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1.  Differential Effects of Dendritic Cell Transfer on Airway Hyperresponsiveness and Inflammation 
Dendritic cells (DCs) are considered to be the most efficient antigen-presenting cells. Intratracheal administration of allergen-pulsed bone marrow–derived dendritic cells (BMDCs) before allergen challenge induces airway hyperresponsiveness (AHR) and inflammation. Ovalbumin (OVA)-pulsed BMDCs from wild-type (WT) mice were transferred into naive WT, CD4−/−, CD8−/−, or IL-13−/− mice. Two days (short protocol) or 10 days (long protocol) after BMDC transfer, mice were challenged with 1% OVA for 3 days and assayed 2 days later. Transfer of OVA-primed BMDCs into BALB/c or C57BL/6 mice elicited AHR in both protocols. Airway eosinophilia, Th2 cytokines, or goblet cell metaplasia were increased in the long but not short protocol. Lung T cells from both protocols produced Th2 cytokines in response to OVA in vitro. Carboxyfluorescein diacetate succinimidylester–labeled BMDCs were observed in bronchoalveolar lavage (BAL) fluid and lung parenchyma at early time points, and were detected in draining lymph nodes 48 hours after transfer. CD8−/− mice developed AHR comparable to WT mice in the short protocol, but decreased levels of AHR, airway eosinophilia, Th2 cytokines in BAL fluid, and goblet cell metaplasia compared with WT mice in the long protocol. CD4−/− or IL-13−/− mice did not develop AHR or airway inflammation in either protocol. These data suggest that allergen-pulsed BMDCs initiate development of AHR that is dependent initially on CD4+ T cells, and at later time periods on CD8+ T cells and IL-13. Thus, the timing of allergen challenge after transfer of allergen-pulsed BMDC affects the development of AHR and airway inflammation.
PMCID: PMC2742748  PMID: 19151321
dendritic cells; CD8+ T cells; airway hyperresponsiveness
2.  Leukotriene B4 Release from Mast Cells in IgE-Mediated Airway Hyperresponsiveness and Inflammation 
Previous studies have shown that leukotriene B4 (LTB4), a proinflammatory lipid mediator, is linked to the development of airway hyperresponsiveness through the accumulation of IL-13–producing CD8+ T cells, which express a high affinity receptor for LTB4, BLT1 (Miyahara et al., Am J Respir Crit Care Med 2005;172:161–167; J Immunol 2005;174:4979–4984). By using leukotriene A4 hydrolase–deficient (LTA4H−/−) mice, which fail to synthesize LTB4, we determined the role of this lipid mediator in allergen-induced airway responses. Two approaches were used. In the first, LTA4H−/− mice and wild-type (LTA4H+/+) mice were systemically sensitized and challenged via the airways to ovalbumin. In the second, mice were passively sensitized with anti-ovalbumin IgE and exposed to ovalbumin via the airways. Mast cells were generated from bone marrow of LTA4H+/+ mice or LTA4H−/− mice. After active sensitization and challenge, LTA4H−/− mice showed significantly lower airway hyperresponsiveness compared with LTA4H+/+ mice, and eosinophil numbers and IL-13 levels in the bronchoalveoloar lavage of LTA4H−/− mice were also significantly lower. LTA4H−/− mice also showed decreased airway reactivity after passive sensitization and challenge. After LTA4H+/+ mast cell transfer, LTA4H−/− mice showed increased airway reactivity after passive sensitization and challenge, but not after systemic sensitization and challenge. These data confirm the important role for LTB4 in the development of altered airway responses and suggest that LTB4 secretion from mast cells is critical to eliciting increased airway reactivity after passive sensitization with allergen-specific IgE.
PMCID: PMC2689918  PMID: 19029019
rodent; T cells; cytokines; lipid mediators; lung
3.  Vγ1+ T Cells and Tumor Necrosis Factor-Alpha in Ozone-Induced Airway Hyperresponsiveness 
γδ T cells regulate airway reactivity, but their role in ozone (O3)-induced airway hyperresponsiveness (AHR) is not known. Our objective was to determine the role of γδ T cells in O3-induced AHR. Different strains of mice, including those that were genetically manipulated or antibody-depleted to render them deficient in total γδ T cells or specific subsets of γδ T cells, were exposed to 2.0 ppm of O3 for 3 hours. Airway reactivity to inhaled methacholine, airway inflammation, and epithelial cell damage were monitored. Exposure of C57BL/6 mice to O3 resulted in a transient increase in airway reactivity, neutrophilia, and increased numbers of epithelial cells in the lavage fluid. TCR-δ−/− mice did not develop AHR, although they exhibited an increase in neutrophils and epithelial cells in the lavage fluid. Similarly, depletion of γδ T cells in wild-type mice suppressed O3-induced AHR without influencing airway inflammation or epithelial damage. Depletion of Vγ1+, but not of Vγ4+ T cells, reduced O3-induced AHR, and transfer of total γδ T cells or Vγ1+ T cells to TCR-δ−/− mice restored AHR. After transfer of Vγ1+ cells to TCR-δ−/− mice, restoration of AHR after O3 exposure was blocked by anti–TNF-α. However, AHR could be restored in TCR-δ−/−mice by transfer of γδ T cells from TNF-α–deficient mice, indicating that another cell type was the source of TNF-α. These results demonstrate that TNF-α and activation of Vγ1+ γδ T cells are required for the development of AHR after O3 exposure.
PMCID: PMC2660562  PMID: 18927346
ozone; airway responsiveness; γδ T cells; TNF-α
4.  Estrogen Determines Sex Differences in Airway Responsiveness after Allergen Exposure 
The female hormone estrogen is an important factor in the regulation of airway function and inflammation, and sex differences in the prevalence of asthma are well described. Using an animal model, we determined how sex differences may underlie the development of altered airway function in response to allergen exposure. We compared sex differences in the development of airway hyperresponsiveness (AHR) after allergen exposure exclusively via the airways. Ovalbumin (OVA) was administered by nebulization on 10 consecutive days in BALB/c mice. After methacholine challenge, significant AHR developed in male mice but not in female mice. Ovariectomized female mice showed significant AHR after 10-day OVA inhalation. ICI182,780, an estrogen antagonist, similarly enhanced airway responsiveness even when administered 1 hour before assay. In contrast, 17β-estradiol dose-dependently suppressed AHR in male mice. In all cases, airway responsiveness was inhibited by the administration of a neurokinin 1 receptor antagonist. These results demonstrate that sex differences in 10-day OVA-induced AHR are due to endogenous estrogen, which negatively regulates airway responsiveness in female mice. Cumulatively, the results suggest that endogenous estrogen may regulate the neurokinin 1–dependent prejunctional activation of airway smooth muscle in allergen-exposed mice.
PMCID: PMC2335333  PMID: 18063836
estrogen; sex; airway hyperresponsiveness; EFS; neuronal activation
5.  IL-2 and IL-18 Attenuation of Airway Hyperresponsiveness Requires STAT4, IFN-γ, and Natural Killer Cells 
IL-18 is known to induce IFN-γ production, which is enhanced when combined with IL-2. In the present study, we investigated whether the combination of exogenous IL-2 and IL-18 alters airway hyperresponsiveness (AHR) and airway inflammation. Sensitized mice exposed to ovalbumin (OVA) challenge developed AHR, inflammatory cells in the bronchoalveolar lavage (BAL) fluid, and increases in levels of Th2 cytokines and goblet cell numbers. The combination of IL-2 and IL-18, but neither alone, prevented these changes while increasing levels of IL-12 and IFN-γ. The combination of IL-2 and IL-18 was ineffective in IFN-γ–deficient and signal transducer and activator of transcription (STAT)4-deficient mice. Flow cytometry analysis showed significant increases in numbers of IFN-γ–positive natural killer (NK) cells in the lung after treatment with the combination therapy, and transfer of lung NK cells isolated from sensitized and challenged mice treated with the combination significantly suppressed AHR and BAL eosinophilia. These data demonstrate that the combination of IL-2 and IL-18 prevents AHR and airway inflammation, likely through IL-12–mediated induction of IFN-γ production in NK cells.
PMCID: PMC1899318  PMID: 17038663
IL-2; IL-18; STAT4; IFN-γ; airway hyperresponsiveness
6.  RANTES (CCL5) Regulates Airway Responsiveness after Repeated Allergen Challenge 
RANTES (CC chemokine ligand 5) contributes to airway inflammation through accumulation of eosinophils, but the exact role of RANTES (CCL5) is not defined. C57BL/6 mice, sensitized by injection of ovalbumin (OVA) on Days 1 and 14, were challenged with OVA on Days 28, 29, and 30 (3 challenges, short-term–challenge model) or on Days 28, 29, 30, 36, 40, 44, and 48 (7 challenges, repeated–challenge model) and evaluated 48 h later. Anti-mouse RANTES was given intravenously, and recombinant mouse RANTES or PBS was given intratracheally. These reagents were given on Days 28, 29, and 30 in the short-term–challenge study and on Days 44 and 48 in the repeated-challenge study. After short-term challenge, there were no effects after administration of anti-RANTES or RANTES. In the repeated-challenge study, although control mice showed a decrease in airway hyperresponsiveness, administration of anti-RANTES sustained and enhanced airway hyperresponsiveness and increased goblet cell numbers. In contrast, administration of RANTES normalized airway function but reduced goblet cell numbers. IL-12 and IFN-γ levels in BAL decreased in the anti-RANTES group and increased in the RANTES group. IFN-γ–producing CD4 T cells in lung, and IFN-γ production from lung T cells in response to OVA in the anti-RANTES group, were significantly decreased but were increased in the RANTES group. Anti–IFN-γ, administered with RANTES, decreased the effects of RANTES on AHR after repeated challenge. These data indicate that RANTES plays a role in the regulation of airway function after repeated allergen challenge, in part through modulation of levels of IFN-γ and IL-12.
PMCID: PMC2643254  PMID: 16528011
airway hyperresponsiveness; IFN-γ; IL-12; RANTES (CCL5)
7.  Syk Activation in Dendritic Cells Is Essential for Airway Hyperresponsiveness and Inflammation 
We evaluated the role of Syk, using an inhibitor, on allergen-induced airway hyperresponsiveness (AHR) and airway inflammation in a system shown to be B cell– and mast cell–independent. Sensitization of BALB/c mice with ovalbumin (OVA) and alum after three consecutive OVA challenges resulted in AHR to inhaled methacholine and airway inflammation. The Syk inhibitor R406 (30 mg/kg, administered orally, twice daily) prevented the development of AHR, increases in eosinophils and lymphocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid, and goblet cell metaplasia when administered after sensitization and before challenge with OVA. Levels of IL-4, IL-5, and IFN-γ in BAL fluid and allergen-specific antibody levels in serum were not affected by treatment. Because many of these responses may be influenced by dendritic cell function, we investigated the effect of R406 on bone marrow–derived dendritic cell (BMDC) function. Co-culture of BMDC with immune complexes of OVA and IgG anti-OVA together with OVA-sensitized spleen mononuclear cells resulted in increases in IL-13 production. IL-13 production was inhibited if the BMDCs were pretreated with the Syk inhibitor. Intratracheal transfer of immune complex-pulsed BMDCs (but not nonpulsed BMDCs) to naive mice before airway allergen challenge induced the development of AHR and increases in BAL eosinophils and lymphocytes. All of these responses were inhibited if the transferred BMDCs were pretreated with R406. These results demonstrate that Syk inhibition prevents allergen-induced AHR and airway inflammation after systemic sensitization and challenge, at least in part through alteration of DC function.
PMCID: PMC2644204  PMID: 16339999
AHR; dendritic cells; eosinophils; mice; Syk

Results 1-7 (7)