Airway hyperresponsiveness, late allergic response, and eosinophilia are reversed with mycobacterial antigens in ovalbumin-presensitized mice

Pretreatment with mycobacterial Ags has been shown to be effective in preventing allergic airway inflammation from occurring in a mouse model. Because most asthmatics are treated after the development of asthma, it is crucial to determine whether mycobacterial Ags can reverse established allergic airway inflammation in the presensitized state. Our hypothesis, based upon our previous findings, is that mycobacteria treatment in presensitized mice will suppress the allergic airway inflammation with associated clinical correlates of established asthma, with the noted exception of factors associated with the early allergic response (EAR). BALB/c mice sensitized and challenged with OVA were evaluated for pulmonary functions during both the EAR and late allergic response, and airway hyperresponsiveness to methacholine. Following this, sensitized mice were randomized and treated with placebo or a single dose (1 x 10(5) CFUs) of bacillus Calmette-Guérin (BCG) or Mycobacterium vaccae via nasal or peritoneal injection. One week later, the mice were rechallenged with OVA and methacholine, followed by bronchoalveolar lavage (BAL) and tissue collection. Mice treated with intranasal BCG were most significantly protected from the late allergic response (p < 0.02), airway hypersensitivity (p < 0.001) and hyperreactivity (p < 0.05) to methacholine, BAL (p < 0.05) and peribronchial (p < 0.01) eosinophilia, and BAL fluid IL-5 levels (p < 0.01) as compared with vehicle-treated, sensitized controls. Intranasal M. vaccae treatment was less effective, suppressing airway hypersensitivity (p < 0.01) and BAL eosinophilia (p < 0.05). No changes were observed in the EAR, BAL fluid IL-4 levels, or serum total and Ag-specific IgE. These data suggest that mycobacterial Ags (BCG>M. vaccae) are effective in attenuating allergic airway inflammation and associated changes in pulmonary functions in an allergen-presensitized state.     

Attenuation of antigen-induced airway hyperresponsiveness and inflammation in CXCR3 knockout mice

Background

CD8+ T cells participate in airway hyperresponsiveness (AHR) and allergic pulmonary inflammation that are characteristics of asthma. CXCL10 by binding to CXCR3 expressed preferentially on activated CD8+ T cells, attracts T cells homing to the lung. We studied the contribution and limitation of CXCR3 to AHR and airway inflammation induced by ovalbumin (OVA) using CXCR3 knockout (KO) mice.

Methods

Mice were sensitized and challenged with OVA. Lung histopathological changes, AHR, cellular composition and levels of inflammatory mediators in bronchoalveolar lavage (BAL) fluid, and lungs at mRNA and protein levels, were compared between CXCR3 KO mice and wild type (WT) mice.

Results

Compared with the WT controls, CXCR3 KO mice showed less OVA-induced infiltration of inflammatory cells around airways and vessels, and less mucus production. CXCR3 KO mice failed to develop significant AHR. They also demonstrated significantly fewer CD8+ T and CD4+ T cells in BAL fluid, lower levels of TNFα and IL-4 in lung tissue measured by real-time RT-PCR and in BAL fluid by ELISA, with significant elevation of IFNγ mRNA and protein expression levels.

Conclusions

We conclude that CXCR3 is crucial for AHR and airway inflammation by promoting recruitment of more CD8+ T cells, as well as CD4+ T cells, and initiating release of proinflammatory mediators following OVA sensitization and challenge. CXCR3 may represent a novel therapeutic target for asthma.     

IL-5-induced hypereosinophilia suppresses the antigen-induced immune response via a TGF-beta-dependent mechanism

Although eosinophils play an essential role in allergic inflammation, their role has recently been under controversy. Epidemic studies suggest that hypereosinophilia induced by parasite infection could suppress subsequent Ag sensitization, although the mechanism has not been fully clarified. In this study, we investigated whether eosinophils could suppress the Ag-specific immune response in the airway. BALB/c mice were sensitized and airway challenged with OVA. Systemic hypereosinophilia was induced by delivery of an IL-5-producing plasmid. IL-5 gene delivery suppressed the Ag-specific proliferation and cytokine production of CD4+ T cells in the spleen. IL-5 gene delivery before OVA sensitization significantly suppressed airway eosinophilia and hyperresponsiveness provoked by subsequent OVA airway challenge, while delivery during the OVA challenge did not suppress them. This IL-5-induced immune suppression was abolished in eosinophil-ablated mice, suggesting an essential role of eosinophils. IL-5 treatment increased the production of TGF-beta1 in the spleen, and we demonstrated that the main cellular source of TGF-beta1 production was eosinophils, using eosinophil-ablated mice and depletion study. TGF-beta1, but not IL-5 itself, suppressed the Ag-specific immune response of CD4+ T cells in vitro. Furthermore, IL-5 treatment enhanced phosphorylation of Smad2 in CD4+ T cells. Finally, a TGF-beta type I receptor kinase inhibitor restored this IL-5-induced immune suppression both in vitro and in vivo. These results suggest that IL-5-induced hypereosinophilia could suppress sensitization to Ag via a TGF-beta-dependent mechanism, thus suppressed allergic airway inflammation. Therefore, hypereosinophilia could reveal an immunosuppressive effect in the early stage of Ag-induced immune response.     

Immune Modulatory Effects of IL-22 on Allergen-Induced Pulmonary Inflammation

IL-22 is a Th17/Th22 cytokine that is increased in asthma. However, recent animal studies showed controversial findings in the effects of IL-22 in allergic asthma. To determine the role of IL-22 in ovalbumin-induced allergic inflammation we generated inducible lung-specific IL-22 transgenic mice. Transgenic IL-22 expression and signaling activity in the lung were determined. Ovalbumin (OVA)-induced pulmonary inflammation, immune responses, and airway hyperresponsiveness (AHR) were examined and compared between IL-22 transgenic mice and wild type controls. Following doxycycline (Dox) induction, IL-22 protein was readily detected in the large (CC10 promoter) and small (SPC promoter) airway epithelial cells. IL-22 signaling was evidenced by phosphorylated STAT3. After OVA sensitization and challenge, compared to wild type littermates, IL-22 transgenic mice showed decreased eosinophils in the bronchoalveolar lavage (BAL), and in lung tissue, decreased mucus metaplasia in the airways, and reduced AHR. Among the cytokines and chemokines examined, IL-13 levels were reduced in the BAL fluid as well as in lymphocytes from local draining lymph nodes of IL-22 transgenic mice. No effect was seen on the levels of serum total or OVA-specific IgE or IgG. These findings indicate that IL-22 has immune modulatory effects on pulmonary inflammatory responses in allergen-induced asthma.     

Airway activation of formyl peptide receptors inhibits Th1 and Th17 cell responses via inhibition of mediator release from immune and inflammatory cells and maturation of dendritic cells

Formyl peptide receptors (FPRs) are chemoattractant receptors that mediate inflammatory cell responses to infection. Recent evidence indicates that noneosinophilic asthma phenotypes can be developed by both Th1 and Th17 cell responses when exposed to LPS-containing allergens. In this study, we evaluated the effects of airway activation of FPRs by their synthetic agonist, Trp-Lys-Tyr-Met-Val-D-Met (W-peptide), on the development of Th1 and Th17 cell responses in a noneosinophilic asthma mouse model. A noneosinophilic asthma mouse model was generated by intranasal sensitization with 10 μg of LPS plus 75 μg of OVA on days 0, 1, 2, and 7. Mice were then challenged with 50 μg of OVA alone on days 14, 15, 21, and 22. W-peptide was administered during the sensitization period, and immune and inflammatory responses were evaluated after OVA challenge. Lung inflammation after OVA challenge was partly abolished by airway activation of FPRs during sensitization. Maturation of dendritic cells (DCs) and migration of DCs from the lung to lung-draining lymph nodes were inhibited by FPR activation. In addition, airway activation of FPRs inhibited allergen-specific T cell proliferation in the lymph nodes. Production of IL-12 and IL-6 (Th1- and Th17-polarizing cytokines) from lung DCs was decreased by airway activation of FPRs. This effect resulted in the inhibition of allergen-specific Th1 and Th17 cell responses. Airway activation of FPRs during sensitization effectively prevents the development of Th1 and Th17 cell responses induced by LPS-containing allergens via multiple mechanisms, such as inhibition of DC maturation and migration and the production of Th1- and Th7-polarizing cytokines.     

Tryptase inhibition blocks airway inflammation in a mouse asthma model

Release of human lung mast cell tryptase may be important in the pathophysiology of asthma. We examined the effect of the reversible, nonelectrophilic tryptase inhibitor MOL 6131 on airway inflammation and hyper-reactivity in a murine model of asthma. MOL 6131 is a potent selective nonpeptide inhibitor of human lung mast cell tryptase based upon a beta-strand template (K(i) = 45 nM) that does not inhibit trypsin (K(i) = 1,061 nM), thrombin (K(i) = 23, 640 nM), or other serine proteases. BALB/c mice after i.p. OVA sensitization (day 0) were challenged intratracheally with OVA on days 8, 15, 18, and 21. MOL 6131, administered days 18-21, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 22; intranasal delivery (10 mg/kg) had a greater anti-inflammatory effect than oral delivery (10 or 25 mg/kg) of MOL 6131. MOL 6131 reduced total cells and eosinophils in bronchoalveolar lavage fluid, airway tissue eosinophilia, goblet cell hyperplasia, mucus secretion, and peribronchial edema and also inhibited the release of IL-4 and IL-13 in bronchoalveolar lavage fluid. However, tryptase inhibition did not alter airway hyper-reactivity to methacholine in vivo. These results support tryptase as a therapeutic target in asthma and indicate that selective tryptase inhibitors can reduce allergic airway inflammation.     

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.     

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.     

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.     

Pathogenesis of murine experimental allergic rhinitis: a study of local and systemic consequences of IL-5 deficiency

Recent studies have demonstrated an important role for IL-5-dependent bone marrow eosinophil progenitors in allergic inflammation. However, studies using anti-IL-5 mAbs in human asthmatics have failed to suppress lower airway hyperresponsiveness despite suppression of eosinophilia; therefore, it is critical to examine the role of IL-5 and bone marrow responses in the pathogenesis of allergic airway disease. To do this, we studied the effects of IL-5 deficiency (IL-5(-/-)) on bone marrow function as well as clinical and local events, using an established experimental murine model of allergic rhinitis. Age-matched IL-5(+/+) and IL-5(-/-) BALB/c mice were sensitized to OVA followed by 2 wk of daily OVA intranasal challenge. IL-5(-/-) OVA-sensitized mice had significantly higher nasal mucosal CD4(+) cells and basophilic cell counts as well as nasal symptoms and histamine hyperresponsiveness than the nonsensitized group; however, there was no eosinophilia in either nasal mucosa or bone marrow; significantly lower numbers of eosinophil/basophil CFU and maturing CFU eosinophils in the presence of recombinant mouse IL-5 in vitro; and significantly lower expression of IL-5Ralpha on bone marrow CD34(+)CD45(+) progenitor cells in IL-5(-/-) mice. These findings suggest that IL-5 is required for normal bone marrow eosinophilopoiesis, in response to specific Ag sensitization, during the development of experimental allergic rhinitis. However, the results also suggest that suppression of the IL-5-eosinophil pathway in this model of allergic rhinitis may not completely suppress clinical symptoms or nasal histamine hyperresponsiveness, because of the existence of other cytokine-progenitor pathways that may induce and maintain the presence of other inflammatory cell populations.     

The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

Background

H89 is a potent inhibitor of Protein Kinase A (PKA) and Mitogen- and Stress-Activated protein Kinase 1 (MSK1) with some inhibitory activity on other members of the AGC kinase family. H89 has been extensively used in vitro but its anti-inflammatory potential in vivo has not been reported to date. To assess the anti-inflammatory properties of H89 in mouse models of asthma.

Methodology/Principal Findings

Mice were sensitized intraperitoneally (i.p.) to ovalbumin (OVA) with or without alum, and challenged intranasally with OVA. H89 (10 mg/kg) or vehicle was given i.p. two hours before each OVA challenge. Airway hyperresponsiveness (AHR) was assessed by whole-body barometric plethysmography. Inflammation was assessed by the total and differential cell counts and IL-4 and IL-5 levels in bronchoalveolar lavage (BAL) fluid. Lung inflammation, mucus production and mast cell numbers were analyzed after histochemistry. We show that treatment with H89 reduces AHR, lung inflammation, mast cell numbers and mucus production. H89 also inhibits IL-4 and IL-5 production and infiltration of eosinophils, neutrophils and lymphocytes in BAL fluid.

Conclusions/Significance

Taken together, our findings implicate that blockade of AGC kinases may have therapeutic potential for the treatment of allergic airway inflammation.     

Phosphatidylinositol 3-kinase inhibitor suppresses inducible nitric oxide synthase expression in bronchiole epithelial cells in asthmatic rats

Inducible nitric oxide synthase (iNOS) is known to produce nitric oxide (NO), which is a main contributor to asthmatic airway inflammation. Recent studies have shown that phosphatidylinositol 3-kinase (PI3K) is ubiquitously expressed in airway epithelial cells and its inhibition could relieve airway inflammation and hyperresponsiveness. This study aimed to explore the interaction of PI3K and NO signaling in allergic asthma. We investigated the effects of PI3K inhibitor wortmannin on iNOS expression in bronchiole epithelial cells and NO, IL-4 and IFN-γ levels in lung tissues of asthmatic rat model, which was prepared by 10% OVA solution sensitization and 1% OVA aerosol challenge. Our results showed that the ratio of eosinophils to total cells in BALF, PI3K activity, NO and IL-4 levels in lung tissues was increased after OVA sensitization and challenge, but then was attenuated by the administration of wortmannin. In contrast, IFN-γ level in lung tissues was decreased after OVA sensitization and challenge and increased after the administration of wortmannin. The expression of iNOS protein in bronchiole epithelial cells, iNOS mRNA level and iNOS activity in lung tissues was markedly upregulated after OVA sensitization and challenge, but the upregulation was significantly antagonized by wortmannin. Taken together, these data provide evidence that PI3K functions upstream to modulate iNOS/NO signaling, which then promotes the development of airway inflammation in asthmatic animal model. PI3K inhibitor wortmannin could lead to reduced iNOS expression and NO production, therefore inhibiting airway inflammatory responses.     

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.     

Connexin 43 Upregulation in Mouse Lungs during Ovalbumin-Induced Asthma

Background

Connexin (Cx)-based gap junction channels play important roles in the inflammatory response. Cx43 is involved in the pathogenesis of some lung diseases such as acute lung injury. However, the Cx43 expression in asthma is unclear. In the present study, we used a murine model of ovalbumin (OVA)-induced allergic airway disease to examine the levels of Cx43 and analyze the relationship between Cx43 and airway inflammation in allergic airway disease.

Methods

Asthma was induced in mice via sensitization and challenge with OVA. Cx43 mRNA and protein expression levels were investigated via QT-PCR, western blot, and immunohistochemistry 0 h, 8 h, 1 d, 2 d and 4 d after the first challenge. The relationship between Cx43 protein levels and inflammatory cell infiltration, cytokine levels was analyzed.

Results

The OVA-induced mice exhibited typical pathological features of asthma, including airway hyper-responsiveness; strong inflammatory cell infiltration surrounding the bronchia and vessels; many inflammatory cells in the bronchoalveolar lavage fluid (BALF); higher IL-4, IL-5 and IL-13 levels; and high OVA specific IgE levels. Low Cx43 expression was detected in the lungs of control (PBS) mice. A dramatic increase in the Cx43 mRNA and protein levels was found in the asthmatic mice. Cx43 mRNA and protein expression levels increased in a time-dependent manner in asthma mice, and Cx43 was mostly localized in the alveolar and bronchial epithelial layers. Moreover, lung Cx43 protein levels showed a significant positive correlation with inflammatory cell infiltration in the airway and IL-4 and IL-5 levels in the BALF at different time points after challenge. Interestingly, the increase in Cx43 mRNA and protein levels occurred prior to the appearance of the inflammatory cell infiltration.

Conclusion

Our data suggest that there is a strong upregulation of Cx43 mRNA and protein levels in the lungs in asthma. Cx43 levels also exhibited a positive correlation with allergic airway inflammation. Cx43 may represent a target to treat allergic airway diseases in the future.     

IL-10- and IL-12-independent down-regulation of allergic sensitization by stimulation of CD40 signaling

Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.     

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.     

Suppression of Th2-driven, allergen-induced airway inflammation by sauchinone

Sauchinone, a lignan compound isolated from the root of Saururus chinensis, has been recently demonstrated to exhibit anti-inflammatory activity via the suppression of NF-kB p65 activity in vitro. In an effort to evaluate the in vivo anti-inflammatory function of sauchinone, we have evaluated the effects of sauchinone on allergen-induced airway inflammation using a murine model of allergic asthma. We observed that marked eosinophilic and lymphocyte infiltration in the BAL fluid were suppressed to a significant degree by sauchinone, and that mucus-secreting goblet cell hyperplasia and collagen deposition in the airways were also ameliorated by administration of sauchinone treatment. Moreover, gene expression of the inflammatory cytokines, IL-13, and IL-5 and eotaxin in the lung, and IL-5 in the draining lymph node were significantly decreased in sauchinone-treated mice. We demonstrated that sauchinone repressed Th2 cell development in vitro and IL-4 production by Th2 cells, and also inhibited GATA-3-mediated IL-5 promoter activity in a dose-dependent manner. Collectively, sauchinone ameliorated allergen-induced airway inflammation, in part, by repressing GATA-3 activity for Th2 cell development, indicating the possible therapeutic potential of sauchinone in airway inflammatory diseases including allergic asthma and rhinitis.     

IFN-γ-mediated efficacy of allergen-free immunotherapy using mycobacterial antigens and CpG-ODN

Epidemiological and experimental evidence supports the notion that microbial infections that are known to induce Th1-type immune responses can suppress Th2 immune responses, which are characteristics of allergic disorders. However, live microbial immunization might not be feasible for human immunotherapy. Here, we evaluated whether induction of Th1 immunity by the immunostimulatory sequences of CpG-oligodeoxynucleotides (CpG-ODN), with or without culture filtrate proteins (CFP), from Mycobacterium tuberculosis would suppress ongoing allergic lung disease. Presensitized and ovalbumin (OVA)-challenged mice were treated subcutaneously with CpG, or CpG in combination with CFP (CpG/CFP). After 15 days of treatment, airway inflammation and specific T- and B-cell responses were determined. Cell transfer experiments were also performed. CpG treatment attenuated airway allergic disease; however, the combination CpG/CFP treatment was significantly more effective in decreasing airway hyperresponsiveness, eosinophilia and Th2 response. When an additional intranasal dose of CFP was given, allergy was even more attenuated. The CpG/CFP therapy also reduced allergen-specific IgG1 and IgE antibodies and increased IgG2a. Transfer of spleen cells from mice immunized with CpG/CFP also reduced allergic lung inflammation. CpG/CFP treatment induced CFP-specific production of IFN-γ and IL-10 by spleen cells and increased production of IFN-γ in response to OVA. The essential role of IFN-γ for the therapeutic effect of CpG/CFP was evidenced in IFN-γ knockout mice. These results show that CpG/CFP treatment reverses established Th2 allergic responses by an IFN-γ-dependent mechanism that seems to act both locally in the lung and systemically to decrease allergen-specific Th2 responses.