Flt-3 ligand reverses late allergic response and airway hyper-responsiveness in a mouse model of allergic inflammation

Flt3 ligand (Flt3-L) is a growth factor for dendritic cells and induces type 1 T cell responses. We recently reported that Flt3-L prevented OVA-induced allergic airway inflammation and suppressed late allergic response and airway hyper-responsiveness (AHR). In the present study we examined whether Flt3-L reversed allergic airway inflammation in an established model of asthma. BALB/c mice were sensitized and challenged with OVA, and AHR to methacholine was established. Then mice with AHR were randomized and treated with PBS or 6 microg of Flt3-L i.p. for 10 days. Pulmonary functions and AHR to methacholine were examined after rechallenge with OVA. Treatment with Flt3-L of presensitized mice significantly suppressed (p < 0.001) the late allergic response, AHR, bronchoalveolar lavage fluid total cellularity, absolute eosinophil counts, and inflammation in the lung tissue. There was a significant decrease in proinflammatory cytokines (TNF-alpha, IL-4, and IL-5) in bronchoalveolar lavage fluid, with a significant increase in serum IL-12 and a decrease in serum IL-5 levels. There was no significant effect of Flt3-L treatment on serum IL-4 and serum total IgE levels. Sensitization with OVA significantly increased CD11b(+)CD11c(+) cells in the lung, and this phenomenon was not significantly affected by Flt3-L treatment. These data suggest that Flt3-L can reverse allergic airway inflammation and associated changes in pulmonary functions in murine asthma model.     

Contribution of antigen-primed CD8+ T cells to the development of airway hyperresponsiveness and inflammation is associated with IL-13

The role of Th2/CD4 T cells, which secrete IL-4, IL-5, and IL-13, in allergic disease is well established; however, the role of CD8(+) T cells (allergen-induced airway hyperresponsiveness (AHR) and inflammation) is less clear. This study was conducted to define the role of Ag-primed CD8(+) T cells in the development of these allergen-induced responses. CD8-deficient (CD8(-/-)) mice and wild-type mice were sensitized to OVA by i.p. injection and then challenged with OVA via the airways. Compared with wild-type mice, CD8(-/-) mice developed significantly lower airway responsiveness to inhaled methacholine and lung eosinophilia, and exhibited decreased IL-13 production both in vivo, in the bronchoalveolar lavage (BAL) fluid, and in vitro, following Ag stimulation of peribronchial lymph node (PBLN) cells in culture. Reconstitution of sensitized and challenged CD8(-/-) mice with allergen-sensitized CD8(+) T cells fully restored the development of AHR, BAL eosinophilia, and IL-13 levels in BAL and in culture supernatants from PBLN cells. In contrast, transfer of naive CD8(+) T cells or allergen-sensitized CD8(+) T cells from IL-13-deficient donor mice failed to do so. Intracellular cytokine staining of lung as well as PBLN T cells revealed that CD8(+) T cells were a source of IL-13. These data suggest that Ag-primed CD8(+) T cells are required for the full development of AHR and airway inflammation, which appears to be associated with IL-13 production from these primed T cells.     

Role for IgE in airway secretions: IgE immune complexes are more potent inducers than antigen alone of airway inflammation in a murine model

IgE is present in airway secretions from human patients with allergic rhinitis and bronchial asthma. However, the contribution of IgE present locally to the overall airway inflammation is not well understood. We hypothesize that Ag-specific IgE can capture airborne Ags and form immune complexes. These immune complexes may function as potent inducers of immune responses in the lung, contributing to the perpetuation of airway inflammation. BALB/c mice were first sensitized with OVA in alum systemically and then challenged with nebulized OVA. Bronchoalveolar lavage (BAL) fluid from these mice contained significant amounts of IgE, of which >50% was Ag specific. The IgE levels in airway secretions remained elevated for more than 15 days after the termination of Ag exposure. Significant amounts of IgE-OVA immune complexes were detected in BAL fluid from the OVA-challenged mice. For comparison of IgE immune complexes vs Ag alone, we treated OVA-immunized mice with intranasal administration of trinitrophenyl-OVA or trinitrophenyl-OVA-anti-DNP IgE. Those treated with the immune complexes showed significantly higher levels of IL-4 and more pronounced eosinophilia in BAL fluid than did those receiving the Ag alone. The IgE immune complexes did not augment the inflammatory response in high affinity IgE receptor (FcepsilonRI)-deficient mice. We conclude that IgE present in the airways can capture the Ag and that the immune complexes thus formed may augment allergic airway response in an FcepsilonRI-dependent manner. Thus, IgE present in airway secretions may facilitate Ag-mediated allergic airway inflammation.     

Interleukin-10 does not mediate inhalational tolerance in a chronic model of ovalbumin-induced allergic airway disease

OBJECTIVE: IL-10 is a potent anti-inflammatory cytokine, and IL-10-producing regulatory T cells are effective inhibitors of murine asthmatic responses. This study determined whether IL-10-dependent mechanisms mediated the local inhalational tolerance seen with chronic inhalational exposure to antigen. METHODS: Wildtype and IL-10(-/-) mice were sensitized with ovalbumin (OVA) and then challenged with daily OVA inhalations for 10 days or 6 weeks. RESULTS: The 10-day animals developed allergic airway disease, characterized by BAL eosinophilia, histologic airway inflammation and mucus secretion, methacholine hyperresponsiveness, and OVA-specific IgE production. These changes were more pronounced in IL-10(-/-) mice. The 6-week IL-10(-/-) and wildtype animals both developed inhalational tolerance, with resolution of airway inflammation but persistence of OVA-specific IgE production. CONCLUSION: IL-10 may have anti-inflammatory effects in the acute stage of murine allergic airways disease, but the cytokine does not mediate the development of local inhalational tolerance with chronic antigen exposure.     

Long-term protective and antigen-specific effect of heat-killed Mycobacterium vaccae in a murine model of allergic pulmonary inflammation

This report examines the effect of heat-killed Mycobacterium vaccae in a mouse model of allergic pulmonary inflammation. The s.c. administration of M. vaccae 3 wk before the immunization significantly reduced Ag-induced airway hyperreactivity and the increase in the numbers of eosinophils observed in the bronchoalveolar lavage fluid, blood, and bone marrow, even though no detectable changes in either cytokine (IL-4, IL-13, IL-5, and IFN-gamma) or total IgE levels were observed. Furthermore, transfer of splenocytes from OVA-immunized and M. vaccae-treated mice into recipient, OVA-immunized mice significantly reduced the allergen-induced eosinophilia by an IFN-gamma-independent mechanism, clearly indicating that the mechanism by which M. vaccae induces its inhibitory effect is not due to a redirection from a predominantly Th2 to a Th1-dominated immune response. The protective effect of M. vaccae on the allergen-induced eosinophilia lasted for at least 12 wk after its administration, and the treatment was also effective in presensitized mice. Moreover, the allergen specificity of the inhibitory effect could be demonstrated using a double-immunization protocol, where M. vaccae treatment before OVA immunization had no effect on the eosinophilic inflammation induced by later immunization and challenge with cockroach extract Ag. Taken together, these results clearly demonstrate that M. vaccae is effective in blocking allergic inflammation by a mechanism independent of IFN-gamma, induces long term and Ag-specific protection, and therefore has both prophylactic and therapeutic potential for the treatment of allergic diseases.     

Mucosal immunotherapy with CpG oligodeoxynucleotides reverses a murine model of chronic asthma induced by repeated antigen exposure

Murine models of acute atopic asthma may be inadequate to study the effects of recurrent exposure to inhaled allergens, such as the epithelial changes seen in asthmatic patients. We developed a murine model in which chronic airway inflammation is maintained by repeated allergen [ovalbumin (OVA)] inhalation; using this model, we examined the response to mucosal administration of CpG DNA (oligonucleotides) and specific antigen immunotherapy. Mice repeatedly exposed to OVA developed significantly greater airway hyperresponsiveness and goblet cell hyperplasia, but not airway eosinophilia, compared with those exposed only twice. CpG-based immunotherapy significantly reversed both acute and chronic markers of inflammation as well as airway hyperresponsiveness. We further examined the effect of mucosal immunotherapy on the response to a second, unrelated antigen. Mice sensitized to both OVA and schistosome eggs, challenged with inhaled OVA, and then treated with OVA-directed immunotherapy demonstrated significant reduction of airway hyperresponsiveness and a moderate reduction in eosinophilia, after inhalation challenge with schistosome egg antigens. In this model, immunotherapy treatment reduced bronchoalveolar lavage (BAL) levels of Th2 cytokines (IL-4, IL-5, IL-13, and IL-10) without changing BAL IFN-gamma. Antigen recall responses of splenocytes from these mice demonstrated an antigen-specific (OVA) enhanced release of IL-10 from splenocytes of treated mice. These results suggest that CpG DNA may provide the basis for a novel form of immunotherapy of allergic asthma. Both antigen-specific and, to a lesser extent, antigen-nonspecific responses to mucosal administration of CpG DNA are seen.     

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses

Background

Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.

Methods

BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.

Results

Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.

Conclusion

These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.     

Effect of microbial heat shock proteins on airway inflammation and hyperresponsiveness

Microbial heat shock proteins (hsp) have been associated with the generation and induction of Th1-type immune responses. We tested the effects of treatment with five different microbial hsp (Mycobacterium leprae, Streptococcus pneumoniae, Helicobacter pylori, bacillus Calmette-Guérin, and Mycobacterium tuberculosis) in a murine model of allergic airway inflammation and airway hyperresponsiveness (AHR). Mice were sensitized to OVA by i.p. injection and then challenged by OVA inhalation. Hsp were administered to each group by i.p. injection before sensitization and challenge. Sensitized and challenged mice developed increased serum levels of OVA-specific IgE with significant airway eosinophilia and heightened responsiveness to methacholine when compared with nonsensitized animals. Administration of M. leprae hsp prevented both development of AHR as well as bronchoalveolar lavage fluid eosinophilia in a dose-dependent manner. Treatment with M. leprae hsp also resulted in suppression of IL-4 and IL-5 production in bronchoalveolar lavage fluid, while IL-10 and IFN-gamma production were increased. Furthermore, M. leprae hsp treatment significantly suppressed OVA-specific IgE production and goblet cell hyperplasia/mucin hyperproduction. In contrast, treatment with the other hsp failed to prevent changes in airway responsiveness, lung eosinophilia, or cytokine production. Depletion of gamma/delta T lymphocytes before sensitization and challenge abolished the effect of M. leprae hsp treatment on AHR. These results indicate selective and distinctive properties among the hsp, and that M. leprae hsp may have a potential therapeutic role in the treatment of allergic airway inflammation and altered airway function.     

CC chemokine receptor-2 is not essential for the development of antigen-induced pulmonary eosinophilia and airway hyperresponsiveness

Monocyte chemoattractant proteins-1 and -5 have been implicated as important mediators of allergic pulmonary inflammation in murine models of asthma. The only identified receptor for these two chemokines to date is the CCR2. To study the role of CCR2 in a murine model of Ag-induced asthma, we compared the pathologic and physiological responses of CCR2(-/-) mice with those of wild-type (WT) littermates following immunization and challenge with OVA. OVA-immunized/OVA-challenged (OVA/OVA) WT and CCR2(-/-) mice developed significant increases in total cells recovered by bronchoalveolar lavage (BAL) compared with their respective OVA-immunized/PBS-challenged (OVA/PBS) control groups. There were no significant differences in BAL cell counts and differentials (i.e., macrophages, PMNs, lymphocytes, and eosinophils) between OVA/OVA WT and CCR2(-/-) mice. Serologic evaluation revealed no significant difference in total IgE and OVA-specific IgE between OVA/OVA WT mice and CCR2(-/-) mice. Lung mRNA expression and BAL cytokine protein levels of IL-4, IL-5, and IFN-gamma were also similar in WT and CCR2(-/-) mice. Finally, OVA/OVA CCR2(-/-) mice developed increased airway hyper-responsiveness to a degree similar to that in WT mice. We conclude that following repeated airway challenges with Ag in sensitized mice, the development of Th2 responses (elevated IgE, pulmonary eosinophilia, and lung cytokine levels of IL-4 and IL5) and the development of airway hyper-responsiveness are not diminished by a deficiency in CCR2.     

Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model

Mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in the activation of inflammatory cells. Recent findings revealed that the activity of p42/44 MAPK (also known as extracellular signal-regulated kinase (ERK)) in the lungs was significantly higher in asthmatic mice than in normal controls. We hypothesized that inhibition of ERK activity may have anti-inflammatory effects in allergic asthma. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of VCAM-1 expression, and airway hyperresponsiveness. Intraperitoneal administration of U0126, a specific MAPK/ERK kinase inhibitor, significantly (p < 0.05) inhibited OVA-induced increases in total cell counts, eosinophil counts, and IL-4, IL-5, IL-13, and eotaxin levels recovered in bronchoalveolar lavage fluid in a dose-dependent manner. U0126 also substantially (p < 0.05) reduced the serum levels of total IgE and OVA-specific IgE and IgG1. Histological studies show that U0126 dramatically inhibited OVA-induced lung tissue eosinophilia, airway mucus production, and expression of VCAM-1 in lung tissues. In addition, U0126 significantly (p < 0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine in a dose-dependent manner. Western blot analysis of whole lung lysates shows that U0126 markedly attenuated OVA-induced tyrosine phosphorylation of ERK1/2. Taken together, our findings implicate that inhibition of ERK signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.     

Allergen-induced airway hyperresponsiveness mediated by cyclooxygenase inhibition is not dependent on 5-lipoxygenase or IL-5, but is IL-13 dependent

Cyclooxygenase (COX) inhibition during allergic sensitization and allergen airway challenge results in augmented allergic inflammation. We hypothesized that this increase in allergic inflammation was dependent on increased generation of leukotrienes that results from COX inhibition, as leukotrienes are important proinflammatory mediators of allergic disease. To test this hypothesis, we allergically sensitized and challenged mice deficient in 5-lipoxygenase (5-LO). We found that 5-LO knockout mice that were treated with a COX inhibitor during allergic sensitization and challenge had significantly increased airway hyperresponsiveness (AHR) (p < 0.01) and airway eosinophilia (p < 0.01) compared with 5-LO knockout mice that were treated with vehicle. The proinflammatory cytokines have also been hypothesized to be critical regulators of airway inflammation and AHR. We found that the increase in airway eosinophilia seen with COX inhibition is dependent on IL-5, whereas the increase in AHR is not dependent on this cytokine. In contrast, the COX inhibition-mediated increase in AHR is dependent on IL-13, but airway eosinophilia is not. These results elucidate the pathways by which COX inhibition exerts a critical effect of the pulmonary allergen-induced inflammatory response and confirm that COX products are important regulators of allergic inflammation.     

Regulatory role of B cells in a murine model of allergic airway disease

Mice sensitized to OVA and subjected to acute OVA aerosol exposures develop allergic airway disease (AAD). However, chronic continuous Ag exposure results in resolution of AAD and the development of local inhalational tolerance (LIT). Because we have previously observed the persistence of B cells in the bronchoalveolar lavage (BAL) and hilar lymph nodes (HLN) at the resolution stage of this model, we investigated the role of B cells in the modulation of AAD. Although B cell-deficient mice developed LIT, adoptive transfer of HLN B cells from LIT mice to OVA-sensitized recipients resulted in attenuated AAD following subsequent OVA aerosol exposure, as determined by reduced BAL leukocytosis and eosinophilia, decreased tissue inflammation, and absent methacholine hyper-responsiveness. In similar adoptive transfer studies, HLN B cells from AAD mice were without effect. The protection transferred by LIT HLN B cells was Ag specific and was associated with accumulation of Foxp3(+) T regulatory cells regionally in BAL and HLN, but not systemically in the spleen. Fluorescent labeling of LIT HLN B cells before adoptive transfer demonstrated that these cells had the capacity to migrate to local inflammatory sites. In vitro assessment demonstrated that the LIT HLN B cells exerted this regulatory effect via TGF-beta induced conversion of CD4(+)CD25(-) T effector cells into functionally suppressive CD4(+)CD25(+)Foxp3(+) T regulatory cells. These findings illustrated a novel regulatory role for regional B cells in AAD and suggested a possible contributory role of B cells, along with other cell types, in the establishment of LIT.     

Endogenous and exogenous IL-6 inhibit aeroallergen-induced Th2 inflammation

Chronic Th2-dominated inflammation and exaggerated IL-6 production are characteristic features of the asthmatic airway. To understand the processes that are responsible for the chronicity of this response and the role(s) of IL-6 in the regulation of airway Th2 inflammation, we compared the responses induced by OVA in sensitized wild-type mice, IL-6 deficient (-/-) mice, and transgenic mice in which IL-6 was overexpressed in the airway (CC10-IL-6 mice). When compared with wild-type mice, IL-6-/- mice manifest exaggerated inflammation and eosinophilia, increased levels of IL-4, IL-5, and IL-13 protein and mRNA, exaggerated levels of eotaxin, JE/monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha and -2, and mRNA, increased bronchoalveolar lavage (BAL) TGF-beta1, and exaggerated airway responses to aerosolized methacholine. In contrast, CC10-IL-6 mice, on both C57BL/6 and BALB/c backgrounds, manifest diminished inflammation and eosinophilia, decreased levels of IL-4, IL-5, and IL-13 protein and mRNA, and decreased levels of bronchoalveolar lavage TGF-beta1. IL-6 also decreased the expression of endothelial VCAM-1 and airway responsiveness to methacholine in these animals. These alterations in the IL-6-/- and CC10-IL-6 mice were not associated with significant decreases or increases in the levels of IFN-gamma, respectively. These studies demonstrate that endogenous and exogenous IL-6 inhibit aeroallergen-induced Th2 inflammation and that this inhibition is not mediated by regulatory effects of IFN-gamma. IL-6 may be an important anti-inflammatory, counterregulatory, and healing cytokine in the airway.     

Toll-Like Receptor Ligands LPS and Poly (I:C) Exacerbate Airway Hyperresponsiveness in a Model of Airway Allergy in Mice,Independently of Inflammation

It is well-established that bacterial and viral infections have an exacerbating effect on allergic asthma, particularly aggravating respiratory symptoms, such as airway hyperresponsiveness (AHR). The mechanism by which these infections alter AHR is unclear, but some studies suggest that Toll-like receptors (TLRs) play a role. In this study, we investigated the impact of TLR3 and TLR4 ligands on AHR and airway inflammation in a model of pre-established allergic inflammation. Female BALB/c mice were sensitised and challenged intranasally (i.n.) with either PBS or ovalbumin (OVA) and subsequently i.n. challenged with poly (I:C) (TLR3) or LPS (TLR4) for four consecutive days. The response to methacholine was measured in vivo; cellular and inflammatory mediators were measured in blood, lung tissue and broncheoalveolar lavage fluid (BALF). OVA challenge resulted in an increase in AHR to methacholine, as well as increased airway eosinophilia and TH2 cytokine production. Subsequent challenge with TLR agonists resulted in a significant increase in AHR, but decreased TLR-specific cellular inflammation and production of immune mediators. Particularly evident was a decline in LPS-induced neutrophilia and neutrophil-associated cytokines following LPS and poly (I:C) treatment. The present data indicates that TLRs may play a pivotal role in AHR in response to microbial infection in allergic lung inflammation. These data also demonstrate that aggravated AHR occurs in the absence of an exacerbation in airway inflammation and that allergic inflammation impedes a subsequent inflammatory response to TLRs. These results may parallel clinical signs of microbial asthma exacerbation, including an extended duration of illness and increased respiratory symptoms.     

Effects of Mycobacteria Major Secretion Protein,Ag85B,on Allergic Inflammation in the Lung

Many epidemiological studies have suggested that the recent increase in prevalence and severity of allergic diseases such as asthma is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination. However, the underlying mechanisms by which mycobacterial components suppress allergic diseases are not yet fully understood. Here we showed the inhibitory mechanisms for development of allergic airway inflammation by using highly purified recombinant Ag85B (rAg85B), which is one of the major protein antigens secreted from M. tuberculosis. Ag85B is thought to be a single immunogenic protein that can elicit a strong Th1-type immune response in hosts infected with mycobacteria, including individuals vaccinated with BCG. Administration of rAg85B showed a strong inhibitory effect on the development of allergic airway inflammation with induction of Th1-response and IL-17and IL-22 production. Both cytokines induced by rAg85B were involved in the induction of Th17-related cytokine-production innate immune cells in the lung. Administration of neutralizing antibodies to IL-17 or IL-22 in rAg85B-treated mice revealed that IL-17 induced the infiltration of neutrophils in BAL fluid and that allergen-induced bronchial eosinophilia was inhibited by IL-22. Furthermore, enhancement of the expression of genes associated with tissue homeostasis and wound healing was observed in bronchial tissues after rAg85B administration in a Th17-related cytokine dependent manner. The results of this study provide evidence for the potential usefulness of rAg85B as a novel approach for anti-allergic effect and tissue repair other than the role as a conventional TB vaccine.     

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.     

Limonene-induced activation of A2A adenosine receptors reduces airway inflammation and reactivity in a mouse model of asthma

Animal models of asthma have shown that limonene, a naturally occurring terpene in citrus fruits, can reduce inflammation and airway reactivity. However, the mechanism of these effects is unknown. We first performed computational and molecular docking analyses that showed limonene could bind to both A_2A and A_2B receptors. The pharmacological studies were carried out with A_2A adenosine receptor knock-out (A_2AKO) and wild-type (WT) mice using ovalbumin (OVA) to generate the asthma phenotype. We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) and NECA (nonselective adenosine analog) by administering limonene as an inhalation prior to OVA aerosol challenges in one group of allergic mice for both WT and KO. In whole-body plethysmography studies, we observed that airway responsiveness to MCh in WT SEN group was significantly lowered upon limonene treatment but no effect was observed in A_2AKO. Limonene also attenuated NECA-induced airway responsiveness in WT allergic mice with no effect being observed in A_2AKO groups. Differential BAL analysis showed that limonene reduced levels of eosinophils in allergic WT mice but not in A_2AKO. However, limonene reduced neutrophils in sensitized A_2AKO mice, suggesting that it may activate A_2B receptors as well. These data indicate that limonene-induced reduction in airway inflammation and airway reactivity occurs mainly via activation of A_2AAR but A_2B receptors may also play a supporting role.

     

Downregulation of leukotriene biosynthesis by thymoquinone attenuates airway inflammation in a mouse model of allergic asthma

Chronic airway inflammation is a key feature of bronchial asthma. Leukotrienes are potent inflammatory mediators that play a role in the pathophysiology of asthma, and their levels are elevated in the airways in response to allergen challenge. We examined the anti-inflammatory effect of thymoquinone (TQ), the active principle in the volatile oil of Nigella sativa seeds, on leukotriene (LT) biosynthesis in a mouse model of allergic asthma. Mice sensitized and challenged with ovalbumin (OVA) antigen had an increased amounts of leukotriene B4 and C4, Th2 cytokines, and eosinophils in bronchoalveolar lavage (BAL) fluid. In addition, there was also a marked increase in lung tissue eosinophilia and goblet cell numbers. Administration of TQ before OVA challenge inhibited 5-lipoxygenase, the main enzyme in leukotriene biosynthesis, expression by lung cells and significantly reduced the levels of LTB4 and LTC4. This was accompanied by a marked decrease in Th2 cytokines and BAL fluid and lung tissue eosinophilia, all of which are characteristics of airway inflammation. These results demonstrate the anti-inflammatory effect of TQ in experimental asthma.     

Enhanced airway Th2 response after allergen challenge in mice deficient in CC chemokine receptor-2 (CCR2)

To evaluate the role of CCR2 in allergic asthma, mutant mice deficient in CCR2 (CCR2(-/-)) and intact mice were sensitized with i.p. OVA with alum on days 0 and 7, and challenged by inhalation with nebulization of either OVA or saline. Airway hyperreactivity, measured by the methacholine-provoked increase in enhanced pause, was significantly increased (p < 0.05) in OVA-challenged CCR2(-/-) mutant mice, compared with comparably challenged CCR2(+/+) mice. OVA-challenged CCR2(-/-) mutants also were also found to have enhanced bronchoalveolar lavage fluid eosinophilia, peribronchiolar cellular cuffing, and Ig subclass switching, with increase in OVA-specific IgG(1) and IgE. In addition, RNase protection assay revealed increased whole lung expression of IL-13 in OVA-challenged CCR2(-/-) mutants. Unexpectedly, serum monocyte chemotactic protein-1 levels were 8-fold higher in CCR2(-/-) mutants than in CCR2(+/+) mice sensitized to OVA, but OVA challenge had no additional effect on circulating monocyte chemotactic protein-1 in either genotype. Ag stimulation of lymphocytes isolated from OVA-sensitized CCR2 mutants revealed a significant increase (p < 0.05) in IL-5 production, which differed from OVA-stimulated lymphocytes from sensitized CCR2(+/+) mice. These experiments demonstrate an enhanced response in airway reactivity and in lung inflammation in CCR2(-/-) mutant mice compared with comparably sensitized and challenged CCR2(+/+) mice. These observations suggest that CC chemokines and their receptors are involved in immunomodulation of atopic asthma.