Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated ovalbumin-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared to sham-infected, ovalbumin-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13 and IFN-γ. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemistry showed eotaxin-1 in the lung macrophages of virus-infected, ovalbumin-treated mice, and confocal fluorescence microscopy revealed co-localization of rhinovirus, eotaxin-1 and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from ovalbumin-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from ovalbumin-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2 and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from ovalbumin-sensitized and -challenged mice reduced eosinophilic inflammation and airway hyperreactivity following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.
Allergic asthma is a chronic inflammatory lung disease that is characterized by airway hyperresponsiveness (AHR) to allergens, airway oedema, increased mucus secretion, excess production of T helper-2 (Th2) cytokines, and eosinophil accumulation in the lungs. Corni fructus (CF) is a fruit of Cornus officinalis Sieb. Et. Zucc. (Cornaceae) and has been used in traditional Korean medicine as an anti-inflammatory, analgesic, and diuretic agent. To investigate the anti-asthmatic effects of CF and their underlying mechanism, we examined the influence of CF on the development of pulmonary eosinophilic inflammation and airway hyperresponsiveness in a mouse model of allergic asthma.
In this study, BALB/c mice were systemically sensitized to ovalbumin (OVA) by intraperitoneal (i.p.), intratracheal (i.t.) injections and intranasal (i.n.) inhalation of OVA. We investigated the effect of CF on airway hyperresponsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production, and OVA-specific immunoglobulin E (IgE) production.
The CF-treated groups showed suppressed eosinophil infiltration, allergic airway inflammation, and AHR via reduced production of interleuin (IL) -5, IL-13, and OVA-specific IgE.
Our data suggest that the therapeutic effects of CF in asthma are mediated by reduced production of Th2 cytokines (IL-5), eotaxin, and OVA-specific IgE and reduced eosinophil infiltration.
Corni fructus; Asthma; Eosinophil; IL-5; CCR3
Eosinophil-produced cytokines have been shown to participate in the maintenance of antigen-specific plasma cells (PC) in bone marrow (BM), suggesting that eosinophils are required in the development and/or maintenance of alloantibody responses post-transplant.
To test this hypothesis, we sensitized eosinophil-deficient ΔdblGATA1 mice and WT control mice with allogeneic splenocytes or with allogeneic heart grafts and compared the kinetics and titers of serum donor-specific antibodies (DSA), as well as BM and spleen CD130+B220low PC populations between groups.
Spleen cells from naïve ΔdblGATA1 BALB/c mice contained higher percentages of PC than WT without detectable differences in BM PCs. After sensitization with allogeneic splenocytes, BALB/c ΔdblGATA1 mice contained fewer BM PCs but more splenic PCs compared to controls. These differences were associated with modestly lower titers of serum DSA 4 and 12 weeks after sensitization but secondary immunizations induced similar increases in both groups. Moreover, the kinetics and strength of DSA did not differ in WT and ΔdblGATA1 BALB/c mice transplanted with B6 cardiac allografts, nor did they differ in transplanted ΔdblGATA1 and WT mice on a B6 background.
Therefore, eosinophils are not required for alloantibody formation or maintenance in mice and are thus unlikely to be effective targets for antibody desensitization.
eosinophil; donor-specific antibodies; GATA-1; transplant
Background and Objective. The features of asthma are airway inflammation, reversible airflow obstruction, and an increased sensitivity to bronchoconstricting agents, termed airway hyperresponsiveness (AHR), excess production of Th2 cytokines, and eosinophil accumulation in the lungs. To investigate the antiasthmatic potential of hesperidin as well as the underlying mechanism involved, we studied the inhibitory effect and anti-inflammatory effect of hesperidin (HPN) on the production of Th2 cytokines, eotaxin, IL-17, -OVA-specific IgE in vivo asthma model mice.
Methods. In this paper, BALB/c mice were systemically sensitized to ovalbumin (OVA) followed intratracheally, intraperitoneally, and by aerosol allergen challenges. We investigated the effect of HPN on airway hyperresponsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production and OVA-specific IgE production in a mouse model of asthma. Results. In BALB/c mice, we found that HPN-treated groups had suppressed eosinophil infiltration, allergic airway inflammation, and AHR, and these occurred by suppressing the production of IL-5, IL-17, and OVA-specific IgE. Conclusions. Our data suggest that the therapeutic mechanism by which HPN effectively treats asthma is based on reductions of Th2 cytokines (IL-5), eotaxin, OVA-specific IgE production, and eosinophil infiltration via inhibition of GATA-3 transcription factor.
Here we describe a novel method via which ex vivo cultured mouse bone marrow-derived eosinophils (bmEos) can be adoptively transferred into recipient mice in order to study receptor-dependent recruitment to lung tissue in vivo. Intratracheal instillation of recombinant hCCL24 prior to introduction of bmEos via tail vein injection resulted in a ~four-fold increase in Siglec Fpos/CD11cneg eosinophils in the lungs of eosinophil-deficient ΔdblGATA recipient mice compared with controls. As anticipated, bmEos generated from CCR3-gene-deleted mice did not migrate to the lung in response to hCCL24 in this model, indicating specific receptor dependence. BmEos generated from GFPpos Balb/c mice responded similarly hCCL24 in vitro and were detected in lung tissue of BALB/c wild-type as well as BALB/c ΔdblGATA eosinophil-deficient recipient mice, at ~four-fold (at 5 h post-instillation) and ~three-fold (at 24 h post-instillation) over baseline respectively. Comparable results were obtained with GFPpos C57BL/6 bmEos responding to intratracheal hCCL24 in C57BL/6 ΔdblGATA recipient mice. The use of ex vivo cultured bmEos via one or more of these methods offers the possibility of manipulating bmEos prior to transfer into a wild-type or gene-deleted recipient host. Thus, this chemotaxis model represents a novel and robust tool for pharmacological studies in vivo.
bone morrow-derived eosinophils; CCL24; chemotaxis; immunology; mouse model
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.
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.
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.
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.
Neutrophil; Elastase; Airway; Hyperresponsiveness; Asthma
Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high expression of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.
allergy; chemokines; eosinophils; lung; mouse
Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.
allergy; chemokines; eosinophils; lung; mouse
Enhanced eosinophil responses have critical roles in the development of allergic diseases. IL-5 regulates the maturation, migration and survival of eosinophils, and IL-5 and eotaxins mediate the trafficking and activation of eosinophils in inflamed tissues. CD4+ Th2 cells are the main producers of IL-5 and other cells such as NK also release this cytokine. Although multiple signalling pathways may be involved, STAT6 critically regulates the differentiation and cytokine production of Th2 cells and the expression of eotaxins. Nevertheless, the mechanisms that mediate different parts of the eosinophilic inflammatory process in different tissues in allergic airway diseases remain unclear. Furthermore, the mechanisms at play may vary depending on the context of inflammation and microenvironment of the involved tissues.
We employed a model of allergic airway disease in wild type and STAT6-deficient mice to explore the roles of STAT6 and IL-5 in the development of eosinophilic inflammation in this context. Quantitative PCR and ELISA were used to examine IL-5, eotaxins levels in serum and lungs. Eosinophils in lung, peripheral blood and bone marrow were characterized by morphological properties. CD4+ T cell and NK cells were identified by flow cytometry. Antibodies were used to deplete CD4+ and NK cells. We showed that STAT6 is indispensible for eosinophilic lung inflammation and the induction of eotaxin-1 and -2 during allergic airway inflammation. In the absence of these chemokines eosinophils are not attracted into lung and accumulate in peripheral blood. We also demonstrate the existence of an alternate STAT6-independent pathway of IL-5 production by CD4+ and NK cells that mediates the development of eosinophils in bone marrow and their subsequent movement into the circulation.
These results suggest that different points of eosinophilic inflammatory processes in allergic airway disease may be differentially regulated by the activation of STAT6-dependent and -independent pathways.
Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is enriched in normal airway lining fluid, but is significantly reduced in airway epithelium exposed to a Th2 cytokine milieu. The role of SPLUNC1 in modulating airway allergic inflammation (e.g., eosinophils) remains unknown. We used SPLUNC1 knockout (KO) and littermate wild-type (C57BL/6 background) mice and recombinant SPLUNC1 protein to determine the impact of SPLUNC1 on airway allergic/eosinophilic inflammation, and to investigate the underlying mechanisms. An acute ovalbumin (OVA) sensitization and challenge protocol was used to induce murine airway allergic inflammation (e.g., eosinophils, eotaxin-2, and Th2 cytokines). Our results showed that SPLUNC1 in the bronchoalveolar lavage fluid of OVA-challenged wild-type mice was significantly reduced (P < 0.05), which was negatively correlated with levels of lung eosinophilic inflammation. Moreover, SPLUNC1 KO mice demonstrated significantly higher numbers of eosinophils in the lung after OVA challenges than did wild-type mice. Alveolar macrophages isolated from OVA-challenged SPLUNC1 KO versus wild-type mice had higher concentrations of baseline eotaxin-2 that was amplified by LPS (a known risk factor for exacerbating asthma). Human recombinant SPLUNC1 protein was applied to alveolar macrophages to study the regulation of eotaxin-2 in the context of Th2 cytokine and LPS stimulation. Recombinant SPLUNC1 protein attenuated LPS-induced eotaxin-2 production in Th2 cytokine–pretreated murine macrophages. These findings demonstrate that SPLUNC1 inhibits airway eosinophilic inflammation in allergic mice, in part by reducing eotaxin-2 production in alveolar macrophages.
SPLUNC1; asthma; alveolar macrophage; Th2 cytokines; eotaxin-2
Over 40% of chronic stable asthma patients have evidence of respiratory Mycoplasma pneumoniae (Mp) infection as detected by polymerase chain reaction (PCR), but not by serology and culture, suggesting a low-level Mp involved in chronic asthma. However, the role of such a low-level Mp infection in regulation of allergic inflammation remains unknown.
To determine the impact of a low-level Mp infection in mice with established airway allergic inflammation on allergic responses such as eosinophilia and chemokine eotaxin-2, and the underlying mechanisms (i.e., prostaglandin E2 [PGE2] pathway) since PGE2 inhalation before allergen challenge suppressed eosinophil infiltration in human airways.
BALB/c mouse models of ovalbumin (OVA)-induced allergic asthma with an ensuing low-dose or high-dose Mp were used to assess IL-4 expression, BAL eosinophil, eotaxin-2 and PGE2 levels, and lung mRNA levels of microsomal prostaglandin E synthase-1 (mPGES-1). Primary alveolar macrophages (pAMs) from naïve BALB/c mice were cultured to determine if Mp-induced PGE2 or exogenous PGE2 down-regulates IL-4/IL-13-induced eotaxin-2.
Low-dose Mp in allergic mice significantly enhanced IL-4 and eotaxin-2, and moderately promoted lung eosinophilia, whereas high-dose Mp significantly reduced lung eosinophilia and tended to decrease IL-4 and eotaxin-2. Moreover, in both OVA-naïve and allergic mice, lung mPGES-1 mRNA and BAL PGE2 levels were elevated in mice infected with high-dose, but not low-dose Mp. In pAMs, IL-4/IL-13 significantly increased eotaxin-2, which was reduced by Mp infection accompanied by dose-dependent PGE2 induction. Exogenous PGE2 inhibited IL-4/IL-13-induced eotaxin-2 in a dose-dependent manner.
This study highlights a novel concept on how differing bacterial loads in the lung modify the established allergic airway inflammation, and thus interact with an allergen to further induce Th2 responses. That is: Unlike high-level Mp, low-level Mp fails to effectively induce PGE2 to down-regulate allergic responses (e.g., eotaxin-2), thus maintaining or even worsening allergic inflammation in asthmatic airways.
asthma; Mycoplasma pneumoniae; eotaxin-2; PGE2; alveolar macrophages
Eotaxin is a member of the C-C family of chemokines and is related during antigen challenge in a guinea pig model of allergic airway inflammation (asthma). Consistent with its putative role in eosinophilic inflammation, eotaxin induces the selective infiltration of eosinophils when injected into the lung and skin. Using a guinea pig lung cDNA library, we have cloned full-length eotaxin cDNA. The cDNA encodes a protein of 96 amino acids, including a putative 23-amino acid hydrophobic leader sequence, followed by 73 amino acids composing the mature active eotaxin protein. The protein-coding region of this cDNA is 73, 71, 50, and 48% identical in nucleic acid sequence to those of human macrophage chemoattractant protein (MCP) 3, MCP-1, macrophage inflammatory protein (MIP) 1 alpha, and RANTES, respectively. Analysis of genomic DNA suggested that there is a single eotaxin gene in guinea pig which is apparently conserved in mice. High constitutive levels of eotaxin mRNA expression were observed in the lung, while the intestines, stomach, spleen, liver, heart, thymus, testes, and kidney expressed lower levels. To determine if eotaxin mRNA levels are elevated during allergen-induced eosinophilic airway inflammation, ovalbumin (OVA)-sensitized guinea pigs were challenged with aerosolized antigen. Compared with the lungs from saline-challenged animals, eotaxin mRNA levels increased sixfold within 3 h and returned to baseline by 6 h. Thus, eotaxin mRNA levels are increased in response to allergen challenge during the late phase response. The identification of constitutive eotaxin mRNA expression in multiple tissues suggests that in addition to regulating airway eosinophilia, eotaxin is likely to be involved in eosinophil recruitment into other tissues as well as in baseline tissue homing.
Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.
Eosinophilic inflammation is closely related to angiogenesis in asthmatic airway remodeling. In ovalbumin-sensitized mice, bone marrow-derived pro-angiogenic endothelial progenitor cells (EPCs) are rapidly recruited into the lungs after ovalbumin aerosol challenge, and promptly followed by mobilization and recruitment of eosinophils.
We hypothesized that bone marrow-derived EPCs initiate the recruitment of eosinophils through expression of eosinophil chemoattractant eotaxin-1.
EPCs were isolated from ovalbumin murine model of allergic airway inflammation and from asthma patients. Endothelial and smooth muscle cells were isolated from mice. Eotaxin-1 expression was analyzed by immunofluorescence, real-time PCR or by ELISA. In vivo recruitment of eosinophils by EPCs was analyzed in mice.
Circulating EPCs of asthmatic individuals had higher levels of eotaxin-1 as compared to controls. In the murine model, ovalbumin allergen exposure augmented eotaxin-1 mRNA and protein levels in EPCs. The EPCs from ovalbumin-sensitized and challenged mice released high levels of eotaxin-1 upon contact with lung endothelial cells from sensitized and challenged mice, but not from control animals, and not upon contact with cardiac or hepatic endothelial cells from sensitized and challenged mice. Intranasal administration of the eotaxin-rich media overlying cultures of EPCs caused recruitment into lungs, confirming functional chemoattractant activity.
Bone marrow-derived EPCs are early responders to environmental allergen exposures, and initiate a parallel switch to a pro-angiogenic and pro-eosinophilic environment in the asthmatic lungs.
eosinophils; allergy; airway inflammation; angiogenesis; bone marrow; eotaxin
We have recently devised a culture method that generates large numbers of eosinophils at high purity from unselected BALB/c mouse bone marrow progenitors [Dyer et al., 2008. J. Immunol. 181: 4004–9]. Here we present the extended scope of this approach, as we have used this method successfully to generate eosinophil cultures of virtually 100% purity from bone marrow from C57BL/6 mice, and from TLR2, TLR3, TLR7 and TLR9-gene-deleted mouse strains on the C57BL/6 background. Both wild-type and TLR3 gene-deleted bone marrow eosinophils (bmEos) are functional, releasing peroxidase in response to the secretogogue, platelet activating factor. We have also used this method to re-evaluate production of eosinophils in bone marrow cultures from ΔdblGATA mice, a strain that is eosinophil-deficient in vivo. Interestingly, bmEos can be detected in the ΔdblGATA cultures (5% of total cells at day 10), although ~80-fold fewer bmEos are detected in ΔdblGATA than in parallel wild-type (BALB/c) bone marrow cultures. Overall, we find that generation of large numbers of eosinophils at high purity from unselected bone marrow progenitors proceeds efficiently in a variety of wild-type and gene-deleted strains, and as such this approach shows promise as a universal method for the study of eosinophil structure and function.
Cytokines; interleukin-5; toll-like receptors; mouse; hematopoiesis
Pulmonary eosinophilia is one of the most consistent hallmarks of asthma. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase, which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild-type hematopoietic cells to generate chimeric CYBB mice. In response to ovalbumin (OVA) challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFNγ, or eotaxin), or numbers of T cells, neutrophils, or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation. Moreover, these studies provide a basis for targeting VCAM-1-dependent signaling pathways in asthma therapies.
endothelium; gp91 phox; eosinophils; VCAM-1
Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO) and major basic protein-1 (MBP-1), during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL), we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity.
Eosinophil recruitment is a classic characteristic of both allergic and parasitic helminth diseases. Elucidation of the role of eosinophils in these diseases is of pivotal importance for understanding the mechanisms of protection and the development of pathology. In the last few years, the part played by eosinophils in helminth-defence has been dissected using in vivo models and their importance in protection has been shown to be highly specific to the host-parasite combination. This study dissects the role of eosinophils during infection with the human lymphatic filarial parasite, Brugia malayi, which causes the major neglected tropical disease, lymphatic filariasis. In particular, we study the role of the eosinophil as a double–edged sword in generating both protection and pathology. We definitively confirm the importance of eosinophils in protection against B. malayi microfilariae and show that protection is not mediated by release of the eosinophil granule proteins, major basic protein or eosinophil peroxidase alone. Overall, we reveal that during an infection with B. malayi microfilariae, eosinophils are critical for primary protective responses. However, eosinophils contribute to nematode-induced lung dysfunction, while additionally, eosinophil granules are important negative regulators of parasite-induced lung inflammatory and some adaptive immune responses.
The role played by the β-galactoside-binding lectin galectin-3 (Gal-3) in airway remodeling, a characteristic feature of asthma that leads to airway dysfunction and poor clinical outcome in humans, was investigated in a murine model of chronic allergic airway inflammation. Wild-type (WT) and Gal-3 knock-out (KO) mice were subjected to repetitive allergen challenge with ovalbumin (OVA) up to 12 weeks and bronchoalveolar lavage fluid (BALF) and lung tissue collected after the last challenge were evaluated for cellular features associated with airway remodeling. Compared to WT mice, chronic OVA challenge in Gal-3 KO mice resulted in diminished remodeling of the airways with significantly reduced mucus secretion, sub-epithelial fibrosis, smooth muscle thickness, and peribronchial angiogenesis. The higher degree of airway remodeling in WT mice was associated with higher Gal-3 expression in the BALF as well as lung tissue. Cell counts in BALF and lung immunohistology demonstrated that eosinophil infiltration in OVA-challenged Gal-3 KO mice was significantly reduced compared to WT mice. Evaluation of cellular mediators associated with eosinophil recruitment and airway remodeling revealed that levels of eotaxin-1, IL-5, IL-13, FIZZ1 and TGF-β were substantially lower in Gal-3 KO mice. Finally, leukocytes from Gal-3 KO mice demonstrated decreased trafficking (rolling) on vascular endothelial adhesion molecules compared to WT cells. Overall, these studies demonstrate that Gal-3 is an important lectin that promotes airway remodeling via airway recruitment of inflammatory cells, specifically eosinophils, and the development of a Th2 phenotype as well as increased expression of eosinophil-specific chemokines, pro-fibrogenic and angiogenic mediators.
Lovastatin is an effective inhibitor of cholesterol synthesis. A previous study demonstrated that lovastatin can also suppress airway hyperresponsiveness (AHR) in murine model of asthma. We aimed to investigate the effect of lovastatin on mucus secretion and inflammation-associated gene expression in the lungs of murine model of asthma.
Female BALB/c mice were sensitized and challenged with ovalbumin (OVA) by intraperitoneal injection, and orally administered lovastatin from days 14 to 27 post-injection. Gene expression in lung tissues was analyzed using real-time polymerase chain reaction. AHR and goblet cell hyperplasia were also examined. BEAS-2B human bronchial epithelial cells were used to evaluate the effect of lovastatin on the expression of cell adhesion molecules, chemokines, and proinflammatory cytokines in vitro.
We showed that lovastatin inhibits the expression of Th2-associated genes, including eotaxins and adhesion molecules, in the lungs of murine model of asthma. Mucin 5AC expression, eosinophil infiltration and goblet cell hyperplasia were significantly decreased in the lung tissue of murine model of asthma treated with lovastatin. Furthermore, lovastatin inhibited AHR and expression of Th2-associated cytokines in bronchoalveolar lavage fluid. However, a high dose (40 mg/kg) of lovastatin was required to decrease specific IgE to OVA levels in serum, and suppress the expression of Th2-associated cytokines in splenocytes. Activated BEAS-2B cells treated with lovastatin exhibited reduced IL-6, eotaxins (CCL11 and CCL24), and intercellular adhesion molecule-1 protein expression. Consistent with this, lovastatin also suppressed the ability of HL-60 cells to adhere to inflammatory BEAS-2B cells.
These data suggest that lovastatin suppresses mucus secretion and airway inflammation by inhibiting the production of eotaxins and Th2 cytokines in murine model of asthma.
Asthma; cytokine; eosinophil; eotaxin; lovastatin; MUC5 AC
Asthma is one of the most common chronic airway inflammatory diseases. The clinical hallmarks of asthma include elevated serum levels of immunoglobulin E (IgE), eosinophilic inflammation and airway hyper-responsiveness (AHR). Arsenic trioxide (As2O3) is considered a carcinogen; however, it has also been used to treat diseases, such as syphilis, in traditional Chinese and Western medicine. Today, As2O3 is used as one of the standard therapies for acute promyelocytic leukemia (APL). Previous studies have indicated that As2O3 can induce apoptosis in eosinophils. However, the effect of As2O3 on asthma has not been investigated. We used ovalbumin (OVA)-immunized mice as a model for asthma and treated mice with As2O3 at doses of 2.5 and 5 mg/kg. The mice were then monitored for OVA-specific IgE production, airway inflammatory cell infiltration and AHR. We found that administration of As2O3 in OVA-immunized mice abrogated airway eosinophil recruitment by downregulating eotaxin expression but did not alter serum IgE or IL-5 levels in bronchoalveolar lavage fluid (BALF). Furthermore, the development of AHR and cellular infiltration into the airway were reduced by treating mice with As2O3. In vitro data suggested that low concentrations of As2O3 could induce only a small degree of apoptosis in primary pulmonary cells but could significantly inhibit the secretion of eotaxin by these cells. These results indicate that the administration of As2O3 to OVA-immunized mice can suppress lung allergic inflammatory responses. As2O3 might therefore have therapeutic potential in treating allergic airway inflammatory diseases.
arsenic trioxide; asthma; eosinophils
Tissue recruitment and activation of eosinophils contribute to allergic symptoms by causing airway hyperresponsiveness and inflammation. Shape changes and mediator release in eosinophils may be regulated by mammalian Rho-related guanosine triphosphatases. Of these, Rac2 is essential for F-actin formation as a central process underlying cell motility, exocytosis, and respiratory burst in neutrophils, while the role of Rac2 in eosinophils is unknown. We set out to determine the role of Rac2 in eosinophil mediator release and F-actin-dependent shape change in response to chemotactic stimuli.
Rac2-deficient eosinophils from CD2-IL-5 transgenic mice crossed with rac2 gene knockout animals were examined for their ability to release superoxide through respiratory burst or eosinophil peroxidase by degranulation. Eosinophil shape change and actin polymerization were also assessed by flow cytometry and confocal microscopy following stimulation with eotaxin-2 or platelet-activating factor.
Eosinophils from wild-type mice displayed inducible superoxide release, but at a small fraction (4–5%) of human eosinophils. Rac2-deficient eosinophils showed significantly less superoxide release (p < 0.05, 26% less than wild type). Eosinophils lacking Rac2 had diminished degranulation (p < 0.05, 62% less eosinophil peroxidase) and shape changes in response to eotaxin-2 or platelet-activating factor (with 68 and 49% less F-actin formation, respectively; p < 0.02) compared with wild-type cells.
These results demonstrate that Rac2 is an important regulator of eosinophil function by contributing to superoxide production, granule protein release, and eosinophil shape change. Our findings suggest that Rho guanosine triphosphatases are key regulators of cellular inflammation in allergy and asthma.
Superoxide; Eosinophil peroxidase; Exocytosis; Calcium ionophore; Eotaxin-2; Platelet-activating factor
Rationale: The D6 chemokine receptor can bind and scavenge several chemokines, including the T-helper 2 (Th2)–associated chemokines CCL17 and CCL22. Although D6 is constitutively expressed in the lung, its pulmonary function is unknown.
Objectives: This study tested whether D6 regulates pulmonary chemokine levels, inflammation, or airway responsiveness during allergen-induced airway disease.
Methods: D6-deficient and genetically matched C57BL/6 mice were sensitized and challenged with ovalbumin. ELISA and flow cytometry were used to measure levels of cytokines and leukocytes, respectively. Mechanical ventilation was used to measure airway reactivity.
Results: The ability of D6 to diminish chemokine levels in the lung was chemokine concentration dependent. CCL17 and CCL22 were abundant in the airway, and their levels were attenuated by D6 when they were within a defined concentration range. By contrast, airway concentrations of CCL3, CCL5, and CCL11 were low and unaffected by D6. Allergen-challenged D6-deficient mice had more dendritic cells, T cells, and eosinophils in the lung parenchyma and more eosinophils in the airway than similarly challenged C57BL/6 mice. By contrast, D6-deficient mice had reduced airway responses to methacholine compared with C57BL/6 mice. Thus, D6 has opposing effects on inflammation and airway reactivity.
Conclusions: The ability of D6 to scavenge chemokines in the lung is dependent on chemokine concentration. The absence of D6 increases inflammation, but reduces airway reactivity. These findings suggest that inhibiting D6 function might be a novel means to attenuate airway responses in individuals with allergic asthma.
chemokines; lung; D6; allergic; transforming growth factor–; β
Steroid treatment of allergic eosinophilic airway diseases is considered to attenuate cell recruitment by inhibiting several chemokines and to cause eosinophil clearance through inducement of apoptosis of these cells. However, roles of these mechanisms in the actions of steroids in vivo have not been fully established. Also, as regards clearance of tissue eosinophils other mechanisms than apoptosis may operate in vivo.
This study explores anti-inflammatory effects of steroids instituted during either development or resolution of airway allergic inflammation.
Immunized mice were subjected to week-long daily allergen challenges (ovalbumin). Steroid treatment was instituted either amidst the challenges or exclusively post-allergen challenge. CC chemokines, goblet cell hyperplasia, occurrence of eosinophil apoptosis, and airway tissue as well as lumen eosinophilia were examined at different time-points.
Daily steroids instituted amid the allergen challenges non-selectively attenuated a range of chemokines, permitted egression of tissue eosinophils into airway lumen to increase, and reduced development of lung tissue eosinophilia. Steroid treatment instituted post-challenge selectively inhibited the CC-chemokine regulation upon activation, normal T cell expressed and secrted (RANTES), permitted continued egression of eosinophils into airway lumen, and resolved the tissue eosinophilia. Eosinophil apoptosis rarely occurred at development and resolution of the allergic eosinophilic inflammation whether the animals were steroid treated or not. However, anti-Fas monoclonal antibodies given to mice with established eosinophilia post-challenge produced apoptosis of the tissue eosinophils indicating that apoptotic eosinophils, if they occur, are well detectible in vivo.
Airway tissue eosinophils are likely eliminated through egression into airway lumen with little involvement of apoptosis and phagocytosis. Our data further suggest that therapeutic steroids may resolve airway inflammation by permitting clearance of tissue eosinophils through egression and inhibiting RANTES-dependent cell recruitment to lung tissues.
apoptosis; asthma; chemokines; glucocorticoids
In this investigation we have used a mouse model containing certain phenotypic characteristics consistent with asthma and IL-4- and CD40-deficient mice to establish the role of this cytokine and allergen-specific immunoglobulins in the initiation of airways hyperreactivity and morphological changes to the airways in responses to aeroallergen challenge. Sensitization and aerosol challenge of mice with ovalbumin resulted in a severe airways inflammatory response which directly correlated with the induction of extensive airways damage and airways hyperreactivity to beta-methacholine. Inflammatory infiltrates were primarily characterized by the presence of CD4+ T cells and eosinophils. In IL-4-deficient mice, the recruitment of airways eosinophils was impaired, but not abolished in response to aeroallergen. Moreover, the characteristic airways damage and hyperreactivity normally resulting from allergen inhalation were not attenuated. Induction of these structural and functional changes to the airways occurred in the absence of ovalbumin-specific IgE and IgG1, but IgG2a and IgG3 were detected in the sera of IL-4-deficient mice. CD4+ T cells isolated from both wild-type and IL-4-deficient mice given ovalbumin produced significant levels of IL-5 after in vitro stimulation. Treatment of IL-4-deficient mice with anti-IL-5 mAb before aeroallergen challenge abolished blood and airways eosinophilia, lung damage, and airways hyperreactivity. These results indicate that IL-4 is not essential for the development of IL-5-producing CD4+ T cells or for the induction of eosinophilic inflammation and airways damage and hyperreactivity. In response to sensitization and aerosol challenge, CD40-deficient mice did not produce ovalbumin-specific IgE, IgG isotypes, or IgA, and airways inflammation and hyperreactivity were not attenuated. Our results suggest that allergic airways disease can occur via pathways which operate independently of IL-4 and allergen-specific immunoglobulins. Activation of these pathways is intimately associated with IL-5 and eosinophilic inflammation. Such pathways may play a substantive role in the etiology of asthma.
Phosphoinositide 3-kinase (PI3K)/Akt pathway is linked to the development of asthma. Anti-malarial drug artesunate is a semi-synthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua, and has been shown to inhibit PI3K/Akt activity. We hypothesized that artesunate may attenuate allergic asthma via inhibition of the PI3K/Akt signaling pathway.
Female 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. 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. Artesunate dose-dependently inhibited OVA-induced increases in total and eosinophil counts, IL-4, IL-5, IL-13 and eotaxin levels in bronchoalveolar lavage fluid. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, IL-17, IL-33 and Muc5ac in lung tissues, and airway hyperresponsiveness to methacholine. In normal human bronchial epithelial cells, artesunate blocked epidermal growth factor-induced phosphorylation of Akt and its downstream substrates tuberin, p70S6 kinase and 4E-binding protein 1, and transactivation of NF-κB. Similarly, artesunate blocked the phosphorylation of Akt and its downstream substrates in lung tissues from OVA-challenged mice. Anti-inflammatory effect of artesunate was further confirmed in a house dust mite mouse asthma model.
Artesunate ameliorates experimental allergic airway inflammation probably via negative regulation of PI3K/Akt pathway and the downstream NF-κB activity. These findings provide a novel therapeutic value for artesunate in the treatment of allergic asthma.