Serum OVA-specific IgE Levels Research Articles

United airways: circulating Th2 effector cells in an allergic rhinitis model are responsible for promoting lower airways inflammation

Allergic rhinitis (AR) and asthma often coexist and are referred to as 'united airways' disease. However, the molecular and cellular pathways that are crucially involved in the interaction between upper and lower airways remain to be identified. We sought to assess whether and how AR exacerbates lower airway inflammation upon allergen challenge in mice. We previously developed an intranasal ovalbumin (OVA)-driven AR model, characterized by nasal eosinophilic inflammation, enhanced serum levels of OVA-specific IgE and Th2 cytokine production in cervical lymph nodes. In OVA-sensitized mice with or without AR, a lower airway challenge was given, and after 24 h, lower airway inflammation was analysed. We found that AR mice were more susceptible to eosinophilic inflammation following a lower airway OVA challenge than OVA-sensitized controls. AR mice manifested increased numbers of eosinophils in bronchoalveolar lavage fluid and increased inter-cellular adhesion molecule-1 (ICAM-1) expression on lung endothelium, when compared with OVA-sensitized controls. Depletion of T cells in OVA-challenged AR mice completely abrogated all hallmarks of lower airway inflammation, including enhanced IL-5 and tissue eosinophilia. Conversely, adoptive transfer of Th2 effector cells in naïve animals induced lower airway eosinophilic inflammation after challenge with OVA. Blocking T cell recirculation during AR development by the spingosine-1 analogue FTY720 also prevented lower airway inflammation including ICAM-1 expression in AR mice upon a single lower airway challenge. Our mouse model of 'united airways' disease supports epidemiological and clinical data that AR has a significant impact on lower airway inflammation. Circulating Th2 effector cells are responsible for lung priming in AR mice, most likely through up-regulation of ICAM-1.

Effect of oral tolerance in a mouse model of allergic rhinitis

Objective Induction of oral tolerance (OT) is known to prevent allergic inflammation in models of asthma. This study investigated the preventive effect of OT and airway remodeling in a mouse model of allergic rhinitis (AR). Study Design An in vivo study using an animal model. Setting Catholic Research Institutes of Medical Science. Methods Forty six-week-old, female BALB/c mice were divided into four groups: control, AR, low-dose OT, and high-dose OT. To induce OT, mice were fed ovalbumin (OVA) before sensitization with OVA/aluminum hydroxide, 1 mg for six days in the low-dose OT group and a 25 mg single dose in the high-dose OT group. Mice in the AR group were fed phosphate-buffered saline. After sensitization followed by challenges with OVA during six weeks, nasal behaviors, interleukin (IL)-13 and interferon gamma (IFN-γ) levels in nasal lavage (NAL) fluids, as well as OVA-specific IgE levels in serum, were measured. The degree of goblet cell hyperplasia and thickness of lamina propria were observed in nasal tissues by periodic acid-Schiff and Masson's trichrome stain. A P value < 0.05 was accepted as statistically significant. Results Both OT groups showed a significant decrease in inflammatory cells, IL-13 and IFN-γ in NAL fluids, as well as OVA-specific IgE levels in serum compared with the AR group. In addition, the degree of goblet cell hyperplasia and thickness of lamina propria were attenuated in both OT groups compared with the AR group. Further, these alterations did not differ significantly between the two OT groups. Conclusion These results suggest that OT may effectively reduce allergic inflammation as well as airway remodeling in a mouse model of AR.

The Effects of Lactobacillus rhamnosus on the Prevention of Asthma in a Murine Model

PurposeLactobacilli are probiotic bacteria that are effective in the management of allergic diseases or gastroenteritis. It is hypothesized that such probiotics have immunoregulatory properties and promote mucosal tolerance. Our goal was to investigate whether Lactobacillus casei rhamnosus Lcr35 could inhibit airway inflammation in an ovalbumin (OVA)-induced murine model of asthma.MethodsBALB/c mice aged 6 weeks were used in the present study. Lactobacillus casei rhamnosus Lcr35 was administered daily, starting 1 week prior to the first OVA sensitization (group 1) and 2 days before the first 1% OVA airway challenge (group 2). Mice that received only saline at both sensitization and airway challenge time points were used as negative controls (group 3), and those that had OVA-induced asthma were used as positive controls (group 4). Airway responsiveness to methacholine was assessed, and bronchoalveolar lavage (BAL) was performed. At the endpoint of the study, total IgE as well as OVA-specific IgE, IgG1 and IgG2a in serum was measured by enzyme-linked immunosorbent assay. Lung pathology was also evaluated.ResultsAirway hyperresponsiveness, total cell counts and the proportion of eosinophils in BAL fluid were significantly decreased in group 1 compared with group 4 (P<0.05). Total serum IgE levels were also significantly decreased in group 1 compared with group 4. Serum levels of OVA-specific IgE, IgG1 and IgG2a were not significantly influenced by treatment with Lcr35. There was significantly less peribronchial and perivascular infiltration of inflammatory cells in group 1 compared with group 4; however, there were no significant differences in methacholine challenge, BAL, serology or histology between groups 2 and 4.ConclusionsOral treatment with Lcr35 prior to sensitization can attenuate airway inflammation and hyperreactivity in a mouse model of allergic airway inflammation. These results suggest that Lcr35 may have potential for preventing asthma.

Inhibition of Acidic Mammalian Chitinase by RNA Interference Suppresses Ovalbumin-Sensitized Allergic Asthma

Asthma, a chronic helper T cell type 2-mediated inflammatory disease, is characterized by airway hyperresponsiveness and inflammation. Growing evidence suggests that increased expression of acidic mammalian chitinase (AMCase) may play a role in the pathogenesis of asthma. In the present study, we sought to develop an RNA interference approach to suppress allergic asthma in mice through silencing of AMCase expression. Mice sensitized with ovalbumin (OVA) were intratracheally administered a recombinant adeno-associated virus expressing short hairpin RNA (rAAV-shRNA) against AMCase. In OVA-sensitized mice, the development of allergic symptoms was significantly associated with elevated AMCase expression. After administration of rAAV-shRNA, there was a significant reduction of AMCase expression in the lung and in bronchoalveolar lavage fluid (BALF) cells of sensitized mice. Sensitized mice receiving rAAV-shRNA showed a significant improvement in allergic symptoms, including airway hyperresponsiveness (AHR), eosinophil infiltration, eotaxin, interleukin-13 secretion in BALF, and serum OVA-specific IgE level. Our data suggest the hyperexpression of AMCase in asthma can be suppressed by rAAV-mediated shRNA. Silencing AMCase expression by shRNA may be a promising therapeutic strategy in asthma.

Anti-Malarial Drug Artesunate Attenuates Allergic Airway Inflammation in A Murine Asthma Model (140.18)

Abstract Artesunate is a derivative of artemisinin isolated from a Chinese herb Artemisia annua L. It is used clinically for the treatment of malaria. There are recent evidences supporting an immune-regulatory role by artesunate. We investigated potential anti-inflammatory actions of artesunate in a mouse asthma model. BALB/c mice sensitized and challenged with OVA developed airway eosinophilia and mucus hypersecretion, and elevations in serum IgE level and in bronchoalveolar lavage (BAL) fluid cytokine level. Artesunate significantly inhibited OVA-induced increases in BAL fluid eosinophil count, and IL-5 and IL-13 levels. It also reduced the serum level of OVA-specific IgE. Histologic analysis reveals that artesunate suppressed OVA-induced inflammatory cell infiltration and airway mucus production. Besides, artesunate markedly attenuated OVA-induced mRNA expression of ICAM-1, VCAM-1, Muc5ac and chitinases. Furthermore, artesunate inhibited TNF-α-induced phosphorylation of phosphoinositide 3-kinase (PI3K) and its downstream Akt and p70 S6 kinase, as well as nuclear factor (NF)-κ B DNA binding activity in normal human bronchial epithelial cells. Taken together, our results clearly demonstrate anti-inflammatory effects of artesunate in a mouse asthma model, and it may act by inhibiting PI3K/Akt signaling pathway. (This work was supported by a BioMedical Research Council grant BMRC06/1/21/19/443)

PGI2, and its analog iloprost, regulate CD4+ Th2 lung inflammation and B cell production of IgE (140.11)

Abstract Our previous findings have shown that PGI2 plays an important role in regulating Th2-mediated lung inflammation. Here, we have examined the mechanism by which PGI2, and its analog iloprost, limit allergic inflammation and the IgE response. Effector DO11.10 CD4+ Th2 cells, generated in the presence of the iloprost, produced normal levels of IL-4 and IL-5, but expressed higher levels of ICOS and CD62L than untreated Th2 cells. Adoptive transfer of iloprost-treated Th2 cells into BALB/c mice caused a markedly reduced airway inflammation following OVA inhalation, compared to untreated Th2 cells. CFSE labeling of CD4+ Th2 cells prior to injection revealed that the iloprost-treated cells proliferated less effectively in vivo in response to inhaled OVA. Moreover, intranasal instillation of FITC-OVA showed that iloprost-treated Th2 cells were less effective at promoting dendritic cell migration to the draining lymph nodes and subsequent expansion OVA-specific T cells. These effects were confirmed using mice lacking the IP receptor for PGI2 (IPKO). Following OVA aerosol challenge, primed IPKO mice developed increased eosinophilic inflammation, with elevated levels of airway CD4+ T cells and serum levels of OVA-specific IgE, compared to WT mice. Collectively, these data demonstrate that generation of PGI2, during allergic lung inflammation serves to limit both Th2 cell expansion and IgE production by B cells. A critical role for IL-4 in promoting expression of PGI2 receptor, IP, on Th2 cells and B cells that produce IgE is proposed.

A Novel Anti-Inflammatory Role for Andrographolide in Asthma via Inhibition of The Nuclear Factor-kappaB Pathway (140.17)

Abstract Persistent activation of nuclear factor (NF)-κB has been associated with the development of asthma. Andrographolide, the principal active component of a medicinal plant Andrographis paniculata, has been shown to inhibit NF-κB activity. We hypothesized that andrographolide may attenuate allergic asthma via inhibition of the NF-κB signaling pathway. BALB/c mice sensitized and challenged with OVA developed airway inflammation. Andrographolide inhibited OVA-induced increases in total cell count, eosinophil count, and IL-4, IL-5 and IL-13 levels in bronchoalveolar lavage fluid, and reduced serum level of OVA-specific IgE. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, chitinases, Muc5ac and inducible nitric oxide synthase in lung tissues, and airway hyperresponsiveness to methacholine. In human lung epithelial cells, andrographolide blocked TNF-α-induced phosphorylation of inhibitory κB (IκB) kinase-β (IKKβ), and downstream IκBα degradation, p65 subunit of NF-κB phosphorylation, and p65 nuclear translocation and DNA-binding activity. Our findings implicate a potential therapeutic value of andrographolide in the treatment of asthma and it may act by inhibiting NF-κB pathway at the level of IKKβ activation. (This work was supported by a BioMedical Research Council grant BMRC06/1/21/19/443)

Induction of Th1 cytokines by Leuconostoc mesenteroides subsp. mesenteroides (KCTC 3100) under Th2-type conditions and the requirement of NF-κB and p38/JNK

Leuconostoc mesenteroides subsp. mesenteroides (LMM) KCTC 3100, is one of the prominent species in the fermentation of kimchi, a traditional Korean food. In the present study, we investigated the capacity of this microorganism in inducing Th1 cytokines in the presence of Th2 signals in vitro and in vivo and the requirement of NF-κB and MAPK signaling. Stimulation with heat-killed LMM in mouse splenocytes induced the expression of IFN-γ, which was dependent on IL-12 production by LMM. Pre-treatment with LMM in vitro augmented the production of IFN-γ and IL-4 in response to anti-CD3 plus recombinant IL-4 (rIL-4). LMM administration to mice, beginning either before or after the development of OVA sensitization, increased OVA-restimulated IFN-γ production in the splenocytes and reduced serum total and OVA-specific IgE levels. However, only the pre-sensitization treatment induced a slight reduction in IL-4 from the same cells, but the post-sensitization treatment did not. Induction of IL-12 by LMM in peritoneal macrophages involved NF-κB, p38 and JNK, but not ERK1/2. In conclusion, our data presented the upregulation of IFN-γ by LMM under the pro-Th2 conditions and the requirement of NF-κB, p38 and JNK for IL-12 production. These observations suggest that this microorganism can be a useful Th1-inducing agent in modulating the Th1/Th2 imbalance.

Single-Walled and Multi-Walled Carbon Nanotubes Promote Allergic Immune Responses in Mice

The adjuvant effect of particles on allergic immune responses has been shown to increase with decreasing particle size and increasing particle surface area. Like ultrafine particles, carbon nanotubes (CNTs) have nano-sized dimensions and a large relative surface area and might thus increase allergic responses. Therefore, we examined whether single-walled (sw) and multi-walled (mw) CNTs have the capacity to promote allergic responses in mice, first in an sc injection model and thereafter in an intranasal model. Balb/cA mice were exposed to three doses of swCNT, mwCNT, as well as ultrafine carbon black particles (ufCBPs, Printex90) during sensitization with the allergen ovalbumin (OVA). Five days after an OVA booster, OVA-specific IgE, IgG1, and IgG2a antibodies in serum and the numbers of inflammatory cells and cytokine levels in bronchoalveolar lavage fluid (BALF) were determined. Furthermore, ex vivo OVA-induced cytokine release from mediastinal lymph node (MLN) cells was measured. In separate experiments, differential cell counts were determined in BALF 24 h after a single intranasal exposure to the particles in the absence of allergen. We demonstrate that both swCNT and mwCNT together with OVA strongly increased serum levels of OVA-specific IgE, the number of eosinophils in BALF, and the secretion of Th2-associated cytokines in the MLN. On the other hand, only mwCNT and ufCBP with OVA increased IgG2a levels, neutrophil cell numbers, and tumor necrosis factor-alpha and monocyte chemoattractant protein-1 levels in BALF, as well as the acute influx of neutrophils after exposure to the particles alone. This study demonstrates that CNTs promote allergic responses in mice.

Therapeutic target validation of protein kinase C(PKC)-ζ for asthma using a mouse model

Protein kinase C (PKC) is a complex family consisting of many types of isoenzymes, of which PKC-zeta, an atypical isoform, has been reportedly implicated in the regulation of apoptosis and NF-kappaB, as well as control of T-dependent responses. Based on the recent report that PKC-zeta controls TH2 response, the current study was aimed to evaluate PKC-zeta as a potential therapeutic target for asthma using a mouse model. Mouse allergic asthma was induced by repeated sensitization followed by intranasal challenge with OVA and PKC-zeta pseudosubstrate inhibitor (PPI) was intratracheally instilled before each OVA challenge. Airway hyperreactivity (AHR) was measured by beta-methacoline-induced airflow obstruction. Cellular and cytokine profile in bronchoalveolar lavage fluid (BALF) and level of serum IgE as well as cytokine production by draining lymph node cells were compared. AHR and numbers of eosinophils in BALF were significantly lowered by PPI, indicating that blocking of PKC-zeta activation alleviates asthmatic manifestations. Additionally, PPI instillation decreased IL-5 and IL-13 levels in BALF to approximately 20% of controls, but not IFN-gamma level. Instillation of PPI also caused a marked fall in the level of TNF-alpha, another NF-kappaB-dependent, proinflammatory cytokine. Serum OVA-specific IgE level and ex vivo IL-4, IL-5 and IL-13, but not IFN-gamma, production by peribronchial lymph node cells was also considerably lower in PPI-treated mice. In conclusion, blockade of PKC-zeta signals by intratracheal instillation of PPI alleviates allergen-specific TH2 response as well as asthmatic manifestations and hence PKC-zeta is a promising target for treatment of asthma.

Allergic airway hyperresponsiveness, inflammation, and remodeling do not develop in phosphoinositide 3-kinase γ–deficient mice

Bronchial asthma is characterized by chronic airway inflammation caused by inflammatory cells. Phosphoinositide 3-kinases (PI3Ks) are known to play a prominent role in fundamental cellular responses of various inflammatory cells, including proliferation, differentiation, and cell migration. PI3Ks therefore are expected to have therapeutic potential for asthma. Although some investigations of the involvement between the pathogenesis of asthma and PI3K have been performed, it is unknown whether PI3Kgamma, a PI3K isoform, is involved in the pathogenesis of asthma. We investigated the role of PI3Kgamma in allergen-induced allergic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling with PI3Kgamma-deficient mice. After ovalbumin (OVA) sensitization, wild-type (WT) and PI3Kgamma-deficient mice were exposed to aerosolized OVA 3 days per week for 5 weeks. In OVA-sensitized and OVA-challenged (OVA/OVA) PI3Kgamma-deficient mice, levels of airway inflammation, AHR, and airway remodeling were significantly decreased compared with those in OVA/OVA WT mice. On the other hand, no significant differences were detected in serum OVA-specific IgE and IgG1 levels and CD4/CD8 balance in bronchoalveolar lavage fluid between OVA/OVA WT mice and OVA/OVA PI3Kgamma-deficient mice. To determine in which phase of allergic responses PI3Kgamma plays a role, we transferred splenocytes from OVA-sensitized WT or PI3Kgamma-deficient mice to naive mice of either genotype. Similar increased levels of eosinophils were induced in both WT recipient mice but not in both PI3Kgamma-deficient recipient mice. PI3Kgamma might be involved in allergic airway inflammation, AHR, and airway remodeling by regulating the challenge/effector phase of allergic responses.

A Novel Antiinflammatory Role for Andrographolide in Asthma via Inhibition of the Nuclear Factor-κB Pathway

Persistent activation of nuclear factor (NF)-kappaB has been associated with the development of asthma. Andrographolide, the principal active component of the medicinal plant Andrographis paniculata, has been shown to inhibit NF-kappaB activity. We hypothesized that andrographolide may attenuate allergic asthma via inhibition of the NF-kappaB signaling pathway. BALB/c mice sensitized and challenged with ovalbumin (OVA) developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and cytokine and chemokine levels. Serum IgE levels were also determined. Lung tissues were examined for cell infiltration and mucus hypersecretion, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Andrographolide dose-dependently inhibited OVA-induced increases in total cell count, eosinophil count, and IL-4, IL-5, and IL-13 levels recovered in bronchoalveolar lavage fluid, and reduced serum level of OVA-specific IgE. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, chitinases, Muc5ac, and inducible nitric oxide synthase in lung tissues, and airway hyperresponsiveness to methacholine. In normal human bronchial epithelial cells, andrographolide blocked tumor necrosis factor-alpha-induced phosphorylation of inhibitory kappaB kinase-beta, and downstream inhibitory kappaB alpha degradation, p65 subunit of NF-kappaB phosphorylation, and p65 nuclear translocation and DNA-binding activity. Similarly, andrographolide blocked p65 nuclear translocation and DNA-binding activity in the nuclear extracts from lung tissues of OVA-challenged mice. Our findings implicate a potential therapeutic value of andrographolide in the treatment of asthma and it may act by inhibiting the NF-kappaB pathway at the level of inhibitory kappaB kinase-beta activation.

1α,25-Dihydroxyvitamin D3 Potentiates the Beneficial Effects of Allergen Immunotherapy in a Mouse Model of Allergic Asthma: Role for IL-10 and TGF-β

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a potent inhibitor of NF-kappaB expression, can prevent the maturation of dendritic cells in vitro leading to tolerogenic dendritic cells with increased potential to induce regulatory T cells. Herein, we investigated whether the combination of allergen immunotherapy with 1,25(OH)(2)D(3) potentiates the suppressive effects of immunotherapy and whether the immunoregulatory cytokines IL-10 and TGF-beta are involved in the effector phase. OVA-sensitized and challenged BALB/c mice displayed airway hyperresponsiveness (AHR) and increased serum OVA-specific IgE levels, bronchoalveolar lavage eosinophilia, and Th2 cytokine levels. In this model, the dose response of allergen immunotherapy 10 days before OVA inhalation challenge shows strong suppression of asthma manifestations at 1 mg of OVA, but partial suppression of bronchoalveolar lavage eosinophilia, IgE up-regulation, and no reduction of AHR at 100 microg. Interestingly, coadministration of 10 ng of 1,25(OH)(2)D(3) with 100 microg of OVA immunotherapy significantly inhibited AHR and potentiated the reduction of serum OVA-specific IgE levels, airway eosinophilia, and Th2-related cytokines concomitant with increased IL-10 levels in lung tissues and TGF-beta and OVA-specific IgA levels in serum. Similar effects on suboptimal immunotherapy were observed by inhibition of the NF-kappaB pathway using the selective IkappaB kinase 2 inhibitor PS-1145. The suppressive effects of this combined immunotherapy were partially reversed by treatment with mAb to either IL-10R or TGF-beta before OVA inhalation challenge but completely abrogated when both Abs were given. These data demonstrate that 1,25(OH)(2)D(3) potentiates the efficacy of immunotherapy and that the regulatory cytokines IL-10 and TGF-beta play a crucial role in the effector phase of this mouse model.

Endogenous IL-18 in experimentally induced asthma affects cytokine serum levels but is irrelevant for clinical symptoms

T cells and T cell derived cytokines are involved in the complex pathogenesis of asthma. The role of the cytokine IL-18 however, is not clearly defined so far. On the one hand side IL-18 induces Th1-type cytokines and thereby might counter-regulate Th2-mediated allergic asthma. On the other hand IL-18 also bears pro-inflammatory effects possibly enhancing experimental asthma. In order to elucidate the role of IL-18 in allergic pulmonary inflammation typical symptoms were compared after induction of experimental asthma in IL-18 −/− and in wild type mice. Asthma was induced using ovalbumin (OVA) as allergen for sensitization and challenge. Sham sensitized and OVA challenged mice served as controls. Bronchoalveolar lavage-fluid cytology, leukocyte infiltration in lung tissues, serum levels of OVA-specific IgE and cytokines, and lung function were analyzed. Clear differences could be observed between control and asthmatic mice, both in wild type and IL-18 −/− animals. Surprisingly, no differences were found between asthmatic wild type and IL-18 −/− mice. Thus, in contrast to conflicting data in the literature IL-18 did not suppress or enhance the pulmonary allergic immune response in a murine experimental model of asthma.

High dose of lipopolysaccharide pre-treatment prevents OVA-induced anaphylactic decreases in rectal temperature in the immunized mice

It remains unclear whether lipopolysaccharide (LPS) pre-treatment, which prevents Th2-type responses via Toll-like receptor 4 (TLR4), inhibits anaphylaxis. To determine the dose-dependent effects of LPS pre-treatment on anaphylactic decreases in rectal temperature caused by ovalbumin (OVA) re-exposure in immunized mice, C3H/HeN mice were divided into vehicle/OVA (0mg/kg LPS), L-LPS/OVA (0.5mg/kg LPS), M-LPS/OVA (1.0mg/kg LPS) and H-LPS/OVA (3.0mg/kg LPS) groups. After receiving these treatments, the mice were systemically immunized with OVA. Negative control mice were not immunized with OVA (N-OVA). After measuring the serum levels of OVA-specific IgE and IgG1 antibodies, the mice were examined for changes in their rectal temperature and plasma histamine concentration after OVA re-exposure. The allergen-specific IgE and IgG1 concentrations in sera from L-LPS/OVA, M-LPS/OVA and H-LPS/OVA mice were significantly lower than those in sera from vehicle/OVA mice despite OVA immunization. However, the antibody levels in all OVA-immunized mice, with the exception of the IgG1 levels in H-LPS/OVA mice, were significantly higher than those in N-OVA mice. Interestingly, H-LPS/OVA mice were the only group that did not exhibit a decrease in rectal temperature, since the rectal temperatures in vehicle/OVA, L-LPS/OVA and M-LPS/OVA mice were significantly decreased by OVA re-exposure. Furthermore, the decrease in rectal temperature after OVA re-exposure in L-LPS/OVA mice, which did not exhibit an increase in the plasma histamine concentration, was significantly prevented by treatment with a platelet-activating factor (PAF) receptor antagonist alone. Taken together, the present results indicate that high-dose LPS pre-treatment may prevent anaphylaxis in OVA-immunized mice, and that this mechanism may depend on inhibition of the IgG-PAF pathway rather than the IgE-histamine pathway.

The Role of Sphingosine Kinase in a Murine Model of Allergic Asthma

Asthma is an allergic disease characterized by chronic airway eosinophilia and pulmonary infiltration of lymphocytes, particularly of the Th2 subtype, macrophages and mast cells. Previous studies have shown a pivotal role for sphingosine kinase (SphK) on various proinflammatory cells, such as lymphocyte and eosinophil migration and mast cell degranulation. We therefore examined the roles of SphK in a murine model of allergic asthma. In mice previously sensitized to OVA, i.p. administration of N,N-dimethylsphingosine (DMS), a potent SphK inhibitor, significantly reduced the total inflammatory cell infiltrate and eosinophilia and the IL-4, IL-5, and eotaxin levels in bronchoalveolar lavage fluid in response to inhaled OVA challenge. In addition, DMS significantly suppressed OVA-induced inflammatory infiltrates and mucus production in the lungs, and airway hyperresponsiveness to methacholine in a dose-dependent manner. OVA-induced lymphocyte proliferation and IL-4 and IL-5 secretion were reduced in thoracic lymph node cultures from DMS-treated mice. Moreover, similar reduction in inflammatory infiltrates, bronchoalveolar lavage, IL-4, IL-5, eotaxin, and serum OVA-specific IgE levels was observed in mice with SphK1 knock-down via small interfering RNA approach. Together, these data demonstrate the therapeutic potential of SphK modulation in allergic airways disease.

Leukotriene A4 Hydrolase Inhibitors Decrease Allergic Airway Inflammation and Hyperreactivity by Modulating Leukocyte Recruitment and IL‐13 Production

Leukotriene A4 hydrolase (LTA44H) produces the pro‐inflammatory mediator LTB4, a potent chemoattractant of allergy‐associated leukocytes such as mast cells, T cells, eosinophils, neutrophils and monocytes. The potential utility of a selective, LTA44H inhibitor was examined in vivo using a murine, mast cell‐dependent model of allergic airway inflammation. Treatment with a LTA44H inhibitor significantly decreased eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) compared to vehicle‐treated animals. Cytokines such as IL‐4, IL‐5, IL‐13 and MCP‐1 were also decreased in the BALF. Airways hyperreactivity, measured by the forced oscillation technique, was significantly reduced in LTA44H inhibitor‐treated animals, as were serum levels of ova‐specific IgE and IgG1. Ex vivo antigen stimulation of peribronchiolar lymph node cells from inhibitor‐treated animals resulted in decreased IL‐13 production. Mechanistically, LTA44H inhibitors decreased CD4+ and CD8+ T cell infiltration into the lung and migration of Ova+ dendritic cell into draining lymph nodes. An associated decrease in T cell cytokine production and dendritic cell function in the lung potentially explains the decrease in multiple aspects of airway inflammation and hyperreactivity. These results demonstrate that inhibition of LTA44H has potential therapeutic utility in the treatment of allergic airway disease.

Down-regulation of Caveolin-1, an Inhibitor of Transforming Growth Factor-β Signaling, in Acute Allergen-induced Airway Remodeling

Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-beta (TGF-beta). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-beta, perhaps by forming membrane invaginations that enfold TGF-beta receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-beta signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-beta signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-beta signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-beta and thereby ameliorating pathological airway remodeling.

Indoleamine 2,3-dioxygenase–dependent tryptophan metabolites contribute to tolerance induction during allergen immunotherapy in a mouse model

The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been implicated in immune suppression and tolerance induction. We examined (1) whether IDO activity is required during tolerance induction by allergen immunotherapy or for the subsequent suppressive effects on asthma manifestations and (2) whether tryptophan depletion or generation of its downstream metabolites is involved. Ovalbumin (OVA)-sensitized and OVA-challenged BALB/c mice that display increased airway responsiveness to methacholine, serum OVA-specific IgE levels, bronchoalveolar eosinophilia, and TH2 cytokine levels were used as a model of allergic asthma. Sensitized mice received subcutaneous optimal (1 mg) or suboptimal (100 microg) OVA immunotherapy. Inhibition of IDO by 1-methyl-DL-tryptophan during immunotherapy, but not during inhalation challenge, partially reversed the suppressive effects of immunotherapy on airway eosinophilia and TH2 cytokine levels, whereas airway hyperresponsiveness and serum OVA-specific IgE levels remained suppressed. Administration of tryptophan during immunotherapy failed to abrogate its beneficial effects toward allergic airway inflammation. Interestingly, administration of tryptophan or its metabolites, kynurenine, 3-hydroxykynurenine, and xanthurenic acid, but not 3-hydroxyanthranilinic acid, quinolinic acid, and kynurenic acid, during suboptimal immunotherapy potentiated the reduction of eosinophilia. These effects coincided with reduced TH2 cytokine levels in bronchoalveolar lavage fluid, but no effects on IgE levels were detected. During immunotherapy, the tryptophan metabolites kynurenine, 3-hydroxykynurenine, and xanthurenic acid generated through IDO contribute to tolerance induction regarding TH2-dependent allergic airway inflammation.

Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo

Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-alpha, IL-1beta, and LPS resulted in significant ROS production and NF-kappaB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-alpha levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b+ macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.