Rationale: Respiratory syncytial virus (RSV) bronchiolitis in infants may be followed by the development of asthma-like symptoms. Age at first infection dictates consequences upon reinfection. Reinfection of mice initially exposed as neonates to RSV enhanced development of airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus hyperproduction. RSV lower respiratory tract disease is associated with activation of the leukotriene pathway.

Objectives: To determine the effects of montelukast (MK), a cysteinyl leukotriene (cysLT) receptor antagonist, in primary and secondary RSV-infected newborn and adult mice.

Methods: BALB/c mice were infected with RSV at 1 week (neonate) or 6 to 8 weeks (adult) of age and reinfected 5 weeks later. MK was administered 1 day before the initial infection and through Day 6 after infection. Seven days after primary or secondary infection, airway function was assessed by lung resistance to increasing doses of inhaled methacholine; lung inflammation, goblet cell metaplasia, and cytokine levels in bronchoalveolar lavage fluid were monitored.

Measurements and Main Results: RSV infection induced cysLT release in bronchoalveolar lavage fluid. MK decreased RSV-induced AHR, airway inflammation, and increased IFN-γ production in primary infected adult and neonatal mice. MK, administered during initial infection of neonates but not during secondary infection, prevented subsequent enhancement of AHR, airway eosinophilia, and mucus hyperproduction upon reinfection.

Conclusions: MK attenuated the initial responses to primary RSV infection in both age groups and altered the consequences of RSV reinfection in mice initially infected as neonates. These data support an important role for cysLT in RSV-induced AHR and inflammation.

BACKGROUND

Although implicated in the disease, the specific contributions of FcεRI and IL-13 to the pathogenesis of peanut-induced intestinal allergy are not well defined.

OBJECTIVES

To determine the contributions of FcεRI, IL-13, and mast cells to the development of intestinal mucosal responses in a mouse model of peanut-induced intestinal allergy.

METHODS

Sensitized wild-type (WT), FcεRI-deficient (FcεRI−/−), and mast cell-deficient (KitW-sh/W-sh) mice received peanut orally every day for 1 week. Bone marrow-derived mast cells (BMMC) from WT, FcεRI−/−, IL- 4−/−, IL-13−/−, and IL- 4/IL-13−/− mice were differentiated and transferred into WT, FcεRI−/−, and KitW-sh/W-sh recipients. BMMC from WT and UBI-GFP/BL6 mice were differentiated and transferred into WT and KitW-sh/W-sh mice. Blockade of IL-13 was achieved using IL- 13Ra2-IgG fusion protein.

RESULTS

FcεRI−/− mice showed decreased intestinal inflammation (mast cell and eosinophil numbers) and goblet cell metaplasia, and reduced levels of IL-4, IL-6, IL-13, and IL-17A mRNA expression in the jejunum. Transfer of WT BMMC to FcεRI−/− recipients restored their ability to develop intestinal allergic responses compared to transfer of FcεRI−/−, IL-13−/−, or IL-4/IL-13−/−BMMC. FcεRI−/− mice exhibited lower IL-13 levels and treatment of WT mice with IL-13Rα2 prevented peanut-induced intestinal allergy and inflammation.

CONCLUSIONS

These data indicate that the development of peanut-induced intestinal allergy is mediated through a mast cell-dependent, IgE-FcεRI-IL-13 pathway. Targeting IL-13 may be a potential treatment for IgE-mediated peanut allergic responses in the intestine.

Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract diseases (bronchiolitis and pneumonia) during infancy and early childhood. There is increasing evidence which indicates that severe pulmonary disease caused by RSV infection in infancy is associated with recurrent wheezing and development of asthma later in childhood. However, the underlying mechanisms linking RSV infection to persistent airway hyperresponsiveness and dysfunction are not fully defined. To study these processes in ways which are not available in humans, animal models have been established and have provided valuable insight into the pathophysiology of RSV-induced disease. In this paper, we discuss experimental models of RSV infection in mice and highlight a new investigative approach in which mice are initially infected as neonates and then reinfected later in life. The findings shed light on the mechanisms underlying the association between early severe RSV infection and development of asthma later in childhood.

Naturally occurring CD4+CD25+Foxp3+ T regulatory cells (nTregs) regulate lung allergic responses through production of IL-10 and TGF-β. nTregs from CD8−/− mice failed to suppress lung allergic responses and were characterized by reduced levels of Foxp3, IL-10, and TGF-β, and high levels of IL-6. Administration of anti–IL-6 or anti–IL-6R to wild-type recipients prior to transfer of CD8−/− nTregs restored suppression. nTregs from IL-6−/− mice were suppressive, but lost this capability if incubated with IL-6 prior to transfer. The importance of CD8 in regulating the production of IL-6 in nTregs was demonstrated by the loss of suppression and increases in IL-6 following transfer of nTregs from wild-type donors depleted of CD8+ cells. Transfer of nTregs from CD8−/− donors reconstituted with CD8+ T cells was suppressive, and accordingly, IL-6 levels were reduced. These data identify the critical role of CD8–T regulatory cell interactions in regulating the suppressive phenotype of nTregs through control of IL-6 production.

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.

Background

An important aspect of the innate immune response to pathogens is the production of anti-microbial peptides such as cathelicidin-related antimicrobial peptide (CRAMP), the murine homologue of human cathelicidin LL-37. In this study, mechanisms regulating LPS-induction of CRAMP gene expression in mast cells were investigated. NF-κB and MAPK pathways were the focus of investigation.

Methods

Mouse bone marrow-derived mast cells were grown in culture and stimulated with LPS. MAPKs and NF-κB were monitored by immunoblot analysis. ERK, JNK and p38 MAPK were inhibited using siRNAs or a pharmacological inhibitor. Accumulation of the p65 component of NF-κB was inhibited by siRNA and NF-κB activation was inhibited by overexpression of IκBα. MEKK2 or MEKK3 were overexpressed by transfection. The effects of all of these treatments on CRAMP gene expression were monitored by RT-PCR.

Results

Inhibition of ERK, JNK or p38 MAPK had little discernible effect on LPS-inducible CRAMP gene expression. Overexpression of MEKK2 or MEKK3 likewise had little impact. However, inhibition of the accumulation of p65 NF-κB prevented LPS-induced CRAMP mRNA. An important role for NF-κB in CRAMP gene expression was confirmed by overexpression of IκBα, which reduced both basal and induced levels of CRAMP mRNA.

Conclusions

NF-κB, but not MAPKs, plays an important role in LPS-mediated induction of CRAMP gene in mast cells. Defects which inhibit NF-κB activity may increase susceptibility to bacterial and viral pathogens which are sensitive to cathelicidins.

Asthma currently affects the lives of more than 30 million Americans from infancy to the elderly. In many ways, pediatric asthma differs from adult asthma, including childhood-onset adult asthma. Despite many advances in our understanding of the disease, the natural history of asthma is not well defined, especially in different subsets of patients. For many with allergic asthma the disease has its origins in early childhood, associated with early sensitization to aeroallergens and exposure to repeated viral infections. These early life exposures, coupled with genetically determined susceptibility, have a major impact on the natural history of the disease. A number of risk factors during the critical early stages in the initiation of asthma have been associated with subsequent outcomes. In addition, protective factors linked to early life experiences have also been delineated which may impact the development of atopy and asthma and reduce the prevalence of these diseases. Cumulatively, the data highlight the critical nature of this early period in which immune/inflammatory responses in the lung are initiated and serve to maintain the disease in subsequent years.

Asthma is a complex syndrome with many clinical phenotypes in children and adults. Despite the rapidly increasing prevalence, clinical investigation and epidemiological studies of asthma, the successful introduction of new drugs has been limited due to the different disease phenotypes and ethical issues. Mouse models of asthma replicate many of the features of human asthma, including airway hyperreactivity, and airway inflammation. Therefore, examination of disease mechanisms in mice has been used to elucidate asthma pathology and to identify and evaluate new therapeutic agents. In this article, we discuss the various animal models of asthma with a focus on mouse strains, allergens, protocols, and outcome measurements.

Activation of the alternative pathway of complement plays a critical role in the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in mice. Endogenous factor H, a potent inhibitor of the alternative pathway, is increased in the airways of sensitized and challenged mice, but its role in regulating inflammation or AHR has been unknown. We found that blocking the tissue-binding function of factor H with a competitive antagonist increased complement activation and tissue inflammation after allergen challenge of sensitized mice. Conversely, administration of a fusion protein that contains the iC3b/C3d binding region of complement receptor 2 (CR2) linked to the inhibitory region of factor H (CR2-fH), a molecule directly targeting complement activating surfaces, protected mice in both primary and secondary challenge models of AHR and lung inflammation. Thus, although endogenous factor H does play a role in limiting the development of AHR, strategies to deliver the complement regulatory region of factor H specifically to the site of inflammation provide greater protection than that afforded by endogenous regulators. Such an agent may be an effective therapy for the treatment of asthma.

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.

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.

Rationale: Ambient particulate matter concentrations have been positively associated with urinary leukotriene E4 (LTE4) levels and albuterol usage in children with asthma but interactions with environmental tobacco smoke (ETS) exposure have not been demonstrated despite obvious exposure to both pollutants in an urban setting.

Objectives: To assess the health effects of concurrent ETS and ambient particulate matter exposure in children with asthma.

Methods: Albuterol usage and LTE4 levels were monitored in 82 urban schoolchildren with asthma over three consecutive fall to spring school periods. Concentrations of morning maximum ambient particulate matter <2.5 μm in aerodynamic diameter (mmPM2.5) and urine cotinine levels were also measured daily.

Measurements and Main Results: Albuterol usage and LTE4 were related to mmPM2.5 concentrations on days when urine cotinine levels were low (<10 ng/ml/mg creatinine); on these days, mean albuterol usage and LTE4 increased up to 5 or 6% per 10 μg/m3 increase in mmPM2.5. In contrast, no significant relationship was observed when cotinine was high, although mean albuterol usage and LTE4 levels were greater in this case. Model fits for LTE4 levels as a function of mmPM2.5 concentrations were improved when mmPM2.5 concentrations were logged, suggesting a nonlinear dose–response relationship between particulate matter exposure concentrations and airway mediators of asthma, for which the relationship tends to flatten at higher concentrations.

Conclusions: This study suggests that ETS modifies the acute effects of low-level ambient PM2.5 exposure on childhood asthma. This negative interaction, the smaller effect of particulate matter exposure in children exposed to higher ETS, may be related to a nonlinear dose–response relationship between asthma mediators and particulate exposures.

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.

Background

Notch signaling pathways govern immune function and the regulation of Th1 and Th2 differentiation. We previously demonstrated essential interactions between Notch on CD4+ T cells and Jagged1 on antigen-presenting cells in Th2 differentiation for the full development of allergen-induced airway hyperresponsiveness (AHR) and allergic airway inflammation.

Methods

Bone marrow-derived dendritic cells (BMDCs) were differentiated and incubated with different preparations of ovalbumin (OVA), including lipopolysaccharide (LPS)-depleted and LPS-spiked preparations. In some experiments recipient mice also received soluble Jagged1-Fc in addition to allergen-pulsed BMDCs. Ten days following transfer of BMDCs, mice were exposed to three airway challenges with OVA, and airway responsiveness to inhaled methacholine, airway inflammation and cytokine production were monitored 48 h later. Notch ligand expression was assessed by real-time PCR.

Results

Induction of Jagged1 expression on antigen-pulsed BMDCs was dependent on low-dose endotoxin. In vivo, transfer of endotoxin-free, antigen-pulsed BMDCs failed to induce AHR or airway eosinophilia on allergen challenge. However, administration of exogenous Jagged1-Fc together with endotoxin-free, allergen-pulsed BMDCs fully restored the responses to allergen challenge.

Conclusions

These data demonstrate that LPS regulates the expression of Jagged1 on BMDCs, which is essential for the full development of lung allergic responses.

Chemokine receptor (CCR) 5 is expressed on dendritic cells, macrophages, CD8 cells, memory CD4 T cells, and stromal cells, and is frequently used as a marker of T helper type 1 cells. Interventions that abrogate CCR5 or interfere with its ligand binding have been shown to alter T helper type 2–induced inflammatory responses. The role of CCR5 on allergic airway responses is not defined. CCR5-deficient (CCR5−/−) and wild-type (CCR5+/+) mice were sensitized and challenged with ovalbumin (OVA) and allergic airway responses were monitored 48 hours after the last OVA challenge. Cytokine levels in lung cell culture supernatants were also assessed. CCR5−/− mice showed significantly lower airway hyperresponsiveness (AHR) and lower numbers of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid compared with CCR5+/+ mice after sensitization and challenge. The levels of IL-4 and IL-13 in BAL fluid of CCR5−/− mice were lower than in CCR5+/+ mice. Decreased numbers of lung T cells were also detected in CCR5−/− mice after sensitization and challenge. Transfer of OVA-sensitized T cells from CCR5+/+, but not transfer of CCR5−/− cells, into CCR5−/− mice restored AHR and numbers of eosinophils in BAL fluid after OVA challenge. Accordingly, the numbers of airway-infiltrating donor T cells were significantly higher in the recipients of CCR5+/+ T cells. Taken together, these data suggest that CCR5 plays a pivotal role in allergen-induced AHR and airway inflammation, and that CCR5 expression on T cells is essential to the accumulation of these cells in the airways.

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.

Rationale: There is conflicting information about the development and resolution of airway inflammation and airway hyperresponsiveness (AHR) after repeated airway exposure to allergen in sensitized mice.

Methods: Sensitized BALB/c and C57BL/6 mice were exposed to repeated allergen challenge on 3, 7, or 11 occasions. Airway function in response to inhaled methacholine was monitored; bronchoalveolar lavage fluid inflammatory cells were counted; and goblet cell metaplasia, peribronchial fibrosis, and smooth muscle hypertrophy were quantitated on tissue sections. Bone marrow–derived dendritic cells were generated after differentiation of bone marrow cells in the presence of growth factors.

Results: Sensitization to ovalbumin (OVA) in alum, followed by three airway exposures to OVA, induced lung eosinophilia, goblet cell metaplasia, mild peribronchial fibrosis, and peribronchial smooth muscle hypertrophy; increased levels of interleukin (IL)-4, IL-5, IL-13, granulocyte-macrophage colony–stimulating factor, transforming growth factor-β1, eotaxin-1, RANTES (regulated on activation, normal T-cell expressed and secreted), and OVA-specific IgG1 and IgE; and resulted in AHR. After seven airway challenges, development of AHR was markedly decreased as was the production of IL-4, IL-5, and IL-13. Levels of IL-10 in both strains and the level of IL-12 in BALB/c mice increased. After 11 challenges, airway eosinophilia and peribronchial fibrosis further declined and the cytokine and chemokine profiles continued to change. At this time point, the number of myeloid dendritic cells and expression of CD80 and CD86 in lungs were decreased compared with three challenges. After 11 challenges, intratracheal instillation of bone marrow–derived dendritic cells restored AHR and airway eosinophilia.

Conclusions: These data suggest that repeated allergen exposure leads to progressive decreases in AHR and allergic inflammation, through decreases in myeloid dendritic cell numbers.

Rationale: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor–mediated signaling.

Objective: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo.

Methods: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcɛRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow–derived mast cells (BMMCs) before cross-linking with allergen.

Results: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor α, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge.

Conclusion: This study delineates a functional role for Syk in the development of mast cell– and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma.

Rationale: Leukotriene B4 (LTB4) is a rapidly synthesized, early leukocyte chemoattractant that signals via its cell surface receptor, leukotriene B4 receptor 1 (BLT1), to attract and activate leukocytes during inflammation. A role for the LTB4–BLT1 pathway in allergen-induced airway hyperresponsiveness and inflammation is not well defined. Objectives: To define the role of the LTB4 receptor (BLT1) in the development of airway inflammation and altered airway function. Methods: BLT1-deficient (BLT1−/−) mice and wild-type mice were sensitized to ovalbumin by intraperitoneal injection and then challenged with ovalbumin via the airways. Airway responsiveness to inhaled methacholine, bronchoalveolar lavage fluid cell composition and cytokine levels, and lung inflammation and goblet cell hyperplasia were assessed. Results: Compared with wild-type mice, BLT1−/− mice developed significantly lower airway responsiveness to inhaled methacholine, lower goblet cell hyperplasia in the airways, and decreased interleukin (IL)-13 production both in vivo, in the bronchoalveolar lavage fluid, and in vitro, after antigen stimulation of lung cells in culture. Intracellular cytokine staining of lung cells revealed that bronchoalveolar lavage IL-13 levels and numbers of IL-13+/CD4+ and IL-13+/CD8+ T cells were also reduced in BLT1−/− mice. Reconstitution of sensitized and challenged BLT1−/− mice with allergen-sensitized BLT1+/+ T cells fully restored the development of airway hyperresponsiveness. In contrast, transfer of naive T cells failed to do so. Conclusion: These data suggest that BLT1 expression on primed T cells is required for the full development of airway hyperresponsiveness, which appears to be associated with IL-13 production in these cells.

CD4+ T cells have been shown to play a role in the development of airway hyperresponsivness (AHR) and airway eosinophilia in mice using ablation as well as adoptive transfer experiments. However, as other T cell subsets (CD8, NKT) may play a role in these models, we examined the responses of sensitized CD4-deficient mice after either primary or secondary airway allergen challenge. After sensitization, CD4-deficiency in mice was not associated with airway eosinophilia, allergen-specific IgE, or elevated levels of interleukin (IL)-4 or IL-13. Increases in lung CD8 T cells and IL-5 were observed and shown to be essential for AHR as demonstrated after CD8 T cell depletion or anti–IL-5 treatment. In contrast to the response of sensitized CD4-deficient mice to primary allergen challenge, they failed to develop AHR after secondary allergen challenge. Although the importance of this CD4+ T cell–independent pathway in normal mice is unclear at this time, these studies identify the diversity of the cellular pathway, which may contribute to the development of AHR after primary allergen exposure of sensitized mice.

Most of the studies investigating the effectiveness of blocking the leukotriene B4 (LTB4) receptor 1 (BLT1) have been performed in models of primary or acute allergen challenge. The role of the LTB4-BLT1 pathway in secondary challenge models, where airway hyperresponsiveness (AHR) and airway inflammation have been established, has not been defined. We investigated the effects of blocking BLT1 on early- and late-phase development of AHR and airway inflammation in previously sensitized and challenged mice. Female BALB/c mice were sensitized (Days 1 and 14) and challenged (primary, Days 28–30) with ovalbumin. On Day 72, mice were challenged (secondary) with a single OVA aerosol, and the early and late phases of AHR and inflammation were determined. Specific blockade of BLT1 was attained by oral administration of a BLT1 antagonist on Days 70 through 72. Administration of the antagonist inhibited the secondary ovalbumin challenge–induced alterations in airway responses during the late phase but not during the early phase, as demonstrated by decreases in AHR and in bronchoalveolar lavage neutrophilia and eosinophilia 6 and 48 hours after secondary challenge. The latter was associated with decreased levels of KC protein, macrophage inflammatory protein 2, and IL-17 in the airways. These data identify the importance of the LTB4-BLT1 pathway in the development of late–phase, allergen-induced airway responsiveness after secondary airway challenge in mice with established airway disease.

Early-phase reactions (EPRs) and late-phase reactions (LPRs) are characteristic features of bronchial asthma, although the pathogenetic mechanisms responsible for each of the responses are not fully defined. A murine model of EPRs and LPRs was developed to investigate the role of IL-5 and eosinophils in development of both responses. After initial intraperitoneal sensitization and airway challenge to ovalbumin (OVA), mice were provoked by additional exposure to OVA. An EPR, characterized by a transient increase in airway responsiveness, was observed 5–30 minutes after antigen provocation. This response was followed by an LPR that reached its maximum at 6 hours after challenge and was characterized by increased airway responsiveness and significant lung eosinophilia. The EPR was blocked by cromoglycate and albuterol, whereas the LPR was abolished by cromoglycate and hydrocortisone. Before provocation with allergen, administration of anti–IL-5 antibody prevented the influx of eosinophils into the lung tissue and abolished the LPR but not EPR. These results suggest that IL-5 and eosinophils are essential for development of the LPR, but not EPR, in this model.

Background

Children with asthma exposed to second hand tobacco smoke (SHS) may be at higher risk for severe exacerbations but biomarkers of susceptibility to SHS exposure have not been previously reported.

Objectives

To assess the ability of urinary leukotriene E4 (uLTE4) levels to predict increased risk of severe asthma exacerbations requiring emergency room (ED) or urgent care (UC) visits.

Methods

44 schoolchildren with moderate to severe asthma receiving inhaled corticosteroids were followed for 5 months with repeated measurements of urine LTE4 and monitoring of ED and UC visits. SHS exposure status was determined by pre-study questionnaires and repeated measurements of urinary cotinine during the study.

Results

Nine of 20 (45%) children with SHS exposure experienced a severe exacerbation requiring an ED or UC visit compared to three of 24 (12.5%) children without significant SHS exposure (relative risk =3.6, 95th confidence interval (CI) 1.1-11.5, p=0.02). Urinary LTE4 was a significant predictor of exacerbation risk in children exposed to SHS (area under the curve 0.85, p= 0.003). Other predictors such as nighttime symptom frequency, pre and post bronchodilator lung function and exhaled nitric oxide were not related to exacerbations in this group. Urinary LTE4 levels at or above 106 pg/mg achieved 67% (6/9) sensitivity and 100%(11/11) specificity for predicting children with SHS exposure who required an ED or UC visit.

Conclusions

Children exposed to SHS are at increased risk for severe asthma exacerbations despite use of inhaled corticosteroids. Urinary LTE4 levels identify children exposed to SHS at high risk for asthma exacerbations.

Alterations in nuclear factor kappa B (NF-κB) essential modulator (NEMO; HUGO-approved symbol IKBKG) underlie most cases of ectodermal dysplasia with immune deficiency (EDI), a human disorder characterized by anhidrosis with diminished immunity. EDI has also been associated with a single heterozygous mutation at position Ser32 of the NF-κB inhibitor IκBα, one of two phosphorylation sites that are essential for targeting IκBα for proteasomal degradation and hence for activation of NF-κB. We report a novel heterozygous nonsense mutation in the IKBA (HUGO-approved symbol, NFKBIA) gene of a 1-year-old male child with EDI that introduces a premature termination codon at position Glu14. An in-frame methionine downstream of the nonsense mutation allows for reinitiation of translation. The resulting N-terminally truncated protein lacks both serine phosphorylation sites and inhibits NF-κB signaling by functioning as a dominant negative on NF-κB activity in lymphocytes and monocytes. These findings support the scanning model for translation initiation in eukaryotes and confirm the critical role of the NF-κB in the human immune response.

To test if manipulating TCR complex-mediated signaling (TCR signaling) could treat autoimmune disease, we generated the double SKG Src-like adapter protein (SLAP) knockout (DSSKO) mouse model. The SKG mutation in ZAP70 and SLAP have opposing functions on the regulation of TCR signaling. The combination of these two mutations alters TCR signaling in the context of a defined genetic background, uniform environmental conditions, and a well-characterized signaling disruption. In contrast to SKG mice, DSSKO mice do not develop zymosan-induced chronic autoimmune arthritis. This arthritis prevention is not due to significant alterations in thymocyte development or repertoire selection but instead enhanced numbers of regulatory T cells (Tregs) and decreased numbers of Th17 cells skewing the ratio of Tregs to autoreactive effector T cells. Treg depletion and/or functional blockade led to the development of arthritis in DSSKO mice. In vitro suppression of effector T cell proliferation was also enhanced, demonstrating that DSSKO mice have increased numbers of Tregs with increased function. Understanding how TCR signals influence development, expansion, and function of Tregs in DSSKO mice could advance our ability to manipulate Treg biology to treat ultimately autoimmune disease.