 24 h in the presence of allantoic fluid (control), SeV (5 x 106 pfu/well), or an equivalent amount of ultraviolet-inactivated SeV (UV-SeV), and IL-18 levels were measured in the supernates by ELISA. Data represent mean values for triplicate cultures from individual rats of each strain (control and SeV: n = 7; ultraviolet-inactivated SeV: n = 4) and are shown as box plots. Whiskers represent the 10th and 90th percentiles. (B) Splenic macrophages from weanling BN (open circles) and F344 (filled circles) rats (n = 8) were incubated for 24 h with allantoic fluid (control) or SeV (2.5 x 107 pfu/well), and supernate IL-18 levels were measured. Bars indicate medians. (A, B) Dotted line represents the lower limit of detection of the IL-18 ELISA (4 pg/ml). *P < 0.001, ANOVA; **P < 0.007, Mann-Whitney test indicate a significant difference between BN and F344 responses. {dagger}Significant difference between responses to SeV and those to allantoic fluid (P < 0.001; ANOVA). {ddagger}Significant difference between responses to SeV and those to ultraviolet-inactivated SeV (P < 0.001; repeated measures ANOVA).

 
Splenic macrophages were evaluated as a potential source of IL-18. In the presence or absence of SeV, the levels of IL-18 secretion from BN macrophages were significantly lower than those from F344 macrophages (Figure 6B; P < 0.007). Although for six out of eight F344 rats, IL-18 levels were higher in macrophage cultures incubated with SeV than in those that had been incubated with allantoic fluid, there was no significant treatment effect for either rat strain.

Role of IL-18 in SeV-Induced IFN-{gamma} Production by Splenocytes
Because IL-18 was secreted by BN and F344 splenocytes, we investigated whether IL-18 was involved in SeV-induced IFN-{gamma} production in these cultures using IL-18BPa/Fc, which specifically binds to IL-18 and prevents it from binding to its receptor (15). The levels of SeV-induced IFN-{gamma} production from BN or F344 splenocytes incubated in the presence of IL-18BPa/Fc were significantly reduced compared with those from splenocytes incubated under either of the control conditions (Figure 7A ; P < 0.001). IFN-{gamma} levels for cultures treated with control IgG1 did not significantly differ from those that were untreated. The median percent reductions in SeV-induced IFN-{gamma} levels in BN and F344 splenocyte cultures containing IL-18BPa/Fc, compared with parallel cultures that were untreated, were 92% and 90%, respectively, which showed that this IFN-{gamma} production was IL-18–dependent.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 7. Effects of IL-18BPa/Fc and IL-12 p40 homodimer on SeV-induced IFN-{gamma} production by splenocytes from weanling BN and F344 rats. Splenocytes from weanling BN (open boxes) and F344 (filled boxes) rats were incubated for 24 h with 5 x 106 pfu/well of SeV, and supernate IFN-{gamma} levels were measured by ELISA. Data represent mean values for triplicate cultures from individual rats of each strain (A, n = 3; B, n = 7) and are shown as box plots. Whiskers represent the 10th and 90th percentiles. Little or no IFN-{gamma} was detected in supernates from BN (< 16 pg/ml for all rats) and F344 (median: 44 pg/ml) splenocytes incubated with allantoic fluid. Dotted line represents the lower limit of detection of the IFN-{gamma} ELISA (16 pg/ml). (A) Incubated in the presence of either medium alone, human IgG1 (1 µg/ml), or IL-18BPa/Fc (1 µg/ml). (B) Incubated in the presence of either medium alone, IL-12 p40 homodimer (0.2, 2, or 20 ng/ml), or IL-12 p40 homodimer (20 ng/ml) in combination with either human IgG1 (1 µg/ml) or IL-18BPa/Fc (1 µg/ml). (A, B) There was a significant difference between BN and F344 responses to SeV (P < 0.001; ANOVA). *Significant difference between responses of cultures with either medium alone or human IgG1 and those with IL-18BPa/Fc (P < 0.001; ANOVA). **Significant difference between responses of cultures with medium alone and those with IL-12 p40 homodimer (P < 0.001; ANOVA). {dagger}P < 0.001, {ddagger}P < 0.01 indicate a significant difference between responses of cultures containing IL-12 p40 homodimer (20 ng/ml) with or without human IgG1 and those containing both IL-12 p40 homodimer (20 ng/ml) and IL-18BPa/Fc (paired t tests).

 
Dependence of SeV-Induced IFN-{gamma} Production on IL-12 and IL-18
IL-12 p40 homodimer binds to, but does not activate, the IL-12 receptor, serving as a competitive antagonist of IL-12–mediated signaling and cell activation (16). The addition of 0.2, 2, or 20 ng/ml of IL-12 p40 homodimer to these cultures resulted in a significant, dose-dependent inhibition of SeV-induced IFN-{gamma} production (Figure 7B; P < 0.001), with median percent reductions of 58, 72, and 90% for BN splenocytes and 50, 63, and 89% for F344 splenocytes, respectively. The level of inhibition of IFN-{gamma} production mediated by 20 ng/ml of IL-12 p40 homodimer was comparable to that mediated by neutralizing anti–IL-12 antibody.

Inhibition of the binding of either IL-12 or IL-18 to their respective receptors markedly inhibited SeV-induced IFN-{gamma} production by BN and F344 splenocytes. However, the levels of SeV-induced IFN-{gamma} production in the presence of IL-12– or IL-18–specific antagonists were still 10–20% of the levels in the absence of antagonists. Therefore, we investigated the effects of the combined inhibition of IL-12 and IL-18 on SeV-induced IFN-{gamma} production. SeV-induced IFN-{gamma} production was completely inhibited in BN (100% inhibition; P < 0.01) and F344 (99% inhibition; P < 0.001) splenocyte cultures incubated with both IL-12 p40 homodimer and IL-18BPa/Fc compared with those incubated with IL-12 p40 homodimer (Figure 7B). Addition of control IgG1 did not have a significant effect on IFN-{gamma} production in the cultures. Therefore, IFN-{gamma} production in response to SeV was dependent on both IL-12 and IL-18, and these cytokines acted in a synergistic manner.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NK cells are likely to be an important early source of IFN-{gamma} during the innate host response to viral infections (17). NK cell responses to SeV infection may be especially relevant to our model because the development of the postbronchiolitis asthma-like phenotype in BN rats involves the infection of naive hosts, in whom there will be a distinct lag period before the generation of adaptive antiviral immune responses, which would likely involve CD8+ T cells as an another important source of IFN-{gamma} (18). Our hypothesis, that the SeV-induced IFN-{gamma} production in splenocyte cultures represented an innate response, was confirmed by cell depletion studies, which clearly demonstrated that NK cells were the primary source of SeV-induced IFN-{gamma} production in these cultures. Together with our previous work (11), these results demonstrated that mechanisms contributing to the reduced ability of SeV-stimulated splenocytes from BN weanlings to secrete IFN-{gamma} include the diminished IFN-{gamma}–producing capacity and the decreased frequency of the primary cell source for this cytokine, the NK cell.

We extended these findings by evaluating the contributions of two IFN-{gamma}–inducing cytokines, IL-12 and IL-18, to SeV-induced IFN-{gamma} production by NK cells. We demonstrated that incubation of splenocytes with SeV resulted in the induction of IL-12 p70 production. We also observed an SeV-induced enhancement of IL-18 secretion in these cultures, which may be related to a previous report that SeV could induce caspase activation in human monocyte–derived macrophages, leading to proteolytic cleavage of proIL-18 and secretion of mature, biologically active IL-18 (19). Furthermore, the ability of weanling BN splenocytes to secrete IL-12 and IL-18 in response to SeV was significantly attenuated in comparison with that of weanling F344 splenocytes. The addition of specific antagonists of IL-12 and IL-18 activity to these cultures showed the IL-12 and IL-18 dependence of SeV-induced IFN-{gamma} production, which was completely abrogated by the simultaneous inhibition of IL-12 and IL-18 activity. Furthermore, the effects of IL-12 and IL-18 on IFN-{gamma} production were synergistic, which is consistent with other work demonstrating synergism between IL-12 and IL-18 with regard to the induction of IFN-{gamma} secretion (15), and with our previous data showing that incubation of purified NK cells from BN and F344 weanlings with IL-12 and IL-18 resulted in the synergistic enhancement of IFN-{gamma} production (11). Therefore, the reduced capacity of weanling BN splenocytes to generate the IFN-{gamma}–inducing cytokines upon which the SeV-induced IFN-{gamma} production was dependent, along with our previous data showing the decreased ability of weanling BN NK cells to produce IFN-{gamma} in response to either IL-12 or a combination of IL-12 and IL-18 (11), represent additional mechanisms contributing to the attenuated innate response of BN weanlings to SeV.

SeV-induced IFN-{gamma} production by splenocytes apparently occurred via an indirect innate mechanism, whereby SeV stimulated professional antigen-presenting cells to secrete IFN-{gamma}–inducing cytokines that, in turn, stimulated NK cells to produce IFN-{gamma}. The observation that SeV could induce secretion of IL-12 by splenic macrophages provides support for this mechanism. In addition, incubation of purified splenic NK cells with SeV in the absence of a source of accessory cells did not result in IFN-{gamma} production (L. A. Rosenthal, unpublished observations). Other investigators have shown that rhinovirus and influenza A virus could induce IFN-{gamma} production by indirect innate mechanisms, which also involved eliciting the secretion of IFN-{gamma}–inducing cytokines from mononuclear phagocytes (20, 21). Consistent with the data from splenocyte cultures, BN splenic macrophages produced significantly less SeV-induced IL-12 than did F344 splenic macrophages. In addition, BN macrophages secreted significantly less IL-18 than did F344 macrophages. However, there was no significant increase in IL-18 secretion by splenic macrophages in the presence of SeV. Although our data demonstrate that splenic macrophages could serve as a source of IFN-{gamma}–inducing cytokines, other antigen-presenting cells, such as splenic dendritic cells, also may have been important contributors of these cytokines. The discrepancy between splenocyte and macrophage cultures with regard to the effect of SeV-stimulation on IL-18 production could reflect contributions of dendritic cells in the splenocyte cultures. Also, it is possible that differential production of IFN-{gamma} in the BN and F344 splenocyte cultures may accentuate differences in the secretion of IFN-{gamma}–inducing cytokines through a positive feedback mechanism. This type of mechanism could help to explain why it was more difficult to detect IL-12 and IL-18 in BN macrophage cultures, which have no IFN-{gamma} production, than in BN splenocyte cultures, where relatively low levels of IFN-{gamma} are induced.

Our data clearly show that IL-12 p70 production was induced in splenocyte and splenic macrophage cultures by stimulation with SeV. However, we cannot rule out the possibility that IL-23, a recently described cytokine (22), may have also been produced in response to SeV. IL-23 is a heterodimeric cytokine consisting of the p40 subunit of IL-12 and, rather than the IL-12 p35 subunit, a second chain known as p19 (22). In addition, the IL-12 receptor ß1 chain is a component of the heterodimeric IL-23 receptor (23). IL-23 shares some activities with IL-12, including the ability to induce IFN-{gamma} expression (22). In our experiments, the activity of SeV-induced IL-12 p70 was inhibited by either neutralizing anti–IL-12 antibody or IL-12 p40 homodimer. Both of these inhibitors would prevent the binding of SeV-induced IL-12 p70 to its receptor, albeit by different mechanisms. However, it is possible that these inhibitors may also prevent the interaction of any IL-23, which may have been generated, with its receptor. Before the recent availability of reagents to specifically measure rat IL-12 p70, our reagents only permitted us to measure both IL-12 p70 and p40. At least for F344 splenocytes and BN and F344 splenic macrophages, the levels of SeV-induced IL-12 p70/p40 measured were typically higher than those of SeV-induced IL-12 p70, which leaves open the possibility that some IL-23 may have been generated in these cultures. Consistent with this possibility, a recent report described the induction of both IL-12 and IL-23 from human monocyte-derived macrophages in response to incubation with SeV (24).

Our results demonstrated the importance of NK (NKR-P1A+ CD3–) cells as the primary source of SeV-induced IFN-{gamma}. However, in our cell depletion experiments, when we depleted NKR-P1A+ cells from splenocytes we also depleted splenic NKT (NKR-P1A+ CD3+) cells. NKT cells, a T cell lineage distinct from NK cells, are also capable of secreting IFN-{gamma} (25). The frequency of NKT cells in the spleens of weanling rats was substantially lower than that of NK cells (L. A. Rosenthal, unpublished observations). The fact that depletion of CD3+ cells, which would include T cells and NKT cells, had little or no effect on SeV-induced IFN-{gamma} production strongly suggests that the contribution of NKT cells to the levels of IFN-{gamma} secreted was, at best, minor.

Experiments to determine whether the induction of cytokines by innate mechanisms required infectious SeV yielded results that were dependent on the dose of virus. At the lower SeV dose, ultraviolet inactivation almost completely inhibited IFN-{gamma} and IL-12 production, whereas at the higher SeV dose, this inhibition was significant, but incomplete for the BN IFN-{gamma} responses, the F344 IFN-{gamma} and IL-12 responses, and the BN and F344 IL-18 responses. The reasons for the partial refractoriness of cytokine production to the effects of SeV inactivation at the higher viral dose are unclear. It may be that an increase in the number of viral particles interacting with cell-surface receptors compensated in some manner for a reduced efficiency in triggering cellular activation pathways. However, it is clear that infectious SeV was markedly more effective than noninfectious virus in stimulating cytokine production by splenocytes, which is consistent with the observation that Sendai virus can establish productive infections in weanling rat alveolar macrophages (26). Similarly, our results showed that SeV productively infected splenic macrophages from BN weanlings, which are a source of SeV-induced IFN-{gamma}–inducing cytokines in the splenocyte cultures. For reasons that are unclear, we were not able to demonstrate productive infection of splenic macrophages from F344 weanlings, although initial SeV levels did persist at 24 h postinoculation before declining. It is possible that the increased levels of SeV-induced cytokine production in F344, compared with BN, macrophage cultures may have enhanced the activity of antiviral cellular pathways.

The mechanisms responsible for the diminished capacity of weanling BN splenocytes to secrete IL-12 and IL-18 in response to SeV remain to be determined. However, these reduced responses would be consistent with a dysregulation of a pattern recognition receptor pathway. The innate immune system employs a diverse array of pattern recognition receptors, which may function as cell surface, intracellular, or secreted receptors (27). These receptors recognize structural elements, known as pathogen-associated molecular patterns or PAMPs, that are unique to bacteria, viruses, and fungi (27). Interaction of host cell surface pattern recognition receptors with their respective ligands can trigger cell activation and the secretion of cytokines, including IL-12 and IL-18 (28). Prominent examples of pattern recognition receptors, which have been implicated in antiviral responses, are the toll-like receptors (27). For example, it has been reported that the innate host response to RSV, another paramyxovirus, involves the pattern recognition receptors, toll-like receptor 4 and CD14 (29). Studies are currently underway to investigate the role of pattern recognition receptors in innate responses to SeV and the possibility that these pathways are attenuated in BN weanlings.

Two important aspects of the postbronchiolitis asthma-like phenotype in BN rats are that the phenotype is generated by a single exposure to virus, and that this exposure must occur during a relatively narrow developmental window early in life (6–9, 11), which parallels the potential role of RSV-induced bronchiolitis in the development of childhood asthma (1–4). Recently, exposure of C57BL/6 mice to a single inoculation with SeV has been shown to induce an acute bronchiolitis, which is followed by chronic airway dysfunction lasting at least one year (30). Also, the age at which BALB/c mice are first infected with RSV has been shown to have marked effects on their subsequent responses to rechallenge with RSV, with an increase in eosinophilia and a more Th2-biased response being observed in mice that had originally been infected as neonates (31). In another mouse model, the relative timing of RSV infection and allergic sensitization had marked effects on pulmonary inflammatory and physiologic outcomes (32). Therefore, distinct models of virus-induced airway dysfunction have revealed that a single respiratory viral illness can have profound and long-lasting effects on airway inflammation and function, and that the timing of the viral infection may be a critical factor in determining the eventual outcome of the illness.

The primary target for SeV infection in our rat model is the respiratory system. In the present work, splenocytes were used as a convenient source of immune system cells, akin to the use of human PBMC, to investigate mechanisms regulating SeV-induced IFN-{gamma} production. Now that we have identified deficits in innate mechanisms of SeV-induced IFN-{gamma} production that may contribute to the attenuated IFN-{gamma} responses observed in BN weanlings during acute SeV infection, we have begun to extend our studies into the lung. Our preliminary in vitro experiments suggest that similar deficits in innate responses to SeV can be observed in mononuclear cells isolated from the lungs of BN weanlings (L. A. Rosenthal, unpublished observations).

Our results appear to parallel several observations in humans. Cord blood mononuclear cells, compared with adult PBMC, have been noted to produce less IL-12 mRNA and protein, but IFN-{gamma} production in response to IL-12 was similar (33). A recent study has shown that, in addition to cord blood mononuclear cells, PBMC from 5- and 12-yr-old children also have a diminished capacity for IL-12 production in comparison with that of PBMC from adults (34). Therefore, alterations in IFN-{gamma} production in the developing infant and child may be a reflection, at least in part, of IL-12–mediated regulation of IFN-{gamma} production. Furthermore, adult patients with atopic asthma have a deficient IL-12 response when whole peripheral blood cultures are stimulated with Staphylococcus aureus (35). In addition, heterozygosity for an IL12B promoter polymorphism (IL12B encodes the IL-12 p40 subunit) has been associated with increased asthma severity in childhood and decreased IL-12 p40 mRNA and IL-12 p70 protein production (36). Taken together, these findings suggest that the observed decreases in IFN-{gamma} production in patients with atopy and/or asthma may be related to abnormalities in positive regulatory factors such as IL-12. The parallels between these observations and the attenuated innate responses in BN weanlings lend further support to the relevance of this rat model of virus-induced chronic airway dysfunction to studies of innate immune responses that contribute to the inception of childhood asthma.


   Acknowledgments
 
The authors thank Claire Dick for helpful discussions and Kathleen Schell for assistance with flow cytometry. This work was supported by National Institutes of Health grant AI50500.

Received in original form May 7, 2003

Received in final form October 2, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gern, J. E., R. F. Lemanske, Jr., and W. W. Busse. 1999. Early life origins of asthma. J. Clin. Invest. 104:837–843.[Medline]
  2. Castro-Rodríguez, J. A., C. J. Holberg, A. L. Wright, M. Halonen, L. M. Taussig, W. J. Morgan, and F. D. Martinez. 1999. Association of radiologically ascertained pneumonia before age 3 yr with asthmalike symptoms and pulmonary function during childhood: a prospective study. Am. J. Respir. Crit. Care Med. 159:1891–1897.[Abstract/Free Full Text]
  3. Sigurs, N., R. Bjarnason, F. Sigurbergsson, and 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. 161:1501–1507.[Abstract/Free Full Text]
  4. Stein, R. T., D. Sherrill, W. J. Morgan, C. J. Holberg, M. Halonen, L. M. Taussig, A. L. Wright, and F. D. Martinez. 1999. Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet 354:541–545.[CrossRef][Medline]
  5. Renzi, P. M., J. P. Turgeon, J. E. Marcotte, S. P. Drblik, D. Bérubé, M. F. Gagnon, and S. Spier. 1999. Reduced interferon-{gamma} production in infants with bronchiolitis and asthma. Am. J. Respir. Crit. Care Med. 159:1417–1422.[Abstract/Free Full Text]
  6. Kumar, A., R. Sorkness, M. R. Kaplan, W. L. Castleman, and R. F. Lemanske, Jr. 1997. Chronic, episodic, reversible airway obstruction after viral bronchiolitis in rats. Am. J. Respir. Crit. Care Med. 155:130–134.[Abstract]
  7. Uhl, E. W., W. L. Castleman, R. L. Sorkness, W. W. Busse, R. F. Lemanske, Jr., and P. K. McAllister. 1996. Parainfluenza virus-induced persistence of airway inflammation, fibrosis, and dysfunction associated with TGF-ß1 expression in Brown Norway rats. Am. J. Respir. Crit. Care Med. 154:1834–1842.[Abstract]
  8. Sorkness, R. L., W. L. Castleman, A. Kumar, M. R. Kaplan, and R. F. Lemanske, Jr. 1999. Prevention of chronic post-bronchiolitis airway sequelae with interferon-{gamma} treatment in rats. Am. J. Respir. Crit. Care Med. 160:705–710.[Abstract/Free Full Text]
  9. Rosenthal, L. A., R. L. Sorkness, and R. F. Lemanske, Jr. 2003. Origin of respiratory virus-induced chronic airway dysfunction: exploring genetic, develpomental, and environmental factors in a rat model of the asthmatic phenotype. In Respiratory Infections in Allergy and Asthma. S. L. Johnston and N. G. Papadopoulos, editors. Marcel Dekker, Inc., New York. 353–376.
  10. Sorkness, R. L., J. E. Gern, K. A. Grindle, A. G. Mosser, L. A. Rosenthal, L. D. Mikus, and R. F. Lemanske, Jr. 2002. Persistence of viral RNA in 2 rat strains differing in susceptibility to postbronchiolitis airway dysfunction. J. Allergy Clin. Immunol. 110:607–609.[CrossRef][Medline]
  11. Mikus, L. D., L. A. Rosenthal, R. L. Sorkness, and R. F. Lemanske, Jr. 2001. Reduced interferon-{gamma} secretion by natural killer cells from rats susceptible to postviral chronic airway dysfunction. Am. J. Respir. Cell Mol. Biol. 24:74–82.[Abstract/Free Full Text]
  12. Sorkness, R. L., W. L. Castleman, L. D. Mikus, A. G. Mosser, and R. F. Lemanske, Jr. 2002. Chronic postbronchiolitis airway instability induced with anti-IFN-{gamma} antibody in F344 rats. Pediatr. Res. 52:382–386.[CrossRef][Medline]
  13. Castleman, W. L., L. J. Brundage-Anguish, L. Kreitzer, and S. B. Neuenschwander. 1987. Pathogenesis of bronchiolitis and pneumonia induced in neonatal and weanling rats by parainfluenza (Sendai) virus. Am. J. Pathol. 129:277–286.[Abstract]
  14. Tomura, M., X.-Y. Zhou, S. Maruo, H. J. Ahn, T. Hamaoka, H. Okamura, K. Nakanishi, T. Tanimoto, M. Kurimoto, and H. Fujiwara. 1998. A critical role for IL-18 in the proliferation and activation of NK1.1+ CD3- cells. J. Immunol. 160:4738–4746.[Abstract/Free Full Text]
  15. Novick, D., S.-H. Kim, G. Fantuzzi, L. L. Reznikov, C. A. Dinarello, and M. Rubinstein. 1999. Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response. Immunity 10:127–136.[CrossRef][Medline]
  16. Gillessen, S., D. Carvajal, P. Ling, F. J. Podlaski, D. L. Stremlo, P. C. Familletti, U. Gubler, D. H. Presky, A. S. Stern, and M. K. Gately. 1995. Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist. Eur. J. Immunol. 25:200–206.[Medline]
  17. Biron, C. A., K. B. Nguyen, G. C. Pien, L. P. Cousens, and T. P. Salazar-Mather. 1999. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu. Rev. Immunol. 17:189–220.[CrossRef][Medline]
  18. Hussell, T., and P. J. Openshaw. 1998. Intracellular IFN-gamma expression in natural killer cell precedes lung CD8+ T cell recruitment during respiratory syncytial virus infection. J. Gen. Virol. 79:2593–2601.[Abstract]
  19. Pirhonen, J., T. Sareneva, I. Julkunen, and S. Matikainen. 2001. Virus infection induces proteolytic processing of IL-18 in human macrophages via caspase-1 and caspase-3 activation. Eur. J. Immunol. 31:726–733.[CrossRef][Medline]
  20. Gern, J. E., R. Vrtis, E. A. B. Kelly, E. C. Dick, and W. W. Busse. 1996. Rhinovirus produces nonspecific activation of lymphocytes through a monocyte-dependent mechanism. J. Immunol. 157:1605–1612.[Abstract]
  21. Sareneva, T., S. Matikainen, M. Kurimoto, and I. Julkunen. 1998. Influenza A virus-induced IFN-{alpha}/ß and IL-18 synergistically enhance IFN-{gamma} gene expression in human T cells. J. Immunol. 160:6032–6038.[Abstract/Free Full Text]
  22. Oppman, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, F. Zonin, E. Vaisberg, T. Churakova, M. Liu, D. Gorman, J. Wagner, S. Zurawski, Y. Liu, J. S. Abrams, K. W. Moore, D. Rennick, R. de Waal-Malefyt, C. Hannum, J. F. Bazan, and R. A. Kastelein. 2000. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13:715–725.[CrossRef][Medline]
  23. Parham, C., M. Chirica, J. Timans, E. Vaisberg, M. Travis, J. Cheung, S. Pflanz, R. Zhang, K. P. Singh, F. Vega, W. To, J. Wagner, A. M. O'Farrell, T. McClanahan, S. Zurawski, C. Hannum, D. Gorman, D. M. Rennick, R. A. Kastelein, R. de Waal-Malefyt, and K. W. Moore. 2002. A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rß1 and a novel cytokine receptor subunit, IL-23R. J. Immunol. 168:5699–5708.[Abstract/Free Full Text]
  24. Pirhonen, J., S. Matikainen, and I. Julkunen. 2002. Regulation of virus-induced IL-12 and IL-23 expression in human macrophages. J. Immunol. 169:5673–5678.[Abstract/Free Full Text]
  25. Arase, H., N. Arase, and T. Saito. 1996. Interferon {gamma} production by natural killer (NK) cells and NK1.1+ T cells upon NKR-P1 cross-linking. J. Exp. Med. 183:2391–2396.[Abstract/Free Full Text]
  26. Castleman, W. L., P. J. Northrop, and P. K. McAllister. 1989. Replication of parainfluenza (Sendai) virus in isolated rat pulmonary type II alveolar epithelial cells. Am. J. Pathol. 134:1135–1142.[Abstract]
  27. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20:197–216.[CrossRef][Medline]
  28. Seki, E., H. Tsutsui, H. Nakano, N. M. Tsuji, K. Hoshino, O. Adachi, K. Adachi, S. Futatsugi, K. Kuida, O. Takeuchi, H. Okamura, J. Fujimoto, S. Akira, and K. Nakanishi. 2001. Lipopolysaccharide-induced IL-18 secretion from murine Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1ß. J. Immunol. 166:2651–2657.[Abstract/Free Full Text]
  29. Kurt-Jones, E. A., L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg. 2000. Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat. Immunol. 1:398–401.[CrossRef][Medline]
  30. Walter, M. J., J. D. Morton, N. Kajiwara, E. Agapov, and M. J. Holtzman. 2002. Viral induction of a chronic asthma phenotype and genetic segregation from the acute response. J. Clin. Invest. 110:165–175.[CrossRef][Medline]
  31. Culley, F. J., J. Pollot, and P. J. M. Openshaw. 2002. Age at first viral infection determines the pattern of T cell-mediated disease during reinfection in adulthood. J. Exp. Med. 196:1381–1386.[Abstract/Free Full Text]
  32. Peebles, R. S., Jr., K. Hashimoto, R. D. Collins, K. Jarzecka, J. Furlong, D. B. Mitchell, J. R. Sheller, and B. S. Graham. 2001. Immune interaction between respiratory syncytial virus infection and allergen sensitization critically depends on timing of challenges. J. Infect. Dis. 184:1374–1379.[CrossRef][Medline]
  33. Lee, S. M., Y. Suen, L. Chang, V. Bruner, J. Qian, J. Indes, E. Knoppel, C. van de Ven, and M. S. Cairo. 1996. Decreased interleukin-12 (IL-12) from activated cord versus adult peripheral blood mononuclear cells and upregulation of interferon-{gamma}, natural killer, and lymphokine-activated killer activity by IL-12 in cord blood mononuclear cells. Blood 88:945–954.[Abstract/Free Full Text]
  34. Upham, J. W., P. T. Lee, B. J. Holt, T. Heaton, S. L. Prescott, M. J. Sharp, P. D. Sly, and P. G. Holt. 2002. Development of interleukin-12-producing capacity throughout childhood. Infect. Immun. 70:6583–6588.[Abstract/Free Full Text]
  35. van der Pouw Kraan, T. C. T. M., L. C. M. Boeije, E. R. de Groot, S. O. Stapel, A. Snijders, M. L. Kapsenberg, J. S. van der Zee, and L. A. Aarden. 1997. Reduced production of IL-12 and IL-12-dependent IFN-{gamma} release in patients with allergic asthma. J. Immunol. 158:5560–5565.[Abstract]
  36. Morahan, G., D. Huang, M. Wu, B. J. Holt, G. P. White, G. E. Kendall, P. D. Sly, and P. G. Holt. 2002. Association of IL12B promoter polymorphism with severity of atopic and non-atopic asthma in children. Lancet 360:455–459.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0181OCv1
30/5/702    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rosenthal, L. A.
Right arrow Articles by Lemanske, R. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rosenthal, L. A.
Right arrow Articles by Lemanske, R. F., Jr.

HOME HELP FEEDBACK